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IILIL'IIILLLL‘ LLL'LLL'LIL‘LTLL 1““LLLLILIILLH LLII'LI LLLIL ILL ‘LL'LI'L'LILLLILL-LIL‘LL .LIL'ILILLLLI LLLLLLLLLLLL LL‘LLLLLLLQILLI ”..ILLLIILL L‘ L L .IILLLL’LILLH IILIIII.L .LL..LLLL IIILI .LIL .“.I LI'L L L -I“III‘LLI‘ILIL|-.LL LL LL LL ILIIILIL II'xIL LL IIIIII .L." LIL ..ILl. I‘ILIIIL. LILII-L II LL‘LI"|.lILI.II" .IL’ILLII’UIILIIIIIIL LIIILLLI 'IIIL IILILIIII‘ ‘IIL. L313?“ LI“ LIIDIIIILLLLIkLI If. L'LI LI LLLLLLL ILLLLLLI 'LILL‘ILLL'LLLL LLLLLL‘ LL LIIL ' LL'LLLLLLLL LIIL L I L . ..L“ ‘L. I .II I LII II I L l? LILILISMILIIIIIIIIIIIIIL.:. '.(LIIJIIIIII' L' IIIIIMLIILIIII LI LL L L LL LLL L LL II L LII. "' L'Ld L‘L‘LI “'.. .LLL‘FLL- mm l1 \ \llllfllllllfllllllllllll 3 1293 00088 4310 Y‘HES'S ‘ imam“ " Michigan Stan: " University This is to certify that the thesis entitled EFFECT OF GENOTYPIC VARIATION AND DELAYED HARVEST UPON SEED QUALITY IN PHASEOLUS VULGARIS L.. UNDER CONDITIONS OF INTERNAL SEED—BORNE FUNGAL INFECTION. presented by Krishna Prasad Shanna has been accepted towards fulfillment of the requirements for M. S . Plant Breeding degree in Department of Crop and Soil Sciences 4. Major professor 1- tiff! 7 V”? #19262; WM ma: 25¢ per 40 per it. RETUMXIG LIBRARY MATERIALS: Place in book return to rmve charge from ctrculatlon records EFFECTS OF GENOTYPIC VARIATION AND DELAYED HARVEST UPON SEED QUALITY IN PHASEOLUS VULGARIS L, UNDER CONDITIONS OF INTERNAL SEED-BORNE FUNGAL INFECTION By Krishna Prasad Sharma A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Sciences 1979 ABSTRACT EFFECTS OF GENOTYPIC VARIATION AND DELAYED HARVEST UPON SEED QUALITY IN PHASEOLUS VULGARIS L, UNDER CONDITIONS OF INTERNAL SEED-BORNE FUNGAL INFECTION By Krishna Prasad Sharma Potential genetic variability and effect of delayed harvest after normal maturity for: the extent of external and internal seed infection by fungi, jn_vitro seed germination, field emergence, specific fungus-genotype interactions, and genetics of resistance to internal seed-borne fungi in dry bean (Phaseolus vulgaris L) were studied in the laboratory, green-house, and in the field from l977 to 1979. Forty-two bean genotypes were used in preliminary screening for resistance to internal and external seed-borne fungi. Non—surface sterilized and surface-sterilized seeds were incubated in SPDA plates for 5-6 days under normal light and temperature to study external and internal seed infection, respectively. Seeds were surface-sterilized for one minute in lzl NaOCl solution (2.6% a.i.) and blotted dry. Crosses between San-Fernando (resistant) and Tuscola (sus- ceptible) cultivars were made in the green-house. F1 and seven other selected genotypes including the parents were planted in the field. Plots were sprayed with a mold spore su5pension at physiological maturity. Seeds were harvested at three different times: normal maturity, two weeks after, and three weeks after normal maturity. Four hundred surface-sterilized seeds/genotype/harvest date and 200 seeds/plant for fifteen F1 plants harvested two weeks after normal Krishna Prasad Sharma maturity were used for internal seed infection study. In_xjt§g_seed germination and field emergence tests were made by using 400 non- surface-sterilized seeds/genotype/harvest date. No genotypic variation for external seed infection and a large genotypic variation for internal seed infection by fungi were observed. Harvest date X genotype interaction was highly statistically signifi- cant. San-Fernando, Nap-2, Turrialba #l, and Ex-Rico-23 showed nega- tive effects while Tuscola, BTS, and Seafarer showed positive effects of delayed harvest. Eight fungi: Alternaria, Rhizoctonia, Fusarium, Penicillium, Cladosporium, Epiccoccum, Chaetomium, and Rhizopus were isolated from surface-sterilized seed; the first two fungi showed specific fungus X genotype interactions. Many pairs of genes with additive effects or showing partial dominance over susceptibility are postulated to confer resistance. Significant genotypic variation for i vitro seed germination and field emergence was observed. Harvest delay did not show a significant reduction in jn_vitro seed germination of the resistant parent. Black and thick seed coated genotypes showed superiority to white and thin seed coated genotypes for field emergence. ln_vitro seed germination overpredicted the field emergence of Nap-2. To my Parents ii ACKNOWLEDGMENTS The author wishes to express his sincere appreciation and grat- itude to his major professor, Dr. M. w. Adams for his valuable guidance, moral support and encouragement at all times during study and thesis preparation. Equal thanks and gratitude are due to Dr. A. w. Saettler, member of my committee, for his guidance, moral support, friendship, appreciation, encouragement and for providing laboratory facilities during the conduct of the experiments. Appreciation is expressed to Dr. G. L. Hosfield and Dr. L. O. Capeland for serving as guidance committee members and for their help- ful discussions and suggestions during thesis preparation. Thanks are also extended to Dr. M. A. Ellis University of Puerto Rico, for his suggestion in the conduct of this study, to Dr. Barbara Dyko for helping identify fungi, to J. L. Taylor for his assistance in the field, and to Kailash Pyakuryal and S. P. Dhital for their help in various ways during my thesis preparation. A grateful acknowledgment is also extended to the MUCIA/NEPAL Project for financial support and to IAAS Tribhuwan University Nepal, for granting me study leave throughout my study at Michigan State University. To the ones very special to me, my wife Anju and daughters Sinju and Munu, my thanks for their conStant love, understanding, and sacri- fices given to me for the entire two years of my study. Without their dedication, my graduate study could never have been possible. TABLE OF CONTENTS Page LIST OF TABLES ......................... v LIST OF FIGURES ........................ vii INTRODUCTION .......................... 1 LITERATURE REVIEW ....................... 3 CHAPTER I PRELIMINARY STUDY ON GENOTYPIC VARIATION FOR EXTERNAL AND INTERNAL SEED CONTAMINATION BY FUNGI IN DRY BEAN (PHASEOLUS VULGARIS L.) ................. 15 II SECTION A: EFFECT OF DELAYED HARVEST ON INCIDENCE OF INTERNAL SEED-BORNE FUNGI IN DRY BEAN (PHASEOLUS VULGARIS L.) ...................... 27 SECTION B: PRELIMINARY OBSERVATIONS ON THE NATURE OF INHERITANCE FOR RESISTANCE T0 INTERNAL SEED-BORNE FUNGI IN DRY BEAN (PHASEOLUS VULGARIS L.) ......... . . 50 III SECTION A: EFFECT OF DELAYED HARVEST 0N LABORATORY SEED GERMINATION IN DRY BEAN (PHASEOLUS VULGARIS L.) GENOTYPES ..................... .. . . 56 SECTION 8: EFFECT OF DELAYED HARVEST 0N FIELD EMERGENCE IN DRY BEAN (PHASEOLUS VULGARIS L.) SEED ........ 62 DISCUSSION ........................... 57 SUMMARY AND CONCLUSIONS .................... 76 LITERATURE CITED ........................ 79 iv Table 10. ll. 12. 13. LIST OF TABLES Percentage external infection of dry bean (Phaseolus vulgaris L) seeds by fungi ................ Percentage internal infection of dry bean (Phaseolus vulgaris L) seeds by fungi ................ Percentage internal infection of dry bean (Phaseolus vulgaris L) seeds by fungi ................ Effect of genotype and delayed harvest on internal bean (Phaseolus vulgaris L) seed infection by fungi ...... Comparison of genotypic and harvest time means for internal bean (Phaseolus vulgaris L) seed infection by fungi .......................... Effect of delayed harvest on internal fungal infection of seed of 'Seafarer' bean genotype ........... Comparison of three harvest time means for internal fungal infection of seed of 'Seafarer' ............. Incidence of total internal fungi and fungal genera in seeds of six dry bean (Phaseolus vulgaris L) genotypes . . Percentage internal fungal seed infection of fifteen F1 plants of the cross between 'San-Fernando and Tuscola' bean genotypes ...................... Effect of genotype and delayed harvest on laboratory seed germination of dry bean (Phaseolus vulgaris L) seed ........................... Comparison of genotypic and harvest time means for A laboratory seed germination of dry bean (Phaseolus vulgaris L) seed ..................... Effect of delayed harvest on laboratory seed germination of Seafarer bean genotype ................ Comparison of harvest time means in Seafarer bean genotype for laboratory seed germination ......... V Page 17 24 26 30 53 59 60 61 Table Page l4. Effect of genotype and delayed harvest on field emergence of dry bean (Phaseolus vulgaris L) seeds ......... 63 15. Comparison of genotypic and harvest time means for field emergence of dry bean (Phaseolus vulgaris L) seeds . . . . 64 l6. Effect of delayed harvest on field emergence of Seafarer seed ...................... 66 l7. Comparison of harvest time means in Seafarer for field emergence ........................ 66 vi LIST OF FIGURES Figure I. 5A. SB. SC. 7A. 7B. 7C. 9A. Degree of external seed contamination by fungi in forty—two bean genotypes ................. Genotypic variation in percent internal seed contamina- tion by fungi in forty-two bean genotypes ........ Genotypic variation in percent internal seed contamina- tion by fungi in fourteen selected genotypes ....... Pod molding in San-Fernando bean genotype harvested at normal maturity (l), two weeks after (2), and three weeks after (3) normal maturity ................ Internal fungal infection of seed of San-Fernando genotype harvested at normal maturity .......... Internal fungal infection of seed of San-Fernando genotype harvested two weeks after normal maturity . . . . Internal fungal infection of seed of San-Fernando genotype harvested three weeks after normal maturity . . . Pod molding in Tuscola bean genotype harvested at normal maturity (1), two weeks after (2), and three weeks after normal maturity ..................... Internal fungal infection of seed of Tuscola genotype harvested at normal maturity ............... Internal fungal infection of seed of Tuscola genotype harvested two weeks after normal maturity ........ Internal fungal infection of seed of Tuscola genotype harvested three weeks after normal maturity ....... Pod molding in Nep-2 genoty e harvested at normal matur- ity (l), two weeks after (2 , and three weeks after (3) normal maturity ..................... Internal fungal infection of seed of Nep-Z genotype harvested at normal maturity ............... vii Page 21 23 34 36 Figure 98. Internal fungal infection of seed of Nep-Z genotype harvested two weeks after normal maturity ........ 9C. Internal fungal infection of seed of Nep-Z genotype harvested three weeks after normal maturity ....... lo. Pod molding in Ex-Rico-23 bean genotype harvested at normal maturity (1), two weeks after (2), and three weeks after (3) normal maturity ................ llA. Internal fungal infection of seed of Ex-Rico-23 genotype harvested at normal maturity .............. llB. Internal fungal infection of seed of Ex-Rico-23 genotype harvested two weeks after normal maturity ........ 12. Pod molding in Turrialba #l bean genoty e harvested at normal maturity (1), two weeks after (2 , and three weeks after (3) normal maturity ................ 13A. Internal fungal infection of seed of Turrialba #l geno- type harvested at normal maturity ............ 138. Internal fungal infection of seed of Turrialba #l geno- type harvested two weeks after normal maturity ..... l4. Pod molding in Black Turtle Soup bean genotype harvested at normal maturity (1), two weeks after (2), and three weeks after (3) normal maturity ............. ISA. Internal fungal infection of seed of Black Turtle Soup genotype harvested at normal maturity .......... 153. Internal fungal infection of seed of Black Turtle Soup genotype harvested two weeks after normal maturity 15C. Internal fungal infection of seed of Black Turtle Soup genotype harvested three weeks after normal maturity 16. Pod molding in Seafarer bean genotype harvested at normal maturity (1), two weeks after (2), and three weeks after (3) normal maturity ................... l7A. Internal fungal infection of seed of Seafarer genotype harvested at normal maturity .............. l7B. Internal fungal infection of seed of Seafarer genotype harvested two weeks after normal maturity ........ l7C. Internal fungal infection of seed of Seafarer genotype harvested three weeks after normal maturity ....... viii Page 37 37 38 38 39 39 40 4o 4T 4T 42 42 43 43 44 44 Figure Page l8. F2 germs of the cross between San-Fernando and Tuscola showing highest (A), and lowest (B) internal fungal infection of seed of two F] plants ............ 52 T9. Normal (A) and abnormal (B) dry bean seedlings in ger- mination test. Abnormal seedlings developed from seed internally infected with fungi .............. 57 ix INTRODUCTION After ripening, mold can be a major problem in the successful production of seed of dry beans (Phaseolus vulgaris L,) both in tropical and temperate zones of the world. The molded plants are usually coated with a fine layer of blackish dust which represents infected pods (Figure 10), leaves, and plant as a whole. The fungi causing mold may be external and/or internal seed-borne. The later type is more important from seed quality standpoints. The fungi get pulverized during threshing and the seeds are surface contaminated with viable micro-organisms. They alter the bio-chemical composition of the stored bean reducing commercial value (140). These fungi have a tremendous effect reducing seed germination and field emergence. High temperature (around 85°F), high relative humidity, and a delay in harvest past maturity provide congenial conditions for mold in- vasion. The fungi which are internally seed-borne have been reported to reduce dry bean production as much as 50 percent and complete crop failure is not unusual (34) in some individual cases. The yield reduction in dry beans and soybeans in the tropics by internally seed-borne fungi has been reported by many workers. The seed is one of the most basic elements to any crop production program and without high seed germination and seedling emergence in the field there is no efficient crap production. One of the major problems facing increased dry bean production in the trapics is a reliable 2 source of high quality disease free seeds for planting (3). The same is true in the temperate regions when harvest time is accompanied by rainfall which causes a delay in harvesting. Seed deterioration is highly associated with the percentage of internally-borne fungi. How- ever, colored-seeded genotypes have been reported to do better under such conditions often out-yielding the white—seeded counterpart, color preferences have caused complications in bean production (4,5,10,26, 28,55,92,115,124). The fungus Aspergillus flavus, which causes molding, produces the toxin "aflatoxin" which has potent carcinogenic proper- ties (140). The toxin also causes death in poultry and abortion in higher animals. Michigan, a leading dry bean producing state with 34 percent of the national total, produced 6,440,000 hundred weight (cwt) clean beans, 14 percent larger than in 1977 of which 90 percent were navy beans (88). Growers increased the planted acreage over 1977 only by 4 percent. Low yields in 1977 were due in part to crop abandonment after rainfalls during time of harvest. The crap was abandoned because of the high degree of mold invasion. Seed quality may be completely deteriorated if the storage conditions are favorable for the mold which is already present in the seeds, to develop. The objectives of this study were: (1) to determine the pos- sible existence of genetic variation for incidence of internal seed infection by fungi: (2) to identify the fungi involved and determine possible Specific genotype-fungus interactions (3) to determine the effect of harvest date on seed quality, and (4) to study the genetics of resistence for incidence of mold invasion. LITERATURE REVIEW The importance of seed-borne fungi in different crops was recog- nized when harvested products (seeds) stored under high temperature and moisture conditions develOped mold which decreased seed quality and viability. Infected seed exhibited poor field emergence and stand when planted. This fact prompted researchers; 1) to study the genera and species of fungi involved, 2) to study proper storage con- ditions and; 3) to improve seed quality and viability. There are two types of seed-borne fungi; 1) external seed-borne fungi and 2) internal seed-borne. Internal seed-borne fungi constitute the major problem because fungicidal treatments are not effective. The original seed quality problems can be attributed to two major stages; a) Seed production and b) Seed storage (3). Problems encount- ered in the production of high quality seed include; seed infection in the field by micro-organisms, damage to pods and seeds from feeding insects; unfavorable growing conditions for the crap including attack from diseases, insects, and weeds, and unfavorable climatic conditions (rain and high temperature) at time of harvest. Seed storage problems are mainly due to conditions of high temperature and relative humidity during storage of seed already contaminated with viable micro- organisms. Poor seed quality is generally reflected by low laboratory seed germination and field emergence (61). Low moisture content is a key 4 factor in maintaining seed viability (148). I. Dry Beans (Phaseolus vulgaris L,) Numerous fungi have been reported to be borne internally in seeds of bean;(Acrostalomus §p_(24,44); Alternaria §p_(24,34,41,44, 83,127); Aspergillus §p_(18,24,44,84); Botrytis §E_(24,44); Cladospor- jgm1§p_(24,4l,44); Colletotrichum §p_(24,34,44); Fusarium §p_(24,34, 39,41,43,44,64,102,127); Isariopsis §p_(24,44); MacrOphoma §p_(24); Macrophomina §p_(34,41,44,102); Monilia §p_(24,41,44); Nigrospora §p_ (41); Penicillium §p_(24,41,46); Pestalotia §p_(24,41); Peyronellaea 33 (24); Phomopsis s2(24,34,39,41,44,64,114); Phoma g; (34.41); Rhizoctonia s3 (24,28,29,34,41,44,46,79,102,112,127); Rhizogus s2 (24); Sclerotinia §p_(15,24,44); and Trichothecium §p_(34). Incidence of internal seed-borne fungi is negatively correlated with seed quality, seed germination, emergence and seedling vigor in Phaseolus vulgaris (24,29,34,39,43,44,46,123,163). 0f seed that con- tained fungi, 71% did not germinate (24). Dingra in Brazil (123) con- cluded that dry and snap bean seed grown during the rainy season and harvested at normal maturity had very poor germination. Seed infected internally with fungi germinated only 68%. There are high correlations between moisture content, relative humidity, mold count, and duration of storage (13,18,120). Lopez et- a1, (84) in a study of moisture content, number and type of fungi pre- sent after three months storage at 18.5-22% moisture, found no positive correlations between degree or rate of moisture absorption and fungal infection. Defective seedlings such as "baldhead" and "snakehead" eventually resulted from invasion of the seeds by storage fungi (18). Invasion occurred through the hilum and micropyle as well as through cracks in the seed coat: the embryo root, stem, and growing point appeared to be attacked in preference to the cotyledon (18). Preliminary pathogenicity tests indicate that the fungi could penetrate through an uninjured pod wall and infect developing seed (34). He also reported that the fungi grew out through the hilum and micropyle or through the cracks in the seed coat but never through the unbroken seed coat (18), and concluded that if the coats of the seeds invaded by storage fungi were punctured, the fungi grew out through these punc- tures by increasing the mold invasion. Discoloration causes by one pathogen is indistinguishable from that caused by others (34), and such discoloration not only reduces seed quality but also reduces the commercial value of the seed for human consumption. Harvesting at proper maturity (113,132,133) increases seed qual- ity in dry beans. Prolonged rain after full pod set prediSposes pods and seeds to fungal invasion by allowing prolonged periods of high humidity and pod surface wetness (34,39). However, occurrence of fungi infecting dry bean seed varies from location to location and cultivar to cultivar (34). One problem in the production of high quality dry bean seed is pod contact with soil (23,41). Percent internal seed-borne fungi among cultivars with pods in contact with soil ranged from 64-92% as compared to 3-30% with pods not in contact with soil (41). The mean percent germination and field emergence of seeds from pods not in contact with soil was significantly higher than that of seed from pods in contact with soil. Seed treatment had no effect on germination of high quality seed but greatly improved that of poor quality seed (22,45). Plants having many pods in contact with soil are therefore considered poor architectural types, and such plants are also prone to seed-borne diseases. Varietal differences in degree of fungal invasion, seed germina- tion and field emergence have been reported for the bean. Varietal differences for Rhizoctonia solani resistance were observed by Prasad et_al, (112); resistance was highly heritable and associated with colored seed (29).‘ Snap bean cultivars (Phaseolus vulgaris L,) with colored seeds produced stronger and more vigorous seedlings than those with white seed (68). Cultivars with colored seed appeared to adapt much better to adverse conditions than white seeded cultivars. Deakin (28) reported that the mean percentage of emergence showed no signifi- cant differences among genotypes although some variability existed. Lines (genotypes) by color interactions were also nonsignificant. Emergence of color seeded lines was almost always superior to that of their white seeded counterpart; such superiority was thought due to resistance to Rhizoctonia solani. Cesar, gt_al, (18) also found varietal differences in percent seed germination and number of abnormal seedlings among three bean genotypes; Amarillo, Jamapa, and Bayomex. The superiority of Jamapa was thought due to sound seed coats with no detectable breaks or cracks. Much of the seed of the Bayomex and Amarilo 153 varieties had obvious breaks and cracks in the coats and these cracks probably allowed ready invasion of fungi. Hard seed coats appear to inhibit invasion by micro-organisms (78). Seed treatment has little effect on internal seed-borne fungi in dry bean, particularly when conditions 7 are Optimal for disease development (79). II. Soybean (glycine max) Poor soybean seed quality in the northern USA is primarily the result of infection with a species of PhomOpsis (Diaporthe phaseolorum var sgjae) and Diapgrthe phaseolorum var caulivora (126). These fungi can infect seed still contained in the pod, but cause the greatest damage through latent seed infection which leads to seedling rot dur- ing germination. Seed germination is inversely related to percentage seed infection. Infection remains latent until pods begin to mature during wet weather. Poor seed quality is also a major problem of soy- bean production in tropical conditions (138). High temperature and humidity at planting time (101) and rain at or during the maturity period (33,101,145) were favorable for seedling rot and pod diseases leading to seed damage. Many fungi have been reported to be internally seed-borne in soybean; Alternaria §p_(3,40,56,73,89,98,147,163,l59); Arthinum §p_ (3); Asperigillus §2.(3,32,38,56,73,74,9l,98,145,149,159); CerCOSpora §E_(3,33,56,57,70,71,74,98,140,156,158,159); Cephalosporium §p_(3,74); Chaetonium §p_(3,98,159); Chaetgphoma §p_(3,74); Chanephora §p_(3); Colletotrichum §p_(3,40,74,163); Corynespora cassilicola (3); Fusarium s2 (3,40,56,57.73,74,89,98,l21,163,159); Gliocladium 52 (3,74); Glomerella s2 (3.74); HelminthOSporium 3:3 (3); Lasidiplodia _s_p_ (3,74); Leptosphaerolina §p_(3,74); Macrophoma sp.(3,74); Macrophomina §p_ (2,40,74,163); Myrothecium §p_(74); NoduloSporium §p_(3); Penicillium §p_(3,56,59,73,74,98,159); Pestalotia §p_(3,74,98,159); PhomoEsis §p_ (3,6,16,19,27,36,40,50,56,57,75,74,99,106,140,151,152,163,162); Ehgmg_ §p_(3,74); Pogulina §p_(3); Rhizoctonia §p_(3,57,74); Rhizopus §p_ 8 (3,74,98,159); Sclerotinia §p (98,99,159); Sclerotium §E_(3,74); Syncephalastium §p_(3); Thielaviopsis basicola (98,159); Trichocladium §p_(3); Trichoderma §p_(3,74). The majority of fungi isolated from soybean seeds appear to be saprOphytes which have no noticeable effect on seed germination (3). It was concluded that, of thirty-five fungi tested, only nineteen significantly reduced germination in vitro and the most pathogenic were Nodulosporium s3, Sclerotium s9, Leptosphaerulina sp, Phomogsis sp, Lasidiplodia s9, Aspergillus s2, Colletotrichum sp, MacrOphoma sp, Macrophomina s2, and CephalOSporium s2, Fourteen fungi signifi- cantly reduced emergence in sand tests. Pod and stem blight caused by Phomopsis §p_are among the principle diseases associated with re- duced seed germination, and viability and seed quality deterioration in many soybean production areas (6,19,27,36,75,106,l40,151,152); how- ever, the fungus did not reduce jn_vjtrg_germination or field emergence when compared to Sclerotinia §p_(99). Nilcox (157) and Ellis (40) reported that increases in percentage of soybean seed infected by internally seed-borne fungi such as Phomopsis, Fusarium, and Alternaria s2 were accompanied by increases in percentage seed germination and decreases in field emergence. Aspergillus sp, a major problem in stored soybean has a significant negative correlation with seed viability, germinability, and also causes seedling blight (32,38,73,9l,l45,l49) particularly under condi- tions of high “temperature and moisture (90). Many other workers (2,93,96,97,ll7,l34) have reported that micro-organisms infecting seed reduce seed quality and often cause low germinability and seed deter- ioration in soybeans. 9 Cerc05pora §p_causes purple stain in soybean seed and is asso- ciated with poor seed quality (73,138,156). This diesease may reduce yield considerably, and decrease quality and market value of the pro- duct. Incidence of the purple stain disease is most severe when the period of seed maturity occurs during wet weather conditinos (73,140). Resistance to purple stain is highly heritable (H=.9l) (158). Delayed harvest did not significantly affect soybean yield but reduced percentage seedling emergence (101) when harvested seed was ‘J planted. Delay in harvest also significantly reduced emergence (104, \l 156) and the average sand emergence percentages were 95, 88, and 74 percent and field emergence percentages were 90, 77, 57 percent for the non-delayed, two weeks-delayed and four weeks-delayed harvest dates respectively (l32) It was concluded that incidence of internal J seed-borne fungi increased significantly when harvest was delayed be- yond normal maturity; and that the incidence was negatively correlated with germination and field emergence (40,104,157)J’ However, length of storage is the major factor influencing seed germination while de- layed harvest had less but a more variable effect (154). V Plants maturing under dry conditions had less fungal infection so that harvest delays and fungicidal treatments had no effect on in- cidence of fungal seed infection and germination (153). However, de- layed harvest of plants maturing under wet conditions resulted in seed viability reductions of 25% with significant cultivar X harvest time interaction. Alexander £3.31, (1) in experiments conducted for four years found that, a relatively low percentage of soybeans was infected and germination was excellent at normal harvest. At each succeeding harvest, percentage infection increased and germination 10 decreased. At the last harvest most beans were infected and only few germinated normally. The presence of PhomOpsis sojae, Fusarium semitectum and Colletotrichum dematium f. truncata in soybean plantings was signifi- cantly correlated with weed development, suggesting that weeds may serve as alternate hosts or provide a microclimate of prolonged high humidity favoring seed infection (33). However, the occurrences of Macrophomina phaseolina and Cercospora kukuchii were not affected by weed development. Paschal gt_al, (154) found consistent differences in germinabil- ity among soybean lines with some small seeded lines from southeast Asia maintaining more than 50% germination after eight months of stor- age under ambient environmental cOnditions. Seed size was negatively correlated with field emergence and positively correlated with inci- dence of internal fungi (104). Smaller-seeded genotypes had higher emergence percentage and less internally seed-borne fungi. Seed lots of twelve soybean cultivars harvested from different growing locations over a three year period showed significant differ- ences in percentage germination between years, and in occurrence of seed-borne micro-organisms between four locations and years (38,56). The occurrence of seed-borne micro-organisms was influenced more by growing location than by different planting dates, harvesting dates (145) or method of harvest (98,99). Tedia (l45) found that year to year fluctuations in incidence of seed-borne micro-organisms in soy- bean and the associated decline in seed vigor prior to physiological maturity were due to high temperatures during seed maturation. The differences in occurrence of micro-organisms between growing locations ll was expected because of differences in rainfall near harvest (38). It has been concluded that in the absence of soybean cultivars resistant to pod and seed infection, seed should be harvested as soon as possible after normal maturity (104,135,156). There is a great variation in seed quality among soybean culti- vars (38,50,104,138). Percentage seed infection and jg_vitrg_germina- tion ranged from 0-100% (104). Percentage germination and total fungi differed significantly among the seed lots between regions for Wayne but not for Amsoy cultivars (146). Cultivar interactions were noted in germination incidence of total seed-borne fungi (56). Soybean cultivars Hardee and PI 205 912 inoculated with Phomopsis spore suspensions showed no significant difference in percentage Phomopsis infection of harvested seed. However, seeds of PI 205 912 which were infected by Phomogsis had a significantly higher ig_vitrg_ germination percentage than infected seeds of Hardee, suggesting that P1 205 912 may possess tolerance against seed or seedling decay (50,162). III. Pea (Pisum sativum) Storage fungi reduce germination percentage of pea (Pisum sativum) seed (53). A genetic study of tolerance to Aphanomyces root rot found resistance to be associated with the presence of pigmentation in flowers and seeds (85). In a study on the importance of testa color in resistance to Pythium ultimum, Stasz (142) reported that resistance was found only in plants where seed possessed color testa other than white. Alternaria is reported to cause a seed Spot in pea. With 12 increasing temperature, moisture content and time, percentage of in- ternally seed-borne micro-organisms such as Asperigillus §p_increases (35). Increased p0pulations of certain pathogens could then increase infection of the host (12). The association of production disease has also been reported (12). IV. Pigeon Pea (Cajanus cajan) Internal seed-borne fungi play a major role in reducing the quality of pigeon pea (Cajanus cajan) seed (42,47). Ellis gt_al, (48) reported that differences in pigeon pea seed quality were due to the temperature and humidity of the growing location. Low percentage seed germination was associated with poor physical appearance and higher incidence of internally seed-borne fungi. Presence of Phomopsis, Fusarium and Lasidiplodia was negatively correlated with germination and field emergence (47,48). The incidence of Alternaria was not significantly correlated with jn_vitro germination or with field emergence indicating that Alternaria does not adversely affect seed germination (47); also, incidence of Aspergillus was not correlated with field emergence (48). Penicillium and Rhizopus §p_were also isolated from pigeon pea seed. Seed infection increased with time after normal maturity. When seeds were harvested at normal maturity, populations of internal seed-borne fungi were very low and emergence in the field was greater than 90% (47){ This shows the importance of timely harvest)’ Breeding for uniform maturation is suggested for pigeon pea as pods do not mature simultaneously in present cultivars. Different seed lots (150) of pigeon pea were tested for 13 seed-borne fungi and it was found that 53.7% of seeds in original un- selected bulk were infected as compared to 41.7% infection in brown seeded bulk, and 78.7% in light seeded bulk progenies. Brown colored seed were healthier and yielded 14.4% greater. Light brown colored seed were small, yielded less and were more heavily infested with fungi. V. Cow Pea (Vigna unguiculata) Ellis gt_al_(65) harvested cow pea (Vigna unguiculata) seed at normal maturity and at one, two, or three week thereafter. Alternaria, CladOSporium, Fusarium, Lasidjplodia, and Phomopsis were isolated from surface dis-infected seeds. Incidence of internal seed-borne fungi increased and the percent seed germination jn_vjtrg_and field emergence decreased with each delay in harvest; Fungicidal sprays did not pre- vent the decline in seed quality. When harvested at normal maturity, plants yielded high quality seed. Crops harvested when 75-80% of the.“J pods were dry (103) yielded well and harvested seed was of good quality. Percent of high (103,115) quality seed was least when pods were allowed to dry completely before harvest. VI. Miscellaneous (other legumes) VBennett gt_gl, (14) reported that good quality seeds could be '1 obtained by harvesting seeds of rough pea (Lathyrus hirsutum) about one week earlier than usual and drying them. Delay in harvest ac- companied by high temperature and humidity increased mold invasion. Seeds of black gram (Phaseolus mungo), lentil (Lens esculenta), and moth bean (Phaseolus acotinifolius) were found infected with a variety of fungi (144). Seed-borne fungi isolated by the blotter 14 method were Species of Alternaria, Cladosporium, Macrophomina, and Fusarium; artificial innoculations with these fungi reduced seed vigor and viability. While seed treatment with fungicides did not completely eliminate the seed-borne fungi, it did reduce number of fungi. Singh et_al, (136) isolated the following fungi from surface- sterilized black gram seed; Aspergillus, Fusarium, Phoma, (which had no effect on germination) Curvularia, which affected both seed ger- mination and seedling vigor, and Penicillium, which resulted in poor root development. Singh gt_al, (137) reported association of the following fungi with seed of chick pea (Cicer arietinum); Cladosporium §p_(l9%), Curvularia §p_(52%), Fusarium §p_(19%), Penicillium §p_(18%),. Pleospgra s2 (41%), Rhizopus §p_(5%), and Trichothecium §p_(10%). All fungi were pathogenic on seeds and seedlings except Curvularia, Penicillium, and Rhizogus. Pleosggra caused severe seed rot and dark root lesions which later caused seedling death. Trichothecium de- creased seedling vigor. Cladosporium caused a stubby root condition and poor root growth vigor. Fungicide treatments only increased per- centage germination from 2-20%. CHAPTER I PRELIMINARY STUDY ON GENOTYPIC VARIATION FOR EXTERNAL AND INTERNAL SEED CONTAMINATION BY FUNGI IN DRY BEANS (Phaseolus vulgaris L) Materials and Methods Sample Collection Heavy rains and hail in September 1977 damaged a large portion of the Michigan dry bean cr0p. The wet weather was associated with high temperatures and resulted in considerable molding of pods and seeds on crop plants still standing in the field. Molded pods of forty-two genotypes obtained from an international Bean Yield Nursery grown at the Saginaw, Michigan Bean and Beet Research Farm were col- lected to study the extent of internal and external seed contamination by fungi. Pods were hand-threshed and stored at room temperature for about 60 days until tested. Media Preparation Thirty-nine grams of commercial Difco Potato Dextrose Agar (PDA) were added to one litre of distilled water. The mixture was put in a steamer at 100°C until the PDA was completely dissolved. The solution 2 steam was autoclaved for twenty minutes at l32.2°C and 1.1 kg/cm pressure. One ml of a 200 ppm streptamycin sulfate solution was added to each 100 ml of sterilized PDA. Streptomycin potato dextrose 15 16 agar (SPDA) was poured in sterile 100 x 15 mm Dispo Petri Dishes and incubated for thirty-six hours; plates showing growth of contaminants at this time were discarded. Assay Technique Twenty non-surface sterilized seeds of each genotype were placed in SPDA plates (10 seeds per plate) to determine incidence of external seed contamination by fungi. Seeds were incubated at 24 :_1°C in the laboratory, and incidence of fungi growing from the seed was recorded after 5-6 days. To determine incidence of internal seed contamination twenty un- cracked seeds from each genotype were randomly taken and surface- sterilized by soaking for two minUtes in 1:1 aqueous bleach solution (2.6% NaOCl). The seeds were immediately dried on paper towelling and transferred individually to SPDA plates. Instruments used in handling seeds were dipped in 95% ethyl alcohol and thoroughly flamed between individual seeds. Seed transfers and all culture work were performed in a standard transfer chamber. Seeds were incubated at 24 :_1°C in the laboratory and number of infected seeds were recorded after 5-6 days. The same assays were repeated in two replications for thirteen promising and one susceptible genotype; fungal counts were averaged and are expressed in percentage of infected seeds. Results The results show no significant genotypic variation in degree of external seed contamination by fungi. Contamination ranged from 80-100% as shown in Table 1. Of forty-two genotypes tested, 17 Table 1. Percentage external infection of dry bean (Phaseolus vul- garis L) seeds by fungi Percent infection 21-M-(3F5) ‘Genotype Seed coat color (20 seeds) Campbell Soup 105 white 80 Porrille Sintetico black 85 Campbell Soup 109 white 90 PI 313 868 black 90 Linea 29 black 90 Ex-Rico-23 white 90 MSU Line 20489 white 90 Campbell Soup 106 white 90 Campbell Soup 107 white 90 Campbell Soup 103 white 90 Campbell Soup 101 white 90 ICA Huasano black 95 Jalpatagua 72 black 95 PI 310 333 black 95 Jamapa black 95 PI 169 299 white 95 Mexico 12-1 dark brown 95 C 63 S 630-8 beige 95 Campbell Soup 104 white 95 25-M—(3F5) black 100 I-968 black 100 black 100 Table 1. (Continued) 18 Percent infection Genotype Seed coat color (20 seeds) Collection 168 N black 100 Pecho Amarillo black 100 M-Gearais black 100 P1 310 740 black 100 San-Pedero Pinula 72 black 100 R-345-LRK OZ pink lOO R.K. 7690 red 100 Nep-2 white 100 PI 284 703 light brown 100 Lamaniere purpure mottled 100 Mshuizaico red 100 Brasil 2 brown 100 Redkloud pink 100 Tuscola white 100 Sanilac white 100 Campbell Soup 110 white 100 Campbell Soup 102 white 100 Atlas R-9 white 100 PI 196 936 white 100 San-Fernando (S-182 N) balck 100 19 twenty-three genotypes had 100% contamination. The remaining geno- types were contaminated in the range of 90% and above, although two showed less than 90% contamination (fig. 1). Of twenty-three geno- types with 100% seed contamination, fifteen were color-Seeded. Seed coat color apparently had little or no effect upon external seed con- tamination. The frequency distribution of genotypes and percent con- tamination is shown in Figure 1. All the genotypes were then tested for internal seed infection by fungi. Internal seed contamination ranged from 0-90% (Table 2) and there was considerable genotypic variation. San-Fernando, a black seeded genotype, was entirely free of internal seed contamination. Thirteen genotypes ranged from 15-30% contamination of which nine were colored seeded and four white seeded. In general, colored seeded genotypes showed overall superiority to the white seeded genotypes. Some of the white seeded lines, such as Ex-Rico-23 and Campbell Soup 109 did as well as the other colored seeded lines. Black seed coat color showed general superiority to other seed coat colors. However, one black seeded genotype, 21-M-(3F5), showed 70% internal seed contamination. Of the forty-two genotypes tested for internal seed contamina- tion, fourteen were selected for further study; thirteen showed very low and one showed very high infection levels. The percentage internal seed contamination ranged from 1-75% (Table 3) which suggests large genotypic variation to internal seed infection. 0f the fourteen geno- types tested three were contaminated in the range of 0-15% (all color seeded); five genotypes were in the range of 16-30%, of which four were colored and one white. One genotype, white-seeded Sanilac, 20 showed 75% infection. San-Fernando again showed only 1% internal seed contamination even though external seed contamination was 100%. 21 25-w 23 20-» f8 :37 1 5 1‘ m 1 5.. u. .0 E. 10-4 9 5’ 8 8 to (.9 5.” 2 O 80 85 9O 95 100 Percent External Contamination Figure 1. Degree of external seed contamination by fungi in forty-two bean genotypes 22 Genotypes* Colored White, A = 100% 0% B = 76.92% 23.08% C = 75% 25% D = 54.54% 45.46% E = 20% 80% F = 50% 50% 13-) B 12-4 0 11-4 m a, 104 g 91 C no 8‘1‘ “5 7-» 8 6+ .2 5.5 E g- 4‘i F 3-. 2-4 1 A 0 L 15 30 45 60 75 90 Percent Internal Contamination Figure 2. Genotypic variation in percent internal seed contamin- ation by fungi in forty-two genotypes *Most of the colored genotypes included under A, B, C are black seeded and under 0, E, F are pink and brown seeded. 23 Genotypes Colored White A = 100% 0% B = 80% 20% C = 75% 25% D = 100% 0% E = 0% 100% B 8 C D. a 4" O 5 i... O ‘8 '8 2-4 C S 8 1— D E .3: O 15 30 45 60 75 Percent Internal Contamination Figure 3. Genotypic variation in percent internal seed contamin- ‘ ation by fungi in fourteen selected genotypes 24 Table 2. Percentage internal infection of dry bean (Phaseolus vulgaris L) seeds by fungi Percent infection Genotype Seed coat color (20 seeds) San-Fernando (S-182 N) black 0 Jalpatagua 72 black 20 Collection 168N black 20 Pecho Amarrillo black 20 Jamapa black 20 Linea 29 black 20 R-345 02 pink 20 Ex-Rico-23 white 20 Campbell Soup 109 white 20 M. gerais black 30 San Pedro Pinula 72 black 30 Lamaniere purple mottled 3O Redkloud pink 30 .Campbell Soup 107 white 30 25-M-(3F5) black 40 ICA Huasano balck 4O I-968 black 40 P1 313 868 black 40 P1 169 299 red 40 C 63 S-630-B beige 40 Campbell Soup 105 white 40 Atlas R-9 white 40 Table 2. (Continued) 25 Percent infection Genotype Seed coat color (20 seeds) Porrillo Sintetico black (brownish) 50 P1 310 740 black 50 P1 201 333 black 50 R.K. 7690 pink 50 Nep-2 white 50 Mexico-12-1 dark brown 50 Brasil 2 brown 50 Campbell Soup 101 white 50 Campbell Soup 102 white 50 Campbell Soup 106 white 50 Campbell Soup 103 white 60 21-M-(3F5) black 70 Tuscola white 70 Campbell Soup 104 white 70 Campbell Soup 110 white 70 PI-l96 936 brown 70 MSU Line 20489 white 80 Pl 284 703 light brown 80 Nahuizaico red 80 Sanilac white 90 26 Table 3. Percentage internal infection of dry bean (Phaseolus vulgaris L) seeds by fungi Percent infection Genotype Seed coat color (20 seeds) San-Fernando black 1 Brasil 2 brown 15 Jamapa black 15 Collecion 168 N black 20 Ex-Rico-23 white 20 Jalpatagua 72 black 30 Pecho Amarillo black 30 Linea 29 black 30 R 345-LRK OZ pink 35 C 63 S-630-B beige 45 Pl 169 299 red 45 Campbell Soup 109 white 45 Mexico-lZ-l dark brown 50 Sanilac white 75 CHAPTER II SECTION A EFFECT OF DELAYED HARVEST 0N INCIDENCE OF INTERNAL SEED BORNE FUNGI IN DRY BEANS (Phaseolus vulgaris L) Materials and Methods Field Technique Seven genotypes were grown at the Saginaw, Michigan Bean and Beet.Research farm during the summer of 1978. The genotypes were as foloows: Genotype £912; 1. San-Fernando black 2. Ex-Rico-23 white 3. Tuscola white 4. Nep-Z white 5. Seafarer white 6. Turrialba #1 black 7. Black Turtle Soup black Genotypes were used as main plots with three harvest dates as subplots within each genotypes. The harvest dates were: (a) normal crop maturity, (b) two weeks after maturity and, (c) three weeks after maturity. Thus the experiment was set up in a Split-plot design with four replications. The experimental unit consisted of four five 27 28 meter rows per plot with 50 cm between rows. Because dry hot weather prevailed as plants approached physio- logical maturity, the entire planting was sprayed three times at three day intervals with a heavy suspension of fungal spores obtained by washing molded pods obtained from earlier maturing beans. Because of earlier maturity, Seafarer was harvested ten days earlier than the other genotypes. Two meters from each of the two center Seafarer rows were hand-harvested on 8 September 1978 (normal harvest). The second Seafarer harvest and the first harvest of the remaining genotypes were made in a similar manner in 19 September. The third Seafarer harvest and the second harvest of the other genotypes were made on 10 October. The third harvest for six genotypes was made on 17 October. All harvest samples were hand-thrashed and the seed stored in a cold room at 4°C until use. Moisture content of seed was not measured. Laboratory Assay Technique The assay technique for detection of internal seed contamination by fungi was identical to that used in 1977 except that seeds were soaked for one rather than two minutes in the 2.6% NaOCl. One hundred seeds from each treatment were tested for internal fungal contamination. Data Analysis The data for Seafarer harvested earlier than the other six geno- types were analysed as a randomized block design with the three har- vests as treatments and four replications. The other genotypes were analysed as split-plot design. 29 Fungi Isolation and Identification Fungal hyphae growing from infected seeds were transferred into fresh SPDA plates and incubated for about one week. Fungal isolates were then identified to genus with the aide of Barnett and Hunters Guide (9). Results There were highly significant genotypic differences at a 17.0856 F value for incidence of internal seed-borne fungi (Table 4). Delay in harvest past normal maturity did.not significantly affect internal seed infection. However, pod molding increased as harvest was delayed (figs. 4, 6, 8, 10, 12, 14, 16). Genotype x harvest time interaction was highly significant indicating that some genotypes might have had a significant increase in seed infection due to the delayed harvests. Since Analysis of Variance (Table 4) showed no significant effect of harvest date, genotypic means at each harvest date were compared to determine if there was a significant effect within genotype (Table 5). Internal seed infection in San-Fernando was not affected by harvest dates (figs. 5A, B, and C). First harvest means were significantly different from second and third harvest means in the Ex-Rico-23, Nep-2, and Turrialba #1 geno- types; however, differences between the second and the third harvest means were not significant (figs. 9, 11, and 13). Tuscola and Black Turtle Soup showed similar results. Second and third harvest means were not significantly different from each other but differed signifi- cantly from first harvest means. Seed infection in both genotypes increased as harvest was delayed (figs. 7 and 15A, B and C). 30 Table 4. Effect of genotype and delayed harvest on internal bean (Phaseolus vulgaris L) seed infection by fungi Analysis of Variance Source df Sum of squares Mean square F Total 71 9149.523 -- Blocks 3 228.585 76.195 Genotypes 5 4401.089 880.2178 17.0856** Error(a) 15 772.771 51.5180 Harvests 2 121.850 60.925 1.5517 Genotypes X Harvests lO 2211.818 221.1818 5.6335** Error(b) 36 1413.41 39.2613 Note: The data were transformed by the arcsine method. Genotypes used were: San-Fernando Ex-Rico-23 Tuscola Nep-2 Turrialba #1 Black Turtle Soup 31 .mo.o u a an cmgao some soc; acmeoemwc apucmumewemwm no: men mesapou we» cw msmuump p—esm osmm we» can mzog we» cw memuump pouwaeu mama me» An uozop—ou memo: .mcome mezzpou one com memuamp pmem we» use memos so; on» meageou cu vow: mew mcouum_ Feamamu .asom appeae xumpm u mhm ”mount umm>cmz u .m mmwaauocmu u .< "muoz e¢~.e u A.> ~_.m~ x _m.m¢ z mp.mm >~._~ mmaam>< x am.om nu No.9m no mm.¢~ a< mm.o~ nu No.Fm aamom Nm._m a< em.kp 5: Lance mesa: awash x m.m~ no Nw.em page me.e~ nu 8.:~ am mm.me aou< oe.e~ au< mo.P~ 5: emote mama: axe x _m.~m so mm.- mm ._.mm am N..~m am a.~m am m~.o¢ ao< mm.¢~ supaapas Pesto: ome.m "A.mVDmu_ me.m u A.< Cw .mv Q m a. S: u a 5 .5 a m a .m mmmcm>< mpm p* ma_mwcesh N-amz m_oomze mmuouwm-xm oucmccmulcmm .< Vacs» an cowuummcw vmmm am mwcemfla> mapommmcav cums pmccwucw com memos we?» umm>cmg use uwaxuoemm mo comwcmgeoo am apnee 32 SAN FERNANDO Figure 4. Pod molding in San-Fernando bean genotype harvested at normal maturity (1), two weeks after (2), and three weeks after (3) normal maturity. SAN FERNANDO Figure 5A. Internal fungal infection of seed of San—Fernando geno— type harvested at normal maturity. 33 SAN FERNANDO Figure 5B. Internal fungal infection of seed of San-Fernando geno- type harvested three weeks after normal maturity. SAN FERNANDO Figure 5C. Internal fungal infection of seed of San-Fernando geno- type harvested three weeks after normal maturity. 34 Figure 6. Pod molding in Tuscola bean genotype harvested at normal maturity (1), two weeks after (2), and three weeks after (3) normal maturity. TUSCOlA Figure 7A. Internal fungal infection of seed of Tuscola genotype harvested at normal maturity. 35 Figure 78. Internal fungal infection of seed of Tuscola genotype harvested two weeks after normal maturity. TUSCOLA Figure 7C. Internal fungal infection of seed of Tuscola genotype harvested three weeks after normal maturity. 36 Figure 8. Pod molding in Nep-2 bean genotype harvested at normal maturity (1), two weeks after (2), and three weeks after (3) normal maturity. Figure 9A. Internal fungal infection of seed of Nep-2 genotype harvested at normal maturity. 37 Figure 98. Internal fungal infection of seed of Nep-2 genotype harvested two weeks after normal maturity. Figure 9C. Internal fungal infection of seed of Nep-2 genotype harvested three weeks after normal maturity. 38 EX-RICO‘? J Figure 10. Pod molding in Ex-Rico-23 bean genotype harvested at normal maturity (1), two weeks after (2), and three weeks after (3) normal maturity. EXrRICCD-2I3 Figure 11A. Internal fungal infection of seed of Ex-Rico—23 genotype harvested at normal maturity. 39 Figure 118. Internal fungal infection of seed of Ex—Rico-23 genotype harvested two weeks after normal maturity. IUR RIALBA Figure 12. Pod molding in Turrialba #1 bean genotype harvested at normal maturity (1), two weeks after (2), and three weeks after (3) normal maturity. 40 TURRJALBA 1 Figure 13A. Internal fungal infection of seed of Turrialba #1 geno— type harvested at normal maturity. TuRRIALBA Figure 138. Internal fungal infection of seed of Turrialba #1 genOw type harvested two weeks after normal maturity. 41 Figure 14. Pod molding in Black Turtle Soup (BTS) bean genotype harvested at normal maturity (1), two weeks after (2), and three weeks after (3) normal maturity. Figure 15A. Internal fungal infection of seed of Black Turtle Soup genotype harvested at normal maturity. 42 Figure 158. Internal fungal infection of seed of Black Turtle Soup bean genotype harvested two weeks after normal maturity. Figure 15C. Internal fungal infection of seed of Black Turtle Soup genotype harvested three weeks after normal maturity. 43 Figure 16. Pod molding in Seafarer bean genotype harvested at normal maturity (1), two weeks after (2), and three weeks after (3) normal maturity. o‘é‘T V I.’ .f SEAFARER 1 Figure 17A. Internal fungal infection of seed of Seafarer genotype harvested at normal maturity. 44 SEAFARER Figure 17B. Internal fungal infection of seed of Seafarer genotype harvested two weeks after normal maturity. Figure 17C. Internal fungal infection of seed of Seafarer genotype harvested three weeks after normal maturity. 45 Comparison of the six genotypic means at normal maturity showed significant variation (Table 5). San-Fernando and Black Turtle Soup were not significantly different from each other, but differed sig- nificantly from Ex-Rico-23, Tuscola, Nep-2, and Turrialba #1. Comparison of the genotypic means at second harvest showed several significant differences. San-Fernando, Ex-Rico-23, Nap-2, and Turrialba #1 were not found significantly different from each other. Infection in Tuscola was significantly higher than in other five geno- types. Ex-Rico-23, Turrialba #1, and Black Turtle Soup were not sig- nificantly different; however, theinfection in Black Turtle Soup was significantly higher than in San-Fernando and Nep-2. Comparison of genotypic means at third harvest showed that San- Fernando and Nep-2 were not significantly different from each other. They differed significantly from Ex-Rico-23, Tuscola, Turrialba #1, and Black Turtle Soup. Turrialba #1 differed significantly from Black Turtle Soup.and Tusoola. Ex-Rico-23 differed significantly from Tus- cola. Black Turtle Soup and Ex-Rico-23 did not differ significantly from each other. Infection in Tuscola was the highest and differed significantly from Black Turtle Soup which showed the second highest level of infection. Average means comparison of six genotypes at three different hafVest dates did not show any significant effect of harvest date. Sim- ilarly the average means comparison of the three harvest times in six genotypes showed genotypic variation in rate of seed infection. San- Fernando and Nep-2 did not differ significantly from each other and neither did Ex-Rico-23, Turrialba #1 and Black Turtle Soup. The 46 average infection in Tuscola was the highest among six genotypes and was significantly different. Seafarer, a white seeded genotype showed a highly significant effect of delay in harvest (Table 6); the F value was 1769.500. The three harvest means were compared and showed a linear increase in seed infection from 0.25% to 77.75% to 88.25%, at first, second and third harvest dates, respectively (Table 7). Table 6. Effect of delayed harvest on internal fungal infection of seed of 'Seafarer' bean Analysis of Variance Source df Sum of squares Mean square F Total 11 18550.917 -- Blocks 3 38.917 12.972 Harvest dates 2 18480.667 9240.333 l769.500** Error 6 31.333 5.222 Table 7. Comparison of three harvest time means for internal fungal infection of seed of 'Seafarer' Harvest dates Means Normal maturity 0.25 a TWo weeks after normal maturity 77.75 b Three weeks after normal maturity 88.25 c LSD 0.05 3.952 Means followed by the same letter are not significantly differ- ent from each other at P = 0.05 by Duncans multiple range test. 47 Incidence of total fungi isolated from surface-sterilized dry bean seed and occurrence of fungi by genera are presented in Table 8. Fungi isolated most frequently were: Alternaria §p_and Rhizoctonia s3, Fungi isolated less frequently included Fusarium sp, Penicillium s2, Epicoccumggp, Cladosporium s2, Chaetomium 52, Rhizopus s2, and several other unidentified isolates. Alternaria §p_was isolated most frequently from Tuscola seed, followed by Turrialba #1, relatively few Alternaria §p_were isolated from San-Fernando, Nep-Z, Ex-Rico-23, and Black Turtle Soup. Incidence of Rhizoctonia §p_was highest in San-Fernando, and lowest in Tuscola and Turrialba #1. Incidences of internal seed infection by Rhizoctonia §p_in Ex-Rico-23, Nep-Z, and Black Turtle Soup were almost equal. Alternaria s2, and Rhizoctonia sp.showed some genotypic specificity while Fusarium §p_and other fungi did not. San-Fernando: Occurrence of Alternaria sp increased from 61.4 to 63.6 to 75.0% as harvest was delayed from normal maturity to two weeks and to three weeks after normal maturity, respectively. However, in- cidence of total fungi was decreased by delayed harvest. Occurrence of Rhizoctonia sp in this genotype decreased with delay in harvest. Ex-Rico-23: Incidence of Alternaria §p_increased from 64.5 to 71.9 to 72.8% at normal maturity, two weeks after and three weeks after normal maturity, respectively. On the other hand, total incidence and occurrence of Rhizoctonia decreased from 32.5 to 25.8, to 17.5% at the same harvest times. Tuscola: Incidences of Alternaria §p_and Rhizoctonia §p_were very similar at all harvest dates. The highest occurrence of Alterneria and Rhizoctonia in this genotype were 89.4 and 14.2% 48 .— tween mxmo: omega umumm>eeg mummm u m up cmuem mxom: oz» coamm>cmg mummm u N maupcaume nose —useo= an woumm>cmz mummm u pm .Pacse um_emucmuw:= maoocmppoumwz u umpz ”mamoNsz u gem “Espsoummsu n amuse “ancoawoumpu [ u umpu mazouoUPmm u Pam usawpppumcma u can me:_tem=d u mam NewcouuoNpgm u ngm Newcmcemup< u up<~ .mpec umo>ea;\maxuocmm\mumom ooe soc; nmcpeuao mama p N.o o o N.o N.o N.o 4.. o.NN N.Nm m.em m o o o o o c N.N ..PN N.NN N.NN N asom m.ua=e Nua_m o e._ o o o m._ o N.NN m.NN m.m_ _ m._ o o o m.F o o m.oN N.NN o.NF N o o N._ o ¢.N o ¢.N N.N. N._N m.ON N Na aa_a_aase o o o o o e._ o e.m m.am m._m _ o o o o.N o o o.N c.4N o.NN m.N_ N o o N.N o o o o o.PN ¢.NN N.N N N-amz o 4._ o N.o o N.o o ..om _.Ne m.em _ o o o ¢.o N., o N.o N.¢p N.NN N.Nm N o o o o o o N.N o.N ¢.NN N.Nm N apoumsh o m._ o o o o o N.N. o.mN N.NN _ o o o m.o e.N o 4.4 m.N_ N.NN m.NN N N.N o o o o o o N.mN N..N N.NN N NN-oo_N-xm o N._ o o o N._ o m.NN m.¢e N.Na _ N.N o o o o N.N o N.NN o.NN o.m N o o N._ c N.N o N._ m.om «.mo N.NN N oacacaaa-eam a._ o o o o o 4.. N.NN e._e N.NF P .umaz .aem .Amaeo .ua_u .sam .ema .msa .Nwem .u_< Amaze Nausea moaxuocmw Nucmucmq :_ memcma an Nana» Co muconwocm —muoe umm>cmz meaxuocaa «A mpemmps> mapommmsav coma zen xmm we mummm cm memcmm Pmmczm use wacaw pmccmucw pupa» mo.mocmupucm .m mpamh 49 respectively. Neg-2: Incidence of Alternaria §p_increased from 67.1 to 78.4% as harvest was delayed to two weeks after normal maturity and then de- creased to 72.0% at the third harvest date. Rhizoctonia §p_did not show any change in incidence due to harvest time. Cladosporium spp were isolated most frequently in this genotype. Turrialba #1: This genotype exhibited a specific harvest date response to incidence of Alternaria §p_and Rhizoctonia sp, Alternaria §p_decreased from 89.6 to 81.7 to 77.9% as harvest was delayed from normal maturity to two and three weeks after normal maturity, respec- tively. However, Rhizoctonia §p_increased from 5.6 to 12.2 to 20.6% for the same harvest dates. Black Turtle Soup: This genotype was similar to Turrialba #1. Occurrence of Alternaria §p_decreased from 77.5 to 75.2 to 57.24% as harvest was delayed. Rhizoctonia §p_increased from 19.7 to 21.1 to 38.6% due to delay in harvest. SECTION 8 PRELIMINARY OBSERVATIONS ON THE NATURE OF INHERITANCE FOR RESISTANCE TO INTERNAL SEED-BORNE FUNGI IN DRY BEAN (Phaseolus vulgaris L) Materials and Methods Seeds of bean cultivars San-Fernando (black seeded) and Tuscola (white seeded) harvested during the Fall of 1977 were taken from bulk seed sources and planted in a green-house at Michigan State University for making hybrids. San-Fernando and Tuscola were considered as resis- tant and susceptible parents respecitvely on the basis of results ob- tained from the internal fungal seed infection test conducted in 1977. Reciprocal crosses were made in the green-house during the winter of 1978. . Seeds containing F1 germs and of the parents were planted at the Saginaw Michigan Bean and Beet Research Farm during the summer of 1978. Only the parents were replicated, with four rows in each plot. They were sprayed at physiological maturity with heavy suspen- sions of mold spores, as mentioned in Chapter II, Section A. All F15 and two rows of the parents were hand harvested two weeks after normal maturity. Seeds were stored at about 4°C until tested. Two hundred seeds from each of fifteen F1 plants were taken for the internal fungal seed contamination test. Four hundred seeds from 50 51 each parent were used for the fungal infection test. The laboratory technique was the same as mentioned in Chapter I and Chapter II, Sec- tion A. Degree of internal seed infection by fungi was expressed in per- centages for the genetic study. Results Internal seed infection of fifteen F1 plants showed great plant to plant variation (fig. 18), indicating major environmental effects. Infection in seed of the fifteen plants ranged from 7.5 to 57.5%, which is less than the mean percentage infection in the resistant parent but more than the mean percentage infection in the susceptible parent re- spectively (Table 9). Variation in degree of internal seed infection between the F1 plants is thought due to two main factors: (1) Stage of plant development and maturation was not identical for all F1 plants. But all were harvested at the same time irrespec- tive of stage of maturity. Plants which germinated, developed, and matured earlier were exposed to hot wet weather for a relatively longer period after normal maturity than those which did not germinate and mature early. Differences in the stage of maturity and duration of exposure to hot wet weather caused great variation. This conclusion is supported by the trend of internal seed infection in Tuscola and San-Fernando. Delayed harvest after normal maturity decreased seed infection in San-Fernando but infection was increased in Tuscola. (2) Difference in plant architecture may have caused variation in extent of seed infection within the genotype. Percent internal 52 mu”: I. .9. t 2 3.! , .... ' v showing highest (A) and lowest (B) internal fungal in- F2 germs of the cross between San-Fernando and Tuscola fection of seed of two F] plants. Figure 18. 53 Table 9. Percentage internal fungal seed infection of fifteen F1 plants of the cross between 'San-Fernando' and 'Tuscola' bean genotypes . . . Percent infection in parents Percent infection (200 seeds in F1 (400 seeds in each) 57.5 54.0 47.5 36.36 31.5 31.19 29.0 27.5 27.5 26.94 21.13 21.0 14.5 13.5 San-Fernando Tuscola 7.5 Mean 29.77 13.75 56.5 54 seed infection by fungi among dry bean cultivars with pods in contact with soil (41) is always higher than among cultivars with pods not in contact with soil. There are distinct variation in plant vigor among the fifteen F.I plants; some were more vigorous and lodged with many pods in contact with soil, resulting in high percentages of mold in- vasion in seed. Other plants were less vigorous and erect with no pods in contact with soil, resulting in less infection. Further speculation on the fact the fifteen F15 showed such a range of infection can be illustrated as follows: Assume San-Fernando carries dominant genes for resistance to (say) Penicillium and recessive genes for resistance to (say) Rhizopus, then we would expect the F1 to be resistant to Penicillium and suscep- tible to Rhizopus. Now, suppose that in the field the natural distri- bution of Penicillium and Rhizopus was non—random or irregular so that some F.I plants were exposed only to Penicillium spores, in which case those particular plants would show resistance, and some F.l plants would be exposed only to Rhizopus spores, in which case those F‘s would show susceptibility like Tuscola. The mean percentage infection of fifteen F], San-Fernando and Tuscola were 29.8, 13.8, and 56.5% respectively: 13.8 29.8 35.2 , 56.5 1 l is I i 1 I ‘*r San-Fernando F1 mid value Tuscola Very provisionally, it may be suggested that genes affecting seed infection under these conditions display additivity to slightly partial dominance for the resistant response. This is indicated by the fact that mean percentage seed infection in F1 was only slightly 55 displaced from the mid parental value and towards the resistant par- ent. Resistance cannot be said to be specific for there are many fungi which are internally seed-borne. Since the F1 plants had black seed coats and yet showed infection as high as 57%, resistance may be independent of seed coat color. CHAPTER III SECTION A EFFECT OF DELAYED HARVEST ON LABORATORY SEED GERMINATION IN DRY BEAN (Phaseolus vulgaris L) GENOTYPES Materials and Methods Seed samples of the seven genotypes which had been harvested at three different times in the fall of 1978 were stored at about 4°C until April 1979. Germination tests were performed in a germinator maintained at 26 :_1°C. Kimpack, a germination media, was placed over wax paper in a germination tray. Four replications of 100 seeds each for each genotype and each harvest date were tested. Media was lightly watered and 200 seeds were placed on each tray. Seeds were covered with wet paper towelling. Seed germination was counted after 7 days of incubation. Criteria of Germination Counts SEEdITHQS‘Whlch possessed a) strong primary roots and/or secondary roots (fig. 19) sufficiently healthy to support the seedling and b) long hypocotyls with at least half of both cotyledons and with normal plumule were considered to have germinated in a normal manner. Abnormal germination types included a) decayed seeds, and b) seedlings with very short and thickened hypocotyls with no strong primary or 56 57 Figure 19. Normal (A) and abnormal (B) dry bean seedlings in germination test. Abnormal seedlings developed from seed internally infected with fungi. 58 secondary roots, and no plumule. Data Analysis Since results were taken as percent germination, data were trans- formed by the arcsin method. Six genotypes were analysed in split- plot, and Seafarer as a randomized block. Results Genotypes showed significant variation for jg_vitrg_germination. Delay in harvest did not result in any significant decrease in ig_yjtrg_ germination (Table 8). Genotype-harvest interactions were not sig- nificant.. Comparison of germination means at three harvest dates in six genotypes is presented in Table 9. All six genotypic means at first and second harvest did not differ significantly. Germination in Tuscola seed from the third harvest decreased significantly compared to the other five genotypes. Average germination means of six genotypes over three different harvest dates did not show any significant reductions in seed germina- tion. However, average germination means of the three harvest dates over six genotypes showed genotypic variation. Delay in harvest past normal maturity caused significant reduction in laboratory seed ger- mination only in Tuscola. Seed germination in Seafarer for the three harvest dates did not show any significant differences due to delay in harvest (Table 10). However, harvest means comparisons (Table 11) showed significant reduction in germination from first harvest to the third harvest sug- gesting that delay in harvest may cause germination reduction in this 59 genotype. Table 10. Effect of genotype and delayed harvest on laboratory seed germination of dry bean (Phaseolus vulgaris L) seeds Analysis of Variance Source df Sum of squares Mean square F Total 71 3202.6664 -- Blocks 3 156.4657 52.155 Genotypes 5 570.7594 114.152 7.161** Error (a) 15 239.1269 15.942 Harvests 2 119.6421 59.820 1.162NS Genotypes x Harvests 10 264.9426 26.494 0.51NS Error (6) 36 1851.7297 51.437 Data are transformed by the arcsin method. 0 6 “magma umm>cmx u .m “mmaxuocoo u .< .mo.o u a «a ucmcowewu xpucmuvmpemwm no: men mesa—cu on» :P memaamp ppm2m mamm we» use mzos use cw mcmuump poupamo maem use an cozoppoe memo: toe memuump pmem use memos so; on» mgmaeou op cum: use mgmuump Feuwamu .mcmme asapou use mo compemaeou may .aaom mpuczh gumpm n men .vogume wewmoga me» x; custommcmeu men mean. “ouoz a~e.m u om; 3> mm.um 3> m~.mm 3> ¢F.Nm x me.om 3 m~.em > mm.mm x Pm.mm o< m_.Nm c< m~.~m m< -.cw mm Ne.m~ m< mm.om e< o.om x m¢.¢w e< No.~w e< ma.~m m< Pm.~m m< «N.Nw m< Nm.om o< Nm.ww x a~.~m e< o.om m<.~m.mw m< m_.mm w< em.mw m< mp.nw e< o.om pmewu meN.o_ u A.< e? .NV ems va.¢ u om; «we.m u A.m cw . mapommmgav coma New we cowum:_2emm comm aeoumeonmp toe names we?“ umm>ems one uwazuocmm eo compemnsou .pp m—ame 61 Table 12. Effect of delayed harvest on laboratory seed germination of Seafarer bean seed Analysis of Variance Source df SS MS F Total 11 658.144 -- Block 3 131.973 43.99 Harvests 2 264.276 132.138 3.027 NS Error 6 261.895 43.649 Table 13. Comparison of harvest time means in Seafarer bean genotype for laboratory seed germination Means First harvest 90 a Second harvest 84.30 ab Third harvest 78.50 b LSD (0.05) 11.4315 Means with the same letter in common are not significantly different from each other at P = 0.05. SECTION B EFFECT OF DELAYED HARVEST ON FIELD EMERGENCE IN DRY BEAN (Phaseolus vulgaris L) SEED Materials and Methods Seven genotypes as indicated in Chapter II, Section A were har- vested at three different dates in the fall of 1978 and were used for field emergence tests. Seeds were planted at the Crop Science Re- search Farm, East Lansing, Michigan on 14 June 1979. Genotypes were used as main plots with three different harvest dates on sub-plots within each genotype. Thus the experiment was a split plot design with four replications. The experimental unit consisted of two four meter rows with 50 seeds/row. Because hot dry weather prevailed before and after planting, rows were manually watered at five days of planting. No additional watering was necessary thereafter. Number of seeds emerged were counted 20 days after planting. Seafarer was analysed as a random block and remaining genotypes (San-Fernando, Ex-Rico-23, Tuscola, Nep-2, Turrialba #1, and Black Turtle Soup) as a split-plot design. Data were obtained as percentage emergence and transformed by the arcsin method. 62 63 Results There was highly significant genotypic variation for field emergence (Table 14). Delay in harvest did not have any significant effect; genotype x harvest date interactions were also not significant. Table 14. Effect of genotype and delayed harvest on field emergence of dry bean (Phaseolus vulgaris L) seeds1 Analysis of Variance Source df Sum of squares Mean square F Total 71 3402.602 1 -- Blocks 3 279.280 93.09 Genotypes 5 1229.786 245.95 5.58** Error(a) 15 661.217 44.08 Harvest dates 2 31.086 15.54 0.54 NS Genotypes X Harvest 10 179.588 17.95 0.63 NS Error (b) 36 1021.042 28.36 1Data are transformed by the arcsin method. Comparison of genotypic and harvest time means is presented in Table 15. San-Fernando and Turrialba #1 did not differ significantly from each other but showed significantly higher field emergence than Nep-2 for seed harvested at normal maturity.. Ex-Rico-23, Tuscola, Nep-2, and Black Turtle Soup did not differ significantly from each other for field emergence for seed harvested at normal maturity. San-Fernando, Turrialba #1, and Black Turtle Soup were not significantly different from each other but had significantly higher 64 m—ucsp xum_m u mem ”mount amm>emz u .m “mmazaocmw u .< .mo.o u a an emcee comm soc» ucocmwemc appcmu_e_:mwm yo: man mesapou msu cw mewuump __msm as» can mzoe.o;u cw mcouump —euwaeu mEMm me» an umzop—oe memo: .memme :E=_ou as» to compemaeou on» com mgmuum. ppoEN ecu memos so; use memQEou op new: one mgmuump Penance .qaom .uogums :Nmucm mgu xa umsgoemcmeu wee sumo p eNN.m u A. No.mm > mm.mm > mm.mm x Nm.me . oaeee>< x Ne.ce e< Nm.Ne e< N_.ee mum om.mm eN< em.am em N_.mm e< No.ee Neweseee Pesto: Levee mxmmz mots» x PN.Ne ea oe.Ne e< mm.Ne em NN.em ema em.Nm em< ON.mm e< N_.me seeeseee _eeLoe swan; 3.003 03. eN< am.Ne e< _o.me em Nm.mm em< me.oe eN< eN.Ne ea mm.me soeeseee pesto: No..m o_e.N u A.< e. .Nv om; u om; mFN.N u N.N ea .< men pa unpaveeah Nunez m—oumzp mwioowguxm oucmceomieam .< mspommazav :mmn zen eo oucmmewEm upmmw Low memos we?“ umm>cmn use pmummm A; mmgompz> umqauocmm yo commemqeou .mp m_nmh 65 field emergence than Nep-2 for seed harvested two weeks after normal maturity. Ex-Rico-23, Tuscola, and Nep-2 again did not differ signifi- cantly from each other. Comparison of genotypic means for field emergence for seed har- vested three weeks after normal maturity showed no significant differ- ences among Tuscola, Ex-Rico-23, and Nep-2. San-Fernando, Turrialba #1, and Black Turtle Soup had higher emergence than Nep-2, Tuscola and Ex-Rico-23. Average mean comparison of six genotypes over three different harvest dates showed general superiority of black seeded types for field emergence. All three black seeded genotypes (San-Fernando, Turrialba #1, and Black Turtle Soup) had significantly higher field emergence than white seeded types. 1 Mean comparison of three different harvest dates within indi- vidual genotypes did not show any significant effect of delayed har- vest on field emergence. Similarly, average mean comparison of three different harvest dates over six genotypes did not show significant effect of harvest dates for field emergence. Seafarer, a white seeded genotype showed a significant effect of delayed harvest on field emergence at the 5 percent level of signifi- cance (Table 16). Comparison of the three different harvest date means (Table 17) showed no significant difference between the seeds harvested at normal maturity and two weeks after normal maturity for field emergence. However, field emergence decreased significantly at the third harvest suggesting that delay in harvest decreased field emergence in this genotype. 66 Table 16. Effect of delayed harvest on field emergency of Seafarer seed Analysis of variance Source df Sum of squares Mean square F Total 11 374.161 -- Blocks 3 119.744 39.91 Harvest dates 2 168.330 84.165 5.873* Error 6 14.33 1Data are transformed by the arcsin method. Table 17. Comparison of harvest time means in Seafarer genotype for field emergence Harvests Means Normal maturity ’ 61.08 a TWo weeks after normal maturity 63.29 a Three weeks after normal maturity 54.47 b LSD .05 6.550 Means followed by the same letter are not significantly differ- ent from each other. DISCUSSION Pod and seed molding in dry bean (Phaseolus vulgaris L) are not equal in all genotypes, and there is genotypic variation for the ex- tent of molding. Molding is favored by hot humid or wet weather after normal plant maturity. Degree of pod molding in all genotypes in- creased as the plants were exposed to hot wet weather after maturity (figs. 4, 6, 8, 10, 12, 14, and 16). Heavily molded plants and pods are characterized by a brownish-black covering composed of mold growth and spores. I There was no genotypic variation for the extent of external seed contamination by fungi and this is best explained by the fact that ex- ternal seed contamination by fungi takes place during the process of harvesting, threshing, and seed handling (120). Healthy appearing seed may carry 100% external contamination. These results suggest no possibility of obtaining seed free from externally seed-borne fungi when weather favors mold growth. Seeds already contaminated with fungal spores start developing mold when temperature and humidity are high in storage. High correlations between moisture content and dura- tion of storage have been reported (13,18,120). There is considerable genotypic variation for percent incidence of internal seed infection by fungi. Results obtained from a prelim- inary screening test in 1977 indicated that San-Fernando and Sanilac were the most resistant and most susceptible genotypes, respectively, 67 68 for internal seed infection by fungi. Genotypes with pigmented seed coat colors, particularly black were generally more resistant than white seeded genotypes (Tables 2 and 3). Varietal differences for the degree of mold invasion have been reported in bean (Phaseolus vulgaris L) (18), soybean (Glygjgg max) (38,50,104,138,154,162) and pigeon pea (Cajanus cajan) (140): We also found significant differences for internal seed infection by fungi among seven dry bean genotypes. Consistent genotypic variation suggested that genetic factors controlled resistance. Delay in harvest after normal maturity did not affect all geno— types in the same manner. Many researchers (1,40,104,113,133,157) have reported that incidence of internal seed-borne fungi increases as harvest is delayed. Our results differed from these reports; differ- ent dry bean genotypes responded differently. Internal infection in Seafarer, Black Turtle Soup and Tuscola increased as harvest was de- layed beyond normal maturity. However, infection in San-Fernando (black), Nep-2 (white seeded mutant from San-Fernando), and Turrialba #1 (black seeded) decreased as harvest was delayed. These results suggest that seed of all genotypes of dry bean harvested at normal maturity under wet weather are not necessarily high quality in rela- tion to internal fungal seed infection. High temperature and humidity during the period of crop maturity are believed responsible for increased molding. Plants matured under dry weather conditions and harvested at normal maturity produce seed of excellent quality. Results with seed of susceptible Seafarer, har- vested at normal maturity showed essentially no internal fungal in- fection. However, Seafarer seed harvested two and three weeks after 69 normal maturity was heavily infected internally with fungi. There are specific genotype x fungus interactions. Alternaria §p_followed by Rhizoctonia spp dominate other fungi and are the most commonly isolated fungi in dry beans grown at Saginaw Michigan. The two fungi averaged gerater than 70 percent seed incidence. Tuscola and San-Fernando were the most and least congenial hosts for occurrence of Alternaria species, respectively. San-Fernando and Tuscola were the most and least preferred hosts for the occurrence of Rhizoctonia species, respectively. Species of Cladosporium, Fusarium, Epicoccum, Chaetomium, Rhizopus, and Penicillium, were also internally seed-borne in dry bean grown in Michigan. This is the first report of Epicoccum, and Chaetonium spp as internal contaminants in dry bean seed. It is believed that different bean genotypes may synthesize dif- ferent kinds of organic compounds which either inhibit or stimulate the development of a particular fungal species. Resistance of bean plants to fungal seed and seedling pathogens has been reported to be associated with colored seed coat (29,30,87, 109,110,lll,l41). Pigmented scales in onion confer resistance to smudge (Colletotrichum lindemuthianum) (82). Sorghum cultivars with pigmented testa are also reported to have favorable traits such as bird resistance, inhibition of preharvest seed germination, and weather- ing resistance. Such pigmentation is due to the presence of phenolic compounds. The superiority of pigmented genotypes for several favor- able traits is related to phenolic compounds such as tannic and/or shikimic acids (4,11,49,66,87,109,141). Our results show that some white seeded genotypes such as Nep-2, and Ex-Rico-23 are as resistant 70 as the black seeded genotype from which they were developed through mutation. Several black seeded genotypes, particularly Black Turtle Soup, are as susceptible to fungal infection as white seeded lines. Therefore pigmentation is not the sole determinant of resistance in dry beans to internal seed-borne fungi. That some compound(s) may be involved in resistance to internal seed-borne fungi is suggested by the decreasing and increasing trends of infection due to delays in harvest. However, this hypothesis re- mains to be tested. Phenol, a compound which could condition resistance to internal seed-borne fungi is known to be under genetic control (21,52,66,71,86, 116) and its presence in seed is heritable (31,94,95). Deakin gt_al, (29) reported that attempts to obtain white seeded snap bean lines with resistance to Rhizoctonia solani were not successful due to epistatic effects. The authors hypothesized that one homozygous (pp) recessive gene blocks the pathway of synthesis of phenolic ocmpounds such as phaseolin and shikimic acid. However, phaseolin is colorless and one should be able to develop white seeded lines with a level of phaseolin high enough to confer good disease resistance and seedling vigor (28). Yu Ma gt_al, (161) found that white seeded strains of dry beans contained no detectable amounts of tannin and that, when pre- sent in colored seeds, tannin was located in the testae. Although dark colored seeds of Phaseolus vulgaris L contained the highest levels of tannin, the authors found no strong relationship between tannin content and seed coat color. Heritability studies indicated a high broad sense heritability for tannin content. Phenolic compounds, colored or colorless, are generally found 71 in the form of tannic acids, schikimic acids and phaseolin. Genotypes resistant to internal seed infection by fungi perhaps contain high level of phenolic compounds. Since the compound is in seed coat and fungi get in the seed, the mechanism of phenolics for fungal growth inhibition in seed is not well understood. However, it is speculated that fungal mycelia growing in seed produce certain metabolities which hydrolyse the phenolic compounds present in seed coat and the hydro- lysed product diffuses from seed coat into the cotyledon and becomes inhibiting. Smaller seeded genotypes have been reported to contain fewer internally seed-borne fungi than large seeded ones (104). This is explained by the fact that most tannin is located in the seed coat and smaller seed usually have more seed coat area, by weight, than large seed, and therefore may also have a higher tannin concentration. How- ever, correlations between tannin content and seed size were not sig- nificant (161) suggesting that tannin content is independent of seed size. In the present study, San-Fernando and Nep-2 were relatively small seeded compared to other genotypes and showed less internal in- fection.by fungi. Nevertheless, large seeded Turrialba #1 showed less infection than the smaller seeded Tuscola and Seafarer. Results from the preliminary genetic study suggests the possibil- ity of breeding for resistance to internal seed infection by fungi. If resistance is caused by one or more phenolics in the seed-coat or cotyledons and resistance is concentration dependent, then the F1 which shows approximately mid-parental response does so because it is producing phenolics at about half the rate or level of the resistant parent. 72 It would also be necessary to assume that, since San-Fernando shows resistance to several fungi at the same time, its level of phenolics and the particular array or classes of phenolics produced confer generalized resistance. Many pairs of genes for resistance are postulated, so it should be possible to develop white seeded dry bean lines with sufficient genes to confer acceptable resistance. This conclusion is supported by the results obtained with the two isogenic lines San—Fernando (black) and Nep-2 (white) which showed equal levels of internal seed infection. Bean genotypes with hard, thick, and intact seed coats or with thin soft seed coat were reported to exhibit resistance or suscepti- bility to fungal infection respectively (78). This is not the case with Nep-2 (thin soft seed coats) and Black Turtle Soup (thick, in- tact, seed coats). Nep-2 had less internal infection than Black Turtle Soup. Resistance therefore is not totally determined by the seed coat but by the seed itself. Internal fungal infection in seeds harvested from F1 plants further supports this conclusion; all of the seeds (F2 germs) possessed thick seed coats, were black, and showed internal infections as high as 57 percent. If a genetically controlled, colorless, phenolic compound such as tannin does control resistance, the commercial value of dry bean types containing this compound might be reduced. This is because tannins alter the nutritional quality of plant products (125), have a negative correlation with digestion coefficient for crude pro- tein (81), and eventually reduce weight gains in poultry and other animals (20,60,65,72,80,118,119). Results obtained in laboratory seed germination and field 73 emergence tests showed genotypic variation. Harvest delay did not affect field emergence. However, in genotypes like Seafarer and Tus- cola, delay in harvest reduced 13 11359 seed germination. The con- flicting results between ig_yitgg_germination and field emergence can best be explained by the fact that temperature and humidity are rigidly controlled in the germinator (ig_gjtrg_germination). Such conditions favor the germination and development of fungi before seed germination; eventually seed is decayed by fungal growth. In the field, temperature and moisture (humidity) conditions fluctuate considerably and could have been inhibitory to fungi. Only the Tuscola and Seafarer genotypes showed significant nega- tive correlations between incidence of internal seed-borne fungi and ig_!itrg_seed germination due to delayed harvest; this agrees with other reports (24,29,34,39,43,44,46,123) for dry bean and (40,104,157) for soybeans. I1 3.1132 seed germination in several genotypes was not affected by delayed harvest. All of the fungi isolated in the present study appeared to be saprophytes, with no observable negative effect on seed germination. None of the fungi which were reported to reduce jg.yjtrg_soybean'seed germination (3) were isolated from bean seed grown in Michigan. Instead, Alternaria and Fusarium spp which were isolated in this study, were reported to be accompanied by increases in percentage seed germination and decrease in field emergence (40, 157). Our findings agree with others (28,68) in that black seeded geno- etypes have general superiority to white seeded types for field emer- gence. The superiority can be explained as follows: (a) Black seeded genotypes can adapt much better to adverse 74 environmental conditions than white seeded cultivars, and consistently possess an advantage over white when planted in cold, wet, or warm soil (28). (b) Black seeds have greater seed coat dry weight and thickness than white seeds, and these traits are negatively correlated with per- meability and rate of osmosis (160). Osmosis through black seed coats may be slowed by a physical barrier of greater cell numbers, by differences in cell density or by some chemical reaction (i.e., phen- olic oxidation) unique to colored seeds. Slower absorption of water by colored seeds may permit more uniform swelling of the cotyledons, thereby reducing seed coat and/or cotyledon cracking which are import- ant to germination and early seedling growth (62,63,107,160). Differ- ences in field emergence of two isogenic lines differing only in seed coat color support the importance of seed coat color. (c) Superiority of black seeded genotypes may be due to resis- tance to Rhizoctonia root rot (which may have) contributed to differ- ences in emergence. Other lines, however, have reported resistance to this organism and the superior performance of their colored sub- lines must be attributed to other physiological factors (28). This conclusion is also supported by the difference in field emergence of San-Fernando and Nep-2 both of which were almost equally resistant to Rhizoctonia on the basis of internal seed infection tests. Seafarer, a genotype with high internal seed infection showed a negative corrleation between delayed harvest and field emergence. This negative effect in some genotypes could be related to the ability of roots of a particular genotype to exude ogranic compounds. Exuda- tion of organic compounds like amino-acids, sugars, and protein from 75 plant roots and germinating seeds could supply the energy required for a seedling parasite to grow (77,105,109,128,130,155). Seeds of pea varieties most susceptible to damping-off exuded greater amounts of amino-acids and sugars during germination (54,129) than resistant varieties. Bean genotypes with colored seeds produced stronger and more vigorous seedlings than those with white seeds. This effect probably involves phenolic metabolism of the seedlings. The importance of phenolic compounds in resistance of bean plants to seedling diseases has been reported to be manifested during the early stages of plant development (141). This supports the statement made by Deakin (28) "yield advantages conferred by color seed are largely effective at the early stages of growth and are probably related to superior emergence and seedling vigor." SUMMARY AND CONCLUSIONS A two year series of experiments were conducted in the labor- atory and green house, Michigan State University, and in the field at the Saginaw, Michigan Bean and Beet Research Farm, and at the Crop and Soil Science Research Farm, East Lansing, Michigan. Objectives: (a) to study the effect of genotype and harvest date on inci- dence of external and internal seed-borne fungi in dry bean (Phaseolus vulgaris L) as affected by delayed harvest; (b) to determine specific fungus X genotype interactions; (c) to study the effect of genotype and delayed harvest on jg_> yjtrg_seed germination and field emergence; (d) to study in the preliminary way the genetics of resistance to internal seed-borne fungi. Summary of Results: 1. Degree of pod molding increased as time of harvest was de- layed after normal maturity. 2. No genotypic variation was observed for the degree of sur- face infestation of seed by fungi. 3. There is genotypic variation for seed infection by intern- ally seed-borne fungi. San-Fernando and Seafarer were the most and least resistant of the several dry bean genotypes tested, respectively. 76 77 4. Delayed harvest after normal maturity does not affect all bean genotypes in the same way. There is increase in internal seed infection in susceptible genotypes such as Seafarer and Tuscola due to increasing harvest delays. Internal seed infection decreased in resistant genotypes like San-Fernando harvested after normal maturity. 5. Eight fungi were isolated from surface-sterilized bean seed such as: species of Alternaria, Rhizoctonia, Fusarium, Penicillium, Cladosporium, Epicoccum, Chaetomium, and Rhizopus. Alternaria and then Rhizoctonia spp were the most frequently isolated fungi. 6. Two specific fungus x genotype interactions were found. Alternaria was isolated most frequently from Tuscola and least fre- quently from San-Fernando; the reverse was true for RhizoCtonia. 7. Genes affecting seed infection under wet weather conditions display addivity to slightly partial dominance for the resistant re- sponse. 8. Genes for resistance are independent of seed coat color. San-Fernando and Nep-2 isogenic except for seed color, showed no sig- nificant difference in internal seed infection. F1 seeds (F2 germs) of San-Fernando and Tuscola, although black in color, showed higher infection than the resistant parent (San-Fernando). 9. Turrialba #1, a genotype of larger seed size than Seafarer, showed less internal seed infection than Seafarer; seed size may not always be strongly associated with resistance. 10. To obtain high quality seed of susceptible genotypes, seed should be harvested as soon as practical after normal maturity. 11. Black seeded genotypes exhibit better field emergence than white seeded genotypes. Incidence of internal seed-borne fungi and 78 field emergence were not correlated in all genotypes. Genotypes which exhibited high jg_yjtrg_seed germination showed very poor field emergence. This discrepancy has to do with intact seed coat and seed coat thickness; genotypes with thick seed coats have higher field emergence. In_yitrg_seed germination may not be a good method to predict field emergence. 12. There is significant genotypic variation for in_!itgg_seed germination and field emergence. Delay in harvest after normal matur- ity does not affect ig_yjtrg seed germination and field emergence the same in all genotypes. Delayed harvest had a negative effect on 1g vitro seed germination in susceptible genotypes. LITERATURE CITED 10. 11. LITERATURE CITED Alexander, L. 0., P. Decker, and K. Hinson. .1979. 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