wt! .rw'fl—~_fl'“- 4' *wt‘fi wm—v ’ — v—_'— L v PYTHIUM ROOT ROI AFFECTING PEAS IN MICHIGAN Thesis. for the Down 0‘ M. 5. MICHIGAN STATE UNIVERSITY Cesar A. Escobar 1965 LIBRARY Michigan State University Room USE 0w 5,"; s H. ' ’ "‘1 ,\ .aud‘ ABSTRACT PYTHIUM ROOT ROT AFFECTING PEAS IN MICHIGAN by Cesar A, Escobar Pythium Spp. were isolated from pea plants grown in 20 of 25 soil samples collected in pea fields in the "thumb" area and near Jackson in Michigan. All 21thium isolates were able to pro- duce considerable root rot disease severity on pea plants in greenhouse tests“ Three of 5 22thium isolates identified were 2, ultimum, The other 2 isolates were g, debaryanug and g, monospermum. A11 5 fungus isolates showed a similar degree of pathogenicity on peas in greenhouse tests. g, ultimum produced a considerable root rot damage on several commercial pea varieties, pea introductions and legumes other than peas. Cereal plants tested as well as tomato, cucumber, Gomphrena globosa, gicotiana glutinosa, and Chenopodium amaran- ticolor, although diseased, were less susceptible than the legumeso Pea plants inoculated with g, ultimum 1, 8, 15, 22 and 29 days after seeding, showed a similar degree of susceptibility, a possible slightly increased susceptibility at 22 and again at 29 days. When pea plants were inoculated with A, euteiches at the same ages, more severe disease symptoms were observed in those plants inoculated 1 day after seeding soil, but plants became more resistant to the pathogen with each increase in age, Cesar A. Escobar An additive effect was produced when pea plants were in- fected with both g. ultimum and E. solani. The damage produced by these 2 pathogens was almost equivalent to that produced by A, euteiches, which is considered as the most damaging root rot fungus on peas. An even greater enhancement of root rot symptoms occurred when plants previously infected with either BYMV or PMV were inoc- ulated with g, ultimum. Disease severity produced by g. ultimum on Miragreen peas increased as soil temperatures increased from 120 to 280 C. The optimum temperature for growth of the mycelium of Pythium sp. in culture medium was 24° C. Therefore Pythium root rot of peas is most severe in soil at temperature above the optimum for growth of mycelium of the fungus in culture medium and growth of the host in soil. PYTHIUM ROOT ROT AFFECTING PEAS IN MICHIGAN By Cesar A. Escobar A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1965 ACKNOWLEDGEMENTS I wish to express sincere thanks and appreciation to the Institute of International Education for financial aid. Special thanks are extended to my major professor, Dr. J. L. Lockwood, for encouragement and guidance in this work. I am greatly indebted to Dr. E. S. Beneke, Dr. H. H. Murakishi and Dr. C. Pollard for reading the manuscript and making valuable suggestions. Finally, I wish to thank Rodrigo Duarte for aid in the statistical analyses. ii TABLE OF CONTENTS INTRODUCTION. . . . . . . . . . . LITERATURE REVIEW . . . . . . . . MATERIALS AND METHODS . . . . . . Isolation . . . . . . . . Preparation of inoculum. . Selection of substrate for Inoculation method . . . . grow1ng Evaluation of disease severity . RESULTS 0 O O O O O O O O O O O O Pea root rot survey. . . . Pathogenicity of the gythium isolates. Identification of Species of Pythium isolates. Susceptibility of some commercial peas, foreign pea O introductions and other plants to Pythium ultimum. . . Effect of soil temperature on disease severity . . . . Effect of the temperature on growth of mycelium in culture medium . . . . . . Effect of age of pea plants on susceptibility to Eythium ultimum and Aphanomyces euteiches. 0 Effect of combinations of Aphanomyges euteiches, Fusarium solani and gythium ultimum on severity of pea root rot 0 O O C O C 0 Increased root rot severity of Miragreen peas infected with a virus and Pythium ultimum. DISCUSSIONO O O O O O O O 0 O O 0 LITERATURE CITED. . . . . . . . . iii 0 Page \I \Dmmww 10 10 10 12 13 15 19 20 24 32 4O 46 Table LIST OF TABLES Disease indices of Miragreen peas infected by different Pythium isolates Disease indices of commercial peas. foreign pea introductions and other plants infected with Pythium ultimgm in a greenhouse test Effect of soil temperature on pathogenicity of g. ultimum, isolate 8; g. debaryaggm, isolate 15; and 2, monosPermum, isolate 16, on Miragreen peas Effect of temperature on growth of mycelium of three Pythium isolates in potato-dextrose broth Effect of age of pea plants on susceptibility to Pythium ultimum and Aphanomyces euteiches Effect of combinations of Aphanomyces euteiches, Fusarium solani and Pythium ultimum on pea root rot severity Increased root rot severity on Miragreen peas infected with a virus and Pythium ultimum iv Page 11 l4 16 21 33 36 Figure LIST OF FIGURES Effect of soil temperature on pathogenicity of g. ultimum, isolate 8; P. debaryanum, isolate 15; and g, monospermum, isolate 16, on Miragreen peas. Differences between the mean values for three Pythium isolates at each different temperature were statis- tically significant at the 1% level Effect of soil temperature on pathogenicity of Pythium ultimum on.Mirggreen peas. Lsft to r ght disease symptoms at 12 16 200 , 24 and 28 C Effect of temperature on growth of mycelium of P. ultimum. isolates 8 and 9; and P. debaryanum, isolate 15, in potato- -dextrose broth Mean xalues of three Pythium isolates, at 160 20 and 28 C did not differ at the 5% level of significance Effect of soil temperature on disease severity com- pared with the effect of temperature on growth of mycelium Effect of age of pea plants on susceptibility to Py_hium ultimum and Aphanomyces euteiches. Index values for P ultimum at 8,15,22 and 29 days were statistically different at the 57 level of signifi- cance. Values for l and 8 days were not statisti- cally different Effect of age of Miragreen peas on susceptibility to Pythium ultimum and Aphanomyces euteiches. Symptoms when plants were inoEElated one day after sowing. (left) g. ultimum, (center) control and (right) A. euteiches Effect of age of Miragreen peas on susceptibility to Pythium ultimum and Aphanomy_es euteiches Symptoms when lants were inoculated 8 days after sowin (lefty P. ultimum, (center) control and (right A. euteiches Page 17 22 23 26 27 28 Figure 10. 11. 12. 13. 14. Page Effect of age of Miragreen peas on susceptibility to Pythium ultimum and Aphanomyces euteiches. Symptoms when plants wefe inoculated 15 days after sowing. (left) g. ultimum, (center) control and (right) A. euteiches 29 Effect of age of Miragreen peas on susceptibility to Pythium Elgimgm and Aphanomyces euteiches. Symptoms when plants were inoculated 22 days after sowing. (left) P. ultimum, (center) control and (right) A. euteiches 30 Effect of age of Miragreen peas on susceptibility to Pythium ultimum and Aphanomyces euteiches. Symptoms when plants were inoculated 29 days after sowing. (left) 2. ultimum, (center) control and (right) A. euteiches 31 Effect of combinations of Aphanomyces euteiches, Fusarium solani and Pythium ultimum on pea root rot severity. Treatments underscored by a line did not differ statistically at the 5% level of significance 34 Increased root rot severity of Miragreen peas infected with a virus and Pythium ultimum. Treatments under- scored by a line did not differ statistically at the 1% level of significance 3? Miragreen peas infected with: (left) Bean Yellow Mosaic Virus, (center) Pythium ultimum and (right) Bean Yellow Mosaic Virus and g. gltimum 38 Miragreen peas infected with: (left) Pea Mosaic Virus, (center) Pythium ultimum and (right) Pea Mosaic Virus and P, ultimum 39 vi INTRODUCTION Peas are important in the diet of a large percentage of the population of the United States. They are grown to some extent in almost every part of the country for home use, for canning, for freezing, for seed, and for fresh market (41). However, root rots are the limiting factor in pea production in Michigan as well as in other pea growing states (25). Aphanomyces euteiches Drechs., has been reported as the most damaging fungus affecting roots of peas in Michigan followed in importance by Fusarium solani f. pigi (F. R. Jones) Snyder and Hansen (23, 25). A breeding program was begun by Lockwood and Ballard (25), to obtain pea varieties resis- tant to these pathogens. Selected lines of peas showing some degree of resistance in greenhouse tests sometimes failed to show resistance in the field. Two factors possibly reaponsible for lack of resistance in the field could be that different strains of these pathogens occur in the field or that fungi other than A. euteiches and E. solani are involved in the pea root rot complex. In 1956, Lockwood (23) also obtained pathogenic isolates of Rhizoctonia sp. and Pythium sp. from root rot infected peas, but these are generally considered to be unimportant in connection with pea root rot. In 1963, a survey was made in Michigan pea fields in the "thumb" area and near Jackson in order to determine the prevalence 1 2 of Pythium sp. Studies were also made with selected Pythium iso- lates found during the survey. These included the effect of soil temperature on severity of the disease, effect of temperature in growth of mycelium in pure culture, effect on the host of combined infections with Pythium and other root rot fungi or viruses, effect of the age of plants on susceptibility to this pathogen, and host range. LITERATURE REVIEW Although the pea (Pisum sativum) is subject to numerous types of diseases, root rots are generally considered to be the most important factors limiting pea yields and quality in Michigan and other parts of the United States (19, 20, 22, 23, 26, 31). Pythium species, mainly P. ultimum Trow., have been fre- quently reported to produce root rots in peas, or to be associated with other pathogens which also cause infection in this host, there- by denoting a potential importance of this pathogen in the pea root rot complex (8, 16, 29, 30, 31, 35, 41). In surveys made during ten years in commercial pea growing areas in New York, fourteen different fungi were associated with pea root rot. However, the following organisms, listed in order of importance were primarily responsible for disease: Fusarium solani (Mart.) Appel and Wr., Aphanomyces euteiches Drechs., Pythium ultimum Trow., Rhizoctonia solani KGhn, and Ascochyta pinodella L. K. Jones (16). In 1925, Jones and Linford reported that Aphanomyces root rot was the most severe disease affecting peas in Wisconsin and that root rots produced by Fusarium sp. and Pythium sp. were less important (19). However, Pythium isolates from diseased pea plants were capable of preventing germination of pea seeds and caused a 3 4 considerable degree of root rot. P, ultimum was reaponsible for pre-emergence damping-off and root rot of pea in Toluca, Mexico (10). Lockwood (23) also considered that fungi other than A. euteiches and E3 gglggi_were involved in the pea root rot complex in Michigan, and obtained pathogenic isolates of Rhizoctonia sp. and Pythium sp. from root rot-infected peas. McDonald and Marshall (28) found a high percentage of suscep- tibility to P, ultimum in most of 450 introductions or varieties of peas, with colored flowers which, as a group, seemed to be more resistant to other root rot diseases. Sometimes, interactions between two pathogens attacking roots of plants have been found. Kerr (20) reported a marked interaction between Fusarium oxysporum f. 2131 (Linford) Snyder and Hansen and Pythium ultimum affecting peas in Australia. Either pathogen caused no serious damage when acting alone, both fungi together produced severe wilt symptoms about 6 weeks after sowing and finally caused death of seedlings. On the other hand, Bateman (5) found that Thielaviopsis sp. and Pythium sp. in combination caused less root rot of poinsettia plants than was expected on basis of the expected additive amount of damage caused by the two pathogens separately. However, the amount of damage produced in combination was greater than that produced by either one alone. Virus infection may predispose pea plants to increased 5 infection by root rot fungi. Farley and Lockwood (11) reported more severe root rot symptoms when pea plants were infected with any of several viruses and either A, euteiches or E, solani f. 2151 than when peas were infected with a fungus alone. Wheat plants infected with barley yellow dwarf virus were also more susceptible to some root rot pathogens (37). An increment in post~emergence dampinga off of cucumber by Rhizoctonia sp. occurred in seedlings infected with cucumber mosaic virus (4). Pythium app. have been reported as producing economic damage of different levels of importance to many different commercial plants. For example, this pathogen was found to be the most common cause of seed decay and seedlings diseases in corn (18). It also produces damping-off of cotton seedlings (7), and was responsible for poor emergence and most damping-off of alfalfa seedlings (33, 34) as well as in red clover (15). Intensity of root rot diseases is affected by soil fertility, moisture level, and temperature. Although P. ultimum has been iso- lated from soils and susceptible hosts collected from many different places in the world, this pathogen frequently causes no important injury (30). Jones and Linford (19) considered that the most obvi- ous explanation for the failure of this fungus to produce more ser- ious injury in plants is that a comparatively high soil temperature is required for its activity. The optimum temperature for Pythium 6 disease of various plants generally ranged between 24° and129o C. (13, 19, 34, 35, 38). Maximum development of Pythium mycelium in culture medium usually occurs at temperatures between 220 and 310 C. (6, 30). On alfalfa and poinsettia plants, a similar effect of temperature upon fungal growth rate and disease intensity was found (6, 27). Pythium Spp. often become progressively less aggressive as plants develop underground lignified tissues (29). Although this pathogen has been reported as responsible for root rot of older and mature plants, the attack is often restricted to immature parts of the root system (9, 13). An increased degree of intensity of Pythium root rot in hosts such as peas and poinsettia has occurred under wet soil conditions (3, 35, 40). Another factor which may influence disease severity of this pathogen is that Pythia which produce pea root rot belong to several Species differing somewhat in pathogenicity and frequency of occur- rence (18, 31, 35). Also, lack of a sufficient inoculum level of fungus in certain areas may allow plants to escape disease (13). MATERIALS AND METHODS Isolation. Miragreen pea plants were grown in soil samples collected in a survey made in different fields of peas in'Michigan. Pathogens infecting the roots of these plants were isolated by the agar plate method. The tap and lateral roots and the lower stem were washed with running water for 45 minutes, then cut into pieces approximately 2 cm. long and surface-sterilized by immersion in.0.51 sodium hypochlorite (Clorox) for 5 minutes. The disinfected pieces were then washed with sterile distilled water. Small portions of tissue from the margin of a disease lesion were excised, dried on sterile filter paper and then placed in Petri dishes on a medium containing 20 g agar and 10 mg Streptomycin per liter (17, 36). The inoculated plates were incubated for three days at 240 C. Mostly, three different kinds of mycelial growth were observed on the surface of the plates. Selected pieces of mycelium from differ- ent fungal colonies were transferred to test tubes containing potato-dextrose broth (PDA) (an infusion from 200 g potatoes, 20 g dextrose and 20 g agar per liter). 1 Preparation of inoculum. To prepare inoculum for pathogen- icity tests and another experiments, isolates of Pythium'were grown for 5 days at 243C in 500 Erlenmeyer flasks containing 100 ml of potato-dextrose broth. The mycelia were washed 3 times with 7 8 distilled water and fragmented in a Waring Blender for 15 seconds. Distilled water was added to each mycelial suSpension to make 80 m1 volume which was used for inoculation of 4 pots. Selection of substrate for_growing:peas. A soil mixture com- posed of 3 parts compost soil and 1 part each of muck and sand was used in all experiments. Soil was autoclaved and placed in pots or glazed crocks. Ten or 12 Miragreen pea seeds were surface-sterilized with a 10% Clorox solution for 15 minutes, then sown in each pot or crock. Seedlings were inoculated 8 days later. This soil mixture was a suitable substrate for production of a uniform root rot on pea plants. Another soil mixture, 1 part each of sand and peat, was tried in same way but results were less satisfactory. In a seedling test for evaluating resistance of peas to Aphanomyces and Fusarium root rots, Lockwood and Ballard (25) found white sand to be a suit- able substrate for this purpose. This growing medium was tried for Eythium root rot but disease development was not satisfactory. Inoculation method. An injection method was used for inocu- lating plants in autoclaved soil (ll, 27). TWenty m1 of inoculum were added to each pot by applying 4 ml portions in 5 different positions with a hypodermic syringe inserted to a depth of l in. below the soil surface. Another trial for soil infestation con- sisted in mixing the same amount of inoculum with the soil before planting pea seeds. This method did not produce as uniform infection 9 of the pea roots as did the injection method. Evaluation of disease severity, Evaluation of disease sever- ity was done by estimating increasing severity using a scale from 0-9 which was developed by Lockwood and Ballard (24, 25). It con- sists of grading separately the tops of the plants, epicotyls and roots. Slight, moderate and severe disease symptoms were rated as l, 2 and 3, reapectively. The separate ratings from each part of the plant were totalled. The ratings were made for the groups of plants in each pot and an average was obtained for all replications of each treatment. This was designated as the disease index. Each treatment was replicated with 3 or 4 pots and analysis of variance was applied to the individual pot ratings. The multiple range test developed by Duncan was used for ranking the treatment means (21). RESULTS Pea root rot survey. A survey was made in 1963 of 25 differ- ent fields of peas in the eastern part of Michigan in the area of the "thumb" and near Jackson in the south-central Michigan. Pea root rot in those fields ranged from slight to severe. Before peas were harvested, soil samples were collected from several locations in each field where pea plants showed disease symptoms. Collected soil samples were tranSported in polyethylene bags and then placed in 6 in. clay pots in the greenhouse. Ten surface-sterilized Mira- green pea seeds were sown in each pot. After 3 weeks most of the plants showed root rot disease symptoms. Isolations made from the diseased roots showed that Fusarium solani was present in peas grown in each of the 25 soil samples, Aphanomyces euteiches from 6 and Pythium sp. from 20 different soil samples. Pathogenicity of the Pythium isolates. Pathogenicity of 20 different Pythium isolates from fields sampled in the survey Was tested. The isolates tested all produced definite symptoms of root rot. The most severe symptoms of disease observed in Miragreen pea plants were rated as 7 and the least as 3.5 (Table 1). In general, the more severe disease symptoms were confined to the epicotyls and tap and lateral roots of the plants. Symptoms of the disease at an 10 11 Table 1. Disease indices of Miragreen peas infected by different Pythium isolates. Pythium isolate Disease Uninoculated index* plants '1 6.0 o 2 7.0 0 3 5.0 0 4 4.5 0 5 5.0 0 6 5.0 0 7 3.5 0 8 6.5 0 9 6.0 0 10 5.0 0 11 3.0 0 12 4.0 0 13 5.5 0 14 6.0 0 15 6.0 O 16 6.5 0 17 4.0 0 18 5.0 O 19 6.0 0 20 5.5 0 *Disease index was based on a scale of increasing severity from 0-9. Each figure is a mean index of 3 pots each with 10 plants. 12 early stage were characterized by a somewhat water-soaked and soft- ened diseased tissue extending above and below the seed attachment. Parts of affected roots showed numerous superficially necrotic spots. In advanced stages of the disease the shrunken tissue presented a black-brown necrosis of the cortex of the tap and lateral roots and lower stem below the soil. The leaves of some plants developed slight symptoms of wilting; the highest rating for foliage damage of plants was 1.5 of a maximum of 3.0. Identification of species of Pythium isolates. An attempt was made to identify some of those Pythium isolates more pathogenic to peas. For this purpose, isolates l, 8, 9, 15 and 16 were selected. Most of these isolates failed to produce both sexual and asexual or- gans when growing on PDA medium. A synthetic medium was used for obtaining sexual organs. The formula for this medium was supplied by Dr. A. F. Schmitthenner of the Ohio Agricultural Experiment Station. This medium contains sucrose 1.2 g, L-asparagine 0.13 g, K2HP04 0.15 g, MgSO4 0.05 g, EDTA (Naz) 0.029 g, ascorbic acid 0.01 g, agar 20 g, distilled water 1000 m1 and 5 m1 of a trace mineral con- taining FeCl3 0.048 g, CaCl2 0.278 g, MnCl2 1.438 g, ZnCl2 0.836 g per liter of distilled water. Another synthetic medium, MP 5, con- taining 4 g maltose, 1 g peptone and 20 g agar per liter was used for production of sporangia. Small blocks of this medium holding mycelium growth were suspended in sterile distilled water and half 13 a boiled hemp seed was added to promote the production of those asexual organs. Descriptions compiled by Middleton (30) and Gilman (14) were used for identification of the Pythium species. Isolates number 1, 8 and 9 are Pythium ultimum Trow., 15 is Pythium debarygnum Hesse and 16 is Pythium monospermum Pringsheim. Susceptibility of some commercial peas, foreign pea intro~ ductions and other plants to Pythium ultimum. Disinfected seeds of 10 commercial pea varieties, 4 pea introductions and 15 plants other than peas were sown in autoclaved soil mixture (Table 2). Pea seed- lings were inoculated with g, ultimum 8 days after sowing. All of the commercial pea varieties and pea introductions developed root rot in the infested soil. The intensity of disease in those plants ranged from 4 to 5.5. Possibly, the pea introductions were slightly more resistant than commercial varieties. Other legumes showed a similar level of disease symptoms. Cereals were more resistant to Pythium root rot, the disease intensity ranging between 2 and 3.5. Nicotiana glutinosa was as susceptible as legume plants but Cheap: podium amaranticolor, Gomphrena globosa, Cucumber (National Pickling) and Tomato (Bonny Best) were as resistant as cereal plants. In general, symptoms of disease were located mostly in the lower stem and on the tap and lateral roots of the plants. However, the foliage appeared healthy except in some pea varieties which showed symptoms of wilting and slight stunting. 14 Table 2. Disease indices of commercial peas, foreign pea introductions and other plants infected with Pythium ultimum in a greenhouse test. Host Disease index* Pea Introductions 166159 169604 180693 175232 Commercial Pea Varieties Early Perfection Miragreen Alaska 94003 Freezer 33 Early Perfection 63579 Alderman Bliss Everbearing Pacific Freezer Perfected Wales Dark Skin Perfection Other Plants Corn.(Michigan 400) Sweet Corn (Golden Cross Bantam) Oats (Gopher) Barley (Kindred) Wheat (Little Club) Beans (Bush Wax Pod King) Soybeans (Keye) Beans (Prince) COWpea (Black) Beans (Pinto) Tomato (Bonny Best) Cucumber (National Pickling) Gomphrena globosa Nicotiana glutinosa Chenopodium amaranticolor bbbb omub Ln-I-‘LJ'IUIU'IL‘UIUIM-p UlU'IOCOMU‘IUIOO u:c>u:c>u:c>c>E>Lau1E>E>Labuba 0 HwaNbNb-D-P‘NwNF-‘w *Disease index was based on a scale of increasing severity from 0-9. Each figure is a mean of 3 pots each with 10 plants. plants of each kind remained healthy. Uninoculated 15 Effect of soil temperature on disease severity. Autoclaved soil mixture was placed in glazed crocks and 12 surface-sterilized Miragreen pea seeds were sown in each crock. After 8 days, soil was infested with mycelia of isolates 8, 15 and 16, respectively, P. ultimum, g, debaryanum and E, monospermum. The crocks were kept in tanks containing water at constant temperatures of 12? 16? 20? 240 and 28°C for 2 weeks. Increases in severity of the disease occurred with each increase in temperature from 12° to 280 C. (Table 3; Figs. 1, 2). The highest disease severity was obtained on those plants grown at a soil temperature of 280 C, and P, ultimum produced the highest disease index, 8.5, at this temperature. Each of the 3 Pythium species behaved in a similar way regarding the increment of disease as a response to higher temperatures in the soil. The mean disease indices for all 3 species of Pythium in soil at 12°, 16°, 20°, 240 and 280 C were 1.0, 2.3, 3.6, 5.6 and 7.5, respectively. Differences between these values were statistically significant at the 1% level. Although all 3 isolates behaved similarly, the dis- ease index for Pythium isolate 16 was higher than those of isolates 8 and 15 at 200 C; the index for isolates 15 and 16 were higher than that of isolate 8 at 240 C, and values for isolates 8, 15 and 16 at 280 C all differed at the 5% level of significance. A simi- lar experiment was run twice more using P. ultimum only. Crocks holding uninoculated plants were kept as controls for each tempera- 16 Table 3. Effect of soil temperature on pathogenicity of g, ultimum, isolate 8; g. debaryanum, isolate 15; and P, monospermum, isolate 16, on Miragreen peas. 0 Disease index for indicated isolate* Temperature, C g. ultimum g, debaryanum g. mongspermum ‘Mean (8) (15) (16) O 12 1.0 1.0 1.0 1.0 16° 2.5 2.0 2.5 2.3 20° 3.5 3.0 4.5 3.6 24° 5.0 6.0 6.0 5.6 28° 8.5 6.5 7.5 7.5 *Disease index was evaluated using a scale of increasing severity from 0-9. Each figure is a mean of 4 crocks, each with 12 plants. cl Differences between the mean values were statistitally significant at the 1% level. INDEX DISEASE 17 s ISIS s ISIS s 1516 s :5 l6 s l5l6 |2° IS‘ 20' 24° 28° SOIL TEMPERATURE , “C Fig. 1. Effect of soil temperature on pathogenicity of P. ultimum, isolate 8; g, debaryanum, isolate 15; and P. monospermum, isolate 16, on Miragreen peas. Differences between the mean values for 3 Pythium isolates at each different temperature were statistically significant at the 1% level. 18 .0 owN a“ O‘NN «CON «OOH «ONH U“ QgUQgQ oumomau «unugu on amen .mmom noouwmuaxnuo Susanna asanumm mo muqouoowonuum no unaucuonaou Hwom mo uommmm .N .wwm .. . x . .. ,\\. x. . ,_ m '5'. . .mmfilac.‘ . .36.... /r. ,e .. 4 19 ture. Statistically significant increases in disease severity with each increase in temperature were again found. Control plants pre- sented slight root damage rated as 0.5 and 1.0 at temperatures of 240 and 280 C, respectively, but not at lower soil temperatures. Fresh weights of uninoculated plants, at temperatures of 12°, 16°, 200, 240 and 280 C were 44 g, 38 g, 33 g, 27 g and 26 g per pot, reSpectively. Thus, pea plants grew best at cooler temperatures. Nevertheless, plants appeared healthy at all temperatures used. Therefore, the increased disease indices which occurred as tempera- ture increased were in fact due to increased root rot symptoms and not to a direct effect of unfavorable temperature on the host. This ‘was confirmed by fresh weights of diseased plants which were 32 g, 28 g, 23 g, 17 g, and 14 g over the same increasing temperature range. It can be seen that the greatest percentage losses in fresh weights occurred at the higher temperatures. Effect of the temperature on growth of mycelium in culture medium. The effect of the same 5 different temperatures on the mycelial growth of g, ultimum, isolates 8 and 9; and g. debaryanum, isolate 15; was determined. Erlenmeyer flasks, each containing 50 ml of potato-dextrose broth were seeded with an 8 mm diameter PDA disk containing a uniform mycelial growth of the fungus. Three flasks containing each isolate were incubated at 12°, 16°, 20°: 24o o and 28 C for 5 days. The mycelial mat from each flask was rinsed 20 through a Buchner funnel at low air pressure with 40 ml of distilled water. Mycelial mats were placed in.pre—weighed aluminum cups and dried for 24 hr. at 1000 C. Thus, total mycelium growth of each fungus isolate was measured by weight of oven-dried mycelium. The optimum temperature for growth of mycelium of the 3 Pythium isolates was 24° C (Table 4; Fig. 3). At temperatures higher or lower than 24° C mycelial growth was significantly less. A poor development of the fungi was produced at 12° C, but there was a significant in- crement of growth at 16° C. No significant difference was found among the mycelial weights at temperatures of 16°, 20° and 28° C. Differences between isolates at different temperatures were not significant. The optimum temperature for mycelial growth in arti- ficial medium was below the soil temperature which produced the highest intensity of the disease (Fig. 4). Disease severity was always increased in soil from 12°to 28°C, and although mycelial growth increased at temperatures from 12°to 24°C, it decreased at 28°C. Effect of age ofgpea plants on susceptibility to Pythium ultimum and Aphanomyces euteiches. Sixty clay pots filled with autoclaved soil mixture were planted at different time intervals with 10 surface-sterilized Miragreen pea seeds. Soil in all pots was infested at the same time with either 2, ultimum or A, guts;- ches when pea plants were 1, 8, 15, 22 and 29 days old. 21 Table 4. Effect of temperature on growth of mycelium of three Pythium isolates in potato-dextrose broth. 0 Temperature, C Mean dry wt. of mygelium,gmg* ‘2. ultimum g, ultimum P. debaryanum Mean (8) (9) (15) O 8 12 38 43 38 40 o b 16 191 200 196 196 200 213 222 217 217b O C 24 255 248 270 258 o b 28 218 239 210 222 *Each figure is a mean of 3 dried mycelial mats. Means followed by different letters differed statistically at the 5% level of significance. Differences between isolates at the same tempera- tures were not significant. WEIGHT OF MYCELIUM, MG. 270 230 I90 I50 IIO 70 3O 22 1 A O O C 12° 16° 20 24 28 TEMPERATURE, °C. Fig. 3. Effect of temperature on growth of mycelium of E. ultimum, isolates 8 and 9; and g. debaryanum, isolate 15, in potato-dextrose brotho Mean values of the 3 Pythium isolates, at 16°, 20 and 28 C did not differ at the 5% level of significance. WEIGHT 0F MYCELIUM, MG 270 230 I90 I50 IIO 30 23 j i I J L - 8 A‘ MYCELIAL WT. " 7 I pd”’o’ "‘ 6 _ x I, 5 w I C) DISEASE mosx _ 4 E m (D - 3 4 11.1 S’.’ .. 2 o I - l I l2“ 16° 20‘ 24" 23" TEMPERATURE, ’C Fig. 4. Effect of soil temperature on Pythium disease severity compared with the effect of temperature on growth of mycelium. WEIGHT 0F MYCELIUM, MG 270 230 I90 I50 NO 30 23 - 8 /A\\ MYCELIAL WT. " 7 do” °° a” _. 6 _. X I, 5 w I D DISEASE mosx _ 4 E U (D " 3 < “J ‘L’ _ 2 o ’ - l I I I I I '2’ 16’ 20° 24° 28° TEMPERATURE, °c Fig. 4. Effect of soil temperature on PZthgfl disease severity compared with the effect of temperature on growth of mycelium. 24 Uninoculated plants were kept as controls for each age. Seventeen days after inoculation, plants were examined and the intensity of the disease was evaluated. Disease index of Aphgpomyges root rot was 8.8 in those plants inoculated 1 day after seeding soil, but the disease intensity decreased in older plants (Table 5; Figs. 5, 6, 7, 8, 9, 10). The lowest disease index, 4.5, occurred in plants inoculated at 29 days of age. 0n the other hand, pea plants grown in soil infested with g. ultimum showed increases in disease severity with each increase in age of the plants from 1 to 29 days. Disease severity was rated as 3.8, 4.1, 4.6, 5.6 and 6.2 in soil infested when plants were 1, 8, 15, 22 and 29 days old, respectively. Statistically significant differences (5% level) were obtained between disease indices 4.6, 5.6 and 6.2. Control plants presented some root rot decay which was assessed as 0.3, 0.8, 1.1 and 1.2 in plants at 8, 15, 22 and 29 days of age. Simi- lar results were obtained in another similar experiment. However, in this experiment the disease index in plants inoculated when 29 days old was not significantly different from that at 22 days. Effect of combinations of Aphanomyces euteiches, Fusarium solani and Pythium ultimum on severity of pea root rot. Standard inoculum suspensions of mycelia of A. euteiches, E, §21§p§_and g. ultimum were used to infest soil containing 8 day-old pea plants. One-half the usual quantity of inoculum was used for some treatments 25 Table 5. Effect of age of pea plants on susceptibility to Pyphium ultimum and Aphanomyces euteiches. Age of plants at Disease index* inoculation, days .3. ultimum A, euteiches Control 1 3 8 8.8 0 a 8 4.1 8.2 0.3 b 15 4.6 7.0 0.8 c 22 5.6 6.1 1.1 29 6.2d 4.5 1.2 *Disease index was evaluated using a scale of increasing disease severity from 0-9. Each figure is a mean index of 4 pots, each with 10 plants. Values followed by different letters differed statistically at the 5% level of significance. INDEX DISEASE 26 _ APHANOMYCES EUTEICHES -1 "' '1 - PYTHIUM ULTIMUM " - H CONTROL I T/fik I I 8 l5 2 2 29 AGE OF PLANTS AT INOCULATION, DAYS Fig. 5. Effect of age of pea plants on susceptibility to Pythium ultimum and Aphanomyces euteiches. Index values for g. ultimum at 8, 15, 22 and 29 days were statistically different at the 5% level of significance. Values for l and 8 days were not statistically different. 27 Fig. 6. Effect of age of Miragreen peas on susceptibility to Pythium ultimum and Aphanomyces euteiches. Symptoms when plants were inoculated one day after sowing. (left) 2. ultimum, (center) control and (right) A, euteiches. 28 Fig. 7. Effect of age of Miragreen peas on susceptibility to Pythium ultimum and Aphanomyces euteiches. Symptoms when plants were inoculated 8 days after sowing. (left) 2, ultimum, (center) control and (right) A, euteiches. 29 Fig. 8. Effect of age of Miragreen peas on susceptibility to Pythium ultimum and Aphanomyces euteiches. Symptoms when plants were inoculated 15 days after sowing. (left) g. ultimum, (center) control and'(right) A. euteiches. Fig. 9. Effect of age of Miragreen peas on susceptibility to Pythium ultimum and Aphanomypes euteiches. Symptoms when plants were inoculated 22 days after sowing. (left) g. ultimum, (center) control and (right) A. euteiches. 31 Fig. 10. Effect of age of Miragreen peas on susceptibility to Pythium ultimum and Aphanomyces euteiches. Symptoms when plants were inoculated 29 days after sowing. (left) 2, ultimum, (center) control and (right) A. euteiches. 32 for better comparison with single fungi. Pots were inoculated with E. solani alone, A, euteiches alone, 3. ultimum alone, 2, ultimum plus E, solani (1/2 quantity each), 2, ultimum plus E, solani (usual quantities), P, ultimum plus A, euteiches (1/2 quantity each) and a, ultimum plus A. euteiches (usual quantities). Disease was estimated 2 weeks after inoculation (Table 6, Fig. 11). Respective disease indices for E, solani, P. ultimum and A. euteiches were 4, 4.5 and 8. Disease severities ranging between 7.5 and 8.7 were obtained in those treatments in which any 2 pathogens acted together. Signifi- cant differences were not found between these values, thus, similar results were produced whether half or full quantities of inoculum were used. Of particular interest was the additive effect of com- bined inoculation with g. ultimum and E, gglgpi. These pathogens together caused a disease severity almost equivalent to the sum of disease indices produced by the same pathogens acting separately. Other pathogens combinations did not produce this effect. Another similar experiment gave the same results. Increased root rot severipy of Miragreenppeas infected with a virus and Pythium ultimum. The 2 first leaves of 8 day-Old pea plants were inoculated with a virus by dusting them with carborun- dum, then rubbing twice with a cotton swab which had been dipped in sap of pea plants containing either bean yellow mosaic virus (BYMV) or pea mosaic virus (FMV). Soil was infested with g. ultimum 3 days 33 Table 6. Effect of combinations of Aphanomyces euteiches, Fusarium solani and Pythium ultimum on pea root rot severity. Treatment Mycelial mats Disease per 4 pots index* Pythium 1 4.58 Fusarium 1 4.08 Aphanomyces 1 8.0b Pythium + Fusarium 1/2 + 1/2 7.5b Pythium + Fusarium l + l 8.0b Pythium + Aphanqmyces l/2 + 1/2 8.0b Pythium + Aphanomyces l + l 8 7b - 0.0c Control *Disease index was evaluated using a scale of increasing disease severity from 0-9. Each figure is a mean index of 4 pots, each with 10 plants. Means followed by different letters differed statis- tically at the 5% level of significance. INDEX DISEASE Fig. 11. Effect of combinations of Aphanqmyces euteiches, Fusarium solani and Pyphium ultimum on pea root rot severity. Treatments underscored by a line did not differ statistically at the 5% level of signifi- cance . 35 after the fungus was applied. The highest disease index, 7.8, was obtained in plants infected with both a virus and the fungus (Table 7; Fig. 12, 13, 14). The effect produced by g, ultimum alone was rated as 4.4, a value significantly lower than that produced in plants infected with either virus or the fungus. Disease levels in plants infected with either BYMV or PMV alone were significantly lower than those in plants infected by the fungus alone. Disease severity in virus-fungus infected plants was considerably higher than the sum of disease indices of plants infected by both the fungus alone and either virus alone. It can be noted, in Figs. 13 and 14, that disease severity in virus-fungus infected plants was increased in all parts of the plants and was characteristic of a severe root infection. Similar results were obtained in another experiment. 36 Table 7. Increased root rot severity of Miragreen peas infected with a virus and Pythium ultimum. Treatment Disease index* Pythium ultimum 4.3a BYMV 0.3b b PMV 0.2 c E, ultimum + BYMV 7.7 c g, ultimum + PMV 7.7 b Control 0.0 *Disease index was evaluated using a scale of increasing severity of the disease from 0-9. Each figure is a mean index of 4 pots, each with 10 plants. Values followed by different letters differed statistically at the 1% level of significance. INDEX DISEASE 37 Fig. 12. Increased root rot severity of Miragreen peas infected with a virus and Pythium ultimum. Treatments underscored by a line did not differ statistically at the 1% level of significance. 38 .5533 .M pad 25.; cacao: 30:9». zoom Aunmwuv was Eon—UH: aafinuhm Auoucoov .maufir oammoz 30:3. anon AumoC "£33 pmuommaw moon dogmas: .nH .wwm 39 .eaaauaa .M pom maua> oammoz mom Aunmwuv cam assauaa aswzufim AumuGQUV .maua> owmmoz mom Aumoav .suas noncomaa noon noouwmuwz .¢H .wwm DISCUSSION Pythium sp. may be an important component in the pea root rot complex in Michigan. The results of this survey show that Pythium sp. was present in soil samples from 20 out of 25 different pea fields in Michigan in the area of the "thumb" and near Jackson. Moreover, all isolates of this fungus were able to produce consider- able root rot disease on pea plants in greenhouse tests. In 1960, Lockwood (23) considered that fungi other than A. euteiches and E, solani involved in the pea root rot complex in Michigan, and ob- tained pathogenic isolates of Pythium sp. and other pathogens from root rot infected peas. Three of 5 Pythium isolates were identified as 2. ultimum. According to the literature, when Pythium spp. are reported as causing damage on pea plants, 2, ultimum is frequently mentioned as producing the disease, or associated with other pathogens which also cause infection in this host (16, 35, 41). The other 2 isolates identified were 2, debaryanum and g. monospermum. These were as virulent in greenhouse tests as were the isolates of g, ultimum. It would be of interest to identify other isolates of Pythium patho- genic to peas in order to establish which species of thium are most frequently associated with pea root rot. 40 41 E, ultimum isolated from infected pea plants produced con- siderable damage to roots of several commercial pea varieties, pea introductions and legumes other than peas. McDonald and Marshall (28) found a high percentage of susceptibility to this fungus in about 450 pea introductions and varieties. In the present study cereal plants, tomato (Bonny Best), cucumber (National Pickling), Gomphrena globosa, Nicotiana glutinosa and Chenppodium amaranticolor were less susceptible to this fungus. However, isolates of P. ultimum from peas, in this work, were more pathogenic on legumes in general than on other plants tested, suggesting some degree of host specificity with respect to virulence. Christensen has reported Pythium sp. as reSponsible for root rots of older plants of wheat, oats and barley, but with damage restricted mainly to immature parts of the root system (9). Al- though Pythium Spp. often become progressively less aggressive as plants develop underground lignified tissues results obtained with pea plants inoculated with g. ultimum l, 8, 15, 22 and 29 days after seeding, showed a similar degree of susceptibility, a possible slightly increased susceptibility at 22 and again at 29 days. In peas, damage was not restricted to younger parts of the root system. When pea plants were inoculated with A, euteiches, most severe disease symptoms were observed in those plants inocu- lated 1 day after seeding, but plants became more resistant to the 42 pathogen.with each increase in age. Kerr (20) considered a synergistic interaction which occurred between F, oxysporum f. pig; and g, ultimum as an important factor limiting the production of peas in Australia. In the present work an additive effect was produced when pea plants were infected with both 2. ultimum and F. ggigpi. Damage produced by these 2 pathogens together was almost equivalent to that produced by A. euteiches alone. Since the latter pathogen is considered the most damaging root rot fungus on peas (23), it should be considered that combined infection by P. ultimum and E, gplgpi, both of which are very free quently found in agricultural lands, may produce a disease severity similar to that producedby A. euteiches. An even greater enhancement of disease occurred in plants infected with a virus and g. ultimum. Miragreen peas infected with either BYMV or PMV and g. ultimum showed more severe symptoms than peas infected with the fungus alone. Final symptoms shown by the virus-fungus-infected plants of peas were characteristic of those produced by the fungus alone. Farley and Lockwood (11) reported more severe root rot symptoms when pea plants were infected with any of several viruses and either A. euteiches or E, solani f. pigi than when peas were infected with either fungus alone. Several cases of synergism between virus-fungus-infected plants have been reported in legumes other than peas and in cereals (4, ll, 37). 43 Since virus diseases are second in importance to pea root rot in Muchigan (26), the synergistic reaction found in.those virus-fungus- infected plants of peas suggest the desirability further of work to better understand the interaction between these 2 pathogens. The practical importance of this interaction is supported by the work of Watson and Guthrie (39) who reported a severe root rot epiphytotic on pea plants associated with wideSpread virus infection. The root fungi isolated were unable to reproduce the original syndrom when tested either singly or in combination. The actual mechanism which brings about the increase in sus— ceptibility in virus-infected plants to root rot pathogens is as yet little understood. One possibility is that changes in the rhizo- sphere microflora due to altered host metabolism may result in more suitable conditions for the fungi infecting roots of peas (32). Sadasivan (32) also found that rhiZOSpheres of the legume Dolichos lablab showed a considerable increment of bacteria and actinomycetes after inoculation.with Dolichos enation mosaic virus. He also found an increase in the free amino acid contents of the infected plants. Possibly, this might result in a larger amount of amino acids in exudations from roots of such plants, and provide a more suitable source of nutrition for the pathogens. Moreover, Farley and Lockwood (11), through a transplanting experiment, found evidence which sug- gested that virus infection in pea plants increased fungus inoculum 44 potential at the root surface. The possibility also exists that the host resistance is decreased as a result of virus infection. In most cases, diseases caused by Pythium are reported to be unfavorable for growth of the host, with little relation to the temperature favoring fungus growth (7). Increased disease severity produced by g. ultimum on Miragreen peas increased with soil temper— atures from 12° to 28° C. On the other hand, the optimum tempera- ture for growth of the mycelium of Pythium sp. in culture medium was at 24° C, a temperature below that producing most severe disease on pea plants, but above the optimum for growth of the host which is reported to be between 15° and 18° C (7). Thielaviopsis basicola behaves in a similar way with respect to the relation between soil temperature, growth temperature and root rot severity on peas. Thielavippsis root rot in high temperature plants such as tobacco, orange and poinsettia was most severe at low soil temperatures (6, 27). Analogous results can be cited for Pythium sp. and other pathogens of cotton seedlings which grew best at 280 C (2). In general, disease severity was greatest at 21° C whereas the optimum for the crop Was 300 C (12). In view of fact that isolates of Pythium sp. pathogenic to pea occur in most pea fields in.Michigan, further research on this root rot disease would be profitable. If the Pythium disease on peas is an important factor limiting production of this legume, it 45 would be important to search for possible sources for resistance for use in breeding. LITERATURE CITED Arndt, C. H. 1943. Pythium ultimum and the damping-off of cotton seedlings. Phytopathology 33: 607-611. Arndt, C. H. 1957. Temperature as a factor in the infection of cotton seedlings by ten pathogens. Plant Disease Reptr. Suppl. 246. 84 p. Bateman, D. F. 1961. The effect of soil moisture upon develop- ment of poinsettia root rots. Phytophathology 51: 445-451. Bateman, D. F. 1961. Synergism between cucumber mosaic virus and Rhizoctonia in relation to Rhizoctonia damping-off of cucumber. (Abstr.) Phytopathology 51: 574-575. Bateman, D. F. 1963. Factorial analysis of environment and pathogens in relation to development of the poinsettia root rot complex. Phtopathology 53: 509-516. Bateman, D. F., and A. W. Dimock. 1959. The influence of temperature on root rots of poinsettia caused by Eggpl- aviopsis basicola, Rhizoctonia solani and Pythium ultimum. Phytopathology 49: 641-647. Boswell, V. R. and H. A. Jones. 1941. Climate and vegetable crops. lg Climate and Man. U.S.D.A. Yearbook 1941: 373-399. 46 10. 11. 12. 13. 14. 15. 47 Bywater, Joan and C. J. Hickman. 1959. A new variety of Phytophthora epythroseptica, which causes a soft rot of pea roots. Trans. Brit. Mycol. Soc. 42: 513-524. Christensen, T. T. 1953. Root rots of wheat, oats, rye and barley. Ip_Plant diseases. U.S.D.A. Yearbook 1953: 321-328. Ewing, E. 1959. Factors for resistance to pre-emergence damping-off in pea (Pisum sativum L.) incited by Pythium ultimum Trow. In Diss. Abst.20: 1518-1959 (Rev. Appl. Mycol. 39: 521-522). Farley, J. D., and J. L. Lockwood. 1964. Increased suscep- tibility to root rots in virus-infected peas. Phyto- pathology 54: 1279-1280. Fulton, N. D., and K. Bollenbacher. 1959. Pathogenicity of fungi isolated from diseased cotton seedlings. Phyto- pathology 49: 684-689. Garrett, S. D. 1960. Biology of root-infecting fungi. Cambridge Univ. Press. 293 p. Gilman, J. C. 1959. A manual of soil fungi. Iowa State Univ. Press, Ames. 450 p. Hanson, E. W., and K. W. Kreitlow. 1953. The many ailments of clover. In Plant diseases. U.S.D.A. Yearbook 1953: * 217-228. 16. 17. 18. 19. 20. 21. 22. 23. 24. 48 Haskins, R. H. 1963. Morphology, nutrition, and host range of a Species of Pythium. Can. J. Microbiol. 9: 451-457. Hine, R. B., and L. V. Luna. 1963. A technique for isolating Pythium aphanidermatum from soil. Phytopathology 53: 727-728. Hoppe, P. E. 1953. Infections of corn seedlings. 13 Plant diseases, U.S.D.A. Yearbook 1953: 377-385. Jones, F. R., and M. B. Linford. 1925. Pea disease survey in Wisconsin. Wis. Agr. Exp. Sta. Res. Bull. 64: 30. Kerr, A. 1963. The root rot-Fusarium wilt complex of peas. Austral. J. Biol. Sci. 16: 55-69. Li, J. C. 1964. Introduction to statistical inference. Edwards Brothers, Inc., Ann Arbor, Michigan. 658 p. Linford, M. B. 1929. Pea diseases in the United States in 1928. Plant Dis. Reptr. 67: 14. Lockwood, J. L. 1960. Progress and problems in breeding peas resistant to root rot. Mich. Agr. Exp. Sta. Quart. Bull. 43: 358-366. Lockwood, J. L., and J. C. Ballard. 1959. Factors affecting a seedling test for evaluating resistance of pea to Aphanomyces root rot. Phytopathology 49: 406-410. Lockwood, J. L., and J. C. Ballard. 1960. Evaluation of pea introductions for resistance to Aphanomyces and Fusarium root rots. Mich. Agr. Exp. Sta. Quart. Bull. 42: 704-713. 26. 27. 28. 29. 30. 31. 32. 33. 49 Lockwood, J. L., D. J. DeZeeuw, A. L. Andersen, and D. J. Hagedorn. 1957. Pea diseases in Michigan, 1955-1956. Plant Dis. Reptr. 41: 478-480. Lloyd, A. D., and J. L. Lockwood. 1963. Effect of soil tem- perature, host variety, and fungus strain on Thielaviopsis root rot of peas. Phytopathology 53: 329-331. McDonald, W. C., and H. H. Marshall. 1961. Resistance to pre- emergence damping-off in garden peas. Canad. Plant Dis. Survey. 41: 275-279. McNew, G. L. 1953. The effects of soil fertility. LA.Plant diseases. U.S.D.A. Yearbook 1953: 100-114. Middleton, J. T. 1943. The taxonomy, host range, and geo- graphic distribution of the genus Eyghigm. Mem. Torrey Bot. Club 20: 171. Reinking, 0. A. 1942. Distribution and relative importance of various fungi associated with pea root-rot in commercial pea growing areas in New York. N. Y. Agr. Exp. Sta. Tech. Bull. 264. 43 p. Sadasivan. T. S. 1963. Physiology of virus-infected plants. J. Indian Bot. Soc. 42: 339-357. Schmitthenner, A. F. 1962. Isolation of Pythium from soil particles. Phytopathology 52: 1133-1138. 34. 35. 36. 37. 38. 39. 40. 41. 50 Schmitthenner, A. F. 1964. Prevalence and virulence of Phytophthora, Aphanomyces, Pythium, Rhizoctonia, and Fusarium isolated from diseased alfalfa seedlings. Phytopathology 54: 1012-1018. Schroeder, W. T. 1953. Root rots, wilts, and blights of peas. U.S.D.A. Yearbook 1953: 401-408. Singh, R. 8., and J. E. Mitchell. 1961. A selective method for isolation and measuring the population of Pythium in soil. Phytopathology 51: 440-444. . Smith, H. C. 1962. Is barley yellow dwarf virus a prediSposing factor in the common root rot disease of wheat in Canada? Canad. Plant Dis. Survey. 42: 143-148. Walker, J. C., and W. W. Hare. 1943. Pea diseases in Wisconsin in 1942. Wis. Agr. Exp. Sta. Res. Bull. 145. 29 p. Watson, R. D., and J. w. Guthrie. 1964. Virus-fungus inter- relationships in a root rot complex in red clover. Plant Dis. Reptr. 48: 723-727. Wilhelm, S. 1959. Parasitism and pathogenesis of root-disease fungi. ‘lp Plant pathology, problems and progress, 1908- 1958. Univ. Wisconsin Press, Madison, p. 356-366. U.S.D.A. Dept. of Agr. Zaumeyer, W. J. 1962. Pea diseases. Handbook 228. 30 p. 11131111119113111111111111111111111111111