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Study of Plasmodiophora brassicae Wor. DeWolfe, Moyra Kathleen 1962

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A STUDY OP PLASMODIOPHORA BRASSICAE tfOR. by MOYRA KATHLEEN DEWOLFE B.S.A., U n i v e r s i t y o f B r i t i s h C o l u m b i a , I 9 6 0 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE i n t h e D i v i s i o n P l a n t S c i e n c e We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September, 1 9 6 2 In presenting this thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P l a n t S c i e n c e The University of British Columbia, Vancouver 8, Canada. Date September 12, 1962 i i ABSTRACT Reports concerning the l i f e history of Pla smo di op hora brassicae Wor. are highly conflicting. The contradictions in the literature on the subject and the d i f f i c u l t i e s encountered in the i n i t i a l phase of the present investigation brought about an inquiry into the techniques employed by previous workers. Repetition of these techniques indicated that many previous experiments had not taken into considera-tion the problems inherent in an in vitro study of Plasmodiophora  brassicae. Conclusions drawn from such experiments are thus of doubtful value. Methods described as successful by earlier investigators were employed in an attempt to stimulate germination. The germination process was not observed, and contaminants intro-duced with the resting spore material made i t impossible to designate any one motile organism as P. brassicae. Attempts were made to obtain root hair infection. Pour methods described by previous investigators proved unsuccess-f u l . A limited degree of infection was obtained by placing young cabbage and cauliflower seedlings in a buffered nutrient solution containing washed P. brassicae resting spores. In spite of the fact that artifacts were readily produced, early infection stages were observed and photographed and the existence of a zoosporangial stage in the root hair was confirmed. The infection rate was too low to permit intensive i i i observations of the development of the parasite within the root hair. A proteolytic enzyme preparation was somewhat successful in increasing the infection rate. This was taken as an indi-cation that a combination of enzymes could provide the neces-sary germination stimulus. Decay of the host tissues i s apparently necessary to the maturation of the resting spores. Present experiments indi-cated that i t i s not feasible to separate the contaminants from these spores. As i t was not possible to draw conclusions from contaminated cultures, i t i s concluded that the only approach of value i s to provide an a r t i f i c i a l germination stimulus to spores from clean clubs, so that the development of the parasite may take place under sterile conditions. v i ACKNOWLEDGMENTS The a u t h o r w o u l d l i k e t o t h a n k t h e f o l l o w i n g p e r s o n s f o r t h e i r a s s i s t a n c e d u r i n g t h e p r e p a r a t i o n o f t h i s t h e s i s : D r . N.A. MacLean, A s s i s t a n t P r o f e s s o r , D i v i s i o n o f P l a n t S c i e n c e , U n i v e r s i t y o f B r i t i s h C o l u m b i a , u n d e r whose s u p e r v i s i o n t h i s t h e s i s was c a r r i e d o u t . Dr . R . J . B a n d o n i , A s s i s t a n t P r o f e s s o r , D e p a r t m e n t o f B i o l o g y a n d B o t a n y , a n d D r . D.P. Orrarod, A s s i s t a n t P r o f e s s o r , D i v i s i o n o f P l a n t S c i e n c e , U n i v e r s i t y o f B r i t i s h C o l u m b i a , whose g u i d a n c e t h r o u g h o u t t h e c o u r s e o f t h e i n v e s t i g a t i o n was i n v a l u a b l e . D r . W.L. Seaman, P l a n t P a t h o l o g i s t , Canada D e p a r t m e n t o f A g r i c u l t u r e , R e s e a r c h B r a n c h , Ottawa, who k i n d l y o f f e r e d many s u g g e s t i o n s w h i c h p r o v e d h e l p f u l . The members o f t h e G r a d u a t e Committee a n d t h e F a c u l t y o f A g r i c u l t u r e who have o f f e r e d a d v i c e a n d a s s i t a n c e t o t h e a u t h o r d u r i n g h e r g r a d u a t e s t u d i e s . iv TABLE OF CONTENTS Page INTRODUCTION " 1 REVIEW OF THE LITERATURE 2 Economic Significance of Plasmodiophora brassicae 2 Symptoms of the Host Plant l± Distribution and Host Range of Plasmodiophora brassicae . . . . 5 Relationships of Plasmodiophora brassicae 6 Life History of Plasmodiophora brassicae . . 7 Germination of the resting spore 7 F i r s t motile stage 11 Penetration and zoosporangial formation . . 13 Possibility of a sexual phase i n the l i f e cycle . . . 16 Growth and distribution of the parasite within the host tissues, and formation of resting spores 19 EXPERIMENTAL WORK 21 Materials 21 Methods 22 Source of clubbed tissue 22 Preparation of resting spore inoculum 21j. Germination of resting spores 2 5 The parasite within the root hair 28 D I S C U S S I O N y? LITERATURE CITED APPENDIX Table I. Relative Effectiveness of Nine Methods of Obtaining Root Hair Infection by Plasmodiophora brassicae  Figure I. Resting Spores of Plasmodiophora brassicae  Figure II."-. Young Plasmodium of Pla smodiophora brassicae i n Root Hair • Figure III. Young Plasmodia of Plasmodiophora brassicae i n Root Hair Figure IV. Cluster of Zoosporangia i n Root Hair 1 A STUDY OP PLASMODIOPHORA BRASSICAE WOR. INTRODUCTION Club root of crucifers, caused by Plasmodiophora  brassicae Wor., is undoubtedly one of the greatest problems now facing growers of cruciferous crops. The organisms attacks cabbage, cauliflower, turnip, Brussels sprouts, rutabaga, rape, and mustard, as well as wild cruciferous hosts, causing the plant to die, or f a i l to form a marketable product. The occurrence of this disease has been recognized for several centuries, and during this time many and varied hypotheses have been put forward to explain i t s cause and development. In spite of the extensive attention which has been given to this problem, a truly effective control measure is yet to be found. Contemporary workers are coming to be of the opinion that clues to the control of the disease l i e in the l i f e cycle of the parasite i t s e l f . The l i f e cycle of P. brassicae, however, i s the subject of much controversy and at the present time there are many conflicting hypotheses and few observed facts. The search for an effective control measure has been directed chiefly at the resting spore stage, and i t could be, in the words of Grace M. Waterhouse (Ijl}.), 2 " t h a t t o o much f u n g i c i d e i s c h a s i n g t o o many s p o r e s . " T h e r e must s u r e l y be a much more v u l n e r a b l e p h a s e i n t h e d e v e l o p -m e n t a l s e q u e n c e o f t h e o r g a n i s m t o w a r d s w h i c h c o n t r o l m e a s u r e s c o u l d be e f f e c t i v e l y d i r e c t e d . U n t i l t h e l i f e c y c l e o f t h e o r g a n i s m i s c l e a r l y o u t l i n e d , however, t h i s a p p r o a c h t o t h e c o n t r o l o f t h e d i s e a s e c a n n o t be s u c c e s s f u l l y u t i l i z e d . I t was i n i t i a l l y t h e i n t e n t i o n o f t h e a u t h o r t o o f f e r a c o n t r i b u t i o n t o t h e c l a r i f i c a t i o n o f t h e p r o b l e m o f t h e l i f e c y c l e o f P. b r a s s i c a e . However, so many c o n t r a d i c t i o n s were f o u n d i n t h e l i t e r a t u r e a n d so many d i f f i c u l t i e s i n t e c h n i q u e n o t m e n t i o n e d b y p r e v i o u s w o r k e r s were e n c o u n t e r e d i n t h e c o u r s e o f t h e i n v e s t i g a t i o n , t h a t i t was d e c i d e d t h a t t h e c h i e f p u r p o s e o f t h e i n v e s t i g a t i o n s h o u l d be t o p o i n t o u t t h e c o n t r a -d i c t i o n s , t o s e e k t h e r e a s o n s f o r t h e c o n f l i c t i n g e v i d e n c e , a n d t o s u g g e s t p o s s i b l e s o u r c e s o f e r r o r . I t i s t o be h o p e d t h a t t h i s a n a l y s i s w i l l p r o v i d e a g u i d e t o f u t u r e work on t h e p r o b l e m a n d t h u s p r e v e n t t h e r e p e t i t i o n o f e r r o r s w h i c h have c o n t r i b u t e d t o t h e c o n f u s i o n w h i c h s u r r o u n d s t h e p r o b l e m o f P l a s m o d i o p h o r a b r a s s i c a e t o d a y . REVIEW OF THE LITERATURE E c o n o m i c S i g n i f i c a n c e o f P l a s m o d i o p h o r a b r a s s i c a e C l u b r o o t may be c o n s i d e r e d a s t h e most i m p o r t a n t d i s e a s e o f c r u c i f e r o u s c r o p s i n t h e t e m p e r a t e zone ( 1 0 ) . The d i s e a s e i s w i d e s p r e a d g e o g r a p h i c a l l y a n d b e c a u s e t h e c a u s a l o r g a n i s m c a n p e r s i s t i n t h e s o i l f o r y e a r s , i t p r e s e n t s a r e a l p r o b l e m t o t h e m a r k e t g a r d e n e r a n d t o o t h e r s who grow c r u c i f e r s i n t e n s i v e l y . 3 In 1869 half the cabbage crop in the St. Petersburg region of Russia was destroyed by club root and i t was a plea on the part of the Royal Russian Gardening Society in 1872 which instigated the classic investigation by W.oronin (I4.8) into the cause of the disease. Similar d i f f i c u l t y was experienced in Great Britain during the nineteenth century causing inquiries to be made into the cause and control of the disease. As early as 1893 Halstead (18) estimated the loss in the United States as being represented by millions of dollars annually. P. brassicae attacks cruciferous crops in Canada in more or less serious proportions each year. The disease has become widespread in the coastal regions of British Columbia. In 19i).7 particularly severe infections of club root were found on both cabbage and cauliflower in Chinese market gardens on the Fraser River delta (ij.). In 1950 heavy losses were again sus-tained in the lower Fraser Valley, especially i n delta truck gardens (5). Since that time the Fraser Valley and the delta region have suffered yearly losses of cabbages, cauliflowers, and turnips. The disease has almost eliminated the production of crucifers i n home gardens in the lower mainland. In 1955 club root was reported for the f i r s t time in the Terrace d i s t r i c t of the province (7). Growers in Fort William, Ontario, reported in 195lj- that they were unable to raise cabbages because of the disease (6). In recent years, the disease has severely limited crucifer production in southern Quebec and i s considered the worst problem facing vegetable growers i n the area. A special survey made i n 19M 5 revealed club root i n every cabbage f i e l d inspected (3). In 19I|.7 at Riviere des P r a i r i e s , Quebec, every turnip i n a ten acre planting was affected and the crop almost a complete loss i n spite of the ap p l i c a t i o n of powdered cyana-mide as a control measure (ij.). Club root i s general i n the Montreal d i s t r i c t , causing losses of from f i v e to sixty per cent of the crop i n cabbage, cauliflower, Chinese cabbage, turnip, and r a d i s h • A t h i r t y per cent reduction i n y i e l d , as a r e s u l t of loss of i n d i v i d u a l plants, as well as general growth retardation, i s common yearly i n the Maritime provinces, where common crop r o t a t i o n of c r u c i f e r s , hay, and potatoes does not permit the heavy a p p l i c a t i o n of lime that would discourage club root but encourage potato scab ( 6 ) . Symptoms of the Host Plant A l l symptoms on the above ground portions of the plant are a r e s u l t of hypertrophy and hyperplasia caused by the development of the parasite within the root tissue. The severity of clubbing and hence the severity of the above ground symptoms depends on the species and va r i e t y of the host, age of the host at the time of i n f e c t i o n , and s o i l conditions such as temperature and moisture (10). The f i r s t i n d i c a t i o n of i n f e c t i o n seen on the above ground portions of the plant i s usually tem-porary w i l t i n g during warm periods, generally followed by stunted, unproductive growth. I f i n f e c t i o n occurs early i n the development of the plant, death may r e s u l t before maturity i s 5 reached. Thesa symptoms are a result of the disruption of the tissues which effect the absorption and translocation of water and mineral salts. Infected roots enlarge relatively rapidly to form excresences which take on a variety of shapes. This rapid growth of clubbed tissue i s inimical to the normal develop-ment of cork cambium and secondary invasion by low grade para-sites always results in early decay (10). Anatomically the parasite affects the cortical parenchyma most conspicuously. One of the most striking appearances i n sections of diseased tissue i s the presence of more or less Isolated groups of hypertrophied cells which Nawaschin (3i|.) named "Krankheitsherde". The medullary ray cells become hyper-trophied and form large bands of pathological tissue which spl i t and force the xylem tissues apart, u n t i l the latter become dis-torted and shifted out of position. Separated from each other in this manner the vascular bundles are no longer able to function normally. The parasite very seldom invades the differentiated vascular tissue, but in young roots the cambium i s a center of abnormal growth. Distribution and Host Range of Plasmodiophora brassicae Although club root disease is Regarded as reaching i t s most serious proportions in the temperate zone, i t i s by no means confined to these regions. It i s known to occur with con-siderable severity in tropical regions of Asia, Africa, and South America (11). The disease appears to have been f i r s t re-corded on cultivated members of the Oruciferae, but by the end 6 of the l a s t century i t was recognized that various wild c r u c i f e r -ous plants could also be attacked by P. brassicae. This l a t t e r group serves as perpetuating hosts of the parasite. Up u n t i l X9I4.9 a l l the plants proved to be susceptible were members of the Cruciferae. At t h i s time i t was reported that P. brassicae was capable of i n f e c t i n g the root hairs of some non-cruciferous plants although the t y p i c a l swellings did not occur (l|5). Colhoun (10) presents an extensive l i s t of susceptible and non-susceptible cruciferous plants as well as a l i s t of ten non-cruciferous plants i n which zoosporangia have been observed. Relationships of Plasmodiophora brassicae Plasmodiophora brassicae belongs to the family Plas-modiophoraceae i n the order Plasmodiophorales. The r e l a t i o n -ships of t h i s order and of the genera and species within i t are uncertain, and the group has been assigned various positions i n schemes of c l a s s i f i c a t i o n . Because of inadequate data concerning the order these assignations have been l a r g e l y speculative, and a review of the l i t e r a t u r e shows that few workers have agreed on the systematic p o s i t i o n of P. brassicae. Woronin (fy.8) considered that the genus P l a smo d i op hora was intermediate between the Myxomyceteae and the Chytridiaceae, possessing some of the characters of each group. DeB'ary (3) described P. brassicae as a doubtful member of the myxomycetes. Saccardo (39) included the Plasmodiophorales as a main subgroup under the Myxomycetes. F i t z p a t r i c k (17) assigned the group to 7 t h e r a n k o f a f a m i l y w h i c h he i n c l u d e d i n t h e o r d e r C h y t r i d i a l e s o f t h e c l a s s P h y c o m y c e t e s . S p a r r o w (1 .^3). &ad C l e m e n t s a n d S h e a r (9) a l s o i n c l u d e d them among t h e p h y c o m y c e t o u s f u n g i . Z o o l o g i s t s have a l s o c l a i m e d t h e P l a s m o d i o p h o r a l e s a n d have i n c l u d e d t h i s o r d e r a s a s u b c l a s s o f t h e Myxomycetes among t h e P r o t o z o a ( 2 1 ) . The P l a s m o d i o p h o r a l e s a p p e a r t o have, t h e r e f o r e , some d e v e l o p m e n t a l p h a s e s a n d c y t o l o g i c a l c h a r a c t e r i s t i c s i n common w i t h t h e Myxomycetes, t h e C h y t r i d i a l e s , a n d t h e P r o t o z o a . W h e t h e r t h i s o r d e r has o r i g i n a t e d d i r e c t l y f r o m s u c h g r o u p s o r d e v e l o p e d a l o n g p a r a l l e l l i n e s w i t h them f r o m a d i s t a n t common a n c e s t o r , however, i s s t i l l u n c e r t a i n . Our knowledge o f t h e c r i t i c a l s t a g e s i n t h e l i f e c y c l e s o f t h e P l a s m o d i o p h o r a l e s i s t o o i n c o m p l e t e t o w a r r a n t d e f i n i t e c o n c l u s i o n s a t p r e s e n t . F u r t h e r i n t e n s i v e s t u d y o f t h e l i f e c y c l e s w i l l d o u b t l e s s i n v a l i d a t e many o f t h e p r e s e n t l y h e l d b e l i e f s c o n c e r n i n g t h i s g r o u p a n d p o i n t t o more d e f i n i t e l i n e s o f r e l a t i o n s h i p s . L i f e H i s t o r y o f P l a s m o d i o p h o r a b r a s s i c a e G e r m i n a t i o n o f t h e r e s t i n g s p o r e . The r e p o r t s i n t h e l i t e r a t u r e o f t h e g e r m i n a t i o n p r o c e s s a n d f a c t o r s a f f e c t i n g i t a r e h i g h l y c o n t r a d i c t o r y . The l a c k o f a c c u r a t e i n f o r m a t i o n on t h i s p h a s e o f t h e d e v e l o p m e n t o f t h e o r g a n i s m a p p e a r s t o be t h e r e s u l t o f two f a c t o r s . The c h i e f p r o b l e m a p p e a r s t o be t h a t w o r k e r s have f o u n d i t e x t r e m e l y d i f f i c u l t t o g e r m i n a t e t h e r e s t i n g s p o r e s u n d e r c o n d i t i o n s w h i c h a l l o w o b s e r v a t i o n o f t h e g e r m i n a t i o n p r o c e s s . S e c o n d l y , t h e s p o r e s a r e so m i n u t e t h a t 8 they must be observed under an o i l immersion lens with the accompanying disadvantages of a small f i e l d and shallow depth of focus. Slight movement on the part of the germinating spore removes i t from view. Several workers have given accounts of their attempts at in vitro germination of P. brassicae resting spores. Woronin (I4.8) gave a brief description and a series of i l l u s -trations of the germination process. His technique consisted of growing cabbage seedlings in shallow watch glasses with a quantity of mature spores. Woronin's observations and drawings have provided a basis for a l l later studies of P. brassicae. Wellman (lj.6), however, suggested that Woronin's observations and illustrations were partly based upon his knowledge of the saprophytic myxomycetes. Eycleshyraer ( 1 5 ) placed pieces of tissue containing spores under a eover glass and tapped the glass so as to free spores. The slide was then placed in a moist chamber for five to twenty-four hours, after which he claimed to have observed swarm c e l l s . Chupp (8) observed at least part of the process of germi-nation. He found that spores would not germinate in d i s t i l l e d water, and recommended germinating spores obtained from frozen o roots in a muck s o i l f i l t r a t e and incubating them at 28 G. He reported that germination dropped rapidly as the incubation temperature was lowered and he was unable to get germination at room temperature. He found that in the presence of a young seedling, infection took place at a temperature of from 16 to . 9 20 °G . In contrast to this, Honig (23) found that germination o occurred! below 21 C in the absence of seedlings. Cook and Schwartz (12) found results in germinating the spores to be most satisfactory i n Knop's solution with one and two per cent glucose. They admitted that i t was not possible to say definitely that the amoeboid bodies seen had originated from the germination of P. brassicae spores. Wellman (lj.6) teased spores out of previously frozen clubbed roots into sterile d i s t i l l e d water. Excess debris was removed, and the suspension centrifuged. Spore suspensions were made into hanging drop cultures in d i s t i l l e d water or tap water and the author reported seeing hundreds of swarm spores, and in very few cases, the actual germination process. Ellison (U4.) experienced considerable d i f f i c u l t y i n inducing germination of P. brassicae resting spores. He attempted to obtaincgermination by many methods, including those recom-mended as successful by previous workers. Sterile excised root tips were placed with resting spore suspensions in the hope that the presence of the liv i n g host might have a stimulatory effect on the spores. Rain and snow water were tried, the pH was manipulated, the media oxygenated. Results were either entirely negative or germination so very slight that i t was impossible to obtain zoospores. Some degree of germination was obtained from Infected roots which had been repeatedly frozen and thawed over a two month period, then macerated i n a mortar. Water was added, and the spore suspension was repeatedly centri-fuged in order to reduce the bacterial population. The washed o spores were l e f t to germinate in a Syracuse watch glass at 25 G. 10 On the third day, Ell i s o n reported, a " f a i r " amount of germi-nation had taken place. Only once was a zoospore observed escaping from the spore case. He found from his experience with the resting spores of many of the lower fungi that there are no generalized methods which are effective in inducing production of the swarm spores. Each specimen must be treated by the laborious process of t r i a l and error. Germination of spores in tap water was reported to occur readily by Ayers (1). He found that i t took place at room temperature i n from one to ten days depending on the maturity of the spores. He found that the best spores from the standpoint of germination were obtained from old clubs, heavily invaded by secondary organisms. Successive washings of the spore material in tap water apparently was satisfactory in removing contaminants. Smith (I4.2) i n an extensive study of the host-parasite physiology of the disease, was uniquely fortunate in obtaining uncontaminated spore suspensions. He reported that the germi-nation of resting spores isolated from minced tissues of surface ste r i l i z e d galls varied greatly from one spore prepa-ration to the next, depending in part on the age of the gal l tissue from which spores were isolated. Significantly better germination was obtained in non-sterile s o i l extracts, and he f e l t that the factor responsible for increased germination was associated with the microorganisms in the s o i l and was of a rather unstable nature. His work indicated that digestion of the resting spore preparation by proteolytic enzymes gave an 11 increase in the infection rate. No observations of the germi-nation process or the resultant zoospores were made. Partially disintegrated clubbed roots of cabbage stored in a frozen condition for several weeks were used by Seaman et a l (If-l) in an attempt to obtain P. brassicae zoospores. The clubs were macerated, the coarse debris removed, and the spore suspension repeatedly washed by centrifugation. After the f i n a l washing, spores were resuspended in sterile tap water and incu-bated at 2lj.°C or at room temperature. The occurence of fl a g e l -lated cells in such suspensions was unpredictable and variable. Seaman (personal communication) acknowledged the fact that possession of heterokont flagella i s not confined to any one type of motile c e l l found in the spore supernatant. The germi-nation block i s , i n his opinion, physiological although he believes that the need for " f u l l y mature" spores from rotted clubs may not be as necessary as some have believed. Fi r s t motile stage. The majority of workers who have reported seeing the zoospore of P_. brassicae do not give a com-plete description of the essential points: morphology of the flag e l l a , their point of attachment to the zoospore, their position while the c e l l i s moving freely in suspension. This lack of detail i s l i k e l y due to the d i f f i c u l t y with which zoo-spores are obtained, and fo their small size and great motility. It i s to be hoped that the present day use of the phase contrast and electron microscopes w i l l result in a more clearly docu-mented record of zoospore morphology. 12 Most investigators agree that each resting spore gives rise to one zoospore, however, the description given for the zoospore varies widely. Woronin {ILQ) described the myxamoeba when released from the spore wall as possessing a somewhat elongated spindle shaped body, provided with a rather long, whip-like flagellum at i t s beaked, sharply pointed anterior end. The motion was stated to be due in part to the l i v e l y weaving from side to side of the supple beak and also by the character-i s t i c movement resembling creeping, the shape changing con-stantly. Chupp (8) considered the shape of the zoospore to be more or less pyriform, although he concurred with Woronin on many other morphological details. The presence of flagella was disputed by Honig (23) who was of the opinion that only amoebae were produced. He believed that such amoebae could survive for a long time outside the plant and increased In size during that time. It has been suggested (10) that he may have confused some other organism with certain stages in the l i f e history of P. brassicae. The same criticism has been leveled at the investi-gations of Jones (2lj.) who claimed that in addition to the single zoospore, other spores germinate to give rise to as many as twenty smaller bodies which may be uniflagellate or amoeboid. Karling (25) suggested that spores germinating under certain conditions may, instead of producing flagellate zoospores, give rise to bodies In an amoeboid state, however Ledingham (28), Ayers (1), and Ellison (llj.) reported that the zoospores are consistently biflagellate and heterokont. Ayers (1) found the fixed and stained primary zoospores to vary considerably in size. The smallest were as small as the resting spore, but the majority 13 were Intermediate to moderately large. This variation was taken as an indication that growth of the zoospores occurs after their emergence from the resting spores. Seaman et a l (ij.1) examined zoospores found in association with club root tissue. Observations with a phase contrast microscope indicated that the zoospores vary from elongate to pyriform or globose in shape and are anteriorly biflagellate and markedly heterokont. Examination of the zoospores with the electron microscope showed that the longer flagellum was of the tin s e l type, with the shorter flagellum bearing no appendages. Seaman later granted (personal communication) that i t would be highly unusual, phylogenetically for the primary zoospore to possess a tinsel-type flagellum i f the secondary zoospore possesses a t r a i l i n g whiplash flagellum as reported (12). Considerable variation exists in the recorded sizes of zoospores. The following measurements have been given: 1.7- 3.5)1 (8); ij-.0x2.0u or 1.0x0.5)* (21*.); 3.0-3.5u (12); 2.8- 5.9u (1); 3.0x3.0-3.9x1^.8^ (ip.). Penetration and zoosporanglal formation. The d i f f i c u l t i e s involved in observing the actual process of host penetration have resulted in much conflicting evidence concerning the event. Although i t i s generally conceded that penetration takes place through the root hair (8, 12, l+O, 1), there i s disagreement as to the possibility that infection takes place through the epi-dermal cells as well. Although Chupp (8) believed that seldom i f ever does direct penetration of epidermal cells occur, a 111. number of investigators ( 2 6 , 2 0 , 1) have stated that infection is not limited to the root hair region of the root. The work of Samuel and Garrett (I4.O) indicated that infection takes place only when the root hair i s young and f u l l of protoplasm. It would appear unlikely that penetration of older tissues of the plant takes place ( 2 7 ) . According to most investigators the zoospores come to rest on the host and enter as amoebae through the root hair. Only twiee has the penetration process been observed and described, and these two reports are rather discrepant. Honig ( 2 3 ) reported that the amoeba resulting from the resting spore became closely applied to the root hair, and then passed through a hole which appeared at the point of attachment. The hole disappeared immediately following penetration. Rochlin ( 3 8 ) showed that the c e l l wall of the root hair became swollen where a spore became attached. Treatment with chloroiodide of zinc indicated the absence of cellulose in the wall at this deformed spot. The penetrating amoeba assumed a spherical plasmatic form and was slightly larger than the spore. The protoplast passed through the swollen and gelatinized region and entered the host c e l l . Small colorless, uninucleate bodies in the root hairs and epidermal root cells are taken as evidence that penetration has been effected. Ayers (1) found that when multiple infection of a root hair occurs these bodies show no tendency to fuse with one another. It was believed by Chupp ( 8 ) that resting spores develop 15 from the Plasmodia in the root hairs. This conclusion was later shown to be incorrect by Cook and Schwartz (12) when they recorded a hitherto unknown phase in the l i f e history of P. brassicae and showed that zoosporangia are formed in the root hair at this stage. These investigators reported that the nucleus of the infecting body divided so that a small Plas-modium was formed. This Plasmodium may eontain over one hundred nuclei (16). The multinucleate Plasmodium, according to Cook and Schwartz, then cleaved into thin walled zoospor-angia, each containing one nucleus. The nucleus of each divided to give four, or sometimes six, nuclei and a small mass of protoplasm collected around each. The zoospores eventu-a l l y were thought by Cook and Schwartz to make their way to the exterior. The diameter of the zoosporangium was recorded as from 6.0-6.5u while the zoospores were about 1.5j* in length and 0 .5 "to 0»7p. in diameter. No flagella could be seen. Ayers (1) confirmed the existance of the zoosporangial stage and provided further information regarding i t s development. He found that small Plasmodia formed only a few zoosporangia, but large ones were transformed into a large number which may be arranged in compact, irregular aggregations. Ayers reported that shortly after formation,of zoosporangia, the nucleus of each divided to produce four to eight zoospores which were shown to be biflagellate and heterokont with a diameter of from 1.9 to 2.3u. Zoospores were observed escaping from the zoosporangia only three times. Apart from their smaller size these secondary zoospores closely resembled those thought to 16 escape from the resting spores. The mature zoosporangia, according to Ayers, became attached to the c e l l wall and open-ings developed at the point of attachment. Apparently the zoospores escaped to the exterior through these openings for their discharge Into the root hair was never observed nor were they ever found free within the root hair. Under moist condi-tions the zoospores were completely discharged. It was found that zoospore discharge from mature zoosporangia took place In a short time in tap water. According to Ayers, from two to eight days, depending on conditions of moisture and temperature, may elapse between the occurrence of infection and the f i n a l zoospore discharge. Nothing has been ascertained of the subse-quent role of the discharged zoospores. Possibility of a sexual phase in the l i f e cycle. The lack of information on sexuality i n P. brassicae helps to explain the diverse interpretations of i t s l i f e cycle. Nothing i s known about sexual reproduction in P. brassicae, yet most workers have assumed i t occurs. It i s generally agreed that there Is a reduction division in the formation of the spores, but no one i s certain of the position of syngamy. The extremely small size of the nuclei of the organism has imposed limitations on attempts to study nuclear phenomena. This, combined with the fact that primary and secondary zoospores are d i f f i c u l t to obtain, has caused the hypotheses set forward to support the existence of a sexual phase to be of a varied nature. Prowazek (37) contended that the incipient spore segments fused in pairs following cleavage, after which the zygote or 17 binucleate spore began to encyst. One of the so-called gametic nuclei then underwent a reduction division and formed a variable number of reduction bodies. Meiosis was followed almost at once by karyogamy. Prowazek*s account was refuted by Maire and Tison ( 3D who fa i l e d to find any evidence of plasmogamy and karyogamy following cleavage. They nevertheless believed that sexual fusion occurs in P. brassicae and postulated that i t might take place between two amoebae from germinating spores. Winge (1+7) agreed with this view and assumed that the motile cells from resting spores copulate; In pairs to form small "myxaplasma" which penetrate the host and develop into Plasmodia. The diploid phase was assumed to persist u n t i l the second sporo-genous division where reduction occurs. Lutman (29), Chupp (8), and Milovidov (33) although uncertain as to the time and place of plasmogamy and karyogamy, believed that the reduction i n chromosome number which takes place during the f i r s t sporogenous division was evidence that fusion must occur at some stage. Wawaschin (31+) postulated that fusion occurs outside the host c e l l between pairs of zoospores. The unorthodox views of Jones (2I4.) have been seriously questioned by later workers and his hypothesis of unequal-sized gametes arising from the resting spores has been refuted by most investigators. Cook and Schwartz (12) suggested a fusion of the second-ary zoospores in the very young cortical c e l l s , or in the root hair. Their hypothesis was based entirely on the observation of zoospores lying side by side in pairs, and the presence of 18 binucleate amoebae. Fedorintschik (16) postulated a fusion of gametes from zoosporangia or gametangia after a period of vege-tative budding within the host. He also believed that two reductions occur in P. brassicae, one during the f i r s t d i v i -sion of the sporangium nucleus and another at sporogenesis. However, he,reported only one nuclear fusion in his account. Helm (19) after extensive studies of the nuclear behav-iour of P. brassicae concluded that fusion of nuclei takes place in the older Plasmodia in the root tissues, plasraogamy having occured upon fusion of zoospores. These Plasmodia, according to Heim, represent the diploid stage and do not ramify. It i s this stage, during which the chromatin' i s barely discernible upon staining, which corresponds to the akaryote stage of some authors. The diploid nuclei, after a resting period, divide three times to provide the nuclei for future spores. She noted that the f i r s t division had a l l the characteristics of raeiosis and had the same appearance as the f i r s t nuclear division in an ascus or basidium. The divisions within a single Plasmodium were simultaneous. After the f i r s t division of the fusion nucleus the cytoplasm condensed around the daughter nuclei, and the pleurinucleate Plasmodium became a mass of uninucleate Plasmodia which i n turn divided mitoti-cally. Heim claimed that the tiny c e l l s resulting from the second division rounded up and were capable of becoming amoeboid. The third and f i n a l division gave rise to the resting spores. These findings have not yet been confirmed by other workers. 19 The question of the existance and position of plasmogamy, karyogamy and reduction division thus remains in an uncertain state and w i l l doubtless remain so u n t i l extensive monospore studies have been made. Growth and distribution of the parasite within the host  tissues, and formation of resting spores. Woronin (ILQ) was uncertain as to whether amoebae fuse to form Plasmodia, or whether each Plasmodium arises from a single amoeba but he f e l t the f i r s t possibility was the more plausible. Wawaschin (3I4.), Prowazek (37), and Pedorintschik (16) supported the theory that Plasmodia or myxamoebae flow together within the host, but Maire and Tison (3D and Ghupp ( 8 ) did not agree. According to Cook and Schwartz (12) amoebae in their early stages may combine to form large Plasmodia although this i s i n no way a conjugation but only a simple joining up of proto-plasts. Some workers ( 2 9 , 26, 16) believed the amoebae or young Plasmodia to be capable of dividing repeatedly In the host c e l l so that their numbers may be rapidly increased. A few workers (12, 2l\.) contended that these Plasmodia may encyst under unfavourable conditions but this i s thought unlikely (10). It Is now generally agreed that the spread of the para-site in the host tissues occurs i n two ways: by migration of amoebae and young Plasmodia from c e l l to c e l l , and by passive distribution of the parasite through repeated divisions of infected c e l l s . Lutman ( 2 9 ) , Chupp ( 8 ) , Kunkel (26), Honig (23), and others have demonstrated in fixed and stained tissue the passage of small Plasmodia from c e l l to c e l l . Cook and 20 Schwartz (12), more than a decade later, expressed doubt as to this occurence and postulated that only the secondary zoospores, which i n their opinion function as gametes, possess the faculty of passing from one c e l l to another. Fedorintschik (16) believed that in the early stages of the disease migration of amoebae i s the principal method of distribution in the host tissues, but, after the Plasmodia have formed and begun to mature, further spread i s by division of infected c e l l s . While i t i s now generally believed that division of the host c e l l greatly increases the number of infected c e l l s , i t nevertheless appears to play a minor role in the distribution of the parasite through-out the root. Chupp (8) stated that the fungus i s distributed horizon-t a l l y and v e r t i c a l l y through the cortex, c e l l division taking place as fast as invasion occurs. Studies made by Kunkel (26), Larson (27), and others showed that the parasite can reach the cambium and thereafter cells formed from the invaded cambium are also infected as they are formed. Kunkel (26) has shown also that many of the cortical cells become infected by the move-ment of the parasite outwards from the cambium. The cells of the medullary rays are usually severely attacked but only rarely Is the parasite found in the xylem elements (26, 12). When a Plasmodium has become established in a c e l l suitable for development, the amoeboid movement ceases. Rapid growth, accompanied by nuclear divisions takes place at the expense of the food reserves of the c e l l . According to Woronin (k-Q)» the mature Plasmodium becomes vacuolate and the protoplasm i s divided by a fine lattice-work of vacuoles. 21 The vacuoles then begin to disappear and the granular proto-plasmic substance lying between them collects into small sphaeroid aggregations which are the future spores. Woronin noted that at f i r s t a spore was naked, but soon a thin, unmarked wall was l a i d down around i t . At no time was the spore mass ever enveloped by a common membrane. The resting spore walls contain chitin but no cellulose (36). At maturity the resting spores are nearly spherical. Considerable variation in their size has been reported. The following spore diameters have been recorded: 1.6u (Ij.8); 1.9-I}..3P- (8); 1.7u (I4.6); 2-3u and [j..6x6.Op (12); 3.9u (23). The mature spores remain in the clubbed tissue u n t i l they are released to the s o i l by decay. EXPERIMENTAL WORK Materials Severely clubbed cauliflower roots were obtained in the f a l l of I960 from a garden in Vancouver. These clubs were a i r dried and kept in this condition u n t i l experimental work began the following spring. Three races of the organism were obtained from the Canada Department of Agriculture Experimental Farm at Charlottetown, Prince Edward Island. Greenhouse tests with s o i l grown cabbage and cauliflower plants showed the Vancouver race to be much more pathogenic than the three eastern races, so the latter were not used in the following experiments. When i t became necessary to grow infected plants, the dried roots were soaked in d i s t i l l e d water and macerated in a Waring blendor. 22. The macerate was mixed into loam s o i l prepared for greenhouse use at the rate of approximately five grams of macerate per kilogram of s o i l . This s o i l was put in six-inch pots. Six-week old,cabbage and cauliflower seedlings were transplanted from f l a t s to the pots of infested s o i l . Varieties used during the course of the experimental work were Snowball X, a susceptible variety of cauliflower, and Winter King Savoy, a susceptible variety of cabbage. Methods Source of clubbed tissue. Seedlings were grown in pots of infested s o i l in the greenhouse during the summer months with a temperature range of 65 to 95°P« As i t was found that club development was at a minimum in the greenhouse during the winter, the Infected plants were, from October to May, grown o in growth chambers at 70 P.- with about 1000 foot candles of white fluorescent light. The clubbed tissue was then kept frozen u n t i l used. The majority of clubs obtained from these plants were badly decayed by the time they reached a size suit-able for use. This invasion by secondary organisms was hastened by the maintenance of a high level of moisture in the s o i l , a precaution which was made necessary as a result of the inefficiency with which the distorted root systems conducted water. Because the s o i l grown clubs became decayed at an early stage in their development, attempts were made to grow clubbed plants in soil-less culture in the hopes of obtaining large, clean clubs from which to prepare inoculum. Two-week old 23 cabbage seedlings which had been germinated on moist f i l t e r r paper and then exposed to a heavy suspension of resting spores i n watchglasses for four days were supported above half-pint jars containing Knop's nutrient solution (2) with microelements added (22). Only the root system of each plant was submerged. Aeration was provided to each jar for two-hour periods three times daily. The nutrient solution was renewed twice weekly. In spite of the aeration, the roots of the seedlings became covered by a heavy growth of phycomycetous water molds, intro-duced with the spore material. This mold growth rapidly destroyed the root system with the consequent death of the plant. "Moldex" (methyl parohydroxybenzoate) was added in an attempt to retard mold growth but was ineffective. The application of a higher concentration of this inhibitor was not attempted because i t was thought that this could inhibit the development of both P. brassicae and the host plant. Another series was set up i n the laboratory, the plants being grown in s i l i c a sand with nutrient solution being supplied by a subirrigation system. Gallon crocks, connected at the base to a large carboy containing nutrient solution, were f i l l e d with washed s i l i c a sand. The nutrient solution saturated the sand when the carboy was raised, and drained upon lowering the vessel, drawing air through the sand at the same time. Half the seedlings transplanted to this sand had been germinated and grown for two weeks in infested s o i l , the remaining plants had been germinated on moist f i l t e r paper and exposed i n watchglasses to a heavy spore suspension, consisting of washed, macerated, decayed club tissue. The plants were grown in the sand for 120 days. A l l seedlings i n i t i a l l y exposed to infested s o i l developed clubbed roots, however none of those exposed to spores in aqueous suspension did. Either infection did not take place at a l l , or the root hairs once infected were k i l l e d by the action of contaminant microorganisms. Although at an early stage of development the sand grown clubs were clean, decay soon set i t and the clubs at the age of two months were not much cleaner than those grown in s o i l . This was l i k e l y due to a carry-over of microorganisms on transplanting from the s o i l and to the necessity of keeping the sand nearly saturated with nutrient solution in order to prevent the clubbed plants from wilting. Preparation of resting spore inoculum. Two methods were used in the preparation of resting spore inoculum for in vitro studies of P. brassicae. I n i t i a l l y the method of Ayers (1) was followed, using s o i l grown clubs which were heavily invaded with secondary organisms. These clubs were macerated for three minutes in a Waring blendor and, after allowing the coarse debris to settle out, the supernatant was centrifuged and the material which had been spun down washed three times in tap water. The resulting spore paste was stored i n glass flasks at 5°C Secondly, in an attempt to obtain cleaner spores, the following procedure was carried out. Young clubs which had a minimum amount of decay were used. A l l fibrous roots and any decayed areas were cut away and the clubs washed in running tap water for one hour to remove any s o i l particles which had lodged in crevices of the hypertrophied tissue. The clubs were 25 then soaked in a ten per cent solution of "Chlorox" (5*25 per cent commercial calcium hypochlorite) for fifteen minutes to reduce the surface bacteria. After being rinsed three times in sterile d i s t i l l e d water, the clubbed tissue was macerated in a sterile Waring blendor for three minutes. The macerate was f i l t e r e d through cheesecloth and the f i l t r a t e centrifuged at 3300 r.p.m. for fifteen minutes. The material which was spun down was resuspended in sterile d i s t i l l e d water and recentrifuged three times. The wet spore paste was then stored in sterile flasks at 5°C Care was taken to minimize aerial contamination but i t was not feasible to carry out the entire procedure under completely aseptic conditions because the required equipment was not available. Germination of resting spores. As very l i t t l e infor-mation exists as to the germination requirements of P. brassicae resting spores, as many methods as possible were employed in the hope that one would produce the necessary germination stimulus. Spores, from both clean and decayed clubs, were sus-pended in sterile tap water, sterile d i s t i l l e d water, heat sterilized crude s o i l extract, and non-sterile crude s o i l ex-tract. The s o i l extract was prepared by pouring water through a clay pot f i l l e d with garden loam. The pH of these media was adjusted to l\..0, 6 . 0 , and 8 .0 . Half the flasks containing the spores were kept at room temperature and half kept in an incu-bator at 2 5°C Of those kept at room temperature, half were wrapped in aluminum f o i l to exclude light, the remainder were exposed to daylight. During the day small amounts of the sus-pension were withdrawn at four-hour intervals and examined under 26 a L e i t z phase contrast microscope. A solution of methyl c e l l u -lose was used as an a l t e r n a t i v e mounting medium to slow down the movements of the motile organisms. A .IN solution of iodine i n a two per cent solution of potassium iodide i n water was i n t r o -duced under the cover s l i p a f t e r examination of the l i v i n g organ-isms, and t h i s was found to f i x and s t a i n f l a g e l l a i n such a manner that the number and approximate length could be deter-mined, although d i s t o r t i o n was too severe to observe any further morphological d e t a i l s . In none of the treatments were P. brassicae zoospores p o s i t i v e l y i d e n t i f i e d . Neither temperature, l i g h t , nor pH had any apparent effect on the stimulation of germination. No one treatment had a high concentration of a heterokont b i f l a g e l l a t e organism of a size that would cause i t to be considered as a P. brassicae zoospore. There were markedly more f l a g e l l a t e organisms i n f l a s k s containing spores obtained from soil-grown, decayed clubs. In a l l the non-sterile s o i l extracts, as well as i n a l l media containing spores from decayed tis s u e , a b i f l a g e l l a t e heterokont zoospore was observed frequently, of a size ranging from 3.0 to 10.Ou i n length. Of p a r t i c u l a r i n t e r e s t i s the observation that several of these zoospores were t e t r a f l a g e l l a t e , each c e l l bearing two long and two short f l a g e l l a at I t s anterior end. The p o s i t i o n of the f l a g e l l a , however, indicated that the t e t r a f l a g e l l a t e organism was a r e s u l t of incomplete cleavage of protoplasm i n spore forma-t i o n rather than of fusion. Although i t was d i f f i c u l t to make out the i n t e r n a l structure and d e t a i l s of f l a g e l l a r morphology, 27 the biflagellate form of the zoospore matched the description given by previous authors (1, 28) except that the wide variation i n size would seem to lessen the possibility of i t s being a P. brassicae zoospore. Within two days bacterial contamination was so high as to preclude accurate observation with the phase contrast microscope. In an attempt to eliminate the bacterial contamination, two bacteriocidal agents, rose bengal at .067 per cent as reco-mmended by Martin (32), and streptomycin sulfate at £00 parts per million were added to the non-sterile s o i l extract contain-ing spores from decayed clubs. Although both substances retarded bacterial growth, the growth of water molds was extremely rapid as a result of the elimination of competition for the substrate. Because of the possibility of the phycomycetous organism releasing biflagellate zoospores which could be confused with the P. brassicae zoospores, attempts to reduce bacterial con-tamination by the use of bacteriocidal agents were not repeated. Although the number of motile organisms and bacteria was considerably less in flasks containing spores from clean clubs, the non-sterile s o i l extract medium contained several biflagel-late organisms. None of these, however, resembled those seen in flasks containing spores in non-sterile s o i l extracts and spores from decayed clubs. In a l l the flasks containing clean spores there were heavy concentrations of starch grains which hampered observations. It would appear that these starch grains are broken down by the microorganisms responsible for the decay of the clubbed tissue. Attempts to separate the starch grains 2 8 from the resting spores were unsuccessful as the two are of a similar size and density. Microscopic examination of these spores revealed them to have a less dense cytoplasm than those obtained from decayed club tissues and as this was taken as an indication of immaturity, the spores from young, clean clubs wiere not used in the experiments which followed. Resting spores from old clubs are shown in Figure I. The parasite within the root hair. On the basis of the above observations, and i n view of the d i f f i c u l t i e s involved in germination of the resting spores under in vitro conditions, i t was f e l t that attention should be directed at that portion of the l i f e cycle which takes place in the root hair of the plant. It was also hoped that an examination of the micro-environment of the root hair growing in a liquid medium would reveal some-thing of the processes of germination and penetration. Conse-quently, several attempts were made to obtain root hair infection in young cabbage and cauliflower seedlings. I n i t i a l l y , the method for obtaining root hair infection described by Samuel and Garrett (liO) was employed. Seedlings were germinated and grown In tumblers of infested s o i l for one week. They were then rinsed in d i s t i l l e d water and stained in acetocarmine. At the same time attempts were made to obtain root hair infection following the method described by Palm and McNew ( 3 5 ) . Seedlings were sown in tumblers of infested s i l i c a sand supplied with Knop's nutrient solution ( 2 ) . Seedlings were removed after six days and placed in one per cent aceto-carmine. No infections were observed in either method. In 29 both methods, particularly that of Samuel and Garrett, i t was extremely d i f f i c u l t to remove adhering particles of s o i l and sand without at the same time removing or damaging the root hairs containing the parasite. It was found that adhering parti-cles of sand and s o i l prevented the coverslip from lying evenly on the stained root and thus hampered observations. Another disadvantage to these two methods i s that the damage caused to the root hairs upon removal from the s o i l and on prolonged rinsing resulted in changes in the cytoplasm and consequent dis-ruption and obliteration of any stages of the parasite present. A solution culture technique described by Macfarlane (30) was tried. - Spores obtained from decayed clubs were suspended in a modified Hoagland and Snyder's solution as given by Macfarlane. Small vials were used to contain the spores and cauliflower seedlings were supported in the vials on fine mesh nylon gauze, in which holes for the roots had been punched. Macfarlane claims to have obtained abundant infections i n these vessels within four to six days. In the present experiment, however, the seedlings died within forty-eight hours, apparently from attack of the root system by bacteria and water molds intro-duced with the spores. This decay of the roots was hastened by lack of aeration in the narrow via l s . An attempt was made to obtain infection under aseptic conditions following the method of Chupp ( 8 ) . Diseased roots that contained spores but were not far enough invaded by bacteria were surfaced st e r i l i z e d in .001 per cent mercuric chloride, rinsed, transferred to agar slants i n test tubes, and minced finely. These were incubated for four days and any showing bacterial contamination were discarded. Then a few drops of sterile s o i l f i l t r a t e and a young cabbage seedling, which had been grown under sterile conditions in a petri plate, were added. After one week the seedlings were stained i n aceto-carmine and examined. Drops of the liquid surrounding the root hairs were also examined microscopically. This procedure proved to be very laborious and the great majority of slants were con-taminated. No root hair infection was observed, nor were any motile cells seen. It was apparent that very few spores were released upon mincing and i t was possible that there were insufficient spores to cause infection. Attempts were then made to obtain root hair infection of seedlings under conditions of minimum contamination. Spores were deposited on f i l t e r paper using a Buchner funnel and suction flask and washed several times with ster i l e , d i s t i l l e d water. One-third of these spore-laden f i l t e r paper disks were soaked in .001 per cent mercuric chloride for five minutes and rinsed three times with ster i l e , d i s t i l l e d water, and the re-maining third were used directly. Week-old cabbage and cauli-flower seedlings, grown under sterile conditions in petri plates, were placed on the f i l t e r paper bearing the spores in sterile petri plates. A few drops of ste r i l e , d i s t i l l e d water were added to provide the necessary moisture. In ten days time the seedlings were removed for examination. Prior to examination the seedlings were stained, then mounted in a semi-permanent fashion to preserve the roots for comparison to seedlings grown under similar conditions but not exposed to P. brassicae spores. 30 minced finely. These were incubated for four days and any showing bacterial contamination were discarded. Then a few drops of sterile s o i l f i l t r a t e and a young cabbage seedling, which had been grown under sterile conditions in a petri plate, were added. After one week the seedlings were stained in aceto-carmine and examined. Drops of the liquid surrounding the root hairs were also examined microscopically. This procedure proved to be very laborious and the great majority of slants were con-taminated. No root hair infection was observed, nor were any motile cells seen. It was apparent that very few spores were released upon mincing and i t was possible that there were insufficient spores to cause infection. Attempts were then made to obtain root hair infection of seedlings under conditions of minimum contamination. Spores were deposited on f i l t e r paper using a Buchner funnel and suction flask and washed several times with sterile, d i s t i l l e d water. One-third of these spore-laden f i l t e r paper disks were soaked in ten per cent "Chlorox" for ten minutes and rinsed three times in steril e , d i s t i l l e d water, one-third were soaked in .001 per cent mercuric chloride for five minutes and rinsed, and the remaining third were used directly. Week-old cabbage and cauli-flower seedlings, grown under sterile conditions in petri plates, were placed in sterile petri plates on the f i l t e r paper bearing the spores. A few drops of steril e , d i s t i l l e d water were added to provide the necessary moisture. In ten days time the seedlings were removed for examination. Prior to examination the seedlings were stained, then mounted i n a semi-permanent fashion to preserve the roots for comparison to seedlings grown under similar conditions but not exposed to P. brassicae spores. 3 1 Several stains were tried: one per cent solution of cotton blue in lacto-phenol, . 0 5 per cent aqueous Giemsa tissue stain, aqueous aniline blue at a 1 : 5 0 0 dilution, seventy-five per cent lactic acid followed by one per cent cotton blue in lacto-phenol, and one per cent aqueous aceto-carmine. Of these, aceto-carmine proved most satisfactory, staining the mature zoosporangia b r i l -liant red, and the young Plasmodia faintly pink. Seedlings were placed in aceto-carmine for three days, after which the root systems were mounted in the stain and heated almost to boiling. Sufficient pressure was then placed on the cover slip to flatten the radicle without damaging the root hairs. This was found necessary i n order to have the root hairs on a single plane for observation and photography. The coverslip was then sealed to the slide with a mixture of two parts vaseline to two parts lanollne to one part paraffin. This sealing compound was readily applied and kept the slides in good condition for up to two months. Infections were extremely few and sporadic (Table I ) . Although the seedlings in the "Chlorox" and mercuric chloride treated plates showed no greater incidence of infection than those untreated, the former plates remained free from decay several days longer than those untreated. A major disadvan-tage of this method was that the root hairs tended to adhere to the f i l t e r paper and were consequently damaged when the seedlings were removed for examination. The most successful method for obtaining root hair infection by P_. brassicae was found to be one in which week-old cabbage and cauliflower seedlings were placed in sterile 32 petri plates containing a thin layer of Knop's solution with trace elements, to which was added a .5 molar phosphate buffer containing spores. The spores were obtained from decayed club tissue and washed thoroughly by centrifugation. The seedlings were removed daily, rinsed in an isotonic buffer solution, and stained in the usual manner. Over five hundred roots were mounted in the manner previously described. Infection took place sporadically, but the number of infections was much greater than In any other method tried (Table I ) . Unfortunately, infection stages were seldom observed before ten to fourteen days after exposure to the spores, and by this time the seed-lings were no longer vigorous and healthy, the contaminants introduced with the spores having destroyed some of the root system. Phycomycetous water molds continued to be a problem as they obliterated the view of many root hairs. Early infection stages appeared in root hairs u n t i l the seedlings died at about three weeks. The young Plasmodia stained so faintly in aceto-carmine as to be indiscernible except in phase contrast, while mature zoosporangia stained very darkly and distinctly. It appeared as though the a f f i n i t y for the stain increased with maturity. Nuclei of the host c e l l stained deeply, but were in most cases a distinctive spindle shape, although occasional spherical nuclei were observed. Infection was apparently confined to the root hair, never having been seen i n the epidermal c e l l s . In only a few Instances was distortion of the root hair containing the parasite observed. Control seedlings grown under identical conditions but without 33 t h e s p o r e s were u s e d t h r o u g h o u t t h e o b s e r v a t i o n s f o r c o m p a r a t i v e p u r p o s e s . The d i s t o r t i o n s s e e n p e r i o d i c a l l y i n i n f e c t e d s e e d l i n g s were observed.', w i t h e q u a l r e g u l a r i t y on t h e c o n t r o l s e e d l i n g s . Y o ung P l a s m o d i a were s e e n c h i e f l y i n t h e t i p a n d m i d -s e c t i o n o f t h e r o o t h a i r ( F i g u r e s I I . a n d I I I ) . Anywhere f r o m one t o s e v e n P l a s m o d i a c o u l d be f o u n d i n a s i n g l e r o o t h a i r . T h e y were a t t i m e s somewhat i r r e g u l a r i n o u t l i n e , a l t h o u g h t h e m a j o r i t y a p p r o a c h e d b e i n g s p h e r i c a l i n s h a p e . The s i z e r a n g e d f r o m 3*7^ t o 7.1+n i n d i a m e t e r . B e c a u s e t h e n u c l e i o f t h e P l a s -m o d i a were n o t d i s c e r n i b l e I t was n o t p o s s i b l e t o d e t e r m i n e t h e number o f n u c l e i p e r P l a s m o d i u m . No l a r g e r P l a s m o d i a were s e e n a l t h o u g h o c c a s i o n a l l y c l u s t e r s o f o v e r l a p p i n g , s p h e r i c a l , l i g h t l y s t a i n i n g b o d i e s were o b s e r v e d . I t was n o t p o s s i b l e t o t e l l w h e t h e r t h e s e g r o u p s were f u s i n g P l a s m o d i a o r i n c i p i e n t z o o s p o r -a n g i a . The m a t u r e z o o s p o r a n g i a l c l u s t e r s were u n m i s t a k a b l e , c o n -s i s t i n g o f f r o m s i x t o t h i r t y s p h e r i c a l z o o s p o r a n g i a ( F i g u r e I V ) . E a c h z o o s p o r a n g i u m m e a s u r e d f r o m 5.0 t o Q.l\.p. i n d i a m e t e r , a n d i n many, z o o s p o r e c l e a v a g e l i n e s c o u l d be r e a d i l y s e e n . M e a s u r e -ments o f s t a i n e d z o o s p o r a n g i a were t h e same a s t h o s e o f l i v i n g z o o s p o r a n g i a . A n a t t e m p t was made t o m a i n t a i n a s e c t i o n o f i n f e c t e d r o o t i n a b u f f e r s o l u t i o n u n d e r t h e m i c r o s c o p e i n o r d e r t h a t t h e e s c a p e o f z o o s p o r e s f r o m t h e z o o s p o r a n g i a m i g h t be o b s e r v e d . However, t o o few i n f e c t i o n s were o b t a i n e d t o p e r m i t t h i s t o be r e p e a t e d u n t i l s u c c e s s f u l . A s t r i k i n g f e a t u r e n o t e d i n a l l t h e mounted r o o t s was t h e number o f r e s t i n g s p o r e s w h i c h a d h e r e d t o e a c h r o o t h a i r i n s p i t e o f t h e w a s h i n g e a c h r o o t s y s t e m r e c e i v e d p r i o r t o 31* staining (Figure IV). Because i t had been noted that the spores obtained from clubbed tissue tended to adhere together, an attempt was made to break down the matrix which held them by the use of trypsin, a proteolytic enzyme. Accordingly,J1:100 trypsin powder was dissolved in 0.85 P©r cent sodium chloride at 5000, 500, 50 and 5 parts per million. Two m i l l i l i t e r s of enzyme solution, two m i l l i l i t e r s of .5 molar phosphate buffer of pH 6.2, and six m i l l i l i t e r s of a heavy P. brassicae spore suspension which had been repeatedly washed in a centrifugation process, were incu-bated for fourteen hours at 25°C In f i f t y m i l l i l i t e r flasks, the f i n a l enzyme concentration being 1000, 100, 10, and 1 parts per million. A check with no enzyme was run at the same time. At the completion of the digestion period, the spores in each treatment were centrifuged and washed three times in .5 molar buffer of pH 6.2. Following this they were suspended in eight m i l l i l i t e r s of modified Knop's solution with two m i l l i l i t e r s of .5 molar phosphate buffer of pH 6.2. Six-day old cabbage and cauliflower seedlings, grown under sterile conditions in a petri plate, were placed in petri plates containing ten m i l l i -l i t e r s of nutrient-spore-buffer mixture, with the shoots sup-ported on one-quarter inch diameter glass rods. Seedlings were removed daily for examination in the usual manner. Again infection was sporadic and infrequent, however there were indications that spores exposed to the strongest concentration of enzyme had a slightly higher rate of infection than did the controls (Table I), however, a more extensive experiment would have to confirm this finding. 35 The relative effectiveness of the various methods of obtaining root hair infection by P_. brassicae, as carried out -in this investigation, i s presented in Table I. DISCUSSION It i s rather d i f f i c u l t to explain the confliction in reports which various workers have made concerning their success in germinating the resting spores of Plasmodiophora brassicae. Several methods reported as successful by other authors were carefully repeated i n these experiments with entirely unsatis-factory results. It would seem strange that many of the prob-lems and complications which were encountered by the present author were not mentioned by the majority of workers reporting success. It i s f e l t by this author that many of the techniques employed by previous investigators did not take Into considera-tion the problems inherent in an in vitro study of P. brassicae. The principle factor which leads to confusion i s the presence of a wide variety of contaminant microorganisms in the same micro-environment as the P_. brassicae resting spores. Several of these contaminants are similar in morphology to that reported for P_. brassicae zoospores. It i s apparent that very few investigators have realized the significance of this factor and thus the majority of conclusions which have been made on the morphology of the. germination product have been drawn from unsound evidence. Although in this study a biflagellate, hetero-kont organism resembling the description given by others was seen, It i s , in the author's opinion, entirely impossible to state that an organism present in a highly contaminated medium 3 6 i s a P. brassicae zoospore unless i t has been observed escaping from a P. brassicae resting spore. This process has been observed and described only three times in the entire history of the study of the organism and the reports are highly contra-dictory (ij.8, ij.6, II4.). This failure to associate the zoospore with the resting spore could account i n part for the conflicting descriptions of both morphology and size of the zoospore. It is granted that close examination of the organism under observa-tion is made d i f f i c u l t by the small size and rapid movement of the motile organisms. It would seem, however, that i f the high rate of germination obtained In various media by some workers (15, i f 6 , 1) actually occured, i t would take only continued observation to see the germination process as frequently as was necessary to obtain an accurate description of both the germination process and the zoospore i t s e l f . It i s indeed d i f f i c u l t to reconcile the extremely high germination rate and long v i a b i l i t y of P_. brassicae resting spores under natural conditions in the s o i l , with the very low, Inconsistent rate of germination in in vitro laboratory studies. The wide geographical distribution of the pathogen, i t s occur-rence in many s o i l types, i t s high resistance to drastic control measures and extreme temperatures, would indicate that the spores of this parasite are remarkably tolerant of abuse. The failure to obtain a good rate of germination and subsequent infection under carefully controlled in vitro conditions would then seem to indicate the lack of some specific germination factor present under natural conditions in the s o i l , but lacking under the circumstances necessary for the study and observation 3 7 of germination and infection in the laboratory. It i s entirely possible that the factor responsible for the maturation of the resting spore i s closely concerned with the decay of the root tissue by the s o i l microorganisms which are ultimately responsible for contamination in culture. It i s logical to suggest that the same process which aids in dispersing the organism should be effective in preparing It for germination, in somewhat the same manner that the spores of some coprophilous fungi are prepared for germination by passage through the digestive tract of an animal. The various enzyme complexes responsible for the break-down of host tissues could readily act as germination promoters. That this germination stimulus may be an enzymatic process i s suggested in these experiments by the increase, albeit slight, in the infection rate by spores exposed to the proteolytic enzyme, trypsin. Besides the proteolytic enzymes such as trypsin and pepsin, many others take part in the normal breakdown of plant material In the s o i l . Cellulase, pectinesterase, poly-galacturonase, and the Q enzyme, a l l could conceivably have a role in the preparation of the resting spores for germination. It remains to be seen whether a synthetic complex of enzymes applied to the spores could successfully duplicate the natural germination factors and induce a high germination rate. If the germination stimulus could be duplicated In such a manner, i t would then be possible to use spores from clean clubs, thus eliminating contamination by microorganisms. D i f f i c u l t i e s in technique are not confined to the germi-nation of the resting spores. This investigation illustrated that plants grown and infected in s o i l and sand are of l i t t l e 38 v a l u e i n t h e s t u d y o f P. b r a s s i c a e , a s i t i s n o t p o s s i b l e t o remove p a r t i c l e s f r o m t h e r o o t s y s t e m w i t h o u t c a u s i n g damage t o t h e t i s s u e . Damage t o t h e r o o t h a i r s c a n n o t be t o l e r a t e d i f t h e l i v i n g t i s s u e i s t o be examined, a n d i s t o be a v o i d e d i f s t a i n e d t i s s u e i s t o be u s e d . I d e a l l y , t h e e x a m i n a t i o n o f t h e p a r a s i t e w i t h i n t h e h o s t p l a n t , a n d I n p a r t i c u l a r t h e r o o t h a i r s , s h o u l d be c a r r i e d o u t on l i v i n g t i s s u e i n o r d e r t h a t a r t i f a c t s b r o u g h t a b o u t by f i x i n g , s e c t i o n i n g , a n d s t a i n i n g t e c n i q u e s may be a v o i d e d . The u s e o f l i v i n g t i s s u e a l s o p e r m i t s t h e e x a m i n a t i o n a n d o b s e r v a t i o n o f t h e d e v e l o p m e n t o f t h e p a r a s i t e , i f o n l y f o r s h o r t p e r i o d s o f t i m e . A l t h o u g h most w o r k e r s have u s e d s t a i n i n g t e c h n i q u e s (12, 8, 16, 20)' i n o r d e r t o s t u d y t h e p a r a s i t e I n t h e r o o t h a i r , none have m e n t i o n e d t h e ea s e w i t h w h i c h a r t i f a c t s a r e p r o d u c e d i n t h e s e t i s s u e s . T h i s i n v e s t i g a t o r f o u n d t h a t t h e r e i s a p r o n o u n c e d t e n d e n c y o f t h e c y t o p l a s m t o p u l l away f r o m t h e c e l l w a l l when t h e r o o t h a i r i s m e c h a n i c a l l y i n j u r e d o r e x p o s e d t o a t o x i c s u b s t a n c e s u c h a s a n y o f t h e commonly u s e d s t a i n s . S u c h c o a l e s c e d clumps o f c y t o p l a s m t a k e up s t a i n t o a g r e a t e r o r l e s s e r d e g r e e a n d c o u l d be m i s t a k e n f o r a r o o t h a i r p a r a s i t e o f a p l a s m o d i a l n a t u r e . A l t h o u g h r o o t h a i r n u c l e i a r e g e n e r a l l y o f a c h a r a c t e r i s t i c s hape, t h e o c c a s i o n a l a b n o r m a l n u c l e u s s e e n i n t h e s e e x p e r i -ments i s o f t h e same s i z e a n d shape a s a young P l a s m o d i u m . The d i s t o r t i o n o f t h e r o o t h a i r , t a k e n by most a u t h o r s (I4.8, 1) t o be a r e a c t i o n on t h e p a r t o f t h e h o s t p l a n t t o t h e f u n g u s , i s most p r o b a b l y c a u s e d by some o t h e r f a c t o r , a s i t o c c u r e d i n t h e s e e x p e r i m e n t s w i t h e q u a l f r e q u e n c y I n u n i n f e c t e d r o o t s . W i t h r e g a r d t o t h e e l u c i d a t i o n o f t h e l i f e c y c l e o f t h e 39 parasite, this study has done l i t t l e more than confirm the existence of an early amoeboid or plasmodlal stage and a later zoosporangial stage. Because no flagellate organisms arising from P_. brassicae resting spores were observed, and because spores adhered in large numbers and with tenacity to the root hair exterior, the suggestion i s put forth here that, In some cases at least, a protoplast i s exuded from the spore directly into the host through the c e l l wall at the point of contact, without the existence of a primary flagellate stage. This, however, is no more than a hypothesis, and along with the hypo-theses regarding the function of the so-called secondary zoo-spores and the existence of a sexual phase, remains to be sub-stantiated by irrefutable evidence. It is apparent from this study that such evidence w i l l be obtained only when satisfactory techniques have been developed. A method of obtaining high germination rate of the resting spores under conditions of minimum contamination i s essential In order that the product of germination may be studied. Neces-sary also i s a technique which w i l l allow the infection of the 4 host plant and growth of the parasite under conditions favorable for observation of the li v i n g parasite. Until these problems in technique are overcome, further work on Plasmodiophora  brassicae i s l i k e l y only to add to the confusion which already surrounds the problem, without in any way adding to our know-ledge of the l i f e cycle of the parasite. ho LITERATURE CITED 1. A y e r s , G.W. 1914J.. S t u d i e s on t h e l i f e h i s t o r y o f t h e c l u b r o o t o r g a n i s m P l a smodi opho r a b r a s s i c a e . Canad. J . R e s . , S e c t . C. 22:lij.3-1^9. 2. B o n n e r , James, a n d A.W. G a l s t o n . 1952. P r i n c i p l e s o f P l a n t P h y s i o l o g y . W.H. Freeman a n d Company, San F r a n s i s c o . 3. C a n a d i a n P l a n t D i s e a s e S u r v e y . 191*6. T w e n t y - s i x t h A n n u a l R e p o r t . D i v i s i o n o f B o t a n y a n d P l a n t P a t h o l o g y , S c i e n c e S e r v i c e , D o m i n i o n o f Canada D e p a r t m e n t o f A g r i c u l t u r e . 1*. C a n a d i a n P l a n t D i s e a s e S u r v e y . 19if7» T w e n t y - s e v e n t h A n n u a l R e p o r t . D i v i s i o n o f B o t a n y a n d P l a n t P a t h o l o g y , S c i e n c e S e r v i c e , D o m i n i o n o f Canada D e p a r t m e n t o f A g r i c u l t u r e . 5. C a n a d i a n P l a n t D i s e a s e S u r v e y . 1950' T h i r t i e t h A n n u a l R e p o r t . D i v i s i o n o f B o t a n y a n d P l a n t P a t h o l o g y , S c i e n c e S e r v i c e , D o m i n i o n o f Canada D e p a r t m e n t o f A g r i c u l t u r e . 6. C a n a d i a n P l a n t D i s e a s e S u r v e y . 195h> T h i r t y - f o u r t h A n n u a l R e p o r t . 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APPENDIX TABLE I R e l a t i v e E f f e c t i v e n e s s o f N i n e Methods o f O b t a i n i n g R o o t H a i r I n f e c t i o n by P l a s m o d i o p h o r a b r a s s i c a e No. o f No. o f M e thod R o o t s I n f e c t i o n s E x a m i n e d Seen 1. a f t e r Samuel a n d G a r r e t t 20 0 2. a f t e r P a l m a n d McNew 13 0 3. a f t e r M a c f a r l a n e i * . a f t e r Chupp \\$ 0 5. s p o r e s on f i l t e r p a p e r washed w i t h " C h l o r o x " 100 2 6. s p o r e s on f i l t e r p a p e r washed w i t h m e r c u r i c c h l o r i d e 95 2 7. s p o r e s on f i l t e r p a p e r washed w i t h w a t e r 101 1 8. s p o r e s i n p e t r i p l a t e s w i t h o u t t r y p s i n 531 35 9. s p o r e s i n p e t r i p l a t e s w i t h t r y p s i n 95 17 FIGURE I RESTING SPORES OF PLASMODIOPHORA BRASSICAE ( X 2000) FIGURE II YOUNG PLASMODIUM OF PLASMODIOPHORA, BRASSICAE IN ROOT HAIR (X 2000) FIGURE I I I YOUNG PLASMODIA OF PLASMODIOPHORA BRASSICAE I N ROOT HAIR ( x 3500) FIGURE IV CLUSTER OF ZOOSPORANGIA IN ROOT HAIR (X 11*00) 

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