DWWSOFPEXSEWSWGW Dissertation for the Degree at Ph. D. MICHEGAN STATE UNEVERSEY WED E. SHORT 1975 1.; f This is to certify that the thesis entitled Determinants of Pea Seed and Seedling Rot presented by Gerald E. Short has been accepted towards fulfillment of the requirements for Ph.D. degreein Plant Pathology 922/27ij 0%; floor profuse! Date 3 //oz / 7 .s/ / / / {2/ 0-7639 ABSTRACT DETERMINANTS OF PEA SEED AND SEEDLING ROT By GERALD E. SHORT The mechanisms by which biotic and abiotic factors interact to affect incidence of seed and seedling rot in pea (Pisum sativum L.) were investigated. Exudates from germinating seeds stimulated Spore germination of pathogenic fungi in Spermosphere soil. This phenomenon has been called the spermosphere effect. The effects of soil moisture, soil temperature, seed age, seed color, and seed treatments on seed exudation, spore germination, and incidence of disease were investigated. 1. A method was developed for directly observing germina- .tion of Fusarium solani f. sp. pisi chlamydospores in l-mm incre- ments of distance from the pea seed coat. The maximum distance from seeds at which spores germinated was greatest at 10 C and 50% soil moisture, and never exceeded 7 mm under any conditions tested. More spores germinated at 50% than at 20% soil moisture. More germ- ination occurred near the emerging radicle than in other areas of the spermosphere. Spore germination decreased with increasing temperature at 50% soil moisture, but at 20% soil moisture a higher percentage of spores germinated at 22 than at 10 or 30 C. The Gerald E. Short wrinkled-seeded pea cultivar Miragreen supported more spore germina- tion in the spermosphere than did the smooth-seeded cultivar Alaska. When Miragreen seeds were soaked in aerated water for 48 hours prior to planting, spores germinated only in the millimeter of soil near- est the seed and the spore germination was only one-sixth as great as in the same zone near unsoaked seeds. It is suggested that spore germination is a function of the availability of nutrients exuded by the seed. II. Exudates from surface-sterilized pea seeds were col- lected during 4 days of germination in sterile leaching systems of glass beads. Total carbohydrates were measured using a modified anthrone analysis. The amount of carbohydrate exuded from pea seeds was influenced by incubation temperature, cultivar, seed age, and also by factors associated with seed color and rate of water imbibition. The greater part of the carbohydrate was exuded during the first l8 hours of seed germination at 22 and 30 C, but signifi- cant exudation persisted for about 48 hours at 10 C. Total exuda- ‘tion over a 4 day period was greater at 10 C than at higher tempera- tures. Wrinkled-seeded Miragreen peas exuded more carbohydrates than the smooth-seeded Alaska cultivar, and yellow ("blond") Mira- green seeds exuded more carbohydrates than green Miragreen seeds under most experimental conditions. Eight-year-old Miragreen seeds exuded up to ten times more carbohydrates than one-year-old seeds, and seeds which required only 5 hours to complete swelling in water exuded more nutrients than seeds needing 8 or 12 hours to complete swelling. Gerald E. Short III. Wrinkled-seeded Miragreen and smooth-seeded Alaska pea seeds were planted in soil artificially infested with Fusarium solani f.sp. pisi and naturally infested with Pythium ultimum. Incidence of seed and seedling rot was determined in growth chambers. Tem- peratures were maintained at 10, 22, or 30 C, or were alternated every l2 hours, starting with a low of 10 C and a high of 25 C. Every other day for l0 days the alternating lows and highs were each increased by l degree until the final range was from 15 to 30 C. Incidence of seed rot in the same soil in field plots was also determined. Miragreen seeds rotted more frequently than Alaska seeds. Incidence of rot was greater with yellow than with green Miragreen seeds. More seeds rotted at high than at low soil mois- ture. Seed and seedling rot were most severe when temperatures were alternated between the lower temperatures favorable for disease development by E, ultimum and the higher temperatures favorable for f, solani f. sp. pj§i_infection. Soaking Miragreen seeds in water at 22 C for 48 hours prior to planting reduced the incidence of 'seed and seedling rot; however, soaking seeds at l0-l5 C for 48 hours usually increased seed and seedling rot, perhaps because of low temperature injury. However, in the absence of such putative seed injury, incidence of seed and seedling rot at any soil tempera- ture tested was directly related to the magnitude of the spermos- phere effect, which in turn was influenced by the amount of seed exudation and by soil moisture. DETERMINANTS OF PEA SEED AND SEEDLING ROT By 1.1-“~ d Gerald E. Short A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1975 To Karen 1'1 ACKNOWLEDGMENTS Sincere appreciation is expressed to Dr. M. L. Lacy for his guidance, encouragement, and friendship during my tenure as a gradu- ate student. I gratefully acknowledge the other members of my guidance committee, Drs. J. L. Lockwood, w. G. Fields, J. M. Tiedje, and N. E. Good, for their helpful suggestions and critical evaluation of the manuscript. Thanks are also due to Drs. A. H. Ellingboe, A. L. Jones, J. L. Lockwood, J. M. Vargas, C. R. Trupp, and Mr. C. L. Zehr for their assistance in providing equipment needed to complete this study. I am grateful to the Michigan Agricultural Experiment Sta- tion for financial assistance during this investigation. I am particularly grateful for the prayers and discipline received fromnn'late father, and my mother; their efforts were most influential in helping me persevere in attaining personal goals. Lastly, the love, understanding, and patience, of my wife, Karen, were a constant source of encouragement while I was in gradu- ate school. In more subtle ways Laura and Rachael provided their own inspiration. TABLE OF CONTENTS LIST OF TABLES AND FIGURES GENERAL INTRODUCTION LITERATURE CITED . PART I GERMINATION 0F FUSARIUM SOLANI F. SP. PISI CHLAMYDOSPORES IN THE SPERMOSPHERE 0F PEA INTRODUCTION MATERIALS AND METHODS Selection and Preparation of Seeds Source, Preparation, and Infestation of Soil Adjustment of Soil Moisture . Determination of Spore Germination RESULTS . Effect of Different Regions of the Spermosphere Effect of Soil Moisture . . Effect of Cultivar . Effect of Temperature . Effect of Soaking Seeds DISCUSSION . LITERATURE CITED . PART II CARBOHYDRATE EXUDATION FROM NRINKLED AND SMOOTH PEA SEEDS IN RELATION TO TEMPERATURE, AGE, AND SEED COLOR INTRODUCTION MATERIALS AND METHODS iv Page vi (I) 0000000000 00 CDNNN \I \l —J —l 13 15 Source and Treatment of Seeds Collection of Exudates Analysis of Exudates RESULTS . Effect of temperature . Effect of Cultivar . Effect of Aging. . . Relation to Imbibition. Rate . Effect of Soaking Seeds Relation to Seed Color DISCUSSION . LITERATURE CITED . PART III FACTORS AFFECTING PEA SEED AND SEEDLING ROT IN RELATION TO SEED EXUDATION AND THE SPERMOSPHERE EFFECT INTRODUCTION MATERIALS AND METHODS Selection and Treatment of Seeds . . Source, Preparation, and Infestation of Soil Control of Soil Moisture and Temperature Assessment of Seed and Seedling Rot 'RESULTS . Effect of Soil Moisture Effect of Cultivar . Effect of Seed Color Effect of Temperature . Effect of Soaking Seeds DISCUSSION . LITERATURE CITED . CONCLUSIONS Page LIST OF TABLES AND FIGURES PART I Table l. Apparent germination of Fusarium solani f. sp. pisi chlamydospores at 22 C in the spermospheres of two pea cultivars at various times after planting in soil at 20% and 50% moisture . . . Figure l. Germination of Fusarium solani f. sp. pisi chlamydospores in the spermospheres of pea culti- vars 'Miragreen' and 'Alaska' . . PART II Table l. Effect of temperature and cultivar on water imbibition and carbohydrate exudation by pea seeds during 4 days of germination in a sterile, moist glass bead environ- ment . . . 2. Relation of cultivar, seed color, age, and imbibition rate to carbohydrate exudation . . 3. Seed color and imbibition rate of Miragreen peas in relation to carbohydrate exudation during 24 or 48 hours soaking in sterile distilled water at 22 C, and during a subsequent 24 hours at 22 C in petri dishes containing sterile, moist glass beads . . Figure l. Sterile leaching systems used to collect carbohydrate exudates from surface-sterilized pea seeds during 4 days of germination in glass bead environments 2. Apparatus used to condense hourly leachings from individual pea seeds . . . . . . . vi Page l0 23 24 25 19 21 Figure Page 3. Amount of carbohydrate exuded from Miragreen and Alaska pea seeds at hourly intervals during the first 4 days of seed germination at three temperatures . . 22 PART III Figure 1. Effect of soil moisture, temperature, cultivar, and seed treatment on incidence of pea seed and seedling rot in soil naturally infested with Pythium ultimum and artificially infested with Fusarium solani f. sp. pisi . . . . . . . . . . . . . . . . . 45 2. Germination of Fusarium solani f. sp. pisi chalmydo- spores in the spermosphere at incremental distances from pea seeds . . . . . . . . . . . . . . 47 vii GENERAL INTRODUCTION In l904 Hiltner (2) observed that microorganisms were more abundant in soil surrounding plant roots than in soil remote from roots, and called this zone of soil the rhizosphere. In 1947 Slykhuis (13) observed that microbial activity in soil surrounding germinating seeds was greater than in soil distant from seeds, and Verona (l4) later referred to this zone of soil as the spermosphere. Amino acids, simple sugars, and presumably other substances are exuded from roots and seeds (8,l0) providing a food supply readily utilized by many soil microorganisms. This loss of nutrients can be beneficial to the plant if symbionts such as mycorrhizal and rhizobial species are stimulated. However, seedling exudates may also stimulate plant pathogenic microorganisms, initiating a series of events which frequently result in a weakening or killing of the -host. Techniques used to measure root and seed exudates and result- ant microbial activity have had serious limitations. For example, it has not been possible to directly measure exudation in soil, since sugars and amino acids are readily utilized by the indigenous soil microflora (4). Hence, exudates have been collected from microbe-free plants jn_yjtrg_under conditions quite different from the conditions of the soil environment (5). Imprecise methods of sampling soil in close proximity to roots and seeds have hindered attempts to define quantitatively and qualitatively the rhizosphere and spermosphere. Usually, roots have been carefully removed from soil, and soil firmly adhering to roots was defined as rhizosphere soil (3,9). Soil firmly adhering to seeds was called spermosphere soil (l3). Seeds or roots with adhering soil were then vigorously agitated in sterile distilled water to suspend the soil particles and microorganisms in solution. Aliquotes of the soil suspension were serially diluted and plated out on agar media for determining microbial populations as a measure of microbial activity (3). With this method it was impossible to determine differences in magnitude of microbial activity at defined distances from seeds or roots. A better understanding of the nature and extent of the rhizosphere and spermosphere would likely further our understanding of the mechanisms involved in seed and root rot diseases (1,4,6). For several reasons seeds seemed more suitable than roots for an initial attempt at refining techniques of measuring exudation and 'resultant pathogenic activity in soil. It seemed likely that the location of microbial activity around a single, stationary, hypoge- ously germinating seed could be measured more precisely than about an elongating, branching root system. In addition, exudation from seeds is transitory in contrast to the continual loss of nutrients from growing root tips (7,ll,l2); thus, it also seemed likely that the time course of exudation and the Special distribution of exudates could be assessed more accurately with seeds than with a growing, fibrous root system. Spores of many seed-rotting fungal pathogens germinate in response to seed exudates, particularly the sugar components (8,10, ll). The purposes of this investigation were: (i) to refine jg_ yitrg_techniques of collecting and analyzing carbohydrate exuded during seed germination; (ii) to develop a technique for directly measuring pathogenic fungal spore germination in soil surrounding seeds; (iii) to determine how various seed characteristics, environ- mental factors, and cultural practices affect seed exudation and spore germination; and (iv) to relate seed exudation and the conse- quent germination of pathogenic spores to incidence of seedling mortality with the hope of ultimately using the information obtained to control seed and seedling diseases. 10. LITERATURE CITED--GENERAL INTRODUCTION BAYLIS, G. T. S. 1941. Fungi which cause pre-emergence injury to garden peas. Ann. Appl. Biol. 28: 210-218. HILTNER, L. 1904. Uber neuere Erfahrungen und Probleme auf dem Gebiet der Boden-bakteriologie und unter besonderer Berficksichtigung der GrUndUngung und Brache. Arb. Deut. Landw. Ges. 98: 59-78. JOHNSON, L. F., AND E. A. CURL. 1972. Methods for research on the ecology of soil-borne plant pathogens. Burgess Publishing Company, Minneapolis, Minn. 247 p. KERR, A. 1964. The influence of soil moisture on infection of peas by Pythium ultimum. Austr. J. Biol. Sci. 17: 676- 685. MATTHEWS, S., and R. WHITBREAD. 1968. Factors influencing pre- emergence mortality in peas. 1. An association between seed exudates and the incidence of pre-emergence mortal- ity in wrinkle-seeded peas. P1. Path. 17: 11-17. PADWICK, G. W. 1938. Complex fungal rotting of pea seeds. Ann. Appl. Biol. 25: 100-114. PEARSON, R., and D. PARKINSON. 1961. The sites of excretion of ninhydrin-positive substances by broad bean seed- lings. Plant Soil 13: 391-396. ROVIRA, A. D. 1965. Plant root exudates and their influence upon soil microor anisms, pp. 170-184. In_K. F. Baker and W. C. Snyder Ied.), Ecology of soil-borne plant pathogens. Univ. California Press, Berkeley. ROVIRA, A. D., and C. B. DAVEY. 1974. Biology of the rhizos- phere. pp. 153-204. In_Carson, E. W. (ed.), The plant root and its environment. Univ. Press of Virginia, Charlottesville. SCHROTH, M. N., and D. C. HILDEBRAND. 1964. Influence of plant exudates on root-infecting fungi. Ann. Rev. Phyto- pathology 2: 101—132. 4 ll. 12. 13. 14. SCHROTH, M. N., and W. C. SNYDER. 1961. Effect of host exudates on chlamydospore germination of the bean root rot fungus, Fusarium solani f. phaseoli. Phyto- pathology 51: 389-393. SIMON, E. W., and R. M. RAJA HARUN. 1972. Leakage during seed imbibition. J. Exp. Bot. 23: 1076-1085. SLYKHUIS, J. T. 1947. Studies on Fusarium culmorum blight of crested wheat and brome grass seedlings. Can. J. Res. 25: 155-180. VERONA, 0. 1963. Interaction entre la graine en germination et 1es microorganismes telluriques. Ann. Inst. Pasteur (Paris) 105: 75-98. PART I GERMINATION 0F FUSARIUM SOLANI F. SP. PISI CHLAMYDOSPORES IN THE SPERMOSPHERE OF PEA Germination of F usarium solani f. sp. pisi Chlamydospores in the Spermosphere of Pea G. E. Short and M. L. Lacy Graduate Assistant and Associate Professor, respectively, Department of Botany and Plant Pathology, Michigan State University, East Lansing 48824. Journal Series Article No. 6443, Michigan Agricultural Experiment Station. Accepted for publication 5 November 197 3. ABSTRACT A method was developed for directly determining amount of spore germination in pea spermospheres in l-mm increments of distance from the seed coat. The greatest distance at which Fusarium solani f. sp. pisi chlamydospores germinated was established within 24 h at 22 or 30 C, and never exceeded 7 mm under any conditions tested. More spores germinated, and the spermosphere was larger at 50% than at 20% soil moisture. More germination occurred near the emerging radicle than in other areas of the spermosphere. Greatest distances from seeds at which spores germinated Additional key words: agar-embedded soil columns. decreased with increasing temp at 50% soil moisture. At 20% soil moisture, a higher percentage of spores germinated at 22 than at 10 or 30 C. The wrinkle-seeded pea cultivar ‘Miragreen' supported more spore germination in the spermosphere than did the smooth-seeded cultivar ‘Alaska’. lf Miragreen seeds were soaked in aerated water for 48 h prior to planting, spores germinated only in the millimeter of soil nearest the seed and percentage germination was one-sixth that of spores in the same zone near unsoaked seeds. Phytopathology 64:558-562. Spores of most plant pathogenic fungi do not germinate in soil unless provided with some external stimulus (10). Germinating seeds exude nutrients capable of stimulating microbial activity, including spore germination (17). The zone around the seed into which exudates diffuse and microbial activity increases, has been designated the spermosphere (24) or spermatosphere (22). Fusarium solani f. sp. phaseoli chlamydospores were reported to germinate as far as 10-12 mm from germinating bean seeds (23). Attempts to ascertain sporangial germination (23) and populations (21) of Pythium ultimum at varying distances from seed surfaces, were less successful. Inadequate techniques of observing activities of microorganisms in soil has been a major obstacle in determining quantitative dimensions of the spermosphere. The objective of this investigation was to determine the amount of chlamydospore germination of F. solani (Mart.) App. & Wr. em. Snyd. & Hans. f. sp. pisi occurring in different regions, and at various distances from germinating seeds of Pisum, sativum L. as influenced by: (i) cultivar; (ii) soil temp; (iii) soil moisture; and (iv) soaking of seeds prior to planting. A preliminary report has been published (19). MATERIALS AND METHODS—Selection and preparation of seeds.——Wrinkle-seeded (‘Miragreen’) and smooth-seeded (‘Alaska’) pea cultivars obtained from Ferry-Morse Seed Co., Mountain View, Calif. were used. Individual seeds were selected on the basis of uniformity in size and color (yellow in the case of Miragreen; green for Alaska) and freedom from spots or cracks on the seed coat. Prior to planting, seeds were surface disinfested for 10 min in 0.5% sodium hypochlorite containing 1 ml of Tween 20 (polyoxyethylene sorbitan monolaurate) per liter, followed by 5 min of rinsing in sterile distilled water. In one experiment, seeds were soaked for 48 h in aerated water prior to planting. Source, preparation, and infestation of sod—Conover loam soil from the Michigan State University farm, collected from an area free from recent pesticide application, was' used in all experiments. Soil was stored at 15-20% moisture at 22-25 C in closed plastic containers. Prior to use, soil was air-dried and passed through a 30-mesh sieve. Water-holding capacity of this soil was 61%, organic matter content was 3.4%, and pH was 6.6. The soil contained 18% clay, 15% silt, and 67% sand. ' Chlamydospores of F. solani f. sp. pisi were produced in shaken liquid culture as follows: Macroconidia were removed from potato-dextrose agar (PDA) plates in 25 ml of sterile distilled water, combined with 40 ml of potato-dextrose broth, and agitated on a reciprocal shaker for 48 h. The germinated conidia were washed and suspended in 40 ml of sterile soil extract [prepared by mixing 1 liter of water with 1 kg of Conover loam, allowing the mixture to stand for 48 h, and filtering the supernatant through a 2.211 Gelman membrane filter (1)]. Germinated conidia agitated in soil extract produced an abundance of chlamydospores free from mycelium within 7 days. Chlamydospores were washed, resuspended in distilled water, and agitated at low speeds for 1-2 h in a Sorvall Omni-Mixer to break up aggregates of chlamydospores. The chlamydospore suspension was adjusted to 2.5 X 107 chlamydospores/m1 using a hemacytometer. Air-dried, sieved soil was placed in a mixing apparatus, and the spore suspension was applied with an atomizer until a final concn of 1.6 X 106 chlamydospores/g dry wt of soil and a 20% soil moisture were simultaneously attained. Adjustment of soil moisture—Infested soil was placed in 2.5-cm-diam Pyrex glass tubes with a fine screen fastened to the base. Uniform compaction in the upper 2.5 cm of soil was attained by dropping the tubes from a height of 15 cm until further 8 April 1974] compaction ceased. One pea seed, with the liilum oriented downward, was centered in the upper 2.5 cm of each soil column prior to the addition and compaction of the uppermost 1.25 cm of soil. The lower ends of the columns were immersed to a l-mm depth in a water bath, permitting the upward inovcmcnt of water by capillary action. Moisture levels of 20% and 50% wcrc established in the uppcr 2.5 cm of soil by using 33 and 8.1-cm soil columns, respectively. This system maintaincd a constant moisturc level in the upper 2.5 cm of soil during all stages of seed germination. Determination ofsporc germination. ~ Spores were incubated at 10, 22, or 30 C in soil columns for 24-96 h following sced placement. Columns wcrc then dried with a stream of air by applying‘a vacuum to thc lower ends, infiltrated with 2% molten water agar, cooled, and the agar hardened by immersing in ethanol for 12 h. Agar-cmbcddcd soil columns wcrc extruded from the l’yrcx tubes and a 2-mm-thick cross-section in the vicinity of tlic sccd was removed with a razor blade. A small sharpened spatula was used to scrially remove blocks of soil at millimeter incrcmcnts from the sccd surface in the area of radicle emergence and opposite the radicle area (Fig. l-l"). Soil corcs above and below the .sccd wcrc removed with a 4-min-diam cork borer, and serially sectioned in millimeter increments with a razor blade. Each block of soil was placed on a microscope slide, and a drop of 5 N llCl addcd to dissolve the agar. Soil smears wcrc made with 0.1% aniline blue in lactic acid, similar to thc tcchniquc of Nash ct :11. (I3). The smears were examined for chlamydospore germination at a magnification of X430. Fifty chlamydosporcs wcrc counted pcr slide. Treatments were rcplicatcd fivc limcs and cxpcrimcnts were repeated oucc with similar results. Statistical differences between treatments wcrc dctcrmincd using a two-way analysis of va‘riancc following angular transformation of data. RlZSULTS.- Preliminary experiments were carricd out to determine the optimum time after planting to sample chlamydosporc germination in the spcrmosphcrc. Germination at 22 C was dctcrmincd after 24. 4'2, 48, or 72 h. using both pca cultivars and moisturc levels of 20% and 50% (Table I). The grcatcst distance from the seed at which spores germinated in any trcatincnt was established within 24 h. Percentage germination was grcatcst 42 h after planting at 50% soil moisture and 48 h after planting at 20% soil moisture. lixtcnsivc mycclial growth after 42 h at 50% moisture obscured additional germination; however. this did not occur at 20% soil moisture. During preparation of soil smears from the 50% moisture samples. many ungcrminatcd spores appeared to become dislodged from entangled hyphac of germinated spores, possibly rcsulting in an underestimate of spore germination. (icrm tube lysis further rcduccd apparent spore gcrmination 72 h after planting at 22(‘('l'ablc 1). No lysis was observed up to 96 h after planting at 10 C, but at 30 C lysis was already evident .10 11 after planting. In later experiments (Fig. 1), soil columns were incubated for SllORl AND LACY: l’FA SPERMOSPHERE 24 h at 30 C; for 42 and 48 h at 50% and 20% soil moisture, rcspcctivcly, at 22 C;and for 96 h at 10 C. Effect of different regions of the spermosphere.--Sporc germination was always greater near the emerging radicle than in other regions of the spcrmosphcrc (Fig. l-A, B, E, F). However, no differences in gcrminat ion in the other areas (hilum, opposite hilum, and opposite radicle) were found; hcnce, only data from the radicle and opposite radicle rcgions were plotted. At 50% soil moisture and 22 C. sporcs germinated with greater frequency at a given distance from the seed near the radicle than in other regions of thc spcrmosphcrc. At 20% soil moisture, sporc germination near the emerging radicle of either cultivar was consistcntly greater than in other areas only in the millimeter of soil directly adjacent to the seed. Maximum spore germination observed was 70% in the millimctcr of soil (50% moisture) adjacent to the radiclc arca of the cultivar Miragreen. Spore gcrmination dcclincd with increasing distance from the seed. and was never dctcctcd further than 7 mm from the sccd (Fig. l-C). Iz'j'j'cct of soil moisture. «Spores germinated 2-5 mm further from the seed when soil moisture was incrcased from 20% to 50%, regardless of cultivar or tcmp uscd (Fig. I-A, B, C, D). Spore germination at a given distancc from the seed was also considerably greater at 50% than at 20% soil moisture. Effect of cultirar.- Sporcs germinated at greater distances and in higher t'rcquencies at comparable distances from the Miragrccn than the Alaska cultivar under all soil moisture and temp conditions (Fig. l-A, B, C, D). I:']_‘/'act of temperature—The region of the spcrmosphcrc sampled was opposite the radicle (Fig. l-F), since the amount of spore germination in this area was indicative of that in most other regions of the spermosphcrc. At 50% soil moisture, the maximum distance from the seed at which chlamydosporcs germinated decreased with increasing temp with both cultivars (Fig. 1C, D). However, at 20% soil moisture, germination was greatest at 22 C and was least at 10 C. Effect of soaking secdr.--Spore germination was confincd within 1 mm of the surface of Miragreen pea sccds soaked in aerated water for 48 11 before planting (Fig. 14".). Germination within 1 mm of soaked seeds was one-sixth that of spores in the same zone near unsoaked sccds. DISCUSSION.~The method of embedding and sectioning soil described cnablcd quantitative measurement of spore germination in the spermosphere with a precision heretofore not possible (14, 21. 22, 23). This technique could also be employed for dctcrmining spore germination in the rhizosphere. Undoubtedly, amount and rapidity of exudation are two very important considerations in sclccting a host for study. If amount of exudation rcstrictcd sporc germination to within 1 mm of the sced or root, this technique would not be as applicable. We have not bccn able to remove soil scctions in increments of less than 0.5 mm. Thc idcal time for assessing spore germination in 9 PHYTOPATHOLOGY [VOL 64 7° '""‘“~._ cv. MIRAGREEN 70" Cv. ALASKA “a 22 c R, 22 C 60 - '-._ — oo - ‘-\ — . '5, -— 20 15 Soil Moisture — 20; Soil Moisture 50 I ‘3'. 3‘ mm 50 5 Soil Moisture 50 .‘.._.. K'.‘ “"" 50 I Soil Moi-ture '-_ '-, I Near Radicle ‘3 'u‘ - Near Radicle ‘ '- '-. = ‘-. '. g 40 F "-,_ '5. 0 Opposite Rodicle 2 ‘0 ._ ‘o‘ '3. O Opposite lladicle .5 'i .g '-. ". E " s, E "- ,3 30 - ‘X ‘-t_ 5 30 - Q, 2 \ 's e 5. '. o ‘-. 3 £20 - 320 - .,._ I “3. it ‘i... l _‘ ‘._ _ a. '-._. oi- .\ R, A 10 h ‘ B 2., '-. "5 O _ ~~..,..I--........ o . '0" ‘-. l I I l l 1 I I 1 0-1 1-2 ' 2-3 3-4 4'5 5‘6 6’7 7'3 0" 1'2 2‘3 3-4 4-5 5'6 H 7‘3 Dutance from Seed (mm) Distance from Seed (run) 7° Cv. MIRAGREEN 7°” Cv. ALASKA 60 — 20! Soil Moisture 60' — 20! Soil Moisture --- SOS Soil Moisture "r“- SOI Soil Moisture 50 0 IO C 50 e 10 c D 22 C D 22 C 45.0 esoc 5‘0 e 30c 3 2 s -- 5 E (3 30 6 30 3 9. o o 320 -. 320 R "‘_ I 10 . a“... C 10 D H’- "'n 0 -.._.~'. \.~\ 0 1 1 1_1 1 0" l-2 2-3 3" 4- 5-6 6‘7 7'8 0" l'? 2'3 '4 4'5 5'6 6'7 7'8 Distance from Seed5 (""10 Distance From Seed (mm) 7° ':""'-" Cv. MlRAGREEN o""-. 22 c 60- $3.... '3‘". 50! Soil Moistur: 50" '3‘ "- — Seeds Soaked 3.. .'-._ """ Seed: Not Soaked .5 ‘0 _ "- 3. e Near Radicle 3 ". '-. .2 '-.. '-.‘ . Opposite Rodicle 3 30 _ I'.‘ .' Radicle Area 3 $20- ‘ 10- 0 l l A 04 1-2 - ”-4 4-5 5-6 7-8 Distance from Seed (mm) Fig. 1 10 April 1974] SHORT AND LACY: PEA SPERMOSPHERE TABLE 1. Apparent germination of Fusarium solani f. sp. pisi chlamydospores at 22 C in the spermospheres of two pea cultivars at various times after planting in soil at 20% and 50% moisture Time . . after Chlamydospore germination (%) Soil moisture planting at incremental distances from radicle (mm) Cultivar (%) (h) 0-1 1-2 2-3 3-4 4-5 5-6 6-7 Miragreen 50 24 61 5 3 38 23 13 2 0 42 7O 68 49 27 9 2 0 48 50 34 23 8 3 0 72 a .. Alaska 50 24 45 33 21 12 O 42 65 50 25 8 1 0 48 61 38 13 2 O 72 45 13 1 0 Miragreen 20 24 50 31 4 0 48 66 26 2 0 72 45 13 l 0 Alaska 20 24 22 8 0 48 32 4 0 72 22 l 0 3No data due to extensive germ tube lysis. soil is when the last germ tube has appeared. Unfortunately, by that time some germ tubes have lysed, and at 22 C and 50% soil moisture extensive mycelial growth also had occurred and made counting of germinated spores difficult. Both chlamydospore germination and germ tube lysis were less at the lower soil moisture (Table 1), in agreement with Cook and Flentje (5). It was possible, however, to accurately ascertain the greatest distance from seeds at which spores germinated. Lysis, mycelial growth and delayed spore germination were most pronounced within 2 mm of the seed. Thus, the values in Fig. 1 probably underestimate the final extent of chlamydospore germination close to the seed, but become increasingly more accurate toward the periphery of the spermosphere. Severity of pre-emergence damping-off in peas has been directly correlated with amount of carbohydrate exudation during seed germination (7, 12). Wrinkle-seeded cultivars exuded more sugars than the less-susceptible smooth-seeded cultivars (7). Pre-emergence damping-off and carbohydrate exudation were greater in wet than dry soils (6, 7. 8, 9). The data (Fig. l-A, B) suggest that at constant temp, spermosphere size is directly related to carbohydrate exudation. The considerably larger spermosphere at 50% soil moisture is likely due to(i) an increase in exudation, and (ii) a facilitated diffusion of these sugars through soil water (23). The greater spermosphere effect near the radicle than in other areas of the seed (Fig. l-A, B) is also 6 likely due to greater amounts of nutrient exudation. The micropyle is a major portal through which seeds imbibe water (11) and simultaneously exude sugars (18, 20). Exudation from soybean (4), bean (16), and broad bean (15) was greatest in the micropylar zone, where the radicle penetrated the seed coat. Schroth et al. (18) reported a 50% increase in carbohydrate exudation from seeds of the pea cultivar Alaska as temp was increased from 15-30 C. Bacterial competition for these nutrients would also increase with temp in the range of 10-30 C (2). Thus, at 10 C, the slow rate of bacterial consumption of these exudates may have enabled their diffusion further from the seed than at warmer temp. Hence, spermosphere size appeared to be inversely related to temp at the 50% soil moisture level (Fig. l-C, D). However, at 20% soil moisture, the spermosphere was larger at 22 C than at 10 C, conceivably due to an insufficient increase in microbial activity to consume the greater amount of exudates as quickly. But when temp was further increased to 30 C. the smaller spermosphere effect was likely due to increased microbial competition. Any direct effect of temp on spore germination seems unlikely, since chlamydospore germination on PDA was greater than 90% at 10, 22, or 30 C. Size of the spermosphere in which pathogenic spores germinate may be directly related to disease severity. Pre-emergence rotting of peas is most severe in cool wet weather (3, 8). conditions which resulted in a large spermosphere (Fig. l-C, D). The Miragreen Fig. l-(A to F). Germination of Fusarium solani f. sp. pisi chlamydospores in the spermospheres of pea cultivars ‘Miragreen’ and ‘Alaska.’ A & B) Effect of soil moisture in two regions of the spermosphere. C G. D) Effect of temp and soil moisture. Region of sampling was opposite the radicle. E) Effect of soaking seeds for 48 h prior to planting. F) Regions of the spermosphere sampled. ll I’I1Y'1‘()1’A’1'11()l,()(iY cultivar had a larger spermosphere than the less susceplible Alaska cultivar (Fig. l-C, D). Flentjc and Saksena (7) obtained a S-fold increase in emergence by soaking wrinkle-seeded peas for 20 h prior to planting in soil infested with I’ytliimn. Since little exudation occurred after imbibition of water was complete (approximately 8 h) (20), fully swollen seeds when planted should exude considerably less nutrients than unsoaked seeds. Soaking pea seeds for 48 h prior to planting drastically reduced the. volume and intensity of the spermosphere effect (Fig. Hi), and presumably inoculum potential. Knowledge of the effect of soil moisture. temp, type of cultivar, cultural practices, and other factors in altering spermosphere dimensions may be useful in controlling pre-emergcnce rotting of peas and other agricultural crops. LITERATURI’. ('ITI'ID LALEXANDI‘R. J. V., J. A. lt()URR1".T. A. II. GOLD, and W. C. SNYDER. 1966. Induction of chlamydospore formation by Fusarium solani in sterile soil extracts. Phytopatliology 56:353-354. 2.Al.l’.XANI)I-IR. M. 1961. Introduction to soil microbiology. John Wiley and Sons, New York 472 p. 3.BAYI.IS. (i. '1‘. S. 1941. I-‘ungi which cause pre-cmergence injury to garden peas. Ann. Appl. Biol. 28:210-218. 4. BROWN. (i. 1'... and 11. W. KI'LNNI'IDY. 1966. l"t'fect of oxygen concentration on I'ythium seed rot of soybean. Phytopathology 56:407-411. 5.(‘00K. R. J., and N. T. I'LI'ZN'I‘JI'Z. 1967. (‘ltlalttydosporc germination and germling survival of Fusarium solani f. pisi in soil as affected by soil Water and pea seed e\udation. Phytopathology 57:178-182. 6.1-‘LlfiN'l‘Jl‘. N. T. 1964. Pre-emergence rotting of peas in South Australia. 11. Factors associated with the soil. Austr. J. Biol. Sci. 17:651-664. 7.1-‘L15N1‘J1'T. N. 'I‘., and 11. K. SAKSI‘INA. 1964. Pre-emergcnce rolling of peas in South Australia. 111. Host-pathogen interaction. Anstr. J. Biol. Sci. 17:665-675. 8.11ULL R. 1937. I‘Tl‘l'ecl of cttvirontltcntal conditions. and more particularly of soil moisture upon the emergence of peas. Ann. Appl. Biol. 24:681—689. ‘1.KI"RR. A. 1964. The inlluence of soil moisture on inlcctiou of peas by Pythium ultimuru. Austr. J. lliol. SCi. 172670-685. [VttL 64 10.1.0CKWOOD. J. 1.. 1964. Soil fungistasis. Annu. Rev. l’liytopathol. 22341-362. 1|.MANOIIAR, M. S., and W. IIIZYDIiCKIiR. 1964. Effects of water potential on germination of pea seeds. Nature 202:22-24. 12.MA1'1‘llI-TWS. S.. and W. T. BRADNOCK. 1968. Relationship between seed esudation and field emergence in peas and french beans. Hortic. Res. 8:89-93. 13.NAS|I. S. M., T. (‘11R18TOU, and W. C. SNYDI‘IR. 1961. lixistence of Fusarium solani f. phascoli as chlamydospores in soil. Phytopathology 51:308-312. l4.PA|’AVIZAS. G. C., and C. B. DAVI'TY. 1961. Extent and nature of the rhizosphere of Lupinus. Plant Soil 14:215-236. 15.1’I-TARSON. R., and I). PARKINSON. I961. The sites of excretion of ninhydrin-positive substances by broad bean seedling. Plant Soil 13:391-396. 16.8(‘11ROT1L M. N., and R. J. COOK. 1964. Seed exudation and its intlucnce on pie-emergence damping-off of bean. Phytopathology 54 :670-673. 17.SC11ROT11. M. N., and W. C. SNYDER. 1961. Effect of host exudates on chlamydospore germination of the bean root rot fungus, Fusarium solani f. phaseoli. Phylopathology 51 :389-393. 18.8(‘11ROT1L M. N., A. R. WliINIIOLD, , and D. S. IIAYMAN. 1966. The effect of temperature on quantitative differences in esudates from germinating seeds of bean, pea, and cotton. Can. J. Bot. 44:1429-1432. 19.SIIORT, G. 1".. and M. L. LACY. 1972. Direct observation of I-‘usarium solani f. pisi chlamydospore germination in the spermosphere of peas. Pliytopathology62:1111 (Abstr.). 20.SIMON, Ii. W., and R. M. RAJA HARUN. I972. Leakage during seed imbibition. J. If\p. Bot. 23:1076-1085. 2|.SINGII. R. S. 1965. Development of Pythium ultimum in soil in relation to presence and germination of seeds of different crops. Mycopathol. Mycol. Appl. 27:155-160. 22.SI.YKIIUIS. J. T. 1947. Studies on Fusarium culmormn blight of crested wheat and brome grass seedlings. (3m. .1. Res. 252155-1811. 23.STANGIII£I.I 1N1, M. 13.. and J. G. HANCOCK. 1971. Radial c\tcnt of the bean spermosphere and its relation to the behavior of Pythium ultimum. I’ltytopathology 61:165-168. 24.V1-iR()NA. (l. 1963. Interaction entre la graine en germination et Ics microorganismes telluriques. Ann. Inst. Pasteur (Paris) 105275-98. PART II CARBOHYDRATE EXUDATION FROM NRINKLED AND SMOOTH PEA SEEDS IN RELATION TO TEMPERATURE, AGE, AND SEED COLOR 12 INTRODUCTION Nutrients exuded during seed germination diffuse into sur- rounding soil where they stimulate an increase in microbial activity known as the spermosphere effect (22,25). Recently, Fusarium solani chlamydospore germination was used as an index for determining the magnitude of the spermosphere effect in soil around bean (24) and pea (22) seeds. Unfortunately, it has not been possible to directly measure the magnitude of seed exudation in natural soil, since sugars and amino acids are quickly utilized by the indigenous soil microflora (1,2,10,12). However, exudation has been measured ig_ 31339 from seeds germinating submerged in water (11.16.18), on moist filter paper (19,20,23) and cheese cloth (9), and in sterile moist sand (20,21). The purpose of this investigation was to refine jg_yjt§9_techniques of collecting seed exudates so that the rela- tionship between seed exudation and the spermOSphere effect could be determined. Pea (Pisum sativum L. "Alaska" and "Miragreen") seeds seemed most apprOpriate for such a study for the following reasons: (i) the magnitude of the spermosphere effect using 5, §glggj_f.sp.gpi§i as an index has been determined for these cultivars (22); (ii) the dramatic decrease in the SpermOSphere effect produced by soaking Miragreen pea seeds prior to planting (22) merited a definitive 13 14 explanation; (iii) the effect of temperature on carbohydrate exuda- tion from wrinkled-seeded pea cultivars has not been examined, and was inclusive with smooth-seeded Alaska peas (21); (iv) pea seed exudation is of sufficient magnitude to enable measuring carbohy- drate exudation from individual seeds (10,18); and (v) the effect of maturation on seed exudation (23), particularly when accompanied by a loss of color (14), has not been investigated under sterile condi- tions. MATERIALS AND METHODS Source and Treatment of Seeds Wrinkled-seeded (Miragreen) and smooth-seeded (Alaska) pea cultivars were purchased from Ferry-Morse Seed Co., Mountain View, Calif.; all seed was less than a year old when used, except in one experiment where 8-year-old seed was also planted. Seeds were selected for use on the basis of weight [220-240 mg (fresh weight)/ seed] and freedom from spotted or cracked seed coats. Miragreen seeds were separated into lots of yellow, yellow-green, and green seeds. All seeds were surface-sterilized for 30 minutes in 0.5% sodium hypochlorite containing 1 ml of Tween 20 (polyoxyethylene sorbitan monolaurate) per liter, followed by a 5 minute rinse in sterile distilled water. Seeds with defective seed coats began to swellduring this pregerminative treatment, and were discarded. Swelling is indicative of imbibition of water, and thus was used .as a visual criterion for determining the apparent amount of time required to complete imbibition in moist glass beads or in sterile distilled water. Collection of Exudates Two sterile leaching systems were used to collect exudates during the first 2-4 days of seed germination at 10, 22, and 30 C. One system consisted of a separatory funnel connected with Tygon tubing to a Pyrex glass cylinder (25—mm-diameter X 90-mm-long) 15 16 with a rubber stopper at each end, containing 20 gm of l-mm-diameter glass beads, and equipped with an air vent plugged with cotton, and a 7-mm-diameter drainage outlet (Fig. 1-A). Following sterilization of this apparatus, sterile distilled water was poured aseptically into the separatory funnel, a single surface-sterilized pea seed was placed aseptically within the glass bead matrix, and the glass cylinder was covered with aluminum foil to exclude light. Water was then percolated through the glass beads at a rate of 10 ml/hour using a peristaltic pump. A fraction collector was utilized for collecting the leaching water at hourly intervals in test tubes con- taining 95% ethanol to prevent possible utilization of exudates by contaminating microorganisms in the collection tubes. The other leaching apparatus (Fig. 1-8) consisted of a modi- fied petri dish (lOO-mmediameter X 80-mm-deep) containing 300 gm of l-mm-diameter glass beads, which was connected to a separatory funnel (containing sterile distilled water) above and a collection flask below. Surface-sterilized pea seeds were individually soaked ‘for 8 hours in 5 ml of sterile distilled water, and grouped accord- ing to the apparent amount of time required to complete imbibition: 5, 6, or 8 hours. Seeds which had completed imbibition after 8 hours were placed in the leaching system for exudate collection. Seeds whith had not completed imbibition after 8 hours were left in water until swelling was visually complete (about 12 hours). In one experiment, surface-sterilized seeds were individu- ally soaked in 5 m1 of sterile distilled water for 24 or 48 hours, l7 and were grouped according to imbibition rate. Ten seeds of a com- parable imbibition rate were aseptically placed within the glass bead bed in each petri dish (Fig. l-B). Exudates were collected at the time of planting and 24 hours after planting. Analysis of Exudates Leachings from both systems were tested for sterility on potato-dextrose agar and contaminated experiments were discarded. Cellular debris was removed from the leachings by filtration through a Millipore filter with a 0.22 pm pore diameter. Leachings from petri dish systems containing 10 seeds were condensed to dryness at 40 C with a Rotavapor high vacuum evaporator, and redissolved in 10-25 ml of distilled water. Leachings collected hourly from indi- vidual seeds germinating in glass cylinders were condensed to dry- ness at 40 C using a manifold to which were attached 40 pasteur pipettes arranged to direct a filtered air stream into test tubes containing leachings (Fig. 2); the exudates were redissolved in l ,mlof distilled water. Total water-soluble carbohydrate was deter- mined by a modified anthrone analysis (17) in which 1 m1 of exudate was mixed with 9 ml of anthrone reagent, placed in a boiling water bath for 10 minutes, and cooled to room temperature. Optical dens- ity at 600nm was measured with a Bausch and Lomb Spectronic 20 Spectrophotometer, and carbohydrate concentrations were determined using glucose as a standard. Experiments in which exudates were collected hourly from individual seeds were repeated 5 times; experi- ments with 10 seeds/petri dish were repeated once. Significant 18 differences between means were determined at the 5% probability level using Ducan's multiple range test (7). The quantitative determination of carbohydrate exuded every hour from individual pea seeds germinating in sterile glass beads represents a refinement of previous methods (9,11,16,18,19,20,21,23) with the following advantages: (i) variability among seeds could be determined, eliminating the possibility that exudation means might be distorted by a few unusually leaky seeds (18); (ii) the probability of contamination was much less when a single surface- sterilized seed was transferred to a sterile environment than when many seeds were involved, and contaminated or non-viable replicates were easily discarded without nullifying the entire experiment; and (iii) seed exudation has usually been studied when seeds germinated submerged in water, whereas a glass bead environment more nearly represented a natural soil environment. Fig. 1-(A & B). 19 Sterile leaching systems used to collect carbo- hydrate exudates from surface-sterilized pea seeds during 4 days of germination in glass bead environ- ments. (A) Using a peristaltic pump, water flowed at 10 ml/hour from the separatory funnel to the glass cylinder (a) where it percolated through the glass beads in which a single seed was germinating; leachings were collected every hour in test tubes on a fraction collector. (B) Exudates from 10 seeds germinating in glass beads in a modified petri dish were collected every 8 hours by twice flooding the glass beads, and allowing the leach- ings to drain into an Erlenmeyer flask. 20 21 Fig. 2. Apparatus used to condense hourly leachings from individual pea seeds. 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' 5'" 3 . 0 Green Seed (LSR ,: 7.9) .0 ' Y Yellow Seed ' S10 .- '° . =2 c 2 ‘ HI ”I I 70 - __ if n n - 60 _ . Untreated Untreated (22C [k tad hr) "mom-mated & Alalka Miragreen (VG) Moraoreen (VG) Miragreen (YG) E 50 I e . 2 ‘0 . - 1974 a E 30 — I K 30 i a ’ Sat. I E20 5. O Si" 2 - 20 )- m I Site 3 w + I g Io .. I use“: 3.9) . n E 7 x .. [1 I a n I n I I V G Y 6 G Y Untreated Untreated Seekeadh') Therm-treat“) » - (22 CM Untreated Sacked "worn—treated Untreated (2mge‘dh‘Thrrarn-treated Aluka Miragreen (YG) Miragreen (VG) Miragreen (Y0) (22 C/24 hr) 47 —wem eee Le>wupee ee peewem Awupee we Homemm Amy .meeem ewe seem mmeeeemwe Pepeesmeeee we exegemeeLeem To ‘ d d d 1 I, u “UoOM 9.32 .>U o ceecoogi .>U - 22:02 20m I on II 9530.2 __om R on I uoyouguueg mods g on .u o_ we eeeumwee .Am e evum .eea AEEV veem 89m 02.230 mun To cum nfw T min a). To 1 d) d d d M ‘4 I 0’ e I I III, ’0’, /I/O I. /o 1 / 0’ I Ill l/l / o T < I // Cl / / e/ // u ’ ex 4 z x I x t // I e. .u.e_ ./ ,, / a If! t 9102 .>U o I/ 1 / ceeeuozi .>U - 22:02 :0m 8 on II 23:02 __0m 8 ON I op on on ow on 00 ON OJOdS g uououguueg [IIIIIIIIII DISCUSSION Pea seed cotyledons germinate beneath the soil surface and remain confined to the spermosphere soil throughout seedling devel- 0pment. This is disadvantageous to the seedling if seed-rotting organisms within the spermosphere have been stimulated to produce an active vegetative mycelium capable of infecting the seed or seedling axis. particularly when soil moisture and temperature are unfavor- able for seed germination and seedling growth. The ability of Pythium ultimum sporangiospores to germinate within 1 1/2 hours after receiving the seed exudate stimulus (32), and the rapid growth rate of the mycelium at 12-30 C(19) produces a prolific growth of Pythium mycelium in the spermosphere responsible for the characteristic "balling" of soil around seeds (6.10). "Balled" seeds may rot prior to emergence (5). produce a seedling which rots 'following emergence (4), or result in a weakened seedling (22). Pea (18) and bean (26) seeds exude nutrients, the greatest proportion of which are simple sugars such as glucose, mannose, sucrose. and fructose. Such sugars are capable of stimulating spore germination and germ tube growth of seed-rotting fungi (6, 26). Incidence of rot has been directly correlated with the quan- tity of carbohydrate exuded by soybeans (13). beans (25). and peas (21). However, amount of nutrient exuded jg_vitro may not 48 49 always be indicative of the extent of pathogen activity in the spermosphere. For example. Miragreen seeds exuded significantly more carbohydrate at 10 C than at 22 C (p. 23). but E, s91ggj_f. sp. pi§i_chlamydospore germination in soil at 20% moisture was con- siderably greater at 22 C than at 10 C (28). It is also conceiv- able that seeds, like roots (3). could exude inhibitors of spore germination and mycelial growth which could affect the activity of the pathogen in the spermosphere. Leach (19) tried to predict the fate of seeds. including peas. planted in Pythium-infested soil by calculating the ratio of the rate of emergence of the host in pasteurized soil to the rate of growth of the fungus in potato-dextrose broth at temperature increments from 4-35 C. Ratios below unity, indicating that the growth rate of the pathogen exceeded that of the host. were asso- ciated with severe pre-emergence infection; as the ratio increased beyond unity, the disease declined accordingly. However. these ratios have not always proven to be useful in predicting damping- -off (7.8), perhaps because the effect of soil moisture on the host and pathogen were not considered (19). For example, raising the soil moisture level increased pea seed exudation. producing a higher incidence of pre-emergence rot (14). A more direct approach to predicting pre-emergence rotting of seeds is possible due to recent progress in measuring the spermosphere effect (28.32). The radial extent and intensity of 50 the spermosphere effect, as measured by E, g91ggj_f. sp. 2151 chlamydospore germination at 10, 22. or 30 C (28). increased with various pea cultivar-soil moisture combinations in the following order (Fig. 2-A,B): Cv. Alaska, 20% soil moisture; Cv. Miragreen, 20% soil moisture; Cv. Alaska, 50% soil moisture; Cv. Miragreen, 50% soil moisture. Incidence of pea seed and seedling rot in growth chambers (Fig. l-A) at 10 C, 30 C. or under alternating tempera- tures increased in precisely the same sequence, indicating a direct relationship between incidence of rot and the magnitude of the spermosphere effect at any particular temperature. A simple relationship between incidence of rot and the spermosphere effect was neither expected nor found when tempera- ture was included as a variable. because temperature affects not only amount of seed exudation (p. 23). but also bacterial competi- tion for exudates (1). growth rates of host and pathogen (19). and rate of disease development (11,19). Optimum temperature for seed and seedling rot caused by Pythium ultimum (12-25 C) is lower than 'for E, sglgfli_f. sp. pj§j_(24-33 C) (11.19.22). Thus, seed and seedling rot at 10 C (Fig. l-A,B) may have been caused primarily by E, gltjmgm, while that at 30 C may have been mainly due to E, §91§gj_ f. sp. Eiéi, The greater incidence of rot under alternating temper- atures than at 10 or 30 C (Fig. 1-A,B) was likely due to tempera- tures favoring disease deve10pment by both pathogens at different times of the day (4.17). When the E, §glggj_f. sp. pj§1_inoculum was increased 2.5 fold (Fig. l-B). incidence of rot was much less at 10 C than at 30 C or when temperatures ranged from 10-30 C. 51 Generally, environmental conditions which adversely affect seedling growth are most conducive to pre-and post-emergence damp- ing-off (l9). Pre-emergence rotting of peas is most severe in cool wet soil conditions in which seedling emergence would be delayed and pathogen spore germination would be greatest due to a large spermosphere effect (2.10.22.28.32). Peas are a cool temperature crop which must be planted early in spring in temperate regions to produce maximum yields (22). Fungicide seed treatments have been widely used to minimize pre-emergence rotting in peas, though they have not always adequately controlled the disease (10.12.20). Since Pythium and Fusarium populations are much higher in spermoSphere than in non-spermosphere soil (30,36), minimizing the spermosphere effect might suppress an increase of these pathogens, as well as disease incidence. Seed germination and seedling emergence were impaired when seeds were soaked in water at high (30 C) or low (1-15 C) tempera- tures (16.23.27), or for more than 48 hours (15,16). Thus. the 'high incidence of rot among Miragreen seeds soaked for 48 hours at 10, 15, or 30 C (Fig. l-A.B) was probably due to temperature injury. However, the spermosphere effect (28) and incidence of seed and seedling rot (Fig. 1-B,D,E) were both considerably reduced by soaking Miragreen peas in water at 22 C for 48 hours before plant- ing. Thus. soaking pea seeds under optimum conditions may be a useful cultural practice by which gardeners can minimize pea seed and seedling rot. 52 The green color of pea and lima bean seeds occasionally fades as seeds mature. a process known as bleaching (24.35.37). Bleached peas appear yellow. and are sometimes referred to as "blonds" (35). Incidence of pea seed and seedling rot among untreated yellow Miragreen peas was greater than among green seeds (Fig. l-C), presumably because yellow seeds exuded more carbohy- drate than green seeds (p. 24). As a result. the spermosphere effect would have been greater with yellow than with green seeds. The spermosphere effect around yellow Miragreen seeds soaked in water at 22 C for 24 hours was only slightly less than around unsoaked seeds (unpublished data). indicating that considerable exudation occurred even after 24 hours of soaking; consequently. incidence of rot among yellow seeds was not reduced by 24 hours of soaking; (Fig. l-C). However. soaking green seeds for 24 hours before planting was effective in reducing incidence of rot, appar- ently because carbohydrate exudation from green seeds had subsided to very low levels prior to planting (p. 25). Lima bean seed rot -and seedling vigor have also been reported (24.37) to be consider- ably greater with bleached than with non-bleached seeds. though the mechanism(s) involved were not determined. The loss of green color in peas and lima beans, and the mechanism(s) by which bleach- ing increases susceptibility to seed decay, merit further study. 10. 11. LITERATURE CITED--PART III ALEXANDER, M. 1961. Introduction to soil microbiology. John Wiley and Sons, New York 472 p. BAYLIS, G. T. S. 1941. Fungi which cause pre-emergence injury to garden peas. Ann. Appl. Biol. 28: 210-218. BUXTON. E. W. 1962. Root exudates from banana and their rela- tionship to strains of the Fusarium causing Panama wilt. Ann. Appl. Biol. 50: 269-282. ESCOBAR. C., M. K. BEUTE. and J. L. LOCKWOOD. 1967. Possible importance of Pythium in root rot of peas. Phytopath- ology 57: 1149-1151. FLENTJE, N. T. 1964. Pre-emergence rotting of peas in South Australia. II. Factors associated with the soil. Austr. J. Biol. Sci. 17: 651-664. FLENTJE, N. T.. and H. K. SAKSENA. 1964. Pre-emergence rotting of peas in South Australia. III. Host-pathogen inter- action. Austr. J. Biol. Sci. 17: 665-675. GRAHAM. J. H., V. G. SPRAGUE, and R. R. ROBINSON. 1957. Damp- ing-off of Ladino clover and lespedeza as affected by soil moisture and temperature. PhytOpathology 47: 182- 185. HAYMAN. D. S. 1969. The influence of temperature on the exuda- tion of nutrients from cotton seeds and on preemergence damping-off by Rhizoctonia solani. Can. J. Bot. 47: 1663-1669. HENDRIX, F. F., JR., and W. A. CAMPBELL. 1970. Distribution of Phytophthora and Pythium species in soils in the continental United States. Can. J. Bot. 48: 377-384. HULL, R. 1937. Effect of environmental conditions. and more particularly of soil moisture upon the emergence of peas. Ann. Appl. Biol. 24: 681-689. JONES, F. R. 1923. Stem and rootrot of peas in the United States caused by species of Fusarium. J. Agr. Res. 26: 459-475. 53 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 54 JONES, L. K. 1931. Factors influencing the effectiveness of organic mercury dusts in pea-seed treatment. J. Agr. Res. 42: 25-33. KEELING. B. L. 1974. Soybean seed rot and the relation of seed exudate to host susceptibility. Phytopathology 64: 1445-1447. KERR. A. 1964. The influence of soil moiSture on infection of peas by Pythium ultimum. Austr. J. Biol. Sci. 17: 676-685. KIDD, F., and C. WEST. 1918. Physiological pre-determination: the influence of the physiological condition of the seed upon the course of subsequent growth and upon the yield. I. The effects of soaking seeds in water. Ann. Appl. Biol. 5: l-10. KIDD, F., and C. WEST. 1919. The influence of temperature on the soaking of seeds. New Phytol. 18: 35-39. KRAFT, J. M., and D. D. ROBERTS. 1969. Influence of soil water and temperature on the pea root rot complex caused by Pythium ultimum and Fusarium solani f. sp. 9151. Phytopathology 59: 149-152. LARSON, L. A. 1968. The effect soaking pea seeds with or without seedcoats has on seedling growth. Plant Physi- ology 43: 255-259. LEACH, L. D. 1947. Growth rates of host and pathogen as factors determining the severity of preemergence damp- ing-off. J. Agr. Res. 75: 161-179. LEDINGHAM. R. J. 1946. The effect of seed treatment and dates of seeding on the emergence and yield of peas. Sci. Agr. 26: 248-257. MATTHEWS, S.. and W. T. BRADNOCK. 1968. Relationship between seed exudation and field emergence in peas and french beans. Hort. Res. 8: 89-93. MC NEW. G. L. 1943. Pea seed treatments as crop insurance. The Canner 96: 16-28. PERRY. D. A.. and J. G. HARRISON. 1970. The deleterious effect of water and low temperature on germination of pea seed. J. Exp. Bot. 21: 504-512. POLLOCK, B. M., and V. K. TOOLE. 1966. Imbibition period as the critical temperature sensitive stage in germination of lima bean seeds. Plant Physiology 41: 221-229. 25. 26. 27. 28. 29. 30. 31. 32. .33. 34. 35. 36. 37. 55 SCHROTH, M. N., and R. J. COOK. 1964. Seed exudation and its influence on preemergence damping-off of bean. Phyto- pathology 54: 670-673. SCHROTH, M. N., T. A. TOUSSOUN, and W. C. SNYDER. 1963. Effect of certain constituents of bean exudate on germ- ination of chlamydospores of Fusarium solani f. phaseoli in soil. Phytopathology 53: 809-812. SCHULZ. F. A., and D. F. BATEMAN. 1969. Temperature response of seeds during the early phases of germination and its relation to injury by Rhizoctonia solani. Phyt0path- ology 59: 352-355. SHORT, G. E.. and M. L. LACY. 1974. Germination of Fusarium solani f. sp. pisi chlamydospores in the spermosphere of pea. PhytOpathology 64: 558-562. SHORT, G. E.. and M. L. LACY. 1974. Effect of soil moisture, temperature. and cultivar on Fusarium seed and seedling rot in peas. Ann. Proc. Amer. Phyt0pathol. Soc. 1: (In Press). SINGH, R. S. 1965. Development of Pythium ultimum in soil in relation to presence and germination of seeds of differ- ent crops. Mycopathol. Mycol. Appl. 27: 155-160. SLYKHUIS, J. T. 1947. Studies on Fusarium culmorum blight of crested wheat and brome grass seedlings. Can. J. Res. 25: 155-180. STANGHELLINI, M. E.. and J. G. HANCOCK. 1971. Radial extent of the bean spermosphere and its relation to the behav- ior of Pythium ultimum. PhytOpathology 61: 165-168. TUKEY, J. W. 1951. Quick and dirty methods in statistics. Part 11. Simple analyses for standard designs. Proc. Amer. Soc. Qual. Contr. 5: 189-197. VERONA, O. 1963. Interaction entre la graine en germination et 1es microorganismes telluriques. Ann. Inst. Pasteur (Paris) 105: 75-98. VITTUM, M. T.. and A. A. DUNCAN. 1964. Blonding in peas. Oregon Vegetable Digest 13(4): 4-7. WATSON, A. G. 1966. The effect of soil fungicide treatments on the inoculum potentials of spermosphere fungi and damping-off. New Zeal. J. Agr. Res. 9: 931-955. WESTER, R. E.. and H. JORGENSEN. 1956. Relation of chloro- phyll fading from cotyledons to germination and vigor of some green-seeded lima beans. Seed World 78 (5 : 8. lll‘l‘llllllllllll III. I CONCLUSIONS 56 ‘|[[[‘l||lll j l [I [IIII CONCLUSIONS Success and efficiency in minimizing plant disease losses are dependent on our understanding of the mechanisms by which biotic and abiotic factors interact to prevent pathogens from locating, penetrating. and invading host tissues. Plant disease control measures are often directed at inhibition of the pathogen after it is in contact with the host. However, an equally import- ant goal is to prevent potential pathogens from even encountering susceptible hosts. Above-ground plant structures are difficult to maintain free of pathogens due to wind. rain, and insect dispersal of pathogen propagules. However, contact between host and pathogen in soil is largely dependent on growth on the host, or the pathogen. or both. Fortunately, most fungal spores do not germinate and grow through soil without an external source of nutrients. Germin- 'ating seeds exude sugars, amino acids, and probably other substances which diffuse into surrounding soil and stimulate spore germination and vegetative growth of fungi, including seed pathogens. The increase in microbial activity in soil around germinating seeds has been called the spermosphere effect. Pea seeds exuded sufficient quantities of pathogen-stimulat- ing carbohydrates to enable refinement of previous methods of meas- uring jfl_vitro carbohydrate exudation and Fusarium solani 57 [I'lll‘lllll 58 chlamydospore germination in the spermosphere. In addition, seed and seedling decay are frequently very severe for crops such as peas which exude large amounts of carbohydrates during germination. and then remain within the spermosphere soil where pathogen activity has been stimulated. Thus, garden peas and Fusarium solani f. sp. 21§1_were a particularly appropriate host and pathogen, respec- tively. for studying the relationship between carbohydrate exuda- tion. the spermosphere effect, and incidence of seed and seedling rot. The total amount of carbohydrate exudation and the magni- tude of the spermosphere effect were directly related to the inci- dence of pea seed and seedling rot at 10 C, 30 C, or when tempera- tures were alternated. More specifically. carbohydrate exudation. the spermosphere effect, and incidence of rot were greater for wrinkled-seeded Miragreen than for smooth-seeded Alaska peas, greater at high than at low moisture conditions, and much less for seeds soaked at 22 C for 24-48 hours than for seeds not soaked prior 'to planting. Carbohydrate exudation and incidence of seed and seed- ling rot were greater with yellow than with green seeds; and exuda- tion was greater from old than from young seeds. Temperature affected not only carbohydrate exudation. but probably microbial competition for exudates, growth rates of host and pathogen, and rate of disease development as well. However. when temperatures were alternated to maximize carbohydrate exudation, the Spermos- phere effect, and rate of disease development. incidence of seed and seedling rot were greatest. 59 Research efforts and recommendations to growers for minimiz- ing pea seed and seedling rot should be based on three principles: (i) minimizing carbohydrate exudation, (ii) minimizing the spermos- phere effect, and (iii) optimizing conditions for seed germination and seedling growth. Peas are a cool temperature crop which must be planted early in Spring in temperate regions to ensure high yields; thus, temperature can be "controlled" only to the extent to which peas can be planted in areas where temperatures are conducive both to high yields and to minimum rot. Similarly. rainfall is usually beyond human control and not entirely predictable; however, pea seed and seedling rot can be minimized by planting seeds in well-drained fields. and at times other than immediately preceding an extended rainy period. Unfortunately, the latter variable is rarely under the growers' control. For centuries man has carefully selected and treated seeds to improve emergence and yields. For example. smooth-seeded pea cultivars are generally less susceptible to seed rot than wrinkled- 'seeded cultivars. But, planting smooth-seeded rather than wrinkled-seeded peas is no real solution to pea seed rot problems because wrinkled-seeded peas generally yield more than smooth- seeded peas, and are much sweeter than smooth-seeded peas. Indeed, it is probably this very sought-after sweetness which is the cause of the greater spermosphere effect and hence the greater rot prob- lem. Thus. growers with large acreages of peas have relied primar- ily on fungicide seed treatments for minimizing seed rot. However, 60 soaking peas prior to planting has been a practice of some home gardeners for many years, perhaps because soaked seeds rotted less frequently and plumules emerged more quickly than when seeds were untreated. Planting soaked seeds may also delay an increase in pathogen propagule inoculum in soils repeatedly cropped to peas. Mechanical devices are not currently available for commercially planting soaked and swollen seeds; however. planting soaked seeds would be feasible for those planting peas by hand. and might result in significantly reducing incidence of seed rot. Recent research has revealed additional seed characteristics which affect incidence of seed and seedling decay. For example. the green color of pea and lima bean seeds occasionally fades as seeds mature, a process known as bleaching. When pea and lima bean seeds were planted in pathogen-infested soil, bleached seeds had a higher incidence of rot than non-bleached seeds. Bleached (yellow) peas exuded more pathogen-stimulating carbohydrate than non-bleached (green) peas; consequently. the spermosphere around bleached peas -would likely be larger than around non-bleached peas. The mechan- isms of bleaching in peas and lima beans have not been elucidated. Nevertheless, environmental factors rather than genetic factors are probably responsible for differences between bleached and non- bleached peas in susceptibility to seed-rotting organisms.