LIBRARY Michigan State University This is to certifg that the thesis entitled ‘ERTICILLIIH‘J 1NILT RESISTANCE IN POTATO presented by Franklin R. Fronek has been accepted towards fulfillment of the requirements for ljh. D. degree in Botany & Plant Pathology ,/ ,. 1' -" /' | . 1A-. "ff" m , . , , .. ,’ 7. ‘4’ L y A' A; V'L’ 31‘ Major professor // /I t L L '\ Date February 26, 1965 0-169 ROOM USE ONLY ROOM USE OM}: ABSTRACT VERTICILLIUM WILT RESISTANCE IN POTATO by Franklin R. Fronek This study was undertaken to determine varietal resistance of potatoes to Verticillium albo-atrum Reinke and Berth. (DM type) in Michigan. Tests were made on both muck and mineral soil types for 2 consecutive years. USDA $41956, Onaway, Early Gem, Green Mountain, and Arenac were the most resistant while Irish Cobbler, IA llOo—S, Cherokee, Tawa and Antigo were the most susceptible. Disease indices calculated from foliage symptoms and those calculated from tuber symptoms were in good agreement suggesting that varietal resistance could be evaluated from data of either foliage symptoms, tuber symptoms, or both. The root_dip, soil—slit, and seed-piece—dip methods of inoculation were compared in an attempt to find the most efficient method of inoculation. The seed-piece—dip method was eliminated after 1 year of testing as the results were not significantly different from those of the control. The soil—slit method was not consistent as the results were not significantly different from those of the controls in l of Franklin R. Fronek 2 years of testing. The results of the root—dip method were significantly different from the results of the controls in all tests. Yields of susceptible varieties were reduced more than those of the resistant varieties when infected by Verticillium wilt. Cuttings of resistant and susceptible varieties were used to determine if the tops of potato varieties exhibited a differential reaction. In filtrates diluted from 1:3 to 1:10, more wilting and vascular discoloration developed in Cobbler and Tawa than in 841956 and Green Mountain. Cuttings of these varieties responded to substances present in culture filtrates similarly to their resistance in field inoculations. Reciprocal cleft grafts were used to determine if the rootstocks controlled or influenced symptom expression of the side branches. 841956 and Tawa were reciprocally grafted as were Green Mountain and Cobbler. Resistant $41956, when grafted as a side branch on a susceptible Tawa plant exhib— ited considerably milder symptoms than did the Tawa top. However, somewhat more severe symptoms developed on the side branch than on the intact $41956 plant. When the reciprocal graft was made, the Tawa side branch on a $41956 plant exhib— ited more severe symptoms than the 841956 top. Somewhat less severe symptoms developed on the side branch as compared to an intact Tawa plant. The same reaction occurred when Cobbler and Green Mountain were reciprocally grafted. Franklin R. Fronek These results suggest there is an inherent resistance present in the tops of different potato varieties. These tops maintain this inherent resistance to the disease regard— less of the resistance of the rootstock. The rootstocks, however, contain a potential for allowing progression of the host—pathogen interaction to a level which may or may not affect the top of the plant. Symptoms of wilt appearing in the top of the plant are then understood as being influenced both by inherent resistance of the root stock as well as the resistance or tolerance of the top to either the fungus or its by—products. VERTICILLIUM WILT RESISTANCE IN POTATO By Franklin R. Fronek A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1965 ACKNOWLEDGEMENT I wish to express my sincere appreciation and gratitude to Dr° W. J. Hooker for his encouragement, guid— ance and help in the course of this research. I would like to thank Drs. E. S. Beneke, W. B. Drew, J. L. Lockwood, N. R. Thompson and M. L. Lacy for their suggestions in the prepara— tion of this thesis. Sincere appreciation is also extended to Mr. Leon Alwood for his help in greenhouse preparations and to other members of this laboratory for their help and encouragement. I am deeply indebted to my wife, Jill and also to daughters, Cynthia and Pamela for their patience, endurance, and encouragement during this time. ii TABLE OF CONTENTS INTRODUCTION LITERATURE REVIEW MATERIALS AND METHODS Varietal Resistance Cutting Reactions Grafting Experiments EXPERIMENTAL RESULTS Varietal Resistance Cutting Reactions Grafting Experiments DISCUSSION SUMMARY LITERATURE CITED iii Page 10 ll l3 13 19 22 35 42 44 Table LIST OF TABLES Varietal resistance to Verticillium wilt at 3 locations in 1958 Varietal resistance to Verticillium wilt as evaluated by 2 methods of inoculation at 2 locations in 1959 Relative resistance of varieties as determined by individual tests in 1958 and 1959 Tuber symptoms of Verticillium wilt following inoculation by 2 methods in 1960 Influence of 2 inoculation methods on potato yield in 1960 Differential response of cuttings of 2 resistant and 2 susceptible potato varieties in culture filtrates iv Page 14 16 18 2O 21 23 Figure LIST OF FIGURES Resistant 841956 (left) and susceptible Tawa (right) cuttings in sterile culture filtrates after 96 hours Susceptible Tawa plant (left) with wilting extending almost to the apex and chlorosis on the lower leaves 30 days after inoculation. Resistant 541956 side branch (right) is not visibly affected . . . . . . . . Susceptible Tawa plant (left) exhibiting severely wilted leaves extending to the apex with the lower leaves dried up and fallen off. Resistant $41956 side branch (right) is only slightly affected after 41 days . . . . . . Susceptible Tawa side branch (left) exhibit— ing severe Verticillium wilt symptoms 61 days after inoculation. Resistant $41956 plant exhibits moderate symptoms but is still actively growing Wilt symptoms (DI) in grafted 841956 and Tawa plants (average from 3 separate tests) Susceptible Cobbler and resistant Green Mountain reciprocal grafts 41 days after inoculation. (A: Cobbler plant (taller) with moderate symptoms while the Green Mountain side branch shows only slight symptoms. B: Green Mountain plant (taller) exhibiting slight symptoms while the Cobbler side branch shows moderate symptoms) Page 24 25 27 28 29 31 Figure Page Cobbler and Green Mountain reciprocal grafts 61 days after inoculation. (A: Green Mountain side branch (right) showing slight symptoms while the Cobbler plant tOp (left) shows severe symptoms. B: taller stem is Green Mountain plant exhibiting moderate symptoms while the Cobbler side branch exhibits severe symptoms) . . . . . . . . . . . . . . . . . . 32 Wilt symptoms (DI) in grafted Green Mountain and Cobbler plants (average from 3 separate tests) . . . . . . . . . . . . . . 34 vi INTRODUCTION Verticillium wilt is a disease of considerable importance in potatoes in Michigan. Little is known regard- ing varietal resistance of potatoes in the state and for this reason varietal trials were undertaken. A survey of Verticillium culture types was also made to determine the naturally occurring types of V. albo—atrum Reinke and Berth. in potato growing areas as this may be important in the resistance or susceptibility of a given variety. No reports concerning suitable field inoculation techniques are available. Several methods of inoculation were used and evaluated during these tests. The relation- ship of foliage symptoms, tuber symptoms, and yield has not been reported. Data were taken to determine the influence of varietal resistance on these characteristics. No observations have been reported as to the rela— tive role that potato roots and stems have in resistance to Verticillium wilt. In order to determine where resistance was located in the plant, resistant and susceptible vari— eties were compared in greenhouse tests. The response of cuttings of resistant and susceptible varieties was deter- mined in cell free culture filtrates. The effect of the potato root system on a plant top in terms of influencing symptom expression of the latter is not known. Resistant plants were grafted with a susceptible side branch to deter— mine if symptoms were influenced by either the root or stem portion of the plant. Reciprocal grafts were also made. LITERATURE REVIEW Considerable confusion exists concerning varietal resistance of potatoes to Verticillium wilt in the various potato growing areas in North America. Sebago has been reported to be fairly resistant to wilt in Idaho (Nielson, 1948), however it was susceptible in Maine (Ayers, 1952). Russet Burbank was resistant in Maine (Akeley, gt _1., 1956), but susceptible in Idaho (McClean, 1955). Menominee is some- what resistant but the variety seems to become more heavily infected in Maine than it does in Idaho (Akeley g: 31., 1956). Houma was reported as resistant in Maine (Akeley E: 31., 1956) whereas it was reported as susceptible in Connecticut (Waggoner, 1956). These inconsistencies may be due to one or more of a number of factors. The type of Verticillium present in an area may be an important factor in the resistance of a given variety. A11 isolates obtained from potatoes grown in Wisconsin and Eastern Canada (Robinson, _£I_1., 1957) were the dark myce— lial (DM) type. Isolates from Idaho and Oregon were pre— dominantly forms producing pseudo-sclerotia (PS) (Nielson, 1948). Both the PS and the DM types have been obtained from Maine but the DM type is more frequently isolated (Folsom, 1957). Russet Burbank was susceptible to the PS type but not to the DM type while Irish Cobbler was susceptible to the DM type but not to the PS type (Robinson, gt 31., 1957). This suggests that a variety may be susceptible or resistant as determined by the type of Verticillium present in a given area. Temperature may have an influence on the type of Verticillium present in a given area. It was determined that the PS type was the only one which grew at 320C while the other types grew at 280C (Robinson, gt 31., 1957). In Idaho, wilt has been reported as being more severe at low elevation where the growing season is longer and warmer than in higher elevations where the season is shorter and cooler (Nielson, 1948). Potato stocks that had an average of 26—30% wilt in the field in Maine had only 0—3% wilt when grown in Florida under much higher temperatures (Gratz, 1930). Other factors such as nutrition, soil type, and soil moisture may also play roles of varying importance in the disease syn— drome. Bewley (1922) made the first observation that a toxic fraction exists in culture filtrates of Verticillium albo—atrum. The toxic substance was precipitated with an equal volume of 95% ethanol. When the toxicant was resus— pended in distilled water, it was destroyed by heating at 1000C for 5 minutes. A complex polysaccharide, heterogenous in molecular size (Porter and Green, 1952), has been obtained from fil— trates. At least 2 fractions were demonstrated, one of which would not pass through a Seitz filter. Later it was demonstrated (Green, 1954) that a proteinaceous material was responsible for host responses other than vascular discolora— tion and that a polysaccharide was responsible for vascular discoloration. Wilting of cotton plants in V. dahliae filtrates was attributed to compounds of high molecular weight which im— peded the upward flow of sap (Karmel and Wood, 1956). Wilt— ing activity of filtrates on tomato was attributed to a thermostable material and not an enzyme (Threlfall, 1959). Verticillium metabolites produced in culture had a non-dif- ferential effect on hop varieties (Talboys, 1956) and fil— trates induced desiccation and leaf necrosis irrespective of the varietal resistance or the pathogenicity of the fungal strain. Scheffer, et a1. (1956) found that Verticillium cul— ture filtrates caused wilting and vascular discoloration when the original solution was diluted up to 1:16. Vascular discoloration did not occur however, if the filtrate was heated at 1000C for 10 minutes. They attributed the cause of wilting to nonspecific materials not associated with disease deveIOpment. A study of the relative role of root and stem in disease severity of hops (Keyworth, 1953a) indicated that the root was responsible for resistance. On susceptible rootstocks, both resistant and susceptible scions reacted as if severely infected. When susceptible and resistant scions were grafted to resistant stocks, only slight disease symp— toms were observed. Keyworth concluded that all stems showed a similar and positive resistance to invasion by Verticillium albo—atrum, and (Keyworth, 1953b) that the site of differential resistance was in the roots. Later (Key— worth, 1963) after experimenting with the effects of Fusarium wilt on grafts of monogenic resistant and suscepti- ble tomato varieties, it was conceded that an incorrect interpretation was made concerning Verticillium wilt resis— tance in hops. Grafting experiments also with the hop (Talboys and Wilson, 1956) suggested that the intensity of symptoms by the plant as a whole depended upon some inherent varietal characteristic of the root system. It was theorized that the roots of resistant varieties do not exert a protective influence on the aerial parts by translocation of substances inhibiting the fungus nor a stimulating resistance mechanism in the stems. It was also theorized that the primary cause of death of the plant may have been due to a reduction in the water absorbing capacity of the root system. Talboys (1956) also observed that symptom intensity is determined by the host~parasite reaction in the root and is not influenced by the stem. Localization of Fusarium wilt resistance in the roots of tomato was first demonstrated by Heinz and Andrus (1945). They found that the resistant Pan American scions were com— pletely susceptible when grafted on susceptible Bonny Best stocks. It was concluded that the resistance to the wilt organism was apparently localized entirely in the root system of the resistant variety and was not transportable. Intergeneric grafts were used by Davis (1953 and 1954) to demonstrate translocatable toxins in the Fusarium wilt disease of tomato. Non—susceptible scions of Solanum tuberosum, S. nigrum, Nicotiana tabacum and Physalis alkekengi were grafted to susceptible Bonny Best stocks. A11 scions showed symptoms of the disease as well as did the susceptible stock. Wilt of the scions was attributed to a translocatable toxin since the fungus could not be recovered from the scion. Susceptibility or resistance was shown not to depend on root produced factors in Fusarium wilt of tomato (Scheffer, 1957). Resistant and susceptible tomato varieties, Jefferson and Bonny Best were grafted in all combinations and inocu- lated with a conidial suspension of Fusarium oxysporum f. 1yc0persici. Inoculation was accomplished by allowing scion spurs to take up the conidial suspension. Only Bonny Best scions developed typical wilt symptoms. It was concluded that resistance of the scion was not altered by the stock and that the resistance present in stems is similar to that in the roots. Cleft grafts were used with Ailsa Craig and Loran Blood (susceptible and resistant tomato varieties respec— tively) in all combinations (Threlfall, 1959). Fusarium caused wilting on the susceptible stocks within 8—15 days, however, grafts on the resistant stocks involved never did wilt. Deese and Stahmann (1960) postulated that suscepti— bility to Fusarium and Verticillium wilts may be associated with greater pectic enzyme formation in the susceptible tissues. Resistance, on the other hand, may be due to a suppression of fungal pectic enzymes in the resistant tissue since there was an increased polygalacturonase activity in expressed sap of inoculated susceptible varieties while there was no such increase in inoculated resistant varieties. MATERIALS AND METHODS Varietal resistance.——Inocu1a were prepared by seed- ing flasks of potato dextrose broth (300 g peeled potatoes and 5 g dextrose per liter) with a conidial—mycelial trans— fer of V. albo—atrum (DM type). This was incubated for 5 days at approximately 220C on a culture shaker. Unless indicated otherwise, inoculum was used in this concentrated form. Potatoes were inoculated using.either the root—dip, seed—piece—dip, or soil—slit method. Root—dipping consisted of removing a l to 4 in. plant from the soil, breaking off the seed piece, dipping the roots and lower stem into the inoculum and replanting. Seed—piece—dipping involved immers- ing the seed—piece into the spore suspension, allowing the suSpension to dry for a short time, placing the seed piece in an open furrow and promptly covering it with soil. The soil-slit method consisted of placing inoculum close to freshly cut roots. For this a spade was inserted into the soil approximately 2 in. from the stem and to a depth approx~ imately 3/4 that of the seed piece. This was done on Oppo— site sides of a 1 to 4 in. plant. The Spade was tilted slightly to produce a slit in the soil approximately 1/2 in. at the top and 10 m1 of the inoculum, diluted 1:4 with water, 10 was poured into the slit. The spade was removed and the opening closed by stepping on it. Disease indices (DI) were calculated for foliage and tuber symptoms. Plants were placed in groups with appropri— ate numerical values on the basis of foliage symptoms: 0 (no symptoms), 1 (slight symptoms), 2 (moderate symptoms), 3 (severe symptoms), or 4 (dead). Foliage DI were calculated by the following formula. O'NO+1°N1+2'N2+3'N3+4'N4 O l 2 3 4 N = number of plants in each group with the subscript numbers indicating the numerical value of the group. Tuber discoloration was rated similarly as follows: 0 (no discoloration), l (slight discoloration), 2 (moderate dis— coloration), or 3 (severe discoloration). Tuber DI were calculated by the following formula. °N +1°N +2-N +3-N O 0 1 2 3 x 100 4 (NO+N1+N2+N3) D1 = Cutting reactions.——Aerial portions of representative resistant (Green Mountain and $41956) and susceptible (Cobbler and Tawa) varieties were used to determine if tops exhibited a differential reaction. Cuttings for work with culture filtrates were obtained by planting eyes cut from tubers in 11 flats as tuber units. Plants were examined for visual evi- dence of virus infection prior to being used in a test. When the plants were about 6 in. high, stems were excised at the ground level. Plants were surface sterilized by immers- ing top first into a 10% chlorine solution, and rinsing under running tap water. A fresh cut was made with a ster— ile knife in the basal sterilized portion of the stem. These cuttings were placed into the appropriate solutions in sterile 10 m1 flat bottom test tubes. Cuttings were main- tained at 220C with a constant light intensity for all tests. Foliage symptoms were evaluated as previously described. Filtrates were prepared by centrifuging and filter- ing the supernatant from DM cultures through a Seitz filter and later diluting with sterile distilled water. Controls of sterile water as well as those of potato dextrose broth were maintained. Grafting experiments.--P1ants were grown as for cut— tings except that when 3 to 5 in. high they were transplanted singly into 3 in. clay pots of sterilized soil. A scion top was cleft grafted on the stock plant 2 to 4 days later, the top of the stock plant left intact. This produced a bifur— cated plant with 2 tops, one of a resistant variety and one of a susceptible variety. For example, if a $41956 top was grafted to an intact Tawa plant, reference will be made to the $41956 side branch on a Tawa plant or reference will be made to the top of the Tawa plant which had a $41956 side 12 branch. Tawa and $41956 (both immune to potato virus X) and Green Mountain and Cobbler (both latent carriers of potato virus X) were reciprocally grafted. Grafts were not made between plants of these types of resistance to PVX because they were incompatible for the purposes of these trials. Inoculum from potato—dextrose broth cultures was ground in a Waring blendor at low speed for approximately 30 seconds to break up mycelial mats. Inoculation was made 1 to 3 days after grafting by the soil—slit method using 10 ml of inoculum per plant. Plants were grown in the greenhouse at approximately 240C. Foliage data were taken as soon as symptoms appeared. Controls consisted of non—inoculated grafted, non—grafted inoculated and non—inoculated plants. EXPERIMENTAL RESULTS Varietal resistance.--Varieta1 resistance was evalu— ated: (1) in a preliminary test in the greenhouse at East Lansing in 1958; (2) in field inoculation tests on mineral soil in 1958 and 1959 at the MSU Experiment Station in Lake City; and (3) in field tests on muck soil in 1958 and 1959 at MSU Experimental Farm in Bath. A more thorough test of inoculation methods was made in 1960 using representative susceptible and resistant varieties on both mineral and muck soil. The DM fungus type was used in all tests, as this was the only type recovered from about 150 V. albo—atrum isola— tions from Michigan potato growing areas. In 1958, the root-dip method was used in all 3 trials (Table 1). In the greenhouse, 12 plants of each variety were inoculated and 3 plants maintained as controls. In the muck soil, 14 plants were inoculated and 6 plants were maintained as controls in a non—replicated trial. In the mineral soil, 12 plants were inoculated and replicated 4 times with one additional replication of 12 plants maintained as non—inoculated controls. Symptoms of both foliage and tuber discoloration were rated numerically to determine if resistance and/or suscepti— bility could be accurately assessed by either or both types 13 14 Table 1. - Varietal resistance to_!gztigillinm_wilt at 3 locations in 1958 Greenhouse Muck Soil Mineral Soil Foliage Tuber Foliagg Tuber Foliagg Tuber Ave. Variety DI-Rank DI-Rank DI-Rank DI-Rank DI-Rank DI-Rank Rank 341956 4-1 4-1 62-1 31-1 6-1 20-1 1.00 Arenac 62-1 12-1 1.00 Early Gem 10-1 4-2 62-1 35-1 19-3 9-1 1.50 Onaway 29-1 31-3 40-1 39-3 0-1 28-1 1.67 Green Mountain 44-3 3-1 66-2 40-3 16-2 21-1 2.00 Red Beauty 23-1 3-1 23-4 28-2 2.00 Triumph 78-3 35-1 . 2.00 Epicure 35-2 0-1 21-3 51-3 2.25 Kennebec 86-5 45-4 2.25 IA 1111-2 69-2 37-2 20-3 33-2 2.25 Katahdin 31-2 11-2 78-3 36-2 18-2 60-4 2.50 Red Lasoda 33-2 20-2 86-5 39-3 15-1 44-3 2.67 Sebago 47-4 50-4 83-4 45-4 27-4 53-3 2.83 Pontiac 83-4 37-2 3.00 Russet Rural 16-2 56-4 3.00 Saco 79-3 41-3 3.00 Delus 38-2 24-3 71-2 47-4 47-5 37-3 3.17 Antigo 46-3 9-2 86-5 56-5 29-4 31-2 3.50 Chippewa 46-3 28-3 58-1* 49-5* 31-5 57-4 3.50 Emmet 79-3 43-4 3.50 Menominee 48-4 64-5 76-2* 51-5* 20-3 31-2 3.50 Merrimack 52-5 73-5 75-2 41-3 19-3 37-3 3.50 Tawa 46-3 70-5 79-3 35-1 35-4 74-5 3.50 IA 1106-5 89-5* 37-2* 3.50 Cherokee 63-5 57-4 83-4 49-5 15-1* 67-5* 4.00 Huron 55-5 33-4 17-2* 63-5* 4.00 IA 1109-9 93-5 41-3 4.00 Cobbler 52-5 53-4 80-4 64-5 33-5 60-4 4.50 Boone 50-4 57-5 36-5 86-5 4.75 *Comparisons with poor agreement between foliage and tuber symptoms. 15 of symptoms. Within each test, varieties were arranged in descending order of resistance. They were divided into 5 groupings of approximately equal numbers of varieties. These groups were assigned a rank number from 1 (resistant) to 5 (susceptible). When varieties tended to group natural— ly with respect to the DI, the number of varieties in the groups was modified accordingly. Generally there was agree— ment between foliage and tuber rank numbers. Differences in foliage and tuber rank values were in poor agreement in only 5 of 64 comparisons. In the 1959 tests (Table 2), the seed—piece—dip and soil—slit method were compared in an attempt to improve the efficiency of field inoculations. The test was made on both muck and mineral soil. There were 12 plants in each treat— ment. Treatments for each variety were side by side and varieties were replicated 6 times in randomized blocks° In the muck test, there were no apparent differences between treatments or between varieties during the early part of the growing season. Later in the season when foliage symptoms normally develop, a Botrytis foliage infection made it impossible to assess the amount of injury attributable to Verticillium wilt and only tuber discoloration data were obtained. On the mineral soil, foliage and tuber discolora— tion symptoms were evaluated. An analysis of variance was used to determine if the treatments were significantly dif— ferent at the 5% level. When significance was established, l6 .HoboH *m on» we hosvo some 502% vcoaouwwp havchwmwumwm who mzofiaonmwmop hovvoa vcohommwp avg: mucoaveoha .vmov munch oanwvase m.:aocsa u mzn \m. a n6 e n n6 a a an a \m.mza om.¢ m-mm m-mm m-mm m-ms m-es m-eo m-Hm s-oe m-mm omxouaso no.2 ¢-nH s-Om s-nm m-on m-me m-ow m-Ha m-mm m-ow 6369 we.« m-wm m-mm m-me e-om s-nm ¢-nm e-NH m-sm fl-mm m-HHHH Hoe-coo men-comm eeHm-Heom Hoe-coo-meo-eomm seam-226m Hoe-coo nan-60mm.ma4wuaqu. xnmm meovmahm ommwaom msovmahw gonna mfiovmahw henna .w>< deem seamen: anew sou: .amma cw mcofivnooH N 90 scavmfisoonfi “o mpocvoa N ha poumaam>o m0 9H“? ESMHH«0wvuo> ov oocmvmflmoh Hopoflhc> .N wands 17 a Duncan‘s multiple range test was used to determine in which treatments the DI were significantly different. In all 3 tests, the D1 of the soil—slit method was significant— ly different from the DI of the non—inoculated control. The DI of the seed—piece-dip method was not significantly differ— ent from either the DI of the soil—slit method or the control. Generally, good agreement was obtained between tuber and foliage symptoms. Overall relative resistance (Table 3) of these vari- eties was determined using the rank numbers as previously described. Rank numbers from all tests in 1958 and 1959 were averaged separately for foliage and for tuber symptoms. These 2 averages were then used to determine the overall relative resistance. $41956, Onaway, Early Gem, Green Mountain, and Arenac were the most resistant while Antigo, IA 1106-5, Cobbler, Tawa, and Cherokee were the most suscep— tible. Selected varieties from these groups were used to compare the effectiveness of inoculation techniques and for greenhouse studies which followed. In the 1960 tests, the soil—slit and root—dip method were compared. The seed—piece—dip method was omitted because it was not significantly different from the control in pre— vious tests. Only 6 varieties were used, 3 resistant and 3 susceptible. Potato tubers were split into 3 pieces, 1 each was used in the control, in the soil-slit, and in the seed— piece—dip inoculation method so that a direct comparison of 18 Table 3. -- Relative resistance of varieties as determined by individual tests in 1958 and 1959. Tuber Foliage Variety symptomsS/ symptomsg/ Average 541956 1.44 1.17 1.31 Onaway 1.44 1 50 1.47 Early Gem 1.56 1.50 1.53 Green Mountain 1.33 2.00 1.67 Arenac 2.43 1.75 2.09 Katahdin 2.56 1.83 2.20 Emmet 3.00 1.50 2.25 Saco 2.57 2.75 2.66 Red Lasoda 2.67 2.67 2.67 Kennebec 3.00 2.75 2.88 Merrimack 2.83 3.33 3.08 Delus 3.33 3.00 3.17 Menominee 3.44 3.17 3.31 IA 1109-9 3.00 3.75 3.38 Chippewa 3.44 3.33 3.39 IA 1111-2 2.88 4.00 3.44 Sebago 3.67 3.33 3.50 Antigo 3.00 4.00 3.50 IA 1106-5 3.29 4.25 3077 Cobbler 3.56 4.50 4.03 Tawa 4.56 3.67 4.12 Cherokee 4.78 4.17 4.44 '2/ average rank number from all individual tests. l9 treatments was possible. Twelve plants were used in each treatment which was replicated 5 times in randomized blocks. Only tuber discoloration and yield data were taken. Tuber vascular discoloration (Table 4) of the root- dip method of inoculation was significantly more severe than that of the soil-slit method or of the control, while the latter 2 were not significantly different from each other. Vascular discoloration in the 3 resistant varieties was significantly different from that in the 3 susceptible vari— eties. Total yield of plants inoculated by the root—dip method (Table 5) was reduced significantly from those of the soil—slit method or of the non—inoculated control. However, the latter 2 methods of inoculation did not differ signif- icantly from each other. Under disease conditions, the per- cent reduction in yield was more for the susceptible vari— eties than for the resistant varieties. Cutting reactions.—-Potato cuttings were placed in culture filtrates to determine if varieties exhibited a differential reaction. Potato dextrose broth controls were changed every 24 to 48 hours because of bacterial growth. Culture filtrates and water controls did not become cloudy, however additional solution was added every 24 to 48 hours. Foliage symptoms were recorded 4 to 7 days after the test began. Stem vascular discoloration data were also obtained using the rating system previously described for tuber 20 Table 4. -- Tuber symptoms of Verticillium wilt following inoculation by 2 methods in 1960. Inoculation by 5011- Root- Soil type Control Slit . Dip Ave. '2/ and Variety DI DI DI DI DMR muck soil 541956 7 7 19 9 a Green Mountain 5 9 20 12 a Onaway ll 13 . 23 15 a Tawa 29 36 47 37 b Cobbler 36 44 52 44 be Cherokee 36 43 6O 47 be Average 20 25 36 DMR'S/ a a b mineral soil Onaway 3 4 13 7 a Green Mountain 3 7 16 8 a 541956 4 9 13 9 a Cherokee 17 21 35 24 b Cobbler 17 21 4O 27 b Tawa 20 25 43 29 b Average 11 15 27 DMR‘E/ a a b .E/ = Duncan's multiple range test. Treatments with different letter designations are significantly different from each other at the 5% level. 21 Table 5. -- Influence of 2 inoculation methods on potato yield in 1960. Soil type Control Soil-slit Root-dip and Variety lbs. 1bs.-%g/ lbs.-%g/ muck soil Green M0untain 29 28-95 20-68 541956 16 14-91 10-66 Onaway 43 39-90 28-66 Tawa 34 29-83 21-62 Cobbler 34 29-84 18-53 Cherokee 35 30-85 18-50 Average 32 28-86 19-61 DMR-B/ a ab b mineral soil Green Heuntain 38 35-91 28-74 Onaway 38 34-88 28-74 541956 35 32-93 22-69 Tawa 29 28-95 16-54 Cherokee 31 24-78 14-44 Cobbler 32 30-93 10-33 Average 34 31-88 20-58 DMR'E/ a a b 2/ yield of inoculated plots expressed as percent of the non-inoculated control plants, in randomized blocks replicated 5 times. '2/ DMR = Duncan's multiple range test. Treatments with different letter designations are significantly different from each other at the 5% level. 22 vascular discoloration. Leaf epinasty appeared on all varieties within 24 to 36 hours after the cuttings had been placed in the fil— trates. This was followed by wilting, chlorosis, and necro— sis which appeared earlier and was more severe on the suscep- tible varieties than on the resistant varieties. These symptoms were very similar to those seen under field testing conditions, however, epinasty was more evident in the cutting reactions than in the field. Results of all 4 tests (Table 6) were very similar. Tawa and Cobbler exhibited moderate symptoms. $41956 was very resistant and symptoms did not progress beyond epinasty, slight wilting and slight chlorosis (Figure 1). Green Moun— tain was intermediate to the above groups in symptom expres— sion. Grafting experiments.-—In the first reciprocal graft— ing test involving susceptible Tawa and resistant $41956, symptoms were first evident 30 days after inoculation (Figure 2). At this time, tops of Tawa plants which had $41956 side branches were wilting and a slight chlorosis was evident on the lower leaves. $41956 side branches were symptomless. In the reciprocal grafts on $41956 plants, no symptoms were present on either Tawa side branches or $41956 tops. 23 MN I me MN I on s I oN o I m omnuo>m ovmuvawm o I OH o I w o I NH O I m o I w 0 20903 a - 2m m - as m - mm o - w o - w o ¢N2 recap mm - we we - so an - no n2 - me o - 02 m2 2.2 6262222m Amhmo ov .¢ vmoB 2 I m N I 02 o I MH 0 I w o I e 0 Love: m - 22 e - w2 m - mm o - 02 o - s o 02.2 spots #2 I ¢N 2N I an 0N I Ne e I m2 0 I 0 N2 0222 ovmthMH 26ame RV .m 6669 o I 02 o I 52 o I m2 0 I w o I o m Lopez N2 I an HN I om mN I am 0 I 2N o I o o m22 ovmhvawm ”whee NV .N puma O I m 0 I w 0 I m 0 I ¢ 0 I o m poem: m - mm m - mm o - me o - 2m 0 - a m mu2 6262-222 Amhmp NV .2 pmoe Ha Ha Ha Ha Ha Ha HO HO Ha Ha #:0590028 20295229 ou020>< Eovm owmflaom Beam de2202 Beam mmwMHOh Eovm MNWMHom hon hoannoo 0309 :«mvcsoz compo mwmaem magmam mowvoflhm> manwvmoomsm mofivthm> vcmvwwmom .movmhpaflm 0259200 :2 mowvowhmb ovmvom wanfiemoomSm N can vcmvmwmoh N we mm:wvvso Ho uncommoh Hmfipcohomwwa .0 02009 Fig. 1. Resistant $41956 (left) and susceptible Tawa (right) cuttings in sterile culture filtrates after 96 hours. 25 Fig. 2. Susceptible Tawa plant (left) with wilting extending almost to the apex and chlorosis on the lower leaves 30 days after inoculation. Resistant $41956 side branch (right) is not visibly affected. 26 After 41 days, tops of the Tawa plants including apical leaflets were severely wilted (Figure 3). At this time only a slight chlorosis was evident on the lower leaves of resistant $41956 side branches. Tawa side branches on $41956 plants in the reciprocal grafts, exhibited moderate symptoms while only slight symptoms were evident on the tops of $41956 plants. After 50 days, tops of Tawa plants exhibited severe symptoms while $41956 side branches had slight to moderate disease symptoms. Tawa side branches on $41956 plants showed moderate to severe symptoms while $41956 tops exhib— ited only slight to moderate symptoms. After 61 days, Tawa side branches on $41956 plants exhibited severe symptoms while $41956 plant tops had only moderate symptoms (Figure 4). Tawa plants in the reciprocal grafts with $41956 side branches were almost dead at this time while $41956 side branches exhibited severe symptoms. In the second and third tests involving grafts of Tawa and $41956, symptom expression was essentially the same as in the first test. Symptoms first appeared on Tawa plants which had $41956 side branches at 24 and 34 days respectively in the second and third tests. Experiments were terminated at 45 and 51 days respectively. Average DI plotted against days after inoculation for these 3 tests is shown in Figure 5. 27 Susceptible Tawa plant (left) exhibiting severely wilted leaves extending to the apex with the lower leaves dried up and fallen off. Resistant $41956 side branch (right) is only slightly affected after 41 days. 28 Fig. 4. Susceptible Tawa side branch (left) exhibiting severe Verticillium wilt symptoms 61 days after inoculation. Resistant $41956 plant exhibits moderate symptoms but is still actively growing. Fig. Disease index (%) 5. 29 80 2 60 b J. / / l/ 40 — Q / «9 Co / 0 q?«’ J. a?“ b?! / <8 ‘0, 9 / _ OQ/ 5,3 / ‘9 C; / O / ‘9 / ‘9 ‘Ofit/ QQO/( __ . be! / ‘Q'g // 20 6;.r.$9 / 0 .‘QQf/b" be / 66 £0 / $0 //’ 4‘5 _ // ‘QZAK' $ / x / // I // __,..r_’_-4" / 0 ——-——- l L ‘ I 25 30 35 40 45 Days after inoculation Wilt symptoms (DI) in grafted $41956 and Tawa plants (average from 3 separate tests). 30 In the first grafting test involving susceptible Cobbler and resistant Green Mountain, symptoms appeared after 30 days. Cobbler plants with Green Mountain side branches were slightly wilted while the resistant side branches were healthy. In reciprocal grafts, the Cobbler side branches and the Green Mountain plant tops were both beginning to show a very slight wilt. After 41 days (Figure 6), Cobbler plants exhibited moderate symptoms while Green Mountain side branches were slightly wilted. In the reciprocal graft, Green Mountain plant tops exhibited very slight symptoms while Cobbler side branches exhibited moderate symptoms. After 50 days, Cobbler plants showed moderate to severe symptoms while Green Mountain side branches showed only slight symptoms. In the reciprocal grafts, Green Moun— tain plants exhibited slight symptoms while Cobbler side branches exhibited more severe symptoms. After 61 days (Figure 7), Cobbler plants with Green Mountain side branches, showed severe symptoms while the side branches showed only slight symptoms. In the recipro- cal graft, Green Mountain plant tops showed only moderate symptoms while Cobbler side branches were severely wilted. In the second and third tests involving grafts of Green Mountain and Cobbler, symptom expression was essen- tially the same as in the first test. First symptoms 31 Susceptible Cobbler and resistant Green Mountain reciprocal grafts 41 days after inoculation. A: Cobbler plant (taller) with moderate symptoms while the Green Mountain side branch shows only slight symptoms. B: Green Mountain plant (taller) exhibiting slight symptoms while the Cobbler side branch shows moderate symptoms. Fig. 7. 32 A Cobbler and Green Mountain reciprocal grafts 61 days after inoculation. A: Green Mountain side branch (right) showing slight symptoms while the Cobbler plant top (left) shows severe symptoms. B: taller stem is Green Mountain plant exhibiting moderate symptoms while the Cobbler side branch exhibits Severe symptoms. 33 appeared after 24 and 34 days, and tests were terminated at 45 and 51 days respectively. The average DI plotted against days after inoculation for these 3 tests is shown in Figure 8. Disease index (%) 34 80 — 6O - \- 009 //’. 93‘ x’ Q; / 40 — «9‘2 8£:’/ .3 09 / ”\r / e} ‘00 C39 ‘60\ 0° £.é‘,’ 00 ,2‘ 00 ll 0‘9// 8- 7.9/ élf V§3f ‘9 / . be / 20 — ”Q” 9" ’ ‘09 o / S ’ a} / '69 " 0 / ‘N’)" B / 69 // 89 / {e / (34C ’- . C’z’ /’ ’ // ,l / ” x’/ 0 5'. l J l I 25 30 35 4O 45 Days after inoculation Wilt symptoms (DI) in grafted Green Mountain and Cobbler plants (average from 3 separate tests). DISCUSSION Sebago, reported as being fairly resistant in Idaho (Nielson, 1948) was low in resistance in Michigan (Table 3). This may possibly be explained on the basis of the type of Verticillium present in these 2 areas. The PS type was the predominant type in Idaho while only the DM type was iso— lated from potatoes in Michigan. Sebago was also fairly susceptible in Maine (Ayers, 1952) where the DM type is predominant. Menominee was somewhat resistant in Idaho but was more heavily infected in Maine (Akeley, 31 31., 1956). This variety also became more heavily infected in Michigan than in Idaho. In this work, there was an agreement between vine symptoms and tuber discoloration. In the 3 field tests in 1958 and 1959, 2 or more observations on both vine wilting and tuber discoloration were made on 22 varieties. Out of total of 82 comparisons, severity of symptoms in vines and tubers was in good agreement except for only 5 instances involving 5 different varieties (Table 1). The extent of yield reduction by Verticillium wilt depends on the resistance of the variety involved. Yields in 1960, following an effective inoculation, were reduced more in the case of susceptible varieties than of resistant varieties. 35 36 Results of the seed—piece-dip method of inoculation were not significantly different from the data obtained from the controls at the 5% level in 1959. Apparently tuber sur— face borne inoculum is not a major cause of yield and qual— ity reduction the first year in the field. It may, however, be important in introducing the organism to soils which had previously been free of Verticillium wilt. Results of the soil—slit method of inoculation were variable suggesting that inoculum placement, distribution, concentration, and timing of the inoculation in relation to plant growth may have been critical. The data obtained from the 1959 test were significantly different from the results of the controls, however they were not significantly differ— ent in 1960. For these reasons this method of field inocula— tion was not considered adequate for varietal resistance trials. Data obtained from the root—dip method of inocula— tion was significantly different at the 5% level from the controls in 1959 (Table 2) and 1960 (Table 4 and 5). By placing a high concentration of inoculum on roots 10 to 14 days after planting, the concentration of naturally infested soil may have been approximated or exceeded. This method differed from naturally infested soil in that plants in the latter would be exposed to the inoculum as soon as the roots emerged from the lower stem (as compared to 10 to 14 days later). The root—dip method appeared to be the only method 37 of inoculation which was severe enough to give a consistent test for field varietal resistance trials. To the author's knowledge, no previous reports have been published concerning the differential reaction of potato varieties to Verticillium culture filtrates. Cut— tings of $41956 (resistant) exhibited very slight foliage symptoms (Figure 1) while showing no stem vascular discolor— ation. At no time in these tests did $41956 symptoms pro— gress past epinasty and a slight chlorosis of the lower leaf- lets. Green Mountain (resistant) exhibited epinasty, slight wilting, and some chlorosis (slight foliage symptoms) and a trace of stem vascular discoloration. Tawa and Cobbler (susceptible) exhibited moderate symptoms, which consisted of epinasty, severe wilting, chlorosis and necrosis. Symptoms of epinasty in cuttings were more apparent than under conditions of field testing. The reason for this is not understood. It is highly probable that metabolites produced in culture filtrates are not the same as those pro- duced in a diseased plant. Scheffer, 33 31. (1956), found polygalacturonase (PG) in Verticillium culture filtrates and suggested that their experiments provided good supporting evidence that such an enzyme is involved in the expression of disease symptoms. Wood (1961) showed that Verticillium culture filtrates with high PG activity produced some of the symp- toms found in naturally diseased tomato plants. Somewhat 38 in contrast to the above, it was reported (Bateman, 1963) that PG produced by Rhizoctonia solani in culture was not the same as that found in Rhizoctonia—infected bean tissue. Deese and Stahmann (1962) observed that Verticillium formed 3 to 10 times the amount of PG in;living susceptible tomato tissue as compared to resistant tissue. It was also shown (Barker, 1961) that there was no correlation between the ability of E. solani isolates to produce pectin methyl— esterase and their pathogenicity on bean. It is apparent that many of the present interpretations made concerning culture filtrates will be modified as new methods of analysis and experimentation are found. In resistant potato cuttings, there may be an insen— sitivity in the plant tissue to the effect of toxic products found in filtrates. $41956 cuttings evidently have a cell physiology which is not affected by chemical materials which caused a response in the susceptible cuttings. The grafting experiments suggest the primary cause of death of the plant is not due to a reduction in the water absorbing capacity of the plant as theorized by Talboys and Wilson (1956). Susceptible plant t0ps in the late stage of disease development with resistant side branches were appar— ently able to translocate sufficient water so that the side branch exhibited only slight symptoms even though the stock plant was nearly dead. If lack of water absorption for 39 translocation were the primary cause of symptoms, it is difficult to understand why the side branches did not respond similarly to the plant top. Keyworth (1953a and 1953b) concluded that in all varieties of hop there was equal and positive resistance in the above ground plant parts to invasion by Verticillium albo—atrum. This conclusion was later conceded to have been an incorrect interpretation of data after working with Fusarium wilt of tomato (Keyworth, 1963). There should be some reservation for a concession of this sort involving 2 different hosts and 2 different wilt disease fungi. Bifurcated grafts of resistant and susceptible vari— eties were made by grafting a side branch to an intact plant. Resistant side branches on susceptible plants maintained their resistance even though the susceptible stock plant showed moderate or severe symptoms (Figures 3, 6, and 7). On the other hand, susceptible side branches on resistant plants exhibited more disease symptoms than the resistant stock plant (Figures 4, 6, and 7). This suggests that the tops of potato plants have a differential resistance to Verticillium wilt and that this level of resistance is main— tained regardless of the resistance of the rootstock. Tawa and $41956 grafts showed a greater difference in symptoms than did the Green Mountain and Cobbler grafts. It is probable if potato varieties with less divergence in 4O resistance had been used, differences between side branches and rootstocks could not have been demonstrated. Certain similarities with Fusarium wilt of tomato may be helpful in elucidating the site of resistance. The early report (Heinz and Andrus, 1945) that resistance was entirely localized in the roots has been shown to be invalid (Scheffer, 1957 and Keyworth, 1963). By inoculating through scion spurs, Scheffer completely by-passed the root system. He concluded the resistance present in the stems was similar to that in roots and that it was not altered by the stock. In potato, apparently a similar situation exists. The root and lower stem did, however, have an influence on the symptom expression of the side branch when inoculation was accomplished through the root. A resistant side branch on a susceptible plant stock exhibited slightly more severe disease symptoms than a resistant intact plant- Also in the reciprocal graft, the susceptible side branch on a resistant plant stock exhibited slightly less severe symptoms than a susceptible intact plant (Figures 5 and 8). Results of these grafting experiments might be interpreted that the root and lower stem portion of a potato plant has a potential for permitting the development of either the fungus inoculum or toxicant. The amount of fungus and toxicant which reaches the top is influenced by the host—parasite reaction in the roots and lower stem. 41 In a resistant rootstock, possibly less fungus inoculum or toxicant is produced and translocated to the top. Thus the susceptible side branch does not exhibit symptoms as severe as those observed in the intact susceptible plant. Simi- larly, in a susceptible rootstock, possibly more fungal product is produced and translocated. Thus the resistant side branch exhibits more severe symptoms than those ob— served in the intact resistant plant. SUMMARY Relative resistance of a number of potato varieties under Michigan environmental conditions was determined by greenhouse inoculation and by 2 years of field inoculation trials in both organic and mineral soils. The most resis- tant of the varieties was $41956, Onaway, Early Gem, Green Mountain, and Arenac. The most susceptible varieties were Cherokee, Tawa, Cobbler, IA 1106—5, and Antigo. In general, varieties resistant as measured by foliage symptoms were also resistant as measured by tuber vascular discoloration. Susceptible varieties usually exhibited both symptoms. Significant differences were present between se- lected resistant and susceptible varieties as measured by both tuber and foliage symptoms. Tests in 1960, showed that when disease was present, the extent of yield reduction was higher for susceptible than for resistant varieties. The root—dip method of inoculation was the only one of 3 inoculation methods tested which consistently gave results significantly different from the results of the non— inoculated control plants. The results of the soil—slit method were significantly different from the results of the controls in 1959, however they were not in 1960. Results of the seed—piece-dip method was not significantly different 42 43 from those of the controls in any of the tests. Cuttings of plants reacted differently when exposed to culture filtrates. $41956 (resistant) showed a trace of foliage symptoms and did not show any vascular symptoms. Cobbler and Tawa (susceptible) exhibited moderate wilting and slight vascular discoloration when placed in the same culture filtrates. Green Mountain (slightly less resistant than $41956) was intermediate, exhibiting slight wilting and a trace of vascular discoloration. Bifurcated grafts of resistant and susceptible vari— eties showed that the plant top maintained its resistance regardless of the susceptibility of the rootstock. Symptom development of the plant top was modified, however, by the resistance or susceptibility of the rootstock. The amount of fungal products which reach the top is probably dependent upon the host—parasite reaction in the roots and lower stem. When these products reach the top of the plant the symptom expression is influenced by the inherent resistance of the plant top. LITERATURE CITED Akeley, R. V., F. J. Stevenson, D. Folsom, and R. Bonde. 1956. Breeding varieties of potato resistant to Verticillium wilt in Maine. Am. Potato J. 33:15-21. Ayers, G. W. 1952. Studies on Verticillium wilt of potatoes. Am. Potato J. 29:201-205. Barker, K. R. 1961. Factors affecting the pathogenicity of Pellicularia filamentosa. Ph.D, dissertation, Univ. Wisconsin. 82 p. Bateman, D. F. 1963. Pectolytic activities of culture filtrates of Rhizoctonia solani and extracts of Rhizoctonia-infected tissues of bean. Phytopathology 53:197—204. Bewley, W. F. 1922. Sleepy disease of the tomato. Ann. Appl. Biol. 9:116—134. Davis, D. 1953. The use of intergeneric grafts to demon— strate toxins in the Fusarium wilt disease of tomato. (Abstr.) Phytopathology 43:470. Davis, D. 1954. The use of intergeneric grafts to demon— strate toxins in the Fusarium wilt disease of tomato. Am. J. Bot. 41:395—398. Deese, D. C. and M. A. Stahman. 1960. Role of pectic enzymes in susceptibility and resistance to Fusarium and Verticillium wilts of plants. (Abstr.) Phyto— pathology 50:633. Deese, D. C. and M. A. Stahman. 1962. Formation of pectic enzymes by Verticillium albo-atrum on susceptible and resistant tomato stem tissues and on wheat bran. Phytopathologische Zeitschrift 46:53—70. Folsom, D. 1957. Verticillium wilt of potatoes in relation to fungicides added to the fertilizer. Am. Potato J. 34:1—5. 44 45 Gratz, L. O. 1930. Disease and climate as pertaining to the Florida and Maine potato sections. Phytopathology 20:267—288. Green, R. J., Jr. 1954. A preliminary investigation of toxins produced 13 vitro by Verticillium albo—atrum. Phytopathology 44:433-437. Heinze, P. H. and C. F. Andrus. 1945. Apparent localization of Fusarium wilt resistance in the Pan American tomato. Am. J. Bot. 32:62—66. Kamal, M. and R. K. S. Wood. 1956. Pectic enzymes secreted by Verticillium dahliae and their role in the development of the wilt disease of cotton. Ann. Appl. Biol. 44:322— 340. Keyworth, W. G. 1953. Verticillium wilt of the hop. VI. The relative roles of root and stem in the determination of wilt severity. Ann. Appl. Biol. 40:344-361. Keyworth, W. G. 1953. Resistance of hop stems to invasion by Verticillium albo—atrum. Nature, Lond. 171:656—657. Keyworth, W. G. 1963. The reaction of monogenic resistant and susceptible varieties of tomato to inoculation with Fusarium oxySporum f. lycopersici into stems or through Bonny Best rootstocks. Ann. Appl. Biol. 52:257—270. McLean, J. G. 1955. Selecting and breeding potatoes for field resistance to Verticillium wilt in Idaho. Idaho Agr. Exp. Sta. Res. Bull. No. 30. Neilson, L. W. 1948. Verticillium wilt of potatoes in Idaho. Idaho Agr. Exp. Sta. Res. Bull. No. 13. Porter, C. I. and R. J. Green, Jr. 1952. Production of exotoxin in the genus Verticillium. Phytopathology 42:472—476. Robinson, D. B., R. H. Larson, and J. C. Walker. 1957. Verticillium wilt of potato in relation to symptoms, epidemiology, and variability of the pathogen. Wisc. Agr. Exp. Sta. Res. Bull. 202. Scheffer, R. P. 1957. Analysis of Fusarium resistance in tomato by grafting experiments. Phytopathology 47:328— 331. 46 Scheffer, R. P., S. S. Gothoskar, C. F. Pierson, and R. P. Collins. 1956. Physiological aspects of Verticillium wilt. Phytopathology 46:83-87. Talboys, P. W. 1956. Mechanism of Verticillium wilt tolerance in the hop. (Abstr.) Brit. Mycol. Soc. Trans. 39:381. Talboys, P. W. and J. F. Wilson. 1956. A study of wilt— tolerance in the hop by means of intervarietal graft complexes. Rep. E. Malling Res. Sta. 1955:126—130. Threlfall, R. J. 1959. Physiological studies on the Verticillium wilt disease of tomato. Ann. Appl. Biol. 49:120—139. Waggoner, P. E. 1956. Variation in Verticillium albo—atrum from potato. Pl. Disease Reptr. 40:429—431. Wood, R. K. S. 1961. Verticillium wilt of tomatoes. The pectic and cellulolytic enzymes. Ann. Appl. Biol. 49: 120—139.