VEPTICILLIUM WILT OF RASPBEREIES by Robert Harry Fulton “N ”N. .A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER CF SCIEHCE Department of Botany and.Plent PathOlOgy 1951 {/1 275/ 571%. \\ {TI' 1 \A a I‘ ‘ ACICI-O‘.'.’J.CC.|_;_;TZCRt Q o o o o o o I. II. III. IV. VI. VII. VIII. IX. X. «r A Bibli Review 05 Literature. ausal Organism . C‘ «w. 4L m o;...aoo.;;s 0.. A. Black Raspberries B. Red.Rasphcrries . Ketnods of Isolation. the Disease 0 . Aqvvv.‘ -'H inELB OF Cunll-.iS A. Preparation of Plant Tissue Specimen. B. Selection of C. Single-Conifi I-‘Zetho dis 0 f I noculation. Hoie of Infection . . Kechanism of Wilting. . Kutrient Theory . Thr mbosis Tlieory chnhjh- O O TOXIN. .Lll 80“". o o Cultural Studies. . . A. Effect of Temrt Mt B. Effect OI Cultm U1 C. Efiect of Acidti D. Enzyme Activity . Control Keasures. . . SUII‘JTLaI‘y o o o o o o :- OLI‘ Sup-21;)? o o o o o o o 0 NE“... s’h‘ifl rdLanulfirfi Teen. 1 .ues. G'CLS F0131? tion '2:-Cory. o o o - o o o o the Agar Xe6_ium for Plating C]. 13018. uionl [U 0 Ch K)! A CKL' CI'TLED 3—1231? T The writer wisles to express his sincere appreciation to Mr. Donald Cation for his assistance, constructive criti— cism, and guidance throughout the problem and during the preparation of the thesis. Above all for stimulating, by personal example, a correct philOSOphy of individual research which the writer has endeavored to gain. Thanks are also due to Mrs. Karie Mooar for her advice and helpful suggestions during he course of this study. In addition, the writer is grateful for tne support and encouragement from Er. C. A. Boyer, Chief of the Bureau of Plant Industry, Michigan State Department of Agriculture. CHAPTER I INTRODUCTION In the past ten years acreage of bramble fruits throughout Michigan, especially in the southwestern section of the state, has steadily increased. A corresponding increase in bramble plant losses, in.part due to a vascular wilt, has also been noted. Isolations indicated that this vascular wilt disease was frequently associated.with the fungus Verticillium albo:atrum Rke. and Bert. (M3). The writer in his investigations has found two factors mainly responsible for these losses. The first factor is the high degree of susceptibility of the commercial raspberry varieties now being grown in Michigan. The other factor is that on all of the farms visited in this investigation there was a history of crop rotations using potatoes, tomatoes, or eggplants at some time prior to the planting of raspberries. These crOps are well noted for increasing the Verticilliup_content of lesser contaminated soils. It appeared desirable to examine existing literature on Verticillium wilt and conduct as many experiments as possible that would lead to veri- fication or refutation of existing theories about the wilting mechanism, infection, and classification of the causal organism. From this extensive study it was heped that an accurate description of the symptoms of Eggtir cillium wilt of red and black raspberries could be interpreted for growers, county agents, and state inspectors. Finally, additional information on possible measures for the control of this disease was desired. CHAPTER II REVIEW OF LITERATURE The earliest recorded.plant species associated with a wilt disease caused by Verticillium is the potato (Solanum tuberosum). In 1879, two German investigators, Reinke and Berthold (M3), isolated a fungus from a potato plant affected with a disease called."Krauselkrankheit". On the basis of Hoffman's (26) findings, they named the causal organism Verticillium albg:§;rum. It was not until 19OM that a case of hadromycosis "(form of disease in which the parasite is confined almost exclusively to the xylem hadrome)" was reported in ginseng (Panex guinguefolium) by Van Hook (55). However, Van Hook reported the causal organism as Aprostalagmus albus which is actually synonymous with the older genus Verticillium (38). In the same year Verticillium was first recorded in Horth.America. Lawrence (31), in the Puget Sound area of western Washington,_described this fungus as the causal organism of "black raspberry (gphgg_ocgidentalis) bluestem”. However, he gave the name éprostalagmus cauIOQQggpg_to the causal organism. This is the earliest record of Yortigillium attacking any species of the genus Eyhug, Lawrence briefly described the symptoms of the disease on the Cumberland variety and directed most of his attention to the study of the causal organism. In 1918, Carpenter (9) referred the bluestem organism to the genus Vertigillium with the implication that it is identical with, or at least closely related to, Verticillium alhgzatgum, In 1923, Hockey (2h) isolated the organism from red raspberry in the Niagara district of Ontario, Canada. h. In his report he supported Carpenter's views, saying, ”The organism differs very little from and probably is identical with Verticillium albo-atrum Reinke and Berthold." In the same year the disease was reported in California (3 and 27), although it had been observed at an earlier date. In 192M, Verticilligm,was reported on red raspberry (Rubus idaeus) in England by Harris (23). He called the disease “blue stripe" for it was a better term than "bluestem" (31) due to the fact that most commercial varieties of the red raspberry have a general bluish coloration normally on the new canes near the end of the growing season. This maturing color- ation is also present on the new canes of black raspberries. The term "blue stripe” has not been adOpted by plant pathologists in horth.America. They reject the term because black raspberries can be infected with a virus that causes a disease,"eastern bluestem" (58), that shows occasional symptoms of pencil-like blue streaks on the new canes. The disease caused by Verticillium has therefore been called "western bluestem" to differ— entiate between the two. ' The term, ”bluestem", is sometimes confusing to plant pathologists because they are not always sure which disease is referred to. Because the organism Verticilligg_causes deep blue stripe or band.symptoms on the canes and because one of the striking characteristics of this disease is a yellowb ing and wilting of the leaves, the general term “blue stripe wilt" disease seems justified. An incorporation of all the terms or part is offered. These terms leave much to be desired since raspberry plants may also wilt from many causes, either physiological, fungus, or of insect origin. The 5. presence of discoloration on the cause is not an infallible proof of the absence of the disease, throughout the remainder of this thesis referred to as "wilt" for brevity. In 1926, Berkely and.Jackson (3) reported a new species of the organism, which they called Verticilligm ovatum, parasitic on five North American red raSpberry varieties (Ruhus strigosus) growing in New York and the Niagara-Ontario district of Canada. In 1931, Rudolph (#6) published a monograph.on.Verticillium albo— aigpm,» His studies on.§ppu§ included methods of inoculation and the host range of Verticillium isolated from raspberry. In 1936, Zeller (65) presented data of eight years research on Vertigillium wilt of cane fruits including symptoms, varietal resistance, field spread, and crop rotation studies. Recent investigations on the Verticillium wilt problem in gubug have been carried on by Wilhelm (60, 61, and 62). His studies included the vertical distribution of Verticillium in soils, varietal resistance studies on both raspberries and blackberries, and types of crops in rela— tion to the infestation of land with the wilt Verticilliug. CHAPTER III T'fl CAU SAL ORGAIII SM The genus Verticillium was created by Nees von.Esenbeck (38) in 1816. In the year 1838, Gerda (11) described the genus Acrostalagmus to accommodate an organism Aprostalagmus cinnabarinus. It was described as an organism that differed from Verticillium by forming its conidia in heads at the tips of the conidiophores. Hoffman (26), in 185%, advanced evidence that there was no actual difference existing in the manner in which the conidia are united in heads. Hoffman noted that the conidia are held together by e. slime which absorbs water in a moist atmOSphere, forming a globule of water at the tip of each sterigma. Within this globule of water the conidia appear to float about. If the humidity of the environment is in excess of the maximum for the moisture drOps to retain there globular form they will soon collapse, leaving in most cases one conidium on the sterigma tip, as described for the genus Verticillium. This finding indicates that there is no basis for a distinction between the genera Verticillium and Acrostalagmus. Verticillium albo-atrum Rke. and Bert. has been classified by Engler and.Prantl (16) in the section Eu-Verticillium of the genus Eggtiy Qillium of the Engaginaceae - Eyalosporae - Verticillieae. The conidiOphores are verticillately branched; conidia are formed at the tips of all branches and fall off readily. The distinction between the three sections of the genus EurVertigillium Sacc., Oggogladium Wallr., and Gliocgphalum Sacc., has not been sharply drawn. In the latter section the conidia are held 70 together by a hygroscOpic slime, while in the former two sections they are not. Therefore, in following Engler and Prantl's classification, if the characters are to be determined in their natural environment then 2331;- cillium albo-atrum would be placed in the section Gliocephalpm Sacc.. If, however, they are examined in water mounts, rarely more than one conidium would be found on each sterigma tip, and the fungus would there- fore be erroneously placed with the section Eu~Verticillium. Carpenter (9), Klebahn (29), and Van der Meer (56) support the view that Saccardo in 1886 (#7) had set aside the section of the genus Eggtir gillium - Glioceohalum for forms where the conidia are held together in heads. In this investigation on the morphology of the causal organism both types of conidia formations on the sterigma tips were noted. Also some of the original isolates made from raspberry began to form resting structures. Possibly similar structures were noted by Reinke and Berthold in their original study of Verticillium, ney noted that a blackening of certain cultures was due to blackened, septate, somewhat swollen hyphae which they named ”Dauermycelium". From these observations it seemed advisable to con- cur in the name of Ierticillium glbo-atrum.Rke. and Bert.. In 1913, Klebahn (29) isolated a Verticilligm from diseased dahlias and found it to be different from the species of Verticillium isolated by Reinke and Berthold. The isolate of Klebahn's produced microsclerotia while that of Reinke and Berthold formed resting mycelium. It was named by the finder Verticillium dahliae Klebahn. Since the classification of a. this possibly new species there has been a controversial taxonomic treat- ment of the two vascular Verticillia. Carpenter (9), Rudolph (M6) in their publications have stated that the original description by Reinke and Berthold was meant to include both types of resting structures. This is substantiated by the fact that Reinke and Berthold illustrated microsclerotium-like forms in their original plates. Rudolph.(h6) and Nelson (39) consider the presence or absence of microscler- otia an unreliable character and relate that the two forms should be classi- fied as Verticillium albo-aprum with the addition of a varietal specificity if indicated. The evidence of existing strains of Verticillium differing in their pathogenicity has been reported by Nelson on peppermint and by Snyder et a1. (51) on different varieties of vegetable crOps. Van der Meer (56), Berkeley, l-Iadden, and Willison (1+), and Ludbrook (33) support the views of'Klebahn and classify the microsclerotial pro- ducing forms as Verticillium dahliae Kleb.. Berkeley et al. (N) made a cultural and taxonomic study of the many strains of Verticillia and from these studies divided them into two groups: (1) Verticillium albo-atrum group which produces the resting mycelium form, but loses the power of producing this resting stage under continued culturing on artificial media; (2) Eertigillium dghlig§_group which produces the microsclerotial type of resting structure even under conditions of continued culturing on artificial media. However, the writer's observations concur with.Nelson's (39) that either one or both types may be present in the same culture. 90 Such fungi as Verticillia are composed of complex mixtures of established.biotypes and repeated isolations from this complex will result simply in separating from the mass either bingle biotypes or new combinations of them. Presley (Ml) reported saltants from monosporic cultures of-Vertigillium albo-atrum. By repeated transfers from a mono- conidial culture the writer has been able to isolate similarly four main biotypes of the Verticillium fungus, which are: 1. Microsclerotial dominant, slight mycelium 2. Fluffy aerial mycelium 3. Appressed mycelium h. Microsclerotial, abundant aerial mycelium Further transferring from biotype-h soon sectors this isolate into any one of the other three biotypes. The biotypes—l, 2, and 3 have re- nained constant in transferring over a ten month period. Throughout this study these three constant biotyoes will be referred to as 81, S2, and S3. (Figure l). Prolonged cultivation in the laboratory induces in many species of the fungi imperfecti an abrupt change in type of growth. This has been reported by many investigators on the genus Verticillium (21, M0, M1, and M6). Hansen and Smith (21) suggested that the basic unit of the individual is the nucleus and not the cell. Therefore, a multinucleate conidia as has been noted in Vertigilliwa (21) is not an individual but a colony, and it will not give rise to a genetically pure culture unless all of its nuclei are genetically identical. From this it may be hypothesized that the FIGURE 1 Biotypes 81, S2, and S3 of Vorticillium albo-atrum 10. variable forms of Verticillium may owe their instability not to mutation but to nuclear heterogeneity (heterocaryosis). It is widely known that the classification of Verticillia cannot be established on the basis of gross cultural characteristics on standard media. Since gross morphological characteristics cannot be used as a means of separation among Verticillium isolates from different species of plants, another means of classification must be found. is hostapathogen relation- ship can be used to distinguish between species as is found in the classi- fication of the vascular Fusaria group (51). This would be of more value in the ease of identification of this pathogenic fungus since no pro- nounced cultural or physiological differences have been reported by other investigators or noted in this study. The Verticillium pathogen isolated from raspberry in this study has a limited host range confined to the genus Eggu§_and a few species of the Solanacioug family. Another factor that could be used to further classify this fungus pathogen is the host - temperature relationship. Bennett (2) and Zeller (65) have reported that generally the most severe outbreaks of the wilt disease on raspberries occur in cool wet seasons and are correlated.with the minimum temperatures and the number of days of below freezing tempera- tures occurring in any'particular winter. This is at variance with the results obtained in this study for both laboratory and field data indicate that the most severe symptoms of Verticillium wilt on raspberry, induced by inoculations with the writer's Verticillium isolate, occurred during periods of high mean daily temperatures. Therefore, it is possible that 11. the isolate used in this study differs in temperature requirements from those used by Bennett and Zeller and thus temperature differences may possibly be used in the classification in the Vertigillia. Since the morpholOgical observations in this study concur with those of Reinke and Berthold and the isolates were not Specific to certain.plant species as reported in Verticillium dahliae Kleb., it seems advisable to name the causal organism in this study Verticillium albo-atrum Rho. and Bert. (M3). CHAPTER IV SYMPTOMS OF THE DISEASE lghglack Raspberries: The following symptoms are described from the Cumberland variety but the symptoms on the other varieties of black raspberry were generally similar. 1. General Symptoms The earliest signs of this disease on the black raspberry variety Cumberland are noticed on the current year's growth in late June or early July; The first symptoms seen are a dull green cast of the lower leaves when compared with the bright normal green color found on leaves of healthy shoots. The entire stool may be affected, but more often the disease is confined to a few of the young shoots. Then too, this disease works rather slowly and usually from one to three years are required to kill a plant. The disease progresses upward contrary to the observations of Czarnecki (l3) and Lawrence (31) who reported that defoliation took place from the top of the plant downward. The symptoms of the disease are yellowing of the first leaves and stunting of growth caused by the shortening of the internodes, and finally the entire cane becomes wilted and the affected shoots usually deve10p a dark blue color, entirely or limited to stripes. In the advanced stages of wilt the vascular tissue is discolored and has a water-soaked appearance. There is a definite line of demar— cation between the vascular tissue and.pith, the former is brownish in color and the latter usually a white normal color. In severe cases the pith may be slightly brownish in color. 13. 2. Leaf Symptoms The amount of leaves which will show symptoms will depend entirely upon the amount of root infection. If the entire root system is infected the whole plant shows visible wilt symptoms, (Figure 2). However, if only part of the root system is infected only that side of the plant above the crown in line with the infected roots will show visible signs of infection in the leaves. Generally, the lower leaves are the first to show signs of the disease. The uppersurface of the leaves develOpes an "off-green“ or yellowish bronze cast which may be manifested by yellow and green, or brown and green stripes. This striped effect is separated by the prominent lateral veins of the leaflets and is generally confined to one-half of the leaflet. .A whole leaf consists of a terminal leaflet and one to two pairs of lateral leaflets. The pattern on the black raspberry leaf extends from the midrib of the leaflet to its margin in a diagonal fashion following the lateral veins. The striping may be confined to one-half of the leaf- let; but it may and does affect other leaflets of the comnound leaf. Coinciding with the striped.pattern symptom, the leaflets occasionally tend to curl upward (noted in both black and red raspberry varieties) thus exposing the silvery underside of the leaflet, (Figure 3). A compound leaf may have one-half of the terminal leaflet and the lateral leaflet on the corresponding side affected with this striped pattern while the other half appears normal. There are many Wilt symptoms in Cumberland raspberry resulting from severe root infection following inoculation with Verticillium albo-atrum, bi type 51. mourn: 3 Upward curling of leaf margins in Indian Summer red raspberry following inoculation with Verticillium albo-atrum, biotype 82. ~ I BAA 11}. possible combinations of the striped pattern effect. This observa- tion corroborates the hypothesis of others that the effects of Vertigillium disease are concerned with the vascular system. Following the beginning ”off-green"color there is generally a marginal chlorosis, the leaflets turning from a yellow-green to a chocolate brown. his type of tissue discoloration may be erratically distributed throughout a single leaflet or envelOpe the entire compound leaf. The progressive pattern of necrotic areas is generally similar in all cases, beginning with a marginal and tip chlorosis of the leaf- lets soon followed by complete necrosis of the affected leaflet. It was also noted that when marginal necrosis occurred the entire leaf had a tendency to curl downward and resist pressing into a flat normal plane of position. Helson (39) in his studies of Verticillium on mint reported similar curling of the leaves. Leaflets additionally infected with one of the leaf spot fungi .(§eptozia rphi) or (Gleosporium veneta) tend to wither and die with more rapidity than noninfected leaflets. The lesions tend to act as focal points for the spread of the necrosis. The petioles may or may not show the blue discoloration char- acteristic of an affected cane. However, a pinching of the base of the petioles prior to actual leaf symptoms has been observed frequently. 3. Primo Cane (current year's growth) Symptoms A discoloration of the primo cane will occur generally at the same time or earlier than the leaf symptoms. This censsymptom may 15. begin at the groundline if only one or more laterals of the secondary roots are infected. The symptom may begin several inches above the groundline if the infection is confined to the small secondary roots which are produced directly from the main crown of the plant. If this latter type of infection takes place the entire crown may be destroyed in one growing season. If the causal organism does not attack the secondary lateral roots the crown is not totally destroyed and will produce small shoots from the adventitious buds. These small shoots will generally become infected before the growing season is over and they may defoliate from the base up or die back from the tip. The intensely blue to purple discoloration extends in varying heights at variable rates in the black raspberry primo canes. These rates which vary from zeroto nine inches per day, according to Lawrence (31), depend upon the environmental factors of temperature and moisture. A.high mean temperature and low soil moisture content generally are conducive to higher daily spread of cane discoloration. The rate of climb is much slower in the fruiting wood than in the prime canes and this may be correlated.with the lower transpiration rate in the former. The discoloration generally forms a longitudinal strips that touches every node along that side of the cane contiguous with infected roots. Occasionally the blue stripe symptom advances into the petioles in varying degrees. The discolored stripe gradually narrows and terminates to a point. 16. The bluish discoloration noted on the prime canes is not due to a.pigpent produced by the fungus or the host but is the result of the brownishpblue discoloration of the diseased bark as seen through the waxy bloom covering the cane. When the bloom is rubbed off, the purple color disappears (31, M6, and writer's observations). The boundary between the normal and discolored.portions, while rather irregular, is quite distinct. Discoloration in the cane may extend all around the cane. When two inches of a cane becomes encircled, the entire cane above the encirclement will wilt and die. In the fall it is necessary to distinguish between the blue stripe symptom and the maturing color of the cane. .As previously noted, blue discoloration appears on the cane near the groundline and is contin— uous in its pattern upwards, whereas change of color due to maturity often takes place irregularly in patches. This also holds true for red raspberry varieties. Generally, the infected growth of the current season often appears healthy if the season has been moderate. However, the follow» ing spring one finds most of these canes dead at the tips and for vary- ing lengths down the cane and to the base. New growth is usually noted at the base of these dead canes and the shoots appear healthy, though dwarfed.or stunted. 4. Fruiting Cane Symptoms The effects of the disease are finally climaxed in the fruiting canes. In the spring, the diseased canes are conspicuous. Some canes 17. are dead, others have shrivelled and poorly deveIOped buds. On some canes the dead buds lie in a longitudinal line. On sectioning such a cane, a strip of dead.wood was found beneath the dead line of buds while the Opposite side of the cane was green. The buds on the green side develOp normally at first but later there is a stunting in both lateral shoot growth and leaves. The wilting symptoms vary in their time of appearance on the fruiting cane. In some experimental plots the symptoms did not appear until the fruit was almost mature. On these canes the leaves become prematurely discolored and die followed by a drying up of the fruit and death of the cane. In other cases as soon as the first leaves had unfolded they became yellow and.withered up with no production of flower buds. Wilhelm (60) observed that brambles are most seriously affected in the first and second fruiting years. If the fruiting canes are not dead before maturity they will usually yield small tasteless fruit. In some cases, the berries, when.partially deveIOped in size, become perfectly dry except for the placentae which contains considerable moisture but is woody in texture. In Red.Raspberries: The following symptoms are described from the Latham variety but the symptoms on the other varieties of red rasp- berry were generally similar and in some cases were more severe (St. Regis and Indian Summer varieties). 18. 1. General Symptoms The red raspberry varieties have many symptoms of Verticillium wilt that are identical with those symptoms found on the blaCk rasp- berry varieties. The initial symptom is generally noticed on the current year's growth in the later part of July or early August. The lower leaves begin to change from a rich green to a light green color with a yellowish margin. Within a period of fourteen days the leaves begin to fall off. A distinguishing characteristic is that the termi- nal tuft of leaves are invariably the last to be affected. These terminal leaves often survive the rest of the leaves for a consider- able period of time. The prime cane discoloration found quite prominent in black raspberries is often absent on red raspberry varieties, or if present it is hard to distinguish from the normal bronze to reddish coloration. The fruiting wood generally has a deep brown to blue normal mature color that would mask any striping effect resulting from a Verticillium in- fection. In the red raspberry varieties a moderate infection of Vertigillium may be present for years without depriving the plantation of its commercial value. This is not the case in the black raSpberry varieties. 2. Leaf Symptoms The striped pattern effect as noted on black raspberry leaves is more noticeable on red raspberry leaves because of the more promi- nent interveinal areas. This pattern has been well described.previously. 19. It may be necessary occasionally to distinguish the defoliation of the Verticillium wilt disease from Spur Blight caused by Di vmella agplanata. In both cases the dead leaflets fall leaving the petioles attached. The petiole may be persistent for three more weeks. In the case of the Spur Blight disease a chocolate brown to darkened blue discoloration develOpes around the base of the petioles on the new canes. Pycnidia of the Spur Blight fungus can be observed with the aid of a hand lens in these discolored areas. In the case of Verticillium the discoloration generally appears in stripes and no fructifications of the fungus can be observed. The symptom of the entire compound leaf curling downward as in the raspberry virus leaf curl (2) was noted occasionally on black raspberries. However, with all the red raspberry varieties tested there was a pronounced downward curling of the terminal leaflet prior to actual color change in the leaflets. This downward terminal leaflet curling was also observed in the laboratory on tip cuttings of red raspberries placed in a filtrate of the toxin produced by the Eggpir gillégm.fungus. 3. Boot Symptoms In the red raspberry varieties as in the balck raspberry the infected vessels of the xylem are usually discolored. In severe cases most of the woody tissue in the roots have a pronounced brownishpred _ to black discoloration. The black raSpberries do not reproduce by rhizomes, consequently a diseased plant is more likely to die faster 20. than those plants of the red raspberry varieties. In these observa- tions the rhizomes frequently were free of the causal organism. How- ever, as the disease spreads in the roots these disease-free rhizomes eventually were attacked. It was also observed in confirmation of reports by Harris (23) and.Rudolph (M6) that red raspberry varieties infected with Verticillium do not sucker as plentifully as those free from this organism. Three red raspberry plants of the Indian Summer variety showing visual wilt symptoms were tagged in comparison with three plants that appeared normal in growth. Counts were made in the late fall of that growing season on the number of suckers produced by the parent plants. he entire plants were then carefully dug up keeping the root-sucker systems intact. Agar plantings were then made from the rhizomes of all sinparent plants at the points of sucker attachment and also from the individual sucker plants. The results from this eXperiment are recorded in Table I. TABLE I Isolations from normal appearing and diseased plants and the reduction of suckers resulting from Verticillium infection Infected.Parent Plants Normal Parent Plant Plants Used 3 3 Total Suckers Present 23 35 Vertigillia isolates from parent plants 3 O SUCKBB.PLANTS Vertigillia isolates 11+ 1+ Other organisms 9 31 21. From the foregoing table it is concluded: (I) Suckers from infected parent plants may be entirely free of the causal organism; (2) When red raSpherries are infected with Verticillium the amount of suckering is reduced. It will be noted from Table I that sucker plants from apparently healthy'parents had become infected. This possibly resulted from natural infections from organisms in the soil. This table suggests a point of interest in regards to field spread of the wilt disease organism. Sucker plants from infected parents can disseminate the fungus over the extent of their radius. The infected sucker plants growing in the aisles may be torn out of the ground by cultivating tools and dragged for some distance which may further spread the causal organism. CHAPTER V RETHDDS OF ISOLATION A characteristic of the Verticillium fungus is that it does not deve10p fast on artificial media. As a result the body of the culture medium may become densely overrun with bacteria and rapidly growing contaminant fungi (Fuaria, Alternaria, Gleosnorium, et cetera) that in- hibit the growth of the Verticillium isolate. The following methods of isolation were tested to determine which method would prove the most efficient to obtain pure cultures of Verticillium from diseased.plant tissues. A. Preparation of Plant Tissue Specimen Since the surface of the diseased tissue is often contaminated with secondary fungi it is desirable to kill or remove these organisms without injuring the pathogen in the inner tissues. .A series of three different chemicals possessing disinfective qualities were tested for their efficiency on surface disinfection of raspberry tissue. The chemicals tested were 0.1 per cent mercuric chloride, 3 per cent sodium hypochlorite, and l per cent Chlorazene. (All were tested in aqueous solutions). Tested in combination with these chemicals were two procedures for the handling of the diseased.plant tissues. 1. Small sections of the plant tissue were soaked in the specific chemical for one minute. Then, using a flame—sterilized scalpel, sections of the cane were cut and transferred to poured agar plates. 2. In addition to the foregoing procedure the plant tissue was run through four series of sterile distilled water rinses - this aids 23. in removing the residual disinfectant and possible surface cons taminants not previo~nly killed by the chemical. Procedure Number 2 gave the least contamination and more Verti- cillium isolates. Sodium hypochlorite or Chlorazene were suitable disinfectants as they were foot acting and left little toxic residue. Kany of the plant tissues used in this isolation work were thin leaflets and fibrous roots that could be penetrated by the disinfectant with the probability of killing the pathogen. Therefore, it was necessary to prepare the tissue without using a chemical disinfectant. Rinsing the plant tissues under running tap water for thirty to sixty minutes and then running the material through a series of four sterile distilled water rinses gave little contamination and proved satisfactory for isolation of the fungus. An 80 per cent increase in pure cultures of the causal organism were noted when the tissue plantings were cut frOm the region on the cane bor- dering the diseased and normal tissue. This area is apparently free from the secondary invaders. Over 75 per cent of all isolations made from cane tissue already discolored failed to yield Verticillium and most of the isolates were fungi of the Fusaria group. The Verticillium fungus could be obtained two to three inches beyond the discolored portion. No isolations of Verticillium were obtained from plant portions higher than one foot above the crown line unless so previously inoculated. Selection of the Agar hedium for Plating Since the Verticillium fungus deve10ps slowly a medium must be used that eliminates bacteria. Such a medium must be lacking in proteins 2L1. and have a pH around 5.0 to prevent excessive bacterial growth. The rapidly growing contaminant fungi thrive on media that are rich in carbohydrates; therefore, reduced carbohydrate formulation is desirable. Five different types of media tested were: (1) 2 per cent malt extract, (2) corn meal, (3) lima bean, (H) potato-dextrose, and (5) prune infusion, in 2 per cent agar. In addition, a natural media consisting of Cumberland black raspberry foliage chOpped into small pieces about three-eighths of an inch square and mixed in 2 per cent agar. The raspberry foliage was first sterilized in prepylene oxide vapors (52). The best type of media for isolation of the Verticillium fungus was prune agar. Prune agar is slightly acid in nature and very low in proteins, two characteristics that limit bacterial growth. The rasp- berry leaf media was fairly satisfactory for Verticillium isolation but proved of most value for microsclerotial production from $1 biotype. The microsclerotia were used in mass inoculations for varietal resistance studies. Single-Conidial Isolation Techniques There are many methods for single-conidial isolations. Two methods used in these Verticillium studies were as follows: 1. A modified.Hansen and Smith (21) procedure: Cultures of the bio- types were maintained on 2 per cent malt extract agar slants. 10 ml. of sterile distilled water was added to a test tube culture and the tube was vigorously shaken. This water which then contained many 25. conidia was transferred to a clean sterile test tube. The apprOXi- mate concentration of a conidial suspension was determined by direct microscepic counts of conidia carried in a M mm. transfer loop. Counts were made using the Howard counting cell. This conidial concentration was diluted by a series of 100p transfers to sterile distilled 10 ml. water blanks. When a 100p suspension contained approximately 50 to 75 conidia, as determined by count, it was transferred to a test tube containing 10 ml. of melted Coon's (M2) medium made with 0.5 per cent agar. This extremely low percentage of agar nade it possible to cover the bottom of three 90 mm. Petri plates with the 10 ml. of inoculated medium giving approximately 15 to 25 conidia per plate. The thinness of the agar in this technique greatly minimized the possibility of conidia being situated directly above one another and was eSpecially advantageous for the extremely small Verticillia conidia. dost of the conidia germinated within a twenty hour period. At the end of this period the Petri plate was placed on the stage of a compound microsc0pe, the cover removed, and the germinating conidia picked up singly on the tip of a flattened inoculating needle and transferred to 2 per cent malt extract agar slants. This technique was used in these studies as it gave a greater possibility of a single cell isolate. The dilution blank technique: Following the conidial dilution 26. procedure as previously described, direct loop transfers are made to previously poured and soliiified agar plates. Small rings previously drawn on the bottom outside of the plate with a wax pencil aid in placing the 100p transfers on the medium and also facilitates the location of germinating conidia for isolation. This technique has one disadvantage for single-conidial Verticillium isolations. The extrenely small conidia of Verti- cillium may lie directly above one another without detection and there is a possibility that the resulting transfer would not be a single-conidial isolate. The single—conidial transfer needle was made in the laboratory. An inoculating needle was heated red—hot and then hammered flat. The needle was then given a very sharp point by grinding on a whetstone. A point of such fineness must be sterilized by chemical means (1 per cent Chlorazene) as it is immediately destroyed in a flame. CHAPTER VI KETHCDS OF IEOCULATIOH The Verticillium fungus is soil borne (61) and_can penetrate the roots directly (39). However, a variation of three plant inoculation methods were studied. The methods used are outlined below. For each series of inoculations conparable check inoculations were made using sterile dis- tilled water.’ The fungus was later reisolated in pure culture from each plant to prove the actual pathogenicity of the fungus according to the principles of Koch's postulates (30). Method Number 1 The entire root system was simply dipped into a microsclerotial or conidial suspension and the plant potted immediately. Method number 2 The plant was potted and inoculated after it had become established. For inoculation a six inch iron rod was pushed into the soil manually near the base of the growing raSpberry plant, making three or four such holes around the plant. The pathogenic suspension was then.poured into each hole and the tOp of the hole covered with soil. Method Number 3 Using a flame-sterilized scalpel, a T—shaped incision was made into a cane to a depth of about one—eighth of an inch. Into this incision was placed a cotton wad previously soaked in a conidial or microsclerotial suspension of the causal organism. The treated incision was then covered with a prOprietary self-sticking latex tape called Sealtex. This tape not only sticks to itself but also seals against air and water loss from 28. the covered area. The relative incubation period using these three methods with three biotypes of the Verticillium fungus are presented in Table II. The shorter the incubation.period the more efficient the method. Table II indicates that the cane wound method has the shortest incuba- tion period of the biotypes used for inoculation. The $1 biotype ex- hibited the highest degree of pathogenicity as indicated by the shortest incubation period. Of further interest, this biotype showed the highest toxicity in laboratory studies. 29, mm : mm s :m mm 3 am : mm mm s m: a em mean mmmnpa mean .mmmuee mean mm mm OH OH NMMtDz peanmm soapsQSosH womb upsmam doanom soapmpaocH damp musmam doahm&.:oameSOsH pomp mpsmam .goapmHSooqw mo mpospma psmmoMMHw momma dam mmmhpoap passe Iwmfid omega mo own one mcfizoaaom soaked nowpmpdosa mbfipmamm HH mnmaa demo? memo Sofifim Hwom mam poem :ofipmaaoosH mo d mpofi CHAPTER'VII MODE OF INFECTION Reinke and Berthold (1+3) demonstrated the ability of the Verticillium mycelium to penetrate healthy root tissue. Their technique was to simply place mycelial bits in direct contact with potato roots grown in humid chambers. However, under similar conditions they were not able to acheive infection with conidial suspensions. Recent work of several investigators (6, 39, and 56) has independently confirmed the view upheld by Reinke and Berthold. Also, Nelson (39) has shown by histological studies that the Verticillium conidia can penetrate roots directly within a period of six hours. Other investigators (3, 9, 13, and 26) have advanced this theory on assumption only as they offer no investi— gational data to support their contentions. .A. Root Infection Studies Experiment Number 1 Nelson (39) showed that Verticillium conidia were attracted to mint roots, presumably by chemical or electrical phenomena. Following Ielson‘s technique, but using Cumberland black raspberry instead of mint, healthy, fibrous roots were placed in a Verticillium conidial suSpension. The roots were then subjected to a series of sterile distilled water rinses and vigorously shaken. After this procedure, slide mounts were made and examined. Many conidia were still found to be appressed to the surface of the roots. Experiment Eumber 2 An experiment was designed to determine whether isolates of 31. Verticillium could.penetrate directly into the roots. Two Cumberland black raspberry plants were grown in sand culture to develOp a more fibrous root system and to enable an easier removal of the roots from the sand with a minimum of injury. Two inch portions of these roots were placed in pairs on thirty- two aseptically clean glass slides in Petri dish moist chambers. A series of eight slides (sixteen inoculations) per biotype was used and bits of mycelium from the three biotypes were placed upon the apparently uninjured root sections. The remaining eight slides were. used as checks, the roots being subjected to small bits of sterilized cotton instead of inoculum. Within a thir y—six hour period, brown lesions develOped next to and under the inoculum in the SE and $3 inoculations. After a sixty hour period similar lesions were noted wi h the 81 biotype which con- sisted mainly of microsclerotia. As microsclerotia are considered to be a resting stage of he fungus (23, M3, and 61), it seems reasonable and is corroborated by the data that this thick-walled structure will take a longer period of time to germinate and infect. Isolations made from the foregoing diseased root portions readily yielded the Verticillium organism. There is little doubt that the organism penetrated for the isolations were made from surface sterilized roots, using the chlorine compounds and water rinses. Further evidence in support of this data is that no Verticillium isolates could be obtained from di- lution plates made of the rinse waters. Following the final rinse, 32. portions of the stelar tissue were teased from the roots by aseptic techniques and.plated. The results of this experiment are recorded in Table III. TABLE III Roots Inoculated Roots Containing Verticillium BIOTIPES NUEBER EUHBER 51 16 10 $2 ' 16 11+ 53 16 13 Check 16 O The above table indicates that all three biotypes are capable of penetrating root tissue. The lower amount infected in the $1 biotype possibly'results from the longer period required for the germination of microsclerotia. ZExperiment Number 3 Two inch sections of healthy fibrous black raspberry roots, obtained 'by the same procedure given in Experiment Number 1, were subjected to the following test. Using a flame-sterilized forceps, a section of the root ‘was picked up by its basal cut-end, the terminal unwounded end then being dipped into a conidial suspension of Verticillium to a one-half inch depth. The inoculated root was then placed on an aseptically clean glass slide in a Petri dish moist chamber. Other roots were similarly treated using eight roots per biotype. A check consisted of eight roots that had been dipped into sterile distilled water. After a two day period necrotic Specks were noted on the treated portions of all roots, while the check roots appeared normal. 0n the fourth day isolations were made and the Vertieillium was obtained in pure culture. This experimental evidence substantiates the results obtained in the foregoing experiment. No table is presented for all roots dipped into a conidial suspension yielded Verticillium upon isolation. Exp eriment Number )4 In 1922, Bewley (6) grew tomato seedlings in a nutrient agar and when the roots were well develOped he added a conidial infusion of Verticilli‘1 . The roots were readily attacked and the plants began to wilt in nine days. Van der Meer (56) conducted a similar experi- ment using cucumber seedlings and.potato tubers. He concluded with the statement, ”the roots were attacked either directly through the epidermal cells or through the root hairs." Using tomato seedlings for the test plants a modified technique from that given above was adepted. Aseptically clean Bonny Best temato seeds were germinated under aseptic conditions in sterile Petri dish moist chambers. After thirty-two of the young seedlings had developed their primary organs they were transferred singly to large sterile test tubes that contained a 15 m1. conidial suspension of the Verticillium fungus. Eight seedlings per biotype were used. The remaining eight seedlings used as checks were placed in sterile test tubes containing 15 ml. of sterile distilled water. The roots were apparently attacked by the organism, evident by the appearance of many small brown spots scattered over the roots submerged in the conidial suspensions. The wilt symptoms, as noted by Bewley (6) in nine days did not appear until the thirteenth day in thi s s tudy. Isolations from the affected root systems of the seedlings yielded the Verticillium organism upon platirc. Following is the data obtained from this experiment on tomato seedlings. TABLE IV Isolations of Verticillium following inoculation of unwounded tomato seedling roots Seedlings Used Yielding Verticillium in Culture BIOTYPE mans min-113113 Sl ‘ 8 h S2 3 6 S3 8 3 Che ck 8 O This experiment shows that the raspberry isolate of Verticillium is not only capable of root penetration but is also pathogenic to tomato (Selenium lycoocrsici). Experiment lIumber 5 A few investigators (13, 23, and M6) have reported actual field cfbservations on the presence of microsclerotia on the epidermis of 35. infected cane tissue. Isolation studies were not included in their investigational data to justify this field observation. Then too, their observations may have been confused with the fruiting bodies of Coniothyrium sp. and Diiymella applanta which are often found on diseased canes of raspberry. Recent investigations have shown that no fructifications of the Verticillium fungus have been observed on diseased canes in the field or greenhouse (61, 62, and 65). his observation, in agreement with the writer's, then eliminates to a great extent the possibility of wind blown infective conidia. However, there is a possibility that microscle- rotia may be disseminated by water or dust erosion of the soil. Micro- sclerotia are highly resistant to descication for they can withstand a constant temperature of 120 F for several months (61) and still be viable. Their average measurement is from twentyhfive to seventy-five microns in diameter so they are not only small in size but capable of being wind blown. Their occurrence in surface cultivated soils sub- stantiates the wind and water dissemination theory (61). A series of three black and three red raSpberries were grown in the greenhouse. After they had become established, the unwounded canes \ of the above plants were atomized with.pathogenic suspensions of the three biotypes. A similar series of chech.plants were atomized.with sterile distilled water. Vaseline barriers were used on all atomized canes to prevent the suspensions from flowing down the canes and into the soil where infection has been proven to take place. 36. All treated plants were placed in a moist chamber for twenty—four hours, as conidial germination studies indicated a 65 per cent germina- tion within twenty hours. After a forty day period no visual wilt symptoms were noted on any of the treated.plants. Isolations were made from the treated areas on the canes with negative results. Previous experimental trials described in Chapter VI show that in- fection will take place on the aerial portions of the plant if the organ- ism is placed in a wound on the cane. This type of infection, if present, would generally be found near the crown line within range of the micro- sclerotia known to be present in surface cultivated soils. CHAPTER VIII EECHAEISM OF WILTING Hutrient Theory In 1592, Atkinson (l) formulated the first important hypothesis on the nature of vascular wilts. He stated that infected plants wilted because the fungus utilized the available nutrients present in the tracheal fluid and thereby starved the host. Klebahn (29) took a similar view and even went as far as stating that the causal organism inhabited the vessels as a saprOphyte. Thrombosis Theory 1. Mycelial Thrombosis This theory is based on a mechanical plugging of the xylem elements by mycelial wefts or strands, eSpecially in the sieve plates and pits present in these elements. The first investigator to advance the idea that plugging of the vessels brought about a wilting symptom was Klebahn in 1913 (29). "Kycelial develOpment in the vessels," he stated, "eventually'plugged them to such an extent tha translocation of water and food materials to the upper parts of the plant was rendered impossible." Other investigators who were found to agree with the myoelial thrombosis theory are: Reinke and Berthold (M3), Van Hook (55), Berkeley and Jackson (3), Pethybridge (no), and I-Zaclean et al (31+). Arguments against this mycelial theory have been noted by Van der Rear (56), Bewley (6), Brandes (7), and others. Their investiga- tional data pointed out that plants suffering from severe "wilt" may have very few hyphae in their vessels. However, those plants showing only a mild form of "wilt" may have a pronounced congestion of their xylem elenents. 2. Gum and Tylose Thrombosis Theory This theory is substantiated by many investigators (15, MO, M9, 56, and others) who state that other types of occlusions, such as the formation of gums or tyloses in the xylem, are responsible for the blockage of the flow of water and food material upwards in plants so affected. Are tylose formations a primary cause of wilting or are they secondary factors? Tyloses are of common occurrence in many of the angiosperms. The deve10pment of tyloses either normally or as a result of wounding may be due to one or combinations of the following factors: (1) difference in pressure in the cells on each side of a pit membrane; (2) reduction of pressure; (3) cessation of conduction in the vessel thus permitting the membrane to expand into the cell. To disprove this theory one needs to only observe a diseased.plant in the field or greenhouse. On the affected plant the lower leaves may be dead or dying but the tOpmost leaves are generally turgid and show no visual ”wilt" symptoms. These former symptoms are associated with a lack of moisture and one would expect the terminal leaves to be similarly affected if an actual blockage did occur in the vascular system. Further investigational data providing strong evidence against the thrombosis theory (mycelial, gum, or tylose) was reported in 1922 39- by Dowson (15). By experimentation, Dowson, inactivated the conduct— ing channels of lilac and privet for long distances without a sub- sequent symptom of wilting. The writer repeated Dowson‘s emperinent on the inactivation of conducting channels using raspberry. Wedge-shaped pieces were cut from the canes of raspberry on alternate sides. This Operation vas done under water so as not to break the capillary flow of water. The cut surfaces were rubbed with vaseline and the canes were then removed from the water and.the wounds were covered with Sealtex tape. After a period of thirty-two days (average Verticillium incubation period) not the slightest trace of wilting occurred on the treated canes although their conducting tissues were inactivated over a long distance by mechanical plugging. Another argument against the thrombosis theory is that the disease ”wilt" is more severe during the cooler portion of the growing season (33). Bennett (2) and the writer have noted in their observations and experiments that Yerticillium wilt of raspberries occurs most during the hottest period of the summer with an apparent recovery during the cooler days in autumn. Bewley (6) stated that greenhouse tomato plants wilted at high temperatures but they recovered at still higher tempera- tures. Using the thrombosis theory as the criterion, how would one emplain why a tomato plant would reqrire less water to retain turgidity at a higher rather than at a lower temperature? #0. Gas Formation Theory Another theory that had been advanced by investigators is that a gas, such as carbon dioxide, is formed by the fungus and results in he formation of a gas pocket in the tracha . Such a formation would thereby break the transpiration stream and a flaccid, wilted condition would result. In 1926, Tochinai (5M) discovered that a large amount of carbon dioxide was produced by a vascular wilt fungus, Fusarium lini. In view of this theory a series of tests were then initiated to see if the biotypes of Verticillium isolated from raSpberry would produce a gas, (Figure h). The Durham type of fermentation tubes were used. This apparatus consists of pyrex test tubes in which are placed in an inverted position small glass vials (two inches long and one-quarter inch in diameter). If a gas is fo med by saccarolytic enzyme action it will displace the nutrient broth in the vial. The amount of gas formed may be roughly estimated but the kind of g.s cannot be determined by the use of the Durham fermentation.tubes. This type of apparatus possesses some advantages over the Smith fermentation tube, if only the presence of gas production is to be noted, as the tubes are easily cleaned, sterilized, and requires no special apparatus other than a test tube rack. To overcome the possibility of the vial restin: on the bottom of the test tube so firmly that it would exclude the fungus, the Open end of the vial was cut off at an angle. The inoculated tubes were periodi- cally shaken. 1+1. The media used for these test consisted of nutrient broth (3.0 gm. beef extract, 5.0 gm. peptone, 1,000 ml. distilled water) to which was added at a l per cent concentration the fermentable sugar. The results of these gas production studies as recorded in Table V show that no gas was formed by any of the three biotypes of the causal organism. However, data was obtained on the production of microsclerotia and the amount of mycelial growth. This is presented in Table V. From these studies it was concluded that no gas was formed by Ierticillium and that the gas production theory is untenable for the wilting resulting from Vorticillium infection. #2. 0 an a mqm