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Xerox University Microfilms 300 North Zaab Road Ann Arbor, Michigan 48108 74 - 10,929 CHOU, Liu-Gei, 1944- FACTCRS AFFECTING FYffllUM ROOT ROT OF SOYBEAN AND GERMINATION OF OOSPORES OF FY1H HM ULTIMUM. Hie Ohio State University, Ph.D., 1973 Agriculture, plant pathology (

University Microfilms, A XERQXCompany, Ann Arbor, Michigan

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. FACTORS AFFECTING ROOT ROT OF SOYBEAN AND GERMINATION OF OOSPORES OF

DISSERTATION

Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

by

Liu-Gei Chou, B.S., M.Sc.

ft ft ft A ft ft

The Ohio State University 1973

Reading Committee I. W. Deep Approved by J. D. Farley

' 'J A. F. Schmitthenner 4 » Adviser Department of Plant Pathology ACKNOWLEDGMENTS

The author expresses his appreciation to his academic adviser, Dr. A. F. Schmitthenner, for guidance, encouragement, and cooperation given during the course of thlis research and throughout his graduate career. Sincere thanics are also extended to: Drs. I. W. Deep, L. E. Williams L. E. Bendixen and J. D. Farley for constructive criticisms made during the preparation of this manuscript; Dr C. R. Weaver for statistical advice; and Mr. Clark Robey for photographic work. Appreciation is also extended to staff members, graduate students, and technicians of the Department of Plant Pathology at the Ohio Agricultural Research and Development Center and The Ohio State University. The author also appreciates the concern of his father and mother and the patience and encouragement of his wife throughout this endeavor. VITA

April 26, 1944...... Born - Chungking, China 1967 ...... B.S., National Taiwan University Taipei, Taiwan, China 1967-1968 ...... Biology Instructor, The First . NCO Academy, Chinese Army, Chungli, Taiwan, China 1968-1970 ...... Graduate Teaching Assistant, Department of Plant Pathology, The Ohio State University, Columbus, Ohio 1970 ...... M.Sc., The Ohio State University Columbus, Ohio 1971-1973 ...... Graduate Research Associate, Department of Plant Pathology, Ohio Agricultural Research and Development Center and The Ohio State University, Wooster, Ohio

FIELDS OF STUDY

Major Field: Plant Pathology Studies in Soil-borne Pathogens and Root Diseases. Professors A. F. Schmitthenner and J. D. Farley Studies in Field Crop Diseases. Professors C. C. Allison, C. W. Ellett; R. E. Partyka, L. J. Herr, and A. F. Schmitthenner Studies in Plant Virology. Professors D. T. Gordon and 0. E. Bradfute I

FIELDS OF STUDY (continued)

Studies in Physiology of . Professor M. 0. Garraway Studies in Plant Physiology. Professors B, S. Meyer and D. Frantianne Studies in Pesticides. Professor L. E. Bendixen

iv CONTENTS Page ACKNOWLEDGMENTS...... ii VITA ...... iii LIST OF TABLES ...... viii LIST OF FIGURES • ...... x PART I: EFFECT OF RHIZOBIUM JAPONICUM AND ENDOGONE MOSSEAE ON SOYBEAN ROOT ROT CAUSED BY PYTHIUM ULTIMUM AND MEGASPERMA VAR. SOJAE...... 1 INTRODUCTION...... 1 MATERIALS AND METHODS ...... 3 RESULTS ...... 9 DISCUSSION...... 14 PART II: FACTORS AFFECTING GERMINATION OF OOSPORES OF PYTHIUM ULTIMUM...... 17 INTRODUCTION...... 17 MATERIALS AND METHODS...... 19 1. Media for oospore production...... 19 2. Media for oospore germination ...... 20 3. Preliminary methods for inducing oospore germination...... 20

v CONTENTS (continued) Page 4. Method for extraction of oospores from liquid culture ...... 22 5. Effect of age on oospore germination. . 22 6 . Effect of light on oospore germination. 23 7. Light treatment of dark incubated o o s p o r e s ...... 23 8 . Interactions of age and light on oospore germination...... 24 9. Effect of exogenous nutrients on oospore germination...... 24 RESULTS...... 25 1. Preliminary attempts to induce oospore germination...... 25 2. Effect of age on oospore germination . . 25 3. Effect of light on oospore germination , 26 4. Activation of dark-incubated oospores by l i g h t ...... 29 5. Interactions of age and light on oospore germination...... 29 6 . Effect of exogenous nutrients on oospore germination...... 30 DISCUSSION...... 36 CONTENTS (continued)

Page SUMMARY...... 40 LITERATURE CITED...... 43

✓

• • VII LIST OF TABLES Table Page 1 . List and abbreviation of treatments used to study interaction of Pythium ultimum with Endogone mosseae, Rhizobium japonicum or Phytophthora megasperma var. sojae races 1 and 3 . T T ...... 8 2 . Mean plant height in cm of 35-day-old soybean plants inoculated with various combinations of Endogone mosseae, Rhizobium japonicum, Pythium ultimum and Phytophthora megasperma var. sojae races 1 and 3 I “ ...... 11 3. Mean shoot dry weight in g of 35-day-old soybean plants inoculated with various combinations of Endogone mosseae, Rhizobium japonicum, Pythium ultimum and Phytophthora megasperma var. sojae races 1 and 3 .I T . 12 4. Mean number of 35-day-old soybean plants killed out of 10 which had been inoculated with various combinations of Endogone mosseae, Rhizobium japonicum, Pythium ultimum and Phytophthora megasperma var, sojae races 1 and 3 13 S. Percentage of 12-week, dark-incubated Pythium ultimum oospores in different stages of development, following treatment with light for different time periods...... 31 Percentage of Pythium ultimum oospores in different stages of development following incubation for varying periods under different light conditions...... 32

viii LIST OF TABLES (continued)

Table Page 7. Percentage germination in various concentrations of sucrose, asparagine and staling substances of Pythium ultimum oospores from 12-week-old cultures incubated under alternating light and dark conditions. . 34

8. Percentage germination in various concentrations of sucrose, asparagine and NH^NO, of Pythium ultimum oospores from 12-week-ola cultures incubated under alternating light and dark conditions...... 35 LIST OF FIGURES

Figure 1. A double styrofoam cup system for studying the effect of Rhizobium japonicum and Endogone mosseae on the seventy of soybean root rot caused by Pythium ultimum and Phytophthora megasperma var. sojae. A portion of the lower, outer cup is removed to show the development of roots in the space below the inner cup...... 2. Sequential stages during the germination of a single oospore of Pythium ultimum. A) Dormant, thick-walled oospore"! BJ Thin-walled, -like oospore in pregermination stage but the oogonial and oospore wall clearly distinguishable. C-G) Taken at 5 min intervals, showing germination ...... PART I

EFFECT OF RHIZOBIUM JAPONICUM AND ENDOGONE MOSSEAE ON SOYBEAN ROOT ROT CAUSED BY PYTHIUM ULTIMUM AND PHYTOPHTHORA MEGASPERMA VAR. SOJAE

INTRODUCTION

Members of the genus Rhizobium upon infection of the appropriate legume, can cause the formation of nodules and participate in the symbiotic acquisition of N2 (l). Most information on nodulation of legumes by Rhizobium is concerned with the nodulation process, fixing of nitrigen and biochemistry of symbiotic N fixation (1). Very few (a reports deal with the relation of Rhizobium to soil-borne pathogens. Schwinghamer and BilkengTen (27) reported that a strain of Rhizobium trifolii inhibited other Rhizobium species. Roslycky (19) found that bacteriocin produced in Rhizobia bacteria can inhibit some other bacteria. Drapeau et al. (6) tested the antifungal activity of three different Rhizobium isolates against eight different fungi in vitro. Inhibition zones were observed in several cases. Similar results were obtained with cell-free extracts of the media on which Rhizobium had grown.

1 2

Ectotrophic mycorrhizal fungi also have reported to affect severity of root rot. Orchids infe.c.ted with mycorrhizal fungi produce a phytoalexin, orchinol, which may protect the plant against Rhizoctonia repens (11). It has also been demonstrated that mycorrhizal seedlings are more resistant to Mycelium radius atrovirens (18). Marx has reported that ectotrophic mycorrhizal roots of pine are resistant to Phytophthora cinnamomi (15). The influence of ubiquitous vesicular-arbuscular mycorrhizae on root invading pathogens has recently been studied by Ross (20). He reported Endogone increased root rot symptoms caused by Phytophthora megasperma var. sojae in a disease-susceptible cultivar. Pythium ultimum has usually been considered to cause a root and stem rot of soybean only in the seedling stage (13). Recently in Ohio P. ultimum has been associated with root rot of Phytophthora-resistant varieties in heavy soils where P. megasperma var. sojae is known to occur (23). However, it has not been possible to induce severe root rot and death of soybeans by Pythium alone in controlled green­ house tests. The effects of Phytophthora on severity of Pythium root rot have not been studied. The purpose of this study was to determine if severity of Pythium root rot could be affected by interaction with E. mosseae, R. japonicum or P. megasperma var. sojae in a Phytophthora resistant variety. MATERIALS AND METHODS

A double styrofoam cup system modified from Meyer et a l . (16) was used in this study. One 10-cm-diam cup with a nylon tulle bottom was filled a perlite; vermiculite; autoclaved muck; and soil mixture (1 :1 :1 :1 , v/v) and nested in a second cup (Pig. 1). Three g of commercial inoculant of Rhizobium japonicum (Kirchner) Buchanan as "Legume Aid" (Agricultural Laboratories, Columbus, Ohio) was mixed thoroughly with 300 g soil mixture for each cup. Endogone mosseae Mosse was provided by J. W. Gerdemann (Univ. of Illinois) and increased in growing-maize roots in an autoclaved sand-soil mixture (12). About 100 g of this inoculum mixture, which contained maize mycorrhizae and spores of E. mosseae were added to each cup for the Endogone treatment. A similar autoclaved portion of this inoculum mixture was added to the control cups, along with a water filtrate from unsterilized inoculum, which had been passed three times through a sieve with 44 u openings to remove E. mosseae spores. This procedure was to ensure that root microorganisms, which can affect nutrient availability and plant growth, would also be present in control cups (21).

3 4

Virulent isolates of Pythium ultimum Trow (P509), s Phytophthora megasperma Drechs, var. sojae A. A. Hildeb. race 1 (P507) and race 3 (P573) were obtained from diseased soybeans in Ohio. Stock cultures were stored at 5 C in 15 ml bottles on 4% V-8 juice agar (V8A) plus 0.01 g cholesterol/liter (25). Petri plate cultures on the same agar were used as inoculum in inoculation. Two-week-old V8A cultures of Pythium ultimum and Phytophthora megasperma var. sojae races 1 (Pms^) and race 3 (Pms,) were added either as a layer 7 cm below the seeds at planting time or between the bottom of the inner and outer cups 2 weeks after planting. Treatments are listed in Table 1. For each treatment of Rhizobium and/or Endogone there were 11 combinations of P. ultimum and/or Pms or Pms . All combinations of Endogone, ™ 3 Rhizobium, Pythium and Phytophthora were tested using Harosoy-63 soybeans (Glycine max [L.] Merr.). Harosoy- 63 soybeans are resistant to Pms but not to Pms,. Before JL O planting, seeds were surface disinfested with 1% NaCIO and dried. Fourteen seeds were planted in each cup. One week after planting, plants were thinned to 10/cup. The soil mixture was kept near saturation during the experimental period. Experiments were run in the greenhouse with day temperature set for 21 C and night temperature for 17 C and exposed to supplemental illumination during the day of s

Fig. 1. A double styrofoam cup system for studying the effect of Rhizobium japonicum and Endogone mosseae on the severity of soybean root rot caused by Pythium ultimum and Phytophthora megasperma var. sojae. A portion of the lower, outer cup is removed to show the development of roots in the space below the inner cup.

5 CK 7 approximately 3,000ft-c from cool and warm white fluore­ scent lamps for 12 hr/day. Thirty-five days after planting, plant height, dry weight of above-ground plant parts and number of dead plants were determined. Mean plant height and dry weight did not include dead plants. The experiment was repeated six times. A standard deviation of the means was computed to compare treatment means. 8

Table 1. List and abbreviation of treatments used to study interaction of Pythium ultimum with Endogone mosseae, Rhizobium japonicum or Phytophthora rma var. sojaesoj races 1 and 3.megasperma

Treatments Symbol

A. Endogone and Rhizobium treatments E. mosseae Em E. japonicum Rj E. mosseae control Emc E. mosseae + R. japonicum Era+Rj E. mosseae control + R. japonicum Emc+Rj Eeither E. mosseae nor R. japonicum None

B. Pythium and Phytophthora treatments P. ultimum in inner cup Pu-ic E. ultimum in outer cup Pu-oc E. megasperma var. sojae race 1 Pms^-ic in inner cup P. megasperma var. sojae race 1 Pms^-oc m outer cup P. megasperma var. sojae race 3 Pms3-ic in inner cup P. megasperma var. sojae race 3 Pms3-oc “ m outer cup P. ultimum in inner cup + P. Pu-ic+Pms^-oc megasperma var. sojae race 1 in outer cup P. ultimum in outer cup + £. Pu-oc+Pms^-ic megasperma var. soj ae race 1 m inner cup P. ultimum in inner cup + P. Pu-ic+Pms3-oc megasperma var. soj ae race 3 m outer cup P. ultimum in outer cup + P. Pu-oc+Pms3-ic megasperma var. sojae race 3 in inner cup Neither Pythium nor Phytophthora None RESULTS

Inoculation by P. ultimum alone resulted in shorter plants in all Rhizobium and Endogone treatments compared to the control (Table 2). P. ultimum plus Pms^ had the same effect as Pms^ alone. However, plants inoculated with P. ultimum plus Pms, in the inner cup had shorter plant height than those inoculated with Pms, alone but only in treatments 3 without Endogone and Rhizobium. Plants infected by Pms_ alone or Pms_ plus P. ultimum resulted in lower dry weight in all Endogone and Rhizobium treatments (Table 3). Pms, infection resulted in less dry weight in sterile soil than in most treatments with Endogone and Rhizobium. The mean dry weight of plants was the same in the P. ultimum and P. ultimum plus Pms^ treatments.

No plants were killed in P. ultimum or P. ultimum plus Pms^ treatments either in sterile soil or in the presence of Endogone or Rhizobium (Table 4). However, more plants were

killed by _P. ------ultimum plus ------Pms_3 than ------Pms_3 alone in most treatments. Also, more plants were killed by Pms infection 3 in sterile soil than in treatments with Endogone and Rhizobium plus Pms,. More plants were killed when a Pms • j 3 culture was placed in the inner cup than in the outer cup.

9 10

Also, lower mean plant height and dry weight were obtained when plants were inoculated by Pms^ in the inner cup compared to the outer cup. 11

Table 2. Mean plant height in cm of 35-day-old soybean plants inoculated with various combinations of Endogone mosseae, Rhizobium japonicum, Pythium ultimum "and Phytophthora megasperma var. sojae races 1 and TT

Pythium and £ Phytophthora Endogone and Rhizobium treatments treatments* Em Rj Emc Em+Rj Emc+Rj None

Pu-ic 34.9b 36.2 37.7 35.9 35.1 34.3 Pu-oc 35.9 37.1 38.6 36.7 39.0 34.5 PmSj^-ic 36.9 38.0 39.9 38.2 40.3 35.0 PmSj-oc 35.7 37.6 38.3 37.9 40.0 34.8 Pms^-ic 17.5 16.1 15.5 19.2 17.1 18.4 Pms^-oc 28.8 30.8 29.3 29.3 25.8 28.1 Pu-ic+Pms -oc 33.7 36.6 40.0 34.4 37.5 33.9 1 Pu-oc+Pms^-ic 34.5 39.0 41.5 36.6 39.4 36.8 Pu-ic+Pms -oc 30.7 30.1 31.3 29.0 31.6 29.2 3 Pu-oc+PmSg-ic 20.6 15.9 17.2 20.2 17.7 10.4 None 37.3 39.3 40.2 37.3 40.7 39.6

See Table 1 for description of treatments.

Mean of six replications of 10 plants/treatment. Standard error difference of means 3 2.5. 12

» Table 3. Mean shoot dry weight in g of 35-day-old soybean plants inoculated with various combinations of Endogone mosseae, Rhizobium japonicum, Pythium ultimum“and Phytophthora megasperma var. sojae races 1 and TT

Pvthium and Endogone and Rhizobium treatments® Phytophthora treatments11 Em Rj Emc EM+Rj Emc+Rj None

Pu-ic 0.56b 0.56 0.55 0.57 0.56 0.55 Pu-oc 0.59 0.58 0.60 0.54 0.59 0.60 Pms^-ic 0.59 0.56 0.58 0.54 0.60 0.60 Pms^-oc 0.59 0.61 0.62 0.58 0.62 0.61 Pms -ic 0.38 0.24 0.28 0.38 0.40 0.20 3 PmSj-oc 0.51 0.46 0.54 0.40 0.55 0.46 Pu-ic+Pms^-oc 0.55 0.53 0.58 0.57 0.56 0.53 Pu-oc+Pms^-ic 0.56 0.57 0.61 0.59 0.58 0.61 Pu-ic+Pms -oc 0.52 0.42 0.50 0.43 0.49 0.46 3 Pu-oc+Pms -ic 0.32 0.26 0.27 0.32 0.32 0.18 3 None 0.65 0.65 0.65 0.64 0.65 0.65

See Table 1 for description of treatments. b Mean of six replications of 10 plants/treatment. Standard error difference of means ■ 0.065. 13

Tabic 4. Mean number of 35-day-old soybean plants killed out of 10 which had been inoculated with various combinations of Endogone mosseae, Rhizobium japonicum, Pythium ultimum and Pliytophthora megasperma var. sojae races 1 and 3.

Pythium and knytophthora Endogone and Rhizobium treatments® treatments11 Em Rj Emc Em+Rj Emc+Rj None b Pms3-ic 5.6 7.1 6.6 4.6 6.6 8.0 o 00

Pms -oc 0.8 1.5 0.8 1.0 • 1.3 3 Pu-ic+Pms -oc 1.0 1.0 1.3 1.0 1.5 1.5 3 Pu-oc+PmSj-ic 5.0 5.5 7.6 5.5 7.3 8.8 o o

All other 0.0 0.0 0.0 • 0.0 0.0 treatments

See Table 1 for description of terms. b Mean of six replications of 10 plants/treatment. Standard error difference of means = 0.40, DISCUSSION

Severity of Pythium root rot was not affected by E. mosseae and R. japonicum, indicating this endotrophic mycorrhizal fungus and the root nodule bacteria did not dispose the host to infection or enhance the severity of root rot. The presence of Pms^ either in the inner or outer cup did not have any effect on Pythium root rot of soybeans. Also, Pythium root rot did not alter resistance of Harosoy-63 soybean to Pms^. Inoculation of Pms in the inner cup was more severe 3 than inoculation in the outer cup. The same observation was reported by Meyer and Sinclair (17) who found root and shoot dry weights increased with depth of inoculum place­ ment. More plants were killed by Pms in combination with

P. ultimum than Pms7 alone in most treatments with Endogone and Rhizobium, but the differences were small. However, more plants were killed by Pms and lower mean dry weights 3 were obtained in sterile soil than with Endogone and Rhizobium treated soil. E. mosseae and R. japonicum may have the same effect on root rot severity commonly attributed to soil microflora (1). Further study may indicate direct effects of exudates produced by mycorrhizae or root nodules on

14 15 pathogenicity of soil-borne pathogens. The fact that fewer plants were killed by Pms in E. mosseae treatments may indicate some effects were exerted by Endogone on Phytophthora, but mycorrhizal development in Phytophthora- resistant soybean still did not completely protect the plants against infection by a host-compatible Phytophthora race. These results differed from those of Ross (20) who reported that soybean plants were killed by Phytophthora in the presence of Endogone, but not in Endogone-free treatments. However, Ross used fumigated field soil and a different type of inoculum, so the conditions for disease development were not similar. Plants infected by P. ultimum and Pms1 in all Endogone an<* Rhizobium treatments developed normal bacterial root nodules. Plants infected by Pms, alone or in combination with P. ultimum developed very fewer root nodules. This may be due to poor formation of the root system, or the presence of Pms^ in the roots may inhibit nodule formation. Drapeau et al. (2) reported no antifungal activity of three strains of Rhizobium against P. ultimum in vitro, but the effect of Rhizobium against Phytophthora megasperma var. sojae has not been reported. Vesicular-arbuscular mycorrhiza of E. mosseae developed in roots of soybean 3 weeks after inoculation, while roots in the control or non-inoculated cups were nonmycorrhizal. 16

Vesicles, arbuscules, and intracellular hyphae were observed in cortical cells of roots after stain with acid-fuchsin or carbo-fuchsin (4). Soybean roots infected by Pythium or Phytophthora both developed mycorrhiza indicating mycorrhizal development was not affected by the presence of Pythium or Phytophthora in soybean roots. PART II

FACTORS AFFECTING GERMINATION OF OOSPORES OF PYTHIUM ULTIMUM

INTRODUCTION

Thick-walled oospores of soil-borne phycomycetes, e.g., Pythium ultimum are commonly considered to be the resistant structures capable of long-term survival in soil. The role of sporangia of P. ultimum as major survival structures and inocula in cultivated soils has been indicated by Stanghellini and Hancock (29). Very few studies have been reported concerning the role of oospores of P. ultimum as inocula in soil as well as factors affecting their germination. Trow (33) was first to observe oospore germination of P. ultimum. His methods for germination consisted of placing oospores in distilled water for some days, then transferring to running water, and finally to cabbage juice. The oospores generally produced one or more germ tubes, but the method did not always succeed. No time periods were indicated for the three steps for germination and his methods could not be duplicated (33). Dreschler (7,8,9) later reported oospores of P. ultimum germinate rather readily if, after-

17 18 ripened for 2 to 4 months under a bell jar, vrere then placed in water in a block of the original agar. Oospores germinated readily, through formation of germ tubes which developed a vesicle and zoospores or wholly vegetative hyphae or a hyphae bearing one or several sporangia capable of giving rise to zoospores (9). Both authors (9,33) agreed that oospores assumed the internal organization usual in conidia after a resting period and could be recognized only by their position inside the wall of the oogonium. More recently very scant information (2,5) has indicated difficulty in germinating viable oospores of P. ultimum. The present investigation was undertaken to develop a standard method for oospore germination of P. ultimum and to elucidate the factors involved.

» MATERIALS AND METHODS

The Pythium ultimum Trow isolate (P509) was obtained from diseased soybean plants in Ohio. It was stored at 5 C in 15 ml bottles on 4% V-8 juice agar (V8A) plus 0.01 g cholesterol/liter (12). 1. Media for oospore production. V8A, corn meal agar (CMA), and a sucrose-asparagine liquid solution (SAS) were used for oospore production. CMA was prepared by heating 30 g of corn meal in 1 liter of distilled water over a water bath until boiling. The mixture was stirred for 1 hr, then filtered through cheese­ cloth, and 20 g of agar was added and dissolved by boiling. The medium was then autoclaved at 15 p.s.i. for 20 min. Agar media were dispended at 15 ml/9-cm-diam petri dishes. SAS consisted of: 2.6 g sucrose; 110 mg asparagine; 150 mg JCH2P04 ; 150 mg K2HP04 ; 100 mg MgS04*7H20; 4.4 mg ZnS04 *7H20; 1 mg FeS04’7H20; 0.07 mg MnCl2.4 H20i 55 mg CaCl^ 2 mg thiamine hydrochloride; 10 mg cholesterol; and distilled water to make a final volume of 1 liter. The SAS medium with a C:N ratio of 50:1 and 0.261 sucrose concentration was the best of a number of C:N ratios and sucrose concentrations tested for oospore production.

19 20

2. Media for oospore germination* Several media were used for oospore germination. These media included SAS; a selective differential medium for Pythium (PBNC) consisting of V8A with 20 mg PCNB, 10 mg benomyl, 100 mg neomycin sulfate and 5 mg Chloromycetin/ liter added before autoclaving (14); Epiagar (Colab Laboratories, Glenwood, Illinois) containing various combinations of sucrose, asparagine and solutions from 12-week-old liquid SAS cultures (staling substances) listed in Table 7; and Epiagar containing different amounts of sucrose, asparagine, and NH^NO^ listed in Table 8. A C:N ratio of 10:1 was used for all carbon-nitrogen compounds. Sterile distilled water or Epiagar containing sterile tap or distilled water were used as controls. The main constituent of Epiagar is a calcium salt of a sulfuric acid ester of a complex polysaccharide. Unless the agar is hydrolyzed vigorously, it is unlikely that it will break down to smaller molecules that can be utilized by fungal spores as an energy source (4). Oospores were incubated for 5 hr at 24 C on the different media then flooded with 20% carbo-fuchsin to stop further growth and oospore counts were made. Three hundred oospores were counted for each of three replicates for each experiment, and each experiment was repeated three times in all germination tests. 3. Preliminary methods for inducing oospore germination. Four methods were used to induce oospore germination 21 on V8A or CMA: (i) light and freezing treatments used by Erwin and McCormick for germination of Phytophthora megasperma var. sojae oospores (10); (ii) passage of oospore through snails following procedures of Stanghellini and Russell (30); (iii) exposure of cultures to near ultra­ violet light followed by nutrients at two different temperatures; and (iv) macerating oospores from agar cultures in a Waring blender, and then treating with various nutrients. For methods (i) and (ii) the procedures cited were followed exactly. For method (iii) 2-week-old cultures grown on V8A or CMA were exposed to light from 15 W black- ray bulb (Ultra-violet Products, San Gabricel, California) for 2 to 36 hr. Cultures were then flooded with sterile 3,000 ug/ml sucrose solution or sterile distilled water and incubated at 5 C or 24 C for 10-15 hr and examined for oospore germination. For method (iv) oospores were collected from 2-week-old V8A or CMA agar culture by scraping the surface mycelium from the medium, suspending in water, and macerating in a Waring blender. The residue after filtration, consisting of oospores, sporangia, and empty hyphal fragments, was resuspended in sterile distilled water. There were several hundred or more oospores per ml of suspension. Five ml of fresh spore suspension were mixed with 5 ml of 10, 100, or 1,000 ug/ml of glucose, sucrose, asparatic acid or glutamic acid and placed in 9-cm-diam petri dishes at 24 C for 5 to 15 hr. The oospores were 22 then examined for germination. 4. Method for extraction of oospores from liquid culture. A standardized method was used in harvesting oospores. Aqueous suspension of oospores were prepared by mincing a mycelial mat from a SAS culture flask with 100 ml of distilled water in a Sorvall mixer at high speed for 5 min, then adding 400 ml of distilled water to the suspension and passing through a 53 u sieve (U.S. Standard Sieve Series, W. S. Tyler Co., Mentor, Ohio). The sieve surface was washed with 1.5 liter distilled water to force maximum number of oospores to pass through the sieve. The total 2 liter suspension was then passed a 20 u fiber screen (Tobler, Ernst and Trabler, Inc., Elmsford, N.J.) placed on the support screen of a hydrosol stainless filter holder (Millipore Corp., Bedford, Mass.). Oospores collected on the screen were washed three times with 100 ml distilled water in order to remove traces of nutrients that might be present. Oospores were then washed from the screen with 20 ml of distilled water and their concentration was adjusted to 10,000 oospores/ml by using a hemocytometer. The suspension consisted of oospores and sporangia nearly free of viable hyphal fragments. Sterile conditions were not maintained during harvest procedures. 5. Effect of age on oospore germination. In initial studies on the effect of age on oospore germination, culture grown in SAS in flasks were placed in 23

the laboratory in a wall cabinets with glass doors at 24 C. After 4 and 12 weeks incubation the oospores were harvested and placed in SAS for germination. 6. Effect of light on oospore germination. To determine if light had an effect on germination, cultures grown in SAS in flasks were incubated at 24 C under three light conditions: (i) alternating light and dark; (ii) continuous dark; and (iii) continuous light. For alternating light and dark treatments, the flasks were placed in a glass door wall cabinets and illuminated when the laboratory lights were on (approximately 35 ft-c for 12 hr/day). For continuous dark treatments, flasks were placed in a steel door wall cabinet in the same laboratory. For continuous light incubation, flasks were placed in a G-26 Psycrotherm controlled environment incubator (New Brunswick Scientific Co., New Brunswick, N.J.) with 1,090 ft-c of light 24 hr/day. The light intensity was measured by a model-756 Weston illumination meter (Weston Electric Corp., Newark, N.J.). Flasks were sampled for oospore germination in SAS or on PBNC media after 4, 10 and 12 weeks incubation. 7. Light treatment of dark incubated oospores. For testing effects of light on germination of dark- incubated oospores, flasks incubated in continuous dark for 12 weeks were exposed to 3,000 ft-c of light in a M-13 growth chamber (Environmental Growth Chambers, Chagrin 24

» Falls, Ohio) at 24 C for 12, 36 and 60 hr. Oospores were harvested and placed on PBNC media to determine germination. 8. Interactions of age and light on oospore germination. For studying interactions of age and light on oospore germination, SAS cultures were incubated for 4, 6, 8, 10, and 12 weeks under the following light conditions: (i) alternating light and dark; (ii) continuous dark, and (iii) continuous dark followed by 24-hr exposure to 3,000 ft-c. At the end of each specified incubation period, the per­ centage of thick-walled dormant oospores and thin-walled activated oospores were determined. The oospores were then harvested and placed on PBNC media for germination. 9. Effect of exogenous nutrients on oospore germination. For nutritional studies on oospore germination, oospores were obtained from 12-week-old cultures incubated in alternating light and dark. The effect of nutrients on oospore germination was studied in two tests. In both, Epiagar containing various additives were used (Table 7 and

.8),

i RESULTS

1. Preliminary attempts to induce oospore germination. Oospores of Pythium ultimum produced and treated as recommended by Erwin and McCormick (10) for oospore germination of Phytophthora megasperma var. sojae (light during formation-cold treated-frozen-harvested-placed in water) did not germinate. Two-4-week-old oospores passed through live water snails as reported by Stanghellini and Russell (30) for , did not germinate. Two-week-old oospores on V8A or CMA plates exposed to ultraviolet light then flooded with sucrose or distilled water failed to germinate. Two-week-old oospores harvested from V8A or CMA plates and placed in solutions of different carbon and nitrogen sources also failed to germinate. In all these tests thick-walled oospores with a fine-grained cytoplasm remained dormant and no changes in the internal organization of the oospores were observed. 2. Effect of age on oospore germination. Four-week-old oospores harvested from SAS cultures did not germinate in SAS. However, approximately 701 of 12-week-old oospores harvested from SAS cultures germinated when placed in SAS. No germination of oospores occurred in sterile distilled water.

25 It was noted that most of the oospores harvested from 12-week cultures did not have a thick-wall (Fig. 2A) but had the appearance of conidia (Fig. 2B) described by Trow (33) and Drechsler (7) and referred to as after-ripened oospores (7) or pregermination stage in Phytophthora oospores (34) or Pythium aphanidermatum (30). Oospores with this appearance will be referred to as activated oospores, following the terminology of Sussman and Halvorson (32). The activated oospores germinate in 2-5 hr as depicted in Fig. 2C-G. Only germ tubes which developed into hyphae were observed as described by Trow (33). 3. Effect of light on oospore germination. In the previous experiment aging resulted in activated oospores that were able to germinate in the presence of nutrients. The cultures were aged in alternating light and dark in the laboratory. To determine if light had an effect, germination of oospores incubated in alternating light and dark, continuous dark, and continuous light were compared. Oospores incubated in continuous light for 4, 10 and 12 weeks produced empty spores, presumably they were aborted oospores. Oospores incubated in alternating light and dark germinated less than 21 in SAS, after 4-week-incu- bation. No germination occurred in sterile distilled water. However, after 10 weeks approximately 601 of the oospores * from this treatment germinated in SAS. Germination

i percentage was low in distilled water. Fig. 2. Sequential stages during the germination of a single mature oospore of Pythium ultimum. A) Dormant, thick-walled oospore. B) Thin-walled, sporangium-like oospore in pregermination stage but the oogonial and oospore wall clearly distinguishable. C-G) Taken at 5 min intervals, showing germination.

27

29

Less than 101 of the dark incubated oospores germinated in the presence of nutrients even after 12 weeks of aging. Most of the oospores retained the typical thick-walled dormant appearance. It was evident from this experiment that only oospores incubated in alternating light and dark were activated and capable of germination. 4. Activation of dark-incubated oospores by light. This experiment was set up to determine if dark incubated oospores could be activated and induced to germinate by short exposure to high intensity light. The 12-week, dark-incubated culture containing oospores were exposed to 3,000 ft-c of light and sampled at 12, 36, and 60 hr for oospore germination (Table 5). After exposure to light, the texture of the cytoplasm of the thick-walled dormant oospores became coarser and the inner oospore wall began to erode, indicating the dormant oospores were activated. Only 71 of the oospores germinated without light treatment, while 62% germinated after a 36-hr light exposure. 5. Interactions of age and light on oospore germination. Treatments listed in Table 6 were set up to determine if the degree of light activation of oospores was dependent on age of oospores. Oospore incubated in three light conditions were harvested, washed, and placed on PBNC media to determine germination percentage. Oospores sampled at 30

4 weeks did not germinate in any of the treatments. A number of thin-walled oospores were present but the cytoplasm had the appearance of immaturity without the coarse texture appearance of activated oospores. Percentage of dormant oospores decreased progressively and that of activated and germinated oospores increased in both alternating light and dark and the high intensity light treatments with increasing incubation time. Most oospores incubated in continuous dark retained a dormant appearance throughout the 12-week period and less than 10% germinated. Apparently one of the characteristic features of the oospore germination process of P. ultimum is that spores can be activated by light and germinate only after they have reached a critical age. Also, treatment with low intensity light for long periods activates oospores as well as short periods of high intensity light, but the age at which they can be activated is not the same for all oospores. 6. Effect of exogenous nutrients on oospore germination. The effect of nutrients on oospore germination was studied in two tests. In both, oospores harvested from 12- week-old SAS cultures incubated in alternating light and dark were placed on Epiagar with various additives. In Table 7, the percentage germination of oospores on sucrose, asparagine, and staling substances are compared. High percentage of oospore germination was obtained only in the presence of sucrose or asparagine. Only 2% germinated on 31

Table S. Percentage of 12-week, dark-incubated Pythium ultimum oospores in different stages 01 development, following treatment with light for different time periods.

% Oospores in Oospores in different different stages stages following light Stages of before lig^t treatment in hra development treatment n J5---- W

Dormant 69 11 18 16 _b _b _b Activated 11 Germinated 0 79 62 55 Disintegrated 20 10 20 29

Three hundred oospores were counted for each of three replicates for each treatment and each experiment was repeated three times. The mean percentage is shown.

Included in dormant count. 32

Table 6. Percentage of Pythium ultimum oospores in different stages of development following incubation for varying periods under different light conditions.

I Oospores in different stages3 Incubation Time r conditions (weeks) Dormant Activated Germinated

Alternating light 6 76 20 7 and dark 8 46 47 24 10 40 47 28 12 16 76 71

Continuous dark 6 91 7 1 8 81 13 2 10 86 5 4 12 82 11 8

Continuous dark 6 67 26 14 plus 24-hr high intensity light 8 32 59 34 10 33 56 36 • 12 18 72 60 a Three hundred oospores were counted for each of three replicates for each treatment and each experiment was repeated three times. The mean percentage is shown. b Percentage of germination recorded after S-hr incubation on PBNC medium at 24 C. 33

Hpiagar alone, indicating a lack of sufficient nutrients to support germination. Table 8 lists the percentage germination of oospores in different carbon and nitrogen media. Germination declined with decreased concentration of sucrose starting with 100 ug/ml and germination was poor on inorganic nitrogen alone. However, addition of inorganic nitrogen may have improved germination at the lower sucrose concentrations. Oospores of P. ultimum appeared to be nutrient dependent and required a carbon source for germination. 34

Table 7. Percentage germination in various concentrations of sucrose, asparagine and staling substances of Pythium ultimum oospores from 12-week-old cultures incubated under alternating light and dark conditions.

Substrate 1 oospore germination*

Sucrose 3,000 ug/ml 50 Asparagine 300 ug/ml 42 Staling substances 8 Sucrose 3,000 ug/ml asparagine 56 300 ug/ml Sucrose 3,000 ug/ml + asparagine 52 300 ug/ml + staling substances Asparagine 300 ug/ml + staling 48 substances Sterile distilled water 2 a Percentage germination recorded after S-hr incubation at 24 C. b Three hundred oospores were counted for each of three replicates for each treatment and each experiment was repeated three times. The mean percentage is shown. 35

Table 8. Percentage germination in various concentrations of sucrose, asparagine and NH.NO. of Pythium ultimum oospores from 12-week*ola cultures incubated under alternating light and dark conditions.

ug/ml ug/ml % Oospores . Substrate C N germination »

Sucrose-asparagine 10,000 1,000 88 1,000 100 87 100 10 66 10 1 39

Sucrose-NH.NO, 10,000 1,000 81 4 3 1,000 100 80 100 10 70 10 1 60

Sucrose 10,000 86 1,000 --- 83 100 --- 46 10 --- 36

NH.NO_ --- 10,000 18 4 3 --- 1,000 15 100 10 10 9 1

Sterile tap water — — 5

Sterile distilled — — 2 water

Percentage of germination recorded after 5-hr incubation a*t 24 C.

Three hundred oospores were counted for each of three replicates for each treatment and each experiment was repeated three times. The mean percentage is shown. DISCUSSION

Evidence presented in this study indicates that oospores of Pythium ultimum are capable of germination and, therefore, probably play a major role in the life cycle of this species. The three stages that appear to be involved in the germination process of P. ultimum oospores are maturation, activation and germination. Oospores germinated after a 6-12 week maturation period, when activated by light and exposed to an exogenous carbon source. Oospores of P. ultimum, apparently, can not utilize nitrogen only for germination, but nitrogen may enhance effects of low concentrations of carbon. Thus, oospores of P. ultimum are both constitutively and exogen­ ously dormant as described by Sussman (31). Apparently, they must be aged before external factors can activate and break dormancy. P. ultimum differs from Pythium aphanider- matum whose oospores germinate in 3 weeks (30) and from Phytophthora megasperma whose oospores will germinate at a maximum after 35 days (22). It is possible that oospores of other members of the Pythiaceae, which do not germinate readily, can be induced to do so if aged longer before being exposed to external activating conditions.

36 37

Light has been reported to stimulate oospore germination in several species of Phytophthora (3,5,14,28,34) and to affect germination of oospores of P. aphanidermatum (24), and some other species of Pythium (5). However, in none of these studies was there a distinction made as to the role of light in activation of oospores. It is not difficult to visualize the relevance of a light-dependent system for breaking dormancy in a soil-borne pathogen such as P. ultimum. There probably is sufficient light in the top few inches of soil to activate oospores. Oospores deep in the soil would be brought to the surface when soil is plowed. However, oospores deeper than plow depth probably would not germinate and contribute to inoculum, unless there are other factors which will activate oospores. The snail system used by Stanghellini and Russell (30) did not work for P. ultimum, but aged oospores were not used. In this study the effects of extended aging of oospores without light were not determined. The effects of high intensity light should be studied further. Oospores in the soil surface may be exposed to higher intensities of light than used in this study. Short exposure of aged, dormant oospores to high intensity light resulted in activation but extended exposure of activated oospores to high intensity light was not studied. Oospores produced in cultures exposed to 1,000 ft-c continuously were 38

empty and may not have developed normally. However, it was not determined whether this effect was due to continuous exposure to light or to high intensity light, since effects of continuous low intensity light and alternating high intensity light with dark were not investigated. Pythium ultimum oospores are nutrient dependent as are those of Pythium aphanidermatum (30), Phytophthora megasperma (22), and probably most other species of both genera. However, information on nutrient dependent germination is not available for most Pythiaceae because germination studies have generally been done on the agar media in which the oospores were formed (3,14). Stanghellini and Hancock (29) have considered sporangia to be the major inocula of P. ultimum in cultivated soil because they are not dormant, will germinate in less than 2 hr in the presence of small amounts of nutrients (30 ug/ml glucose) and are known to survive in soil for 11 months (29). Oospores of P. ultimum probably are also involved in the disease cycle, once they have been aged and activated. Oospores require a slightly longer time (2-5 hr) and higher nutrient concentrations (100 ug/ml sucrose) than sporangia for optimum germination. Information is needed on whether oospores in soil can be aged, activated by exposure to light and stimulated to germinate by naturally occurring carbon compounds such as 39 seed and root exudates. If a combination of aging, light activation, and carbon nutrients is required for P. ultimum oospore germination in soil, oospores probably are most important as long-term survival structures than sporangia. SUMMARY

Two aspects of root rot of soybeans caused by Pythium ultimum were studied: (i) the effects of three associated organisms on disease severity; the root nodule bacterium, Rhizobium japonicum; an endotrophic mycorrhizal fungus, Endogone mosseae; and the host compatible and incompatible root rot pathogen, Phytophthora megasperma var. sojae races 1 and 3; and (ii) factors affecting germination of the long-term survival structures, the oospores.

PART I

A special method (double styrofoam cup system) was used to expose Harosoy-63 soybean roots to all possible combinations of R. japonicum, E. mosseae, P. megasperma var. sojae races 1 and 3, and P. ultimum. No soybeans were killed by P. ultimum alone or in combination with P. megasperma var. sojae race 1. P. ultimum plus P. megasperma var. sojae race 3 resulted in more plants killed than P. megasperma var. sojae race 3 alone. Soybeans infected by P. megasperma var. sojae race 3 alone or in combination with P. ultimum had significantly

40 41

» lower mean plant height and shoot dry weights than soybeans infected by P. ultimum and/or P. megasperma var. sojae race 1. P. megasperma var. sojae race 3 infected soybeans had lower dry weights in sterile soil than in most treat­ ments with E. mosseae and R. japonicum. The latter two organisms had no other effects on mean plant height and shoot dry weights of soybeans infected by P. ultimum and P. megasperma var. sojae in this system. Fewer plants were killed by £. megasperma var. sojae race 3 in Endogone treated soil than treatments without Endogone. Soybeans infected by £. megasperma var. sojae race 3 developed fewer root nodules than those infected by P. ultimum or P. megasperma var. sojae race 1 or the control.

PART II

Oospores of £. ultimum harvested from 12-week-old sucrose-asparagine liquid cultures incubated under alternating low intensity light and dark conditions germinated 70% after 2 to S hr in the presence of carbon sources, but oospores from 12-week-old cultures incubated in the dark germinated less than 10% in the presence of carbon. Following 24-hr exposure to high intensity light sources, dark incubated oospores germinated as well as the oosp\>res incubated under alternating light and dark conditions. 42

Light treatment resulted in a change from dormant thick-walled oospores with a fine-grained cytoplasm to activated thin-walled oospores with coarse-textured cytoplasm (pregermination stage). Activated oospores germinated in the presence of a carbon source. Only germ tubes and hyphae were produced by germinated oospores. Alternating low intensity light and dark, or high intensity light treatment had no effect on oospores from 4-week-old cultures but resulted in increasing activation and germination as age of cultures increased from 6 to 12 weeks. These studies indicate that age of oospores, exposure to light, and availability of nutrients are critical for oospore germination of P. ultimum. LITERATURE CITED

1. Alexander, M. 1962. Introduction to soil microbiology. John Wiley § Sons, Inc. New York. 436p. 2. Bainbridge, A. 1970. Sporulation by Pythium ultimum at various soil moisture tensions. Brit. Mycol. Soc. Trans. 55:485-487. 3. Berg, L.A., 5 M.E. Gallegly. 1966. Effect of light on oospore germination in species of Phytophthora. Phytopathology 56:583 (Abstr.). 4. Burck, K.T. 1973. Personal communication. 5. Cardoso, E.J.R.N. 1971. Influence of light on the oospore germination of several species of Pythium and Phytophthora. Ph.D. Thesis, The Ohio State Univ., Columbus. 74p. 6. Drapeau, R., J.A. Fortin, and C. Gagnon. 1973. Antifungal activity of Rhizobium. Can. J. Bot. 51:681-682. 7. Drechsler, C. 1946. Zoospore development from oospores of Pythium ultimum and and its relation to rootlet-tip discoloration. Plant Dis. Reptr. 30:226-227. 8. Drechsler, C. 1952. Production of zoospores from germinating oospores of Pythium ultimum and Pythium debaryanum. Bull. Torrey Bot. Club 79:431-45(5^ 9. Drechsler, C. 1960. Two root fungi closely related to Pythium ultimum. Sydowia 14:106-114. 10. Erwin, D.C., 6 W.H. McCormick. 1971. Germination of oospores produced by Phytophthora megasperma var. sojae. Mycologia 63:972-977. It tt 11. Gauman, E., J. Nijesch, 6 R.H. Rimpan. 1960. Weitere Untersuchungen uber die chemischen Abwehrreaktionen der Orchideen. Phytopathol. Z. 38:274-308.

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12. Gerdemann, J.W. 1961. A species of Endogone from corn causing vesicular-arbuscular mycorrniza. Mycologia 53:254-261. 13. Hildebrandt, A.A., 6 L.W. Koch. 1952. Observations on a root and stem rot of soybeans new to Ontario, caused by Pythium ultimum Trow. Sci, Agr. 32:574-580. 14. Leal, J.A., 6 B. Gomez-Miranda. 1965. The effect of light and darkness on germination of the oospores of certain species of Phytophthora on some synthetic media. Brit. Mycol. Soc. Trans. 48:491-494. 15. Marx, D.H., 6 C.B. Davey. 1967. Ectotrophic mycorrhizae as deterrents to pathogenic toot infections. Nature 213:1139. 16. Meyer, W.A., P.N, Thapliyal, J.A. Frank, fj J.B. Sinclair. 1971. Detection of phytoalexin in soybean roots. Phytopathology 61:584-585. 17. Meyer, W.A., 6 J.B. Sinclair. 1972. Root reduction and stem lesion development on soybeans by Phytophthora megasperma var. sojae. Phytopathology 62:1414-1416. 18. Richard, C., J.A. Fortin, 6 A. Fortin. 1971. Protective effect of an ectomycorrhizal fungus against the root pathogen Mycelium radius atrovirens. Can. J. For. Res. l:246-25lt 19. Roslycky, E.B. 1967. Bacteriocin production in the Rhizobia bacteria. Can. J. Microbiol. 13:431. 20. Ross, J.P,. 1972. Influence of Endogone mycorrhiza on Phytophthora rot of soybean. Phytopathology 62:636-897. 21. Safir, G.R., J.S. Boyer, 6 J.W. Gerdemann. 1971. Mycorrhizal enhancement of water transport in soybeans. Science 172:581-583. 22. Salvatore, M.A., F.A. Gray, 3 R.B. Hine. 1973. Enzymatically induced germination of oospores of Phytophthora megasperma. Phytopathology 63:1083- 45

23. Schmitthenner, A.F. 1964. Fungi associated with root necrosis of Phytophthora resistant soybeans. Phytopathology 54:906 (Abstr.). 24. Schmitthenner, A.F. 1972. Effect of light and calcium on germination of Pythium aphanidermatum. Phytopathology 62:788 (Abstr.}. 25. Schmitthenner, A.F. 1972. Evidence of a new race of Phytophthora megasperma var. sojae pathogenic to soybean. PTant Disease Reptr. 56:536-539. 26. Schmitthenner, A.F. 1973. Unpublished data, 27. Schwinghmer, E.A., 6 R.D. Bilkengren. 1968. Inhibition of Rhizobium by a strain of Rhizobium trifolii; some properties of the antibiotic and of the strain. Arch. Mikrobiol. 64:130-145. 28. Shaw, D.S. 1967. A method of obtaining single oospore cultures of Phytophthora cactorum using live water snails.Phytopathology 57:454. 29. Stanghellini, M.E., 6 J.G. Hancock. 1971. The sporangium of Pythium ultimum as a survival structure in soil.Phytop a thology 61:157-164. 30. Stanghellini, M.E., 6 J.D. Russell. 1973. Germination in vitro of Pythium aphanidermatum oospores. PKytopatho1ogy 63:133-137. 31. Sussman, A.S. 1966. Dormancy and spore germination, p. 733-764. In C.G. Ainsworth 6 A.S. Sussman (ed.) Th Fungi. Vol. II. Academic Press, New York. 805 p. 32. Sussman, A.S., 6 H.O. Halvorson. 1966. Spores: Their dormancy and germination. Harper 6 Row, New York 6 London. 354 p. 33. Trow, A.H. 1901. Observations on the biology and cytology of Pythium ultimum n. sp. Ann. Bot. 15:269-312. 34. Zentmyer, G.A., 6 D.C. Erwin. 1970. Development and reproduction of Phytophthora. Phytopathology 60:1120-1127.