PLASMODIOPHORALES JOHN S. KARLING Columbia Vnivernty m THE PLASMODIOPHORALES Including a Complete Host Index, Bibliography, and a Description of Diseases Caused by Species of this Order BY JOHN S. KARLING Columbia Unh'ernitif First Edition- PUBLISHED BY THE AUTHOR NEW YORK CITY 1942 COPYRIGHT, 1942, BY THE AUTHOR. ALL RIGHTS RESERVED. THIS BOOK, OR PARTS THEREOF, MAY NOT BE REPRODUCED IN ANY FORM WITHOUT PERMISSION OF THE AUTHOR. Dedicated to R. A. HARPER Oil tlie Occasion of the 80th Anniversary of His Birtliday PREFACE This thkatisk is part of a serios of leoturcs prc- sfiitcd to srailuatc and rosi-arcli students of mycol- ogy at C'oluniliia I iiivcrsity on tlif di'vi'lopnicnt. ori- gin, and pliyloju'iiy of the lower fuiifri. It liad been originally planned to incorporati' tliis material in a general treatment of the simiile. hiHagellate Ooniy- eete-like fungi. I.agenidiales, and Cliytridiales, hut inasnnieli as tlie Plasniodiopliorales at present a])- pear to lie a fairly eolierent iiliylogenetie group, it seems advisable to treat them separately. The Plas- niodiophorales are an important and significant group of organisms from the standpoints of [ilant pathology and phylogeny of the lower fungi. As de- structive parasites of crucifers and potatoes, some species cause serious economic losses of basic food crops. Phylogenetically. they possess certain devel- opmental phases which are strikingly similar to those of the Proteomyxa. Myxoniycetes. and simjile fungi — similarities which suggest either a common origin or parallelism in development. Although the Plasmodiopliorales have been stud- ied for more than a half century, no serious efl'ort to summarize the accumulated data was made until 1<)33 when Cook monographed the group. Cook gave a detailed description of the known genera and spe- cies and also discussed their cytology and develop- ment in relation to phylogeny. Unfortunately, this otherwise worthy and excellent treatise is marred by certain inconsistencies, based on the author's obser- vations, which are confusing and misleading to be- ginners in this field. Since that time, several new- genera and species have been added to the group. Particularly significant is the discovery of I.eding- ham. Couch, et al.. and Barrett that the zoospores are biflagellatc and heterocont and that thin-walled evanescent zoosporangia are a characteristic devel- opmental phase of the Plasmodiophorales. These discoveries have greatly modified our concepts of the group and make the present revision opportune, worthwhile, and essential. This book is intended primarily for graduate and research students of mycology and the lower organ- isms. Nevertheless, botanists and biologists in gen- eral as well as protozoologists and phytopathologists will doubtless find the summarized data, life cycle diagrams, and descri]>tions of diseases of consider- able value. As a treatise for research students, it nec- essarily includes nnicli that is questionable and con- troversial in nature and which ordinarily might be omitted or discussed more briefly. -Some of the data presented are of doubtful value and significance, in the author's opinion, but they are nonetheless in- cluded with as little bias as possible in order that students may draw their own conclusions and inter- pretations. Although the author agrees with Cook and others that lihi-owi/ja, Sorolpidiiim, .-ind .liiiso- mi/.rii are prob.-ibly synonyms of I.iiiiTa, these gen- era are discussed separately as doubtful members. Likewise, full treatment is given to the excluded genera and s])ecies, thereby making these dat.i .ivail- .■ible to research workers. The author's critical atti- tude and seeming skejitieism toward existing data on "akarvosis," extrusion of chromatin, sexuality, meio- sis, and other critical developmental phases of this group is not intended as a direct criticism of the veracitv and accuracy of certain workers, but rather to indicate how inconclusive present-day knowledge and interjiretations are and thereby to stimulate more intensive study of these phases. The Plasmodi- ophorales are unfavorable for cytological study be- cause of the minuteness of the nuclei. Likewise, the intramatrical habitat of all species makes direct ob- servation of gametic fusion, schizogony, etc., ex- tremely diflicult in living material. It is therefore to be expected that many data are conflicting and incon- clusive. Separate bibliographies are provided at the close of each chapter to expedite reference to literature on particular subjects, genera, and species. Since many of the cited papers are general in nature and relate to several genera, they have been listed several times, which makes the bibliograjjhy somewhat re- dundant. A host index is also provided with each spe- cies. Due to war conditions abroad, it has been im- possible to secure many of the publications relating to club root and powdery scab, so that the host index and bibliography of Plasmodiophora Brassicae and Spoiiflospora suhterranea are unfortunately incom- l)lete. In a bibliograjihy of this magnitude errors are likely to occur, and the author will appreciate having mistakes and omissions called to his attention. The glossary is purposely brief and relates almost en- tirely to terms used in the text. Tlie writer has drawn freely from the illustrations of authors in this country and abroad, to whom he is very grateful. The list of contributors is too long for individual mention, but full credit is given in the de- scriptions of the drawings. The life cycle diagrams presented in Chapters III and V have not been copied directly from other authors' illustrations but are based on their descriptions of the successive de- velopmental phases. The author feels particularly grateful to -Miss Amy L. Hepburn, Natural Science Librarian of Columbia University, for her unstinted help with the literature, without which this work would have been impossible. Columbia Vnivkbsitv New York City No\t:mbkr. 19U CONTENTS I'rct'iui' i CHAI'iKH I Introduction 1 (Tlossarv 2 Bibliography 3 CHAPTER II Cvtologv 4 "I'l-oniitosis" -l "Akdrvote" stage 10 Mciosis 12 Schizogony and Cleavage 14 CHAPTER III Sexiialitv and Alternation of Generations 15 I'liisinodiophora 15 Tetramyxa 16 Sorosphaera 16 Sorodiscns 18 Spongospora 18 Ligriicra 18 Pnlijmi/.ra aiiiJ Ortoiiiij.ra 18 Bibliogra])hy 20 CHAPTER IV Classification and Discnjjtion of Species 20 Key to genera 22 Plasmodiophora 22 Biological races 26 Reliition to bacteria 26 Relation to cancer 28 Bibliography .'J.'j Excluded species '.H Bibliography .■J6 Vll 5755H PLASMODIOPHORALES Tetramyxa 37 Octomyaxi 40 Sorosphaera 41 Sorodisciis 46 Membranosorus 52 Spongospora 54 Llgniera 58 Polymyxa 63 Doubtful genera 64 Rhizomyxa 64 Sorolpid'uim 66 Anisomyxa _ . . 68 Trematophlyctis 70 Pyrrhosorus 71 Excluded genera 72 Sporomyxa 74 Peltomyces 76 Cystospora 76 CHAPTER V Phylogeny and relationships of the Plasmodiophorales 78 Historical 78 Relationships with the Myxomycetes 79 Relationships with the Chvtridiales 85 Relationships with Woronina 85 Relationships with the Proteomyxa and Protozoa 88 Bibliography 91 CHAPTER VI • Diseases caused by species of the Plasmodiophoraceae 93 Club root of crucifers 93 Losses due to disease 93 Discovery of disease 93 Symptoms 93 Cellular relations between host and pathogen 95 Entrance and spread of parasite 96 Dissemination of parasite in nature 96 Environmental factors 97 Hosts and degree of infection 99 Control of club root 104 Sanitary practices 104 Seed, seed bed, and seedling disinfection 105 Disinfection of fields 105 lONTKNTS Liming .... Hasio fortili/crs . Soil drainage Croj) rotation Eradication of wild host Resistant varieties of erucifers Nature of suseeptibilitv and resistance . Gcograpliical distril)iition of club root and bibliog Powdery scab of potatoes Significance of disease . Predisposing factors Symptoms Cellular relations between host Control Sanitary practices Seed tuber disinfection . Soil disinfection . Effects of fertilizers . Resistant varieties Distribution and bibliography Species Index Subject Index Author Index nd pathogen raph no 115 117 117 118 118 119 120 129 129 129 130 i:31 132 132 132 133 133 133 137 137 139 C'hapUr 1 Introduction The Plasmodiophoralks iiicliulo one family of or- ganisms wliicli are often referred to as parasitie slime molds heeause tiiey arc i-liaraeterized by a multiniuleate ulasmodial stauv as in the trne slime molds and ))arasitize tilamentous fiingi, alu'ae. eryp- togams. and liiirher plants. While this eomnion name mav lie deseri|)tive. its use is unfortunate, since it suggests a relationsliip witli the Myxomyeetes which has not been definitely established. Most genera of this order have rather complex life cycles which in- clude zoosj)ores, amoebae, sjiorangiosori, zoosjio- rangia. secondary zoospores, plasmodia, cystosori, resting s))orcs, and probably isoniorjihic gametes. Sporangiosori and tliin-walled evanescent zoospo- rangia were first observed by Borzi in Rhizovii/jca hypoi/ea as early as 188 t. and later by Neniec ('11. '13) in Sorolpidiiim and Anis07ni/jra, but at that time the relationship of these genera to the Plasmodi- ojihoraccae was not clearly understood. Zoospo- rangia were subsetjuently rediscovered by Cook ('26). Cook and Schwartz ('30). I.edingham ('33. '3i. '3.5. '39). Fedorintscliik ('3.")), Coueli. et at. ('39) in Lignii-ra, Plasmodiophora, Poli/mi/.ra, Sponr/ospora, and ()ctomi/xa and are now generally believed to be a characteristic developmental phase of the order as a whole. The zoosporangia are re- garded by some workers as gametangia in which meiosis precedes gametogenesis, but this has not been conclusively ])roven. The s])orangial phase is followed by the develop- ment of a conijiaratively large multinucleate sporo- genous Plasmodium in which meiosis is reported to occur before or during cleavage into resting spores. The latter may remain loose and free of each other or unite in more or less compact cystosori. Upon germination, tlie resting spores ))roduee uninucleate amoebae or motile flagellate zoos|)ores. These cells are regarded by many workers as isomorphic gam- etes which fuse in pairs and thus initiate the diploid generation, but so little is known about sexuality in this order that nothing conclusive can be said as yet about the sexual nature of these cells. Some my- cologists contend that a true ])lasmodinm does not exist in the Plasmodiophorales on the grounds that the naked multinucleate tliallus is not formed by the coalescence of numerous mutually attracted amoebae in the manner described by Cienkowski ('63) for the Myxomyeetes. In so doing, these mycologists disre- gard the reports of Woronin ('77), Halsted ('93), Nawaschin ('99), Evcleshvmer ('01). Massee ('08), Osborn (']!), Kunkel ('15). Terby ('2t), Jones ('28). Home ('30). Cook and .Schwartz ('30). Milo- vidov ('31 ). I.edingham ('39). and others that amoe- bae as well as small plasmodia coalesce in Plasmodi- ophora, Sponf/ospora, Pol i/nii/.ra, etc. \\ hether or not these re])orts are accurate may be oi)en to question, because they are not all based on observations of liv- ing material. These data nevertheless exist in the liter.-iturc and must be given serious <'onsideration. I'urtherniore, the above-mentioned reasons for ex- cluding the term ))lasmodiuin from the Plasmodi- ophoraceae would also ])reclude its use in relation to the .Myxomyeetes according to recent data on this group. .lahn (11. '36), Skupienski ('28), Wilson and Cadnian (28), Cadman ('31), and others have shown that the ])lasmodium is initiated by fusion in pairs (if isomorphic gametes and that the zygotes may subsequently ingest unfused lia])loid amoebae as food material. Thus, the conception of a Plas- modium as Cienkowski interjjreted it has undergone considerable modification and is now used jirinci- l)ally as a deseri))tive term for the naked, multinu- cleate, assimilative phase of the slime molds. In this sense it may be equally well employed for the naked multinucleate thallus of the Plasniodioiihorales. Cook's use of the term myxamoeba for this stage is unfortunate, misleading, and obviously unwar- ranted. According to standard dictionaries .md glos- saries, the term myxamoeba relates to the naked, amoeboid, and usually uninucleate protoplasts formed by the germinating resting spores of the Myxomyeetes, and its introduction as a deseri])tive name for the naked multinucleate plasmodial stage of the Plasmodiophorales will lead to nothing but confusion. Likewise, his use of the term "swarm cells ' for the products of spore germination as a dis- tinctive contrast to the name "zoos])ores " for the flagellate cells formed in zoosporangia is not war- ranted at present and should be avoided. I.edingham and Barrett have clearly shown that the zoospores are biflagellate and hcterocont regardless of whether they are formed in zoosporangia or from resting s])ores and that there are no structur.il distinctions between the so-called swarm cells and zoosjiores. If in the future it is found tliat the resting spores form gametes and the s))orangia zoospores, or vice versa, the two products may then be distinguished and designated as gametes and zoospores, respectively. .Mtliough most s|)ecies of this order, except P. lirax.iicae and S. suhti-rratwa, a])pear to be compara- tively rare in occurrence, they are nevertheless world wide in distribution and have been re))orted from North and .South .\merica. .\frica, Kurope, Asia. Australia and several Atlantic and Pacific islands. Three s])ecies occur in fungi, algae, and cry))togams. while the remainder parasitize higher |)lants. All s))ecies, exce)>t members of the genus JAfj- n'tera, cause distortion of the host and marked changes in its cells. These changes involve enlarge- ment and divison of infected as well as of adjacent PLASMODIOPHORALES healtliy cells, with the result that conspicuous ex- cresences and galls are usually formed. However, only two species are economically important as para- sites. Plasmodiophora Brassicae and Spongospora subterranea are destructive pathogens of crucifers and potatoes, respectively, and cause the diseases commonly known as club root and powdery scab. While these diseases had been recognized since early times, their causative agents were not identified until the latter part of the 19th century. The discov- ery of P. Brassicae in hypertrophied roots of cruci- fers by Woronin in 1 877 may be said to have initiated the study of the Plasmodiophorales as a distinct group of organisms. A second genus, Tetramiixa, was found by Goebel in 1881, and in the same year Zopf created a new family, Plasmodiophoraceae, in the zoosporic Monadineae to include these genera. Two additional genera, Spongospora and Soro- sphaera were reported by Brunchorst and Schroeter in 1886, but the relationship of the former genus was not generally recognized until much later. Schroeter ignored Zopf's classification and created a new or- der, Phytomiixini, with one family, Phytomyxaceae, to include these genera as well as the legume tubercle organism which he redescribed as Phiitomijxa legii- minosarum. Inasmuch as Schroeter's Phytomyxinae was later ('97) incorporated in Engler and Prantl's Die Natiirlichen Pflanzenfamilien, it was widely recognized and accepted. Phiitomyxa as well as Plasmodiophora Alni and P. Elaeagni were excluded by Tubeuf and Smith ('97) and other pathologists in their discussions of the parasitic slime molds, but Schroeter's order and family names nonetheless con- tinued to be used. In 1909 Maire and Tison made an extensive review and study of these doubtful species and showed again that P. legiiminosarum, P. Alni, P. Elaeagni, Tylogonus Agavae, and Pseudocommis J'iiis have little or nothing in common with the true plasmodiophoraceous species. Since Phyiomi/.ra had already been excluded, they pointed out that the name Phytomyxaceae was no longer appropriate. They accordingly adopted Zopf's Plasmodiopho- raceae to include Plasmodiophora, Tetraviy.ra, and Sorosphaera and listed Schroeter's Phytomyxinae pro parte and Delage's Protomyxideae zoosporideae as synonyms. Apparently unaware of ]\Iaire and Ti- son's studies, some protozoologists nevertheless still continue the use of Schroeter's Phytomyxinae or some modification of this name. In the meantime, Sporomyra and Peliomyces had been added to the group, and following Maire and Tison's first paper, Ligniera, MoUiardia, Sorodiscus, Ostenfeldiclla, Cystospora, Tremaiophlyciis, Clath- rosorus, Memhranosorus, Polymy.ra and Octomyxa were successively discovered and included in the Plasmodio])]ioraceae. However, many of these gen- era have eitlier been merged or excluded entirely, so that the order includes at present comparatively few valid genera. The group as a whole was finally raised to ordinal rank by Cook ('28, '33), following a sug- gestion made by Schwartz in 191 K Taxonomically, the Plasmodiophorales have been bandied back and forth by protozoologists and my- cologists for more than half a century, and few work- ers are in agreement about tlie taxonomic position and relationships of this order. Its members have been included at various times in the Mycetozoa, Monadineae, Proteomyxa, Rhizopoda, and Chytri- diales. Some mycologists, particularly Gwynne- V^aughan, Barnes, and Cook ('33), have maintained that the Plasmodiophoraceae are not fungi and have arisen along independent lines from more primitive forms. However, the rediscovery within the last two decades of zoosporangia in this order and the ob- servations that biflagellate heterocont zoospores are produced in such sporangia and also from resting spores indicate a closer affinity with the simple fungi than was formerlv believed to exist. Glossary Akaryote stage, a nuclear stage in which little or no chromatin is visible in the nucleus. Binuclearity hypothesis, the theory that the micro- and macronuclei of infusoria contain the idio- and • trophochromatin, respectively, and that the ordi- nary nucleus of higher forms is accordingly a dual "amphinucleus. ' Blepharoplast, the basal granule at the point of in- sertion of each flagellum. Capillitium, sterile filamentous, simple, branched, or net-like tubes or fibers formed among spores in a sporogenous body. Chromidia, trophochromatin granules which are ex- truded from tlie nucleus into the cytoplasm. Chromidia hypothesis, the theory that the nuclei of rhizopods and other similar organisms contain idio- and trophochromatin. the latter of which is extruded into the cytoplasm as chromidia and de- generates or plays a dominant role in the differ- entiation of specialized structures. Chromidial stage, a nuclear stage during which the trophoeliromatin is extruded into the cytoplasm. Cruciform stage, equatorial ring stage of promitosis in the Plasmodiophorales during which the nu- cleole is elongate and forms a cross with the chro- matin ring. Cystosorus, a more or less compact aggregate of cysts or resting spores. Eucarpic, only a portion of the tliallus transformed into a reproductive organ; remainder of thallus vegetative. Extramatrical, outside of host, matrix, or substra- tum. Double-anchor stage, anaphase stage of promitosis in the Plasmodiophorales during which the arched daughter chromatin bands and nucleoli are con- nected by a chromatic strand and form a figure re- sembling a double anchor. Dumb-bell stage, more or less synonymous with double-anchor stage of promitosis. IXTKODUCTION Flaiiitliim, a wlii))-likc protdpl.-isniic ora:;m of loco- motion of zoospores, sw.inusporcs, and motile iiametes. (iaiiirtaiii/iiim, a ditlereiitiateci sac or vesicle which produces gametes. (iarlaiitl xtaf/r, a i>ropliasc stage of meiosis in wliicli the chromatin is aggregated as garlands at the nu- clear poles. UrttTOcoiit. (flagella) of unequal length. lltilocarpic. entire th.illus tr.insformed at maturity into a re]irodiictive organ. Ili/prrplasif, abnormal growth of tissue resulting from undue cell division. 11 fiperirophii, abnormal enlargement of an organ. II tipopla.li/, defective development due to insufficient nourishment and consequent cessation of growth. Ihtmoihallic, gameto])hytic or ha])loid thalli bi- sexual. Ileteroihallic, gametopliytic or liai)loid thalli uni- sexual. Ilnmophi/tic, sporophytic or diploid thalli bisexual. Hctfrophi/tic, sporojiliytic or diploid thalli uni- sexual. Ilaplomoiioccioitx, haploid generation bisexual = luimothallic. Ilaplodioecious, ha])loid generation unisexual ^ lieterothallic. Diplomonoeciotts, diploid generation bise.xual = homophytic. Diplodioecious, diploid generation unisexual = heterophytic. Ilaplosynoecious, haploid generation bisexual = homothallic ^ haplomonoecious. Ilaploheteroecioiis, haploid generation unisexual =: lieterothallic = diplodioecious. Diplosifnoecious, dililoid generation bisexual = liomophytic = dijilomonoecious. Diploheteroecioiis, diploid generation unisexual = heterophytic ^ diplodioecious. Ind'wchromatin, generative chromatin which is con- cerned with reproduction. Intramatrical, witiiin the host, matrix, or substra- tum. Isofjamy, fusion of structurally similar gametes. Isokont, (flagella) of equal length. Isomorphic, similar in shape and form but not in es- sential structure. Kari/ofjami/, fusion of gametic nuclei. Meront, a uni- or multinucleate product of schizo- gony. Planocyte, a motile cell. Plasmodiocarp, an irregular, sinuous, asymmetrical fruiting body or si^orangium of the Myxogastres. Plasmodium, a naked multinucleate protoplast cajia- ble of amoeboid movement. Plasmogamy, fusion of gametes, followed sooner or later by karyogamy. Promitosis, a pritnitive (?) type of intranuclear mi- tosis in lower organisms wliich is characterized by ill-defined cliromosomes and a large constricting, dividing nudeole. Protomilosis, .a variety of promitosis described by .Viexiefl' in which no clearly defined equatorial |)iate is formed. The perii)heral chromatin instead is distributed in a diffuse f.ishion between the polar halves of the divided karyosome. Psriidoplasmodiiim, a false plasmodium or aggre- g.ate of amoebae which retain their individuality; ch.iracteristic of the Acrasieae and l,.iby rinthulae. Psfiuhipodiitm, -.1 temjiorary i)roto))l.isniic extrusion in .•imoebac and jilasmodia which may be retracted or into wliich the whole mass may move. Saturn stage, equatorial ring stage of ])romitosis in the Plasmodiophorales during which the nudeole lies in the center of a ring of chromatin. Schizofiony , a jirocess of simjile or multi])!e division of a schizont. Schizont, a naked inultiiuicleate vegetative tliallus which undergoes simple or multiple division. Sorocarp, the fruiting structure of the Acrasieae. Sorus, a group of sporangia or resting spores. Sporangiosorus, a more or less compact sorus or ag- gregate of sporangia. Sporangium, a sac or vesicle which produces spores endogenously. Sporoci/st, a cyst which produces asexual spores. Sporogonic, relating to spore formation. Sporont, a thallus destined to form spores. Synkaryon, the zygotic nucleus following karyo- gamy. Thallus, the vegetative body of algae and fungi, without differentiation into root, stem, and leaf. Transitional stage, a term used by Winge to describe the transition in nuclear structure between pro- mitosis and meiosis in the Plasmodiophoraceae; synonymous to some degree with the so-called akaryote stage. Trophochromatin, somatic, vegetative chromatin which is active in nutrition. Zoocyst, a cyst in Monadineae which jiroduces amoe- boid or flagellate cells. '/.oosporangium, a s])orangium which produces zoo- s])ores. Zygote, the product of gametic fusion. bibliography: introduction Brunchorst, J. 1887. Berpens Mus. Aarberet. 186(): 219. Bor/.i, A. 1884. Klii/.oinyxa, nuovo ficomicete. Messina. Cadniiui, E. .T. 1931, Trans. Roy. Soc. Edinhurph .57: 93. Cicnkowski, L. 18();i. .lahrb. Wiss. Bot. '.i: +00. Cook, W. U. I. 19J(i. Trans. Brit. Mycol. .See. 11: 191). . I9J8. New Fhytol. 27: 240. . 19.33. Arch. I'rotistk. 80: 179. , and E. ,1. .Schwartz. 1930. Philos. Trans. Uoy. Soc. London 218B: 283. C ouch, .r. N., .1. I.eitner, and .\. WliiflVn. 1939. .lour. Eli.slia .Mitchell .Scl. .Soc. ,7.5:399. Esmarch, F. 1921. Die Kranke Pflanze 1: 169. Eyclesliyincr, A. C. 1901. ,Iour. .Mycol. 7: 79. Erdorintschik, X. S. 1935. Summ. Sci. Wk. Inst. PI. Pro- tect. I.enin).'rad I93,i:69. Fit/.i)atrick, H. .M. 1930. Tlic lower fungi Phycomycetes. New York. PLASMODIOPHORALES Gw.vnne-Vaiif;han, H. C. I., and B. Barnes. 1937. The structure and development of the funiri. 2nd ed. Cam- bridfre. Halsted, B. D. 1893. New Jersey Agr. Exp. Sta. Kept. 1893: 33;.\ Home, A. S. 1930. Ann. Bot. 44: 199. Jahn, E. 1911. Ber. Dciit. Bot. Gesell. :^9: 231. 1936, Ibid. 54: air. Jones, P. M. 1928. Arch. Protistk. 6;2: 313. Kunkel, L. O. 1915. Jour. Apric. Res. 4: iJ65. Ledinfrham, G. A. 1933. Phytopath. 23: 30. . 1934. Xature 133: ,534. 1935, Ibid. 135: 3994. . 1939. Canad. Jour. Res. C, 17: 50. Maire, H., and A. Tison. 1909. Ann. Mycol. 7: 22n. Massee, G. 1908. Jour. Bd. Agric. London 15: 592. Milovidov, P. F. 1931. Arch. Protistk. 73: 1. Nawaschin, S. 1899. Flora 80: 404. Xemec, B. 1911a. Ber. Deut. Bot. Gesell. 29: 48. . 1911b. Bull. Int. Empr. Fran. Joseph Acad. Sci. 10: 09. 1913, Ibid. 18: 18. Osborn, T. G. B. 1911. Ann. Bot. 2.o: 211, 327. Schroeter, J. 1880. Cohn's Krypt. Fl. Schlesiens 3: 133. . 1897. Engler und Prantl, Die Xat. Pflanzenf. 1,1:7. Schwartz, E. J. 1914. Ann. Bot. 38: 227. Skupienski, F. X. 1938. Acta Soc. Bot. Poloniae 5: 355. Terby, J. 1934. Bull. Roy. Acad. Belg. 11:1. Tubeuf, K. F., and W. G. Smith. 1897. Diseases of Plants. London. Wilson, M., and E. J. Cadman. 1938. Trans. Roy. Soc. Edinburgh 55: .555. Woronin, M. 1877. Arb. St. Petersburg Nat. Gesell. 8: 109. Zopf, W. 1884. Die Pilzthiere oder Schleimpilze. Encyklop. der Xaturwiss. 3: 139. Chapter II Cytology "Promitosis" Cytological studies of the Plasmodiophorales dur- ing the pa.st four decades have centered primarily on the type of nuclear division in the plasmodiuni, the so-called "akaryote" stage, nieiosis, karyogamy, schizogony, and cleavage. Nuclear division in the Plasmodium has been described by most workers as promitotic and fundamentally similar to that which occurs in the Umax group of amoebae and other lower organisms. So consistently has this type of division been rei)orted that many students have regarded promitosis as one of the most diagnostic characters of the wliole order, and one which distinguishes the Plasmodiophorales from all other fungi and higher plants. Cook ('28) in particular has stressed this character as follows: "The diagnostic feature which characterizes the Plasmodiophorales is their two methods of nuclear division, and failing to show evi- dence that both promitosis and mitosis occur in the life cycle, and that these two types are separated by a stage iu which at any rate ])art of the chromatin is extruded into the cytoplasm, no new fungus should be included in this group." At the same time, other workers have maintained that these divisions are typically mitotic with well-defined chromosomes, centrosomes, and astral rays. There is thus sharp disagreement concerning karvokinesis in the Plas- modium, and inasmuch as the presence of promitosis has been regarded as an index of relationship to the amoeba, a full discussion of the so-called vegetative divisions in the Plasmodiophorales is essential to an understanding of this order. Nawaschin ('99) was the first to observe the char- acteristic appearance of these divisions in Plasmodi- ophora and to point out that they are different from those which occur immediately before or during spore formation. He nevertheless described the for- mer mitoses as karyokinetic and regarded ('01) the presence of the two types of division as an indication of nuclear dimorphism — a view much in vogue among the protozoologists of that time. Nawaschin's obser- vation was confirmed by Prowazek ('02, '05), Maire and Tison ('09), Blomfield and Schwartz ('10), Schwartz ('10), Winge ('13) and Lutman ('13) for other species and genera. Prowazek, particularly, and later Blomfield and Schwartz, also stressed the resemblance of the vegetative divisions to those which had been described by protozoologists in cer- tain coccidia and amoebae. In the meantime, Nagler ('09) had proposed the term promitosis for the type of nuclear division found in Amoeba froschi, A. lacu.itrh, etc., which he inter])reted to be a transition between amitosis and mitosis. In these divisions neither chromosomes nor well-defined spindles are formed, according to Nag- ler. Division is intranuclear, and the large endo- some or karyosome functions as a division center. The latter elongates, and as it constricts the chro- matin aggregates and forms a band across the equa- tor of the nucleus. The karyosome then divides into two bodies, and as these migrate toward the poles the band of chromatin splits lengthwise. Each half ac- companies a karyosome to the poles, and both are there incorporated in the daughter nuclei. Subse- quent workers, particularly Chatton ('10) and Alexieff ('13) confirmed in broad outlines Nagler's observations, but distinguished and defined other similar and more advanced types of "primitive" mi- tosis in amoebae. Since Nagler's time the term pro- mitosis as a distinctive term has lost much of its ori- ginal significance and has been employed rather gen- erally for mitosis in lower organisms which are char- acterized by an intranuclear spindle and chromatin derived wholly or in part from a large karyosome. In the process of division the latter is said to elongate and divide and function as a nucleo-centrosome. However, with tlie use of more refined and specific fixatives and stains, many of the cases reported for- CYTOI.OUY mcrly as |)romitt)sis in protozoa, fimu;i. and aljiaf have provin to lie tyi)ical mitosis. NcvtTtlulfss. stmli-nts of tlit- I'lasmodiopliorales ininu-diatcly rt-c-ognlzcd tin- similarity of Niiglfr's jironiitotif divisions in Amoeba to tliosi- in tliis vt-jjo- tativf Plasmodium, and in 1!)11 Main- and Tison adopted Niiirlcr's tt-rni as descriptive of these Latter divisions. Sulisecpient workers, ineliidiiiu: Cook (''Jii, "28. '33), Cook and .Seliwart/, (,'-'!). ;}()). I.edingliam ('39). and Coueli <■( al. ('39) have used the term protoniitosis. a variety of promitosis described by Alexieff. Pavillard ('10). Wcrnham ('35). and oth- ers have employed the term "cruciform" division. Althousth they fijjured the same tyjie of division. Neniec ('11. '13), l-'erdinandscn and Wins;e ('20), and Milovidov ('31, '32. '33) avoided extensive use of these terms, while Osliorn ('U) described the ve!i:etati\c division in Spoiif/o.spora as amitotic. His figures and deserijition of the jirocess are nonetheless similar to those of previous and subsequent workers. Favorski objected to the contention that promitosis is specifically characteristic of primitive animals and the lMasniodioi)horaceac and [lointed out that the karyosome and eliromatin may behave in a similar manner during mitosis in hisjlier ))lants. Terby ('32) likewise condemned tlie use of promitosis for these divisions in Plasmndiophura on the grounds that chromosomes are present and the daugliter nucleoli are formed anew from granules in the telophase nu- clei and not by division of a mother nucleole. Home ('30) and Webb ('3.5) also contended that the vege- tative divisions are tyjjically mitotic in Spongospora and Suro.sphaera and thus contradicted all previous workers who m.iintained that distinct chromosomes are not )irescnt. Two main view))oints have thus been ])resented by these cytologists : one that the vegetative divisions are premitotic and fundamentally similar to those in certain amoebae: the other that they are typically mitotic with well-defined cliromosomes. Prowazek, Maire and Tison. .Schwartz, and Cook in ])articular have cmi)hasizcd the former view, and their accounts of the vegetative divisions may be taken as represen- tative of those who held that these mitoses are quite unlike anything present in other fungi and higher plants. Terby, Home, and Webb may be looked upon as re])resenting the other viewpoint. For the sake of com))arison. drawings representative of both views have been brought togetlier in Plate I and contrasted in turn with those illustrating jjromitosis in certain amoebae. The resting nucleus of amoebae and i)l;ismodia of the Plasmodioi)horaceae is quite small, so that its structure is difficult to see and determine with cer- tainty. Nawaschin described the chromatin in Plas- mod iipliDra as a s])Oiigy. faintly-stainable reticulum tliroughout the nucleus, while Prowazek figured the nuch-i as having an alveolar achromatic structure with several interspersed granules and a large cen- tral nucleole lying in a clear zone. In other nuclei the achromatic material was found to be radially ori- ented on the nucleole (fig. 1). giving the nucleus a wheel-like a))|)earance. Fn Hi>ri>iij>hafia .-ind 'I'l-tra- iiu/.ra, Maire and Tison figured tiie rt'sting nucleus as devoid of a chromatin reticulum (fig. 2) with the nucleole lying in a \ acuole-like ele.ir sp.iee filled with hyaloplasm, and numerous granules distributed on the inner periphery of nuclear membrane. They ( '09) did not, however, regard these granules as true I lirom.itin but instead as secretory chromidia derived t'riiui the karyosome .-iiul destined to ))ass out into the i\toplasm. In Spoiii/oxporu, on the other hand. Os- liorn figured .1 wheel-like nucleus with numerous chromatin granules distributed on radially oriented liniu threads (fig. !•), but he likewise believed that these granules had been derived from the karyosome. Of the more recent workers. Cook, and Cook and Schwartz have maintained that in Ligniera and Plax- mod'iophora the chromatin is aggregated solely in a layer around tiie inner i)eripliery of the nucleus (fig. 5) with the result that the nucleole ai)i)ears to lie in a clear vacuolate s))ace. but their observations have not been confirmed. Cook's ('28) studies on Lif/tiiera, however, were made from unsectioned ma- terial stained in toto, which is obviously unfavorable for study of nuclear details. Although there is thus considerable difference of opinion among these cytologists as to the structure of the nucleus and the presence of a chromatin reticu- lum, the "wheel" tyiie of resting nucleus neverthe- less has been figured most often and shown to occur in Plasmodiophora, Sponr/ospora, Soros pharra, Lif/- niera, Sorodiscus, and Polifmyxa. Milovidov's ('32, '33) observations on resting nuclei of P. Brassicae stained by Feulgen's method are particularly jierti- nent in this relation. In such prei5arations the karyo- some. linin. and granules are colorless, and the only visible structure is the faintly-stained luiclear mem- brane. Milovidov, nonetheless, believed that small chromatin bodies are present around the inner peri- phery of the nucleus. According to Nawascliin. the early ])rophases of the vegetative divisions in Plasmodiophora may be recognized by the emergence of distinct granules in the nucleus (fig. (5) wliich have a markedly different staining reaction from the karyosome and are not in genetic connection with the latter. Their origin is quite distinct from that of the karyosome, in Nawas- chin's o))inion. These granules later unite and form an equatorial ]jlate or band. Newaschin's observa- tions were confirnied by Milovidov's ('32, '33) studies which involved Feulgen's nuclear reaction method. As the nuclei enter the )iro))hases. chromatin granules and threads become visible in the nuclear cavity, and these eventually form an equatori.d ring (fig. .50). Prowazek ('0.5), on the other hand, de- scribed the karyosome or "Inncnkorper" as enlarging and difl'erentiating into a faint-staining achromatic substance and a denser chromatic material (fig. 7). The l.itter sul)stance then separates into a globular luiclcole and a half moon-shaiied row of granules (fig. 8), out of which the equatorial ring is formed (fig. 9). Maire and Tison ('09), like Nawaschin, noted the emergence of gramdes on the linin threads PLASMODIOPHORALES in Soiosphaera during tlie prophases of promitosis (fig. 10, 11), but they contended tliat the granules are derived from the karyosome and subsequently aggregate around the latter as an equatorial ring. Blomfield and Schwartz (10) and Osborn ('11) have figured much the same type of prophases in S. J'erouicae, L. Jitnci, and »S'. suhicrranea. I.utnian likewise reported the presence of chromatin granules in the prophases in Plaxmodiophora. "These gran- ules had been previously concentrated as a hollow sphere enclosing the tropochromatin of the central body" (karyosome), and as the prophases progress the granules of idiochromatin separate from the karyosome and form a spireme, according to I.ut- nian. In Sorodiscu.i Winge also reported a separation of idiochromatin and tropochromatin (fig. 13) in the karyosome in preparation for division, the former giving rise to a thin equatorial plate and the latter forming the nucleole. He believed that in the resting nucleus the idiochromatin may "be partly resolved in the tropochromatin. which later forms the chromo- philous filaments radiating from the caryosome." Cook ('26, '28) and Cook and Schwartz ('30) failed to observe any marked prophase stages in Lir/niera and Plasmodiophora but asserted that the peripheral layer of chromatin which is present in the resting nu- cleus condenses and becomes aggregated in a ring around the karyosome (fig. 14). Shortly thereafter the spindle fibers appear in the nuclear cavity and form a fusiform intranuclear spindle (fig. 1.5) at right angles to the chromatin ring, which in the mean- time lias expanded and drawn away from the central nucleole. Manj- of these cytologists have figured the chromatin ring as a solid continuous band, but Maire and Tison ('11) and Winge reported it to be com- posed of numerous granules and chromosome-like PLATE 1 Fifr. 1. Resting nucleus, P. Brassicae, showing wheel-like structure. Prowazek, '0.5. Fig. -2. Resting nucleus, T. parasitico , with karyosomic granules at peri])hery. Maire and Tison, '11. Fig. 3. Uninucleate amoeba, S. V eronicae , with centro- some and astral rays. Maire and Tison, '09. Fig. -1. Resting nucleus, iS. siibterraiiea, with wheel-like structure. Osborn, '11. Fig. 5. Resting nucleus, L. Juiici, with chromatin around inner periphery of nucleus. Cook, '^8. Fig. a. Early prophase, P. Brasxicae, showing numerous chromatin granules. Nawaschin, '99. Fig. 7. Early prophase, P. Briisslrne. showing separation of idiocbromatin and tropbocliromatin in the karyosome. Prowazek, I.e. Figs. 8, 9. Differentiation of nucleole and chromatin ring. P. Brasnicue. Prowazek, I.e. Figs. 10, 11. Prophases, S. Vfroniciie, showing separation of idiochromatin and its accumulatin on the linin. Maire and Tison, '09. Fig. 12. Early prophase nucleus, L. Juiici, with wheel- like structure. Schwartz, '10. Fig. 13. Separation of idio- and Irophochromatin in karyosome during early prophase, S. Callitrichii). Winge, '13.' Fig. 14. Early prophase, L. Jiinci, showing formation of chromatin ring around nucleole. Cook, 'J8. Figs. 15, 10. "Saturn" stages of promitosis, L. Juiici. Cook, I.e. Figs. 17, 18. "Cruciform" stages with elongating nu- cleoli, L. .IiincI and T. TriS'. xiibterroiiea. Home, I.e. Figs. 40, 41. Late prophases, S. Veronicae, with four elongate chromosomes. Webb, I.e. Figs. 42, 43. Later stages, 8. Veronicae. Chromosomes showing prophase split. Webb, i.e. Fig. 44. Polar view of equatorial plate, S. Veronicae, with four split, twisted chromosomes. Webb, I.e. Fig. 45. Early equatorial plate, S. Veronicae, with four Ll-shaped chromosomes. Webb, I.e. Fig. 48. Equatorial plate or "Saturn-stage," S. Veroni- cae, witb four chromosomes end to end in a ring around the constricted nucleole. Webb, I.e. Fig. 47. Similar stage, P. Brasnicae. with nucleole break- ing up into globules. Terby, "32. Fig. 48. "Saturn-stage," in S. nuhterranea with three of the four chromosomes arranged in a ring. Home, I.e. Fig. 49. Oblique view, N. Veronicae, of same stage. Webb, I.e. Fig. 50. Equatorial plate, P. Bra,.^< © 34 35 36 37 38 39 40 ® 41 42 43 % 44 ^ -=>S (g 49 50 51 52 45 53 46 47 w 48 9^^ ^ 54 ^ 55 56 © 60 61 62 Nuckiir I)i\ isioii 63 ^ v.: 64 \ ^r V 65 8 PLASMODIOPHORALES bodies in Tetramy.ra (PI. 5, fig. 5) and Sorodisciis (PI. 7, fig. 12), which lends support to tiie later views of Terby, Home, and Webb that definite chromosomes are present in the vegetative divisions. The origin of the spindle has not been clearly dem- onstrated in promitosis. Wiiether it originates frcmi achromatic linin material, tropiiocliromatin, or in relation to centrosomes and asters is not sufficiently known. Nawaschin, Favorski, Osborn, Cook and Schwartz, and Webb found no centrosomes and asters during the vegetative divisions in Plasmodio- phora, Lic/niera, and Sponc/ospora, but Prowazek, Maire and Tison, Winge, Lutman, Nemec ('13), Home, and Milovidov ('31) observed them in P/lit ends become visible (fig. t2). indi- cating a splitting of the chromosomes in jirejiaration for division. Uj) to this time the nueleole reni.iiiis more or less globui.ir. but it soon begins to elongate in the direction of the poles. The chromosomes then become arranged end to end in .-in irregular, liroken ring in the equator of the nucleus (fig. 13). \ jiol.ir view of such a stage is shown in figure H- with the s))lit .and twisted chromosomes lying near the i)cripli- ery of the nuclear menibrane. Following this stage, ihev contract and thicken, so th.-it the longitudinal split is no longer \ isible (tig. Ki). The ehroniosomes. nonetheless, retain their iiuiividu.ility . according to Home's and Webb's dr.-iwings. .'is is shown by the breaks in the equatorial ring (fig. K>. IS, li)). This ring stage persists for a comparatively long time and is the one most frequently observed in the vege- tative divisions. ,\eeording to Home's and Webb's figures, the elongate nueleole may become slightly constricted at this stage in preparation for division (tig. Vd. IS). .Miss Terby ('"^3). however, found that the nucleolar changes vary considerably in P. Brassicar. Instead of constricting and dividing more or less equally, it may fragment into two or more unequal parts (fig. Vl) or move intact as a single body to one of the poles. Oftentimes, jiarts of it remain stranded be- tween the daughter nuclei (fig. GK 65) as in higher plants. The metaphase split reapjiears first in the median region of the chromosomes (fig. 51 ) at the conclusion of the equatorial ring stage and travels outward to the ends, which suggested to Webb that the spin- dle fiber attachment is median. As the chromosome halves separate, two daughter rings are formed (fig. 52) which migrate toward the opposite poles (fig. 33-57) until they reach the ends of the elongate nueleole (fig. 56, 58. 59). According to Webb, the nueleole in Sorosphaera does not constrict as a rule until telophase (fig. 57-59). The two parts finally separate and become surrounded by daughter ehro- mosomes (fig. 60) at the poles of the nucleus. The nuclear membrane then constricts and divides in much the same manner as the nueleole .and thus forms the daughter nuclei (fig. 61). The ciiromosomes ad- iiere to the nueleole at first, but later sejiarate from it. Miss Terby. however, maintained that the nucleoli are formed anew at each telophase in P. Brassicae. .\fter the daughter nuclei have been formed, the chromatin mass gives ofl' material which unites to form the daughter nucleoli (fig. 62. 63). .As to the origin of the daughter nuclear areas. Miss Terby ('23) reported that they begin in the projihases as two hyaline vesicles on the jiolar sides of tiie nu- eleole. As the latter elongates, divides, and the two segments separate, the vesicles ])recede them to the |)olcs of the nucleus. The vesicles then pass through the nuclear membrane at the ))oles and expand, and shortly thereafter the d.aughter nuclcol.ar segments and chromatin enter .uid are thus incor|)or;ited in the vesicles. The boundaries of the vesicles become the nuclear membranes and thus constitute the limits of the daughter nuclei. In a later paper, however. Miss Terby ('32j modified this account and rejiorted 10 PLASMODIOP MORALES that the polar vesicles contract to small globular areas surrounded by granules from which astral rays radiate, as noted elsewhere. Thus the vesicles them- selves do not become the nuclei, but tlie daughter nu- clei are formed in the areas occupied by the vesicle before contracting. The type of division described by these three workers is distinctly mitotic and, except for the be- havior of the nucleole, according to Home's and Webb's figures, is fundamentally similar to nuclear division in the higher plants. Miss Terby, as noted before, held that the nucleole also undergoes the same changes as in the higher plants, so that there is no difference in this respect either. On the other hand, the divisions figured by Home and Webb are also similar to tlie promitoses illustrated in figures 1 to 23. The chief difference is the presence of chromo- somes. It is not improbable, as Webb contended, that the earlier workers overlooked the early prophases and the origin of the chromosomes and that their fixation and staining technique did not differentiate chromosomes in the equatorial ring. As noted else- where, the nuclei of the Plasmodiophorales are quite small, and their structure is difficult to interpret. The use of more specific and refined technique in inten- sive study of the early prophases and equatorial ring stages may thus possibly eliminate the present con- troversy on the nature of the vegetative divisions. In tliis relation it is to be noted tliat typical mito- sis without large nucleoli has been reported in the vegetative zoosporangial stage of Ligniera, Plasmo- diophora, Polf/mt/J-a, and Ociom_i/xa by Cook ('26, '28), Cook and Schwartz ("30), Ledingham ('39), and Miss Whiffen ('39). In these as well as other genera the zoospores from germinating resting spores develop into plasmodia which eventually cleave into uninucleate segments — the rudiments of zoosporangia. These segments develop walls, and their nuclei divide twice to several times in a strictly mitotic manner in preparation for zoospore forma- tion. Cook and Schwartz reported that up to tlie time of cleavage into zoosporangial segments the nuclei in the plasmodia of Ligniera and Plasmodiophora di- vide promitotically, but in Polymyxa I^edingham re- ported that division in the thalli which form zoospo- rangia is mitotic from the start. Miss Whiffen also found that the divisions in the zoosporangia of Octo- miiia are mitotic. These authors thus reported a regular alternation of mitosis and promitosis. The zoosporangial stage is characterized by mitosis, then follows a phase of premitotic division in the early de- velo))ment of the sporogenous plasmodium which is terminated by the so-called transitional stage, and finally two meiotic divisions. Inasmuch as the divi- sions in the zoosi)orangia are mitotic and very simi- lar to the two divisions at sporogenesis. Cook ('26, '28, '33) and Fedorintschik ('3.5) concluded that they are meiotic in Ligniera and Plasmodiophora, respectively. In P. Brassicae, however. Cook and Schwartz described them as merely mitotic. In an attempt to explain the alternation of meiosis and pro- mitosis in this species, they proposed the theory that promitosis is characteristic only of diploid nuclei, a theory which is contradicted by their own observa- tion that the first meiotic division of the diploid nu- cleus in spore formation is indirect and not pro- mitotic. Furthermore, if Cook's ('28, '33) previous report is correct that the primary nucleus of the in- cipient zoosporangia in Ligniera is diploid (and un- dergoes meiosis), it should accordingly divide pro- mitotically. However, he described and figured such nuclei as dividing mitotically. The report of typical mitotic divisions during zoo- spore formation, promitosis in the developmental stages of the sporogenous plasmodium, and the re- occurrence of mitosis during the reduction divisions nevertheless raises numerous questions on the signifi- cance of this alternation (if it actually does occur), and it is thus obvious that future studies of karyo- kinesis in the Plasmodiophorales must be closely correlated with the various developmental phases. "Akaryote Stage" The period of vegetative divisions in the develop- ment of the sporogenous plasmodium is reported to be followed shortly by the so-called "enucleate." "akaryote," "chromidial" or "transitional" stage. According to most workers, this phase is charac- terized by a reduction in size and disappearance of the karyosome, comparatively empty, vacuole-like nuclei, and the presence of numerous deeply-stain- able bodies or chromidia in the cytoplasm around the nuclei. Nawaschin first observed this stage in Plas- modiophora in 1899. and since that time it has been reported by most subsequent students for the other genera of this order. In the opinion of many cytolo- gists it is thus as constant and diagnostic a character of the Plasmodiophorales as promitosis. Stages in the development of the akaryote stage are shown in Plates 2 to 13, which illustrate the life cycles of all the plasmodiophoraceous genera, and will not be illustrated separately at this point. After the vegetative divisions have been completed, the karyosome decreases in size as the somatic or tro- phochromatin is extruded into the cytoplasm in the form of secretory chromidia, according to Prowazek ('05), Maire and Tison ('09), and others. Maire and Tison regarded this extrusion as a cleansing process by which the generative chromatin is separated from the nutritive chromatin in preparation for the sporo- gonic divisions which follow. As a result of this ex- trusion, the nuclei, when stained with haematoxylin. appear comparatively empty and devoid of stainable material and frequently have the appearance of vac- uoles in a cytoplasm filled with deeply-stained chro- midia. According to Blomfield and Schwartz, Schwartz, and Osborn, extrusion of chromatin in Sorosphaera, Liqniera, and Spongospora takes place along the linin threads until the chromatin reticulum and karyosome have disappeared. These workers be- lieved that the nuclear membranes also disappear during this stage. In L. Jiinci, Schwartz described the process as follows: "the nuclear membrane dis- t YT()I.<)(iY 11 appc.-irs. ;in(l tlic k.'irvosdiiic (liiuinislios in size ;iikJ finally disappoars also, so that wt- have a number of vacuoles more or lic ehronu)somes are closely associated on the equatorial ])late and in metai)hase and often appear as an irregular band or row of connected globules, so that the ))rofile and ))ol;ir views ;ire not very ciiaracteristic of hetero- ty|)ie divisions. In Spiitifidxpora, however. Home figured the chromosomes .-is short .-md thick with con- s|)icu()us intervening ga))s in the t(i\iatorial jilate, wliieli makes it possible to recognize and count the individual members. At this stage tliey may often show four blunt ends, which indicates their tetrad nature, according to Home. With the excei)tion of Winge, most cytologists held that the homologues separate at metapliase of the first division and move to the ))oles where they are incorporated in the daughter nuclei. In Soro- xphnera, ^A'ebb found the late ana))hase and telo- phase chromosomes to be double, which suggests that the equatorial split for the homeotypic division oc- curs quite early. Cook ('28) failed to see nuclear membranes in the late telo)ihases of L. Jitnci and thus concluded that they are no* formed between the first and second divisions. All previous and subse- quent workers, however, have shown that a well- defined membrane develops around the telo))hase groups of chromosomes and that daughter nucleoli are subsequently formed. Interkinesis is usually short in duration. In P. Brassicae Miss Terby ('2f) reported that the telophase nuclei go directly into the prophases of the next division, but in Spongo- spora wheel-like resting nuclei and distinct pro- ))hases may intervene between the two divisions. The second division is likewise mitotic or indi- rect but considerably smaller in size than the first one. Palling to count the chromosomes. Cook and Schwartz regarded this size difference as ))roof that these two divisions are res])eetively hetero- .ind ho- meotypic. a criterion which is obviously of no critical \alue in this respect. Osborn. Milovidow \\'ernham, Whitf'en. and others also made the s.ime assuiu))tion without counting the chromosomes. On the other hand, Maire and Tison, Winge. Terby. Home, Yendo and Takase. and Webb based their contention on a numerical reduction in chronuisome nmnber dur- ing these divisions. Whetlier or not their chromosome counts are accurate remains, however, to be shown from more intensive study of these divisions. The chromosomes of the PIasm(>dio|)hor.iles are quite small and are not always clearly defined on the equatorial plate, so that it is difficult to make accu- rate counts. Xevertheless. numerous attempts have been made, as is shown in table 1 . The numbers are low multi|)les of 2, with 8 )ire- dominating as the di])loid inniiber. In .S. V iron'icae Maire and Tison reported Hi .and 8 chromosomes, but Webb later found only 8 and i. \\'inge. as noted be- fore, described the first division as vegetative or so- 11 PLASMODIOPHORALES Table 1. Chromosome numbers in the Plusmodiophorales. Species Author Sorosphaera Veronicae Maire and Tison, '09 S. Veronicae Webb, "35 Tetramyxa -parasitica Maire and Tison, '11 T. Elaeac/ni Yendo and Takase, '32 Sorodisciis Callitrichis Winge, '13 S. rudicicohis Cook, '31 S. Heternntherae Wernham, '35 Plasmodiiiphora Brassicae Maire and Tison, '09 P. Brassicae Lutman, "13 P. Brassicae Terby, 'H 4 P. Brassicae Nawaschin, '34 ? P. Brassicae Tones, ^-2S ? Spongospora subterranea Osborn, '11 ? S. subterranea Home, '30 4 Number 3' sy 8 16 4 8 i 8 6 12 4 ■; o 4 » 4- 6 ? 8 ^ 8 6-8 8 8(?) 8 matic and recorded 16 individual cliromosomes asso- ciated in eight pairs on the equatorial plate, which separated and were distributed to the daughter nu- clei. In the second division this number was halved to four. Winge's confusion as to the nature of the respective divisions has led to tlie belief that the chromosome numbers in S. Callitrichis are 16 and 8. but it is evident from Winge's report that 4 should be recorded as the reduced number. In P. Brassicae four workers have recorded 8 as the diploid number, and curiously enough this is the number of bodies figured earlier by Prowazek ('05) in the transitional phase. In Spongospora subterranea, Osborn figured polar views of the second division with 7 chromosomes, which corresponds closely to the number later re- corded by Home for the first division. It is not im- probable, however, that Osborn's figure relates to the first meiotic division. Schizogony and Cleavage Vegetative multiplication of young plasmodia by division, segmentation or fragmentation has been re- ported for all genera of the Plasmodiophorales ex- cept Memhranosorus and Octomifxa. In Ligniera it is said to be lacking entirely or reduced to the forma- tion of a few daughter segments, while in T. Triglo- chinis and P. graminis true "multiple division" has been re])orted. Nawaschin did not observe segmenta- tion in Plasmodiophora, but he believed that its oc- currence is the only plausible explanation of the fre- quent presence of numerous uni- and multinucleate amoebae and plasmodia in a single host cell. Since that time most cytologists have reported its occur- rence, although none of them, with the ])ossible ex- ception of I.edingham, actually observed it in liv- ing material. Like Nawaschin, they found several amoebae and plasmodia in the same cell and assumed that the former were the products of fragmentation. Maire and Tison ('10) found similar stages in Soro- sphaera but interpreted them as fusion stages of amoebae and young plasmodia in the formation of the s))orogenous plasmodium. Brasil, however, sug- gested that these stages relate instead to fragmenta- tion or scliizogony — a suggestion which Maire and Tison adopted. These workers thus introduced the protozoologists' term "schizogony" as descriptive of the vegetative fragmentation of the plasmodium, and since that time it has been rather widely adopted. Pavillard ('10), however, contended that schizogony in Sorosphaera, as described by Maire and Tison, resembles plasmotomy instead of true schizogony as in Trichosphaerium sieholdii (Doflein, '09, '27) and Hepatazoon anis (Wenj'on, '26), for example. He thus restricted the term schizogony to the "multiple division" of Doflein, while Maire and Tison ('11) interpreted it in the broad sense of most protozoolo- gists to include the plasmotomy of Doflein as well as all other methods of simple and multiple divisions. Schizogony in the Plasmodiophorales is reported to occur most frequently during the 8- and 16-nucle- ate stages of the plasmodia or schizonts. A few uni- and multinucleate meronts may be formed as in Lig- niera, or the whole plasmodium may undergo mul- tiple division as in T. Triglochinis and P. graminis. The latter type of complete fragmentation appears to be limited as far as present-day knowledge goes. Most cases so far reported involve primarily the con- striction and cutting off of peripheral uni- and multi- nucleate segments. No cases have yet been described in which all or most of the nuclei migrate to the periphery of the schizont and become enveloped in cytoplasmic buds, which are subsequently pinched off, leaving a central mass of degenerating cytoplasm and nuclei, as in Hepatasoon anis, for example. The mechanics of schizogony are unknown, because the process has not been extensively observed in living material. In Polymiioca graminis, Ledingham merely reported that the pseudopodia are retracted and the protoplasm becomes denser before the thallus splits up into meronts. In cases in which only a few meronts arc formed the remaining portion of the schizont may mature directly into a sporogenous plasmodium or sporont. The delimited meronts grow in size and become mul- tinucleate and may in turn function as schizonts. Otherwise, they develop into sporonts. The destiny of the various portions depends to some extent on the length and activity of the vegetative period. Inas- Sl'.M Al.nv AM) AI.TKIIXATIO.N OF GKNK.IIATIOXS 15 iniu'li as solii/.ojiiiny will hv disciisscil fiirtlicr in tlit' ilcMTiption lit" imli\ iiliial spii-ifs in Chapter IN', fur- tluT discussion of the proc'css nt-od not l>c jircscntiil IllTC. Cytokinesis or division of tin- plasmodiuin or spo- ront into resting spores takes place by cleavajie. and as far as is now known may be closely associated in |)oint of time with the two meiotie divisions. In Soro- xpharra I'l-ronicaf, aceordinj; to Main- and Tison (OS)), cleavaiie begins in tlie late ))rophases of the first division (PI. (i. fig. 33). and by tiie time of the cqu.itorial plate stage, spore mother cells have been completely delimited (fig. 37). These cells divide into two uninucleate segments (fig. 38) in which the second division then takes place (fig. U). At the completion of this mitosis these segments in turn di- vide into the definite spore rudiments (fig. f2). In tills sjiecies at least cytokinesis may follow each mitosis. A similar sequence has been re))orted liy Wingc for Sorodi.icus (PI. 7. fig. 26-30), although the stages do not appear as sharjily defined. Figure 27. however, suggests that the sequence varies and that the first division may be complete before cleav- age begins. .Similar variations have been reported for Pla.smod'iophora also. Lutman ('13) and Milovidov ('31 ) found that the first meiotie division is usually comiilete by the time the initial segments are delim- ited. The latter may be uni-. bi-, or multinucleate, and after the second meiotie division has been com- l)leted (PI. 4, fig. 80. 81) they cleave into uninucle- ate spore segments (fig. 82. 83). Cook ('28). Cook and .Schwartz ('30), however, reported that cleavage in Liqiiiera and Pla.smod'iophora does not begin until both divisions have been completed. I-edinghani's ))hotomicrographs suggest the same sequence of events in Poli/mt/xa. In Tetrami/.ra the peripheral Plasmodium first cleaves (PI. 5, fig. 8) into uninucle- ate segments or sporonts (fig. 9-12). Two meiotie divisions occur in these segments, and these mitoses are usually over (fig. 13-17) by the time cleavage into definite spores is complete. In Octomi/j-a large uninucleate segments are delimited in which the two meiotie divisions occur, and following the completion of the second divisions, which are quadri])ol.ir, the segments cleave into sjiorcs. a<-eording to \\'liirt'en. \'ery little is known about cytokinesis in the other genera. The time relations of clc.iv;ige to the succes- sive meiotie divisiiins doubtless varies in diliercnt species and probably in the same species, so tli.at under varying conditions it may occur during as well as after meiosis. .Marked changes take place in the cytolilasm jirior to cleavage. In /'. /yrn.v.v/cac, according to Nawaschin ('99). Lutman. and .Milovidew NUCLEI fhom O*>0MIDIA P/fCTfiLASUIC BUO nDmNG OUT, OflOMIDIA IN OLD PORT/ON NUCLEI BREAKING UP INTO OtfOMIDIA NUCLEAR BUD DIVIDING. OfKMIDIA IN LCWEP END NUCLEAR BUDDING tew NUCLEAR BUO TEXT-FIG 3 LIFE CrCLE OF P BRASSICAE. according to JONES. 1928. 18 PLASMODIOPHORALES maturation stages of the plasmodium and sporogen- esis, while in Cook's opinion it extends from the time of gametic fusion through scliizogony and "akaryo- sis" to sporogenesis, as is shown in text-figure 7. The data relative to sexual reproduction are even more scanty in Sorodiscus. Winge assumed, as he had for all members of the Plasmodiophoraceae, that gametes from germinating spores copulate in pairs and thus initiate the diploid phase of S. Calliirichis, but he never actually observed fusion. Likewise, plasmogaray and karyogamy have not been seen in S. karlingii. In S. radicicolus, however. Cook ('31) figured and described fusion of amoebae in pairs within the host cell (PL 7. fig. 6). The two gametes here figured are unequal in size, but Cook did not say wliether or not this species is heterogamous. His study was made on fixed material sent from South Africa, and figure 6 shows the only case of pairing observed in sudi material. This may possibly repre- sent only a chance association of amoeba without sexual significance. Obviously, additional data are needed before definite conclusions can be drawn about sexuality in Sorodiscus. Cook ('33), never- theless, believed that fusion of gametes occurs in S. radicicolus and that this species has a well-defined alternation of diploid and haploid generations as is shown in text-figure 8. In Spongospora suhterranea, Massee, Kunkel, and Osborn reported that the sporogenous plasmodium is formed by coalescence of numerous amoebae, but they were uncertain about the origin and sex of the latter. Home was of the opinion that the amoebae are of opposite sex and that in this respect the plas- modium is similar to that of Diciyostelium muco- roides reported by Skupienski ('18). According to Osborn, coalescence is followed by the akaryote stage during which the nuclei disappear. New nuclei are reconstructed de novo, and these subsequently associate in pairs (PI. 10, fig. 28). Karyogamy soon follows as the nuclear membranes break down at the points of contact (fig. 29). Nuclei which do not pair degenerate the manner described by .Tahn for Cera- tiomi/ja. Home confirmed Osborn's report of kary- ogamy before meiosis but maintained that it occurs during instead of after the transitional or akaryote stage. He did not observe paired and fusing nuclei but based his conclusion on the discovery that the chromosome number following the transitional stage is twice that in amoebae and young plasmodia. Ac- cording to Osborn and Home, the diploid phase of S. suhterranea is quite short in duration and includes only the sporogenous plasmodium, as is shown in text-figure 9. Osborn's, and to some extent Home's, observations and reports of karyogamy shortly be- fore sporogenesis in Spongospora are strikingly similar to the earlier accounts of the nuclear fusion in the myxomycetes. In Ceratiomyxa, Arci/ria, and Trichia,0\he ('07) , Kranzlin ('07), and .lahn ('07, '08) described nuclear pairing and fusion in the plas- modium shortly before resting spores are delimited, but these accounts have subsequently been refuted. Cook ('33), on the other Iiand, reported that the zoospores from germinating resting spores pair at the anterior end, retract their flagella, and fuse (PI. 10, fig. 20-22). Plasmogamy is followed shortly by nuclear pairing and fusion (fig. 22). The zygote may become flagellate again, and later, apparently, in- fects the host. Its nuclei divide promitotically, ac- cording to Cook, and at the 6- or 8-nucleate stage the zygote undergoes schizogony. Whether or not the meronts later coalesce and thus form the sporogenous Plasmodium is not apparent from this account. Led- ingham ('35) also observed germination of resting spores and formation of biflagellate zoospores, but he found no evidence of gametic fusion. A few binu- cleate zoospores with four flagella were present in Ledingham's cultures (PI. 10, fig. 9), but he was not certain whether these were the product of fusion or incomplete cleavage. Thus, Cook's report of isog- amy has not been substantiated. He nevertheless be- lieved that the diploid generation of this species em- braces the zygote, schizonts, meronts, and plasmodia, while the haploid phase is limited to the cystosori, resting spores, and gametes, as is shown in text-fig- ure 10. The zoosporangia and zoospores found by Ledingham are apparently a means of rapid vegeta- tive multiplication and doubtless relate to the hap- loid phase, as is indicated in this diagram. Barrett found fusion stages between zoospores or gametes derived from zoosporangia in S. Cotulae, but these relate only to fixed and stained preparations. In Ligniera, Maire and Tison, and other workers, assumed that plasmogamy and karyogamy take place at some stage of development, because the nuclei ap- pear to undergo reduction at sporogenesis. Cook ('26, '33), however, reported a double fusion and reduction in L. Junci. The zoospores from resting spores fuse in pairs at the anterior end and give rise to diploid ])lasmodia. As noted before, these cleave into uninucleate segments, which develop walls and become incipient zoosporangia. The first nuclear di- vision in these sporangia is meiotie, and the zoo- spores or gametes subsequently produced are hap- loid. These fuse in pairs and form the diploid sporo- genous Plasmodium in which meiosis later occurs at sporogenesis. Ligniera Junci thus has two diploid phases each of which is separated by a haploid ])hase, according to Cook, as is illustrated in text-figure 11. Cook neither observed plasmogamy and karyogamy nor counted the chromosomes at meiosis, so that he had no direct evidence for his assumption. It is not improbable that the zoosporangia and zoospores are merely means of vegetative multiplication without sexual significance and relate to the haploid genera- tion, provided an alternation does occur, in much t!;e same manner as is indicated in text-figure 10 of S. suhterranea. No direct evidence of gametes, gametic fusion and karyogamy have been observed in Memhranosorus, Poli/?ni/^-a, Octomyjra, and the doubtful genera, 7?/;;'- somi/xa, Anisomij.ra, and Sorolpidium. Ledingham found a few tetraflagellate binucleate zoospores in Poh/mi/.ra, but he was not certain whether these were SK.VrAl.lTV AND A I.TKH \ ATH).\ OF (iKX K» ATIDN S 19 rvt^noH sKAc fc: *» ' /Q HAPLOID o s\ \ ^ e> 1 ''^^'^^^^^ HAPuyo :<^^:. ®\ v. DIPUD/D .-. ^w^ . © ® ©©■■ '^J ® © S) o' '"■S-^iMvTJ PLtSMD^ :A ■"^h;' S!^^ ^ X p£5' i-rC r:"-^:>- / HAPLOID %m \ <'% DIPUDID aiMKC V12\ 1^ ^ .t^ TEXT-FIG 6 LFE CrCLE OF TETRAMYXA. xcononc to COOK. 1933 TEXT-FIG 7 LIFE CYCLE OF SOROSPHAERA. /cccwing to COOK. 1933. 20 PLASMODIOPHORALES the products of gametic union or incomplete cleav- age. Tetraflagellate zoospores were likewise found bv Couch et al. in Octomi/j-a, but no fusions were ob- served. However, in this genus as well as in Mem- hranosorus Whitten and ^^'ernham each reported re- duction at sporogenesis. which presupposes karyog- amy at some state of development. Miss Whilfen be- lieved that karyogamy occurs during the akaryote stage of O. Achli/ae. It is obvious from this review that the data on sex- uality in the Plasmodiophorales are quite limited. In S. stibierranea, tlie only species in which gametic fusion has actually been observed, the respective gametes are reported to be alike and show no struc- tural, mobile, and physiological differences. In this species at least sexual reproduction appears to be isogamous. Whether it is homo- or heterothallic is not known, since no studies involving monospore cul- tures have yet been made. Therefore, any discussion at present of sex determination, haplosynoecism, haploheteroecism, antithetic alternation of gameto- and sporophytic generations, etc., in the Plasmodio- phorales must be speculative and, in light of the meager present-day knowledge, largely futile. bibliography: cytology and se.xiality Alexieff, A. 1913. Arch. Protistk. 39: 344. Belar, K. 1936. Ergeb. Foitschr. Zool. 6: 335. Blomficld, ,T. E., and E. J. Schwartz. 1910. Ann. Bot. 34: 3.5. Calkins, G. X. 1933. Blolojry of the protozoa. Philadelphia. Chatton, E. 1910. Arcli. zool. Exp. 5 ser. 5: 339, 36T. Cook, W. R. I. 1936. Trans. Brit. Mycol. Soc. 11: 196. . 1938a. New Phytol. 37: 330, 398. • . 1938b. Ann. Bot. 43: 347. . 1931. Ann. .Mycol. 39: 331. . 1933. Arch. Proti.stk. SO: 179. , and E. J. Schwartz. 1939. Ann. Bot. 43: 81. , and . 1930. Philos. Trans. Roy. Soc. London 318 B: 383. Couch, J. X., J. Leitner, and A. Whiffen. 1939. Jour. Elisha Mitchell Sci. Soc. 55: 399. Doflein, E. 1916. Lehrbuch der Protozoenkunde 4th ed. Jena. Favorsky, W. 1910. Mem. Soc. Nat. Kieff 30: 149. Ferdinandsen, E., and O. Winge. 1930. Ann. Bot. 34: 467. Fedorintschik, N. S. 1935. Summ. Sci. wk. Inst. pi. protect. Leningrad 1935:69. Harper, R. A. 1900. Bot. Gaz. 30: 317. . 1914. .\mer. Jour. Bot. 1: 137. Home, A. S. 1911. Rept. Brit. Assoc. Adv. Sci., Ports- mouth, p. 573. . 1930. Ann. Bot. 44: 199. Jahn, E. 1907. Ber. Deut. Bot. Gesell. 35: 23. 1908, Ibid. 36a : 343. Jones, P. M. 1938. Arch. Protistk. 63: 313. Kninzlin, H. 1907. Ibid. 9: 170. Ledingham, G. A. 1939. Canad. Jour. Res. C, 17: 38. Levine, I., and M. Levine. 1933. Jour. Cancer Res. 7: 163, 171. Lutman, B. F. 1913. Vermont Agr. Exp. Sta. Bull. 175. Maire, R., and A. Tison. 1909. Ann. Mycol. 7: 22%. , and . 1910. Bull. Soc. I.inn. Normandie 6 ser. 3:57. , and -. 1911. Ann. Mycol. 9:336. Milovidov, P. F. 1931. Arch. Protistk. 73: 1. . 1933. C. R. Soc. Biol. 109: 170. . 1933. Arch. Protistk. 81 : 138. Niigler, K. 1909. //)W. 15: 1. Nawaschin, S. 1899. Flora 86: 404. . 1901. Kiev Lap. Ohsch. Jest. 17: 1: XXXVI. . 1934. C. R. Acad. Sci. Russie 1934: 173. N'emcc, B. 1911. Bull. Inter. Empr. Fran. Joseph Acad. Sci. 16: 69. 1913, /hi'rf. 18: 18. Olive, E. W. 1907a. Trans. Wise. Acad. Sci., Arts and Let- ters 15:7,53. . 1907b. Sci. n. s. 35: 366. 0.sborn, T. G. B. 1911a. Rept. Brit. A.s.sn. .\dv. Sci. Ports- mouth, p. 573. . 191 lb. Ann. Bot. 35: 371, 337. Palm, B. T.. and M. Burk. 1933. Arch. Protistk. 79: 363. Pavillard, J. 1910. Prog. Rei. Bot. 3: 475. Prowazek, S. 1903. Osterr. Bot. Zeitschr. 53: 313. . 1905. Arb. Kais. Gesundheit 2-2: 396. Schwartz, E. J. 1910. Ann. Bot. 34: 511. Skupien-ski, F. X. 1918. C. R. Acad. Sci. Paris 167: 960. Terby, J. 1933. Mem. Roy. Acad. Belg. 7: 1. '—. 1934. Bull. Roy. Acad. Belg. 5 ser. 10: 519. . 1933. Mem. Roy. Acad. Belg. 11: 1. Webb, P. C. R. 1935. Ann. Bot. 49: 41. Wenyon, C. M. 1936. Protozoology 1 : 66. Wernham, C. C. 1935. .Mycologia 37: 363. Whiffen, Alma. 1939. Jour. Elisha Mitchell Sci. Soc. 55: 343. Winge, (). 1913. Ark. f. Bot. 13, no. 9: 1. Wissenlingh, C. 1898. Jabrb. Wiss. Bot. 31: 619. Vendo, V., and K. Takase. 1933. Bull. Sericult and Silk Inc. Japan 4, no. 3: 4. Chapter IV Classification and Description of Species The Plasmodiophorales include one family, Plas- modioplioraceae, and approximately eight genera and twenty-three species. Numerous other genera and sjiecies have been added at various times, but these liave either been merged with existing genera or excluded entirely as invalid. A natural classifica- tion is well nigh inijjossible at present because so lit- tle is known about tiie critical diagnostic characters of most siJCcies. Furtliermore, the genera are not shar])ly defined and, as Palm and Burke ('33) have so well em])hasized, tend to merge and overlap, so that in certain members generic distinctions are difficult to recognize. The oldest and most frequently used criterion of classification is the grouping assumed by the resting spores at maturity. This criterion was introduced by Schroeter in 1 897, who separated the genera on the basis of whether the spores are free or united in clusters and cystosori. Schroeter also em- ])Iiasized the presence or absence of a soral mem- lirane as a distinctive character of Tetrami/.ra and SE.Xl'AI.lTV AND Al.TKHN ATION (IF (iKNKHATUJNS 21 HAPLOID Dinao ^^v^ < '•[ N, XIOav HAPLOID *KtfiyCir£ STAGE ,^*^;: ■ « ♦ * A TEXT-FIG e LFE CYCLE OF SOROO/SCUS RADtQCOLUS. ACCOKono TO COOK, I93L TEXT-FIC.9 LIFE CYCLE OF SPONGOSPORA SUBTERRANEA, ACCORDINO TO OSBORN, 1911. PLASUoawr •^ ZOOSP0/>/IMJIAL STAB£ HAPLOID DIPLOID ■^ ■■^. ^• r.®^ ,, ^ <^^'-'^B^../:^f% AKwixm sTJce PLASMOGAUr TEXT-FIC 10 LIFE CYCLE OF 5. SUBTERRANEA, accowing to COOK. 1933 TEXT-FIC. II LIFE CYCLE OF LIGNIERA JUNO. AccoKomc n COOK, 1933 22 PL ASMODIOP MORALES Sorosphaern. Although mycologists and protozoolo- gists have clearly recognized the inadequacy of these criteria, they have nevertlieless continued to use tliem as the basis of classification. More recently. Cook ('33) has used the presence of zoosporangia and zoo- spores as another basic distinction. However, since zoosporangia have been subsequently found in nu- merous genera, the mere presence of sucli structures is no longer generically distinctive. Likewise, liis em- pliasis on tlie presence or absence of a membrane around the cystosori as a diagnostic character is open to question, since there is considerable doubt about the occurrence of soral membranes in any of the genera. Palm and Burke, in particular, Iiave severely criticized tlie present-day system of classification and characterized it as artificial. From tlieir obser- vations on the wide variations exiiibited by cystosori of S. 1 eronicae , tliey concluded that Spongoxpora, Lif/niera, Sorodiscus, Ostenfeldiella, Clathrosoriis, and Memhranosorus should be regarded as synonyms of Sorosphaera. On the basis of similarity of life cycles and general structure, they further advised the merging of all known genera except C ystospora into one large genus, presumably Plasmodiophora. The author is in complete agreement with these workers on the low taxonomic value and inadequacy of present-day generic distinctions. However, Palm and Burke's suggestion of reducing the number of genera or merging them does not solve the difficulties of classification in this group. As Ledingham ('39) pointed out, it merely shifts the generic indistinc- tions to tlie species. Further taxonomic distinctions ajjpear to be emerging from the discovery of zoosporangia in old and new genera. When these developmental stages have been fully investigated, the relationship of the various genera will doubtless become clearer, and it may then be possible to separate or merge them with greater accuracy. In the meantime, Schroeter's sys- tem of classification serves as a working basis, and although an unsatisfactory expedient, it may be used to advantage. In the key wliich follows, size, number, and sha])e of zoosporangia are used to some extent in diagnosis, but these characters are of doubtful gen- eric value. Some of the genera — i.e., Memhranosorus and Lif/niera — listed here are obviously questionable and sliould ])erhaps be merged with Sorodiscus and Sorosphaera, but until more is known about the fam- ily as a whole, it may be worth while to treat tliem separately. PLASMODIOPHORACEAE Zopf, 188-i. Die Pilzthiere oder Schleimpilze Thallus a naked. ])lasmodial, multinucleate proto- plast capable of amoeboid movement and undergoing sciiizogony into uni- or multinucleate meronts, wliicli in turn may function as schizonts. Sporogenous thal- lus cleaving into uninucleate spores at maturity. Resting spores loose and free or united in small clus- ters and cystosori ; usually producing one zoospore or amoeba in germination. Zoospores anteriorlj' bi- flagellate and heterocont. Zoosporangia formed di- rectly from zoospores or cleavage segments of young Plasmodia ; free or united in sporangiosori ; produc- ing a few to numerous zoospores which are similar to those formed from resting spores. Key to Genera I. Resting spores not united, free and loose. Zoosporangia few or numerous, small, and producing few zoo- spores 1. PLASMODIOPHORA, p. 22. II. Restin); spores united in small clusters or more or less compact cystosori A. Spores usually in tetrads or dyads. Zoosporangia un- known 2. TETRAMYXA, p. 37. I?. Spores usually in octads. Zoosi)orangia numerous, small, oval, and spherical with or without exit papillae 3. OCTOMYXA, p. 40. C. Cystosorus predominantly spherical to ellipsoidal and hollow; often variable in size and shape. Zoospo- rangia small 4. SOROSPHAERA, p. 41. D. Cystosorus predominantly disc-shaped, two-layered and flattened; often variable in size and shape. Zoosporangia unknown 5. SORODISCUS, p. 46. E. Cystosorus oval, spherical, and s])onge-like, lacking a central cavity but traversed by prominent canals and fissures. Zoosporangia numerous or few, small, oval and spherical, or large and irregular 6. SPONGOSPORA, p. 54. F. Cystosorus indefinite in size and shape 1. Zoosporangia small, oval and spherical; producing few zoospores 7. LIGNIERA, p. 58. 3. Zoosporangia usually large, elongate, lobed and irregular with prominent exit tubes 8. POLYMYXA, p. 63. PLASMODIOPHORA Woronin, 1877. Arb. St. Petersburg Nat. Gesell. 8:169. Osti'iiffliUeUa Ferdinandsen and Winge, 1914. Ann. Bot. 28: 64. (plates 2. 3. 4.) Resting spores lying free in host cell, not united in cystosori, variable in size and shape, usually pro- ducing one zoospore in germination. Zoospores an- teriorly biflagellate and heterocont, becoming inter- mittently amoeboid, infecting the host as an amoeba ( ?), dividing and budding (.''), and eventually form- ing multinucleate plasmodia, which cleave into uni- nucleate segments. Cleavage segments developing into small zoosporangia which produce few zoo- spores. Secondary zoospores reinfecting host and forming additional ])lasmodia. Sporogenous ))lasmo- dium partly or com])letely filling host cell, moving slowly in amoeboid fashion within the host cell and in migrating from cell to cell ; occasionally luider- going schizogony into uni- and multinucleate me- ronts; rarely encysting; cleaving into resting spores at maturity. PLASMOniOIMlOKA 23 Pta.imodiopliora iiirhulcs at prisciit live sjucics, of wliicli only one, /'. lirassicae, is fairly well known. Most of tlu' otluT spci'ics arc so little known that tlu'ir validity as nu-nilnTs of tlu' genus has ht-i-n seri- ously questioned. They nevertheless (jossi-ss the eoni- nion ability of eaiisiiii; eonspieuous galls and malfor- mations of the host tissues. Numerous other organ- isms with )>lasmodial stages have been inehided in the genus from time to time, but eareful reinvestiga- tion has shown them to be invalid. Pla.imodiophora is distinguished from the other genera of the family by the laek of a distinet eystosorus. The resting spores are not united or attaeiied to form a sorus of definite size and sliajx' but lie loose in the host eell. as is shown in figures 88 and 100. When the host eell disintegrates, the spores are liberated into the soil where they may germinate at onee, as in P. Brassi- cae, or remain viable up to seven or eight years (Jorstad, '23), So little is known about the other species of Plasmodiophora that present day discus- sions of the genus must necessarily be based princi- pally on /'. lirassicae. .\lthough this species has been intensively studied for more than .50 years, there is still considerable disagreement and controversy about its critical developmental stages, and doubt- less much remains to be discovered. The resting spores of P. Brassicae normally give rise to a single zoos])ore in germination (fig, 13-17), but it is not imj)rob.ible that more than one may be produced by the occasional large, bi- and multinu- cleate resting spores. Some workers, including Pol- lacei (12) and Honig ('31), have seriously ques- tioned the production of zoospores in this species. Honig, in particular, maintained that only non-flag- ellate amoebae are formed in germination (fig. 16— 18). .\lso, most investigators have figured and de- scribed the zoospores as anteriorly uniflagillate, but Ledingham ('St) clearly demonstrated that they are biflagellate and heterocont (fig. 22, 23). They have also been described in tile literature as varying from oval, pyriform, and fusiform to spherical in shape (fig, 19-23). After emerging from the sjiore case tliey may swim 'apidly away or become intermit- tently amoeboid v ;. 19. 20), during which the ante- rior end may double back and forth and thus jerk the sjiore body along. .\ccording to most students, the zoospores come to rest on the host and enter as amoebae through the root hairs and e))idermal cells, where they soon cause local hypertroi)hy (fig. 28, 29), A few workers, how- ever, have questioned these observations. Kunkel ('18) found nothing but thalli of Olpidium Brassicae in the root hairs of the host sjieeies which he studied and like F.ivorski (10) concluded that the ])revious reports on the occurrence of /'. Brassicae in such host cells were erroneous. However, subsequent investi- gators, including Cook and .Schwartz ('30). Honig ('31), Rochlin ('33). and Fedorintschik ('lio) have clearly shown infection of root hairs. Honig ajipears to have been the first to observe, describe, and figure actual penetration of the parasite into root h.iirs (fig. 27). He maintained that the ))r<)toplasts derived from geruiin.iting resting sjiores are true .iiiioebae without Hagella, a contention which has been re- futed by subsequent observers. Honig found small amoebae .as well as giant ones meas\iring l-2(i jx by 2l'-3(>/( abundant around root hairs and observed tii.it both tyi)es may readily enter the host eell. In so doing they become closely .iiiplicd to the root h.iir, and soon thereafter a hole appe.irs in the wall at the region of attachment, through which they then enter. The hole closes u]> immediately afterwards, so that it is no longer visible after the parasite has entered, Honig also observed that amoebae may live sapro- |)hytieally for weeks in the soil and increase mark- edly in size (fig. 3.")). It is not imiirobable. ;is Hoelilin's ('33) study suggests, that under certain enviroiiment.il condi- tions and particularly when s])ores germinate in eon- tact with the host cell, flagella are not formed, and the jiarasite enters the host almost at once in the amoeboid state. Rochlin found that the resting spores become attached to the root hairs and e))idermal cells of the root and cap and cause localized swelling of the eell wall (fig. 21-26). These regions become gelatinized and show no cellulose reactions when tested with chloro-iodide of zinc, indicating that a chemical change has taken place. .Small jjlastic, spherical protoplasts which presumably emerge from the attached sjiores, pass through these swollen and gelatinized regions (fig. 26) and enter the host cell.' The amoebae and young ))lasmodia in the host cell may bud and divide repeatedly (fig. 30), according to Gay lord (Ol), Chupp (17), Kunkel, Fedorints- chik and others, and thus multiply in number. They may also encyst and develop fairly thick hyaline walls (fig. 44—46) under unfavorable conditions. As the ))lasmodia grow in size and their nuclei multi))lv, they penetrate the walls of the adjacent cells and thus migrate from cell to cell (fig. 31-33), accord- ing to W'oronin ('78), Lutman ('13), Chupp ('17), Kunkel, Honig, Rochlin, Larson ('34), and Fedo- rintschik ('3.5). As to the method of cell wall jiene- tration, Rochlin noted that the plasmodium may be- come closely a))plied to a region of the wall and through lysic action cause localized swelling and gelatinization (fig. 7) of the latter. Passages in the walls are thus formed through which the plasmodium enters. Kunkel believed that only young and small Plasmodia free of oil bodies and other priidiicts are cai)able of migration. Cook .-iiid Schwartz ('30) were uncertain on this jihasc of develoi)ment and some- what vague in their description of it. In one )iart of their paper (p. 287) they stated that the amoebae "have the ])ower of ]>enetr;iting the walls of the host cells and in this way can tr.ivil through the cortical tissues of the host, " but they thought it im))robable ()). 297) that the plasmodia are able to do so. I'inally (|). 301 ) they expressed the belief that oidy gametes from sporangia have the ability of ))assing from cell to cell, .ind after gametic fusion the zygote is dis- tributed onlv bv division of infected host cells. 24 PLASM ODIOPHOR ALES Cook and Schwartz, nonethek-ss, discovered a hitherto unknown stage in tlie life cycle of P. Brassi- cae. The amoebae derived from flaoellate zoospores penetrate root hairs, grow in size, and by regular mitosis become multinucleate plasmodia which soon cleave into uninucleate portions. These segments round up. develop thin hyaline walls, and become in- cipient zoosporangia (Hg. 37). In view of this dis- covery, it seems probable that the small cleaving Plasmodia which Cliupp described and figured (p. •i36, fig. 104H) as stages in resting spore formation in root hairs relate to the development of zoosjioran- gia. The nuclei of the zoosporangia (fig. 38) divide mitotically two or three times, after which the proto- plasm cleaves into uninucleate segments (fig. 39— 42). forming thus four to eight pyriform zoospores (fig. 43). These are smaller than those derived from resting spores, according to Cook and Schwartz, who regarded tliem as gametes. As the walls of the sporangia collapse the zoospores emerge, fuse in pairs, either in root hairs or after migrating into the cortex, and form zygotes which grow into diploid sporogenous plasmodia, as has been described in Ciiapter III. Fedorintschik confirmed Cook and Schwartz's dis- covery of zoosporangia. He reported that individual amoebae in root hairs develop directly into large plasmodia containing up to 100 or more nuclei. These plasmodia cleave into uninucleate segments which develop walls and become rudimentary zoosporan- gia. The first division of their nucleus is meioiic, and then follow a second and sometimes a third mitosis, after which the protoplasm cleaves into four to eight zoospores. A single amoebae in a root hair may, according to Fedorintschik's observations, ulti- mately result in the formation of 400 to 800 zoo- spores. These zoospores become amoeboid and mi- grate into the cortical tissues and multiply rapidly by budding. After the content of the host cell is ex- hausted, they fuse, presumably in pairs (?), and later develop into plasmodia. Fedorintschik believed this fusion constitutes the sexual phase of P. Brassi- cae, and thus confirmed Cook and Schwartz's earlier report of sexual reproduction in this genus. In light of Ledingham's ('3.5, '39), Couch, Leitner, and Whiffen's ('39) studies on Sponc/ospora, Poli/mi/.ra, and Octoviifxa, however, it seems more probable that these so-called gametes are only secondary zoospores which reinfect the host and give rise to an additional amoebae and plasmodia in much the same manner as is indicated in text-figure 10 of Spongospora. Several workers have reported that tlie Plasmo- dium of P. Brassicae may undergo schizogony and give rise to a few or several meronts, whereby the parasite is rapidly multiplied. Nawaschin ('99) did not actually observe the ))roeess, but he believed that the large number of small thalli in a host cell could be explained only on the assumption that they had arisen by division of a preexisting thallus. He thought that the extended pseudopodia of the Plas- modium were cut off as buds, a belief which was later supported by I.utmau and by Henckel ('23). Subse- quently, Maire and Tison ('C9). Chupp, Kunkel, Jones ('28), and Cook ('33) also reported schizog- ony of the Plasmodium of P. Brassicae. It must be noted, however, that many of the early described cases of schizogony in the superficial host cells may possibly relate to the development of zoosporangia. As is shown in figure 36, the meronts may be uni- or multinucleate, and it is not improbable that after a period of growth they in turn may function as schi- zonts and form secondary meronts. With the exception of Cook and Schwartz and Fedorintschik who reported that the sporogenous Plasmodium is formed by the fusion of two gametes, many investigators who studied this phase of devel- opment were of the opinion that the plasmodium arises by the union of several vegetative amoebae or small Plasmodia. Woronin was uncertain whether it originates from a single amoebae or by the fusion of several, although he thought the latter method more plausible. Eycleshymer ('01) observed that if a slide with zoospores and amoebae is kept in a moist chamber, larger ])lasmodia appear, which he assumed had arisen by fusion of amoebae. Honig, however, maintained that the amoebae observed by Evcles- hynier do not relate to P. Brassicae. Halsted ('93) also believed that amoebae coalesce to form large Plasmodia. Nawaschin ('99) tliought that the schi- zonts and meronts remain more or less independent in the host cell until shortly before sporogenesis, when the}' flow together and form a large plasmodium. He admitted also that single amoebae may grow in- dependently into large plasmodia. Gaylord. Ericks- son ('13), Esmarch ('24), Prowazek, and Terby ('24) supported Nawaschin's belief on the union of amoebae, but INIaire and Tison ('09) refuted this contention. They pointed out that although meronts and schizonts may appear to be fused, they are none- theless separate and distinct. They based their view primarily on the lack of synchronism in nuclear di- vision in the closely associated amoebae and plas- modia in the same host cell. Lutman, Chupp, and Kunkel were uncertain about the union of amoebae, but Lutman noted that the nuclei in a plasmodium do not all divide simultaneously, which suggests that they may have been derived from several amoebae of different ages. Later, Jones ('28a, '28b) also re- ported fusion of amoebae and ])lasmodia in cultures of P. Brassicae, but there is considerable doubt about the validity of the organism he had in culture. In ad- dition to describing the origin of the plasmodium from a zygote. Cook and Schwartz reported that in the early stages of development several amoebae and later small plasmodia may fuse vegetatively to form the incipient sporogenous plasmodium. Since that time Milovidov ('31) also reported vegetative fusion of several amoebae. The plasmodium of /'. Brassicae is capable of slow amoeboid movement, and this mobility apparentlv enables it to move from cell to cell. Rochlin reported that the plasmodium first sends out a hyaloplasmic thread (Geissel) in the direction of movement, and shortly thereafter tlie more ajranular mass begins to I'l.ASMODIIU'lldH S 25 niovo. In vouiim: ))l.isiiuHii:i tlio psiiulo|)(i(ls .ire nl.i- tivflv loni: .•iiul tt iiuous. but a> tin- i)l;iMii(i(liuin lu.i- tiircs. tlu-v lu'coiuc Itss oxti-iisivc and more rouniloil at till- ptri))li(r_v. I'ignri- 31 shows a mature Plasmo- dium with several dense, opaque, iiseuihiixidial lobes at the anterior end. The posterior end in eontrast is quite vacuolate, thin, and relatively hyaline. 'I'lu amoebae .-ind younji jdasmodia are hyaline, somewhat transparent, viseous and slimy, and eomparativily free of oil dro))lets and other bodies, but as the i)l;is- modium inere.ises in size, the i)rotoplasm becomes denser, more opacpu'. .md very rieh in oil jjlobuies. Infected hypertrophied host cells are often rich in starch grains, and according to Woronin. Xawaschin ("99). Prowa/.ek ('05). and Lutman. these grains may frequently be found in the folds of the Plasmo- dium. N.iwasehin. I'avorsky ('10), and Henckel ('23) did not believe tiiat amoebae and ))lasmodia are capable of engulfing solid )iarticles, and Nawas- cliin |)ointed out that starch grains, such as those shown in figure 7 !■, are often caught between fusing merouts and thus come to lie within the plasmodiuni. Woronin, Eyeleshymer, and Lutman inferred that the i)lasniodium feeds on these grains, because by the time sporogenesis begins they have almost en- tirelv disai)peared, although a few may occasionally be found later scattered among the sjjores. Although Honig did not observe the plasmodiuni engulfing solid particles, lie nevertheless described it as nour- ishing itself sai)roi)liytically outside of the host for several weeks. In addition to oil globules, starch grains, and other bodies, chondriosomes are quite abundant in the i)lasmodia (fig. 48). according to Von wilier ('18) and Milovidov ('31). They also occur abundantly in the resting spores (fig. 86) and amoebae. L'nder unfavorable environmental conditions plas- modia and segments of the same in P. Brassicae may encyst and develop thick walls, according to Prowa- zek. Cook and .Schwartz, and Milovidov (fig. 46. 47). Prowazek ('05) and .Milovidov regarded these cysts as ))athological and involution forms. Cook and Schwartz described the ])lasmodium as becoming en- veIoi)ed by a distinct wall and then segmenting into several portions which in turn develojied thick walls (fig. 47). With the return of favorable conditions the walls disa))pear, and the plasmodium continues to function normally. Encysted ))lasniodia have also been described in /'. Fici-repeniis by Andreucci ('26). The cysts in this species are globular. 9.15- 73 n in diameter, with sculi)tured. thick w.-ills, and in germination give rise again to plasniodia. The sig- nificance of these cysts as a phase in the life cycle of Plasmodiophora is not clearly understood, but they are doubtles comparable with the sclerotia of the niyxomycetes. The majority of resting spores are iniimuliatc, but occasional globose and irregular ones (fig. 87) have been reported by Prow.azek. Milovidov. and others. Milovidov. in jtarticular. has figured numer- ous tetra-. tri-. and binucleate spores. The binueleate and nndtinucleate condition mav have resulted from the f;iilurc of large cleavage scgMuiits to (li\ ide .-iftcr flu- comi)letion of the second nuclear division. On the other hand, it is not altogether improb.-ible th.it it may have arisen ;is the result of ;i third mitosis in the inci))icut s))orc seguuMits in the maimer described by Maire and Tison and Home in Soro.sphacra I't-ro- iiirar and Sponrjospora siihicrranea, respectively. I.utman ('13) and Tcrby ('2f) also figured biuuele- .itc spores (fig. 95) and believed they had arisen as tiu> result of division in the spore. After the spores h.ive been formed they may remain stuck together for .1 short time by the slimy intercellular substance left from the jjlasmodiuui. Howc\er. they soon develo)j hyaline walls, dehydrate, and sejiarate. .\t no stage are they cnvelol)ed by a common membrane or form a cystosorus of definite structure, size, and shape. According to the rejiorts in the literature the resting spores may vary up to and more than 200 per cent in size. The early investigators found the siiores to be quite small, but measurements by subsequent work- ers have shown them to be considerably larger. Woronin ('78) rejiorted them to be 1.6 /x in diam- eter; Lowenthal ('05), 4 ;u ; Molliard ("09), 1.8- 2.2 /x: Chupp ('17) and Appel ('28), 1.9-4.3 /i, and 2.5-6.9 (u for the irregular ones; Esmarch ('24), 1-2 /J,; Pape ("25) and Honigmann ('26). 2.8-3.3 ix; Wellman ('30), 1.7 /x; Cook and Schwartz ('30), 2-3 /x, and 4.6X6 /x for the oval ones; and Honig ("31), 3.9 jx. The last-named worker made extensive measurements from nimierous hosts grown in differ- ent types of soil and under varying climatic condi- tions and found that the spores did not differ more than 0.5 /x in diameter. According to Wissenlingh ('98) the spore wall consists of chitin and shows no cellulose reaction when tested. The account given above is generally considered to be the usual developmental cycle of P. Brassicae. Henckel and P. M. .lones ('28b). on the other hand, have reported life cycles for this species which vary markedly from the orthodox type. In his study of club root of radishes Henckel described the resting spores as "aplanoamoebae" which by a jirocess of gelatinization or softening give rise to "Umax amoe- bae." These multi))ly outside of the host by budding, and when this jirocess is eomijleted, the numerous daughter amoebae enter the host and form a ))lasmo- dium. At no stage iu the life cycle are zoos))ores or flagellate gametes develojied. according to Henckel. In connection with his account it may be noted that Favorsky also figured and described spore germi- nation in rotten tumors as a process of gelatiniza- tion and softening of the s])ore wall, whereby large /('ma.r-like amoebae are formed. P. .M. .lones re))orted that he had isolated eight |)ure cultures of /'. Brassi- cae from cabbage roots and ui.iintained them in ta]i water under lal)oratory conditions for two months. These cultures caused galls on turnips when used as an inoculum and were subsequently recovered in culture from the diseased roots. .Vccording to .Tones (text-fig. 3). the following successive stages occur within the host: gametes, zygotes, preplasmodia, Plasmodia, cysts and spores ; while in culture outside 26 PLASMODIOPHORALES of the host, gametes, zygotes, cysts, amoebae, pre- plasmodia, and buds are formed. If conditions are favorable, however, P. Brassicae does not develop all of these phases. Jones' account has never been confirmed, and most subsequent investigators have doubted tlie accuracy of his observations. In light of present-day knowledge about P. Brassicae it seems likely that he may have been dealing with develop- mental pliases of more than one organism. Milovidov and Honig contended that some of the stages figured bv Jones relate to Olpidiiim Brassicae and Asiero- cystis radicis. P. BRASSICAE Woronin, I.e., pis. 1-6. 1878. Jahrb. Wiss. Bot. 11: .548. Pis. :39-34. Resting spores globose, spherical 1.6-4.3 /x. aver- age 3.9 jx, oval, ellipsoidal, 4.6X<3jU., sometimes constricted, elongate and irregular, 2. .5-6. 9 /i, with smooth, relatively thin, hyaline walls. Zoospores pyriform, splierical, 2. .5-3.5 /it, swimming rapidly and becoming intermittently amoeboid. Sporangial Plasmodia variable in size. Zoosporangia few or nu- merous, small, oval, spherical, 6-6.5 /^. angular and elongate with thin hyaline walls : producing 4 to 8 zoospores which are liberated by the collapse of the sporangium wall. Sporogenous plasmodia 100-200 /x in diameter, hyaline to pale-grey in color, amoeboid; encysting occasionally, undergoing schizogony into uni- and multinucleate meronts. Parasitic in the roots of wild and cultivated cruci- fers in temperate climates throughout the world, causing spindle-shaped, globose and irregular swell- ings, or galls and occasionally dark sunken spots and lesions. A complete list of hosts, degree of infection, geo- graphical distribution, and bibliography of P. Bras- sicae are given in Chapter VI. Biological Races of Plasmodiophora Brassicae Marked differences in degree of infection have been found in various species and varieties of wild and cultivated crucifers, and this has led to the belief that P. Brassicae may include several biological races or strains which are more or less virulent and specific for certain hosts. Appel and Werth ('10), Ericksson ('13), Hostermann (according to Honig. '31), and Gleisberg ('23) suggested the existence of such races, and numerous attempts have been made to demonstrate their jiresence. Between 1924 and 1929 Honig made six experiments involving a large number of cruciferous liosts from which he ('31) re- ported positive results. A strain of P. Brassicae from kohlrabi was found to be readily transmissible to kohlrabi, cauliflower, rape, turnips, and Cammelina sativa, but could be transmitted only with difficulty to radishes. A cauliflower strain was also discovered which proved to be similar to the one on kohlrabi, but strains from Savov cabbage and radish were found to be distinct. Motte ('33, '35) and MacLeod ('31) believed that they liad obtained evidence of biologi- cal specializ.-ition, but later after making tests of spores from 50 different sources, the latter worker found no evidence to confirm this belief. Motte ('33) found that the form from charlock grew especially well on turnips. Gibbs ('31) likewise tested various inocula for evidence of specialization, but all of his results were negative. In 1939 J. C. Walker observed a high degree of resistance to club root in swedes in Wisconsin, which was contrary to results obtained elsewliere, and thought that this difference indicated a variation in pathogenicity of the causal organism. He accordingly secured spores from widely sepa- rated regions of the United States and tested their virulence on swedes, but found little difference in pathogenicity.' The data in the literature on the ex- istence of biological strains are therefore conflicting, and most investigators, witli the exception of Honig and ^Nlotte. liave doubted tlie presence of such strains in P. Brassicae. 1 However, in a paper presented before the Dallas, Texas, meeting of the American Phytopathological So- ciety, December, 1941 (Phytopath. S2: 18). Walker gave additional data on physiological specialization in P. iJro.v- .licae. Purple Top Milan turnip remahied completely free of club root when grown in heavily infested soil in Wis- consin, but when planted in naturally infected soil in Eng- land, about -'0 per cent of the plants were diseased. On the other hand, an English variety. White Stone, which showed 87 ))er cent infection in an English test, failed to develop clubbed roots in inoculation tests with a representative American isolate of P. Brassicne. Walker accordingly con- sidered this evidence as proof of the existence of physio- logical races. Bacteria in Relation to P. Brassicae The association of bacteria with P. Brassicae in roots of diseased crucifers was noted by Eycleshy- mer in 1894 and confirmed by Pinoy ('05). E. F. Smith ('11). and other early workers. From his pre- PLATES 2, 3, 4 Pldsmndiofihorn Brassicae Fig. 1. Infected cabbage roots with spindle-shaped swell- ings. Woronin, '78. Fig. -2. Beginning of club formation on roots of cabbage inoculated experimentally. Woronin, I.e. Fig. 3. Heavily infected turni]) root. Woronin, I.e. Fig. 4. Cross section of infected cabbage root. Note two compact groups of infected cells on lower right side, the so-called "krankheitsherde." Woronin, I.e. Fig. 5. Nuclear division in an enlarged infected host cell. Lutman. '13. Fig. (i. Division of infected cell. Lutman. I.e. Fig. 7. Enlarged cells of cabbage with mature plasmodia showing the swelling and dissolution of intervening cell walls. Hochlin, "33. Fig. 8. Normal host nucleus. Lutman, I.e. Fig. f)-\2. Enlargement, distortion, and degeneration of liost nuclei in infected cells. Lutman, I.e. Fig. 13. Germination of resting spore. Chuiip, '17. Fig. 14, 15. Same. Woronin, I.e. Fig. l(i-18. Germination of resting spore, and amoebae. Honig, '31. Fig. 19, Jl. .Amoeboid, anteriorly uniflagellate zoospores. Woronin, I.e. Fig. JO. Fixed and stained zoospore with anterior blepho- rophist. Cook and Schwartz, '30. IM.ASMDDIOlMlOltA 27 I'LATl'l •> 28 PL ASMODIOP MORALES vious studies (02, '03) on bacteria in relation to tlie niyxoniycetes. Pinoy (05, 07) concluded that bac- teria were essential to the development of P. Brassi- cae and described the relationsliij) between them as true symbiosis, a viewpoint which Vouk (13) later supported. Pinoy reported that the spores of the fungus will germinate only in the presence of these bacteria, and as the zoospores or amoebae enter the host they are accompanied by cocci which continue to live in constant association with the parasite throughout its entire life cycle. Chupp (17) re- peated Pinoy's experiments to some degree and found that bacteria are absent in small, young swell- ings and do not appear until the galls have become quite large and old. Furthermore, instead of cocci, he found the most prevalent form to be a motile, rod- shajjcd bacillus which forms yellowish, ojjalescent colonies on nutrient media. Chupp concluded from his experiments that bacteria do not enter the host with the amoebae and that the disease must attain a certain advanced stage before the bacteria can enter. According to him, they are not essential to the devel- opment of P. Brassicae, but as Sorauer (08) had previously jjointed out, they may act in decomposing the host cell wall and thus liberating the spores. Naumov ('2.5) likewise failed to find bacteria in young galls, while Fedotowa (30) reported that bac- teria may be present within one and a half to five months after infection. He found that P. Brassicae spores may be easily freed of bacteria by immersing them for five minutes in a .001 per cent corrosive sublimate solution. From diseased roots he isolated one bacillus and two coccus forms which when in- jected into roots in pure culture produced no signs of hy])ertrophy or injury, Fedotowa tlnis showed that bacteria are in no way necessary to spore ger- mination, entrance of the amoebae, or to the nutrition of the Plasmodium. Plasniodiophora Brassicae and Cellular Inclu- sion of Cancer Cells, Small Pox, and Rabies At the close of the last century when animal path- ologists were actively engaged in trying to prove the parasitic nature of the inclusions found in carcinoma Fifr. -2-2, i3. Anteriorly biflagellate, heterocont zoospores. Leclinpliam, '34. FifT. 2i. Restinjr spores attached to root hair tip, small spherical myxamoeba within the cell and two myxamoi-ba entering through a swollen gelatinized region of the wall. Rochlin, '33. Fig. :?.5. Three parasites lying in a swollen gelatinized re- gion of tlie root liair wall. Rochlin, I.e. Fig. 2G. Entry of the parasite through the epidermal cell wall of the root of B. arvensis. Note other swollen and dis- organized regions where additional parasites have entered. Rochlin, I.e. Fig. 27. Entry of an amoeba in root hair. Honig, I.e. Fig. 38. Swollen root hair of cabbage with a living myx- amoeba. Woronin, I.e. Fig. 39. Uninucleate myxamoeba in root hair wliich is locally swollen. Cliupp, I.e. Fig. 30. Division of a myxamoeba by fission. Chupp, I.e. Fig. 31. Early stage in cell wall penetration by a young Plasmodium. Kunkel, '18. Fig. 33. Later stage. Kunkel, I.e. Fig. 33. Young plasmodium passing through cell wall. Kunkel, I.e. Fig. 34. Large living amoeboid plasmodium moving within liost cell. Note pseudopods at the anterior and vacu- oles in the posterior end. Woronin, I.e. Fig. 3.5. Large saprophytic amoebae or ])lasmodia out- side of host. Honig, I.e. Fig. 3(). Root hair filled with meronts, possibly incijiient zoosporangia. Chupp, I.e. Fig. 37. Empty and developing zoosporangia in a root hair wliieli have been formed from a plasmodium. Cook and Schwartz, I.e. Fig. 38. Uninucleate segment of plasmodium which will develop into a zoosporangium. Cook and Schwartz, I.e. Fig. 39. First mitosis (meiotie?) in incipient zoosporan- gium. Cook and Schwartz, I.e. Fig. 40. Binucleate stage of same. Cook and Schwartz, I.e. Fig. 41. Zoosporangium with four ineijiient zoospores. Cook and Schwartz, I.e. Fig. 43. Same with three fully formed zoospores. Cook and Schwartz, I.e. Fig. 43. Nonflagellate zoospores from zoosporangium. Cook and Schwartz, I.e. Fig. 44—46. Encysted myxamoeba and young plasmodia. Milovidov, "31. Fig. 47. Large segmented jilasniodium, the segments of which have encysted. Cook and Schwartz, I.e. Fig. 48. Binucleate plasmodium with numerous cbondrio- somes. Milovidov, I.e. Fig. 49. Resting nuclei of large plasmodium. Cook and Schwartz, I.e. Fig. 50. Same in young plasmodium. Nawaschin, "99. Fig. 51. Same in amoebae with centrosomes and astral rays. Milovidov, I.e. Fig. 53. Early prophase of "promitosis" with chromatin in the form of numerous granules. Nawaschin, I.e. Fig. 53. Equatorial plate stage of "promitosis" with di- vided nucleole. Nawaschin, I.e. Fig. 54. Same stage. Cook and Schwartz, I.e. Fig. 55, 56. "Double anchor" stage of "promitosis." Na- waschin, I.e. Fig. 57, 58. Late anaphase and telophase of "promitosis." Nawaschin, I.e. Fig. 59-61. Successive stages In development of the "akaryote" stage. Cook and Schwartz, I.e. Fig. 63. Akaryote stage. Cook and -Schwartz, I.e. Fig. 03. Siiireme stage of the first sporogonic (meiotie?) mitosis. Lutman, I.e. Fig. 64, 65. Synapsis and i)ossibly diakinesis, respec- tively. Milovidov, I.e. Fig. 66. Early prophase of meiosis. Terby, '34. Fig. 67. Synapsis. Terby, I.e. Fig. 68. Strepsitene. Terby, I.e. Fig. 69, 70. Diakinesis. Terby, I.e. Fig. 71. Polar view of equatorial plate stage showing eight chromosomes. Terl)v, I.e. Fig. 73. Profile view of equatorial plate stage, first divi- sion. Lutman, I.e. Fig. 73. Polar view of same showing eight large chro- matic bodies. Lutman, I.e. ri.A.S.MDUlOl'IlOllA PLATE 3 29 iiv-^'^>^ 65 V-'-' 66 62 63 67 68 IMiisiiiodiojiliorii 69' 70 71 ^64 £^. f o 72 73 30 PLASMODIOPHOHALES cells, numerous parallelisms were drawn between cancer and club root of crucifers. The superficial re- semblance of the tumors on cruciferous roots to can- cerous outgrowths in animals as well as the similarity of the amoeboid stages c P. Brassicae to the cellu- lar inclusions (Plimmer bodies, etc.) in cancer cells led some workers to the belief that fungi, particu- larly tlie myxomycetes and Plasmodiophora, may be associated with, or the cause of cancer in animals. Numerous experiments were accordingly performed in which infected cruciferous tissues were implanted in various kinds of animals. While these studies failed to throw light on tlie cause of cancer, they nonetheless focused attention on club root from the purely pathological viewpoint and are of consider- able liistorical interest. In 1898 and 1899 Behla pointed out the similarity of club root and potato wart to cancer and discussed the possible relation of Plasmodiophora and Sijn- chytrhnn to this disease in animals. After having in- fected animals with these fungi and noted the simi- larity of their developmental stages to certain inclu- sions in carcinoma cells, he concluded in 1903 that cancer is caused by a chytridiaceous organism. In 1900 and 1903 Podwyssotzki reported that he had succeeded in producing tumors in guinea pigs and dogs by subcutaneous and intraperitoneal implanta- tions of infected crucifer tissues. These tumors were about the size of a walnut and resembled large-celled sarcoma, endothelioma, or granuloma. They were mesodermic in origin and had arisen through pro- nounced hypertropiiy and repeated division of the connective tissue cells and endothelium of the peri- vascular fissures. Leucocyte infiltration was quite evident at first but disappeared after 8 to 12 days. Podwyssotzki found furtlier that P. Brassicae pro- duced many other changes in animal cells which were similar to those induced in cells of crucifers. Further attempts to draw analogies between the inclusions of cancerous cells and those produced by P. Brassicae in animal cells were made by Feinberg ('02) and Gaylord ('04). The latter succeeded in infecting animals locally with P. Brassicae, and from his observations on tlie tumors produced he pointed out in detail the parallel cellular symptoms of club root and cancer. Gaylord concluded that can- cer is caused by an amoeboid organism the develop- mental stages of wiiich are very similar to P. Brassi- cae. In 190.5, however, I.owenthal refuted all pre- vious reports that the club root organism produces typical cancerous tumors in animals. He implanted infected crucifer tissues in the stomach, liver, and kidney of dogs and in the skin of white rats, but failed to get tumors or any other specific reactions in the animals. In the same year Prowazek (0.5) made an extensive comparison of P. Brassicae and the in- clusions of carcinoma cells, particularly the Plimmer bodies, and concluded that except for superficial similarities they have very little in common funda- mentally, ^lore recently Levine and I.evine ('22) have made a comparison of the tumors of P. Bras- Fiir. 74. Simultaneous nuclear division (meiotic?) in a large, somewhat vacuolate plasmodium. Note large starch grains lying in clear regions. Xawaschin, I.e. Fig. 75. First meiotic division with centrosome-like bod- ies and astral rays apart from nuclei in the cytoplasm. Terby, I.e. Fig. 7(). Second meiotic division showing four chromo- somes. Terby, I.e. Fig. 77. Second meiotic division showing centrosome-like bodies. Terby, I.e. Fig. 78. Vacuolate stage of plasmodium prior to cleav- age. Lutman, I.e. Fig. 79. Cleavage furrow at edge of plasmodium. Lut- man, I.e. Fig. 80. Nuclear division in a large cleavage segment. Milovidov, I.e. Fig. 81, 8-2, 83. Mitosis and cell division in smaller cleav- age segments. Lutman, I.e. Fig. 84. Fully formed resting spores witli chromatin around inner periplierv of nucleus. Cook and Schwartz, I.e. Fig. 8.5. Mature resting spore with fat droplets. Lutman, I.e. Fig. 86. Resting spores with chondriosomes. Milovidov, I.e. Fig. 87. Variations In size and shape of resting spores. Milovidov, I.e. Fig. 88. Fusion of incipient resting spores. Prowazek, '0,5. Fig. 89. Binucleate resting spore. Prowazek, I.e. Fig. 90. Division of one gametic nucleus. Prowazek, I.e. Fig. 91, 9-3. Formation of "reduction bodies." Prowazek, I.e. Fig. 93. Karyogamv. Prowazek, I.e. Fig. 94. Centrosome separating from nuclear membrane to become the blephoroplast. Terby, I.e. Fig. 95. Resting spore with blephoroplast. Terby, I.e. Fig. 96. Binucleate resting spore. Terby, I.e. Fig. 97. Host cell filled with resting spores. Woronin, I.e. P. J>iphnifherae Fig. 98. Infected plant of Diplanthera U'rif/htii with hvpertrophled bead-like intcrnodes. Ferdinandsen and Winge, "14. Fig. 99. Cleaving plasmodium which fills enlarged host cell and envelops host nucleus. Drawn from photograph. Ferdinandsen and Winge, I.e. Fig. 100. Host cell filled with resting spores. Drawn from photograph. Cook, '33. Fig. 101. Normal and collapsed resting spores. Drawn from photograph. Ferdinandsen and Winge, I.e. /'. Flcl-rej)(ntis Fig. 102. Gall on branch of FIciis repen.-:. Drawn from l)hotograph. Cook, I.e. P. II(ilujihil<(e Fig. 103. Hypertro])hied petiole of IlalophUii ovnVis. Ferdinandsen and Winge, '13. Fig. 104, 105. Normal and collapsed resting spores. Ferdinandsen and Winge, I.e. P. Iiirtaifldtit Fig. 106. Plasmodium t-nvcloping enlarged host mu'leus. Feldmann, '40. Fig. 107. Cleavage into resting spores. Feldman, I.e. Fig. 108. Resting spores. Feldman, I.e. ri. ASMOniOIMIOHA 81 I'LATK 4 --^s::^-,'*w „u ••• vO 80 ^ ^m m 88 89 qO o)(o) ,F'^'^ m O '^qV9 83 I'la>mo(li()i>liora 32 PLASMODIOPHORALES sicae on crucifers and the malignant neoplasia in animal cancer. Analogies have also been drawn between tlie club root organism and the cellular inclusions formed in vaccination against small pox. Gorini ('01) suc- ceeded in producing a slow but marked proliferation of the cornea epithelium in dogs by implanting in- fected cabbage tissues and found that tlie intracellu- lar effects were very similar to those caused in vac- cination. Certain phases of P. Brassicae under these conditions resembled the Ci/torcf/tes bodies associ- ated with small pox. Pollacci ('12) pointed out some of the striking resemblances between the early devel- opmental stages of P. Brassicae and tlie Negri bodies of rabies in dogs and believed that there might be a connection between these two cellular structures. P. DIPLANTHERAE (Ferdinandsen and Winge) Cook, 193-'. Hong Kong Nat. Suppl. 1:34. Oxtf'iifcUUella Diplantherne Ferdinandsen and Winge, 19U. I.e., pi. 45. Fig. 1-4. Resting spores globose, spherical, 4-4. .5 /j,. with fairly thick, brown, smooth walls; germination un- known. Zoospores and evanescent zoosporangia un- known. Plasmodium filling host cell, 125-200^ in diam. ; schizogony doubtful; cleaving directly into uninucleate resting spores during sporogenesis. Parasitic in Diplanthera icrif/htii, St. Croix, West Indies, causing large galls on tlie stems in the re- gion of the internodes. This imperfectly known species was found in 1913 by Ostenfeld who turned over his material to Ferdi- nandsen and Winge for further study. From this scanty and poorly fixed material they created a new- genus, Osienfeldiella, for the species at hand. Cook subsequently examined their prepared slides and concluded that the fungus is a species of Plasmodio- phora. Because its plasmodia cleave directly into resting spores which are not united in cystosori but lie loose and separate in the host cell as in Plasmo- diophora, there is no reason, on the basis of present- day knowledge, for keeping this species in a separate genus. Cook's disposition of it is accordingly fol- lowed here. Ptasmodiophora Diplantherae attacks only the in- ternodes and causes them to enlarge, so that the stem has the appearance "of a string of pearls." as is shown in figure 98. The parasite is restricted to the inner cortex where it leads to marked enlargement of the infected cells. Normal cells measure approxi- mately 35 /i in diameter, while infected ones vary from 12.5 to 200 (u. In the early stages of infection the young liost cells apparently retain their ability to divide, and it is not improbable that the young parasites may be dispersed by division of the host cell. The plasmodium seems to envelop the host nu- cleus (fig. 99), and witli the start of the sporogonic phase tlie nucleus begins to degenerate. The pres- sure exerted by the enlarging cells causes tangential stretcliing of tlie outer cortical elements, and in cases of unilateral infection the central cylinder becomes laterallv displaced. Since the parasite is localized in the inner cortex, infected stems can readily continue to grow and elongate. Whether schizogony or any other division of the parasite within the host occurs in this species is un- certain. So far none has been observed. Ferdinand- sen and Winge nevertheless concluded that the young amoebae divide after each mitosis, because only uni- nucleate stages are to be found in the meristematic areas of the stem. P. HALOPHILAE Ferdinandsen and Winge, 1913. Centralbl. Bakt. Parasitenk. II, 37: 167. Fig. a-c. Resting spores yellowish in mass, hyaline when single, globose, 5 /a, with fairly thin smooth walls. Plasmodia one to several in a cell, variable in size and shape, subglobose. elongate, 30-60 /x long. All else unknown. Parasitic in the petioles of Halophila oralis on the island of Noesa Kembangan near the southern shore of Java, causing conspicuous pea-shaped galls. The diagnosis of this species is based on a study of dried material which Ostenfeld found in a collec- tion of H. oralis in the Botanical Museum of Huana. He believed that the hypertrophied petioles (fig. 103) were parasitized by a species of Plasmodi- ophora but made no study of the organism. The dried material was subsequently sent to Ferdinandsen and Winge who diagnosed the parasite as a new species. It has never since been collected, nor is anything more known about its structure and development. The species which Feldman ('36) found on peti- oles of Halophila BaiUonis in CJuadeloupe, West Indies, may possibly be identical or closely related to P. Halophilae. Feldman merely noted its occurrence witliout describing or identifying the parasite. P. FICI-REPENTIS Andreuoci, 1926. Arch. But. 2: -26. Resting spores spherical. 1.5.5 /x. with thin, hyaline walls; producing pyriform uniflagellate (?) zoo- spores in germination ; flagellum 2.7 jx in length. Thin-walled evanescent zoosporangia unknown. Amoebae and young plasmodia from zoospores varia- ble in shape and size. 6 X 24 ju,, aggregating and fusing into larger plasmodia. which later cleave into irregular segments and finally into spores; some- times encysting to form hyaline, globular, 9.15-73 /x cysts with granular and sculptured thick walls : cysts producing plasmodia in germination. Parasitic on the large and small branches of Ficiis repens in Italy, causing Avoody, brownish-gray, globular, irregular and coral-like tumors up to 5 cm. in diameter (fig. 102). This species differs from 7'. Brassicae ])rimarily bv its smaller resting spores and the fact tiiat it at- tacks aerial organs rather than the roots of its host. It has been recorded but once, and Andreucci unfor- tunately did not illustrate it. However, Cook ('33) examined dried galls, which were unsuitable for cyto- logical study, and described tliis species as a doubt- ful member of the genus. l'l..\SMllllll)l'lll)Il.\ 88 P. BICAUDATA l-Vldmiin, ISUd. Hull. So.-. Hist. Nut. AfriiiiK- Noril M : ITH. Fip. 1. -'. Kcstiiig spores ovoid ami slitw. 10: 17(i. Hehla. K. 189K. Ontralhl. Hakt. Parasitk. 2i: HJ9. . 1899. /..itschr. Hy;:. 3J: i:«. . 1903. Die I'flan/.eiiparasltare L'rsadic des Krel)ses und die Krebspriipliylaxe. Berlin. Cliupp. C. 1917. Cornell I'nlv. .\gr. Kxp. .Sta. Bull. IWT. Cook. W. K. I. 19i6. Trans. Brit. .Mycol. Soc. 11: 19(i. . 19.>8a. Bull. Soc. .Mycol, France 44: 10,i. . 19:;8b. .Ann. Bot. 4J: 347. . 19;!3. .\reli. I'rotisdv. Ml: 179. , und K. J. Schwartz. 19;{(). I'liil. I r.iiis. Uny. Scic. Ser. B. .MS: -'H;1. Courh, .1. N'., ,1. I.eitner. and .\. WliitTrn. l!i:i!i. .lour. Klislia Miteliell Scl. Soc. .Vi: S99. I'.rickssoii. ,1. 19IS. Die Pflanzcnkr. l.aiuhv. KiilturpM.iiize. I.fii>zitr. Esmurch, F. 19-'4. Die Kranke I'flanze 1: 169. KycleshyiiHT, A. C. 191)1. .lour. Mycol. 7: 79. Fiivorsky, \V. 1910. Mem. Soc. N'iif. Kieflf. M): 149. Frdotott-a. T. 19:J0. I'liytiipatli. /eitsclir. I: 19.V Fr. F.inlKTf:, I.. 190.'. Her. Deut. Bot. (Jes. 19:533. Fildniann, .1, 19:i(). Bull. Bot. .Soc. France S3: Ons. Caylord, 1904. /eitsclir. Krehsf. 1: 93. Ciihbs, ,J. G. 1931. New Zealand .lour. .\)rr. 4.': I, 193. Gleisberjr, W. 19:33. Nachrbl. Deut. I'tianzenscli'd. 4:10. Corini. D. C. 1901. Centralbl. Bakt. Parasitk. abt. I. 39: .589. Ilalsted, B. D. 1H93. New Jersey .Apr. Exp. Sta. Kept. 1893: 33 J. Henckel. X. G. 19.'3. Bull. Inst. Hecli. Biol. P.rnie, fasc. 2. Hoiiifr. F. 1931. CJartenbauwiss. 5; 11(>. Houifrniann. 19:2(». Pflanzenbau. Halbnischr. f. Saatwesen Anbau u, Pflege d. Kulturjiflanz. 2: 29U. Home. A. S. 1911. Jour. H.iy. Hort. Soc. 37: 303. . 1930. Ann. Bot. 44: 199. Jones, P. M. 19-'Sa. Arch. Protistk. 62: 313. . 19.'8b. Bot. tiaz. 81 : 441). Jorstad, I. 19-'3. Norsk. Havetid. 39: 126. Kunkel, L. O. 1918. Jour. Agr. Ke.s. 14: .543. I.arsen, H. H. 1934. Ihirl. 49: (>07. I.edinfrham. G. A. 1934. Nature 133: 534. 193.5. IhhI. 135: 3994. . 1939. Canadian Jour. Res. C, 17: 38. I.evine, I., and M. Lcvine. 19:2:?. .lour. Cancer Ros. 7: 163. Ihl(>: .'3. .Motte, M. H. 19.33. Jour. d'Afrr. Prat. 97: 177. 1935, IhM. 99: 93. Naumov, N. A. 19.V.. .Morbi Plant. 14: 49. Nawaschin, S. 1899. Flora 8(i: t04. . 1901, Kiev, Zap. Obseb. Jest. 17: 3(J. , 19;?4, C. R. .\cad. Sci. Russie 19J4: 173. Pape, H. 193.5. Pflanzenbau -': 173. Pinoy, E. 1903. Bull. .Soc. .Mycol. France IS: 388. . 1903. C. R. Acad. Sci. Paris 137: 580. . 1905. C. R. Soc. Biol. 58: 1010. . 1907. Ann, Inst. Pasteur 31 : (iSfi. Podwyssozki, W. 1900. Centr.ilbl. Bakt. Parasitk. abt. I, 37: 97. . 1903. Zeitsebr. klin. .Med. 47: 199. Pollacci, G. 1913. ,\tti Inst. Bot. Univ. Pavia. 3 ser. 15: 391. Prowazek, S. 1905. .\rb. Kais. Gesund. 22: 39(i. Rocblin, E. 1933. Pbytopatb. Zeitsebr. 5: 381. .Smith. E. F. 1911. Bacteria in relation to pl.mt disease 3: l(i9. Sorauer, P. 1908. Ilandb. Pflanzrnkr. Berlin. Terby, J. 1933. .Mem. Roy. .\ead. Belg. 7: 138. . 1934a, Bull. Roy. Sr>c. Bot. Bel);i(|ue 5(i: 48. . 1934b. Bull. Roy. Acad. Belp. 5 ser. 10: 519. . 1933. .Mem. Roy. Acad. Belp. 1 1 : 1-30. 31 PLASMODIOPHORALES Van Wissenlingh, C. 1898. Jalirb. Wiss. Bnt. 31: 619. Vonwiller, P. 1918. Arcli. Protistk. 38: 279. Vouk, V. 1913. Die Naturw. 1: 81. Walker. J. C. 1939. Jour. Agr. Res. .59: 815. Wellman, F. L. 1930. U. S. Dept. .\pr. Tech. Bull. 781. Winge, O. 1913. Ark. Bot. 1-2, no. 9: 1. EXCLUDED SPECIES P. ALNI ( Wor.) Moeller, 1885. Ber. Deut. Bot. Ges. 3: 10:3. Figs. 1-4. 1890. Ibid., 8: .'15. In 1866 Woronin found an organism in galls on the root.s of Alniis gliifinosa which he named Schin:ia Alni and believed to be a hyphomycetou.s fungus. Gravis discussed its identity in 1879. and in 188.5 Moeller placed it in Plasmod'wphora where it was subsequently retained by Schroeter ('86, '97). Since that time its identity and relationship have been tlie subject of extended controversy. In 1886 Brunchorst made an intensive study of the galls of Alnus spe- cies and found no evidence of a plasmodium. Instead, he found a mycelioid fungus with numerous sporan- gia wliich he named Frankia .iiibtilis and believed to be related to the Mucorales or Saprolegniales. This led Moeller ('90) to restudy the causal organism, after which he retracted his former view and con- firmed Woronin's and Brunchorst's observations on the presence of a mycelium in the host cells. Frank confirmed these observations in 1891, but lie was un- certain as to the nature of Frankia subtUis. While he pointed out that it might well be a form of Lepio- thrix, he was nonetheless inclined to the view that it is a mycorrhizal fungus. In 1900, according to Maire and Tison ('09, p. 242), Chodat studied the organism in question and asserted that it is a species of Plasmodiophora. Two years later, after an inten- sive study of the tubercles on AIniis roots, Schibata came to tlie conclusion that no hyphomvcetous fun- gus was present and tliat tlie galls are caused by an organism with bacterium-like filaments which even- tually become bacteroid and deformed. In 1901 Bjorkenheim figured and described fungus hyphae in the galls, but three years later Keissler reported the organism again as Plasmodiophora Alni. Finally, in 1909, Maire and Tison undertook a cytological study of tlie tubercles and confirmed the observations of Shibata. They found an abundance of partially digested mycelial filaments, the ends of which became vesicular and later segmented into a large number of irregular chromatic bodies. Maire and Tison changed the name of the organism to FrankieUa Alni, but since that time Keissler and I.ohwag ('37) have reported it again as Plasmodio- phora Alni on Alnus species in China. P. ELAEAGNI Schroeter, 1889. Cohn's Krypt. Fl. Schle- siens 3: 134. 1897, Engler u. PrantI, Nat. Pflanzenf. 1:7. Schroeter gave this name to an organism wliich he believed to be the cause of galls on the roots of Elacafintis angtistifolia. It seems that he was not aware that Brunchorst ('86) had already found the same organism on F. anr/nstifolia, F. argi-niea, F. pitnc/ens, and Hippophae rhamnoides and named it Frankia subtilis. Claire and Tison ('09) also ob- served similar galls on the //. rhamnoides, and since they found the causal organism to be of the same tvpe as F. Alni, they renamed it FrankieUa Flacagni. It has subsequently been reiJorted as Plasmodio- phora Flaeac/?ii hy J aap ('07) and Sydow ('21) from Switzerland and New Zealand. P. VITIS Viala and Sauvageau, 189;2. C. R. Acad. Sci. Paris 114: 1.558; Jour, de Bot. 6:355, pi. 13. This species was described by Viala and Sauva- geau as causing the "brunissure " disease of grape leaves in Europe and the U. S. A. The disease had previously been observed by several workers, and in 1891 Pastre gave an account of its external symp- toms. It was subsequently reported in England (Anony., '93; Cooke, '93; Massee, '93), Italy (Briosi, '94; Briosi and Cavara, '94; Cuboni, '94; Solla, '01), Germany (Moritz and Busse, '94; Beh- rens, '99), Algeria (Debray, '94a, '94b). Holland (Bos, '95), and France (Frilleiux, '95; Roze, '99). Cooke believed that the clubbing of vine roots also was due to this organism. The presence or absence of a causal organism in this disease as well as its identity and relationship have been the subject of much debate. In 1894 De- bray pointed out that the organism was more closely related to Ceratium than to Plasmodiophora, and in the following year he established a new genus, Pseu- docommis, to include it. In 1895 Massee reported that the plasniodia and amoebae figured by ^'iala and Sauvageau were nothing but vacuolated tannin vesi- cles and the reticulate primordial utricle of the host cell. Behrens likewise questioned the presence of a causal organism in this disease, but in the same j'ear Roze reported that P. litis occurs widely and is almost a universal parasite. Ducomet ('03, '07) con- firmed Massee's view that "brunissure " is the result of certain environmental and physiological condi- tions. Maire and Tison (09) also reported that no organism was present in the diseased tissues which they examined, and they thus concluded that the so- called plasniodia were products of cell degeneration. P. CALIFORNICAE Viala and Sauvageau, 1893. C. R. Acad. Sci. Paris 115:67-69. Viala and Sauvageau believed this s])ecies to be the cause of a vine disease in California, and it was subsequently reported as such by Casali and Fer- raris ('00) in Italy. Massee ('95) pointed out that the disease is physiological and that the rejiorted amoebae and plasniodia are nothing more than tan- nin vesicles and reticulated host jirotoplasm. Ravaz KXl I.rUKl) Sl'KHES 35 ('()(>) also r»-|)ortf(l that /'. Calif iiruiae is not an or- K.-iiiisin Init only (Icjicntratfd cliloroiiliyll. and in IJ>0!l .Main- and Tison conliniud the (indinys of Imtli of tlu'sc in\ (stiirators. P. ORCHIDIS MasMO. l!>!)j. Ami. Bot. !): 170, IJl-U'i), 1>1. 1."). L'ndir this nanu- Ma.ssec reported an organism whieli lie believed to be tlie cause of spot diseases of orehid leaves. Later in the same year after more in- tensive stndy he retracted this view and sliowed that what he had previously believed to be s))ores were nothing more than tannin vesicles. P. TOMATI .\l)l»y. IWI."). .lour. Hurt. M-A ser. 30:.%a. .Vbliey st.ited in a letter to the .lourii;il of Horti- culture tiiat this organism is probably the cause of a disease of tomatoes, but as far as the present writer is aware he never gave a description of the parasite. Massee ('9.5, p. 427) believed that Abbey's disease is not due to a ))arasitic organism but to certain rapid changes in environmental conditions. P. HUMULI Kirk. lS()(i. Utpt. Dept. Agric. New Zealand 11: 337. On the roots of hops in New Zealand Kirk found galls which were similar to those produced by P. Brassicae on crueifcrs. and without seeing the causa- tive agent, he assumed the disease to be caused by a species of Plasmod'tophora to which he gave the name P. huniuli. In 1922 Nicholls reported a disease of hojis in Tasmania which showed the "take all " symp- toms, and he assumed it to be caused by the same organism without examining the tissues microscopi- eally. Later, in his correspondence with Miss Mc- Lennan ('31. p. 12) he stated that he had found a mvxoniycete which he took to be P. humiili. In study- ing the "take all" disease of hops more intensively in Tasmania, McLennan concluded that it may be caused by a virus. In some of the diseased plants, however, she found a })lasniodial organism which was later isolated and grown in pure culture and turned out to be a proteomyxean species. Leptomyxa reticulata var. hiimuli. .She also examined preserved material of diseased hops labelled 1'. humiili which belonged to the Department of .\griculture, Mel- bourne, but found no evidence of a myxomycete. Miss McLennan accordingly concluded that tiie tumors described by Kirk were crown galls caused liy Pseii- dnmonas tumrfacicns and that P. huniuli is no longer valid. P. VASCULARUM Matz, lO.'O. .Jour. Dcyit. Afrric. Porto Kifo J: I.), fi(:s. 7-9. Liiniifra rn»rulnrum M. T. Cook, Ifl.'B. Ihiil. 13: l!t. Pis. 3-6. Soroiiphaera vimculiirum M. T. Cook. 1937. Ihiil. :31:S1. Pis. 5-7. This species was described by Mat/. ('20, '21, '22) as causing the dry top rot of sugar cane, Sac- fhanim officinal i, in Porto Hieo. and in lilSl it wa.s re))orted on the s.ime liost in \'ene/,uel;i by Chardon .ind Toro. .M . '1'. Cook triiusfcrred the org.-inisin to the genus Lii/nicra because it does not cause hyper- trophy of the host. Later ('32), W. R. I. Cook m.adc an intensive study of the organism from material sent by M. T. Cook and found that the disease was caused by two protozoan ))arasitcs to which he gave the names Amocbo.iporus t'asculariini .uid ./. Sac- charinum. M. T. Cook later ('37) tr.insfcrrcd it to the genus Snro.s'pliarra. P. TABACl Jones, 19-'(>. I?ot. Gaz. xl: Uli, pis. 31-37. Kig. 1. -'. /'. lulu, cum .Tones, Ifl.'fi. Pliytopatb. Ifi: fi7. In tobacco. |)otato and tomato plants ati'ected with mosaic-like symptoms and leaf roll, Jones found a plasmodiaceous organism which he believed to be a species of Plasmodiophora. Infected cells become necrotic and adjacent ones hyperplastic, and all tis- sues except bast fibers and xylem are invaded by ))lasmodia which pass from cell. .lones found only Plasmodia in the host plants, but when these are cultured in Knop's solution, they give rise to amoebae and uniflagellate organisms, both of which may or may not encyst. The amoebae which eontiiuie to de- velop discharge ehromidia from the nucleus into the cytoplasm, and these chromidial bodies soon aggre- gate to form daughter nuclei, thereby making the enlarged amoebae multinucleate. Such amoebae give rise to motile uniflagellate isogametes which fuse shortly, forming a uniflagellate zygote. The zygote then divides into two zoospores which in turn form amoebae. The flagellate cells and amoebae which encyst pro- duce amoebae on germination, and these fuse to form the Plasmodium. According to .Tones, hundreds of amoebae may flow together in this manner and make a huge Plasmodium which creeps along ra})idly like a giant amoeba ingesting food in its path. S])orogen- esis in this species is unlike that of any known mem- ber of the Plasmodioi)horaceae. As the ])lasmodium moves along, oval and s|)lierieal sjiorcs are delimited in rows and left behind. The nuclei of these spores soon enlarge, discharge chromidi.i. and eventually disappear, while the ehromidia in the cytoi)lasm ag- gregate and form daughter nuclei. Walls develop around these nuclei, and in this fashion 3 to 15 endogenous spores are formed. Plasmodiophiira ta- haci has a very complex life history, according to .Tones, but he was not certain as to the sequence of stages. Since he also found cert.-iin flagellate and amoeboid stages which could not be fitted into any known life cycle, it is not ini])robable that he may have had more than one organism at hand. Miss Mc- Lennan ('31) believed that the plasmodial stage may relate to a proteomvxean-like organism of the Lcpfnmi/ja reticulata type. In IStSI Cook expressed a similar opinion in st.iting that P. iahaci is prob- ably a species of amoeba which had temporarily en- tered the tobacco leaf, but in 1933 he suggested that 36 PLASMODIOPHORALES the stages which Jones found in mosaic diseased plants may relate to excitation and degeneration products of the kind described by Kunkel, Goldstein, Holmes. Sheffield, and others." At any rate P. tabaci has but little in common with other known members of genus, and the author agrees with Cook that there is little if any justification at present for in- cluding it in the Plasmodiophoraceae. Since he was able to produce mosaic-like symp- toms in plants by inoculation with cultures, .Tones believed his organism relates to the cause of tobacco mosaic. Later in the same year, however, he. Link, and Taliaferro ('2G) found that the organism could be cultivated from healthy as well as diseased plants. Furthermore, upon inoculation, mosaic-like symp- toms appeared only when the amoebae and Plas- modia came from diseased plants. They accordingly concluded that P. tabaci is not the cause of mosaic but may be a carrier of the causative agent. In 1937 .Tones retracted his previous views about P. tabaci and redescribed it as the soil amoeba. Nae- gleria gruberi, wliich he claimed is not an amoeba proper but a stage in the life cycle of a myxomycete. He excluded it from the Plasmodiophorales on the grounds that: (1) several amoebae fuse and form a large multinucleate plasmodium; (2) the nuclei di- vide promitotically as in an amoeba; (3) the plasmo- dium forms an aggregate of separate resting spores ; and (i) it does not parasitize jilants. He furthermore reported that N. gruberi may be an alternate host for the mosaic producing organism in tobacco. .Tones' above-cited reasons for excluding this organism from Plasmodiophora are obviously no more critical than those presented previously for including it in this genus. The additional data which he has presented do not clarify its taxonomic jiosition or relationsliip. P. SOLANI Brehmer and Biirner, 1930. Arb. Biol. Reich- sanst. f. Land-u. Forstwirtsch. 18: 1-S4, pi. 1, fifr. 1-32. Brehmer and Earner gave this name to an oval. 4.3.5-.5..5 /i X 2.9 fi, amorphous, pale yellowish-green organism with a distinct refringent sheath which they found in older portions and parenchymatous tis- sues of potato stems sliowing leaf roll, mosaic, and other degenerative symptoms. The thallus divides into as many as eight daughter cells, and tliese in turn give rise to vesicles or spores which are subse- quently liberated by the breakdown of the daughter cells. The spores produce a filamentous zoospore, 5 jx in length and a fraction of a micron in diameter. Brehmer and Barner found all of these stages in fil- tered juices of diseased plants as well as in plants to which virus symptoms had been communicated by grafting and concluded tlierefrom that P. Solaiii is the cause of potato virus. These authors pointed out the similarities of tlieir organism to .Tones' parasite Calkinsi and the so-called "X" bodies of various in- 1 Kunkel, L. O. 1921. Hawaiian Sugar Planters' Assoc. Bot. Ser. 3: 108. Goldstein. B. 192T. Bull. Torrev Bot. Club 54: 285. Holmes. F. O. 1938. Bot. Gaz. 86: 50. Sheffield, F. M. I>. 1931. Ann. Appl. Biol. 18: 471. vestigators and considered it to be either an inde- pendent amoeboid entity capable of spore formation or a Plasmodium living in symbiotic relationshi)) with the plastids. They regarded it as a member of a hitherto unknown group of the Archimycetes allied to the Plasmodiophoraceae. It has subsequently been reported by Moesz ('38) on potatoes in Hungary. There is little in the life cycle of this organism, as described by Brehmer and Barner, which indicates relationship to Plasmodiophora, and it is accordingly excluded from the genus. P. THEAE Fitzpatrick, 1930. The lower fungi-Phycomy- cetes. New York. See Sorosphaera Theae Speschnew. bibliography: e.\cluded species Anony. 1893. Card. Chron. 74: 217. Behrens, .1. 1899. Weinb. u. Weinh. no. 33. Bjiirkenheim, C. G. 1904. Zeitscli. Pflanzenkr. 14: 1 «l. Bos. R. 1895. Zeitschr. Pflanzenkr. 5: 286. Briosi, G. 1894. Boll. Notiz. Agr. 16: 5-22. , and F. Cavara. 1894. Essiccati, delineati e descritti. Fasc. X. Brunchorst. 1886. Untersuch. Bot. Inst. Tiibingen 2: 151. Casali. C, and T. Ferraris. 1900. Giorn. Vitic. Entolog. 8: 10. Chardon, C. E., and R. A. Toro. 1934. Monogr. Univ. Porto Rico, Phys. Biol. Sci. ser. B, no. 2: 75. Cook, M. T. l'929a. Phytopatli. 19: 91. . 1929b. Ann. Rept. Insular Exp. Sta. Dept. Agr. Labor. Porto Rioo 1927/'28. 1929: 59. Cook, W. R. I. 1931. Ann. Protist. 3: 197. . 1932: Jour. Dept. Agr. Porto Rico 10: 407. Cooke, M. C. 1893. Gard. Chron. 13: 711. Cuboni, G. 1894. Boll. Notiz. .-Vgr. 16: 378. Debray, F. 1894a. C. R. Acad. Sci. Paris 119: 110. '. 1894b. Rev. Vitioult. nos. 35, 38. . 1895. C. R. Acad. Sci. Paris 120: 943. Ducomet, V. 1903. C. R. Assn. Franc, avanc. Sci. 32 sess. Anglers 2nd pt. pp. 697-707. . 1907. Ann. lecole d'Agric. Rennes 1: 1-284. 1908, Ihkl 3: 1-70. Frank, B. 1891. Ber. Dent. Bot. Ge.sell. 9: 244. Gravis, A. 1879. Bull. Roy. Soc. Bot. Belgique 18: 50. Jaap, O. 1907. Ann. Mycol. 5: 246. Jones, P. M. 1928. Arch. Protistk. 62: 307. . 1937. Amer. Nat. 71: 488. Keissler, K. 1907. Ann. Mycol. 5: 220. , and H. Lohwag. 1937. Fungi Symb. Sinicae 2: 2. Link, G. K., P. M. Jones, and W. H. Taliaferro. 1926. Bot. Gaz. 81: 403. Maire, R., and A. Tison. 1909. Ann. Mycol. 7: 242. Massee, G. 1893. Gard. Chron. 14: 282. Matz, J. 1921. Esta. Exp. Insular Porto Rico 8: 63. . 1922. Jour. Dept. Agr. Porto Rico 6: 28. McLennan, E. I. 1931. Australian Jour. Exp. Biol. Med. Sci. 8:9. Moesz, G. 1938. Ann. Hist. Nat. Mus. Hungary 31: ,58. Moritz, J., and W. Busse. 1894. Zeitschr. Pflanzenkr. 4: 257. Nicholls, H. M. 1922. Ann. Rept. Govt. Microbiol. Dept. Agr. Stock, Tasmania 1920-21 and 1921-22. Prillieux, E. 1895. Maladie des Plantes Agric. 1: 47. Ravaz. 1906. Rajip. Caise Rech. Paris 1906: 173. TETU.\MV\A Ho/.<-. M. K. IS!)!I. Hull. S(K-. Myoil. Fraiu-o lo: S7. Shibiitii, K. im:. .Iiihrl). wiss. Hot. :{7: (i«. S|ioschiu'w, N. N. 1!'07. nil- ril/.|iariisilcn assively distributed to the res|)ective d.iughter cells (fig. !•). This ap|)ears to be the ))rimary me.-ms of dispersal of the parasite within the host tissue, although Cook be- lieved that the amoebae are capable of migrating from cell to cell. The relation between host and l)athogcn is very intimate, according to ISIaire and Tison. and no antagonism is exhibited. The cyto- plasm of both often ap|)e.irs to be confluent, and it is frequently impossible to determine the boundaries between them. Although the nuclei of the host cells may be enveloped by the jdasmodium (fig. 2). they do not become enlarged and deformed or divide ami- totically as in Triglochin palustre parasitized by T. Triglochinis. When young, the infected cells contain a fairly large amount of starch, but this usually dis- ajjpears after the sporulation of the Jiarasite. The nucleus remains intact for some time later, but even- tually degenerates. Cook ('33) re])orted that this s])ecies had been col- lected by Boyd and Haddon in Scotland and F'.ng- land and that a diseased specimen of R. rosteUata in the Father Reader Herbarium, University of Bristol, had been collected as early as 1885 in Hampshire. Maire and Tison (11) found T. parasitica in abun- dance on R. rostellaia, which grew in close associa- tion with Z. palu.siris var. pedicellata. The latter host was not infected. Claire and Tison accordingly ex- pressed doubt about Hissinger's report of the j)ara- site's occurrence on L. polycarpa, because many au- thors regard this species as only a form of Z. pa'its- tris. T. TRIGLOCHINIS .Molliard. 1909. l?iill. -Soc. Hot. France .56: i4. Mntli„ril!fi Trii/lorhinis (.\I,ill.) Main- and Tison. 1911. .Ann. .Mycol. 9: J.W. Pi. 1.', fig. +7 ()3; PI. 13. fig. G5-()7. Resting s])ores, zoos|)ores. and zoosjiorangia un- known. Plasmodia small, usually numerous in a host cell: undergoing niulti|)le division into several oval, elongate, sickle-slia))ed uninucleate meronts which grow in size, and during the two- to eight-nucleate stage function in turn as schizonts. .\11 else unknown. Parasite on the stems, flowering stalks, stamens, ovaries, but rarely on the leaves of Triqlochin paliis- fra and 7'. maiifiniim in France (Molliard. Maire .iiul Tison. I.e. ) and 7'. maritiinim in Kngland (Cook. '33). causing small fusiform, ov.al .-ind irregular galls. No resting spores have been observed in this s])e- cies. so that its relationship to the other members of the Plasmodiophoraceae is obscure. Because of the lack of resting spores, Maire and Tison regarded 38 PLASMODIOPHOHALES it as representative of a new genus, but as Cook pointed out. there are no good reasons for introduc- ing a new genus until more is known about the life history of this species. It is accordingly retained provisionally in Tetramyia. Maire and Tisons cyto- logical study, nonetheless, indicates its similarity to T. parasitica in the type of vegetative nuclear divi- sion and the presence of centrosomes and astral rays. The effects of this species on the host are striking. According to Molliard. the parenchyma cells of the stem and flowering stalk are greatly hypertrophied and divide irregularly, while development of the sclerenchyma is inhibited Likewise the flowers in an infected region are sterilized. The infected cells may enlarge to four times their normal diameter, while their nuclei become enormous and deformed (fig. 20 j. The nucleoli also increase markedly in size and become deeply basophyllic. At the same time nu- merous deep-staining, chromosome-like chromatic bodies develop in the nuclear cavity. Furthermore, infected cells may often become multinucleate (fig. 19) as the result of amitosis. according to Maire and Tison. The nuclei of adjacent cells may also become enlarged and deformed. The presence of the parasite further stimulates starch formation in the cells sur- rounding infected regions. T. ELAEAGNI Yendo and Takase. 1933. BulL Sericult Silk — Ind. Japan 4, no. 3: 5. Plasmodium inter- and intracellular, segmenting into uninucleate spore mother cells which divide twice to form tetrads of resting spores. Amoebae formed from germinating resting spores. .Sporangia and zoospores unknown. In the roots of Elaeaf/nui multiflora in Japan. This species causes tubercles or nodules which in exceptional cases may attain the size of a man's fist on old trees. The parasite occurs most abundantly in the cortex and causes marked hypertrophy of the in- fected cells as well as enlargement and distortion of the nuclei. Yendo and Takase found that the percent- age of nitrogen in the nodules was almost twice that of the normal cortex, and for this reason they be- lieved that there is a definite symbiotic relationship between host and parasite. So little is known about this species that its valid- itv as a member of the Plasmodiophoraceae is very doubtful. Yendo and Takase reported that the Plas- modium spreads over the host cells and fills the in- tercellular spaces. Furthermore, the resting spores are said to be capable of forming fine, curled, non- septate, branched germ tubes or filaments instead of amoebae. The formation of germ tubes suggests that Yendo and Takase may have had spores of another fungus at hand. ADDITIOXAL BIBLIOGRAPHY: TETEA.MVXA Cook. W. R. I. \9%2. Honp Kong Nat. Suppl. no. 1: 38. ■ -. 1933. .\rch. Protistk. 80: :?16. Maire, R., and \. Tison. 1910. C. R. .\cad. Sci. Paris 50: 1768. .Schwartz. E. J. 1911. .\nn. Bot. 35: 79+. Winge, O. 1913. .\rk. f. Bot. 13, no. 9: 36. PLATE O Tetramyxa para»itiea Fig. 1. Galls on stems of Rvppia rottelhita. Goebel, I.e.; Maire and Tison, '11. Fig. 3. Multinucleate plasmodium surrounding host nu- cleus. Maire and Tison, I.e. Fig. 3. Plasmodium consisting of two multinucleate me- ronts which appear to be fusing: nuclei dividing in one and at rest in the other. Maire and Tison. I.e. Fig. i. Division of infected cell by which the meronts have been passively divided and distributed. Maire and Tison, I.e. Fig. 5. Equatorial ring stage of "promitosis'' in which distinct chromosomes are evident. Maire and Tison, I.e. Fig. 6. .\naphases of same. Maire and Tison, I.e. Fig. 7. Plasmodium becoming parietal and cleaving into uninucleate spore-mother cells or sporonts. Centrosomes and astral rays present at poles of some nuclei. Maire and Tison. I.e. Fig. 8. 9. Prophases of meiosis in sporonts. Maire and Tison, I.e. Fig. 10. Equatorial plate stage of first meiotic division. Maire and Tison, I.e. Fig. 11. Binucleate sporont with conspicuous astral rays. Maire and Tison, I.e. Fig. 13. Equatorial plate stage of second meiotic divi- sion. Maire and Tison, I.e. Fig. 13. Cleavage into tetrads. Fig. 14. Tetrad of resting spores. Fig. 15. Enlarged host cell with resting spores isolated and single, in linear series, in diads. triads and tetrads. Large resting spores binucleate. Maire and Tison, I.e. Fig. 16. Four resting spores in linear series. Goebel, I.e. Tetramyxa Tri{/lochinit Fig. 17. Galls on Triglochin palu»tre caused by T. Tri- glochinh. Maire and Tison, I.e. Fig. 18. Enlarged host cell with spherical multinucleate and fusiform uninucleate meronts. Maire and Tison, I.e. Fig. 19. Enlarged host cell with uninucleate meronts in vacuoles. Host cell tetranucleate: nuclei with numerous densely chromatic bodies. Maire and Tison. I.e. Fig. 30. \n enlarged, deformed host nucleus. Maire and Tison, Lc. Fig. 31. Uninucleate fusiform meronts. Maire and Tison, I.e. Fig. 22-2i. Equatorial plate, anaphase and telophase stages of "promitosis." Fig. io, 36. Bi- and multinucleate thalli. Maire and Tison, I.e. Oetomyra Arhlyae Fig. 37. Habit sketch of Achlya glomerata showing effects of parasite on the hvphae. Couch, et al.. '39. Fig. 38. Early infection stage showing large parasite nu- cleus in host cell. Fig. 29. Binucleate thallus surrounded by host proto- plasm: nuclei dividing "promitotically." Fig. 30. Large plasmodium in a vacuolate area of hyphal tip. Fig. 31. Sporangiosorus of nearly mature zoosporangia. Fig. 33. Zoosporangia with emerging zoospores. Fig. 33. 34. Biflagellate heteroeont zoospores. Fig. 35. Zoospore killed in osmic acid fumes and stained with gentian violet. Drawn from photomicrograph. Fig. 36. Large tetraflagellate zoospore. Fig. 37. Sorus of resting spores. Fig. 38-40. Groups of resting spores. TETRAMYXA 89 ri.ATK 5 Tetranivxa, Octomvxa 40 PLASMODIOPHORALES OCTOMYXA Couch, Leitner, and Whiffen, 1939. Jour. Eli.sha Mitchell Sci. Soc. 55: 400. Whiffen, 1939. Ibid. p. 243. (PL.^TE .5, FIG. 27—10) Re.sting spore.s usually adhering in groups of eight, sometimes in groups of six to nine; forming zoospores whicli infect the host and develo]) into vegetative plasmodia. Such plasmodia cleaving into sporangiosori composed of numerous small zoospo- rangia, which are sometimes conjoined by narrow isthmuses ; exit papillae lacking on some zoosporan- gia. Zoospores anteriorly bifiagellate and hetero- cont. Sporogenous plasmodia cleaving into small segments which in turn divide into eight uninucleate spores. This monotypic genus is characterized by resting spores wliich are grouped usually in clusters of eight (fig. 38). As in other genera, the zoospores enter the host hyphae directly and completely without leav- ing a spore case on the outside. Infection may occur at any place along the hyphae, but hypertrophy of the host occurs only at or near the tip (fig. 27). The young, naked parasite is surrounded by the host pro- toplasm (fig. 28-29) and soon develops into a multi- nucleate Plasmodium. As the latter develops, the hyphal tip swells and attains its maximum size be- fore the parasite is completely mature. As a result. the Plasmodium lies in a vacuolate region (fig. 30) of the swelling, surrounded by radiating strands of host protoplasm along which small particles may be seen moving toward the parasite. The latter thus lives within and in intimate contact with the host protoplasm, and in the early stages of development the two protoplasts are indistinguishable. The plas- modium usually develops from a single zoospore, but Couch et al. believed several small plasmodia may fuse to form a large one. The mature plasmodium, however formed, may give rise to sporangesori or cytosori, but the latter do not usually appear until the cultures are several days old. The zoosporangia (fig. 31) are delimited as globose or ovoid masses which soon develop thin, hyaline walls. Sometimes cleavage may be incom- plete, so that several sporangia are joined by nar- row isthmuses. As the sporangia mature, exit papil- lae are formed on those adjacent to the host wall and on some in the center of the group or sorus. As a re- sult, the zoospores may be discharged (fig. 32) di- rectly to the outside or within the host cell. They emerge from the zoosporangia singly and slowly, and after moving about sluggishly for a few seconds at the mouth of the exit papillae swim away. The two unequal flagella are attached at or near the anterior end, and during motility the shorter one extends for- ward while the longer i)rojects backward. Occa- sional zoospores witli four flagella occur (fig. 36), which ajjpear to be the result of incomplete or un- equal cleavage instead of fusion. The plasmodia which give rise to the resting spores arc indistinguishable from the zoosporangial Plasmodia until after cleavage begins. Miss Whiffen ('39) reported that the two are to be distinguisiied cytologically by the fact that the nuclei of the rest- ing spore plasmodia pass through the so-called akar- yote stage and undergo reduction division. However, she has not yet counted the chromosomes present during the two meiotic divisions. The sporogenous Plasmodia cleave into a number of comparatively large masses, as in Tetrami/.ra, and these in turn usually divide into eight uninucleate segments which soon encyst in groups of two tetrads of resting spores. This grouping, however, may frequently vary from six to nine. Four normal-sized spores and two larger ones may sometimes occur, while nine and seven may be found in other groups. After a short dormant period, the resting spores germinate, each one giving rise to a single zoospore. The structure. type of flagella, and method of swimming of these zoospores are unknown. O. ACHLYAE Couch, et al, I.e., PI. +7, 48. Resting spores spherical, 2. 1—3.2 /x, with smooth, slightly thickened walls. Zoosporangia spherical, ovoid, sometimes flattened by mutual pressure, 6-16 IX in diameter, hyaline and thin-walled; single exit jjapilla on sporangia adjacent to host wall and in the center of gall; deeper lying sporangia often dis- charging zoospores through the peripheral sporan- gia. Zoosjjores 6-11 in a sporangium, discharged di- rectly to the outside and also within the host wall ; oval; flagella attaciied to or near the anterior end, the shorter one extending forward and the larger one backward during swimming. Parasitic in Achli/a r/lomcrata in North Carolina, U. S. A., causing marked enlargement of the hyphal tips. This species appears to be an obligate parasite of A. glomeraia. Couch, et al., attempted to transfer it to Saprolegnia feraj', S. mef/asperma, .J. imperfecta, A. flageUata, A. colorata, A. racemosa, A. deBary- ana, Aphaiiomyces stellatus, Apodachlya brachi/- nema, A. minima, and AUomyces arbii.^ciila, but all results were negative. So far, this is the only known species of the Plasmodiophoraceae parasitic in a fungus. The life cycle of 0. Achli/ae seems to be almost identical with that of JVoronina poli/cijstis as far as both species are known at present, and it is not im- probable that the two may prove to be related. Ac- cording to Couch, et al., 0. Achylae differs from W. polycy.. Colm's Kivpt. Fl. von Sililo- sicns .■{ : l."{."). ( E'l.ATK ()") Cystosori one to several in .1 ii II. pri (louiiii.intly nt' the slia|)0 of hollow sjiheres or ellipsoids, luit often extremely variable in size anil shape; presenee of oonimon eiiveloj)lng membrane doubtful. Resting spores oval, ellii)soidal. j)vriforni, pyramidal and urn-sliapeil with yellowish-brown to brown, thin, smooth or \errueose walls; with or without .ipie.il eollar; produeinj;: a siniilf bitlaiiellate, heteroeont zoos])ore in gerniin.-ition. Kv;nieseent thin-w.illed zoosporansjia sm.ill. I'l.isniodia one to several in a eell. large or small: sehi/.otjony present ( ?) or lack- injr; produeinji a sinj;le cystosorus. This genus includes at present only two species which have been reported in moist and damj) locali- ties in Europe. England, and the U. S. A. Of these two, iS. r eronicac appears to be more common and has been frequently studied cytologically. Nonethe- less, many of its critical develo])niental stages are still im)>erfeetly known, and there has been consid- erable controversy relative to many of its cytologieal details. Germination of the resting spores had not been observed until very recently. Blomficid and .Scliwartz ('10) re|)orted the jjresence of amoebae in a sterile infusion of 1 erunica leaves which had been inoculated with i)ortions of dead tumors. Since this infusion was thus no longer sterile and soon became invaded with bacteria, molds and other organisms from the tumors, the uninucleate amoebae which they found after fourteen days in the bottom of the test tube may not relate to S. J'eronicae at all. In S. radi- cali.s. Cook and .Schwartz likewise failed to observe germination, but among diseased root hairs they found anteriorly uniflagellate zoospores which they assumed relate to their s))eeies. However, they did not follow the development of these zoosjiores into mature thalli. On the other hand. Barrett foinid that the zoospores from sporangia are distinctly bitlagel- late and heteroeont. He also succeeded in germinat- ing the resting spores, but has not yet determined the number of flagella on such zoos))ores. I.edingham ('3S). p. 1-3) found that zoos|)ores from resting s))ores of .S'. t'eronicae also are biflagellate and heteroeont. Cook ('33) stated that the resting s))ores form a sin- gle amoeba or zoospore, but it is quite probable that the multinucleate spores rejiorted by Maire and Tison (fig. 49-.5I) may give rise to several zoo- spores. -Vs the primary uninucleate amoebae of .S'. I'l-ro- nica (tig. 9) increase in size within the host cell, their nucleus divides, and multinucleate |)lasmodia are soon formed (fig. 11-1.5, 22). Hy the time the eight- nucleated stage has been reached, the plasmodia may function as schizonts and split off uni- and multi- nucleate meronts (fig. 23. 2 J), according to Maire and Tison, The multinucleate meronts may in turn undergo schizogony into uninucleate segments be- fore further mitoses occur. The unimicleate meronts are ciiuiv.ilent to the primary amocb.ii' and ni.iy thus begin the eyi'le .mew. while the scliizont from which they are derived ])asses into the s))orogonie jihase of (levelojimcnt, in the opinion of .Maire and Tison. It is to be noted, however, th.-it these workers have ncxcr observed the actu.-il s))litting oft' of meronts, .ind their re])orts on the presenee of schizogony are based only on the .ipiiea ranee of constricted plas- modia (fig. 2.i. 2 1-) .-md the grc.-it abuiulance of uni- luiele.ite amoeb.ie in infected cells. The Latter may well be the result of nuiltiple infection, while stages such as are shown in figures 23 and 21' may possibly rei)resent, as Maire and Tison earlier interpreted them, fusions of uni- and bimicleatc amoebae with nuiltinucleate |)lasniodia. \\'hile the author readily admits the possibility of schizogony, he does not con- sider the evidence so far ))reseiited as sufficiently re- liable to have conclusively settled the jtroblem. In this connection it is significant to note that schizog- ony has not been recorded in species, such as S. radi- cal'is, where the process if present could be readily observed in living material. Tl'.e vegetative phase is terminated by the so- called transitional stage after which follow cleavage and meiosis, as has been described in Chajiters \\ and III. The plastic cleavage segments or incipient resting spores become associated in a globular mass (fig. H) and resemble myxamoebae in a pseudoplas- modium. By mutual readjustment they soon move to the periphery (fig. II) and thus form a hollow s])here or ellipsoid. At this early stage the center of the mass is filled with a viscuous fluid, doubtless a residue of the plasmodium which is not used up in cleavage. Whether or not this substance rejiresents extraneous waste material which is dumped into a central vacuole in the dediiferentiation of the proto- ))lasm ])reparatory to sporogenesis as in various pro- teomyxean species is not certain. Maire and Tison stated that it has an osmotic coerticient and exerts centrifugal pressure on the s))ores whereby they are |)ushed to the periphery of the mass. .Shortly after their arri\al there, the individual s])orcs develop delicate walls wliic'h thicken and turn brown with maturity and often become verrucose. No evidence of cellulose or i)ectin M-as found in these walls by Maire and Tison. By mutual compression the spores usually become iientagon.-illy and hex- agoiially |)yramidal in shaiie with convex external and slightly concave intern.-il surfjices. According to ^\'inge. a eollar is formed .at the ;i))ex or external surface (fig. 18), but this structure has not been re- corded by other workers. Occasional bi- and trinu- cleate spores occur (fig, ■19-.51). which may have arisen by incomplete cleavage or by subsequent di- vision of the spore nucleus (fig. .50). It is to be partieul.irly noted that in none of the figures and descriptions of Blomficid and .Schwartz or Maire and Tison which illustrate the aggregation of incipient resting spores and their transformation into cystosori is there evidence of a distinct, common eiivelo))ing membrane around the sorus. Likewise, it 42 PLASMODIOPHORALES is lacking in Rostrup's, Winge's, and Palm and Burk's figures of cystosori. Cook's ("33, PI. 6, fig. 9) own photographs of S. J'eronicae fail to show a dis- tinct membrane. Nonetheless, he has often contended that it is present and has used ('33) the presence of this structure as one of the distinguishing generic characters of Sorosphaera as well as Sorodisciis. In the original diagnosis of the genus. Schroeter de- scribed the cystosori as being surrounded by a com- mon cuticle, and this may be partly responsible for Nemec's ('H) and Cook's contention as to the pres- ence of a membrane. Webb described it as being formed after the spores had developed their individ- ual walls, but he gave no figures of its development. Winge ('13, p. 30) denied its existence, while Blom- field and Schwartz as well as Maire and Tison, who have so far made the most extensive study of the genus, said nothing about it. It is quite probable that the adjacent lateral walls of the spores become more or less fused by mutual pressure as they de- velop, and this prevents the spores from separating readily at maturity. The best cytological data in the literature to date do not, therefore, support Cook's view on the presence of a membrane, and the use of this structure as a diagnostic generic character is at present open to serious question. The cvstosori of S. J'eronicae are predominantly hollow spheres and ellipsoids, but numerous varia- tions in shape have been noted by Maire and Tison, Trotter, Webb, and others. Palm and Burk in par- ticular found them to be unusually variable in galls on r. americana collected in Colorado, U. S. A. In this material they found the cystosori to be three principal shapes: hollow spheres, flattened ellip- soids, and irregular sponge-like masses, and between these types all degrees of variations and intergrada- tions were observed. As is shown in figures .52 to 57, the Sorosphaera- or hollow-sphere type predomi- nated, but two-layered flattened discs as in Soro- disciis (fig. 53, 54), spongy masses with narrow or wide channels as in Spongospora (fig. 55, 56), and irregular masses of indeterminate shape as in Lig- niera (fig. 57) were not uncommon. Likewise within the same sorus, spores with smooth and verrucose walls were present (fig. 53, 55, 57). Palm and Burk accordingly concluded that the shape of the cysto- sorus and the relative arrangement of the spores are governed largely by environmental conditions and that the size and shape of the host cell are determin- ing factors. They furthermore concluded that since sori typical of those of Spotu/ospora, Sorodisciis, Ligniera, Osienfeldiella, Claihrosoriis, and Mem- hranosoriis may all be found in S. J'eronicae, these genera should be regarded as synonyms of Soro- sphaera. Fitzpatriek ('30) believed that Ligniera also should be incorporated in Sorospftaera on the grounds that the only difference between the two is that the former causes no hypertrophy of the host. In 1907 Speschnew (p. 22, PI. 2, fig. 7-12) de- scribed a species on tea leaves in the Caucasus which he named Sorospliaera tlieae. Two years later, how- ever, Ducommet ('09) reported that no organism is present in the leaves and that the so-called spores are only tannin deposits in the cells. Fitzpatriek re- ferred to this species as PlasmodiopJiora Theae. S. VERONICAE Schroeter, I.e. Tiihirciiiia f'erouicae Schroeter. 1877. Beitr. Biol. Pflanz. ;? : 383. Sorusporiiim Veronicae Winter. 188-t. Die Pilze Deutsch- lands, Oesterreich und der Schweiz 1 : 103. Cystosori bright brown, one to several in a cell, variable in size and siiape, predominantly in the form of hollow spheres, ]8-42(a, occasionally elon- gate, flat and disc-shaped, irregular and indeter- minate, compact or loose and spongy with numerous ramifying channels, composed of from four to 61 spores. Resting spores ovoid, pyramidal, urn-shaped 1-5 IX X 8-9 fx., with brown, smooth or verrucose outer walls, often surmounted by an apical collar. Zoospores biflagellate and heteroeont. Plasmodia one to several in a cell, 20-30 /i, schizogony doubtful; producing a single cystosorus. Zoosporangia un- known. Parasitic in J'eronica hederaefolia, J', cliamae- drifs, and /'. triphifUos in Germany (Schroeter, '77, '86, '97 ; Winter, I.e. ; Diedieke, '11; Grevillius, '13) ; /'. saj-atilis, J', officinalis, J', hederaefolia, J', scii- tellata, J'. Beccahiinga, J'. Anagallis, J', aqiiatica, J'. serpi/Uifolia, and /'. Chaemaedri/s in Finland, Norway and Sweden (Lagerheim: see Winge, '13; Palm, '08) ; J\ hederaefolio in Sehleswig-Holstein and Denmark (Hennings, '91 ; Rostrup, '94) ; T. Chamaedri^s in France (Maire and Tison, '08, '09, '10, '11; Maire, '10); /'. Chamaedrys in England (Blomfield and Schwartz, '10; Cook and Schwartz, '29) ; r. arvensis and J', hederaefolia in Italy (Trot- ter, '04, '16); r. americana and J', arvensis in the U. S. A. (Palm and Burk, '33; Donald, '34), caus- ing tumors up to 5 mm. in diameter on the stems, petioles, and midrib of leaves. This species was first described by Schroeter in 1877 as a member of the Ustilaginales under the name Tuhercinia J'eronicae, and in 1884 ^^'inter transferred it to the genus Sorosporium. In 1886, however, Schroeter created the genus Sorosphaera for it and transferred it to his newly established Phytomyxinae. Rostrup found it in Denmark in 1894 and replaced it in the Ustilaginales, and ac- cording to Winge, Lagerheim found it in Norway and Sweden on a large number of J'eronica species, and as early as 1901, "and knew the correct system- atic position of Sorosphaera." Trotter discovered it in Italy in 1904, and while he questioned its inclusion among the smuts, he also doubted that it is a mem- ber of the Mycetozoa. The subsequent studies of Maire and Tison and Blomfield and Schwartz clearly showed that it belongs in the Plasmodiophoraeeae in close relation to P. Brassicae. The tumors caused by S. J'eronicae vary from pin- head size to 5 cm. in diameter and are usually com- ])osed of a mass of healthy and infected undifferen- tiated cells among which are intersjiersed a few spi- ral and annular vessels. The galls are the result of SOUOSPIIAKRA 13 both cell limit ipliiaf ion and itll cnlarmciiicnt witli tin- lattiT proi-css playiiiii tlif (loniinaiit role in tin- lati-r stages of divclol)nunt. Since the (larasite has a ])redilcetion for the provascular strands in tlic aj)ieal nieristem. the tumors may involve the entire stem in instances of severe infection. In such eases the priuiordia of the stems and leaves are reduced to a mass of cells in which |)ith. cortex, etc., are indis- tingiiish.-ihle. In less extensive infections only small portions of the stem hceome involved, and the normal growth of the plant is not seriously artectcd. .\ccord- ing to Lagerheim. the development of the vascular ring is suppressed in the region of infection, while in the outer cortex the collenchyma is still present. The rem.iining cortical cells become tangentially oriented in growth and greatly enlarged. The ejiider- mal cells become isodiametric. and the gu.ird cells of the stomata are often considerably enlarged, with the pore itself abnormally wide. .\lthough infection has not been observed. S. J'e- ron'icae appears to make its initial entrance in the apical meristem. because the youngest plasmodia and smallest galls occur in or near the apex. Blomtield and .Schwartz succeeded in jiroducing tumors on Ve- ronica seedlings by si)raying them with water con- taining crushed cystosori and found single, isolated infected cells close to the growing point. The amoe- bae of the parasite are apparently unable to pass through the walls into adjacent cells. According to Blomtield and .Schwartz, and Cook ("33). they are passively distributed by the repeated division of in- fected provascular cells. If the young plasmodia un- dergo schizogony, as Maire and Tison reported, the number of amoebae is greatly increased, and by re- peated division of infected cells, large diseased areas are soon formed. In the early stages of the disease the presence of the [larasite apparently does not inhibit cytokinesis of the host cells, but later on after they have become enlarged the latter lose the ability to di- vide. The enormously enlarged nuclei, however, un- dergo several mitoses with the result that the infected cells become multinucleate (fig. 3). Division of the host nuclei is greatest at the close of the vegetative stage of the parasite, but with the onset of the sporo- gonie ])hase mitosis ceases. At this stage the host nu- clei become distorted (fig. .5), more densely stainable, (fig. i). and eventually disintegrate (fig. Vt). By the time the cystosori are mature, only atroi)hied and de- generated nuclei are to be found, according to Bloni- field and Schwartz. On the otlier hand. .Maire and Tison rei)orted that the nuclei as well as ))]astids and starch grains may jM-rsist long after the sori have ma- tured. In the early stages of infection only slight en- largement of the host cells occurs, but as the plas- modia increase in size, marked expansion takes place. In exceptional eases infected cells may enlarge to 20 times their normal diameter. Sorosphaera J'eronicae accordingly not only causes enormous cell enlarge- ment but also prevents cell diflferentiation. .\djacent healthy cells as well as stomatal guard cells may also be stinuilated to enlarge by the presence of the para- site. .\s is shown in figure 2. there ,ai)))ears to be no visible .■intagonisni between tlie proto])l;ism of the host and pathogen. The I.itter lies embedded in the host eytoiilasm and in the young stages may be closely associated with the host nucleus. Infected cells may contain numerous jilastids and starch grains, but these are not so abundant as in the adja- cent healthy cells. According to I.agerheim. ei)idcr- mal cells in the infected regions .ire richer in crystals th.in tiiose in healthy j)ortions of the stem. Slugs frequently feed on the galls, and it is be- lie\ed th.it they play a signifie.int role in spreading the disease. Most tumors soon soften and decay, lib- erating the cystosori into the soil, where the resting sjiores germinate. When new plants push up through the soil, their ajjices a])l)arently become infected. Sorosphaera J'eronicae has never been found jiara- sitizing the roots. Nematodes also may cause galls on Veronica which arc strikingly like those ))roduced by .V. J'eronicae and may easily be mistaken for them. I''or this rea- son Cook ('33) regarded with susijieion the rejjorts of I.agerheim and Winge of the presence of the para- site in a large number of J'eronicae species in Nor- way and Sweden. S. RADICALIS Cook. 1933. .\reh. Protistk. 80: .'01. PI. T, fig. 10, 11. S. nuliriile Cook and Sclnvart/.. lfl-'9. .\iiii. Hot. 43: 86. PI. 2. Cystosori single and Jiartly filling host cell, hol- low, rarely spherical, 20 yu,, usually oval, ellipsoidal .and elongate, 1(5-20 fj. X 20-.57 /u,, bright yellowish- brown ; including up to 500 spores. Resting spores oval, 3 X ^ /i. with thin yellowish-brown, smooth walls ; producing one zoos])ore in germination. Zoo- spores oval and sl)lierical, 2-3 /x. with an anterior flagellum (.'') l—ti/x long. Evanescent zoosjjorangia unknown. Plasmodia single and partly filling host cell. 20-60 /J. ill diameter, producing one cystosorus ; schizogony lacking (.'). Parasitic in the root hairs only of Poa fluiians MoniVia caerulea, Catabrosa aquaiica, and other grasses in England, causing localized enlargement of the infected cells. This species is distinguishable from .S'. J'eronicae jirimarilv by its oval, ellipsoidal and elongate cysto- sori which are also much larger ;iiid com])Osed of a greater number of small resting sjiores. In .addition, its nuclei are considerably smaller. While .V. radicalis may occur in the same vicinity with and infect some of the hosts of Lifjniera J unci as well as L. verrucosa and L. piloriim. Cook and Schwartz maintained that it is ()uite distinct. However, it is to be noted here that these Lifjniera s|)ecies may also occur in locallv ' In recent correspondence with the aiitliiir. Prof. .J. T. Barrett, College of .Agriciiltnre, Califdrnifi rniversity, re- ported that he had fonnil what lie helieves to he S. ridlirnlis in roots of I'oa annua on the eoUejie rani]>iis. In addition to cystosori and resting spores, he ohserved thin-walled spo- ranjria which produce hifhifrellate, hettrocont zcM)spores. Harrett thus confirms I.edingham's previous n-)iort of sudi /,oo,..pores in Sftro.splifiern. 44 PLASMODIOPHORALES swollen root hairs and occasionally form almost spherical, oval and elongate hollow cystosori. Sorosphaera radicalis lias been found only in root hairs and does not attack the other tissues of the root. Hence no external symptoms of the disease are visi- ble on the host plant except a slight reddening of the stem and leaf bases. When the infected root hairs decay, the cystosori are liberated into the soil. In- fection by zoospores api)arently occurs during the early developmental stages of the root hairs. .Although Cook and Schwartz failed to count the chromosomes, they nonetheless believed that meiosis occurs during the first of the two last divisions pre- ceding sporogenesis. No evidence of gametic fusion has been observed. Cook and Schwartz reported that at the conclusion of promitosis "a wall is now secreted around the Plasmodium, and the whole mass passes into a spor- ing stage." If this statement and observation are true, it is obvious that .S'. radicalis differs radically in this respect from all other known species of the Plasmodiophorales. PLATE 6 Sorosphaera Veroiiicae Fig. 1. Vcroiiira chamaedrys with galls caused by S. t'eronicae. Winge, '13. Fig. -2. Hypertrophied host cell with six plasmodia. Note relative sizes of healtliy and infected cells. Blomfield and Schwartz, '10. Fig. 3. Hypertrophied ho.st cell with five plasmodia. Four host nuclei in telophases of division. Blomfield and Schwartz, I.e. Fig. i. Nucleus of parasitized cell witli numerous nu- cleoli. Blomfield and Schwartz, I.e. Fig. 5. Lobed and distorted nucleus of an infected cell. Maire and Tison, "09. Fig. 6. Old host cell with four cystosori; protoplasm almost completely gone. Blomfield and Schwartz, I.e. S. rfi(UcaUs Fig. 7. Hypertrophied root hair with cystosorus in sur- face view. Cook and Schwartz, '29. Fig. 8. Median longitudinal section of an ellipsoidal cystosorus. Cook and Schwartz, I.e. S. f'erontcae Fig. 9. Uninucleate stage of thallus. Maire and Tison, I.e. Fig. 10. Resting nucleus of young parasite. Blomfield and Schwartz, I.e. Fig. 11. Beginning of promitosis of a 4-nucleate Plas- modium with centro.somes and astral rays. Maire and Tison, i.e. Fig. 12. "Saturn stage"' of promitosis. Maire and Tison, I.e. Fig. 13. Early anaphases. Maire and Tison, I.e. Fig. 14. "Double-anchor" stage of promitosis. Maire and Tison, I.e. Fig. 1.5. I.ate anaphases with centrosomes and asters. Maire and Tison, I.e. Fig. Hi. I.ate prophase of vegetative nuclei in Plasmo- dium with four chromosomes. Webb, '3.5. Fig. 17. Later stage sliowing four split chromosomes. Webb, I.e. Fig. 18. Four chromosomes arranged in a ring around constricting nucleole. Webl), I.e. Fig. 19. Metajjhase; daughter chromosomes beginning to separate. Webb, I.e. Fig. 20. Early anaphase. Two rings of four chromosomes each moving apart. Webl), I.e. Fig. 21. Later ana])liase. Webb, I.e. Fig. 22. Telophases of jiromitosis. Maire and Tison, I.e. Fig. 23, 2i. Schizogony of jilasmodium ; uni- and binucle- ate segments respectively being split off. Maire and Tison, I.e. Fig. 25. Beginning of akaryote stage; chromatin passing out into cytoplasm. Blomfield and Schwartz, I.e. Fig. 26. Akaryote stage; nuclei clear and vacuole-like. Blomfield and Schwartz, I.e. Fig. 27. Reconstructed nuclei following akaryote stage. Blomfield and Schwartz, I.e. Fig. 28. Later stage showing reappearance of nucleoli and chromatin. Maire and Tison, I.e. Fig. 29. So-called "garland" stage of reconstructed nu- clei. Maire and Tison, I.e. Fig. 30. Same stage highly magnified. Winge, I.e. Fig. 31,32. Synezesis (?). Maire and Tison, I.e. Fig. 33. Beginning of cleavage into spore mother cells; appearance of nuclei suggestive of diakinesis. Maire and Tison, I.e. Fig. 34. Early diakinesis. Webb, I.e. Fig. 35. Diakinesis with four pairs of homologous chro- mosomes. Webb, I.e. Fig. 36. Equatorial plate stage of heterotypic division during sporogenesis. Cleavage into spore mother cell com- plete. Maire and Tison, I.e. Fig. 37. Same stage. Winge, I.e. Fig. 38. Late anaphases of meiotic division; first division of spore mother cells beginning. Maire and Tison, I.e. Fig. 39. First division of spore mother cells complete. .Maire and Tison, I.e. Fig. 40. Late prophase nucleus of second or homeotypic division with four chromosomes. Webb, I.e. Fig. 41. Equatorial plate stage of second division during sporogenesis. Maire and Tison, I.e. Fig. 42. Second cell division into incipient resting spores. Fig. 43. Incipient resting spores aggregating into a globular mass; initial stage in formation of cystosorus. Maire and Tison, I.e. Fig. 44. Later stage in cystosorus development; spores arranged at periphery with a viscous substance in the cen- ter. Maire and Tison, I.e. Fig. 45. Young cystosorus in median section with well- defined walls around spores; remants of viscous substance in center. Maire and Tison, I.e. Fig. 46. Cystosorus in median section. Blomfield and .Scbwartz, I.e. Fig. 47. Portion of a cystosorus in surface view. Blom- field and Schwartz, I.e. Fig. 48. Urn-shaped resting s])ore witb apical collar. Winge, I.e. Fig. 49. Binucleate resting spore. Maire and Tison, I.e. Fig. 50. Division of nuclei in binucleate resting spore. .Maire and Tison, I.e. Fig. 51. Trinucleate resting spore. Maire and Tison, I.e. sonosiMiAKiiA 45 PLATE 6 V^^ ® ^ AM i I ^ XW i7 4,W ■■■■ » ^11^''' ^* ■ ^ti^' '^;y • -i- ,25 26 33 40 --A^'i C2 #«% %.# 41 46 - 43 48 49" Sorosplidcra m--. .^^.^' 32 pa 44 50 39 46 PLASMODIOPHORALES PLATE 6— Continued Sorosphaera Veronicae Fig. 52-57. Variations of the cvstosori of iS. Veronicae. Palm and Burk, "33. Fig. 53. Typical hollow Sorfinphaera-tike cvstosori with smooth and verrucosa spores. Fig. 54. Flattened Sorodhcus-lihe cystosorus. Fig. 55. Spongy Span gospora-Whe cystosorus. Fig. 56. Loose, spongy Clathrnsonix-hke cystosorus. Fig. 57. Irregular LU/nierri- and globular Sorosphaera- like cvstosori. Smith, A. L., and J. Ramsbottom. 1917. Trans. Brit. Mycol. Soc. ti: 231. Spescbnew, X. X. 1907. Die Pilzparasiten des Teestrauches, p. -22. Berlin. Trotter, A. 1904. Ann. Mycol. 2: 536. . 1916. Marcellia 15: 58. Webb, P. C. R. 1935. Ann. Bot. 49: 41. Winge, O. 1913. Ark. f. Bot. 12, no. 9: 4. ADDITIONAL BIBLIOGRAPHY: SOROSPHAERA Blomficld, J. E., and E. J. Schwartz. 1910. Ann. Bot. 24: 35. Diedicke, H. 1911. Mitt. Thiir. bot. Ver. n. f. 28: 83. Donald, L. 1934. Phytopath. 24: 843. Ducomet, V. 1909. Ann. I'eeole X'at. d'Agric. Rennes 2: 1. Grevillius, A. Y. 1913. Abh. Ver. Nat. Erfors. Niederrheins 1: 165. Henning, P. 1891. Schrift. Xat. Ver. Schleswig-Holstein 9: 235. Ledingham, G. A. 1939. Canadian Jour. Res. C 17: 43. Maire, R. 1909. Ann. Mycol. 7: 226. . 1910. Bull. Soc. Linn. Xormandie 6 ser. 2: 57. . 1911. Ann. Mycol. 9: 226. , and A. Tison.1908. C. R. Acad. Sci. Paris 147: 1410. Palm, B. T. 1908. Svensk. Bot. Tids. 2: 48. , and M. Burk. 1933. Arch. Protistk. 79: 263. Rostrup, E. 1894. Bot. Tidsskr. 19: 201. Schwartz, E. J. 1914. Ann. Bot. 28: 229. SORODISCUS Lagerheini and Winge, 1913. Ark. f. Bot. 12: 23 (plates 7, 8) Cvstosori usually flat, oval, disc-shaped and com- posed of two layers of spores pressed closely to- gether ; often variable in size and shape, rarely hol- low spheres, occasionally an elongate and irregular linear series of spores or reduced to tetrads, triads, diads and rarely monads ; soral membrane doubtful or lacking. Resting sjiores polygonal, angular and urn-shaped or oval and almost hemispherical with hyaline smooth or spiny outer walls ; apical collar and cap present or lacking; remaining attached to- gether or separating at maturity; producing one or SOHOIIISI IS n possibly nioro tli;iii oiio zoospore in ■;crinin;itied cystosori composed of two closely jiressed layers of resting s])ores. has been seriously ques- tioned. In the type species, iS'. Callitrichis, the cysto- sori m;iy sometimes be hollow spheres, while in S. karliiifiii they may vary from hemisi)herical multi- nucleate monads, diads. triads, tetrad, flat discs, and elongate linear series of spores to almost hollow spheres (PI. 8, fig. 11-21). In all of the s])ecies. however, the majority of cystosori are flattened and disc-like. M'hile A\'inge ('13) regarded Sorodixciis as a distinct genus he nonetheless pointed out that its similarity to Soros phaera is so great "tiiat it would seem most reasonable to unite them into one genus." Later, however, in a communication to Cook ('31. p, 318) he said "that the spore masses are so char- acteristic in Snrodi.scu.1 that it would be wrong to put it in the s;ime genus as Soro.iphai'ra." Palm and Burk (33). on the other hand, regarded Sorodisciis as a syiu)nyni of the latter genus. Schizogonv has not been observed in Sorodi-icus, although Winge believed that the widespread distri- bution of amoebae in the galls formed by -S'. Calli- trichis suggests its occurrence. Whether or not a common enveloping membrane is jircsent around the cystosori in all species is uncertain at i)resent. Fur- thermori-, little is known about the origin and devel- o|)mcnt of this membrane in the s]>eeies in which it has been reported to occur. In S. Callitrichis, accord- ing to Winge. the resting "spore-wall divides into two layers of which the outer one merges into that of the neighboring spores (fig. 31. 32) so that it gives one the inij)rcssion of the spores being de- posited in a common substance." According to this statement no distinct and separate wall is formed, and tlie s))ores are merely adherent by the outer layer of their walls. Figure 33, however, shows an enveloping membrane. Cook considered Winge's in- terpretation incorrect and stated that in S. radici- colus a distinct wall is laid down around the cysto- sori. He did not. however, present any evidence about its origin — whether it consists of the original liouuding mcmbr.inc of the plasmodium ])riscut at the time of clcav.-igc or is dc])ositcd subsecpiently by the m.ituring resting sjjorcs. Furthermore, his fig- ures 23 and 21 of mature spore cakes do not show a separate common w.all .-iround the sjjores. In S. kar- lliif/ii no evidence of an enveloping membrane has vet been observed (fig. 11-21). 'I"hc )iresence of such a membrane in the genus as a whole is thus still open to serious question, and if ))rcscnt its origin and method of development arc certainly in need of in- tensive cytological study. ^^'inge and Cook differed also in their observations relative to sporogenesis and the stage at which meio- sis occurs. In -S'. Callitrichis numerous binucleate segments or s|)ore mother cells are formed by ])ro- gressive cleavage of the plasmodium (fig. 27). and these segments (fig. 28 and 29) then divide once to form groups of s|)ores in twos (fig. 30), according to Winge. These groups of incipient resting s])ores soon aggregate together, deposit two-layered walls (fig. 28, 29). and thus form the characteristic cytosori (fig. 33). In iV. radicicolus, however, according to Cook ('33. p. 207). the j)rimary cleavage segments or sjiore mother cells (fig. 20) divide twice to form four instead of two inci])ient resting spores. Cook did not show clearly how these united to form the cystosorus and an enveloping wall. It may be that the two species actually differ in this respect, but further study is necessary to determine this point. If Winge's and Cook's accounts are correct Sorodiscus shows marked similarity to Sorosphaera by the ])resence of s])orc mother cells which divide into diads and tet- rads and subsequently aggregate into sori. S. CALLITRICHIS I.aperheim and Winpe, I.e.. p. 33. PI. 1, fig. 9, 1(1; PI. -'; PI. 3. fi;:. 43-63. Cystosori up to 10 in a cell, usually circular, flat and "disc-shaped. 30-1-5/. X 10-6.5 )x X 12-11/1, rarely spherical and hollow; composed of U|) to 200 resting spores usually arranged in two layers and closely pressed together ; outer layer of spore walls continuous {!). Resting spores urn-shaped in longi- tudinal section and hexagonal in cross section, t- .5 /n X 6-7 //. with smooth hyaline walls surmounted at the al>ex by a collar; germination unknown. Zoospo- rangia and zoosjjores unknown. Plasmodia one to several in a cell, large. 10-00 /x in diam., each form- ing one cystosorus; schizogony doubtful or lacking; cleaving at maturity into binucleate segments or sjjorc mother cells which divide once (?) into two resting s])ores. P.irasitic in Callitriche icrnaVis in Norway ( I.a- gerheim and Winge, I.e.) and C. autiimnalis in Russia ( KareltsehikotT and Ros.inoff. '70) and Sweden (Ostenfeld). causing globular galls uj) to 3X''' mm. on the primary and secondary axes. This species was first recorded in 1870 by Ka- reltschikoff and RosanofT who mistook the cystosori for cystoliths and com))ared them with those present in the L'rticaeeae. although Rosaimrt was of the o|)inion that they might be rcmnauts of a parasitic mycelium. According to Winge, Lagerheim collected 48 PLASMODIOPHORALES this species on C. vernalis in Norway in 1893 and 1900. and altlioua;li lie fixed, sectioned and studied his material lie ])uhlished notliing but passed the mate- rial on to \^'inge. In 1907 Rosenfeld (Anonymous, 08) discovered the fungus on C. autumnalis in Sweden, and since that time it has not been reported. Sorodiscus CaUitrichis has a marked effect on the host. All parts of the stem except the outermost corti- cal tissues and ejjidermis are attacked, and the vascu- lar bundles become displaced and lie scattered about in the tumors or are completely destroyed. Infected cells may often enlarge to 10 times their normal di- ameter, but whether or not they and adjacent healthy ones are stimulated to divide by the fungus is un- known. It is not improbable, however, that the galls are due to both cell enlargement and cell multiplica- tion. The nucleus of the host cell apparently en- larges also and forms several conspicuous nucleoli. So far nothing is known about the site and method of infection. S. RADICICOLUS Cook, 1931. Ann. Mveol. 29: 321. Pis. 1,2. Cystosori one to several in a cell, usually flat and disc-shaped; composed of up to 50 resting spores usually arranged in two layers and closely pressed together; enveloped in a delicate membrane which later disintegrates and frees the individual spores. Resting spores oval, rectangular and jiolygonal in section, 3.8-4..2 ix X 3.2-3.6 fj., with smooth walls, the outer layer of which may be extended to form blunt spines ; separating at maturity and giving rise to zoospores in germination. Zoospores oval pvri- form. 2. .5-3. .5 jj., soon becoming amoeboid. Zoospo- rangia unknown. Plasmodia one to several in a cell, small 15-30 /t in diameter: schizogony doubtful or lacking; each producing a single cystosorus ; at ma- turity cleaving into uninucleate segments or spore mother cells which divide twice into four resting spores. Parasitic in tlie roots of Giinandrops'is penla- phi/lla near Pretoria, South Africa, causing con- voluted, coral-like galls, 3-15 mm. in diameter. Cook's study of this species was based entirely on prepared slides and fixed material sent by Dr. E. M. Doidge from South Africa. It has accordingly never been examined in the living state. Many of the criti- cal developmental stages such as resting spore ger- mination, fusion of gametes, schizogony, alternation PLATE 7 Sorodiiciis (Fig. 1, 3, 9-15, 23-33 after Winge, '13. Remaining figures after Cook, '31. Fig. 8 drawn from portion of a plioto- graph.) Fig. 1. Callltilche viriudis with lunnerous galls caused by .'^. Callitrichii-: Fig. 2. Enlarged gall. Fig. 3. Diagrammatic sketch of cross secticm througli the root and gall of Gjinandrojinis pentaphyUa showing the progressive developmental stages of parasite from the root to the opposite side of tumor. S. radiciroliis. Fig. 4. Anteriorly flagellate zoospore or gamete. .S. rad'i- cicolus. Fig. 5. Amoeboid stage of same. S. radicieoliis. Fig. 6. Fusion of gametes. S. radicicoliis. Fig. 7. Young binucleate Plasmodium after first division of zygote nucleus. .S. rndicicoliis. Fig. 8. Large multinucleate plasmodium. S. toi/iVico/h.*. Fig. 9. Resting nucleus of plasmodium. S. CaVitrirhis. Fig. 10. Prophase, showing differentiation of "tropho" and "idioehromatin." S. ('iillltrlchh. Fig. 11. Equatorial plate stage of "iiromitosis" (?). S. C'allitrichin. Fig. 12. Similar stage from a young plasmodium showing chromatin aggregated into chromosome-like bodies. S. Citl- iitrichin. Fig. 13. Anaphase with conspicuous astral rays. S. Calll- trirhis. Fig. 14. Late telophase showing differentiation of "tropo-" and "idioehromatin." S. Cdllilrichi.i. Fig. 1.5. Daughter nuclei with well-marked centrosome- like bodies and astral rays. .'?. CaUitrichis. Fig. IB. Beginning of cliiomatin discharge into cyto- plasm from nucleus during chromidial stage. /S. radici- foluif. Fig. 17. Later stage showing karyosome broken u]) into granules which lie at the inner peri))hery of nucleus. iS. rndicicoliis. Fig. 18. Final akaryotc stage with all stainable chro- matin discharged from nucleus. S. rridicicoliis. Fig. 19. Prophase of meiosis, the so-called "garland stage." S. radicicoliis. Fig. 20. Equatorial plate stage of meiosis with four chro- mosomes. Plasmodium segmenting into spore mother cells. .S'. radicicoliis. Fig. 21. Binucleate spore mother cell >S'. racliciciihis. Fig. -2-2. Second meiotic division with two chromosomes. S. ratlicicohis. Fig. 23-24. So-called "garland" stages in S. Callitrichi.i. Fig. 35. Equatorial plate stage of the first (homeotypic) division. S. CaUitrichis. Fig. 2(). Equatorial plate stages of meiosis. Plasmodium cleaving into segments. S. CaUitrichis. Fig. 27. Paired daughter nuclei in cleaving plasmodium. S. CaUitrichis. Fig. 28, 29. Binueleate segment of plasmodium. S'. CaUi- trichis. Fig. 30. Four incipient resting spores resulting from cleavage of two binucleate segments. Nuclei quite large. •S'. Callit richis. Fig. 31. Mature spores with two-layered walls, the outer layer merging with that of neighboring spores. S. CaUi- trichis. Fig. 32. Young spores with outer and inner walls. .S'. Cal- lit richis. Fig. 33. Side view of cystosorus of S. CaUitrichis show- ing common enveloping membrane. Fig. 34. Young spore with incompletely formed walls. »?. radicicoliis. Fig. 3.5. Surface view of small cystosorus. S. radicicoliis. Fig. 3(i. Spiny resting spores. S. radicicoliis. Fig. 37. Thick-walled resting spore broken away from cystosorus. S. radicicolus. SOHODISt TS H) ri.ATK 7 Sorodiscus 50 'LASMODIOP MORALES of haploid and diploid generations, ete., are thus in need of further investigation. The method of infection has not been observed, but Cook believed that the amoeboid zoospores or amoebae are capable of passing through the walls from cell to cell and even to the outside of the host where they may infect other roots. As is shown in figure 3 at least two generations of the parasite may occur in large galls during the course of one season, but the host plants are not seriously affected by the presence of the galls and fungus. The central cylin- der of the roots apparently is not attacked, and the galls seem to originate in the cortex, although Cook was not at all clear about their origin. Infected cells do not enlarge greatly, but their nuclei eventually become disorganized and degenerate. The presence of the fungus may possibly stimulate cytokinesis or at least does not ])revent division of infected and ad- jacent healthy cells. The galls are therefore doubt- less due to both cell enlargement and cell multiplica- tion. S. KARLINGII Cook, 1933, I.e. p. 207. Karling, 1938. Am. .Tour. Hot. 1.5: 485. PI. 3;?, fig. 1-9. Cystosori numerous, up to 400 in a cell, quite va- riable in size and shape, often oval, elongate and disc-shaped, 15-30 /x X 15-70 /x, occasionally almost spherical, 10-35 /x in diameter, irregular, or reduced to tetrads, triads, diads and rarely monads; consist- ing of from 1 to 200 spores; enveloping membrane unknown. Resting spores polygonal and angular, •t-9 ;«., when pressed together in large sori, spherical, oval and ellipsoidal when single or in small groups, 5-23 /J. in diameter, uni- or multinucleate with hya- line smooth walls and surmounted by one and oc- casionally two fairly thick caps; germination un- known. Plasmodia one to several in a cell, multi- nucleate, and uj) to 90 /j, in diameter; schizogony unknown. Zoosporangia and zoospores unknown. Parasitic in Chara coniraria and C. delicatiila in New York City, causing marked hypertrophy of the infected cells. This is the only known species which parasitizes algae. Because of the great variation in the size and shape of its cystosori and the lack of a common sur- rounding membrane, it is a doubtful member of Sorodiscus, and until more is known about its life cycle it is retained only provisionally in this genus. Its effect on the host is quite marked and extensive, and all cells appear to be equally susceptible. Hyper- trophied stipules, leaflets, spicules, internodal and cortical cells have frequently been found. As is shown in figures 1 and 2 infected cells may swell to many times their normal diameter and have the ap- pearance of s))herical. oval and elongate green blis- ters. The presence of the plasmodia has no visible effect at first on the streaming of the host jjrotoijlasm and are continually carried along ])assively with the host nuclei and cytoplasm. Individual hypertrophied cor- tical cells have been removed from the leaves and kept alive in hanging drops for ten days, during which period the plasmodia, host nuclei and cj'to- plasm rotated continually. The streaming begins to slow down in about 12 days and ceases entirely within 20 days, after which the cell soon dies. As is shown in figure 3 the host nuclei and cytoplasm ap- pear normal during the actively streaming period, and in spite of the extension which it has undergone the cell wall remains normal in thickness. Later, the host protoplasm is reduced to a thin layer. The jires- ence of the parasite mav also often lead to the for- mation of an abundance of storage starch grains in the plastids. The cystosori, which were previously ('28) called sporangesori by the author, are quite variable in size and shape, and those consisting of a few large multi- nucleate and several small uni-nucleate spores (fig. 15, 21) have possibly arisen by unequal and incom- plete cleavage of the plasmodium. The unusually large multinucleate spores (fig. 19) are probably the result of the encystment of the entire plasmodia which failed to segment. Since such spores are multi- nucleate, it is not improbable that they form several zoospore in germination. ADDITIONAL BIBLIOGRAPHY: SORODISCl'S Anonymous. 1908. Bot. Tidsskr. -28: XXVII. Cook, W. R. I. 1933. Arch. Protistk. 80: 303. KareltscliikoiT, S., and S. Rosanoff. 1870. Mem. Soc. Sci. Nat. Cherbourg. 3nd ser. 5: 12i. Palm, B. T., and M. Burk. 1933. Arch. Protistk. 79: 371. Schwartz, E. .J. 1914. Ann. Bot. 38: 330. PLATE 8 Sornclisrus karliiigil (All figures after Karling) Fig. 1. Hypertrophied internodal cell of C. fhlicnfiiln which has burst the sheath of cortical cells. Fig. 3. An extreme case of parasitism of the cortical cells of C. contraria. Fig. 3. Longitudinal section of an enlarged cortical cell witli twenty-six cystosori and seven plasmodia surrounded by the host protoplasm. The six host nuclei appear normal. Fig. 4-6'. Uni-, bi- and tetranucleate stages of the jiara- site. Fig. 7. A multinucleate vacuolate plasmodium in surface view. Fig. 8. Similar plasmodium in edge view. Fig. 9. Large irregular plasmodium. Fig. 10. Cleavage of plasmodium to form cystosorus. Fig. 11. Surface view of a large flattened cystosorus con- sisting of approximately 300 spores. Fig. 13. An almost spherical cystosorus. Fig. 13. Flattened cystosorus in end view. Fig. 14. Tetrad of resting spores. Fig. 15, 1(). Further variations in size and shape of cysto- sori. Fig. 17, 18. Small resting spores in side and surface views showing the apical caps. Fig. 19. Large isolated multinucleate spore. Fig. 30, 31. Cystosori consisting of two and three spores. SOKOUISllS 51 PT.ATK 8 "^S^^ 10 Sorodiscus karlingi PLASMODIOPHORALES The genus Membranosonts has been regarded as a synonym of Sorodiscus, but inasmuch as its inclusion in this genus as well as in Sorosphaera is highly questionable at present it seems advisable for the time being to discuss it separately. MEMBRANOSORUS Ostcnfeld and Petersen, 1930. Zeitschr. f. Bot. 23:17. (PL.\TE 9) Cystosori one or more in a cell, variable in size and shai)e ; frequently a hollow, single-layered struc- ture wliich covers the inner periphery of the host cell and conforms to the latter's size and shape ; often oval, disc-like and single-layered, rarely double- layered, occasionally composed of an irregular mass of loosely attached spores or a row of spores ar- ranged in a linear series. Resting spores slightly variable in size and shape ; germination unknown. Plasmodia one or more in a cell, variable in size and shape; often in the form of a parietal layer around the host protoplasm; schizogony unknown. Zoospo- rangia and zoospores unknown. In light of present-day knowledge Memhrano- sorus is obviously a doubtful genus which should perhaps be discarded entirely, but until more is known about the Plasmodiophoraceae as a wliole its inclusion in any of the other genera is open to serious question. Wernham's observations have shown that the outstanding character described by Ostenfeld and Petersen, namely, hollow single-layered cysto- sori which line the inner periphery of the host cell and conform to the latter's size and shape, is too va- riable (fig. n-18) to be of significant diagnostic value. The incorporation of Memhranosorus in Soro- sphaera or Sorodiscus is equally questionable if the present-day concepts of these genera are to be main- tained, because only occasionally are cystosori in the form of hollow spheres or double-layered discs developed. By the extreme variability of its cystosori this genus resembles perhaps more closely Lif/niera and Poli/mi/xa. Ostenfeld and Petersen regarded it as closelv related to Sorosphaera and Tetrami/.ia, while Wernham implied tliat it should be incorpo- rated with Sorodiscus. Palm and Burk regarded it as a synonym of Sorosphaera. Cook api)arently overlooked its existence entirely in his monograph of the Plasmodiophorales. M. HETERANTHERAE Ostenfeld and Petersen, I.e., fi)i. 1-6. Sornili.iciig Hi'teranlherae, Wernham, 1935. Mycologia 3T:-212. PI. IT, 18, ftp. 1,2. Resting spores always aggregated in multiitles of four. Globose, ovoid, angular. S.5-5 /x in diameter, hyaline and buff-brown, with smooth. 0.6-1.0 // thick walls : apical ring, collar or operculum lacking. Plas- modia oval, ellipsoidal. 8 ft in diameter, or disc-like, flat and often ribbon-shaped. 28-70 /n in length, and encircling the host protoplasm. Parasitic on Heieranthera dubia in Ontario and Quebec, Canada; Vermont and New York. U. S. A.. causing marked hyjjertrophy of adventitious and true roots. Whether or not the species described by Osten- feld and Petersen, and Wernham. respectively, are identical is not absolutely certain, but since they have the same habitat and distribution, cause the same symptoms, infect the same tissues of identical hosts, and agree closely as to spore size and shape, they are listed herewitli as synonymous. The chief differences so far relate to spore color and variations in the size and shape of the cystosori. Since Osten- feld's and Petersen's material was very scanty they may have missed most of the variations later ob- served by Wernham. Likewise, although Wernham never found a single-layered cystosorus completely lining a host cell, his figure 2 shows that the type of sorus described by Ostenfeld and Petersen was often api)roximated in his material. Tliere is accordingly good evidence that they may have had the same spe- cies at hand. Nothing is known about tlie method by which this parasite gets into the roots, but entrance appears to PL.\TE 9 Membridio.iorus Heterantheroe (Fig. 1-3, 6, 19 and 20 after Ostenfeld and Petersen; re- mainder after Wernham; fig. 5 and IS drawn from photographs.) Fig. 1. Portion of infected stem of //. fliibia with 10 swollen and 5 normal roots. Fie. 2. Early infection stage with small granular para- site attached to host nucleus. Fig. 3. Young bi- and trinucleate parasites in daughter host cells. Fig. 4. Young parasite with three nuclei. Fig. .5. Large parietal plasmodium which almost com- pletely envelopes host protoplasm. Fig. (i. Large multinucleate plasmodium enveloping the host nucleus. Fig. 7. Plasmodium with nuclei dividing promitotically. Fig. 8. Plasmodium in which nuclei are about to undergo reduction division. Fig. 9. Plasmodium with nuclei which have just under- gone reduction division. Fig. 10. Second meiotic divisions. Plasmodium cleaving into resting spores. Fig. 11. Cystosorus of young thin-walled resting spores. Fig. 12. Flat, almost circular cystorus composed of a sin- gle layer of resting spores. Fig. 13. Similar cystosorus with one resting spore pro- jecting beneath. Fig. U. Flat, two-layered cystosorus. Fig. 15. Cystosorus with resting spores In a row. Fig. 16 and 17. Irregular cystosori with loosely attached resting spores. Fig. 18. Single-layered cystosorus incompletely lining Inner periphery of host cell. Fig. 19. Similar cystosorus completely lining inner pe- riphery of the host cell. Fig. 20. Surface view of similar cystosorus. MKMBHA.NOSOIUS 53 ri.ATE 9 Meinbranosorus 54 PLASMODIOPHORALES be effected at or near the tip. Cells of the periblem are more frequently attacked, and the fungus occurs most abundantly in a region approximately 0.5 cm. back of the root tip. The cells of the central cylmder apparently are never infected. According to Osten- feld and "Petersen, the fungus first appears as a small plastic granular body close by or attached to the host nucleus (fig. 2), and as it grows in size and becomes multinucleate it may envelop the host nu- cleus and cytoplasm (fig. 3. 5. 6). There is thus a close association of the protoplasts of host and pathogen, and in Ostenfeld and Petersen's drawmgs it is difficult to distinguish between them. The para- site causes the infected cells to enlarge somewhat but apparently does not stimulate cell division. Figure 3, however, "suggests that infected cells may divide, whereby the' parasites are passively distributed to the daughter cells. The mature plasmodia vary greatly in size, and the large extensive ones may often line the inner periphery of the host cell (fig. 5) as in Tetramiixa. According to Wernham, cruciform nuclear divisions occur (fig. 7) during the vegetative phase of the Plasmodium, and the nuclei undergo meiosis in the first of the two divisions prior to cleavage into rest- ing spores. Although he stated that he had observed numerous meiotic stages and counted four to six pairs of chromosomes, his figures (fig. 8, 9, 10) show nothing of the process. SPONGOSPORA Brunchorst, 1887. Bergens Mus. Aarsberet. 1886: 225. Clathrosorus Ferdinandsen and Winge, 1920. Ann. Bot. 31.: 168. (plate 10) Resting spores usually arranged in hollow or irregularly-channeled spongy, globose balls or cysto- sori! Resting spores loosely or fairly closely packed together, spherical, oval, pentagonal, hexagonal in op'tical section with hyaline, yellowish to yellowish- green, smooth, thin or fairly thick walls ; each spore producing a single ( ?) zoospore; such zoospores giv- ing rise to either plasmodia or zoosporangia. Plas- modia usually large, irregular, amoeboid and multi- nucleate ; partly or completely filling the host-cell ; forming one or more spore balls. Zoosporangia sin- gle or in clusters, variously-shaped. Zoospores from resting spores and zoosporangia similar, small, bi- flagellate and heterocont; flagella attached at or near anterior end. Sponqospora includes at present three species, one of whicii is poorly known and doubtful. The type spe- cies, .S'. siibterraneaMs been repeatedly studied mor- phologically and cytologically. but there is still con- siderable disagreement concerning some of the criti- cal stages of its life history. As noted in Chapter III. these controversies have centered primarily around the stages at which plasmogamy and karyogamy occur, and the manner by which the parasite invades and spreads in the host tissue. Johnson ('07) de- scribed the resting spores as one- to eight-nucleate and giving rise to a corresponding number of zoo- spores in germination, but subsequent workers in- cluding Massee ('08), Kunkel ('15), Cook ('33) PLATE 10 Spongospora sitbterranea (Fig. 7-9, 11 and 23 drawn from photographs) Fig. 1. Potato with shallow powdery scab lesions. Fig. 3. Malformed potato with deep cankerous lesions and excrescences. Fig. 3. Powdery scab galls on roots of potato. Fig. i. Enlarged host cell with eight spongy spore balls or cvstosori. Osborn, "11. Fig. 5. Section through a mature cystosorus. Osborn, I.e. Fig. 6. Uninucleate resting spores. Osborn, I.e. Fig. 7, 8. Zoospores from germinated resting spores. Ledingham, '35. Fig. 9. Tetrafiagellate zoospore. Ledingham, I.e. Fig. 10. Dividing amoeba. Massee, '08. Fig. 11. Irregular ^oosporangium. Ledingham, I.e. Fig. 1:2. Uninucleate amoebae surrounded by host cyto- plasm. Osborn, I.e. Fig. 13. Host cell with three amoebae and numerous starch grains. Osborn, I.e. ,. , ., ^ , Fig. U. Dividing host cells with passively distributed amoebae. Osborn. I.e. Fig. 15. Hypertrophied cells of S. xcarso-n-iczii which have divided": amoebae aggregated around host nuclei. Melhus, et al. "16. Fig. 16. Group of infected enlarged tomato cells; typi- cal "Krankheitsherde." Melhus, et iil. l-c. Fig. 17. Infecting plasmodium pushing down between host cells. Kunkel, "15. Fig. 18. Plasmodium entering host cell and enveloping nucleus. Kunkel, I.e. Fig 19 Coalescence of amoebae to form plasmodium; host nucleus enlarged, irregular, and densely chromatic. Osborn, I.e. Fig. 20. Plasmogamy of two amoebae derived from ger- minated resting spores. Cook, '33. Fig. 21. Karyogamy. Cook, I.e. Fig. 22. Zygote. Cook, I.e. Fig. 23. S'aprophytic plasmodium (?) grown on nutrient agar. Kunkel, I.e. Fig. 24, 25. Vegetative nuclei degenerating and extrud- ing chromidia into cytoplasm. Osborn, I.e. Fig. 26. Akaryote and chromidial stage. Osborn, I.e. Fig. 27. Reconstructed nuclei emerging on new sites. Os- born, I.e. , _ 1 f ■ Fig. 28, 29. Reconstructed nuclei pairing and fusing. Osborn. I.e. Fig. 30. Late stage in karyogamy. Osborn, I.e. Fig. 31. Diploid nuclei. Osborn, I.e. Fig. 32. I>ate prophase of meiosis with eight chromo- somes. Home, '30. Fig. 33. Contraction stage and beginning ot pairing ot homologous chromosomes. Home, I.e. Fig. 31. Dlakinesis. Home, I.e. Fig. 35. Metaphase, first division, showing three of the chromosome pairs. Home, I.e. Fig. 36. Equatorial plate, second division, showing seven chromosomes. Osborn, I.e. Fig. 37. Anaphase, second division, and cleavage. Os- born, I.e. SPONIiOSl'OllA 55 Pl.A'i"K 10 20 21 2Z ^ " \ Spongospora S^. 56 PLASMODIOPHORALES PLATE 10~Continned Spongospora Campanulae Spoil (jospora Ciimpnnulae Fig. 38. Campanula rapiinculoules with numerous jfalls and nodules on roots. Ferdinandsen and Winge, "JO. Fig. 39. Young parasite with nuclei dividing "promitoti- cally." F. and W., I.e. Fig. 40. Multinucleate Plasmodium. F. and W., I.e. Fig. 41. Plasmodium enveloping host nucleus. F. and W., I.e. Fig. i-2. Irregular cystosorus. F. and W., I.e. Fig. 43. Section through a cystosorus. F. and W., I.e. Fig. 44. Section through two resting spores showing finely punctate warty walls. F. and W., I.e. and Ledingliam ('35) observed only one zoospore. Furthermore, all earlier investigators figured and described the zocspores as uniflagellate, but Led- ingliam demonstrated conclusively that they are bi- flagellate and heterocont (fig. 7, 8). Whether the flagella are attached at or near the anterior end is not definitely known. Massee, Kunkel, Osborne ('11 ) and Home ('30) held that the plasmodium is formed by the fusion of several amoebae (fig. 19). but they were not certain whether such amoebae arise by division of a single amoeba within the infected host cell or are the result of infection bv several amoebae. Cook ('33), on the other hand, contended that the plasmodium is initiated by the fusion of gametes in pairs (fig. 20-22). There is also difference of opinion about infection and spread of parasite in the host tissue. Massee and Cook in particular held that the amoebae have the ability to penetrate the host cell walls and thus pass from cell to cell, spreading the infection. Osborne and Home, in contrast, maintained that the amoebae are incapable of boring through the walls and are distributed passively and fortuitously by division of the infected cell (fig. 14). Kunkel, however, reported that the primary infection of young tubers as well as secondary infection of tissues around old sori occurs by invasion of the plasmodium. The latter passes through and between the epidermal cells, and once beneath the epidermis it spreades out in all directions (fig. 17). Johnson ('09) believed that the plasmo- dium may migrate from the diseased parent tubers into the stem and stolons of the young plants, and eventually infect the young tubers. Massee thought that the plasmodium might encyst during the cold winter season and renew its activities when the tubers began to sprout. \\'ild ('29) considered the Sl'ONGOSPORA 57 Icnticils. instead of the imlirokoii ciJidtTinis, to he flif i)riiu-i|)al .-imihu' of initial infection, with sonu" luni'lration tliroiigli wounds. .\feordintirraiii<)rti Solan! Brunchorst, I.e. .S. srnltieg Massee, 1908. Jour. Bd. \^t\v. Knjriand 1.5: .594. Fip. 1-1:?. .S. siihltrrnnm ri(tllriri>hi Blattny, 19.3.5. Hee. Inst. Kecli. .\(rr(in. Rep. tclieeosl. 137: ii. S. .iiilitirriniiii liihi rirohi Blattny. I.e. Resting spore clusters or balls oval, elongate, ir- regular, 19-8.5 p. in diameter, somewhat spongy with numerous irregular channels. Resting spores loosely l)acked together, angular, polygonal, spherical, 3..5- i.a i-i, with smooth, thin, yellow to yellowish-green walls. Plasniodi.i uiiusu;illy large, u)) to 70 /( or more in length, amoeboid, irregul.ir ; giving rise to one or more sjiore w.alls. Zoospor.-ingia single or in clusters, U)) to a dozen or more in a cell, si)herical, oval, elon- gate, lobed and irregular, hyaline and thin-walled ; ojicuing by the rupture of a small pajiilla which bursts through the host cell wall emitting the zoo- spores. Zoospores from resting sjiores and zoospo- rangi.i oval. si)hcric;il, 2. .5-3. .5 /t, with two unequal Hagella. P.irasitic on Sdlaiium tiihi'rosum, S. rcarscczcicsii, S. haematododuin, S. mamusum, S. marc/inatum, S. ciliatum, S. commersomi, S. nigrum, and Li/copersi- coii esciilenitim, causing scabby lesions and cankers on the tubers, and galls on the roots and steins. A further account of the distribution and hosts of this species is given in Chapter W. Spoiigospora xuhtcrranca causes the disease of potatoes commonly known as powdery or corky scab. While it is chiefly a parsite of the potato, it may also infect close relatives of this host. In extensive inocu- lation experiments Melhus, et al. ('16), found that it will infect all but one of the hosts listed above but not S. nigrum, S. mauritianum, S. duplo.iumatori , S. Lohelii, S. heteracanthum, S. srafnrthianum, S. lanciniatum, S. iorviim, and Solatium sj). Ferdinand- sen ('23), however, reported that it is transmissible to iS. nigrum in Denmark. Weber ('22) and I.eding- ham ('35) also found it on tomatoes in Denmark and Canada, respectively. It has also been reported by Rybakova and Nedoshivinia ('36) on Ullucus tii- bero.sus of the Chenoiiodiaceae in Russia. Truscott ('Si) found a Spongoxpora-like organism in the roots of strawberries in Canada, but he was not certain about its identity. Blattny's distinction of two forms of S. suhterranea on the roots and tubers, respec- tively, does not seem justified. The two forms may be transferred readily from one organ to another and do not differ greatly in size and color of their s])ore balls. Blattny, nevertheless, believed that the root form may be inycorrliizal instead of l)arasitic. Rybakova and Nedoshivinia also described an aber- rant form near Moscow which differs from the nor- mal type by the occurrence of its spore balls out- side of the host cells. These balls are faintly brown instead of yellowish-green in color, jilicate or ir- regularlv crumpled on the surface, and m.iy be aggregated in a common mass. They vary in size from 20-2.5 /x by 13-19 /x .and show no cellular struc- ture. Khrobrykh ('38) ex|)erimented with various forms of .v. mihtrrraura from different jiotato va- rieties of different geographical origin and con- cluded that these forms are not biotypes or geo- gra))hical races but ecotypes dependent on the host variety, height, and size of the |nistules. In this con- nection it may also be noted that .Sharpies ('23) de- scribed a disease of the ))etioles ;ind le;if stalks of the cocoanut |);ilin in Mal.-iya which appeared to be associated with a species of Spongospora, but he was not certain about the identity of the causal organism. It probably does not relate to Spongospora at all. 58 PLASM ODIOPH OR ALES Spoiigospora suhterranea was the first species of the Plasmodiophoraceae to be reported in the litera- ture, but it was not recognized as a member of this family until about fifty years later. It was first re- ported, in part by Wallroth in 18-t2, but he had apparently found it tiie year before as is indicated in Bartling's (18H) discussion. As is sliown in the synonomy above, it was rediscovered a number of times shortly afterwards in connection with otlier fungi in scabby lesions of potatoes, and included in various genera. It was not until 1886, however, that Brunchorst first recognized it as a species of the Plasmodiophoraceae. For a considerable number of years a long controversy raged about its identity and synonj'my, which has been fully reviewed by Lagerheim, Massee, Pethybridge and Cook, and need not be discussed further here. S. CAMPANULAE (Ferdinandsen and Winge) Cook, 1933. Arcli. Protistk. 80: 2lo. Clathrosorus Camija indue Ferdinandsen and Winge, Lc. PI. 21. Spore clusters or balls irregular, rounded or elon- gate, 25-.50 /jL in diameter with large irregular chan- nels. Spores spherical, 4-5.5 /jl, oval, irregular, trun- cate, with fairly thick and slightly verrucose walls. Plasmodia solitary in the host cell and only partly filling it. multinucleate, irregular, 30-50 fi in diam- eter, when mature ; segmenting into resting spores which remain attached in a fairly loose spore ball. Zoosporangia and zoospores unknown. Parasitic on the roots of Campanula rapiinculoides in Denmark, causing numerous single or confluent, tubercle-like galls. This species has been reported but once. Whether it belongs in Sponc/ospora, as Cook believed, or rep- resents a new genus is obviously questionable in light of present-day knowledge, but since its spore clus- ters are reported to be loose, irregular, round or elongate balls (fig. 38), it may be conveniently in- cluded here for the time being. It occurs in the cortex of the roots (fig. 38), and although the central cylin- der mav be distorted, it is never parasitized. The infected cells are only slightly if at all enlarged (fig. 40-12) and do not divide, but the presence of the parasite nonetheless stimulates adjacent healthy cells to divide. The galls are thus almost entirely the result of cell multi])lication. The nucleus of the host cell is often enveloped by the parasite (fig. 10), but it does not become greatly enlarged. According to Ferdinandsen and Winge, meiosis occurs during the last two nuclear divisions in the Plasmodium preceding sjiore ball formation. They did not, however, count the number of chromosomes nor observe plasmogamy and karyogamy, .so that their conclusions are not based on adequate observa- tions. Another species of Sponc/ospora was recently re- ported and described by ,1. T. Barrett in a brief paper presented before a joint meeting of the Ameri- can Mycological and Phytopathologieal Societies at Philadelphia, Pennsylvania, December 30. 1910. Dr. Barrett has not completed his study of this species, but he has graciously allowed me to include a few notes on the essential features of its life cycle. This species parasitizes Cotula australis in California and causes cons])icuous galls or nodules on its roots. Barrett accordingly named it .S'. Cotidae. In germina- tion each resting spore produces a single zoospore with two unequal flagella as in c. n. s. 12: 165. Khrohrykb, \. D. 1938. Summ. Sci. Res. Inst. pi. protect. for' 193()- 1938: -21. Kunkel, I.. O. 1915. Jour. Agr. Res. 4: ^65. I.edingham, G. A. 1935. Nature 135: 394. Massee, G. 1908. Jour. Bd. Agrio. England 15: 594. Melhus, I. E., J. Rosenbaum, and E. S. Schultz, 1916. Jour. Agr. Res. 7: -'13. Osborn, T. G. B. 1911. Ann. Bot. 25:211, 337. Rybakova, S., and H. Nedosliivina. 1936. White Russ. Acad. Sci. Inst. Biol. Sci. Minsk 1936: 57. Sharpies, A. 1933. Malayan Agric. Jour. 11: 367. Truscott, J. H. L. 1934. Canad. Jour. Res. 11: 1. Weber, A. 1933. Tomatsygdonime. Copenhagen. Wild, N. 1939. Phytopath. Zeitschr. 1: 367. LIGNIERA Maire and Tison, 1911. C. R. Acad. Sci. Paris 152: 206. (plate 1 1) Resting spores not consistently aggregated in cys- tosori of characteristic shajie and structure ; vari- ously-shaped with relatively thin hyaline or colored, smooth or verrucose walls. Plasmodium relatively small, ])artly or comi)letely filling tlie host cell; seg- menting into either zoos))orangia or one or more cystosori; schizogony reduced or lacking (?). Zoo- sporangia numerous in a cell and usually grouped to- gether, small and variously-shaped ; opening by a rupture of the wall. Zoosj)ores from sporangia ]iyri- forni. Germination of resting spores doubtful or un- known at ])resent. I.KiXIKllA 59 Tliis jji-iuis was cstaMislKd l)_v Main- aiul 'rismi for all i)lasiiionu'nt within a sinirle host eell. and whieli eause no hyiiertropliy of the iiost. As sueh. it is a very questionahle i;eiuis and should iierhajis lie dis- carded, since noiu- of its charai'ters are very distinc- tive and diajjnostic. In the first |)lacc the shape and character of the restinji spore clusters or cystosori are too variable to be of nnich {generic value. Sec- ondly, none of the si)ccies has yet been studied in- tensivclv and sutHciently well to determine whether or not sehizoiiony is well developed, redui'cd. or lackinsr entirely. I'urthcrniore. it is not certain tli;it the l);irasite completes its entire life cycle within one host cell. Fin.illy. the Jircscnce or absence of host hypertrophy is not a structural or eytological char- acter of tlie jiarasite itself, but relates to the reac- tions of host and ])athogen. Even if this latter charac- ter were tenable, it would not be diagnostic for the group as a whole, because I>. plloriim, according to Fron and (iaillat. causes marked local enlargement of the root hairs of Poa annua. t)n the basis of ])res- ent-day knowledge. Lif/niera al)pears thus to be scarcely more than a convenient dumping ground for species which cause little or no hypertrophy. Further intensive studies, however, may reveal a more fundamental basis of distinction. The pyriform uninucleate zoosi)ores of Lif/niera have been described by Cook as anteriorly uniHagel- late (fig. 1). but more careful study will doubtless show them to be biflagellate and heterocont as in Plasmodiophora, Pol_i/mi/da, Sponr/ospora, and Ocfo- mi/xa/ After penetrating root hairs and epidermal cells, they may become flagellate and actively motile again in the host ]iroto])lasm (tig. 2B). according to Cook. The flagellum soon disap|)ears. however, and the parasite becomes amoeboid in sha)3c and motion (fig. 3). Nuclear divisions occur as the amoebae in- crease in size (fig. K 8. 9). until a multinucleate Plasmodium is formed. One or more amoebae and Plasmodia may be present in a host cell, but so far no conclusive evidence has been presented to show- that they coalesce to form a larger structure. As noted before Cook ('33) re))orted that the zoospores are isogametes which fuse in i)airs to form zygotes. but his evidence of plasmogamy or karyogamy is not very conclusive. Host cells usually contain only one plasmodium. which fills them almost eomjiletcly (fig. 12). Very little is known about the feeding habits of the intra- matrical plasmodia. They alijiarcntly absorb the host cvtoijlasra. envelop the nucleus, and lead to the dis- appearance of the starch grains, so that the infected regions of the roots ap])ear quite pale in color. Maire and Tison ('11). however, reported th.it the Plasmodium is cal)able of engulfing large food pnr- • This is suppestrd by Rarretfs discovery of biflajrcllate heterocont zoospores in Khhiinuij-a hiipoijea whieli is re- garded as a combination of Liijniern sp., and another fun- gus species. tides, .-iiid figured ;! yiiuiig p.'irasite with live en- gulled algal cells (fig. .5). Whether or not schizogony incurs in Lii/nii'ra is still questionable. Schwartz ('10) reported that the young plurinuele.ite iil.ismodi.-i of /.. Jiinci function .■IS schizonts. siilitting off smaller uni- or multiiiuele- ;ite daughter segments. .M.iire and 'I'ison (lib), on the other hand, believed that schizogony may be l.iekiiig entirely or is reduciorangia or cystosori. In the former event uninucleate segments (fig. 20) are delimited by ])rogressive cleavage, and their nucleus divides twice and occasionally three times. Cook claimed that the first division is heterotypic and the second homeotypic, liut his evidence is not at all con- clusive (see Chap, III). In resting spore develo])- ment uninucleate amoebae may sometimes undergo two nuclear divisions (fig. 22) and form tetrads of resting spores, according to M;iire and Tison (1 lb). It is not inijirobable. howe\er. that their figures m.iy relate instead to sjioriingia and zoospore develo))- ment. In larger ))lasmodia. the reconstructed nuclei following the so-called "akaryote" stage also divide twice. Following these divisions the jilasmodium cleaves into uninueleate segments, which round u)). 60 PLASMODIOPHORALES become invested with a wall (fig. 31), and mature into resting spores. These spores usually remain at- tached to each other and form cystosori of variable sizes and shapes (fig. 28-40) in accordance usually with the size of the plasmodium and tlie shape of the host cell. L. JUNCI (Schwartz) Maire and Tison, I.e. Sornsphuera Jiiiici Schwartz, 1910. Ann. Bot. 24: 513. PI. 10. S. (/raminh Schwartz, 1911. Ibid. 25: 791. PI. (il. L. graminh (Schwartz) Winge, 1913. Ark. f. Bot. \-2, no. 9: 15. L. rridicdlii Maire and Tison, I.e.; 1911. Ann. Mycol. 9: 333. PI. 11, fip. 34-38. L. Bellidis Schwartz, 1914. Ann. Bot. 38: 333. PI. 13, fig. 7-8. L. Menthae Schwartz, I.e. PI. 13, fig. 1-6. L. AlisnuiUs Schwartz, I.e., p. 333. Resting spores rarely in tetrads, sometimes end to end in a linear series ; more often in irregular masses, solid or hollow, flat, globose or ellipsoidal, cylindri- cal and elongate cystosori. Resting spores spherical oval, angular and polyhedral when compressed to- gether, 4-7 /i in diameter, with relatively thin hya- line smooth walls; apparently giving rise to zoo- spores which infect the host cell. Plasmodium partly or completely filling the host cell ; segmenting into either zoosporangia or one or more masses of rest- ing spores, schizogony questionable or reduced. Zoo- sporangia oval, subglobose, spherical, angular and polyhedral, 15-20/x, in diameter, with thin hyaline smooth walls; method of dehiscence unknown. Zoo- spores from sporangia 4 to 8 in number, pyriform, 3. 5X^-5 jn. Parasitic in the root hairs and roots of Junciis arti- ciilatus, J. obiusiflorus, J. biifoniiis, J. lamprocar- piis, Poa annua, Bellis perennis, Mentha piilegium, Alisma Plantago, CallHriche stagiialis, Votomgeion natans, Polyc/onum H i/dropiper. Iris pseudocorus, Ranunculus circinatus, R. aquatilis, Plantago ma- jor, Cerastium vidgatum, Veronica Beccahunga, and Chri/santhemum leucanthemum in England (Schwartz, '10, 'II, "14; Cook, '26, '27, '28. '33) ; Callitriche stagnalis and Poa annua in France (Maire and Tison, I.e., 'lib); Alisma Plantago in New York, U. S. A. (Karling, '34). Cook ('26) made extensive cross inoculation ex- periments involving 16.5 individuals of different spe- cies, 151 of which became infected with L. Junci after four months. These plants included the hosts of Schwartz's L. graminis, L. Bellidis, L. Menthae, and L. Alismantis, and since Cook found no essen- tial differences between these Ligniera species and L. Junci, he concluded that they are identical. The species which he found in Callitriche stagnalis was likewise capable of infecting the same hosts ; and for this reason he ('33) later concluded that L. radicalis described by Maire and Tison in C. stagnalis in France is also identical to L. Junci. The resting spores of L. radicalis, however, are only 4- .5 /x in diameter, while those of L. Junci range from .5 to 7 /x. This difference is not very great and may not PLATE 1 1 Liffniera Fig. 1. Zoospore highly magnified (Z/. .Tunc!: Cook, '38). Fig. 3a. Zoospore outside of root hair; 3b, after entering host cell (Cook, "36). Fig. 3, 4. Developmental stages of amoebae and young Plasmodium (L. (/ramini.i; Schwartz, '11). Fig. 5. Young thallus with five engulfed alga! cells (L. radicalis ; Maire and Tison, '11). Fig. 6. Two amoebae approaching a central host nucleus (L. fframinin; Schwartz, "11). Fig. 7. Amoebae clustered around host nucleus {L. (/ra- mini.'i Schwartz, I.e.). Fig. 8. Young amoeboid plasmodium (L. (/rfiminis; Schwartz, I.e.). Fig. 9. Young plasmodium in root hair; nuclei with large karyosome and abundant chromatin {L. .Junci; Cook, '3fi). Fig. 10. Possibly schizogony of plasmodium {L. radi- calis; Maire and Tison, I.e.). Fig. 11. "Promitosis" of vegetative nuclei (L. (/rdminis; Schwartz, I.e.). Fig. 13. Single large plasmodium in a host cell. Nuclei entering akaryote stage (L. fframini.i: Schwartz, I.e.). Fig. 13. Akaryote stage; nuclei appear as clear spaces {L. graminis; Schwartz, I.e.). Fig. 14. Akaryote state; cytoplasm with numerous chro- matic granules ; host nucleus densely chromatic in base of cell (L. radicalis; Maire and Tison, I.e.). Fig. 15-18. Successive stages of extrusion of chromatin from the nucleus (L. .fund: Cook, "33). Fig. 19. Prophase of heterotypic division (?) in a recon- structed nucleus {L. .Junci: Cook, "38). Fig. 30. Cleavage of plasmodium into zoosporangia; the two large mitotic figures in upper left segments are equa- torial plate stages of the first heterotypic division (?); the remainder relate to homeotypic division (?) (L. .Junci; Cook, "36). Fig. 31. Cleavage into zoospores {L. .Junci; Cook, "38). Fig. -23. Second mitoses prior to resting spore formation {L. radicalis; Maire and Tison, I.e.). May possibly relate to sporangia and zoospore development like in figure 30. Fig. 33. Zoosporangia {L. Junci: Cook, '38). Fig. 34. Empty zoosporangia {L. .Junci; Cook, '38). Fig. 35. Plasmodium in swollen root hair tip {L. pilo- rutn : Fron and Gaillat, I.e.). Fig. 37. Cluster of empty resting spores in swollen root hair tip (L. graminis: Schwartz, I.e.). Fig. 38, 39. Small groups of resting spores (L. Minthae; Schwartz, "14). Fig. 30, 31. Types of resting spore clusters (i. graminis; Schwartz, '11). Fig. 33. Single resting spore {L. .Junci: Cook, "38). Fig. 33. Resting spore ball filling host cell {L. Jsoetes; Palm, '18). Fig. 34. Cross section of a similar hollow resting spore ball {L. Isoetes: Palm, I.e.). Fig. 35. Loose chain of resting spores (L. Isoetes: Palm, I.e.). Fig. 3(i. Longitudinal section of hollow cylindrical rest- ing spore cluster (L. radicalis: Maire and Tison, I.e.). Fig. 37. Cluster of resting spores with host nucleus inside (L. radicalis: Maire and Tison, I.e.). Fig. 38. Resting spore clusters of L. piloruni in swollen base and tip of root hair (Fron and Gaillat, I.e.). Fig. 39, 40. Types of resting spore clusters (L. verru- cosa; Maire and Tison, I.e.). I.KiNIKltA 61 PLATE 11 Ligniera 62 PLASMOmOPHORALES be sufficient reason for separating the two species. Light appears to be the dominant factor in infection. No infection occurs in roots exposed to light even when other environmental conditions are optimum, according to Cook ('27). In this connection it may be noted that Hildebrand ('34', PI. I, fig. 5) observed cystosori of indefinite size and shape in diseased rootlets of strawberries in Canada. Whether or not these resting spores re- late to Lif/niera or another genus is uncertain at present, since Hildebrand made no further study of the organism in question. L. PILORUM Fion and Gaillat, 193.5. Bull. Soc. Mycol. France H : ,390. PI. 10. Resting spores aggregated into globose and ir- regular clusters or cystosori, or lying end to end in a linear series ; oval, spherical, 4—6 /t, or angular and polyhedral when compressed together, with thin Iiyaline smooth walls. Plasmodium filling the en- larged base or tip of the host cell ; schizogony ques- tionable ; Plasmodium apparently segmenting into either zoosporangia or resting spores. Zoosporangia (?) oval, spherical, angular and compressed, 4— 6 /x (.'') with tliin, smootli hyaline walls, opening by the rujjture of a thin localized area. Zoospores small, pyriform, up to 1 /x (?) in diameter; flagellum of same length as spore body. Parasitic in the root hairs of Poa annua in France, causing marked local hypertrophy ( ?). Fron and Gaillat's drawings and descriptions of the developmental stages of this species are very brief and inadequate, and it is not clear whether the zoos]Jores arise from germinating resting spores or zoosporangia like those described by Cook ('26) for L. Jnnci. The latter view seems more plausible be- cause figures 7 and 8 by Fron and Gaillat show what appears to be several zoospore initials within a single unit of the aggregate; whereas the resting spores of most plasmodiophoraceous species are now rather generally believed to form but one zoospore apiece. If Fron and Gaillat's measurements are correct, this species is characterized by unusually small zoo- spores. Cook ('26, '33) regarded L. piloriim as syn- onymous with L. Jiinci, because it also occurs in Poa annua and agrees with the latter in life cycle and resting spore size. The chief differences are zoospore size and the fact that L. pilorum causes hy])ertrophy of the host cell, according to Fron and Gaillat. Cook maintained that such hypertrophy is not due to the stimulus of the parasite but that L. pilorum may fortuitously infect root hairs which are already swollen. In further support of his belief that the two species are identical, he Jjointed out that L. Junci occasionally attacks swollen hairs also. Schwartz ('11) likewise observed that normally swollen root hairs (fig. 27) may sometimes become infected witli L. Junci. It seems almost too accidental, however, that all the infected root hairs shown in IVon and Gaillat's (fig. 1) are greatly enlarged at the ti)). Nevertheless, it is not entirely improbable that L. Junci and L. pilorum are identical, but until more is known about the latter siiecies and host range, its identity and validity will remain questionable. L. VERRUCOSA Maire and Tison, I.e. 1911, Ann. Mycol. 9: -'35. PI. 11, fig. 39-41; pi. 12, fig. 43-46. Resting spores occasionally aggregated in a linear series, more often in globular, ellipsoidal solid, rarely flattened, and disc-shaped, or hollow balls ; resting spores oval, spherical, 4— .5 /x in diameter, angular and polyhedral when compressed, with fairly thin, hyaline verrucose walls. A})parently giv- ing to rise to zoospores in germination, which infect the host. Plasmodium partly or completely filling the host cell ; giving rise to one or more cystosori ; schi- zogony reduced or lacking entirely. Zoosporangia and zoospores unknown. Parasitic in the root hairs and roots of Veronica arvensis (Maire and Tison, I.e.), Beta vulgaris, Chenopodium album, Bromus sp., and Fcstuca sp. in France (Guyot, '27), without causing hypertrophy of the host tissue. This species is imperfectly known at present, and many of its critical stages remain to be studied. As is sometimes true of the previous species, the shape and structure of the cystosori de])end to a large degree on the character of the host cell. ^^Mlen the cystosori occur in elongate narrow root hairs, they may consist of a linear series of resting spores, but if they de- velop in the cortical parenchyma cells, they usually have the form of more or less solid, globose and ellip- soidal balls. Guyot regarded this species as a variety of L. Junci, because the characters of his specimens of L. verrucosa seemed to merge imperceptibly with those of L. Junci. Cook ('33), after examining material submitted by Guyot. and Claire and Tison found no difficulty in distinguishing L. Junci and L. verrucosa. However, the warts on Guyot's specimens were found to be much less pronounced than those on Maire and Tison's material. Palm and Burk did not regard the presence of warts as a specific character, since in a single species of Sorosphaera on T'eronica americana they found both smooth and warty spores with all degrees of gradation between the two types. Hence, they regarded L. verrucosa as identical to L. radicalis or L. Junci. The development of smooth and warty spores in a single species is not at all uncom- mon among fungi, and Palm and Burk were probably right in their conclusions. !More intensive study of the develojjment, variations, and host range of L. verru- cosa is. however, essential. L. ISOETES Palm, 1918. Svenska Bot. Tidsskr. 12:328. Fig. 1-3. Resting spores sometimes in more or less loosely aggregated clusters, more often in hollow balls which fill the host cell and conform with the latter's shape. Resting spores oval, almost spherical, angular and l)olyhedral when compressed, .5X6—8 fx, with thin, smooth brownish-colored walls. Plasmodia jjartly or POLYMYXA G3 oomiiK'tcIy tilliiii; tin- host cell. ZoDspiiriiiisiia ;m(] zoospores unknown. I';ir;isitii' in tlic Ic.iMs and roots of Isoftts lacii-i- tris in Swi'ilin (^I'alni. l.o. ) and Ni'W .Icrsi-y. U.S.A. (Karling. "Si), (■.•luslns; larjrf. dark spots in tin- iiost tissuo but no hypcrtropliy. This spi'firs is so littK- known at jnxscnt that its identity is very doulitful. As Cook pointed out, it may well he identieal to L. Jiinci, but some of the rest- injl si)ore elusters tifiured by Palm are strikini;ly like those of speeies of Soroxphncra and Mrriihraiid- soru.i. The jiresent writer's observations on this s))e- cies in 1931- were very limited, and sinee then he has not added any further data on its structure ,ind de- velopment. L. VASCULARUM (Matz) M. T. Cook (\'9) does not appear to In- a valid species. See PlimmndUtjihom vtm- ritUivum. .\DDiTiox.AL bibliography: Lic/iiicm Cook, \V. H. J. 19.'(>. Trans. Brit. Myool. Soc. 11 ; 19(>. 19.^, I hill. 12: 2SJ. . 19iSa. lUiU. Soi-. Mycol. France U: 1().>. . 19.'81>. Ann. Hot. i2: 347. -. 19:}-'a. Hoiijr Koiifr Nat. Suppl. No. 1 : 2i). . 19:l.'l). .lour. Dei)t. .\pr. Porto Rico 1(>: +09. , 19:«. tllaniorau County Hist. Nat. Hist. 1: ;.'13. . 19:U. Watson's .Microscope Record — : 'i, 9. Guyot, .\. I.. 19.'7. Rev. path. Ent. Afrr. It: 17(>. Hiidehrand, A. A. 19;U. Canadian .lour. Res. 11: 2i. KarlinfT, .T. S. 1934. Torreya 34: 13. Palm, H. T., and .M. Hurk". 1933. Arcli. Protistk 79: 363, Smith, .\. I.., and .1. Ranisliottom. 1917. Trans. Brit. Mycol. Soc. (>: -'31. POLYMYXA Lcdinglmm, llKiS, Phvtopath, 23: 20. (I'LATF. 12, FICS. 1-22) Cvstosori or restinjj spore clusters indefinite in size and .shajjc, without a common membrane ; formed by cleavage of a naked multinucleate plasinodium. Resting spores few or numerous, variable in shape, Zoos)>orangia conjoined in a more or less linear series : formed by tin- se))t.ition of an elongate, lobed, irregul.-ir and tubular thallus, which may extend through one or more host cells: exit tubes one or more, variable in length, and septate. Zoospores from resting spores and zoosporangia biflagellate and heterocont. Poliimjisa is a monotypic genus, and like Sponc/o- spora, Lifiiiirra, Plasmodiophora, etc., includes zoo- sporangial and naked ))lasni<)dial stages in its life cycle. The zoos))ores a))parently jienetrate the host cell wall directly (fig. (i, 7) aiul lie in the host ])roto- plasm as small globose bodies. As is shown in figure 8, they soon begin to increase in size and elongate, and as growth continues they become lobed (fig. 9, 10), branched, irregular, and tubular, and sometimes extend through the host walls into adj.icent cells. In this manner l.irgi- septate thalli .are deM-loped which ;ire couipletely surrounded from the begiiniiug by ;i thin hyaline w.ill .lud closely resend)lc the thalli of Srplol pidiiim, I.nifcniiliiim, M iizociji'ium, etc. The segments of the th.alli beeonu' zoosjMjrangia (fig. 1 1 ) .-iiul dcvcloj) one or more septate exit tubes of vari- able length. The protopl;ism then undergoes cleav- ;ige into zoosjiores which exhibit considerable move- ment within the /.oospor.angia before emerging. When mature, they emerge fully formed in succes- si(Ui from the exit tubes, become amoeboid for a few nu)uients, and swim away. The zoospores are ))yriform and ov.ite in slia))e, usually uninucleate, and ])Ossess a long and short flagellum attached to the nucleus near the anterior end of the spore body (fig. 1-t). A few binuclcate zoospores with four flagella have been found, but I.edinghain v\'as not certain whether they were the result of unequal cleavage or fusion of two biflagel- late s))ores. During active swimming the flagella may extend out in front, but the zoospores are usually propelled from behind, according to Ledinghani. They rotate on their axes or roll over in swimming, and their motility appears to be somewhat slower than that of most chytrid zoospores. After an active swimming stage of about two to three hours, the flagella disappear, and the zoospores become amoe- boid again (fig. o). In this state they move about by pseudopodia, and may often engulf small food par- ticles or objects. These amoeboid zoosjiores may penetrate and reinfect host cells, but it is not certain from Ledingham's account whether tliey give rise to another crop of zoosjiorangia or develop into large multiinicleate plasmodia. A))parently they ]iossess both ))otentialities. The thallus from which the resting sjiore cluster is formed begins in the host cell as a naked uninu- cleate amoeba (fig. 12), and at no time does it jiossess a membrane or wall. As it increases in size, repeated nuclear divisions occur, and a multinucleate Plas- modium is soon formed. Its shajjc changes constantly as it moves about in the host cell. It may frequently be long and tenuous, extending the full length of the host cell, or form a crescentric mass around the host nucleus with long thread-like, r.idiating pseudo])odia. These pseudo))odia are later retracted as the jjroto- ))lasin becomes denser, and the plasmodium may then segment into a number of portions or meronts (fig. 18) which often lie in rows or closely ))aeked grou))s in the tr.ichcal and cortical cells. Occasionally fusion of several separate ))l.asuu)dia may occur in the same host cell (fig. II-). but I.edingham was not certain whether these were th.alli of ojiposite sex or merely meronts derived by division of a common schizont. He was unable to count the chromosomes in the nu- clear divisions preceding resting s))ore formation and accordingly found no evidence of meiosis at this stage. In the early stages of growth the ))lasuu>dium is very vacuolate, but as devcloiiment |)roceeds the vacuoles decrease in size. As a result the thallus be- comes more granular and refringent in texture and 6* PLASMODIOPHORALES appearance. Very shortly thereafter progressive cleavage (fig. 15) begins and delimits the individual resting spores which remain in continuity as clusters (fig. 16). The resting spores are usually uninucleate, and in germination each gives rise to one zoospore (fig. 21 ) which is similar in size, shape and structure to those formed in the zoosporangia. Polymyxa is strikingly similar to Ligniera in size and shape of its cystosori, size, shape and arrange- ment of resting spores, and by its failure to cause hypertrophy of the host. It differs primarily by the shape and size of its zoosporangia, but this differ- ence may be only specific instead of generic. The lack of schizogony in Polymyxa, which Ledingham cited as an additional difference, may not prove to be of great significance, since its presence in Ligniera also is still quite doubtful. P. GRAMINIS Ledingham, I.e.; 1939. Canadian Jour. Res. C, 17:50. PI. 1-3. Resting spores spherical, polygonal, 4-7 /tt ; con- tents hyaline and ref ringent ; inner wall hyaline, outer wall smooth, yellowish-brown. Zoosporangia lobed. oval, uteriform and irregular; exit tubes of variable length. Zoospores broadly spindle-shaped, ovate, pyriform, I— .5 jj. in diameter; flagella 16—20 jx and 4— .5 /x long respectively ; zoospores emerging fully formed and swimming directly away ; rolling over and over while in motion, intermittently amoe- boid. Plasmodium variable in size and shape, often filling host cell, amoeboid in shape and motion. Parasitic in the roots of Triiicum aestiz'iivi, T. durum, Hordeum vulgare, and Secale cereale in Canada. Ledingham found similar resting spores in roots of species of Agropyron, Scolochloa, Rumex, and Impatiens, but since no sporangia were present, he was uncertain about the relation of this fungus to P. graminis. He reported further that species of Juncus and Poa in which Ligniera parasites occur failed to become infected when grown witli parasit- ized wheat roots. He accordingly regarded P. grami- nis as an obligate parasite. Truscott ('34) also re- ported what he believed to be P. graminis in roots of strawberries in Canada. DOUBTFUL GENERA Under tliis title are presented four genera about which there has been much disagreement and con- troversy. Rhi-omyxa, Sorolpidium, and Anisomyxa occur in the roots of higher plants, do not cause hy- pertrophy, and form cvstosori of indefinite size and shape. In these characters they resemble Ligniera and are regarded by most recent investigators as synonyms of this genus. Trematophlyctis, however, parasitizes leaves and petioles and causes marked hyixrtrojjhy. There is very little evidence in Patouil- lard's account to warrant inclusion of this genus in the Plasmodiophoraceae, but inasmuch as Palm sub- sequently reported it to be "an undoubted member of this family" a brief description of its life cycle is herewith presented. The present writer is in agree- ment with Maire and Tison's, Ciuyot's, Cook's, and Barrett's interpretation of Rhisomyxa, Sorolpidium and Anisomyxa, but further intensive study may pos- sibly reveal distinct generic differences. For this rea- son thev are described and figured separately, so that research students may judge independently the evi- dence of identity and relationships of these genera. RHIZOMYXA Borzi, 1884. Rhizomyxa, nuova ficomicete, Mes- sina. (plate 12, fig. 23-30) Plasmodia partly or completely filling host cell, variable in size and shape ; forming at maturity either single large zoosporangia or sporangiosori composed of small zoosporangia. or cystosori ( ?). Cvstosori and resting spores poorly known or doubt- ful. R. HYPOGEAE Borzi, I.e., pi. 1, 3. Sporangiosori one or more in a cell, spherical, ovoid, irregular, elongate, sometimes made up of lin- PLATE 12 Pnlymy.ra graminis (All figures, except 30 and -21, after Ledingham; fig. (i, 7, 8, 17, 19 and il drawn from photographs.) Fig. 1-3. Biflagellate heterocont uninucleate zoospores. Fig. 4. Large binucleate tetraflagellate zoospore. Fig. j. Living, amoeboid zoospores. Fig. 6. Zoospore on surface of root hair. Fig. 7. Zoospores after entrance Into root hair. Fig. 8. Stained zoospore inside of cortical cell shortly after penetration. Fig. 9-11. Stages in development of zoosporangial thalli. Fig. 12. Mature zoosporangia with exit tubes passing through adjacent cells. Fig. 13. Naked myxamoeba during period of active growth. Fig. 14. Segments or meronts formed by division of Plas- modia. Fig. 15. Same cell as in fig. 13 after meronts have coa- lesced to form a large plasmodium. Fig. K). Plasmodium just I'.rior to cleavage into incipient cystosori. Fig. 17. Cleavage of plasmodium into cvstosori. Fig. \S-2\. Variations of cystosori. (Fig. .'0 and 2\ drawn from material presented by Ledingham.) Fig. 22. Zoospore from resting sjiore stained in Into. Hhh(>myxrolpitliinii liftar lias lieen tlie sulijeet of eon- siderable diseussion sinee tlie time of its discovery liy N'eniee. He described it as a species of the Cliytri- diaccae with close iirtiiiities with tlie l'lasniodi(i])lio- raceae, hut hecause of the jircsenee of lar«e. tliiii- walled 7.oosi)oraiigia he did not l)elie\ e it siiould he included in this family. Since similar zoosjiorangia have subsequently been found in several genera of the l*l;isniodiophoraceae. this obji-etion is no longer significant. The large, thick-walled, stell.-ite resting cysts surrounded by a thin envelope which Neniec figured are now generally recognized as relating to Olpidium, and outside of these cysts there is nothing in the life cycle of Sorolpidium, as described by Nemec. which excludes it from the Plasmodiopho- raceae. The presence of large holocarpie zoospo- rangia and multinucleate resting spores which pro- duce several zoospores is in line with more recent discoveries in other genera of this family. Saccardo ('26) likewise included .S'. Brtae among the Chytri- diales. Winge ('13), however, asserted that it is closely related to Pi/rrho.soriis and the Plasmodio- phorficeae. Subsequent workers, on the other hand, have questioned the identity of Sorolpidium as a dis- tinct genus of this family and contended that it re- lates to Lif/niera. Cook ('2(3) regarded it as a combi- nation of Lif/niera and Asteroci/sii.s, a view which Giiyot sui)])orted in 1927. The latter worker suc- ceeded in inoculating roots of Beta vulgaris with L. verrucosa and Asteroci/stis radicis, and concluded that Nemec's fungus is merely an accidental associa- tion of these two species in the same host tissues. Cook ("32, '33) later incorporated Sorolpidium in Lif/niera and classed S. Betae (pro parte) as a syn- onym of L. J unci. In the sha])e of its cystosori and the fact that it docs not cause hypertrophy of the host tissues .V. Betae is very similar to species of Lif/niera. Should it prove to be a species of this genus its identity to L. Junci and L. verrucosa will none- theless remain somewhat questionable, because Nemec unfortunately did not give any measurements of the sjjorangia and zoospores. The life cycle of .S'. Betae is similar to that of other ])lasmodio])horaceous si)ecies. The earliest recogniz- able stage consists of a uninucleate oval or spherical. highly vacuolate thallus (fig. 1) which us\ially lies in the primordial utricle of the host cell. This thallus is probably the result of zoospore infection, although Nemec was uncertain whether the zoos|)ore enter di- rectly or first become amoeboid. Within the host the thallus grows in size (fig. 2. 3. K and 5), becomes multinucleate and i)lasmodium-like. The division of the nuclei (fig. 3) during this developmental phase appears to be "promitotie,"' according to Nemec's figures, and no sharply-defined chromosomes are formed. One or more plasmodia (fig. I. o) may occur within a single host cell and are usually embedded in ' Cook {'J>i) stated that N'eniee did not figure "promito- sis," but he obviously overlooked figure 3. the host ))rotopIasm or occupy Ihc icntrjil vacuole. They may be spherical. o\al, clongati', or take the shape of the cell which they occupy. Sometimes, the ))lasmodium may form .i broad band or ))latc around the vacuole (fig. 5). .\t maturity the ))lasuu)(iiuui develops .i rrlati\ely thin, enveloping nienibranc and may be transformed directly into a /.oos|)orangiuni. In tiiis respect Sorol- pidium differs from I'lasmodiophora, Lii/iiiera, and Octom/ixa, where the plasmodium is reported to cleave into a number of uninucleate segments which develop into zoosjiorangia. This difference suggests perliaps that the zoosjiorangia (fig. (i, 7) which Nemec described may relate to a species of Olpidium (.isteroci/stis) with large stell.ite resting sjjores. It is to be noted in this connection, however, that the sporangia of Olpidium usually form more or less elongate exit tubes, which are lacking in Nemec's ■S'. Betae. On the other hand, Nemec may have over- looked the cleavage stage of the plasmodium which results in the formation of several zoos))orangia. His text-figure .5 suggests this possibility. At any rate, the protoplasm of the zoos))orangium cleaves into un- inucleate segments (fig. 6, 7) which become zoo- spores and emerge through an irregular opening in the sporangium wall. The zoospores from such spo- rangia are usually uninucleate, oval or pyriform (fig. 8) and unifiagellate {?). Unfortunately Nemec did not say whether they were anteriorly or pos- teriorly flagellate, which would have settled conclu- sively their identity as well as that of the large zoo- sporangia shown in figures 6 and 7. If these zoo- spores relate to a plasmodiophoraceous sjiecies they will doubtless prove to be anteriorly bifiagcllate and heteroeont. In other mature plasmodia which occur in almost emjity host cells. Nemec found that the nuclei lacked nucleoli and were comparatively jjoor in chromatic material (fig. 9). Peripheral chromosomes later ap- peared (fig. 10). and the nuclei divided in regular mitotic fashion (fig. 11-15). The appearance of these nuclei and their manner of division are very similar to what has been described in most of the other genera, and suggests that figures 9 to 1.5 relate to the so-called "akaryote" stage and ])ro))hases of meiosis preceding sporogcnesis. Some of the nuclei in figure 10 have six chromosomes. The same num- bers are present in figures 11 and 15. but whether or not this is the basic number in Sorolpidium is uncer- tain. Nemec described a second mitosis in such plas- modia in which the chromosomes are larger, elongate, and rod-shalied. but it is difficult to reconcile his con- clusions about this division with ))revious and subse- quent descri])tions of the hoineoty|)ic mitosis in other genera. These jilasmodia. nonetheless, devcloji a thin en- veloping membrane and cleave into uninucleate seg- ments (fig. 16), which form fairly thick walls and become resting spores. The envelo))ing membrane soon disappears, but the resting s|)ores remain at- tached and thus form cystosori of various sizes and shapes (fig. 17-21). They may consist of a linear 68 PL ASMODIOP MORALES row of resting spores (fig. 19), double rows (fig. 18, 24), or flat, rounded or irregular masses (fig. 17, 20, 21 ). When first formed the resting spores are usually polygonal (fig. 17), but later they become more glo- bose. As the}' mature they become knobby and some- what stellate (fig. 18, 19, 20, 22, 23) with intercel- lular spaces between them. Single isolated resting spores may be formed occasionally (fig. 23), and among the normal-sized spores in a cystosorus un- usually large ones may sometimes occur as is shown in figure 17. In these respects S. Betae shows the same variations as other genera. Since the resting spores function as sporangia in germination, Nemec called these aggregates spo- rangesori. In germination the resting spores increase in volume and become more rounded in outline (fig. 17), their nuclei divide, and the protoplasm cleaves into uninucleate segments which round up (fig. 22) and become zoospores. An opening in the spore wall is soon formed through which the zoospores emerge (fig. 21). The number of zoospores formed varies with the size of the resting spores. Nothing is known about the size of these zoospores, but they are prob- ably similar to those formed in the large zoospo- rangia. Nemec found no evidence of gametes and sexual fusion in S. Betae. Like sjjecies of Lic/niera, S. Betae causes no hy- pertrophy or other malformations of the invaded tis- sues. In fact, parasitized rhizodermal cells may re- main alive longer than non-infected cells, according to Nemec. The presence of the parasite, however, causes an accumulation of cytoplasm in infected cells and enlargement of the host nucleus (fig. 1, 5). The latter mav often become irregular and develop an unusually large nucleole. As the plasmodia mature the host protoplasm is reduced to a thin parietal layer and eventually disintegrates. The entrance of the zoospore through the cell wall often leads to a marked reaction. As is shown in figure 25 the en- trance hole becomes plugged up and a conspicuous thickening around this plug is formed on the inner peri])hery of the wall. ANISOMYXA Nemec, 1913. Bull. Int. Empr. Fran. Joseph Acad. Sci. 18: 18. (plate 13, FIG. 26-1.5) Plasmodia usually solitary, partly or almost com- pletely filling host cell and conforming with the lat- ter's size and shape ; schizogony unknown : cleaving into groups (sporangiosori) of small and large zoo- sporangia. Sporangiosori usually solitary, rarely more than one in a cell ; indefinite in size and shape ; spring and winter sporangiosori composed of small and large zoosporangia respectively. Zoosporangia variable in size, exit papillae or tubes lacking; pro- ducing four or more uniflagellate ( .'') zoospores. Cy- stosori made up of relatively thick-walled resting- spores ; germination unknown. It is not certain from Nemec's account whether or not cystosori composed of thick-walled resting spores are formed in this genus. He reported that the Plas- modium divides into aggregates or sori of polygonal, hexagonal and oval cells which are quite variable in size. In spring and summer, sori of small and uni- form cells are formed (fig. 41), while those produced in the winter are made up of much larger cells (fig. 43, 44). In both types of sori, however, the cells are uninucleate at first but later become multinucleate. Because they have tiiin walls and produce several PLATE 13 Soi'olpktium Betae (All figures after Nemec) Fig. 1. Cell of Beta vulf/aris with two uninucleate para- sites. Fig. i. Binucleate stage of S. Betae. Fig. 3. Four-nucleate stage; nuclei dividing "promitoti- cally" (?). Fig. 4. Host cell with four plasmodia. Fig. 5. A large band-shaped Plasmodium surrounding the central vacuole. Fig. 6, 7. Large and small zoosporangia with zoospores. Fig. 8. Zoospores from sporangia. Fig. 9. Plasmodium in which the nuclei lack large nu- cleoli (achromatic stage?). Fig. 10. Later stage; nuclei with parietal chromosomes. Fig. 11-1.5. Division stages of such nuclei with six well- defined chromosomes. Fig. 16. Plasmodium cleaving into resting spores. Fig. 17. Young cystosorus (?) with polygonal resting spores. Fig. 18-2-2. Cystosori of various sizes and shapes. Fig. 19, 20, 22. Cystosori of mature knobby, stellate rest- ing spores. Fig. 22. Resting spores with zoospores. Fig. 23. Single, isolated stellate resting spore. Fig. 34. Cystosorus of empty germinated resting spores. Fig. 25. Swollen cell wall at point of entry of zoospore. Anisomy.va Plantaffinh (All figures after Nemec) Fig. 26. Uniflagellate zoospore. Fig. 27. Biflagellate (?) zoospore. Fig. 28. Small uninucleate thallus. Fig. 29. Binucleate thallus with resting nuclei. Fig. 30. Same with both nuclei dividing "promitoti- cally" (?). Fig. 31. Tetranucleate thallus with centrosomes and astral rays. Fig. 32. Equatorial plate stage of "promitosis" with cap-like centrosomes at poles. Fig. 33. Achromatic or "akaryote" (?) stage of nuclei. Fig. 34-36. Prophase of meiosis (?). Fig. 39. Mature multinucleate plasmodium with some of the nuclei associated in pairs. Fig. 40. Zoosporangia cleaving into zoospores. Fig. 41. Spring sporangiosorus composed of small zoo- sporangia. Fig. i2. Sporangiosorus composed of sporangia arranged in a linear series. Fig. 43, 44. Sporangiosori of large multinucleate spo- rangia. Fig. 45. Cell with cyst-like sporangia. nOfTlTFfl. r.^XEPA 69 PLATE 13 & k 26 28 29 S>^ /fi^''^S:^ 30 33 Sorolpidium, Aiiisoinvxa 70 PLASM ODIOPHORALES zoospores Nemec regarded tliem as zoosporangia and like in SorolpidUim named the aggregates spo- rangiosori. It is not improbable, however, that some of these sori may be cystosori of relatively thin- walled resting spores, since in describing the cytol- ogy of Anisomi/xa Nemec reported several nuclear changes and appearances (fig. 11-15) which sug- gest the meiotie prophases which precede sporogen- esis. Although his account of Anisomyxa is fragmen- tary and not altogether clear, it is evident that Nemec was dealing with a species of the Plasmodio- phoraceae. Wliether or not it represents a new and distinct geims, however, remains to be seen from fu- ture studies. Nemec regarded Anisomi/jra as closelj' related to Rhisomifxa and possibly intermediate be- tween the Plasmodiophoraceae and S_vnchytriaceae. Fitzpatriek ('30) discussed it as a doubtful genus, while Cook ('32, '33) merged it with Ligniera and listed A. Plantaginis {pro parte) as synonymous with L. J unci. The latter worker had previously ('26, '27) found L. Junci in roots of Plantago major, which doubtless influenced his belief that A. I'lanta- gi7iis is a combination of L. Junci and a chytrid. A. PLANTAGINIS Nemec. I.e., p. -21. pi. 1, -2. Text-figures 1-.). Spring and winter sporangiosori variable in size and shape ; irregular, elongate, and oval ; consist- ing of a few to numerous sporangia. Zoosporangia usually remaining attached together in a sorus ; poly- gonal, hexagonal, oval or almost spherical with thin, smooth walls ; spring zoosporangia approximately 4.5X6 /i, producing 4 zoospores; winter sporangia 10.5X15 /^) forming numerous zoospores. Zoospores oval, 1.5X1-8 /i, spherical, 1.5 /j. in diameter. Parasitic in the roots of Plantago lanceolata in Czechoslovakia, without causing hypertrophy of the invaded tissues. The zoospores of A. Plantaginis are very small and oval to spherical in shape (fig. 26). Nemec re- ported them as uniflagellate, but he did not state if the flagellum is anteriorly or posteriorly inserted. It is to be noted here that he figured one zoospore (fig. 27) which appears to be biflagellate. It is ac- cordingly quite probable that when this species is studied more intensively the zoospores will prove to be anteriorly biflagellate and heterocont. Nemec pos- tulated that zoospores of two sizes might be pro- duced, because he found cleavage segments of un- equal sizes in several zoosporangia. Penetration of the parasite into the host cell has not been observed. Nemec found small oval uninu- cleate thalli in several host cells (fig. 28, 42) which appear to have come from zoosporangia. Such tlialli apparently grow in size as their nuclei divide and eventually become multinucleate plasmodia (fig. 31, 39). The nuclear divisions (fig. 30, 32) in the devel- oping Plasmodium resemble the so-called "promito- sis" ty|je and are described by Nemec as vegetative mitosfs in which eentrosomcs and astral ravs are usually quite conspicuous (fig. 31, 32). Following completion of the vegetative divisions the nuclei lose their chromatin, and the nucleole is reduced to a small globule (fig. 33). The cytoplasm, on the other hand, becomes filled with small deeply stainable granules. This stage is followed shortly by another in which dense chromatic granules, rods, and bands appear at one side of the nuclei (fig. 36, 37) and sug- gest synaptic phases of meiosis. These stages initiate the reproductive divisions, according to Nemec. However, figures 33 to 38 are strikingly like the "akaryote" phase and prophase stages of meiosis which in other genera have been interpreted as ini- tiating sporogenesis. It is not clear from Nemec's ac- count whether these stages precede the formation of spring or winter sori. The mature plasmodium does not become envel- oped by a wall like in Sorolpidium but cleaves di- rectly into sporangia which remain aggregated and form sori. The zoosporangia are polygonal (fig. 41) at first but later become oval and spherical (fig. 40). In the small spring sporangia, two nuclear divisions of the mitotic tj'pe occur, and the protoplasm cleaves into four segments which become zoospores (fig. 40). In the larger winter sporangia numerous mitoses occur, producing multinucleate zoosporangia (fig. 42—44) which give rise to numerous zoospores (fig. 21, 22). No exit papillae or tubes were observed by Nemec and nothing is known about the emergence of the zoospores from the sporangia. Nemec found no evidence of sexual fusions in Anisomi/.ra, but he pointed out that tlie nuclei in the mature plasmodium (fig. 39) are often associated in pairs, implying perhaps that karj'ogamy may take place. This suggestion is further implied by his fig- ures of synaptic (fig. 36. 37) and diakinetic (fig. 38) division stages. In addition to the two types of sporangiosori Nemec also found several large, sporangium-like oval cysts (fig. 45), 14.5- 20;it X 20-26 fi. which he believed might possibh' be cysto- sori. Whether or not these are large isolated resting spores of A. Plantaginis is not certain. TREMATOPHLYCTIS Pcatouillard, 1918. Bull. Soc. Mycol. France 34: 86, fig. A-G. (plate 14. fig. 1-6) Patouillard established this genus to include a spe- cies, T. Tjcptodesmiae, which parasitizes petioles and leaves of Leptoc/esmia congesta in Madagascar. His diagnosis was based on dried material eollected by V^iguier in 1912, and there is very little evidence in his brief description to warrant inclusion of this spe- cies in the Plasmodioplioraceae at the present time. The infected leaves and petioles become thick, fleshy (fig. 1,2), and reddish in color, and later numerous round or irregular, 0.5 to 3 unn. high, solitary or ag- gregated, open, aceium-likc pustules filled with yel- low spores appear in the infected areas. norirnii. (iKNi'.iiA Tlio farli('>t kiunvn di'vcloiinifntal stajjos of '/'. Lrpldilrsniitir consists of an tlliptical. rouiiil. or irrojjiilar plasinoiliuin (?) which fills tlic hypcrtro- phied host cell (fifc. !•)• Its protoplasm is honio- pciK'ous, brownish, and slightly granular and not enveloped by a distinct membrane With maturity the protoplasm becomes more siramilar, and the en- tire thallus segments into spores, which are at first polygonal but later become oval and si)herical, 12- 1(5^. and develop smooth hyaline walls (fig. 5). When mature they have a yellowish tint, and as the sorus breaks open to the outside of the host it as- sumes the structure and appearance of a cup-like pustule tilled with pulvereseent spores (fig. 2). Ger- mination of these sjiores has not been observed. P;itouillard's figures and description of the sorus. spore formation, and the appearance of the pustules suggest that T. Lcptodtumiae may possibly be a spe- cies of Si/iichi/triiim of the .S. decipifiis type which forms open Jjustules. His figures of a naked plas- modial stage and comparatively thiek-walled spores, however, militates against this view, but in dried herb.arium material it is obviously difficult to deter- mine tlie ])resence or absence of an enveloping mem- brane. .Saceardo ('31 ) listed T. Lepiodc.im'iae among the IMasmodioi)horaceae. but Cook ('33) excluded it. Palm (see Palm and Burke. '33) collected mate- rial of a species closely related, if not identical, to Tremaiophli/ctis on an unnamed host in southern Madagascar, and his statement that it is an "un- doubted member of this family" carries the implica- tion tli.it be believed Patouillard's genus might be valid. Lnfortunatcly Palm has jjublislicd nothing addition.il on this fungus, and the status of Tremaio- phli/cti.i will remain doubtful until more is known about its life cycle. In relation to these doubtful genera a discussion of Pi/rrhosoriis .fuel may be logically presented at this ))oint. although in so doing the author does not imi)ly that it should be included in the Plasmodio- ])horaceae as this family is now recognized. This genus was created by ,Juel ('01) for an orange- colored species. P. marinus, which he discovered in a red alga, Ci/stoclonium piirpiirascens, in Sweden. Since he found it only in dead branches .luel con- cluded that it is a .saprophyte, but Winge ('13) be- lieved that during some of the developmental stages reported by ,Iuel the organism may be ]);irasitic. P.'/r- rhosoru.s- mariniix has never since been observed, but because it includes several ])lasmodi()i)hor.iceous-like stages in its life cycb- it merits consideration in any discussion of the Plasmodiophorales. .luel was uncer- tain about its taxonomic position, relationship, and phvlogeny but jiointed out and discussed the charac- ters it lias in common with Jf'oronina, Iihi:nmi/xa, Trtramjixa, Pnifomi/sa, and other genera of lower organisms. He particularly stressed the similarity of its tvjjc of sjiorogenesis to that of Trtrnmi/.ra. The life cycle of /'. marinus is as follows: In the early developmental stages it consists of small globu- lar thallus lying within the host cell (PI. 11-. fig. 8). Such thalli may often be associated in pairs (fig. 9) or groups, and .luel aeeordiiigly considered it ))os- sible tiiat they may l.-itcr co.ilisee and form a large pl;ismodium. The iminucleate thallus grows in size as its nucleus enlarges (tig. 10) and app.arently di- vides. Mitoses in the iilasmodium have not been ob- served, and .hiel was uncertain as to the manner of origin of the multinucleate stages. A later stage is shown in figure 1 1 of a plasmodium with four large nuclei. The developing plasiiiodia a|)pareiitly Iiavc the .ability to dissolve intervening cell w.ills ( fig. 1 1 ) ■and m.iy eventually occujiy several cells. Although they may be distinctly amoeboid in shajie with nu- merous blunt, pscudopod-like extensions and vacu- oles (fig. 12, 13) it is not certain from Juel's account that they move about and migrate from cell to cell as in Pla.smodiophora, etc. No evidence of schizog- ony was observed by .luel. but \\'inge interjireted some of the uninucleate stages as ))robable meronts. The mature plasmodium is multinucleate, vacuo- late, and usually irregular in shape (fig. 12-1 t), and just before sporulating forms an enveloping mem- brane like Sorolpidiiim. Plasmodia which are exten- sively drawn out and occupy several host cells may accordingly ap))car lobed, irregular and tubular (fig. 18) after the wall has formed. Following this stage the i)roto])lasm divides into uninucleate segments. In this process no distinct cleavage furrows have been observed. The jilasmodium appears to become highly vacuolate (fig. If) during this process, and the cytoplasm accumulates around the nuclei and forms stellate i)roto])lasmie islands which resemble somewhat the sporonts of Teirami/ja. These seg- ments soon become almost spherical or spindle- shajjcd (fig. If), and .luel thought that the latter type of cells arc formed in ])lasmodia which are highly vacuolate and scarce in cytoplasm. In addi- tion to these two kinds of segments, irregular elon- gate, oval and smaller ones may be formed, appar- ently as the result of unequal cleavage, which finally degenerate. The si)herieal. 8 u. in diameter, and siiindle-shaped segments are uninucleate, naked, and never develo)) a distinct wall. They aggregate to form a definite sorus (fig. 1.5) and each cell soon divides into oetads of s))ores as in Octomi/.ra, which led .luel to call them spore-mother cells. In this jirocess of spore forma- tion the nuclei divide mitotically (fig. 21-2f) and each mitosis is followed by cell division. Definite chromosomes (2 to .5) are formed on a sharply-de- fined sjiindle during mitosis, and there is no evidence of "promitosis." according to .Juel's figures. Each of the eight naked s])ores so(Ui becomes tr;ins formed di- rectly into a zoospore without developing ;i thick wall and becoming dormant. The mature zoospores are small, ijyriform. 4..5X2..') /x, with a tajiering end, laterally biHagellate and isocont (fig. 7). In addi- tion they ))ossess ;i brilli;int or.ange-colorcd s))ot or globule which rtsembles the eye sjiot of algae and lies at the point of insertion of the tlagella. The zoo- spores a))]jarently infect the host cells and develop into the small thalli shown in figures 8 and 9. Cysto- 72 PLASMODIOP MORALES sori or resting spores have not been observed in P. marinus. It is apparent from this description that Juel's fungns differs primarily from the valid species of the Plasmodiophoraeeae by its laterally biflagellate, isocont zoosjjores, naked spore-mother cells and spores, lack of zoosporangia, resting spores, and by its saprophytic nature. As .Juel emiihasized, the for- mation of uninucleate spore-mother cells or sporonts by fragmentation of the plasmodium and their sub- sequent division into 4 and 8 cells is strikingly simi- lar to spore development in Tetramy.ra. Had Octo- mi/xa been known at that time .Juel would doubtless have emphasized the relationship of his species with the Plasmodiophorales even further. It is to be noted, however, that in these two genera each mitosis in the sporonts is not immediately followed by cell division as in Pyrrhosoriis, and that the spores which are formed encyst and pass through a dormant period before giving rise to zoospores. It is possible that under the conditions of .Juel's study the spores of P. marinus failed to encyst and become dormant. It is also possible that zoosporangia occur in this spe- cies but were not present in Juel's material. In tiiat event P. marinus would be very similar to Octomyxa. However, its laterally biflagellate isocont zoospores with an orange-colored eye-spot constitute a serious obstacle to including it in the Plasmodiophorales at present, unless, of course, .Juel was mistaken about the relative lengths and insertion of the flagella. These possibilities, however, are purely speculative. On the other hand, the zoospores are similar to those figured for species of the lower biflagellate Oomy- cete-like fungi, but until more is known about P. ma- rinus its relationship will remain obscure. Winge, nonetheless, considered it closely related to the Plas- modiophoraeeae and made extensive comparisons be- tween its life cycle and that of Sorolpidiiim. He re- garded the sporangiosori of the latter genus as homo- logous with the aggregates or sori of spore-mother cells of Pyrrhosorus, and believed that the absence of wall around the sporonts in the latter is of minor importance. Cook ('33), on the other hand, regarded the relationship of Pyrrhosorus with the Plasmodio- phorales as highly questionable. BIBLIOGRAPHY : DOUBTFUL GENERA Barrett, ,T. T. 193 j. Pliytopath. 25: 898. Cook, W. H. I. 19-'6. Trans. Brit. Mycol. Soc. 11: 310. 1937, 11)1(1.1-2:282. . 1933. Hoiifr Koiifr Xat. Suppl. 1 : 38. . 1933. Arcli. Protistk. 80: 333. Fischer, A. 1893. Kabenhorsts Krypt. 1, aht. +:67. Fitzpatriok, H. M. 1930. The lower fungi — Phycomycetes. New York : p. 03. Guyot, A. L. 1937. Bull. Soc. Path. Yep. I'Ent, Agr. France 14: 181. Juel, H. O. 1901a. Bill. K. Svensk. Yet.-.\kad. Hand. 26 afd. Ill, No. U: 1. . 1901b. Rev. Mycol. 34: 111. Maire, R., and A. Tison. 1911. C. R. .\cad. Sci. Paris, 153: 30G. Minden, M. 191.5. Krypt. Fl. Mark. Brandenburg 5: 378. Nemec, B. 1911. Osterr. Ungar. Zeitscbr. f. Zucker u. Landw. 40. Palm, B. T., and M. Burk. 1933. Arcb. Protistk. 79: 363. Rawlins, T. E. 1935. Pliytopath. 15: 737. Saccardo, P. A. 1936. Sylloge fungorum 34, sect. 1: 17. 1931, Ibid. 35: 13. Schroeter, J. 1897. Engler and Prantl, die Nat. Pflanzf. I, 1:5. Wildeman, E. 1893. Ann. Soc. Micro. Beige. 17: 35. Winge, O. 1913. Ark. f. Bot. 13, No. 9: 29. EXCLUDED GENERA Herewith are presented descriptions and illustra- tions of three genera which have been included in the Plasmodiophorales by various workers, prima- rih' for want of a better group in which to place them. Uniflagellate zoospores are reported to occur in Cystospora but are apparently lacking in Sporo- myxa and Peltomyces. Except for a multinucleate plasmodial stage, resting spores, and the occurrence of intranuclear mitosis and schizogony these genera have little in common with the Plasmodiophorales as this order is now generally recognized. They are, nevertheless, described here so that their validity as members of this order may be judged independently. PLATE 14 Trematophli/ctis Leptodesmiae (All figures after Patouillard) Fig. 1. Leaves of L. congestu with galls. Fig. 3. Portion of a branch with a large gall and three open pu.stules. Fig. 3. Section of a gall showing several sori. Fig. 4. Naked plasmodium (?) filling greatlv enlarged cell. Fig. 5. Group of resting spores formed by segmentation of Plasmodium. Fig. 6. Individual resting spores. Pyrrhosorus niariinis (All figures after Juel) Fig. 7. Laterally biflagellate isocont zoospores. Fig. 8. Uninucleate tballus. Fig. 9. Two paired young thalli. Fig. 10. Uninucleate thallus with enlarged primary nu- cleus. Fig. 11. Four-nucleate tballus passing through cell wall. Fig. 13. Jlultinucleate tballus. Fig. 13. Multinucleate amoeboid thallus. Fig. 14. Cleavage of tballus. Fig. 15. A sorus of spore mother cells. Fig. 16. Isolated spore mother cell. Fig. 17. A sorus, the spore mother cells of wbieli liave di- vided into groups of four daughter cells. Fig. IS. Spindle-shaped spore mother cells (?) in a branched tballus. Fig. 19. Spindle-shaped spore mother cells and accessory sterile cells in an elongate host cell. Fig. 30. Sorus with spore motber and sterile cells. Fig. 31. Sorus with spore mother cells undergoing mito- sis. Fig. 33-34. Mitosis and cytokinesis of s|)ore mother cells. TIlKMATOl'llYLCTlS 73 -•«r 20^ ' 23 23' Trematophlyctis, Pyrrhosorus 74 PLASMODIOPHORALES by research workers. Doubtless, there are numerous other plasniodiaceous organisms wliicli resemble the true Plasmodiophoraceae and simple fungi which must eventually be given serious consideration by mycologists and protozoologists, and it is hoped that by presenting the available data here greater inter- est and research may be stimulated in these border- line organisms. SPOROMYXA Leger, 1908. Arch. Protistk. 12: 111. (plate 13, fig. 1-25) Sporomyxa was created by Leger for a virulent parasite, S. Scauri, which he found in tlie coelome of the imago of Scaunts tristis in Algeria. The parasite has a predilection for the adipose tissue and may be found in enormous numbers there. Unlike most plas- modioplioraceous fungi, it destroys infected cells completely without stimulating them to divide or enlarge. The earliest known stage consists of a small, naked, spherical, ovoid. 6—8 /j.. or spindle-shaped body with an unusually large, 5 /a, nucleus and finely granular cytoplasm (fig. 1). It does not appear to have a sharply defined membrane and lies embedded in the host cytoplasm. As it increases in size the nu- cleus divides mitotically with an intranuclear spin- dle (fig. 2), and tlie thallus becomes binucleate. In this stage it may divide by binary fission (fig. 3). Additional nuclear divisions occur (fig. i). and larger, naked, multinucleate plasmodium-like thalli are eventually formed (fig. 6). Leger found no thalli with more than 8 nuclei, and he believed that from this stage on the parasite undergoes schizogony into uninucleate meronts or sporulates, so that thalli with a large number of nuclei are never formed. The mature thallus may be splierical, elliptical, and sometimes amoebiform, according to the jiosition its occupies in the host tissue, and although it may have the shape and appearance of an active amoeba, it does not move or undergo changes in form. Its cy- toplasm is denser toward the center, but no distinct endo- and ectoplasmic laj'ers are distinguisliable. No wall or membrane is present, and the whole thal- lus may be enveloped by the host protoi)lasm (fig. 6). In addition to these thalli, Leger found otiier smaller ones with numerous fat globules and chromatic gran- ules in the cytoplasm and small nuclei which ap- peared to be lacking in chromatin (fig. 7). He be- lieved such thalli occur at the close of the vegetative phase of iS'. Scauri and mark the beginning of sporo- genesis. Unlike the true plasmodiophoraceous genera, no segmentation of the multinucleate thallus into nu- merous separate spores or cystosori lias been ob- served in S. Scauri. Resting spores, however, occur very abundantly in the adipose tissue, but Leger was not certain whether they are formed by eneystment of vegetative uninucleate thalli or are the products of more or less simultaneous schizogony of a multi- nucleate body. He admitted the possibility of both methods, but did not sliow any figures of the latter process. The spores may sometimes occur in groups, but it is not evident that these aggregates have been formed by segmentation of a multinucleate Plasmo- dium as in Plasmodia phora. The only developmental stages of resting spores described by I-eger relate to small, isolated spores. These are apparentlv formed by the eneystment of uninucleate thalli dur- ing which process the nucleus shrinks in size as chro- matic material is extruded from the nucleole into the cytoplasm (fig. 9-13). As this goes on, the wall thickens and differentiates into a thick outer and a thin inner layer. In bi- and multinucleate tlialli, spore formation may be accompanied by nuclear fusions (fig. 12. 13) of the type described by Prowa- zek (0.5) for P. Bras.iicae. Leger interpreted these fusions as representing rudimentary sexuality. The majority of spores are ovoid, 8X 10 M> but they may often be more elongate, iXS/i. spherical, obpyri- form, constricted in the middle, and unusually large, SO-iO /x (fig. 15-17). The small spores are usually PLATE 15 Sporomi/x(t Scanri (All figures after Leger) Fig. 1. Uninucleate thalhis. Fig. J. Mitosis witli intranuclear spindle and minute chromosomes. Fig. 3. Binucleate thallus undergoing binary fission. Fig. 4. Mitosis in a binucleate thallus. Fig. 5. Tetranucleate thallus. Fig. 6. Large, amoebiform, eight-nucleate thallus witliin host cell. Fig. 7. Thallus with chromatic granules in cytoplasm; nuclei without (?) chromatin. Fig. 8-1:3. Successive stages in resting spore formation. Fig. 13, H. Xuclear fusion (?) in resting spore. Fig. 15-17. Large, abnormal resting spores. S. Tertehronis (All figures after Reitschel) Fig. 19-30. Developmental stages of thallus. Fig. 31. Synchronous nuclear division; polar and profile views. Fig. 22. Completion of cleavage into spore rudiments. Fig. 2S. Later stage of same. Fig. 2i, 25. Uni- and binucleate spores. f'i/.s'/o.s'/iora hfitntd (All figures after Elliott) Fig. 30. Resting state. Fig. :37. Amoebae. Fig. 38-33. Nuclear division and multiplication. Fig. 33. Sixteen-nucleate stage of thallus; nuclei of un- equal size. Fig. 3+. Migration of plasmodium through rootlet. Fig. 33. Cells of host with amoebae and plasniodia. Fig. 36. Root tip cells with plasmodium and amoebae; nuclei of unequal size in plasmodium. Fig. 37-41. Stages in cyst formation from a plasmodium. Fig. 43. Row of cysts. Fig. 43, 44. Formation in and Iil)eration of zoospores from cysts. Fig. 4J-47. Degeneration of cysts. EXrU'DKl) OENERA I'LATK ir, Sporoniyxa, Cystospora 76 PLASMODIOPHORALES uninucleate, but the abnormal ones may possess 2 to 30 nuclei scattered about or aggregated in groups. The wall of the spore is hyaline, streaked, and thick, and by treatment with iodine and sulphuric acid it assumes a bluish tint, indicating the presence of cellulose. A second species, S. Tenebriones, was found by Reitschel ('36) in the fat bodies, ovaries, and con- nective tissues of the larvae and imago of Tenebrio molitor. The life liistory and development of this species (fig. 18—25) are similar to tliose of S. Scauri with the exception that the thalli become larger and undergo cleavage at maturity. At the time of sporu- lation they may contain considerably more than a hundred nuclei and are enveloped by a thin mem- brane. The protoplasm cleaves into uninucleate seg- ments (fig. 22, 23) which later round up and become the resting spores as in Plasmodiophora, The soral membrane disintegrates shortly thereafter and frees the spores. These are usually uninucleate (fig. 24'), rarely binucleate (fig. 2.5), hyaline, smooth, and measure 9—13 fi by 4.5—7 fj.. In neither of these spe- cies have spore germination, zoosporangia, and zoo- spores been observed. Leger believed that Sporomyxa may be closely re- lated to Sapphiia because of its method of sporula- tion. Maire and Tison (09) regarded it as of doubt- ful affinity with the Plasmodiophorales and stressed lack of promitosis in nuclear division as a distinctive character. Fitzpatrick ('30) and Cook ('33) ex- cluded it on the grounds of its habitat and ellipsoidal isolated resting spores, but as Palm and Burk ('33) have pointed out, "the circumstance that it attacks an animal host could hardly be taken as a serious objection." However, our knowledge of its life cycle and cytology seems hardly sufficient to justify its inclusion in the Plasmodiophorales at the present time. PELTOMYCES Leger, 1909. C. R. Acad. Sci. Paris 149: 239. Leger founded this genus to include three para- sites, P. hi/alinus, P. Blatella, and P. Forficulae, which occur in the malpighian tubes of Olocrates, Blatella, and Forficula species. His description of the genus was based primarily on the development and life cycle of P. hijalinus, apparently the oiil}^ species which he studied in detail. This species makes its appearance in the epithelium as a small, 2 //., uninu- cleate globular body. Its nucleus multiplies mitoti- cally, and the parasite soon grows into a multinucle- ate disc-shaped plasmodium which subsequently un- dergoes schizogony and forms a large number of small, 2-3 /x, uninucleate sporonts. At the conclusion of schizogony the sporogonic phase begins. Each sporont increases in size while its nucleus divides mitotically several times. Two types of nuclei are thus formed: small, densely- stainable somatic nuclei without membranes, and larger, normal-looking gametic nuclei with well- defined membranes. The former nuclei disintegrate, while the latter become enveloped in a small spheri- cal mass of cytoplasm and are soon transformed into bowl-shaped, 2 jj., gametes. These fuse in pairs after their nuclei have undergone a chromatic reduction, and this is soon followed by karyogamy. The zygotes or incipient diploid resting spores formed in this manner develop a wall and assume a cylindrical, 3X9/^, shape. Each mature sporont thus encloses within its tliin wall 4 to 8 spores arranged side by side and looks like a sporangium. The gametes in the sporonts which fail to fuse develop into par- thenogenetic spores of about half the size of the dip- loid spores. In some cases prematurely formed spo- ronts, instead of producing gametes, form small en- dogenous cells which escape from the sporonts and behave as schizozoites in the host. Leger did not illustrate any of tliese species, and his account of their development is brief and fragmentary. Zoo- spores, sporangia, and cystosori are unknown in Peltomyces. CYSTOSPORA Elliott, 1916. Delaware Agr. Exp. Sta. Bull. 114: 15. (plate 15, FIG. 26-47) This genus was created b}' Elliott for a myxomy- cete-like organism, C. batata, which is reported to cause "soil rot," "pit " or "pox" of sweet potatoes in the United States. Elliott placed it in the Plasmodio- phorales, but its inclusion here is very doubtful, if at all warranted. In fact, some workers (Manns and Adams, '25) have expressed doubt about tlie ex- istence of an organism of this type and asserted that some of the stages figured by Elliott may be nothing more than products of disturbed metabolism of the sweet potato. Tabenhaus (18), however, reported tliat he was able to grow tliis organism in pure cul- ture on sweet potato agar made up according to Elliott's formula. He further confirmed Elliott's ac- count of the life cycle of C. batata. According to these workers, the zoospores are small, 1—2 ft X 1-5-3 /x, globose with tapering ends and possess a short flagellum, but it is not evident from their descriptions whether the flagellum is an- terior or posterior. The zoospores are nonetheless produced in great numbers (fig. 43, 44) and may re- main active from 1 to 7 days in rare instances, ac- cording to Tabenhaus. The period of activity, how- ever, is usually short, often less than half an hour. The zoospores may sometimes fuse in pairs and form round zygotes which later become amoeboid ( fig. 26, 27). According to Elliott, they bore through the cell wall and infect the host as amoebae, but Taben- haus reported that infection may also take place by means of a plasmodium. The nuclei of the young par- asite divide mitotically and simultaneously (fig. 28, 29, 32), but unfortunately Elliott's figures are so small and poorly drawn that it is impossible to deter- KXCI.rnKD OEXKUA 77 niiiio wlu'tlu-r or not tlic divisions riscinlilo tlu- pro- mitosis (Itscriluii for otlifr ) .ilso ineluded the I'lasniodiophorales amoiiir the ehytrids next to the Synehytriaeeae and stated that their eytolo^y as well as the prcscnee of eliitin in the walls indieates a close rel.-itionship. Cadnian ('31) and Bessey ('3.5), however, believed that the Plasniodio])horales show a closer affinity to the Myxomyeetes. and the former worker listed them as a sub-group of the Myxomy- eetes. .Martin ( 3(>) listed the Plasmodio))lioralcs as the lowest order of the Phyeomyeetes but distinct from the so-called niyxochytridiales. On the other hand, Ciwvnne-\augiian .md Barnes ('26, '37) maintained that the Plasniodio])Iiorales and Myxomyeetes are not fungi and have doubtless arisen from lower forms along inde))endent lines. Cook ('26) agreed in general with these mycologists and expressed the view that "it seems very desirable to keep the Plasmodiophorales quite separate from the Chytridiales and other fungi. If there is any rela- tionship, it is most likely through the Mycetozoa." In I92S. however, he held that the Plasmodiophora- ceae and Myxomyeetes originated from a proteomyx- ean eomiilex through the Lobosa and more s])eeifi- cally .Ircella vuhfari.s and Amoeba miiscicola and diverged at slightly different points. On the basis of the type of nuclear division in the vegetative phase. Cook lielieved that the Plasmodiophorales diverge from the Amoeba series at a more distant point than the Mycetozoa. Later ('33) he asserted that "no close relationshi}) with either the fungi or ])roto/.oa is l)robable." and that the Plasmodioiihorales "repre- sent an independent group having their origin in the Proteomyxa. " Cook tiuis revived and su))))orted the earlier views of Zopf , Delage and Herouard. Lankes- ter, and others on the relationship of the Plasmodio- phorales to the Proteomyxa. Zoologists also have asserted their claims to the Plasmodiophorales and included this order as a sub- class of the -Myxomyeetes among the Protozoa, ])ar- ticularly the Khizo))oda. Most protozoologists. how- ever, have continued to use .Sehroeter's term. Phyto- myxinae, for the group, although it has been evident since the beginning of the present century that Phij- tomi)xa, the genus after which Schroeter named the order and family, is no longer tenable and relates to what are now known as bacteria and mycorrhizal fungi. Protozoologists. furthermore, have ignored the discovery and presence of zoosjjorangia and bi- Hagellate, heterocont zoospores in six genera of the Plasmodiophorales and have adhered to the older, outworn conceptions regarding these organisms. De- lage and Herouard ('96) followed Zojjf's disposi- tion of Plasmoiliophorn and Tetrami/ja by including them with (1 iiniiiococciix, P.ieiidospora, etc., in the zoos))orie Proteomyx.i or .Mon.ulini.-ie their first el.ass uniler the Hliizopod.i. Dotlein ('01) listed the Mycetozoa as the fifth and last class of the Rhizo- ))oda next to the Poramiuifera and divided it into two subclasses of equal rank, the Protomyxidea and Mveetozoidea. Plasmodiophora and Teframi/ja were pl.ieed in the zoos])oric grouj) of the Protomyxidea. ,1 classification which eorres])onds essentially with that of ])revious ])rotozoologists. In subsequent edi- tions of his text-book ('Oi), '11. 'Ki) DoHein placed the Mycetozoa next to the Radiolaria and limited the sub-class Protomyxidea to what are now generally known as the azoosporic and zoosporic Proteomyxa. For the plasmodiophoraceous genera he adopted Seiiroeter's Phytomyxinae, made it a sub-class of the Myxomyeetes, and ))laced it between the Acra- siae and ]\Iyxogastres. In the sixth edition, however, which was rewritten by E. Reichenow, the Phyto- my.xine and Acrasiae were excluded from the My- cetozoa proper and were discussed merely as an "anhang " to this class. I.ankester ('8.5. '09) and Hartog ('06, '09, '22, '36) also included Plasmodiophora and Tetra?)iy.i'a in the Proteomyxa along with J ampyrella, Gi/miio- coccus, Pseudo-spora, etc. Hartog. however, assigned Plasmodiophora together with Protomi/.ra, Vampy- rella, and Serumsporidiuvi to the non-flagellate or azoosporic Myxoidea. In 1909 Calkins referred to P. Brassicae as a mycetozoan and later ('33) in- cluded the Phytomyxida (Phytomyxinae) as an or- der in the Myxomyeetes. He believed that the lack of peridia and capillitia in the plasmodiophoraceous s])ecies is due to their parasitic mode of life. Min- ehen ('12, '21) was uncertain of the relationships of the Phytomyxinae and merely discussed them as border-line organisms in relation to the Sarcodina. Hertwig (19) listed the Mycetozoa as the fifth class of Protozoa and of equal rank with the Rhizo- ])oda and included Plasmodiophora among the My.x- omycetes. Galiano (1921) also grouped the Phyto- myxinae as a suborder of the ^lyxomvcetes, while Rumbler ('23-'2.5) reverted to DoHein's ('09, '11) classification. Kudo ('31, '39), on the other hand, included the Phytomyxinae directly in the Myxomy- eetes. It is evident from this review that several ))oints of origin and lines of develoj)ment and relationship have been em|)liasized for the Plasmodiophorales. These suggested relationshi))s involve the Myxo- myeetes, Chytridiales, Protozoa, Sporozoa, and Pro- teomyxa. The evidence in support and against these relationships will now be considered in greater de- tail. Plasmodiophorales and My-xomvcetrs Inasmuch as the belief that the Myxomyeetes and Plasmodio])horales are closely related is rather widely held, this view will be presented in consider- able detail. Proponents of this view have stressed the presence of a large multinucleate plasmodium and 80 PLASMODIOPHORALES anteriorly uniflagellate zoospore in both groups as evidence that they have originated from a common ancestor. Considerable significance has also been at- taclied to the reports that the plasmodia of Sponc/o- spora (Kiinkel, '15) and Plasmodiophora (P. M. Jones, '28) can live outside of the host and may be cultivated on synthetic media like those of the Myxo- mycetes. Careful analysis of Kunkel's paper, how- ever, shows that the saprophytic plasmodia which he described apparently do not relate to Spongo- spora at all, because at maturity they form stalked Dicti/osfelium-like sorocarps instead of spongy cys- tosori. I>ike\vise, the peculiar and abnormal life cycle described by P. M. Jones for P. Brassicae suggests that he may have been studying some other plasmo- dial organism instead of Plasmodiophora. It thus remains to be seen whether or not the plasmodium of the Plasmodiophorales can be cultivated saprophy- tically outside of the host. As to the mode of nutrition, data are accumulat- ing which suggest that it possibly may be very simi- lar in both groups. The zoospores, amoebae, the plasmodia of the Myxomycetes are capable of en- gulfing food particles, digesting them, and discard- ing the extraneous waste material. While this type of nutrition is not particularly evident in the Plas- modiphorales, claims have nonetheless been made that the plasmodium at least engulfs starch grains and masses of host protoplasm. According to Woro- nin. Nawaschin, Prowazek, and Lutman, starch grains may often be found in the folds and vacuoles of the Plasmodium of P. Brassicae. Nawaschin ('99), Favorsky, and Henckel did not believe these had been engulfed, but Woronin, Eycleshymer, and Lutman nevertheless inferred that the plasmodium feeds on these grains. Maire and Tison (11) like- wise reported that the small plasmodia of Ligniera Junci may engulf algal cells. The zoospores of some species also appear to be capable of taking in solid bodies, but how generally it occurs is not known. In Polymijxa (jraminis Ledingham reported that the pseudopods of amoeboid zoospores may flow around and engulf small objects. The evidence of relationship on the basis of simi- larity in zoospore structure is not particularly con- vincing in light of recent discoveries. Until 1931' it was believed that the zoospores of the Plasmodio- phorales were like those of the Myxomycetes in hav- ing one anterior flagellum, but since that time it has been clearly shown that the zoospores of six genera of the former group are anteriorly biflagellate and heterocont. Further study will doubtless show this to be true in the remaining genera of the Plasmodio- phorales also. The structure of the zoospores and the number, position, and relative lengths of the flagella are very significant phylogenetically, and it would seem offhand that the presence of biflagellate, heterocont zoospores in the Plasmodio])horales sepa- rates this order very sharply from the Myxomycetes. It must be remembered, however, that although the majority are uniflagellate, zoospores with two fla- gella are not uncommon in the Myxomycetes also. De Bary ('84) and Vouk ('11) early noted zoospores with two flagella, and since that time numerous re- ports of similar zoospores have appeared. Gilbert ('27) found that 25 per cent of the zoospores of Stemoiiitis fiisca are biflagellate, and his figures le and If show that one of the flagella is considerably shorter. Similar zoospores have been subsequently described and figured by Smith ('29) for Dictyae thalium plumheum, by Howard ('31) for Physarum polycephalum, and by Sinoto and Yuasa ('St) and Yuasa ('35) for Physarella ohlonga, Fuligo septica, and Comatrichia longa var. ftaccida. In the latter species 13 per cent of the zoospores were biflagel- late, and in rare cases triflagellate. As is shown in figures 2 to 5, Plate 17, the flagella are of equal as well as of unequal length. Stosch ('35) also found biflagellate zoospores in Didymium eunigripes, D. xanthopus, D. squamosum, D. difforme, Physarum cinereum, P. nutans, Trichia favoginea, Comatrichia nigra, and Lycogola epidendrum. In most species which normally have uniflagellate zoospores, bi- and multiflagellate cells are usually the result of unequal or incomplete cleavage, and are consequently large and bi- or multinucleate. Such does not appear to be true of the zoospores shown in figures 2 to 6, Plate 17, since there is but one nucleus present regardless of the number of fla- gella and the size of the zoospore. A more funda- mental cause may perhaps be operating in these cases. Of particular interest in these figures are the basal bodies upon which the flagella are oriented. In Ceratiomyxa fructiculosa var. flaccida, Physarella ohlonga, and Fuligo septica, they are double regard- less of whether one or more flagella are present. E. A. Bessey, Professor of Botany, IMichigan State College, believes that this double condition may per- haps be significant phylogenetically. In correspond- ence with the author concerning these zoospores, he asks : "Are these two granules homologous to the basal granules found in algae and .... sperm cells of mosses or ferns, where each flagellum arises from such a granule ? Then do the planocytes with but one flagellum represent cases where there has been a loss of one flagellum in progressive evolution from a normally biflagellate condition, and do tlie biflag- ellate cells of these slime molds represent the an- cestral condition which has not been com])letely lost in this grou]).'' In the Plasmodiophorales, which are probably closely related to the slime molds, the bi- flagellate condition has not yet been lost, though one flagellum is smaller than the other." Bessey thus suggests that the presence of a second basal granule in uniflagellate zoospores may possibly be a relic of the biflagellate condition and that the Plasmodio- phorales are more primitive than the slime molds. However, it remains to be seen how general the double condition is in uniflagellate zoospores. Jahn ('04), Wilson and Cadman ('28), and Cadman ('31) figured and described only one basal granule, while Cotner ('30) and Stosch reported the presence of several bodies at the base of the flagellum. Sinoto and Yuasa's accounts of the presence of two basal PIIYI.OCJKNV AND HK.I-ATIOXSIIII'S 81 Inulifs in tin- M\ ictuzo.i Iiuve aocordiiiiily nut lucii universiilly continnoil. .Ijiliii ( 30) sovcri'ly critiiizid the belief tliat tlio presence of two H.ifjella aic nl niueli sijrnilieaiiee, questioned the presence of more than one liasal jirannle, and rejjarded all liiHauellate zoospores as alinornial. In tlie I'lasniodiopliorales little is known about the l)lei>haro])last and its eoinposition. 'Terhv (lil-a) and Cook and Sehwartz ('30) tiirnred only oiu' t)lei)h- aroplast in the uniHairellate zoospores of /'. Hras- sicar, but later Terby ("'2t-b) reported that the blepharojilast may divide and form two bodies in the incipient spore. Neither Ledingiiain ('31, '35) nor Couch. <-t al. ('39). showed basal fjranules in their fifrures of the biflai;ellate zoosjiores of Plasmo- diophora, Spoiifioxpora, and Octomi/.ra. In I'oli/- mi/ja, on the otiier liand. I.ediniiham ('39. p. t2) figured the two flagella attached directly to the nu- clear membrane without the presence of blei)haro- plasts or basal granules. It is thus obvious that but little is known about the number of basal granules in the zoosj)ores of this order and their relations to the H.igell.i. Nevertheless. Bessey's suggestion con- cerning the significance of basal granules and the occasional occurrence of biflagellate zoosjiores in the slime molds is very stinnilating and merits further investigation. Turning now to other differences within the two groups, it may be noted that sporangia and capil- litia of the tyi)e found in the slime molds are lack- ing in the Plasmodio]iliorales. As has been noted before, mycologists and protozoologists have re- garded this reduction as due to the jiarasitic mode of life adojjted by the Plasmodiophorales. Cook ('33) suggested that the membrane around the cys- tosori in certain ))Iasmodiophoraceous genera, Soro- discus, Sorosphaera, etc.. may be looked upon as equivalent to the sporangium wall of the Myxomy- cetcs. However, there is considerable doubt about the jiresenee of a soral membrane in these genera. The Myxomycetes. on the other hand, lack s])o- rangiosori and thin-walled evanescent zoosporangia, which have recently been shown to occur in most genera of the Plasmodiophorales. These zoosporan- gia may arise directly from zoos])ores which have entered the host or later from small or large, seg- mented, vegetative plasmodia. These differences — lack of thin-walled, intramatrical zoos])orani!:ia in one grou]) and sjiecialized sporangia and capillitia in the other — are of fundamental significance, in the author's opinion, and are difficult to ex))lain wliolly by differences in mode of life. Other develojjmental phases and eytological dif- ferences between the two groups are to be noted here. Schizogony of the young plasmodiimi has been described in most genera of the Plasmodiophorales, but it a])pears to be lacking in tlie Myxomycetes. At least, no conclusive evidence of its occurrence has yet been presented. Furthermore, neither the so- called "promitotic" nuclear divisions nor a marked "akarvote " stage, which are rejiorted to be charac- teristic developmental i)hases of the Plasmodiopho- rales, li.i\ e been found in the Myxomycetes. W'iiether or not tliese (litl'erenees alone are of much phyloge- nitie signiHc.incc, however, is questionable. Comp.-irisons of the two groups on the basis of sexuality, alternation of generations, time and place of meiosis, etc., are difficult to m.ake at present, be- cause so little is kiu)wn about these ])rocesses in the Plasm()dio|)!iorales. In the Myxomycetes also there is considerable disagreement among workers about these devclo|)mental phases. As far as is now known the resting spores of the slime molds usually form more than one zoospore in germination, and these in turn divide once to several times before becoming gametes. In the Plasmodiophorales, on the other hand, it is claimed that only one zoospore is formed, which functions directly as a gamete without di\ id- ing. Cook ('33) empluisized this distinction and stated that it is "the chief difference between the two groups." In light of data in the literature, this statement is obviously open to criticism. Maire and Tison.and Home found an additional or third mitosis after the two meiotic divisions in Sorosphaera and .S' /;0H r/cs/jora, respectively, where by binucleate spores were occasionally ))rodueed. I.utman and Terby also figured binucleate sjjores in P. Brassicac and be- lieved that these arise as tiie result of division of the spore nucleus. It is not improbable that such spores form more than one zoospore or gamete. In addition to such spores, unusually large multinucleate ones ha\e been found in several genera, and it is not un- likely that they also give rise to several motile cells in germination. Likewise. Cook's assertion "that di- vision of the swarm cells does not take ])laee in the Plasmodiophorales i)rior to fusion" is rather dog- matic and premature in light of our meager present- day knowledge of the behavior of the zoospores in this order. The}- have never been cultured with cer- tainty outside of the host, and very little is known about their behavior within the host cells. Cook's assertion is furthermore contradicted by Massee's (PL 10, fig. 10). Osborn's. Home's and Fedorint- schik's accounts of tin- multi))lieation of amoebae or gametes in Spoiu/ospora and Pla.smodiophora by equal division and budding. In maintaining that the gametes are the direct ))roducts of the resting spores. Cook further contradicted his own and Schwartz's ('30) earlier assertion that the gametes of 1'. Kras- sicae are produced in thin-walled zoos))orangia or gametangia. The origin .-uid method of formation of gametes in the Plasmodiophorales are thus some- what doubtful at jircsent, and it seems jirematurc to make definite comparison between the two groups on this basis. Fusion in pairs of isomorphic amoeboid and flag- ellate gametes has been reported to be characteristic of both groui>s, but as noted elsewiiere actual fusion has so far been seen very seldom in tlie Plasmodio- phorales. The respective gametes are alike in size and structure in both groups, but in the Myxomy- cetes certain other differences between gametes of the opposite sex have been reported, .\ccording to Abe ('3f) the male gamete loses its Hagellum as it 82 PLASMODIOPHORALES flows into tlie female, and its nucleus migrates to- wards that of the female gamete. Furthermore, the latter gamete carries a positive charge and has a low oxidation-reduction potential, while the male gamete is the opposite in these respects. Kambly ('39), however, was unable to confirm these results of Abe, and found no marked physiological differ- ences between swarm cells of various species. Gil- bert ('35) and Stosch ('37) likewise reported that the male gamete may be distinguished during fusion by the migration of its nucleus toward that of the female. Such differences have not been reported for the Plasmodiophorales as far as the author is aware. In the Myxomycetes the gametes fuse by their pos- terior ends, while in the Plasmodiophorales, accord- ing to Cook ('33), they fuse at the anterior ends. However, so little is known about gametic union in this order that it is premature to regard the latter type of fusion as characteristic of the Plasmodiopho- rales. Comparison of the two groups on the basis of time and place of sex segregation is also impossible at present, because little is known about sexuality in the Plasmodiophorales. No monospore cultures or infections have yet been made to determine whether the species are homo- or heterothallic. If, as Cook ('33) maintained, the gametes are the direct prod- uct of uninucleate spores and no division occurs in amoebae and zoospores, sex segregation obviously takes place during one of the meiotie divisions be- fore or during sporogenesis. Otherwise, it is pheno- typically determined in the haploid generation, and the species are accordingly haplosynoecious. In the Myxomycetes also, there are but few data relating to sex segregation. Skupienski ('17) believed that in D. difforme it occurs during one of the divisions in the zoospores. Miss Clay ley reported its occur- rence at the second meiotie division in the zoospores of D. tiif/ripes. Schiinemann confirmed her rejjort of haplophenotypic sex segregation in tliis s])ecies and described D. nigripes as haplomonoecious. Miss Cad- man, however, noted no differences, morphological or physiological, between the gametes in lieficularia and D. nigripes and concluded that no sex segrega- tion is necessary or takes place in these species. Stosch, on the other hand, implied by his statement concerning crosses in D. eiinigripes that sex is geno- typically determined. As to the time and place of karyogamy, meiosis, and alternation of liaploid and diploid generations in the Plasmodiophorales, a detailed account of these subjects has been given in Chapter III. As is evident from this description, the majority of workers have assumed that the isomorphic gametes fuse in pairs, after which karyogamy soon occurs. Nuclear fusion in the zygote thus initiates the diploid phase which includes the plasmodial stage up to the last two nu- clear divisions preceding or during cleavage where reduction occurs. Plasmogamy and karyogamy arc accordingly not followed at once by meiosis. The haploid })l!ase includes the cystosori, spores, zoo- spores, and gametes, according to this viewpoint. However, as noted before, exceptions to this view have been presented by Prowazek, Osborn, Home, Webb, and Whiffen. In the Myxomycetes likewise there is considerable disagreement and controversy concerning karyog- amy, meiosis. and alternation of generations. Much of the controversy about meiosis hinges upon the question of whether one or two divisions occur prior to spore formation in the fruiting bodies. Strasburger ('84.). A. Lister ('93), Rosen ('93), Harper ('00), .7ahn ( '07-36 ), Kranzlin ('07), Gilbert ("3.5), and Stosch ('3.5, '37) found only one. while Wilson and Cadman ('28), Cadman ('31), and Schiinemann ('3.5) reported two divisions. In contrast it may be noted here that most workers on the Plasmodiopho- rales are in agreement that two divisions precede spore formation. However, in order to draw com- parisons between the two groups with respect to meiosis, it is essential to outline briefly the differences of opinion concerning this question in the Myxomy- cetes. In the Exosporae, Olive ('07a) found stages re- sembling syna]>sis in the young spores of Ceratin- 7ni)j-a and later ('07b) on observing pairing and fu- sion of nuclei in the pillars, concluded that the two mitoses in the spores of this genus are meiotie. Olive's conclusions on pairing and fusion of gametic nuclei were confirmed in general by Jahn ('07) who, however, held that these processes occur earlier as the Plasmodium creeps out of the wood. On the other hand, he refuted Olive's contention that meiosis oc- curs in the spore and claimed instead that the two divisions which precede cleavage are reductional. The incipient uninucleate spores are accordingly haploid. Jahn ('08) reasserted his observations on nuclear pairing and fusion, but maintained that only one, instead of two, division occurs prior to cleavage. This division is heterotypic, according to .Tahn. and reduction is thus accomplished by one division. The Plasmodium is formed by the fusion or coalescence of numerous haploid myxamoebae, the nuclei of which divide mitotically several times in the plasmodium. Karyogamy is accordingly delayed until the plas- modium creeps out to fructify. In 1911, however. Jahn concluded that his previous observations on nu- clear pairing and fusion in the mature plasmodium were incorrect and that the appearances of karyog- amy were the results of nuclear degeneration. His observations of endosporus species led him to the be- lief that nuclear fusion follows plasmogamy of amoe- bae. Jahn ('1 1, '33, '36) nonetheless persistently ad- hered to his early view that meiosis occurs during the last division before cleavage, as is shown in text- figure 1 2. Gilbert ( '3.5 ) , on the other hand, confirmed Olive (m meiosis in the spore and in addition showed that the haploid motile gametes fuse posteriorly in pairs to initiate the plasmodium (text-figure 13). He also found that karyogamy follows plasmogamy within 21' hours, thus refuting Olive's observations but confirming Jahn's later view. In the Endosporeae, .lahn (07) reported the same tvjje of nuclear pairing and fusion in the young fruit- IMIYI.IKiKNV AM) IlKI.ATIOXSllll'S 88 plasuooma' )cM>o&>ur rctone it^orc rC^^ e a^ PLASuooAf-iy ttcccssopf fusion/ KMrooAMr vExvsrive mitosis TDfT-FielB LFEiytLEorCePATiouirxA.MXOKDna to J-wn. i9it-je. 2ND M D 1ST M O TexT-FidS Life Cvnc OF CeMTioMrxA. ACCOKDiNC to Qlbert. 1935. FWITING BOO- ZY<30Tt MITOSIS. OMXTICNUaXI ■O o*\ FIASMODIUM PLASU03AUY KAKYOOAMY PLASMODIUM 1ST MB /^r~~ S 200SP0f€S SPOKES "' Text-Fic.14 Lfs OrcLE ofD ofFCKne. accofiding to Sklpcnski. i92s. DIPLOID sex SEGPECATION ^ ZND M. D BISEXUAL SPORE Text-Fig 15 Life Cms of D difforme. Accoaoma to CLo-LEy. i929- rouNG PLASMoauu "/-''icO" Division ZOOSPORES Text-Fig 16 Life Cms of D nigrpes. according to Schunemann. 1930. Text-fifjurc Text- Fig 17 Life Cycle OF R LyOiperdon. according toWIlsonano Cm>MN.I923. 12-17 84 PLASMODIOPHORALES ing bodies of Amaurochaete, Reticularia, Trichia, Stemonitis, and Didymium. In these genera karyog- amy is followed by synapsis, and as the spores are delimited, one mitosis, the heterotypic division, oc- curs. Tliis first meiotic division is followed by a long rest period of the spore, and the second or homeo- typic division is delayed until the first mitosis in the germinating spore, according to Jahn. Similar ob- servations were reported by Kriinzlin (07) and Vouk (11) for species of Trichia and Arcyria, but these were later found to be incorrect by Jahn in 1911. For the first time in the Myxomycetes he found that haploid myxamoebae of Physarum didermoidcs fuse in ]iairs to form the zygote. Plasmogamy is fol- lowed shortly by karyogamy. The diploid zygote may engulf hajjloid amoebae, with the result that haploid and diploid nuclei may be found in the young Plasmodia. Likewise, zygotes may fuse with each other to form larger plasmodia, but fusion of the dip- loid nuclei does not occur. ]Meiosis takes place during the last division in the young sporangium and is not followed by a homeotypic division. Jahn ('33) re- ported the same type of meiosis in Badhamia iitricu- laris, and subsequently persisted in this view on the time and nature of reduction division in the Exospo- reae and Endosporeae. Pinoy (08) concluded from his culture experi- ments that Didymium nigripes is heterothallic and forms -|- and — myxamoebae which in turn give rise to -|- and — Plasmodia. Sporangia are formed only wiien both types of plasmodia are mixed. It is not certain that Pinoy used monospore cultures, and be- cause of this his results have been seriously ques- tioned by Kniep ('28) and Schiinemann ('30). Sku- pienski ('17— '28) also reported heterothallism in D. ni(/ripes and D. difforme. In 1928 he asserted that the sjjores of D. difforme are unisexual and that no sporangia will develop in monospore cultures. Ac- cording to him the plasmodium arises by the fusion of two myxamoebae of opposite sex (text-figure It). Other myxamoebae may unite with the zygote, but the gametic nuclei remain separate and divide mitot- ically in the young plasmodium. The daughter nu- clei later unite in pairs and fuse in the older plas- modium. wliile those whicli fail to find partners de- generate. Meiosis occurs during the last two divisions in the sporangium, according to Skupienski. In the same year Wilson and Cadman showed in Reticularia Lycoperdon that haploid motile gametes fuse in pairs by their posterior ends to form a zygote (text-figure 17). Other gametes may coalesce with the zygote, but their nuclei divide amitotically, de- generate, and are digested by the zygote. Karyogamy of the gametic nuclei follows shortly after the coal- escence witli the non-functional gametes, and meiosis occurs during the last two divisions in the sporogenic protoplasm. Miss Clayley ('29) refuted Skupienski's contention of heterothallism in D. difforme, showed that the s))ores are bisexual, and secured sporangia in monosjjore cultures (text-figure l.'j). She also found that plasmogamy takes place between motile gametes instead of myxamoebae, as claimed by Skupienski. Schiinemann likewise secured plasmodia in mono- spore cultures of Skupienski's own D. difforme and thus refuted the latter's contention of heterothallism. In D. .ranihopus, however, neither plasmodia nor sporangia were formed in monospore cultures. In 7^. niciripes, Schiinemann found that several haploid myxamoebae coalesce to form plasmodia but their nuclei remain separate until the plasmodia become older (text-figure 16). Karyogamy eventually oc- curs, and reduction is accomplished during the two divisions preceding spore formation. ScIiUnemann tlius concluded that a true antithetic alternation of generations occurs in I), nic/ripes. Cadman ('31), however, found that karyogamy occurs shortly after plasmogamy, and that the diploid zygote can ingest zoospores and haploid myxamoebae and coalesce with other zygotes. She nevertheless confirmed Schii- nemann on meiosis. In the same year Howard re- ported fusion in pairs of motile gametes in Physarum polycephalum and expressed tlie belief that plasmo- gamy is followed at once by karyogamy. Abe ('33, 'SI) likewise found fusion of motile gametes in Fulif/o septica, Erionema aureum, D. nic/ripes, P. crateriforme, and Stemonitis fusca. The gametes were found to be isomorphic but differ physiologi- cally, as has been noted previously. In D. nigripes, Stosch ('35, '37) reported the dis- covery of two forms, Z). eunigripes and D. aantho- pus, which are hetero- and homotliallic, respectively. In D. eunigripes, sexuality is well defined, while D. jranthopus is apogamous. Didymium squamulosum and Physarum cinereum were also reported to be apogamous, the first report of which Jahn ('36) characterized as fantastic. Jahn furtlier refuted Stosch's report of heterothallism in D. eunigripes and claimed that tlie failure of the gametes to fuse and form plasmodia and sporangia in Stosch's mono- spore cultures was due to the fact that tliey had not gone through tlie encystment and rest period which are necessary before fusion occurs. For sexual spe- cies of the Didymaceae, Stosch reported that motile gametes fuse in pairs to form zygotes, which in turn fuse with other zygotes in the formation of large ])lasmodia. Plasmogamy of gametes is apparently followed shortly by karyogamy. Onlj' one vegetative mitosis occurs before cleavage in the sporangium, and meiosis takes place in the spore, according to Stosch. Separation of homologous chromosomes may occur in the first and second divisions. Stosch thus supported Olive's and Gilbert's contention that meio- sis occurs in the spore instead of before cleavage in the sporangium. In ajjogamous species, he reported that fusion may occur between amoeboid as well as motile gametes, and tliat instead of meiotic divisions in the spore, one or ))erliai)s two vegetative divisions occur which arc followed by amitosis. It is apparent from this survey that there are marked differences in observations and interpreta- tions concerning karyogamy, meiosis, alternation of generations, and sex segregation in the Myxomycetes as well as in the Plasmodioi)horales. Nonetheless, certain fundamental similarities do exist, and if the •IIYI.OUKNY AM) HKI.ATIDN.SIl ll'S 85 diajiranis rfjircsoiitiiiji the life oyi'lcs of tin- I'l.is- niodi(i|)lioraU's in Chapter III an- compartd with those of the Myxoinycctts tliese siniilaritii's bci'onif iiioro strikinir. .Most rciciit workers in hotli iiroii])s aftree that tlie dijjhiiil phase is initiated liy the fusion of anioehoid or motile ijainetes and karyo-ianiy and extends to the time of the last two nuelear divisions preecding sporojjenesis during whieh reduetion oe- curs. while the haploid phase ineludes the sjiores, zoospores, amoebae, and g.-imetes. However, the presenee of :i zoosporanjri.-il st.-ige in the Plasmodio- Jihorales and the possibility th;it tlie zoosporangia may be gametangia eoni|)lie.ites the situation, and until more is known about this developmental ))hase it is impossible to say how elose the Plasmodiopho- rales and Mycetozoa are to each other. PlASMODIOPHORALES AN-D CllVTRIDIALES As has been noted in the historieal review, the sug- gested relationship of the Plasniodiophorales with the Chytridiales involves principally the families Woroninaceae. Synehytriaceae. and certain members of the Olpidiaceae. Reports of relationship with the Synehytriaceae are based ])riiuarily on tlie fact that the thallus in this family functions as a prosorus and segments into a number of zoosporangia as in some genera of the Plasniodiophorales. It must be noted, however, that this thallus is haploid in the Synehy- triaceae. according to Curtis, Kusano, Kohler and others, while in the Plasniodiophorales it is believed by numerous workers to be di]jloid. Outside of the formation of sporangiosori in both families, there is little or no further similarity. The presence of pos- teriorly uniflagellate zoospores and gametes in the Synehytriaceae jirecludes, in my opinion, any close affinity. Furthermore, the presence of a membrane around the mature tliallus, lack of amoebae and naked plasmodia. and the absence of schizogony, as well as the fact that the zygotic thallus does not seg- ment and form numerous resting spores or cystosori are other outstanding differences which are difficult to reconcile. In certain members of the Olpidiaceae, particu- larly species of Rozella, the thallus has been de- scribed as naked, plasmodium-like, and indistin- guishable from the host protoplasm. In the septi- genous species of this genus, the thallus is further- more reported to segment into numerous portions which develoj) into zoosporangia or resting s))ores. However, as the author ('1-2) has pointed out else- where, the ))resencc of a plasmodium with this type of development has not yet been conclusively dem- onstrated for Rozella. In Prinffsheimella, on the other hand, the evidence of segmentation of the thal- lus and the formation of sjiorangiosori is more con- clusive, according to Coucli's ('39) observations. Certain genera of the Olpidiaceae like Rnzt'Ua and Prinrishi-imi'Ua have thus been described as resem- bling species of the Plasmodiophorales in the devel- opment of s))orangiosori. On the other hand, they differ fundamentally by their posteriorly uniflagel- late aoos])ores. The contention of Winge that Sorol- pidiiim lU'tac, Rhlzomij.va hypoijaea, and Aiimomiixa l*lantali/ci/sti.i but relates to the zoospores of Rozi'lla, Olpidiopsis, and IVoronina as a group. Cook and Nicholson ('33), on the other hand, 86 PLASM ODIOPH OR ALES described the zoospores as spherical (fig. 3, I) with two anterior flagella which lasli back and forth in breast-stroke fasliion in swimming. These workers were non-committal as to the relative lengths of the flagella, but most of the figures show them to be equal in length. One of their figures (fig. 3), however, shows flagella of unequal length. If the zoospores are anteriorly biflagellate, as Cook and Nicholson con- tended, and heterocont as Fischer reported, they do not difter fundamentally from those of the Plasmo- diophorales. In view of the wide differences in ob- servations it is not altogether improbable that what is now called W. polycystis may relate to more than one organism or species. Further critical studies on this species are therefore highly essential. So far schizogony has not been reported in Jf. polyci/stis, and nothing is known about the type of nuclear divisions in the vegetative thallus. This para- site has never been studied critically from fixed and stained material, and it is not improbable that fu- ture investigations may reveal the occurrence of schi- zogony and "promitotic" divisions. It should be noted in this connection, however, that the sporangia and resting spores of W. poli/ci/slis give a definite cellulose reaction, while those of the Plasmodiopho- rales do not. Furthermore, in germination the content of the zoospore enters the host through a penetration tube, leaving the empty case on the outside of the host cell as in Olpidiopsis, Rosella, etc. In the Plas- modiophorales the zoospores are reported to enter directly. The latter difference may not be important, but the presence of cellulose is fundamentally sig- nificant, according to present-day students of phy- logeny. The other species of Woronina, W. glomerata, JV. af/c/regata, W. elegans, and W. asterina, are not well known, and it is difficult to compare them with the Plasmodiophorales. Woronina glomerata para- sitizes J'aucheria and causes septation of the fila- ments without hypertrophy. It forms both sporan- gio- and cystosori, but the resting spores and spo- rangia are not closely aggregated and compact like in 11'. polyci/stis. Motile zoospores have not been illustrated, so that nothing is known about the num- ber, position, and relative lengths of the flagella. The zoospores apparently enter the host directly, divide, according to Zopf ('9-i, p. 54), and form amoebae, which may in turn divide. The amoebae feed on the host i^rotoplasm and engulf starch grains, chloro- phyll granules, etc., whereby they may become quite green in color. This food is held in well-defined vac- uoles, according to Scherffel ('25), and shortly be- fore the parasite fructifies, the extraneous waste ma- terial is extruded as in typical proteomyxean species. The amoebae later unite by fine strands or pseudo- pods and form a reticulate plasmodium, which may completely fill the host cell. The amoebae may sepa- rate again, but at maturity tlie plasmodium cleaves into segments or "Theilplasmodien," each of which becomes a sorus of zoosporangia or resting spores. This division of amoebae and plasniodia is sugges- tive of schizogony in the Plasmodiophorales. The resting spores of Jf. glomerata, unlike those of W. polycystis and the Plasmodiophorales, function as zoosporangia in germination and produce numerous zoospores. Because of its type of nutrition, Zopf and Scherffel regarded W . glomerata as an organism with animal and fungal characteristics and included it with tlie zoosporic Myxozoidia or Proteomyxa. It may be noted, however, that W. polycystis also feeds directly upon the host protoplasm by bodily taking in globules of oil, according to Cook and Nicholson. Except for the possession of biflagellate zoospores and an intramatrical holocarpic thallus, the other known genera of the Woroninaceae, with the pos- sible exception of Rosellopsis Karling ('4'2b), do not appear to have much in common with the Plas- modiophorales. In the polysporangiate, septigenous species of Rosellopsis, the thallus has been described as naked and plasmodium-like, and undergoes seg- mentation to form numerous zoosporangia which be- come separated by cross septa in the host. Further- more, in R. simulans the zoospores are anteriorly bi- flagellate and heterocont, according to Tokunga ('33). However, so little is known about the devel- opment and cytology of these species that it is im- possible to draw further comparisons. Tliere are nevertheless striking similarities in the development of the Plasmodiophorales and certain species of the Woroninaceae, particularly W. polycystis, which suggest a close relationship and common origin. Cook ('33), on the other hand, contended that these simi- PLATE 16 Fig. 1, 3. Biflagellate zoospores. Fischer, '83. Fig. 3, 4. Anteriorly biflagellate zoospores. Cook and Nicholson, "33. Fig. 5, G. Early infection stages. Fischer, I.e. Fig. 7. Same. Cook and Nicholson, I.e. Fig. 8-10. Amoeboid changes in shape and position of young parasite in host cell. Fischer, I.e. Fig. II, 13, 13, 15. Successive stages in develojmient of the parasite and its cleavage into a sporangiosorus. Note local hypertrophy and septation of host hypha. Fischer, I.e. Fig. 14. Vacuolate thallus undergoing centrifugal cleav- age. Fischer, I.e. Fig. 16. Sporangiosorus. Cornu, '73. According to Couch ('39) this figure relates to P. dioicii. Fig. 17-30. Maturation, cleavage, and emission of zoo- spores from a sporangium. Fischer, I.e. Fig. 31. Small empty sporangiosorus. Cornu, I.e. Fig. ^2-2. Cleavage of thallus into a eystosorus. Fischer, I.e. Fig. 33. Mature eystosorus. Cornu, I.e. Fig. 34. Septate, locally hypertrophied hypha of Sapro- leffiiid with five cystosori of various sizes and shapes and two emiity sporangiosori. Fischer, I.e. Fig. 35. Elongate irregular eystosorus. Cook and Nichol- son, I.e. Fig. 36. Variously-shaped resting s))ores from a eysto- sorus. Fischer, I.e. Fig. 37, 38. Thick-walled resting spores. Cook and Nicholson, I.e. Fig. 39. Germination of eystosorus. Resting spores swell- ing and vesiculating to become zoosporangia. Fischer, I.e. Fig. 30. Germination of resting spores. Cook and Nichol- son, I.e. lMlYI.()(iKNV AN"1) IlK.I.ATIONSllll'S IM.ATK 1(5 87 Woronina polycystis 88 PLASMODIOPHORALES larities are incommensurable and that the two groups have but little in common. Most of the objections raised by Cook, however, are no longer tenable in the light of more recent discoveries in the Plasmodio- phorales. Plasmodiophorales, Proteomyxa, and Other Protozoa Inasmuch as the names Monadineae, Myxozoidia, and Proteomyxa are more or less synonymous and have been ratlier loosely used in the literature, a brief discussion of tiieir terminology is essential before proceeding to the questions of relationship with and origin of the Plasmodiophorales from this group of simple organisms. The term Monadineae was first employed by Cienkowski ('65) for a number of primitive organisms whose vegetative reproductive cell develops into amoeboid or plasmodial thalli which are capable of engulfing solid food particles. Following the feeding and growing stage the thalli develop distinct membranes, discharge the extrane- ous food material into a large vacuole, undergo cleav- age, and form zoospores or small amoebae. At the conclusion of this phase, resting spores are formed. Cienkowski divided these organisms into two groups, Monadineae zoosporeae and Monadineae tetraplas- tae, depending on whetlier zoospores or Actinophrys- like amoebae are produced. Many of these aquatic monadinaceous species were later included by Klein ('82) in a new family, Hydromyxaceae, but this name was not widely accepted at that time. More re- cently, however this family was emended by Jahn ('28), raised to ordinal rank, and included as the first order of the Myxomycetes. In 1884 Zopf gave an extended account of the Monadineae in his book on the "Pilzthiere or Schleimpilze" in which he con- tinued Cienkowski's terminology for the whole group but changed the division Monadineae tetraplastae to Monadineae azoosporeae. The following ye ir, how- ever, Lankester created a new class, ProtL.imyxa, of protozoa to include the Monadineae of Cienkowski and Zopf as well as Plasmodiophora and Tetramyxa. In 1 893 Klebs pointed out that continued use of the term Monadineae in the sense of Cienkowski would lead to confusion inasmuch as this name had pre- viously been applied to a group of flagellates of which Monas is the type genus. Zopf ('91) accord- ingly proposed an alternate name, Myxozoidia, for Cienkowski's Monadineae. Doubtless because Zopf's paper was not published in a prominent journal, his term did not become generally known. Lankester's term was accepted by most protozoologists and has accordinglv displaced the terms Monadineae and Myxozoidia in the literature on protozoa. Proto- phytologists, however, have continued to use Cien- kowski's term. According to present-day interpreta- tions the Proteomyxa embraces several families of incompletely known rhizopod-like species, which protozoologists include in the sub-class Rhizopoda of the Sarcodina. For the sake of emphasis and clar- ity, relationships with the Proteomyxa will be dis- cussed here apart from the Protozoa in general, but such treatment does not imply that this order is to be excluded from the Rhizopoda. As Zopf early pointed out, the life cycles of cer- tain monadinaceous species, particularly of the fam- ily Gymnococcaceae, are similar in many respects to those of the Plasmodiophoraceae, and for this rea- son he included both families in the same division of the Monadineae. Subsequent studies by de Bruyne ('90), Scherffel ('2.5), and others have supported Zopf's observations and emphasized these similari- ties even more fully. As a result of such studies, some of these proteomyxean species are now known to have anteriorly biflagellate, heterocont zoospores, PLATE 17 Physarella, Fulii/o, and Diclymiiim Fig. 1. Anteriorly uniflagellate zoospore of Physarella oblonc/a with two "basal bodies." Sinoto and Yuasa, '34. Fig. 3. Biflagellate heterocont zoospores of P. ohlonga with two "basal bodies." Note tail piece at end of flagella. Sinoto and Yuasa, I.e. Fig. 3. Biflagellate isocont zoospore of P. ohlonga with two "basal bodies." Sinoto and Yuasa, I.e. Fig. -1. Triflagellate heterocont zoospore of Fuligo sep- tica with two short flagella attached to one "basal body." Yuasa, '35. Fig. 5. Biflagellate heterocont zoospore of F. scptica with "two basal bodies." Yuasa, I.e. Fig. 6. Biflagellate heterocont zoospore of D. Xanthopus with several "basal bodies." Stosch, '35. PgeiidoKporopsis, Amijlophagus. Gymnococnts, and Aphelkliopsts Fig. 7, 8. Anteriorly biflagellate heterocont zoospores of Pseudosporopsis sp. (Bodo ylobosus) with numerous en- gulfed food particles. Short flagellum extending forward. Scherffel, '-'5. Fig. 9, 10. Zoospores of same with contractile vacuoles and nucleus. Scherffel, I.e. Fig. 11. Anteriorly biflagellate heterocont zoospores of Amylophayus algarum with two contractile vacuoles. Long flagellum extending forward. Scherffel, I.e. Fig. li. Amoeboid stage of same. Scherffel, I.e. Fig. 13. Anteriorly biflagellate heterocont zoospore of P. rotatoriorum witb two contractile vacuoles; long flagel- lum extending forward. Scherffel, I.e. Fig. U. Anteriorly biflagellate zoospores of .1 phelidiop- six epithemiae. Scberftel, I.e. Fig. 15. Large plasmodium ( ?), J. epithemiae, with ex- traneous food material in a large central vacuole. Scherf- fel, I.e. Fig. 16. Zoocysts of J. epithemiae. Scherffel, I.e. Fig. 17. Deliquesced zoocysts and emerging zoospores of ./. epithemiae. Scberffel, I.e. Fig. 18. Eleven zoosporangia, five of which are filled with zoospores, from a single tballus of G. Cladophorae : extruded waste material between sporangia. De Bruyne, '90. Fig. 19. Zoocyst of -/. algannn. Scherffel, I.e. Fig. -20. Emergence of zoospore through zoocyst wall in ./. algarum. Scherffel, I.e. Fig. 21. Sporocyst of P. rotatoriorum with six resting spores. Scberffel, I.e. Fig. 22. Resting spores of J phelidiop.iis epithemiae. Scherffel, I.e. I'llVUMiKNV AMI HKl-ATlO.NSIlll'S ri.ATE 17 89 Myxomycetes, Proteomyxa 90 PLASMODIOPHORALES naked plasmodium-like thalli, zoocysts, and sporo- cysts. When aggregated the latter two structures are comparable with the loose sporangio- and cystosori found in plasniodiophoraceous and woroninaceous species. Aphelidiopsis, Gymnococcus, Pseudosporop- sis and Amylophagits may be taken as examples, and for the sake of more concrete comparisons drawings by de Bruyne and Scherffel of the zoospores and some developmental stages of these genera have been brought together in Plate 17. The zoospores of Pseu- dosporopsis sp. {Bodo filobosus Stein, fig. 7-10), Amylophagiis algarum (fig. \\-\2).P.rotatoriorhim (fig. i3). and Aphelidiopsis epithe7nine (fig. li),like those of the Plasmodiophora, Octomyxa, etc., have two unequal flagella at the anterior end. In B. c/lo- hosus and A. cpithemiae the short flagellum extends forward and the longer one backward in swimming, while in the other species the relative positions are reversed. The zoospores may become amoeboid, and engulf solid food particles (fig. 7, 8), and include a well-defined contractile vacuole. In tiie latter two characteristics they appear to differ sharply from the zoospores of the Plasmodioijhoraceae, but as has been noted before the zoospores of Polymyxa gra- minis and the young plasmodia of /,. J unci are said to engulf algae and particles of food. In all these species, except A. epithemiae and Gymnococcus Cladophorae, the developing thallus becomes invested with a membrane and forms one zoocyst or zoosporangium (fig. 19). There is no cleavage into segments and development of a spo- rangiosorus, according to Scherffel. In A. epiihe- miae, on the other hand, the type of development is more like that of the Plasmodioplioraceae. The con- tent of the zoospore enters the host, leaving the empty spore case on the outside, feeds upon the host protoplasm, and develops into an oval vacuolate thal- lus (fig. 15) which appears to be naked or devoid of a well-defined membrane. At maturity this plasmo- dium-like thallus cleaves into from 2 to 8 segments (fig. 16) which round up, form tliin membranes, and become zoocysts. These vary greatly in size and in the number of zoospores they produce. Small zoo- cysts may form only 3 to 4. zoospores. No exit papillae for the emission of zoospores are developed, and at maturity the wall deliquesces and disappears (fig. 17) freeing the zoospores simultaneously. In G. Cla- dophorae, however, the wall is thicker, more perma- nent, and remains after the zoospores have emerged (fig. 18). No exit papillae are present here also, and the zoospores doubtless bore through the sporan- gium wall as in A. algarum (fig. 20). Scherffel did not observe resting spore formation, but his illus- trations (fig. 22) suggest that they may be formed in the same manner as the zoocysts. They lie free in the host cell without an enveloping membrane. In P. rotatoriorum as many as 8 resting s])ores are formed in a sporocyst (fig. 21), but in this species they are held together by a membrane. Germination of the resting spores has not been observed. It is to be particularly noted that the type of nutri- tion in these species is animal-like. The zoospores. amoebae, and developing thalli engulf chlorophyll granules, starch grains, oil globules, etc., apparently digest them in the food vacuoles, and extrude the waste material shortly before sporogenesis. No con- clusive evidence of this type of nutrition has been found in the Plasmodiophoraceae, and this appears to be one of the chief differences between these two groups of organisms at present. Comparison on the basis of sexuality, time and place of meiosis, alternation of generations, etc., cannot be made, because very little is known about these processes in the Proteomyxa. No good evidence of fusion of amoeboid or motile gametes has been ob- served in the biflagellate species. Likewise no evi- dence of schizogony, "promitosis," "akaryosis" or anj^ other reported cytological characteristics of the Plasmodiophoraceae have been observed, but so far Pseudosporopsis, Aphelidiopsis, and other similar genera have not been intensively studied from fixed and stained material. It is accordingly premature to draw conclusions on these grounds. The belief that the Plasmodiophoraceae are re- lated to Protozoa, exclusive of the Proteomyxa which have already been discussed, stems primarily from the views of the protozoologists who have included this family among the primitive animals. Proto- phythologists in general have opposed this view on the grounds that the Plasmodiophoraceae are fungi. There are, nonetheless, certain specific structural, developmental and cytological similarities among the Rhizopoda and Sporozoa on which this belief is based. The suggested relationship with the Sporozoa relate to similarities in life cycles and asexual repro- duction by schizogony, while in the Rhizopoda, ex- clusive of the Proteomyxa, it concerns the occurrence of "promitosis" and the extrusion of chromidia. The Sporozoa are spore-forming parasites of animals, some species of which may cause marked hyper- trophy of the host cell and form galls or cysts. In certain species of the Myxosporidiae the spores give rise to amoebula which penetrate the host tissue, grow in size, and undergo schizogony, cutting off uninucleate schizonts. Each schizont develops into a multinucleate amoeboid plasmodium or trojiho- zoite and divides into sporonts at maturity. The lat- ter grow in size as their nuclei divide several times, become sporoblasts, and form a variable number of spores, which are usually liberated as the host tissue degenerates, and cause secondary infection. In these respects certain sporozoan species resemble the Plas- modiophoraceae, but further than this the similarity is not very striking. However, the occurrence of schi- zogony is particularly noteworthy. This is a common and widespread method of asexual propagation in the Sporozoa, and has also been reported to occur in most genera of the Plasmodiophoraceae. That its oc- currence in both groups together with the production of numerous spores indicates jihylogenetic relation- ship is, however, highly questionable and doubtful, as Maire and Tison ('09) have already pointed out. The contention that the Plasmodioiihoraceae show affinities to the strictly amoeboid Rhizopoda or I'li'i 1 i)(;knv and Hi:i..\rioNfimi"s 91 AnuH'biiia is IkisoI primarily on tlu' nportiil simi- larity hftwccn till' vi-_s{i'tiitivc mu-K'ar divisions in the plasniodiuni and the (iroinitotic divisions in the liina.r srronp of .tmoilui. Cook (^'28), as noted elsewhere, held this similarity to he of ureat ])hyloiienetie sijr- nitieanee and aeeordinijly helie\ed that the I'lasnio- diophorales have oriicinated from the lohosoid amoe- bae. Home (^'30) severely eritiei/ed Cook's view. and after reviewing the variations of nuelear division exhibited by the fungi, algae, and i>rotista. eon- eluded that "the use of criteria relating to the type of nuelear di\ ision is of very doubtful \alue at the present time in diseussing the aetual relationshi)) be- tween grou)) and group." The reported similarity of promitosis in certain amoebae and the Plasmodiophorales has been fully presented in Chapter 1 1 and need not be discussed further at this point. Suffice it to repeat that Home, Terby and Webb have refuted the rci)orts of pro- mitosis in the Plasmodiophorales and described the formation of well-defined chromosomes during the vegetati\ e divisions. Furthermore, Miss Terby found that the mieleole does not persist and divide into two parts which are later ineorjiorated in the daughter nuclei as the new nucleoli. Instead, the nucleole may fragment and portions of it become stranded in the cytoplasm between the nuclei, while the daughter luicleoli are formed anew in the telo|)hases as in higher i)Iants. There is accordingly no universal agreement that jiromitosis, in the strict sense of Xagler. occurs in the Plasmodiophorales. Xor is pro- mitosis, in the modified sense of later workers re- stricted to the lobosoid amoebae. Intranuclear divi- sion with ill- or jjartly-defined chromosomes and large persistent elongating, constricting, and divid- ing nucleoli have been figured and described in spe- cies of the Rhizomastigina. Thecamoebina, Coccidia. Mvxosporidia, Englenoidina and .Siphonales. A simi- lar persistence and behavior of the nucleole during division has been recorded by N'emec (00). Mano ('Oi), Wager {'01). I.undegardh ('12), and Tahara ('1.5) for Alniis, Phaseolus, Solatium, Cucurbiia, and Helianthiis, resi)ectively, where the process has been referred to as ))seudoprotomitosis. On this basis, ac- cording to Cook's line of argument, the Plasmodio- phorales ;ire related in varying degrees to a large number of .inim.il and plant families. Persistence and division of the nucleole in the manner described above, therefore, does not appear to be of much sig- nificance, and as Doflein, Ti.schler ('22), Terby ('24). Belar, and others have |)ointed out. it may be found in various grou])s of organisms under certain conditions. In light of these data it seems highly doubtful that certain similarities in type of nuclear division are an index of ])hylogenetic descent and relationship. It is obvious from this discussion of ijhylogcny and relationshij) that the Plasmodiophorales have some develo))mental phases and cytological char- acteristics in conmion with the Myeetozoa. Protozoa. and jjolysporangiate s])ecies of the \\'oroninaceae. Whet!ier this order has originated directly from such groups or de\el(>peil along |).'ir.illel lines with them from a distant eonnuou ancestor, however, is still UMt'crtain. Our kiunvledge of the critical stages in the life cycle of the Plasmodio])horales as well as in the groujis with which this order shows .-iftinity is too incomplete to w.-irrant detinite conclusions at |)res- ent. I'urther intensive study of these st.iges as wi'll as the discovery of new sjiecics will (buibtless in- validate many of the iiresent-day beliefs concerning the Plasmodiophoraceae. Likewise the similarities this family has in common with other groujis, which now ])oint to definite lines of origin and relationshi|), may in the future ))ro\e to be ])hylogenetically insig- nificant. Xe\ ertheless. the Plasmodiophorales at jiresent appear to be similar to JVoronlna pol i/ci/slis and the biflagellate heteroeont species of the Proteoniyxa in zoospore structure, and general type of development. This similarity, of course, does not necessarily mean a eonnuou origin and close relationship. It may equally well be nothing more than ))arallelism in development from se)iarate ancestors. This rela- tionshi)) has. nonetheless, been emphasized rather strongly in the discussions above, primarily with the hope of encouraging intensive research along these lines. \'ery little can be said at present about relation- ships within the order itself, because the life cycles of many species are not fully known. Furthermore the genera are not sharply defined. As is indicated in Cha))ter III, the relation and arrangement of the resting spores is rather generally regarded as an index of relationships and relative complexity. On this basis Plasmodiophora has been regarded as the most primitive genus, because its resting spores are not united in cystosori. Tetrami/.ra, and Octomi/.ra, with spores in tetrads .and oetads res])ectively, are accordingly next in line. Sorosphaera and Sorodi.sciis at present seem similar to these two genera in that uninucleate spore mother cells or sporonts are de- limited in wliich the meiotic divisions later occur. Whether or not this is an index of relationship is, however, questionable. Poli/mi/xa has the most ex- tensive and complex zoosjiorangial stage of all known genera, but its cystosori resemble those of Lif/niera, a genus which Cook ('33) regarded as primitive. BIBLIOCiHAPHV: I'HVLOOENY Abe, S. 19.33. Bot. and Zool. 1 : 1579. . 1934. Sci. Hept. Tiikyo-Hunrlca-Daifraku. .sec. R. 1: 193. AlexieflF, A. 1913. ,\roli. Protistk. -'9: 344. Hessey, E. A. 193,5. A text-book of mycolofry. Philadelphia. Cadman, Elsie .1. 1931. Trans. Hoy. Soe. Edinburfrh ~i~ : 93. Calkins, c;. X. 1909. Pnitozooliifry. Pbiladelpliia. . 1933. Hicilofry cif tin- |)r(itn/,i)a. .'iid, cd. Pliiliidel- phia. Cavers, K. 191.5. New Phytol. 11: 30t. Chattiin, E. 1910. ,\reli. /ool. Exp. et Gen. ."> ser. 5: ;?39, .'(i7. Cienkowski. L. IHH.). Ark. .Mikro. Anat. 1 : -'03. Clavlrv, D. M. 19;?9. Trans. IJrit. .Mveol. .Soe. 14: 227. 92 PLASMODIOPHORALES Cook, W. R. I. 1926. Ibid. n:-2l0. . 1928. New Pliytol. 27:2i3. . 1933. Arch. Protistk. 80: 245. , and W. H. Nicholson. 1933. Ann. Bot. 47: 851. Cornu, M. 1872. Ann. Sci. Nat. Bot. ser. 5, 15: 1. Cotner, F. B. 1930. Sci. n. s. 71 : 670. Couch. J. N., J. Leitner, and A. Whiffen. 1939. Jour. Elisha Mitch. Sci. Soc. 55:399. Couch, J. N. 1939. Ihkl. 55: 409. De Bary, A. 1884. Vergleichende Morpliologie der Pilze. De Bruyne, C. 1890. Arch, de Biol. 10: 43. Delage, Y., and E. Herouard. 1896. Traite de Zoologie Con- crete 1: 76. Doflein, F. 1901. Die Protozoen als Parasiten und Krank- lieitserreger. Jena. . 1909. Lehrbuch der Protozoenkunde. Jena. 3rd ed. 1911. 4th ed. 1916. -, and E. Reiclienow. 1928. Lehrbuch der Protozoen- kunde. 5th ed. Jena Fischer, E. 1882. Jahrb. Wiss. Bot. 13: 286. Galiano, E. F. 1921. Morfolgia y biologia de los protozoas. Madrid. Gaumann, E. A. 1926. Vergleichende Morphologic der Pilze. Zurich. , and C. W. Dodge. 1928. Comparative morphology of fungi. New York. Gilbert, H. C. 1935. Amer. Jour. Bot. 32: 52. Gwynne-Vaughan, H. C. I., and B. Barnes. 1926. The .struc- ture and development of fungi. Cambridge. 2nd ed. 1937. Harper, R. A. 1900. Bot. Gaz. 30: 217. Hartog, M. 1906. Protozoa. Cambridge Nat. Hist. 1: 89. London. 2nd ed., 1909. 3rd ed., 1922. 4th ed., 1936. Hertwig, R. 1919. Lehrbuch der Zoologie. 12th ed., Jena. Howard, F. L. 1931. Am. Jour. Bot. 18: 116. Jahn, E. 1904. Ber. Deut. Bot. Gesell. 22: 84. 1907, IhuL 25: 53. 1908, Ihid. 26a: 342. 1911, Ibid. 29: 231. . 1914. Zeitschr. f. Bot. 6: 877. . 1928. Engler and Prantl, Nat. Pflanzf. 2: 304. . 1933. Ber. Deut. Bot. Gesell. 51: 377. 1936, Ibid. 54: 517. Jones, P. M. 1928. Arch. Protistk. 62: 313. Kamblv, P. E. 1939. Am. Jour. Bot. 26: 88. Karling, J. S. 1942a. Ibid. 29: 33. . 1942b. Mycologia 34: 205. Klebs, G. 1893. Zeitschr. wiss. Zool. 55: 281. Klein, J. 1882. Bot. Centralbl. 11: 254. Kniep, H. 1928. Die Sexulitiit der niederen Pflanzen. Jena. Pp. 253, 258. Kranzlin, H. 1907. Arch. Protistk. 9: 170. Kudo, R. R. 1931. Handbook of Protozoology. Baltimore. Kunkel, L. O. 1915. Jour. Agr. Res. 4: 265. Lankester, R. 1885. Encyl. Brit. 9th ed. 19: 839. . 1909. A treatise on zoology 1: 11. Lister, A. 1893. Jour. Linn. Soc. 29:529. Lotsy, J. P. 1907. Vortrage iiber Botanische Stammen- geschichte 1 : 403. Jena. Lundegardli, H. 1912. Cohn's Beitr. Biol. Pflanz. 11: 373. Maire, R., and A. Tison. 1909. Ann. Mycol. 7: 248. 1911, Ibid. 9:240. Martins Mano, T. 1904. La Cellule 22: 57. Minchen, E. A. 1912. An Introduction to the Protozoa. Lon- don. 2nd ed. 1922. Nagler, K. 1909. Arch. Protistk. 15: 1. Nawaschin, S. 1924. C. R. Acad. Sci. Russie 1924: 173. Nemec, B. 1900. Funfstiick's, Beitr. Wiss. Bot. 4: 37. Olive, E. W. 1907a. Trans. Wise. Acad. Sci. Arts, Letters 15: 753. . 1907b. Sci. n. s. 25: 266. Pascher, A. 1918. Ber. Deut. Bot. Ges. 36: 377. Pavillard, J. 1910. Progr. Rei. Bot. 3: 475. . 1912. Ann. Mycol. 10: 218. Pinoy, E. 1908. C. R. Soc. Biol. 94: 630. Prowazek, S. 1902. Osterr. Bot. Zeitschr. 52: 213. . 1905. Arb. Kais. Gesundheit. 22: 396. Rhumbler, L. 1923-1925. In Kiikenthal ard Krumbach's Handbuch der Zoologie 1: 106. . 1939. Protozoology. Baltimore. Rosen, E. 1893. Cohn's Beitr. Biol. Pflanz. 6: 237. Scherffel, A. 1925. Arch. Protistk. 52: 1. Schroeter, J. 1886. Cohn's Krypt. Fl. Schlesiens. 3: 133. . 1897. Engler and Prantl. Die Nat. Pflanzenf I, 1 : 7. Schunemann, E. 1930. Planta 9: 645. Sinoto, Y., and A. Yuasa. 1934. Bot. Mag. Japan Bot. Soc. 48: 720. Skupienski, F. X. 1917. C. R. Acad. Sci. Paris 165: 118. 1918, Ibid. 167: 31. 1926, Ibid. 182: 150. 1927, Ibid. 184: 1341. . 1928. Acta Soc. Bot. Poloniae 5: 255. Smith, E. C. 1929. Am. Jour. Bot. 16: 645. Stevens, F. L. 1913. The fungi which cause plant disease. New York. . 1925. Plant disease fungi. New York. Stosch, H. A. 1935. Planta 23: 623. . 1937. Ber. Deut. Bot. Gesell. 55: 362. Strasburger, E. 1884. Bot. Zeit. 42: 305. Tahara, M. 1915. Bot. Mag. Tokyo 29: (1). Tokunaga, Y. 1933. Trans. Sapporo Nat. Hist. Soc. 13: 20. Tubeuf, K. F., and W. G. Smith. 1897. Diseases of Plants. London. Vouk, V. 1911. Oesterr. Bot. Zeitsch. 61: 131. Wager, H. 1904. Ann. Bot. 18: 29. Wettstein, R. 1935. Handbuch der Systematische Botanik. 4th ed., p. 199. Leipzig and Vienna. Wilson, M., and Elsie J. Cadman. 1928. Trans. Roy. Soc. Edinburgh 55: 555. Winge, O. 1913. Ark. f. Bot. 12, No. 9: 26. Woronin, M. 1878. Jahrb. Wiss. Bot. 11: 568. Yuasa, A. 1935. Bot. Jlag. Japan Bot. Soc. 49: 538. Zopf, W. 1884. Encykl. der Naturwiss. 3: 129. . 1888. Abli. Naturf. Ges. Halle 17: 1. . 1894. Phys. Morph. Nied. Organismen 2: 3. CLl"!! HOOT OF inr< IKKRS 98 riiMpt.r VI Diseases Caused by Species of Plasmodiophoraceae ALTHoriiii ALL known species of tliis family arc parasitic only two members are economically ini))or- tant as pathosrens of food crojis. As noted elscNvhcrc. Plaxmodiophora linisxicac and Sponc/ospora siihtcr- ranra cause diseases of crucifcrs and potatoes re- s])ectivcly. which are eonimoidy known as eluh root and powdery seal). The other species jjarasiti/.e fungi, aljiae. cryijtojiams. ;ind wild or seldom culti- vated higher plants. CLIH HOOT OF CRrCIFERS Club root is a destructive root disease of wild and cultivated erueifers which is world-wide in distribu- tion in temperate climates and known throughout the world by a large number of common names. In Eng- land. Scotland, and ^^'ales it is known as finger and toe disease, anbury, hanbury. ambury. club root and clubbing; in Russia as hernia or Kapoustnaja kila ; Kro|)fkrankheit des Koliles. Kohlhernie. Klumpen- fuss. Knotensucht. Fingerkrankheit. Kelch, Galle. Knolle. Huas, Kuss, etc.. in Germany. Switzerland and Austria ; Gros pied, maladic du Chou, and hernie du Chou in France; Tubereulosi dei cavoli and Mai de gozzo dei cavoli in Italy; Knoelvoet in Holland; Kwab. Kwabbe. Kwabbeziekte. Knol. Knolziekte, Kiinker. Knoo]). Knuist, Knobbcl and Kwabbel in Belgium; Kaalbrok in Denmark: Klum))rots juka in Sweden ; Dik Voet in South Africa ; club foot and club root in U. S. A. ; and by various other names in other countries. According to ^'anderyst ('04, p. 518) the name Vingerziekte used by Woronin and numer- ous subsequent writers for the disease in Belgium is unknown in that country. From the economic standpoint club root is the most important disease of cultivated erueifers. In badly infested fields entire cro])s may be destroyed unless stringent control measures are employed. In Ger- many. England. Russia, the U. S. A., and other coun- tries in Europe, Asia, and Africa .50 to 100 per cent destruction of turnips, swedes, cabbages, etc., has been rel)orted ( Brunchorst. '87; Rostrup, '93. '91; Halsted. '93-'99; Eyeleshymer. '94; I.aubert, '0.5; Reniy and Liistner. '11 ; (ieorgeson, '16; Gleisberg. '20; Korff and Boning. '27. and others). \\'oronin ('78) rejjorted that in 18()9 the loss in the vicinity of St. Petersburg alone amounted to more than $20,000,000. while Heri)ers ('25) estimated that the annual loss in Ciermany runs into millions of marks. In New York .State alone a loss of sever;d thousand tons of cabbage were reported by Haskcl and Mar- tin in 1918. Edson. Miller and Wood ("3.5, '3(>. 37) have subsequently rei)orti(l losses of 5 to 100 per cent in cruciferous ero])s throughout the U. S. A. The most significant fact about club root is that it spreads ra))idly, and once it lias become established in the soil, it uiakts the fields almost useless for crueifer cultivation for a number of years. Tlie origin of club root is unknown, but its symp- toms had been well described more than a century before \\'oronin showed it to be due to a ))lasniodio- ])lioraeeous organism. According to Biiliner ('22), the disease is as ancient as its hosts. The occurrence of spongy, fungus-like roots (radices fungosae) of erueifers noted by Albert the (Jreat as early as tlie i;ith century is supposed to relate to club root, and his control practice of avoiding fresh stable manure and the disposal of chaff appears to have been ac- quired from the Roman Pallatius, according to Bohiur. The disease was well known in Spain in the l.)th century where cabbages were described as being sy))hilitic (see \\'oroiiin. '78, p. .552), and the swell- ings were thought to be due to the organism causing syjihilis (Ruiz Diaz de Isla). The first report of its occurrence in England was made by Ellis in 1736, who believed the disease was contagious and due probably to an excess of barnyard manure. Adam discussed its wides])read occurrence in England in 1789. and it was subsequently reported in Scotland from 1829 to 1831 by Farquharson. Abbay. and Birne who thought it to be due to unsatisfactorv soil conditions or unbalanced fertilizer practices. Abbay saw the disease as early as 1801, and Anderson stated tliat it first became troublesome in Scotland about 1813. Renard rejiorted that it was first ob- served on cauliflower in 1820 in France. By 1853 it was fairly abundant around Hamburg. Wurzburg, in the Rhine valley and other |)arts of Germany (N. N. '53), and from 1855 on it appeared in vari- ous parts of Norway (Jorstad, '30). Other workers, including Curtis ('IS), Kiihn ('58), Henderson ('67). Sorauer ('7^), Slingerhand ('94'), and others (see Woronin '78, pp. 552-55 1) believed it to be due wholly or in jiart to various insects and other ani- mals. Buckman ('5i), however, claimed that club root was due to reversion to the original wild forms. By 1872 the disease had become so widespread and destructive around St. Petersburg that the Royal Russian Gardening Soeietv in St. Petersburg offered a iirize for the solution of the cause and control of hernia. \A'oronin began to study the disease inde- ))endeiitly of this ofl'er in 1873. and two years later he announced th;it it is caused by a |)lasniodiopIio- raceous organism to which he subsequently ('78) gave the name I'Jasmudiopliora Brassicae. Symptoms Club root disease is iisu.'illy characterized by marked enlargement of the infected roots (PI. 2. fig. 1 ). and in exceptional eases tlie galls on cabbage may reach the size of a man's fist and appear greasy-gray and pale-yellow in color. In most cases the clubs are 91 PLASJIODIOPHORALES regularly spindle-shaped, but when several infec- tions occur togethtr the swellings may fuse and pro- duce irregular growths or compound spindles (fig. 3). According to Kiister ('11) and M. T. Cook ('23) these galls are kataplasniic, since the affected tissues usually remain parenchymatous and do not undergo differentiation. Other root symptoms have also been reported. According to Appel and ^^'erth ('10), no hypertrophy occurs in radishes, and the disease is here characterized only by darkened and decayed areas. Honig ('31) found similar symptoms on Lu- naria biennis. Ravn ('22) and Pape ('25) likewise reported tlie occurrence of deep wounds or lesions on turnip roots which were filled with spores. Accord- ing to Pape, such symptoms appear when the galls or nodular excrescences on the roots decay. In a study of 101 species from 28 genera, Cun- ningham ('li) found definite types of hypertrophy and symptoms more or less characteristic for certain crucifers and classified them accordingly : 1. Complete clubbing of main and lateral roots. Brassica oleraceae. 2. Clubs on main root, laterals free. Sisi/mbriiim altissitnum. 3. Clubs on lateral roots, main root free. Sisi/m- brium officinale and Erysimum cheiranthoides. 4. Clubs on main and lateral roots with club-free rootlets above the diseased portion. Lepidium sativum. 5. Clubs as tumors of the roots. Raphanus sati- I'US. 6. Dark, decomposing spots on the roots. Rapha- nus satii'us. In the last category true hypertrophy does not occur. The disease is here characterized by cracks, fissures, and darkened areas in the host tissue whicli turn black, decay, and serve as sites of secondary infections by other fungi. As has been noted above, Appel and Werth claimed that these are the charac- teristic symptoms of the disease on radishes, but Cunningliam found them only on the Everlasting radish, in addition to spindle-shaped swellings of the rootlets. Club root disease may also stimulate branching of the roots and shoot and lead to the production of buds where they do not normally occur, as has been de- scribed by Caspary, Woronin, Favorski, and Kunkel. The secondary roots may attain a length of several inches or become stunted as short knobs. On tlie other hand, the production of secondary rootlets may be greatly inhibited, according to Laubert ('0.5) and Schlumberger ('11). The diseased buds on infected roots and shoots are often unable to respond normally to fi;ravitv, and they may grow downward and hori- zontally as well as upward. In the latter instances the infected buds may push uji above the surface of the ground and give rise to tiiick, distorted, fleshy, and abnormally succulent leaves and petioles, so that the disease may occasionally manifest itself above ground in the shoot, petioles, and leaves. In addition to tliese above-ground symptoms, club root causes yellowing of the leaves, wilting on hot days, and in tlie case of cabbage, atrophy, or complete lack of head development. Seedlings which are infected earlv usually die within a few weeks. The wilting of large diseased plants is partly due to hypoplasia of the xylem region and to splitting up of the woody cylinder by infection and expansion of the medullary rays. All galls or swellings on roots of crucifers, how- ever, are not due to P. Brassicae. Nematodes, insects, and other factors may cause malformations which are superficially very similar to club root, and unless microscopic examination of the tissues is made, these galls may be easily mistaken for those of the finger- and-toe disease. Anatomically, the causal organism of club root affects the cortical parenchyma most conspicuously, but it also produces marked changes in the cambium, xylem, and medullary rays. When roots of consider- able size are infected the amoebae and small Plas- modia migrate through the cortical parenchyma into the cambium. Here they follow the path of least re- sistance, according to Kunkel and Larsen, and spread up, down, and around the central cylinder through the delicate thin-walled cambium cells and form tlnis a cylinder of infected tissue. From the cambium they may travel laterally into the cortex, medullary rays, and xylem. Their migration up and down in the cambium ceases after a while, and the distance of the infection in these directions deter- mines the ultimate length of the spindle-shaped club. Each club, in Kunkel's opinion, is a morphological unit which has resulted primarily from the abnormal growth of the cambium. In comparatively old in- fected roots the medullary ray cells divide a number of times and enlarge and thus form large bands of pathological tissue which split and force the xylem tissues apart, until the latter becomes distorted and shifted out of their natural position. Separated from each other in this manner, the vascular bundles grow out fan-wise instead of remaining wedge-shaped and are no longer able to function normally. Plasmodia and amoebae have frequently been found in the tra- cheids, but they do not seem to have any appreciable effect on the normal functions of such differentiated cells. In young roots medullary ray infection is less common, and most of the abnormal growth occurs in the region of the cambium and the cortex. The xylem, nonetheless, may fail to differentiate properly and is often supplanted by a mass of partially differen- tiated cells. As is shown by figure 4, one of the most strik- ing appearances in sections of diseased roots and shoots is the presence of more or less isolated groups of hypertrophied infected cells which Nawaschin named "Krankheitsherde." He believed that these groups arise by repeated anti- and periclinal division of one or more originally infected cells, whereby the ])lasmodia are passively distributed in a radial di- rection around the region of infection. Cliupp also reported that a single amoeba might give rise to as many as six such groups by multi]ilication and migra- I I.rH ll<)t)T OF ( lUl IKKltS 95 tioii from cell to itll. His ;u'COUllt was sllbscqucntlv c'oiiririiicd liy K\inktl who holicvfil tliat a siiiitK' in- foi'tioii may l»'a(l to tlif formation of tlioiisands of sejtaratf and ilistiiu't " Kraiikluit>lurdc." Kiinkil as- sumed that as a plasmodium migrates Irom cell to cell it may divide, whereby portions are left inhind and lieconK- established here and there in the tissue and irive rise to siroiijjs of infected cells. Cclluliir Intenelfitions Hetweeii Host and I'litliogeri Plasmotiuiphora lirassicaf has a iironouneed etfect on infected and healthy cells. Infection may be tem- Jjorary or permanent, and if the iilasniodium mi- grates out of a cell before stimulatinn- mueli change, the latter m.-iv recover and continue to function nor- mally. Permanently infected cells, however, may ex- |)and to more than 10 to 20 times their normal size. In the early stages of infection the presence of the jiarasite does not inhibit nuclear (PI. 2, fig. 5) and cell division (fig. 6), so that some cells may function normally in this respect for a short time. Other cells m;iy begin to enlarge directly after infection with- out dividing. Occasionally, cell division may be affected to the extent that the cell wall is only partly develo))ed across the mother cell (fig. 6). Even- tually the jjower to divide is lost completely, and the infected cell gradually expands to its large size. Prowazek found that karyokinesis may continue after cell division has ceased, resulting in binucle- atc cells. I.utnian also found abnormal types of mito- sis which al)l)eared to be a modified form of amitosis. The first visible effect of the parasite on the host nucleus is an enlargement of the nucleus as a whole followed by an increase in the number of nucleoli, according to Lutman (fig. 8-11). By the time the parasite is mature, the host nucleus has lost its regu- lar outline, and the nucleoli lie (fig. 1 1 ) in clear spaces surrounded by a distinct membrane, an ap- pearance which led Prowazek to assume that smaller nuclei may be formed in a mother nucleus. In the final stages of degeneration the chromatic material collects into irregular strands (fig. 12) and assumes a peripheral jjosition in the distorted and hypertro- phied nuclei. The relation between the protoplasts of host and |)athogin appears to be very intimate, and little or no visible antagonism is exhibited. The amoebae and voung Plasmodia of the parasite lie embedded in the host proto])lasm (fig. .■), (i. 2(5. 28). and in the living condition the two are indistinguishable, according to Woronin, Xawaschin, Lutman, and others. This close association together with the fact that the in- fected host cells may continue to divide and function normally for some time led Xawaschin. f J.iylord. .and Vanderyst to believe that there is a symbiotic rcl/i- tionshi)) between the host and ])atliogcii during the hitter's early developmental stages. The host cyto- plasm has been described as becoming more vacuo- late as the jilasmodium enlargj's, but part of the early change is probably due to the great increase in xolumc of the host cell whcriby the cytopl;isui is tliinni-d out. Later, iiowcx cr. .-is the plasmodia mature .111(1 .ipproacli sporogeiicsis the proto))lasm is .ilmost completely gone. Infected cells develo]) .m unusually large amount of tr.insitory starch. ;iccording to Wal- ker. H;ilsted, and Naw.ischin. whi<-li m.iy be grouped .1 round the nucleus as l.utni.in h.is shown. These grains may later be found in the plasmodium (fig. 7-i) and are apparently wholly or jiartly digested be- fore sporogenesis. Reed ('11) noted an appreciable iiu'rease in calcium, m.-ignesium, ])otassium, phos- phoric acid, sulphuric acid, etc., in diseased cabbage roots. The increase w.is greatest in the case of ))otas- sium. which he attributed to an accumulation of jiro- toi)lasm and starch in diseased tissues. Nicolotf and Stefanova ('22), however, found that roots of dis- eased cabbage plants were high in protein and lower in phosphorus and potassium than those of healthy plants. Noninfected cells are also stimulated to di\ ide by the presence of the parasite and may often enlarge considerably. This is jiarticularly true of medullary rav cells, which may ex])and until they have lost all characteristics as such. The nuclei of these cells en- large also and keep pace to some extent with the in- crease of cell size. According to Kunkel, the stimulus travels in advance of the infection, so that increased cell division may be noted before the parasite reaches a particular, undifferentiated tissue, which suggests that a growth-stimulating substance is released by the causal organism and travels ahead of the plasmo- dium. Nawaschin, on the other hand, believed that the division of noninfected cells around the "Krank- heitsherde" is due to the stimulus of mechanical out- ward pressure exerted by the enlarging parasitized cells. Kunkel suggested that the limitation of the para- site in groups of cells might be due to a ijrotective substance or antitoxin ))rodueed by the infected cell which diffuses out into the adjoining healthy cells and renders them imnmne to attack. Levine and I.e- vine ('22) believed that the surrounding cells are not only immune but present a reactive protective barrier against the spread of the parasite. The ques- tion of whether or not infected jjlants can recover from club root and become immune has often been debated. Woronin ('78), Eycleshymer ('91), Lau- bert ('0.5a) and Miiller-Thurgau and Osterwalder ('23) maintained that recovery is impossible, but Massee ('96), Mathieu-Sanson ('97). Apjiel and Schlumberger ('13). .Schlumberger ('1 !■), and Wahl- ing ('22b) rejiorted varying degrees of recovery when infected ))lauts were treated with a 2 jier cent (jotash solution, milk of lime, planted in ore mud. and sterile soil, and watered with sulfur and solibar solutions. Miiller and Osterwalder transplanted in- fected plants to heavily limed soil, but found no in- hibitory effects or recovery. Honig ('31) believed that if infected i)l:mts are transjjlanted to sterile and d'sinfcctcd soil the progress of the disease may be halted, but such ))lants can recover only if they are sufficiently healthy to begin to grow anew. 9ti PLASMODIOPHORALES Entrance and Spread of P. Brassicae in the Host Actual penetration of P. Brassicae into the host was not observed by the early workers, but most of them assumed that it occurs only when the plants are young and susceptible. Honig and Rochlin, however, subsequently demonstrated its entrance through the walls of root hairs and epidermal cells, although Woronin, Chupp, Cook, Schwartz, and others had previously held that the amoebae gain entrance through the root hairs (fig. 28, 29) and migrate into the deeper lying tissues. W. G. Smith ('SI), on the other hand, maintained that the parasite enters as a Plasmodium. Favorski reported that infection may take place through ordinary epidermal cells and stated that Woronin's figures of amoebae in root hairs relate to Olpidiiim Brassicae. Kunkel found that old plants are as susceptible as young ones and that infection of old roots is very common. He fur- ther refuted the claim that root hairs are of any im- portance as avenues of infection and concurred witli Favorski's belief that Woronin had figured thalli of 0. Brassicae and O. borzii in the root hairs instead of P. Brassicae. Cook and Schwartz, Honig. Roch- lin, and otiiers, however, have subsequently dem- onstrated quite definitely that P. Brassicae occurs in root hairs and thus confirmed the observations of M'oronin and Chupp. Kunkel, nonetheless, showed that old plants are susceptible and may become in- fected as long as they live. Infection through me- chanical wounds and ruptures caused by adventitious roots and by the removal of lower leaf petioles at the time of transplanting is fairly common, according to Larson ('S-l). The enlargements, however, which are formed at the region of injury on the stem are defi- nite spheroid galls in contrast to the spindle-shaped clubs on the roots. As to the spread of the parasite in the host tissues and the channels involved, it is now generally agreed that it occurs in two ways : by migration of amoebae and young plasmodia from cell to cell, and by passive distribution of the parasite through repeated divi- sions of infected cells. Woronin contended that amoe- bae and Plasmodia migrate only througli pits and sieve plates, while Atkinson believed that amoebae are able to spin out into sucli fine tlireads that they can enter the roots along with nutrients in solution. Eycleshymer found plasmodia in xylem vessels and thought therefore that tliey may travel in the fibro- vascular bundles. Nawaschin believed that migra- tion of amoebae from cell to cell is impossible after secondary thickening begins in the roots, and hence distribution by division of infected cells is the princi- pal method of dissemination in old roots. Subse- quently, Lutman figured and described tlie passage of small plasmodia from cell to cell, and since that time Cluipp, Kunkel, Honig, Rochlin, and others (fig. 31-33) have demonstrated its occurrence. Cook and Schwartz, more than a decade later, however, still expressed doubt as to its occurrence. Fedorint- schik ('3.5) believed that in the early stages of the disease, migration of amoebae is the principal method of distribution in tlie host tissues, but after the plas- modia have formed and begun to mature, further spread is by division of infected cells. While it is now generally believed that division of the host cell greatly increases the number of infected cells, it nevertheless appears to play a minor role in distrib- uting the parasite throughout the roots and shoots.' Dissemination of P. Brassicae in Nature The club root organism is readily disseminated in nature in various ways and by numerous agents. It was formerlj' believed (Atkinson, '89; Carruthers, '93; Miiller and Osterwalder, '19) that the motility of the zoospores in moist soil spread the disease, but Chupp ('17) lias presented evidence to show that zoospores and amoebae rarely travel more than five inches. It has also been claimed (Carruthers. Ravn, '08, and otiiers) that wind is an important agent of dissemination, but this factor apparently operates only in the case of light, dry, loose soils and where strong winds prevail. It has been demonstrated in heavier and more compact soils that unless the patho- gen is transferred by some other agent, wind does not usually spread it from one field to another. Rains and water are doubtless more important, particularly on rolling land where the water following a heavy rain runs off quickly and carries the spores to lower-lying fields. According to Naumov ('2.5), however, disper- sal in a radial direction by such means is not very extensive. Miiller-Thurgau and Osterwalder ('23) reported tliat in the course of a year club root does not spread laterally more than 1 Vo to 2 meters in the ground. Earthworms have also been found to be ac- tive in the dissemination of club root in small gardens (Gleisberg, '22; Bremer, '2-t; Fedorintschik, '3.5). The spores may be carried in the mucilage on the skin or in the intestinal tract, and virulent forms of P. Brassicae have been found in the excreta of worms. Ground moles, root nematodes and insects feeding on diseased roots doubtless spread the disease to some extent (Favorsky, '10; Esmarch, '2-1; Beyer. '25; Chupp, '25; Erickson, '26), but how important they are as active disseminators is not known. 1 In a paper presented before the meeting of the Ameri- can Phytopatholofjical Society at Dallas, Texas, December 19+1, Walker reported system infection of cabbage and dis- tortion of buds, stem, and leaves as follows: "Under green- house conditions when cabbage seedlings are grown in soil infested with P. /Jivi.v.fiVfic the pathogen, after infecting the root, may migrate through the cambium into the stem. There is relatively little camhial proliferation in the inter- nodal regions abo\'e the third or fourth leaf. Dormant buds at the leaf sears, however, are stimulated to grow and be- come invaded by the pathogen. They become malformed due to hyperplasia. The organism may reach the growing point in young |)lants and cause extreme distortion of stem and leaves. Wlien ]ilants are incK'ulated at above ground leaf nodes, the pathogen may migrate down the stem, leav- ing no evidence of proliferation in its path until the hypo- cotyl is reached, where a typical club is formed. There is evidence that the reaction of the host is influenced by the nutrient sujiplied to it." (Phytopath. 'iJ: 18) I ( l.llt HOOT OK CRl't'IFERS 97 I)is|HTsal l>y tlif (hmp; of livostoi'k fid with dis- «';isfd roots is wry i-oimiion. 'VUv s])orts rciiiaiii alive diiriiiii passaiTf tliroiia:li tlu- dijivstive tract, and if animals wliicli liavc lucn fed on distascd i-rucifers are let out to i)asturf, the spores are disseminated in the dropi)ings. Gihbs (,'31) found that the s])ores may remain viable in fresh cattle droppinjjs for at least fifteen weeks. They also remain alive for long periods of time in dung piles around stables, and it has long been known that the a])plieation of sueh maiuire to virgin soil introduces the parasite. 'I rans- port of infected soil on farm implements, laborer's, lior.se's and livestock feet. etc.. is also ettcctive in spreading the disease. Numerous wild cruciferous plants are suscejitiblc to club root, as has been shown by Halsted ('96-'99), Ravn ('08), Cunningham ('12), Ssaeharoff ('16), Nauuiova ('26). Gibbs ('32). Rochlin ('33). Jaraa- lianen ('3() ) and others, and these hosts often harbor and peri)etuate the disease in the absence of culti- vated crueifers. Sueh wild infected hosts have been found in grass pastures, wayside ditches, river beds, gardens, and cultivated fields, and their presence on infected soil reduces the effectiveness of crop rota- tion in club root control. Environmental Factors The degree of infection, development and severity of club root depends to a large extent on environ- mental factors, but the manner and extent to which each factor operates are not clearly understood. The disease is connnonly believed to be favored by wet, poorly-drained, acid soils and temperatures slightly higher tlian those optimum for host root development, but reports to the contrary have often been made. Motte ('33). for instance, reported that club root is most prevalent in light soils and during the dry sea- son in Denmark. As to spore germination, many workers have found it occurs mostly abundantly in acid media. Bremer ('23. '2i. '26). however, reported that H-ion con- centration is not the sole determining factor. He found that strong alkalinity inhibits germination of the sjjores without killing them and that germina- tion occurs over a j)H range of .5.1- to 7.-5 but not at pH 8.0. Honig ('31), on the other hand, reported that sijores germinate as well in alkaline as in acid solutions: all of which indicates that other little- known soil factors operating in combination are equallv as ini]>ortant as H-ion concentration. Most workers liave. nonetheless, found a fairly close correlation between incidence of infection and pH range (^Nlassee, '96; Christensen, Harder and Ravn, 11 : Ravn, '12-13; Hiltner and Korff. '16; Neger. 17; Atkins, '22; Bremer, '2J-'28; Lindfors, '2i. '2.5 : Naumov, '2.5 ; Ludwigs, '2.5 ; Riehni, '25 ; Tessenow, '26; Cileisberg, '26; Chupp, '28; Briinnle, '28; Martin. '28 : Blunck. '29; SchafTnit and Meyer. *30; Beaumont and Staniland, '33; Wilson. '3Ketc.). I.indfors ('24) observed a marked decline in per- centage of infection with an increase in soil alkalin- ity. In .1 pH range of 7.1 to 7. .5, 8.5 ))er cent of the plants were diseased while .'it ])H 7.8 to 8.0, all plants remained healthy. Naumov ('2.5) found that infection occurs most rc;i). Laubcrt ('35a) Hiirkhardt ("15). Tricschniann ("17). Hos ('18). Hronu-r ('23-28) Kindshoven (•2n. Hall (10). and .\tkins ('22). Nauniov ('27) found that soils with a lime (in terms of oxide) con- tent of 0. 1 per eent or more are sienerally free of the disease. Init Honig ('31 ) reported tiiat in the vicinity of Munich soils with a 58 per eent linu- content were heavily infested with clul) root. Herjiers ('2!») and Honiir ohstrved tli.it the di.sease is very aliundant in soils which heat up readily. The jihysical character of the soil also influences the infective ability of the fungus spores according to Fedotova ("28). In ordinary grey garden soil with 40.000 s|)ores |>er ec. of soil. 66 l)er cent of the ])lants became infected, wliile in lilack. greenhouse dirt witli 100.000.000 s])ores per ce. only 12.5 per cent of the plants were clubbed. N.uimov ('28) also found that in clayey soils 20.000.000 spores per ce. of soil were necessary for optimum infection, while in humus-rich soils 100.000.000 were essential. He. furthermore, reported that in the vicinity of Leningrad the spores do not remain viable in the soil in the absence of hosts for more than three years unless fairly high tempera- tures and humidity are maintained. The observations of most of the workers men- tioned above were not correlated with exact data on the water-holding capacity and acidity of the respec- tive tvpes of soil, and it is quite probable tliat the in- creased infection and severity of club root reported on clayey, heavy soils and those rich in humus are due not so much to the pliysical nature of the soils as to their high acidity and water-holding capacitv-. Hosts and Dcfjrcc of Infection Club root was first observed on cultivated cru- cifers. but later it became evident that wild species of the mustard family also are susceptible to this dis- ease. Woronin reported hyi)ertrophied roots of Ibi'ri.s- in 1878. and some years later Magmis ('93) and Henning ('96) found other genera and species attacked by /'. Brass'icao. Halsted ('92-'99) ap]jears to be the first to have undertaken a more extensive study of the host range, and since that time this phase of the disease has been intensively investigated in various ])arts of the world. Club root is confined to species of the mustard family, and although reports of its occurrence on plants outside of this family are to be found in the literature, they have subsequently been proven false. The number of hosts is large, and in the following table are listed the cruciferous spe- cies which have been examined for the presence of club root. Included also is the degree of infection found bv investigators who have studied the host range of /'. liraxsictie. Previous authors have usu- ally arranged the genera and species according to sub-families, but for the sake of convenience they are listed in alphabetical order below. Index to .\i-thohs Citko .vxd Deouee of IxKEiTio.v or Hosts .\pi)cl = A)i|><-I iiiul Werth ('1(1) fun. = C'uniiiii;;li!ini {'II) Clint. = Clinton ClUi) Da. = Davis {'-■'>) Erick. = Erirksson ('9(i. ",'()) Gi. = Giblis (•;}-') Gl. = GleisluTf; (V3) F. S.= F. .S. (•-'()) Hal. = Halsted (•<).' 'OO) Ham. = Haininarlund ("l.")) Henn. ^ llinninp ('9()) Hon.= H()nif:C3I) Host. = Hiisterniann ('-1) ,Tam. = Jamalainen {"Mi) Katt. = Katterfeld ('23) Mag. =: .Mapiuis ("93) Mass. ^Massee ("96) Mil. =^ Miiller-Tliurgau and (Jsterwalder ('23) N. N.^ Anonymous ('53) Naum. ^ Naumann ("13) N. = N'aumov (■14-' -'8) Rain.^ Rainio ("303) Ravn. = Ravn ('08) Roc. = Roclilin ('33) Ros.= Rostnip ("93) Schl. = Schleycr ("07) Sit. = Sitensky ("98) Ssach. = SsaciiarofF ('16) Svec. = Svec. ('-'3) Weiss. = Weiss ('18) Wor. ^Woronin ('78) = no infection -f = weak infection -|--|- = medium infection -|--|--|- = severe infection Aethionema arahicum A. hujcbaum'ii A. cappad'icum A. rotuudifolitim AUiaria nffic'inaVis Al i/xsinii al pest re var. odor at u m .1. alpestre A . ahjusoides A. argenteum A. hornmiilleri A. beiithami compactum A. eali/chium A. cam pest re A. condensatum ./. corymbosum A. desertorum A. edeiif Ilium .1. lischerianiim A. r/emonense A. idaeinn A. marilimum := Lobii- laria maritime X. ('21) Katt. N. ('25) N. ('15) 15% Mil. 0; Ravn; Jam. N. ('13) 107f ; Roc. 17% Hal.; Ravn. Gl. 20%;N. ('12)+;N. ('13)4-. Cun. 51.1%; Jam. GI. 0; Jam. Jam. 10.5%-11.6% GI. 71.13% ;N. ('12)+; N. (-13) + Jam. 3.8% Cun. 33.1% Jam. 3l7ri N. ('25)0; Roe. 57% N. ('15) 507o Jam. N. ('15) 5% .lam. 1 l-.5% HaI.4-;Ravn. Cun. 11.2- 50%;X. ('15) 5%; Jam. 100 PLASMODIOPHORALES A. minimum A. moellendorfianum A. monianiim A. podoUcum A. rostratum A. sa.ratile A. serpyllifolitim A. sinuatum A. Strihrnyl A. umbeUatum A. Wierzhichii Arahis alhida A. alhida var. grandiflora A. alhida var. nana A. alhida var. umbrosa A. Allionii A. alpestris A. alpina A. areiiosa A. hellidifolia A. brachycarpa A. Canadensis A. coeridea A. glabra A. halleri A. hirsuta A. holboelli A. laevigata A. muralis var. collina rosea A. pendula A. petraea A. procurrens A. ptimila A.Stelleri A. suecica A. Turriia Aubretia Bougainvillei A. deltoidea A. eyrei A. graeca A. hendersoni A. LeichtUni A. olympica A. pinardi A. purpurea Barbarea arcuata B. hracteosa B. intermedia B. I y rata B. plantaginea B. praecox B. rupicola B. stricta B. verna Gl. i.76%;N. ('15)0 Cun. 100% G1.0;N. ('12)+;Cun. 22.2%; Jam. 18.8% Jam. 3.9% Cun. 86.7% Hal.;N. (■l4)3%;Cun. 32%, Cun. 8.3% N. ('15)0; Gl. 100% N. ('15) Gl.O Cun. N. ('1-t) 0; Jam. N. ('U) 0; Jam. X. ('14) N. ('13) 60% Jam. N. ('13) 50% Cun. 52.4% ; N. ('12)+; Roc. 27% -18% N. ('12) + Jam. 1.9% Hal. + Hal. Jam. Hal. Cun. N. ('12) + ;N. ('13) 80%; Jam. 1.5% Cun. 50% Hal. + ; Ravn. Jam. N. ('14)0 N.('12)^- N. ('15) 0; Jam.O N. ('14) 0;N. ('25) 80% N. ('25) 44%) N. ('15)+;72.2% Jam. 7.4% Cun. Roc. Cun. Cun. Cun. Cun. 37.5% Roc. N. ('15)0;F. S. ('20)-j- Cun. 67.7% Gl.O Roc. Gl.O Roc. Roc. 65 7o G1.0;N. ('12)+; X. ('15)0 Roe. 99% Cun. 4.3-7% ;G1.0:Gi.O Gi. B. vulgaris B. vulgaris fol. variegatis Berteroa incana B. mutabilis B. obliqua Biscutella auriculata B. cichorifolia B. did y ma B. laevigata B. leiocarpa Brassica arvensis B. balearica B. cernua B. chinensis B. cretica B. elongata B. incana B. insularis B. junci B. macrocarpa B. napus B. napus var. oleifera B. napus var. esculenta B. nigra B. oleracea B. oleracea var. acephala B. oleracea hotrytis B. oleracea var. capitata B. oleracea gemmifera B. rapa L. (== B. cam- pestris?) B. pekinensis B. pe-isai B. rapifera B. robertiana B. sabularia B. Tinei B. tournefortii B. sp. 1 JS. sp. 2 Braya alpina Cun. 0-70.1% ; Ham., Gl. 0;N. ('14) 0; X. ('25) 1.6%; Gi. 0; Rain. 66.7%; Jam. 3.5%, Roc. ; Jam. X. ('14)0;N. ('24)0;N. ("25) 7.97c; Jam. N. ('15)0 N. ('15)0 X. ('24)0;X. ('25) 0; Katt. 257c; Roc. 40 7o X. ('15)0;X. ('24) N. ("25) 0;Katt. 137c X. ('14) 0; Jam. 11% X. ('15) Cun. 99.8 7o;Gi. 100 7c; Roc. 1007c X. ('15) 307c Gl. 1007cN. ('25)337o; Katt. 100% X. ('25) 10070 Katt. 10070 X. ('15) 407o Jam. 207c X. ('15) 8O70 X. ('15) 40% Cun. 9970 ; Gl. 1007o ; X. (25) 967o X. ('15) 807o Wor. ; Ravn. ; Cun. 83.7 7o —49.270; Gl.O Cun. 83.77o;Roc. 11 7c; Jam. 84.87: Ssach. ; Roc. Hal. +; Ravn. +; Cun. 28.27o; Gl. 207c; X. ('15) 0; Hon. 4.5- 62.l7o; Roc. 0; Jam. 3.470 Ravn. ; Cun. 94.2 — 81.67c ;G1.16.67o;Gi. 0-100%; Roc. 10070 Cun. 92.87o Cun. 88.8% Cun. 93.170 Cun. 88.2% Wor. -f ; Ravn. ; Cun. 100 — 1.3%; Gl. 0; Ham.; X. ('25) 16.67c; Gi. 75-10070; Roc. 35- 507o; Jam. 62.5% Da.; Ikeno ('29) Cun. 10070 Hal. X. ('15) 50% X. ('15) 1007o X. ('15) 607o X. ('15)0 X. ('14) 307o N. ("14) 2070 Jam. 53.370 CLril HOOT OF C nrclFEHS 101 Riiiiias Dritiilalis Camcliiia deniata C.foetida C. linifolia C. macrocarpa C. sativa C. sp. Capsella bursa pastoris C. heegrri Cardamine heterophylla C. pratensis Carpoceras sibirictts Carrichtera veUa Cbfiranthii.i AUioni ('. alpiniis C. annus C. Cheiri C. incanus C. marifimus C. scoparlus C. semper florens C. Senaneri Clypeola jonthlaspi Cochlear! A morac'ta C . ant/lica C. dan tea C. groenlandica C. officinalis Conrinffia orientalis Coronopus didyma C. prncumhens Crambe abyssin'ica C. cordifolio C. hispanica C. maritima C. lartarica N. ('12)+; N. (-13) 2 outof 2;N. ('11) 0; X. ("25) 0; Jam. 28.6% Cun. 1007c ;G1. 100 fc Katt. 1007o N. {ir,)90'/o Cun. 9 Ko 7c Hal. -\- ; Ravn. ; Cun. 80%; Gl. 1007c; Mii., 637o; N. ('II) 0; Jam. 91.9% X. ('11) ioo7c Wor.; Hal. ++; ^lass.; Ravn. +; Cun. 10.87c; Host. 62 7o; Katt. 0; Mu. 95.77c; N. ('12) + ; N. ('13) +; N. (•24) +;N. ('25) +; Gi. 78-1007o; Rain. 32.57c; Jam. 467c X. ('21) 507 ; Katt. 907c Gi. 50-100% Ravn.; Cun. 1007o;Erik. (•26) X. ('25) N. ('24) 80-1007c;Katt. 1007c; Roc. 1007 Cun. 76.27c ;G1.207c; Mii. 99% Gl.O; Jam. Hal. Hal.; Sit.; Ravn. +; Xaum. -| — I — \- ; Sor. ('21); Host. 85 7o; Mu. 997o; Gl. 1.3%; Gi. 78%; Jam. Cun. Cun. 43.67c N. ('15) 5076 Mu. 1007o X. ('25) 287c X. 2 out of 2 X.X.;Schl. ('07); Roc. Gl. 1007c Gl. 95.59% ; Roc. 757c; Jam. 8.67o Gl. 97.56% Gl. 86.5470; Mii. 50%; X. ('14) 0; Roc. 0; Jam. 107-59.370 Cun. 40.8%, 87c; X. ('15) 207c; X. ('24) 0; Katt. 10070 Gi.O Gi.O X. ('25) 17% ;Katt. 100 7c Cun. 1007c;X. ('12)+; X. (^15)0 Cun. 6.37c ;X. ('lo) + ; Jam. 207-98.1% Cun. 68.27 ;X. ('15)0 Cun. 100% Desviirniiiia sopliia Diptota.ri.s itik aides 1). iiiiiralis D. tenuifolia Draba aizoides var. oblonijata I), aizoon D. altaica 1). ampicjicaulis 1). androsajcea I), aniiata D. borealis D. caroliniana D. Corsica D. dorneri D. fladnizeiisis D. friffida I), oj i/carpa D. cjlacialis D. rupestris 1). f/ra7idiflora 1). hai/naldii I), hirstita = repens ( ?) D.hirta I), hispanica I), incana D. johannis D. nemorosa D. montana D. 7iivalis I), nori'er/ica D. scan- dinavica D. pi/renaica D. subamplejicaulis D. tomentosa D. verna I), xcahleiibergi Eriica cappadocica K. nrthu.sepala E. saliva (vesicaria) Kriica.striim obiitsangu- 1 II m Eri/siminn alfaicuiii E. as per urn E. aiirantiacum E. Barbarea E. cheiranthoides Ham.; Gl. 41.677c; X. (•12)+; N. ('13) +; X. ("24) 20 7o; Katt. 100%; Gi. 75%; Jam. 8.3% X. ('25) 3.570 X. (^24) 10%;Gi. 4- 10070 (ii. 19-10070 X. ('u) X. ('14) X. ('25)0; Jam. 370 Jam. Cun. Cun. X. ('14) Cun.O X. ('14) Roc. 337c Cun. 507c X. ('14)0 Jam. 28.67o-37.57o X. ('14) Jam. 14.770 X. ('15) 0; Jam. 7270 Jam. X. ('12) + X. ('12) + X.('14) X. ('25) 0; Jam. Jam. 9.87c X. ('12)0 Jam. X. ('14) Roc. 26% ; Jam. 1.97o- 60 7f Roc. 637o Roc. 44% ; Jam. 0-107o Jam. 0-22.270 X. ('24) 80% X. ('14) 0;X. ('15) X. ('25)0; Katt. 100% X. ('15) 50% Cun. 96.37c. 637c; Jam. 18%-27.37c; X. ('15) 90%; N. ('15) 507o; X. ('23) 507c; Roc. 307o ; Ravn. ; Sit. Gl. 6.857c ;N. ('25)0; Katt. 1007f X. (•15)0;X. ('25) 2.570 Hal. : Ravn. + : Cun. 50% Jam. 48.9% Cun. 76.3% Hal. -| — |-; Mass.; Ravn.; Cun. 72.1% ; Ham.; Gl. 1007c; X. ('12)+; X. ('13)+; Rain. 13.97c; Jam. 11.1% 102 PLASMODIOPHORALES E. comaium E. crepidifolium E. helveticum E. hieractifolium E. leptophijllum E. ockroleucum E. orientale E. parviflorum E. perowskianum E. piilchellum (E. rupe- stre) E. sfrictum E. virgaium Heliophila amplexicaule Hesperis alpina H. fragrans H. lute a H. matronalis H. matronalis var. nivea H. matronalis var. nana H. runcinata H. tristis H. violacea Iberis amara I. coronaria I. qihraltarica I. hybrida I. intermedia I. lagascana I. odorata I. pinnata I. sempervirens I. taiirica J. Tenoreana I. umbeUata I. zenoreana I. sp. Isatis glauca I. tinctora I. undulata Jonopsidium acanle Koniga libi/ca Lepidiiim apetalum L. campcstre Cun. Sit. ; Ravn. Cun. 2.l7c; Jam. i.8% Gl. 457c;N. ('1-i) 30%; Katt. N. ('15) 20% Cun. 57.270 Gl. 25% Cun. 85.7% Hal. ; Ravn. + ; Naum. ; N. ('15) 0-1.3%; Roc. 62% N. ('15) 0; Jam. 4..370 Appel.;Gl. 14.29% N. ('15) \% N. ('24)+;N. ('25)0; Roc. 46 7o Roc. Roc.O Roc. 100%; Jam. 11.3%; Hal. +; Cun. 32.1 7c; Ham.; Gl. 5.367o ; Mii. 1007c ; N. ('14) 507c; N. ('24) 7570; Jam. 2.8 7o N. ('15)0 N. ('14)0; Roc.O N. (15) Jam. 107c Jam. 23.67c Cun. 877o;Gl. 1007c; N. ('25) 0; Roc. 18-5l7o; Jam. 41.470 Cun. 73.770 Mii. 24.2% Cun. 52.(i 7o Gl. 10070 Cun. 47.370 Cun. 41.67c ;N. ('25)0; Jam. 85.1 7o Gl. 4.4270 ;N. ('25)0; Roc. 827c Cun. 43.5%; N. ("25)0; Jam. 10.570 N. ('15)307o;N. ('25)0 Jam. 34.370 Wor. ; Hal. ++; Ravn. ; Cun. 927c; N. ('15)0; Gl. 99.1470; Roc. 7370; Jam. 60.7 7o Cun. 2.37c Cun. 10070 Cun. 68.47c ;F.S. ('20) 33% ; Roc.O Hal. ; Cun. 42.570; Roc. 1770; Jam. 0-9.570 N. ('25) 0;Roc. N. ('24) N. ('25) Cun. 527c Hal.; Ravn.; Cun. 42.87o; GI.O;Gi. 0-100% L. draba L. hirtum L. intermedium L. latifolium L. menziesii L. micrantum L. montanum L. perfoliatum L. reticulatum L. ruderale L. sativum L. tenuicaule L. virginicum Lunaria biennis Malcomia africana M. flexuosa M. graeca M. maritima Matthiola bicornia M. fenestralis M. incana ( ^ annua) M. oyensis M. tricuspidata Melanosinapis communis Meniocus ehrenbergii Myagrum perfoliatum Nasturtium amphibium N. officinale N. palustre N. silvestre Neslia paniculata Notoceras canariense Peltaria alliacea P. turmena Kaphanus maritimus R.sp. R. niger R. odessanus R. oleiferus R. radicula R. raphani.strum R. rosiratus Jam. N. ('15) Hal. Jam. Hal. ; Ravn. N. ('13) 507o Hal. N. ('15)0 N. ('25) N. ('25)907c;Gi. 50- 7570 Cun. 1.8%.; N. ('13) Katt. 0; Gl. 0; Mu. N. ('25) 0; Roc. Jam. Gi.O Hal. ++; Ravn.; Cun. 23%.; N. ('25) 177o; Gi. 10 7c Hal. ; Cun. 97.2% ; Naum. + ; Mii. 10070; Hon. -|-;Gi. 27-1 007c N. ('25)0 N. ('15)0 N. ('15)0 Ravn. ; Roc. 9% ; Jam. 4.3%-ll.l% Hal. +; Cun. 7.9-4.37o ; Gl. 0; N. ('15) 0; Jam. GI.O Wor. +; Hal. 0; Ravn.; N. ('15) 0;Host. 0;G1. 0;Mii. 0;Gi. N. ('25) Cun. 0; GI.O N. ('24) out of 1 ;N. ('25) N. ('24)+;Katt. N. (15) 1 out of 1 Ham. (after Naumann, '13) Gi.O Hal. ; Ham. ; Ham. ; Gi. Hal. ; Mag. ; Ham. Cun. 100% ;N. ('12) + N. ('24) 75%; Katt. 7570 Jam. Jam. N. ('I5)0;N. ('25) Gi.O N. ('15) N. ('15) 0;Katt. 157c Honig8.5-877o Cun. 53.4%; Ssach. I07c Hen. -\- ; Ravn. ; Ham. ; Mii. 36.47o; Svec. + + +; N. ('12) +; Weiss +; N. ('25, '28); Rain. 14.87o; Jam. 39.1 7c N. ('14) l7o (Ml) HOOT OF < Hl( IFKHS 103 II. satix'us li. sativiis v;ir. li. niger Jiapistriitii hispaiiiciim Ik. prrrnne li. riifiosum Hicolia liinaria lioripa amoracia li. palu,ttri.s ]{. .1 i I fi\) and (Heisberg ('2(>). to avoid or de- stroy all plants from tliat particular bed. since it is only rarely that infected seedlings recover. Seed, Seed Bed and Seedlitifi Disinfection. — Seeds of infected crucifers occasionally bear the fungus spores externally, and in such cases seed steriliza- tion is necessary. Soaking seeds in tillantin B and 0.25 per cent to 0.5 per cent usi)ulun for one-half to one hour before planting has been reported by Mothes ('25). Bronnle ('2(i) and Leines ('26) to reduce the incidence of infection if followed by fun- gicidal treatment of the soil. Such seed treatment, however, is wortliless unless it is followed by seed bed disinfection. ^'arious fungicides and chemicals as well as heat have been used in seed bed disinfection. Heating the soil 1^2 hour at 60° C. or above kills the spores, according to Vladimirskaya, Jorgensen, and Shew- ell-Cooper. Commercial formalin (1 part to 10), 0.05 per cent to 0.2 mercuric chloride (1 to 2 gals. per sq. yd.). 0.1 per cent to 0.5 per cent liquid ceresan. corrosive sublimate (1 oz. in 2-10 gals. water). 0.5 per cent uspulun solution, uspulun and solibar mixed (1 to 5). 10 per cent solution of wash- ing soda, folosan and brassiean ( 1 8 oz. per cubic yard of soil) mixed with lime, carbolic acid, mustard oil, etc.. applied 1 to 5 times to seed beds have been re- ported to reduce or completely control seedling in- fection bv the following workers: Anony. (Australia, ■-to), Somnier ('22). Jorstad ('23). Bremer ('23- '2 1). Darnell-Smith ('24). Kind.shoven ('24). Chupp ('25), Hofferichter ('26). Clayton ('26), Blunck ("28), O.sterwalder ('29). Preston ('30), Hoffman ('32). .Jorgensen ('33). Gibbs ('34), Woodman. Benchley and Hanley ('34), Kiipke ('35). and Smieton ('39). Effective control has been reported from the use of uspulun on seed beds, but some workers have claimed that it is less satisfactory than mercuric chloride. According to Clayton ('26) the spores of /'. Brassicae in the soil are fairly sensitive to mer- curic compounds, but such substances have been found to be more or less toxic to the host, especially in dry hot weather, and may reduce the cro]) to some extent. Wellman ('30). however, found that mercury compounds used according to Clayton's methods were ineffective in Wisconsin unless applied in con- centrations high enough to be injurious to the host. Copper carbonate and sul))hate. and carbonates and sulphates of calcium were likewise ineffective. Hy- drated lime worked into the soil at the rate of 1 .500 pounds to 5 tons per acre gives good control in seed beds, according to Wellman. Motte ('34) found that the fungus spores r;irely exceed a dc|)th of 20 cms. in tlie soil, ;ind .-is a control measure for seed beds he .Khoe.itfil rcmov.d of the upper 25 cms. of soil. .Seedling disinfection alone before or at planting has not i)roven gener.illy i)r,ictic;il in controlling club root. l)ii)|)ing seedlings uj) to the coll.ir in weak solutions of uspulun, mixtures of uspulun ;ind solibar solutions (1:5), mercuric eldoride. 0.1-1.5 i)er cent liquid ceresan, etc., before planting has been recom- mended by Kind.shoven ('24), Preston ('29), Rabbas ('30). Kiipke ('35). and others, but Jamalainen ('.•}6) asserted that seedling treiitment at and after planting is ineffective. While such disinfectants may inactivate the spores in the soil .idhering to the roots and root hairs, they obviously cannot destroy the amoebae and plasmodia within the tissues, if such stages are already jiresent, without killing the host. It is doubtful that enough fungicide will remain on the roots during transplantation to kill or inactivate the siJores which may be present in the plant holes. Seedling treatment, as recommended above, must ob- viously be followed by soil disinfection in the field to be effective. The addition of 1 , 2. and 25 gms. uspulun dust per plant hole (Esmarch, '25; Blunck, '28), 1 liter of .25 per cent uspulun solution, 10 liters of .20 per cent uspulun, tillantin B, and germisan per plant (Lind- fors, '25; Hertel, '26; Rabbas. '30). 10-15 gms. humus carbolineum per plant (Popp, '25), V2 pt. .01 per cent (or 1 oz. in 6 gals, water) corrosive sublimate per plant (Preston. '27; Holmes-Smith, '30). chloropicrin in plant holes (Anony.. Rhode Island. '39), 1/, pint .062-. 1 per cent mercuric chlo- ride per plant (Pre-ston, '29; Olgilvie and Mulligan, '34 ; Smieton. '39), and other chemicals have been re- ported to reduce or completely control infection. Preston ('28) found that Vj pt. per plant of .2 per cent methyl green, malachite green, methyl violet, and Brilliant green applied at planting was ineffec- tive. Likewise clubicide and Clieshunt Brown com- pounds as well as .2-.5 per cent formalin and .2 jjer cent lysol were unsatisfactory for seedling treatment at and after transl)lanting. Soil Disinfection in the Field. — In attempts to combat club root in the field by soil disinfection a wide assortment of chemicals, fungicides and spe- cial remedies have been employed as is shown in table 2 and the accompanying i)ages. In pots, seed beds, small gardens, and ex|)crimental plots these substances are fairly effective, but with the excep- tion perliajjs of usjjulun they have not jiroven com- mercially satisfactory and exi)edient in the field. As Larsen and Walker ('34) have pointed out. green- house pot tests are not always a true index of what may be expected in the field. The cost of materials and expense of apjilication often outweigh the bene- ficial results obtained, and in many instances the fungicides directly injure or reduce the ero]). Accord- ing to Motte ('33) very little is now being done to combat the disease in Denmark beside avoiding ma- nure, using basic fertilizers, and growing resistant varieties. 106 PLASMODIOPHORALES 3 s ^ "^ •a c = 2 "^ E ts. S o %: H ■s ■§ o U 3 S St: £<-^ — it: cr. C ^ °, M -1- o V !t= ::; S ^' Sfc, -c M9 12 ^ g ^ t; '■;» C" <& ^ I' o ^ 1 c s i^ < 3 CI. u o ^- *- ■3 I •/ it= ^ it: it: o c < O j: ." 14 ^ e •= ? S l£ b Cb I 1.111 ROOT OF IBl'lIFEKS 107 ■^1 > i-5 c c l;^ ■ c 5 _• ie •■T £^^£ ;; 5.- X J r t?^ r^ TT vt. V — ■? jt. ' J - u: i '■« - r- 108 PLASMODIOPHORALES 3 •a S o O S to V g .= 3 8a it: a as it: — exit: o s :?; a it: ^- ^ its i s it: 2 S "= I — St: ^' d it: it: Ed it: it: u & •^ i 1. ?i 1. £ X 1 > « t ^1 u: *c E 1- S> " oi =J 3 X •+- «£: o Q W it: it: 'l ^ ^ s c =^ 1= -5 5-." t 5 .t; s Z ^ ^ X r" +-' 7= S > ;? I I Mil HOOT OK < nil IFEHS 109 Till- nsults sliiiwii in t.ilili- 2 ari' t-oiitr;uliitor_v in seviT.'il cases. 'I'liis is iloulitlcss due in many in- stances to outside factors sucli as those wliicli iiiHu- ence tlie cfTectivencss of lime and other basic fer- tilizers. DitTerenees in time .-iiid methods of ai)l)lic;i- tion. de};ree of soil infestation, soil moisture, etc.. ob- viously operate here also. As is shown in table 2 uspulun has been extensively used, especially in Eu- rope, and wiieii applied at rates of 0.5 to 1 gm. per ksr. of soil or 120 to 300 gms. per sq. ni. in the field two weeks or more before }>lanting it is the most etl'ectivc and practical of all fiiiijiicides for the con- trol of club root, according to the data in the litera- ture. On the otiicr iiand. numerous workers have re- ported it to be unsatisfactory. It may be used as a solution and jjoured over the soil or as dust mixed with fertilizers, but Honig ('31 ) stated that its eft'eet is less certain and complete when used in solution. \\licther or not uspulun will jirove practical in large- scale operations is uncertain, according to Blunck ("29). but Honig claimed that its practicability in this respect has already been demonstrated. In com- bination with solibar. lime and other basic fertilizers its use may be greatly extended, but even when mixed with soil alone it is too expensive for practical pur- poses, according to Riehm ('2.5). The effect of uspulun on the parasite and host is not definitely known. Whether it kills the spores or prevents germination is uncertain. Bremer ('23) found that 1 gm. per kg. of soil destroys about one half of the spores, and held that it acts primarily in killing the amoebae. Honig ('31 ) believed that uspu- lun may possibly stimulate spore germination and kills the amoebae as they emerge, or that it increases the resistance of the host, along with a weakening of the amoebae. Mercuric chloride is generally reported to be eflTective. but whether or not it is economically prac- tical in large-scale operations is still uncertain. For- malin has been extensively employed, but the results obtained are very conflicting, as is shown in table 2. Its efficacy in the field is doubtful, and Hammarlund ("I.t). Burkhardt ('15), and Lindfors ('21) .stated that it is too expensive for commercial use. Sulphur has proven ineffective, and in only a few- instances has corrosive sublimate reduced infection, Bordeauj mixture is also of little or no value in com- bating club root. C arbolineum alone and mixed with various types of humus, however, has been reported to be fairly satisfactory. In addition to fungicides listed in table 2 various other chemicals, substances, and remedies have been used in combating club root. These have been used singly or in combination, and with or without alka- line fertilizers, but here again the results obtained are contradictory and generally unsatisfactory. Segetan, a mercury compound, is ineffective ac- cording to Osterwalder ('29). Cresol (2 kg. per 1. of water) applied at the rate of 2.5 1. per c. m. of soil is effective, according to I.ocw (12), but .JO gms. per eu. m. of soil lias no ). IJroMii (,'."i7). Murpliy (^'■"(7) .-ind l$rn- m-tt ('3!»). On tlu- otluT liaiul, unsatisfactory and incoiu'lu- sivo results from tlu- uso of linio as a control meas- ure have been reported l)y the followiiifi: workers: Potter ('in). Hiltner ('08). Naiiniaiin ('12. '13). Appel and Schlumberfier ('13). Sehliiniberger (It). I'ettera ^' 1 7 ). .lanson ('20), Kjrirenieyer ( '20), \'iel- hauer ('20). Vogel ("22). Whiteliead ('22). I.indfors ('21-), Ksmarch ("2.5). Korff and Boninp; ('27), Flachsaiid Kronberger ("30), Vilkaitis ("33), Motte ('33), Bailie and Miiskett ('33). and .Tanialainen ('3(5). The .■iniount of lime used and reeominended by many of these workers varies greatly, and this may partly explain some of the inconsistencies in the re- sults obtained. The investigators listed below have used and advocated the following quantities of lime in the control of club root: .\bluiy (1831), :?.5(i bu. per ;nre. Hunter ("-i'). 14— 1(> tons per acre. Sommerville ('!U), "DO lbs. per acre in drill.s witli seed. .1. A. Voelcker (94), 2 tons ))er acre. Stewart ('9.5), 90 bu. per acre. Mathleii-Sanson ("9"). 400 liters per acre. Hawk ('98), ()-8 tons per acre. Mc.Mpine ('03), 0.3-'-0.(>7 liters per sij. in. I.aubert ("0.5), 1..5 kp. per sq. m. Seliluinherjrer (14), --3 kjr. per sq. ni. Hurkart ('lo), 0.5-0.0' fnn. per sq. m. Neper ("l"), 0.5-1.0 kp. ))er sq. ni. Triesehmann ("17), ;?-3 kp. per sq. m. Popp ('19), 0.5-0.6 kp. per sq. m. Biibner ("--), 1.4 kp. per sq. m. Hosterinann and Noak ("J3), 0.5-0.6 kp. per sq. ni. Darnell-Smith ("-4), 1.50 bu. per acre. Herpers {'-H), O.ij kp. i)er sq. m. Beyer ('-5), 0..5 to 0.6 kp. i)er sq. m. Tessenow (':J6), 400 pms. (ler sq. m. Gleisberp {'26), 0.5-0.6 kp. per sq. ni. Kirsebner ('27), l-J kp. per sq. m. Blunek ('i?9), 1-2 kp. per sq. m. .\lbert ('31), 1—4 tons ])er acre. Anony. (Australia, "40), 2 tons bydrated lime per acre. Stubbs ("41), 1-2 tons per acre. The majority of workers listed above did not specify the kind of lime used, and it is im])ossible to determine whether they used pure calcium hydrate, air-slaked lime, carbonate of lime, etc., or calcium cyanamide. Since all kinds of lime are not equally effective in controlling club root many of the differ- ences in results reported in the literature arc doubt- less due to tliis factor. .Soil difl'erences. degree of spore infestation, environmental conditions, soil moisture, variations in technique and time of lime ap- plication before ))lantiiig, use of manure and acid fertilizers with lime, etc., are factors which may in- fluence the effectiveness of lime, and unless they are kejit as constant as possible in cx|)erimental work, ditfereiices in results are certain to occur. That such factors are im))ortant is well shown by the jirecaii- tions reconnnended for the use of lime. Schlumberger ('14), for instance, claimed that lime is effective only it the soil is thoroughly ;ur,iti(l at the time of .ippli- c.-ition, while I.arscn and Walker ('SI) rcjiorted that acr.-ition in relation to liming increases infection. They also found that fluctuations of soil moisture at a relatively low moisture content influenced the de- gree of infection in limed soils. A|)pel .-iiul .Schlum- berger (11) noted th.it liming becomes less effective on a given i)lot the second year, and I.indfors ('21-) .asserted that lime is ineffective if the disease is already present. If not, lime is a good club root in- hibitor. Murphy ('27) m;iintained that lime does not t