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" "iii-r. .19- .‘ WV,- . ”In“. by?" ,. 1'11." .. .. , 75*; .- .4 ~ "w «r: .r .. - "l' . , ' N " 1v "ma FEM :r w' '. ,- J...q_-w»1 v17“ n - rear“ :Wa‘h‘ijb; — 7”.» ...,I w ' l a I . .4 >- .7.'~'::->‘¢‘:$J;U$' . $5,," I 0:3,.» grim}: .515...» pme-F’ ' 2"‘W.~‘tnm :1 .,. '0“ “mar 121‘.“ ”W ‘E “\Efi“, ’9- 3 ‘ d'lu-Tizmamu ~ (#14019th (; WEN" LIBRARY Michigan State University This is to certify that the thesis entitled THE GENETIC RELATIONSHIP BETWEEN THE REACTION OF DRY BEAN GENOTYPES (PHASEOLUS VULGARIS L.) TO BEAN COMMON MOSAIC VIRUS AND BEAN YELLOW MOSAIC VIRUS presented by MARTIN NYOLOLANI MBEWE has been accepted towards fulfillment of the requirements for Master's CrOp and Soil Sciences degree in Major professor / / May 18, 1989 [)ate 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES retun on or More due out. DATE DUE DATE DUE DATE DUE MAY 2 4 2003 I l—Tl | J l l__= l I % =11 MSU Ie An Affirmative Adlai/Equal Opportunity Institution THE GENETIC RELATIONSHIP BETWEEN THE REACTION OF DRY BEAN GENOTYPES (PHASEOLUS VULGARIS L.) TO BEAN COMMON MOSAIC VIRUS AND BEAN YELLOW MOSAIC VIRUS BY MARTIN NYOLOLANI MBEWE 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 Science 1989 ABSTRACT GENETIC RELATIONSHIP BETWEEN THE REACTION OF DRY BEAN GENOTYPES (PHASEOLUS VULGARIS L.) TO BEAN COMMON MOSAIC VIRUS AND BEAN YELLOW MOSAIC VIRUS BY Martin Nyololani Mbewe Bean common mosaic virus (BCMV) and bean yellow mosaic virus (BYMV) are both highly variable plant pathogens which have eluded all other methods of control except through genetic resistance. Crosses were made between elite breeding lines 885009, C-20 and N84004. F1 progenies, F2 individuals and F3 families were evaluated for reaction to the necrosis inducing NL3 strain of BCMV in the detached-leaf technique (necrosis I test) and the ‘C isolate of BYMV on growing plants. A single recessive gene resistance was shown in both cases. An analysis for independent assortment for the 9:3:3:l ratio using the chi-square test detected a coupling phase linkage in 885009 between the recessive bc-3 gene controlling resistance to BCMV and another recessive gene conferring resistance to the ‘C’ isolate of BYMV. A distinction was made between the newly identified gene and previously reported recessive genes conferring resistance to other strains of BYMV. To My Younger Brother, Benson, and Mr. B. Nkhuwa for taking good care of my son and daughter while my wife and I were away studying. ii ACKNOWLEDGEMENTS I am deeply greatful to my major professor, Dr. James D. Kelly, who gave me unlimited support and encouragement throughout my studies. Even in my worst moments, when I felt hopeless, he was always there to give me his best suggestions and ideas. His advice and constructive critism of my work provided me a healthy atmosphere to learn. He shared with me some of his vast experiences in research strategies and I am deeply honored to have studied under him. I would also like to thank Dr. Alfred W. Saettler who did not only serve on my guidance committee but also gave me free access to his laboratory from where I did all my work. His advice in helping me deal with the pathological aspects of my work is greatly appreciated. I am also thankful for his extensive review of this manuscript. Dr. M. Wayne Adams served on my committee and offered critical review of this manuscript. His suggestions are greatly appreciated. I am thankful to Dr. Charles Cress who also served on my guidance committee and provided valuable reviews to this manuscript. iii Special thanks to my wonderful family, wife and children, for their love and moral support throughout my studies. Finally, I would like to thank the Zambia Agriculture Research and Extension (ZAMARE) project for providing financial support for the first two years of my studies. I also thank the Food and Agriculture Organisation of the United Nations for having supported me during the last six months of my studies. iv TABLE OF CONTENTS TITLE PAGE LIST OF TABLES . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . viii I. INTRODUCTION . . . . . . . . . . . . . 1 II. LITERATURE REVIEW . . . . . . . . . . . 8 III. ‘MATERIALS AND METHODS . . . . . . . . . 31 IV. RESULTS . . . . . . . . . . . . . . 44 1. Confirmation Tests . . . . . . . 44 2. Development of the Necrosis Test for BCMV . . 47 3. Comparative Studies Between Genotypes Carrying the bc-3 Recessive Gene and Those Known to Carry Other Resistance Factors to BYMV . . . 50 4. Reaction of Parental and Segregating Individuals to BCMV (NL3 Strain) and BYMV (‘C’isolate) . . . . . . . . . . 50 5. Association Between Genetic Factors Controllihg Immunity to the NL3 Strain of BCMV and Resistance to the ‘C’ Isolate of BYMV . . . 58 6. Test for Independence Between Genetic Resistance Factors to BCMV (NL3) and BYMV (‘C') . . . 62 7. Genetic Distance Between the Two Linked Genes . 63 V. DISCUSSION . . . . . . . . . . 69 VI. BREEDING IMPLICATIONS . . . . . . . 79 VII. 85 LITERATURE CITED . . . . . . . . . LIST OF TABLES TABLE PAGE 1. Essential Amino Acid Content of Bean Seed Protein (9 amino acid 16g N-l) . . . . . . 2 2 BCMV Strains Reported in the Literature. . . 13 3. Reaction to Specific Strains of BCMV of Recessive Gene Differentials . . . . . 17 4. Observed and Theoretical Reactions to Specific Strains of BCMV of Dominant I Gene Differentials in Combination with Strain-specific Resistance Genes . . . . . . . . . . 18 5. The Pedigrees of the Three Parental Lines used in the Study. . . . . . . . . 34 6. Reaction of the Forty nine Breeding Lines to the NL3 Strain of BCMV based on the Necrosis Test and the ‘C’ Isolate of BYMV Inoculated on the Growing Plants 0 O ‘ O O O O O O O I 45 7. Plant Systemic Infection by the NL3 Strain of BCMV in Relation to Time Between Inoculation and Leaf Detaching in the Necrosis Test . . . . 48 8. Reaction to the ‘C' Isolate of BYMV on Three Check Cultivars and Three Cultivars with known Recessive Gene Resistance to Other BYMV Strains . . . 51 9. Inheritance of Resistance to the NL3 Strain of BCMV Resulting from the Cross of 385009 X C-20 . 54 10. Inheritance of Resistance to the ‘C’ Isolate of BYMV Resulting from the Cross of B85009 X C-20 . 55 11. Inheritance of Resistance to the NL3 Strain of BCMV Resulting from the Cross of B85009 X N84004 O O O O O O I O O O O 56 12. Inheritance of Resistance to the ‘C’ Isolate of BYMV Resulting from the Cross of B85009 X N84004. 57 13. Association Between the Genetic Factors Controlling Immunity to the NL3 Strain of BCMV and Resistance to the ‘C' Isolate of BYMV. . vi .61 14. 15. Reaction to the NL3 Strain of BCMV and the ‘C' Isolate of BYMV on the F Families from F2 Selections Resulting from the Cross of B85009 x C-20. . . . . . . . . . . . 65 Reaction of the NL3 Strain of BCMV and the ‘C’ Isolate of BYMV on the F Families from F Selections Resulting frog the Cross of B8S009 X N84004 . . . . . . . . . . .66 vii LIST OF FIGURES FIGURE PAGE 1. Systemic Mosaic Symptoms Induced on Cultivar Sanilac (genotype = iibc-Z bc-Z) by the NL3 Strain of BCMV. . . . . . . . . lO 2. Systemic Necrosis Induced on N84004 (Genotype = II) by the NL3 Strain of BCMV . . . . . . . ll 3. Systemic Mosaic Symptoms Induced on Bean Breeding Line N84004 (genotype = II) by the ‘C’ Isolate of BYMV . . . . . . . . . . . . . 33 4. Pin-Point Local Lesions and Vein Necrosis Induced on the Detached Leaf of N84004 (genotype = II) by the NL3 Strain of BCMV . . . . . . 37 5. Immune Response Induced on Breeding Line 885009 (genotype = II bc-3 bc-3) by the NL3 strain of BCMV. 38 viii I . INTRODUCTION Beans (Phaseolus vulgaris L.) belong to the plant family Leguminosae and subfamily Phaseoleae. Leguminosae is exceeded only by family Graminae in being the most utilized plant family by humans and their domestic animals. Legumes are used for their chemicals, esthetic value, timber, as a ~cooking fuel, brouwse trees and shrubs, forage crops, pasture crops, cover crops, green manures, and for feed and food. Beans are utilized for food in the form of unripe pods, immature seed or mature dry seed directly or indirectly and represent the single most important pulse crop in the developing world. They provide variety to the human diet and supply dietary protein for many populations lacking sufficient animal or fish protein. Since they are rich in lysine and, to a lesser extent, tryptophan, but poor in methionine content, they complement the reverse amino acid pattern found in cereals (l, 35). Data on mean amino acid values for a standard variety in each of several classes of commercial classes of dry beans are shown in Table 1. OBeans may be boiled, baked, canned, refried, used in soups either alone or with other vegetables, meats and chili pepper, as a fresh salad, or curried. In many African Table 1. Essential amino acid contentlof bean seed protein (3 amino acid 163 N ) (1). commercial class of bean FAO* provisional + Red Tropical Small Amino acid standard Navy Kidney Black Red Arginine - 5.1 5.1 6.7 7.2 Histidine - 2.0 2.6 2.9 3.2 Isoleucine 6.4 4.3 4.2 4.1 3.8 Leucine 11.2 7.5 8.1 7.7 7.7 Lysine 8.6 5.7 6.7 6.7 7.2 Methionine - 1.3 0.9 2.0 1.3 Methionine plus cystine 5.6 2.0 1.9 3.0 2.2 Phenylalanine 9.8 5.0 5.3 5.1 5.4 Threonine 6.4 5.1 4.2 3.1 3.5 TryptOphan 1.6 1.8 1.5 1.5 1.7 Valine 8.0 4.7 5.1 4.9 5.0 *Food and Agriculture Organization of the United Nations pattern of amino acid requirement (1973), FAD/WHO Technical Bulleting Series No. 522. _1 Navy beans are white-seeded types with small (15-22 g 100 seeds), round to oval seeds. - - No known standard. 3 countries immature leaves are harvested as a spinach or sun dried and used as a base for soups during the dry season. Immature pods are used either as snap beans or as a source of green shelled beans (1). Common beans are extensively grown in eastern Africa, north and central America, south America, eastern Asia, and western and south-eastern Europe. They are cultivated either in mixed cropping with maize, cassava, sorghum, amaranth, potato or other tropical subsistence crops under rainfed conditions, or in monoculture sometimes under irrigation. In North America, Europe and in limited areas of other producing regions, beans are extensively I commercialized, utilizing inputs from mechanization, fertilizer and pesticides. In developing countries most dry bean producers cultivate unimproved traditional varieties with different seed types and seed colors in complex multiple-cropping systems making it difficult to use mechanization (l). The 1978 FAO Production Yearbook lists. beans as the most important edible legume with approximately 28% of the total world production of edible legumes (41). Beans are exposed to a large array of yield constraints during their growth cycle, often leading to less than optimal and erratic yields. These constraints include weather conditions, poor soil fertility, diseases and insect pests. Diseases and insects are responsible for serious losses in yield and quality. Among the most important bean disease problems are the virus pathogens such as bean common 4 mosaic virus (BCMV) and bean yellow mosaic virus (BYMV). Bean golden mosaic virus is also an important bean problem but has only been reported to be a major problem in tropical areas, especially in Central America (59). BCMV and BYMV are potyviruses and have been widely regarded as the most ubiquitous and generally destructive virus-induced diseases of dry beans (30). BCMV has been reported from every part of the world where beans are grown and is considered a major disease throughout Africa, Europe, North America and Latin America (18, 59). It is a seed-borne disease and is readily transmitted by aphids from infected susceptible cultivars (27, 33, 69). It causes leaf-malformations, leaf and pod necrosis or local lesions depending on the genotype of the infected plants. In severely infected fields, yields may be reduced by 35-98% (12, 30, 71). BYMV has also been reported in nearly all areas of the world associated with bean production. Unlike BCMV, BYMV is not seed-borne but is transmitted by aphids to beans from stands of perennial forage legumes which are the principal reservoirs of the virus (27, 33, 71). It causes leaf malformations; also systemic necrosis or localized necrotic lesions may be induced by other strains of the virus on infected plants. Attempts to control these viruses have included cultural practices such as altered planting dates, elimination of alternate hosts in areas where BYMV is a 5 major problem, and clean seed production in the case of BCMV to reduce the incidence of infection in susceptible plants (11, 59). Insect control by insecticides has also been attempted to reduce the transmission of the viruses from infected to healthy plants (27, 51, 58, 59). However, the most reliable method of control appears to be the use of plant resistance (19, 70). Due to the greater diversity of the BYMV pathogen, genetic control is more difficult since it is dependent upon the identification of suitable resistance factors. Two types of resistance, known as "recessive" and "dominant" resistance, respectively, have been identified and used in the breeding of resistant cultivars. In the case of BCMV, Drijfhout (18) identified six recessive genes governing resistance: five are strain specific and one is strain non-specific. In the U.S.A., the dominant inhibitor or "I gene" has been the most commonly used form of resistance in breeding programs. Cultivars carrying the I gene exhibit the hypersensitive form of resistance (black root or top necrosis) only when infected with necrotic strains of the virus. This form of resistance results in the death of tissue and thus prevents spread of the virus to neighboring healthy plants. The same I gene lines when infected with non necrosis inducing strains of BCMV, exhibit normal resistant reaction without necrosis or systemic mosaic systems. Bean breeders in regions where necrotic strains of BCMV frequently occur have not relied as heavily 6 on the I gene but have utilized the specific recessive genes to a greater extent (19). As in the control of BCMV, different recessive and dominant resistance genes have also been useful in the control of BYMV. The genetics of resistance to specific strains of BCMV and BYMV has until recently been studied independently. However, recent greenhouse observations at Michigan State University (unpublished), indicated that bean germplasm lines carrying the dominant I gene and the recessive bc-3 gene (I bc-3 gene combination) were not only resistant to the necrotic strains of BCMV but were also resistant to several strains of BYMV at moderate temperatures (IS-20°C). Reports of a single gene conferring resistance to more than one disease include those of Cook (15), Schroeder and Provvidenti (57) and Kyle et a1. (38). The following steps were undertaken to investigate the possibility of a relationship between different genetic factors controlling resistance to BCMV and BYMV in dry beans (Phaseolus vulgaris L.): a) Screen with BYMV MSU's navy and black bean breeding lines known to carry dominant I resistance gene and bc- 3 recessive gene combination. b) Screen the F1 and back-cross progenies, F2 and F3 individuals from crosses between dominant I gene parents and a parent carrying both the dominant I gene and the recessive bc-3 gene for reactions to both BCMV (NL3 strain) and BYMV ‘C' isolate). 7 c) Determine the genetic relationship, if any, between resistance to BCMV (NL3 strain) and BYMV (‘C’ isolate). d) Compare other known sources of resistance to BYMV with the resistance factor(s) associated with the bc-3 gene. II. LITERATURE REVIEW 1. Bean Common Mosaic Virus (BCMV) BCMV was one of the first virus diseases reported in the world when it was observed in the Soviet Union by Iwanowski in 1899 (18). Other names used as synonyms to designate the virus have been: common bean mosaic, bean mosaic virus, bean virus 1, Phaseolus virus 1 (18). 1.1. Transmission and Epidemiglogy BCMV particles may be transmitted mechanically, in pollen grains, ovules, flowers and seed from infected plants. Seed transmission is irregular, depending on stage of growth at the time of infection, cultivar and virus strain. If infection occurs after flowering,the virus does not usually reach the seed (46). In nature, infection seems confined to Phaseolus vulgaris but the following plant species have also been found susceptible to BCMV in artificial inoculations: Phaseolus acutifolius Gray var latifolius Freeman, g; angularis, P; calcaratus Roxb, P; mpnqo, P; coccineus, P; radiates, g; aureus, P. lunatus, Vigna sesquipedalis, V. unguiculata, Vicia faba, Lupinas alba, Nicotiana Clevelandii, Pisum sativum, Medicagg sativa, polichos 9 lablab, Trifolium subterrageum, Chgpgppdium guinqa, Gomphrena globosa and Tetraggnia expanse (58). 1.2 Svmptomgtplogy BCMV may incite three types of symptoms on the host depending upon the genotype of the cultivar, time of infection, strain of the virus and environmental conditions. Mosaic symptoms may appear in systemically infected genotypes not possessing the dominant type of resistance derived from cultivar Corbett Refugee and may cause a mottling, curling, stunting and malformation of primary leaves (Fig. l). Stunting is usually evident and plants infected early are more likely to be stunted, yielding less than plants infected at a later stage. Maturity is delayed; sometimes infected plants remain green until frost. Systemic top necrosis or black root symptoms may appear in cultivars possessing the dominant type of resistance (hypersensitive 1 gene) when infected with necrosis-inducing strains (Fig. 2). Symptoms initially appear as leaf lesions or in the plant apex and young trifoliolates which wilt, become dull-green and then black. Eventually the entire plant or a portion of the plant wilts and dies. A characteristic necrosis (reddish-brown to. black) of the vascular system may be evident in leaves, stems, roots and pods (18, 71). Grogan and Walker (25) 10 Fig.1. Systemic mosaic symptoms induced on cultivar Sanilac (genotype = ii bc-2bc-2) by the NL3 strain of BCMV. 11 Fig.2. Systemic necrosis induced on N84004 (genotype = II) by the NL3 strain of BCMV. 12 reported the absence of systemic mosaic in plants with dominant resistance after inoculation with necrosis- inducing strains of BCMV. The virus may also incite local or brownish "pin-point" restricted local lesions above the leaf veins but systemic necrosis never develops. These symptoms are expressed in genotypes carrying the dominant I gene and the recessive bc-22 gene from cultivar Great Northern 31. 1.3 BCMV Strains The majority of BCMV strains reported in the literature are shown in Table 2. Richards and Burkholder (50) reported the type and New York-15 (NY-15) strains of the virus in 1943. The identification of the type strain preceeded that of NY-lS. Navy bean cultivars Michelite and Robust were found to be resistant to the type strain but resistance of these two cultivars was later broken down by a new strain (NY-15) collected from Batavia in New York State. In 1954, Dean and Wilson (16) reported an Idaho or 8 strain which infected cultivars Great Northern-123 (GN-123) and GN-3l. In 1961, Skotland and Burke (61) described a virus in the western United States that attacked cultivar GN-123 but was non-pathogenic on cultivars Michelite, Sanilac, Pinto UI-lll, Red Mexican 34 (RM 34) and GN-31. In 1969, 13 Table 2. BCMV Strains reported in the Literature*. Year Strain name Described Reference Type 1943 Richards and Burkholder (50) New York-15 1943 Richardsand Burkholder (50) Idaho or B 1959 Dean and Wilson (16) Western 1961 Skotlandand Burke (61) Florida 1963 Zaumeyer and Goth (68) Mexican 1969 Silbernagel (60) Imuna (NL-2) 1963 Hubbeling (42) Michelite (NL-3) 1963 Hubbeling (42) Great Northern (NL-4) 1963 Hubbeling (42) Jolanda (NL-S) 1972 Hubbeling (34) Colana (NL-6) 1972 Hubbeling (34) NL-7 1977 Drijfhout and 803 (19) NL-8 1977 Drijfhout and 803 (19) * From Morales (42). 14 Silbernagel (60) designated this strain as the Western strain (WBCMV). In 1964, Zaumeyer and Goth (69) reported the Florida strain which caused more severe symptoms on susceptible cultivars than did the Type, NY-15, or Idaho strains. Cultivar Stringless Green Refugee was susceptible to Florida strain but cultivars Pinto UI-lll, Michelite, Sanilac, RM-34, GN-123 and GN-3l were resistant. Silbernagel (60) isolated the Mexican strain from a Mexican bean line (PI 197690 S) in 1969. This strain differed from those previously reported in that it was seed-transmitted through cultivar Red Mexican-35 and by its inability to infect cultivar Improved Tendergreen. This strain also induced top necrosis in plants of cultivar Topcrop when subjected to temperatures of 32°C for three days. In the Netherlands, strains with wider host ranges have been isolated that induce systemic necrosis at moderate temperatures (18°C - 20°C). They comprise the group called the necrotic inducing strains. In 1963, Hubbeling (42) described the Imuna (NL-2), Michelite (NL-3) and Great Northern strains (NL-4) isolated from cultivars Imuna, Michelite and GN-123, respectively. He compared these strains on cultivars Dubbele Witte, Imuna, Michelite, GN-123, and Widusa. All three strains attacked cultivar Dubbele Witte. The Imuna and Michelite strains each produced symptoms both in Imuna and Michelite. These two 15 strains did not affect GN-123, which was susceptible only to the Great Northern strain. Michelite strain differed from Imuna in giving local and systemic necrosis at 20°C in Widusa and other cultivars with dominant resistance; thus this strain produced systemic necrosis at the moderate temperature. In addition, Hubbeling (34) isolated two strains in 1972 from pods of Jolanda and Colana cultivars that were known to carry dominant resistance. The Jolanda (NL-S) strain, like the Michelite (NL-3) strain, produced local and systemic necrosis at 20°C in cultivars with dominant resistance. It differed from the Michelite strain in inducing rapid systemic necrosis at 20°C in cultivar Jubila (a new added differential), in which Michelite strain induced only local vein necrosis. Colana (NL-6) strain differed from Great Northern strain in its inability to infect GN-31 and its ability to induce systemic necrosis in Jubila, and from Jolanda and Michelite strains in not attacking cultivars Michelite and Sanilac. Finally, two strains, coded NL-7 and NL-8, were isolated by Drijfhout and 805 (19) in 1977. Strain NL-7 did not attack I gene differentials Jubila, Topcrop, Improved Tendergreen 40031, Widusa, Black Turtle Soup, and Amanda. It produced typical mosaic symptoms in the differentials Dubbele Witte and Stringless Green Refugee, both susceptible to all strains. Strain NL-8, in addition to infecting cultivars Dubbele Witte and Stringless Green 16 Refugee, also infected cultivars Sanilac, Michelite and RM- 34. It induced local necrosis at 20°C in all differential cultivars with dominant I gene, but systemic necrosis only in Widusa and Black Turtle Soup. 1.4. Differentiation of BCMV Strains Drijfhout et a1. (20) developed a group of differential cultivars which were used to distinguish and classify BCMV strains. The cultivars were classified into eleven host groups based on the reaction induced by different virus strains (Tables 3 and 4). The virus strains were regrouped into seven pathogenicity groups and subgroups based on the variation in symptoms produced on the differential cultivars. Drijfhout also renamed the strains on the NL (Necrotic Lesion) basis. 1.5. Genetics of Resistance to BCMV Drijfhout (18) presented a comprehensive background review of the genetics of resistance to BCMV in beans. Until then, inheritance of resistance to BCMV had been studied using one strain at a time while his studies involved all different strains. 17 Table 3 . Reaction to specific strains of BCMV of recessive I gene differentials . Virus strains Resistance Recessive US2NL5 USS Differentials group ggnes NLl NLZ NL3 NL4 NL6 NL7 NL8 Dubbele witte 1 * + + + + + + + Immune 2 bc-l - + + + + + - RG-B 3 bc-l 2 - - + + + - - Michelite 4 bc-Z - + + - - - + Pinto 114 5 bc-lbc-Z - + + - - - _ an 31 6 bc-lzbc-ZZ - - - + - - - IVT 7214 7 bc-Zbc-3 - - - - - - - # From Drijfhout (18). * - No recessive genes present. + - Susceptible. - - Resistant. RG-B - Redlands Greenleaf B. GN 31 - Great Northern 31. 18 Table 4. Observed and expected reactions to specific strains of BCMV of dominant I gene diffeientials in combination with strain- specific resistance genes . Virus strains II Resistance Recessive USZ USS NL3 Differentials ggoup genes NLl NL2 NL3 NLS NL6 NLB Widusa 8 * - - + + + + Jubila, 9a bc-l - + + - + - Topcrop 9b bc-l - + + - + - Amanda 10 bc-i2 - — - + _ _ IVT 7233 11 bc-lzbc-ZZ - - - - _ - # From Drijfhout (18). * ' No recessive gene combination. - - Resistant. + - Top necrosis. 19 Based on NL grouping, he performed diallel crosses among the groups of differential cultivars carrying the recessive allele of the I gene. The F1 and F2 generations were tested with strains from all pathogenicity groups. After studying the segregation ratios he proposed the use of recessive genes identified in specific hosts: bc-l (Imuna gene), bc-l2 (RG-B gene). bc-2 (Michelite gene). bc-z2 (cu-31 gene), bc-3 (IVT 7214 gene), all of which are strain specific, and bc-u gene which is strain non-specific. Based on this interpretation, the genotype of each differential was determined and is shown in Table 3. In addition, the II differentials (Table 4) were also crossed with different bc genes, i.e, bc-l (Imuna), bc-2 (Michelite), and bc-22 (GN-31). The F2 generations of these crosses were tested with appropriate virus strains and the genotype of the dominant I gene differentials was also determined. Further, Drijfhout determined the pathogenicity genes on a gene-for-gene host/virus parasite relationship-basis and suggested that twelve (if some are allelic) or sixteen strain genotypes are possible. Seven of these are types presently known, while within the remaining nine, five are likely to be detected with the known set of differentials. 20 1.6. Gene Combination for Resistance to BCMV Burke and Silbernagel (12) advocated the incorporation of the dominant type of resistance, conferred by the I gene, to control BCMV in all been types. This gene hinders virus production, prevents mosaic development but allows systemic necrosis in the presence of necrotic strains of BCMV. Interestingly, necrotic strains of BCMV had not been identified in the United States when the strategy was suggested. However, in 1984 Kelly et a1. (37) isolated a necrosis inducing strain called Sanilac-Necrotic strain which caused top necrosis in commercial cultivars carrying dominant I gene resistance. They developed a strategy of incorporating additional resistance using the recessive bc-22 gene found in the Monroe and GN-31 cultivars. Drijfhout (18) suggested the pyramiding, into all II genotypes, the -strain-specific bc genes not overcome by necrosis-inducing or temperature-independent BCMV strains. In order to provide the plant with a more durable resistance against simultaneous mutations of more than one pathogenicity gene that would breakdown more than one corresponding gene, he recommended the incorporation of one or two bc genes. Resistance factors not yet overcome by the pathogen such as the recessive bc-3 gene present in cultivar IVT 7214 (be—2, bc-3 i) which is effective against all known strains, were recommended: Based on these considerations, 21 he recommended a host genotype of bc-u, bc-12, bc-22, bc- 3, I as the best combination for complete and durable resistance. 2. Bean Yellow Mosaic Virus BYMV was one of several mosaic diseases described in legumes around the 1930’s (8). It was isolated from cv. Red Valentine at Madison, Wisconsin, in 1931, and was identified to be different from BCMV by producing distinct symptoms and infecting varieties Corbett Refugee, Great Northern UI No. l and Robust, which were resistant to BCMV (45). BYMV is currently worldwide in distribution and known to exist in many countries where legumes are grown. Other names used as synonyms to designate the virus have been: Bean Virus 2, Phaseolus Virus 2, Gladiolus Mosaic Virus, and Pea Mosaic Virus (7). 2.1. Transmission and Epidemiology BYMV particles may be easily transmitted mechanically but more commonly the disease is transmitted by insect vectors, primarily aphids. More than twenty aphid species have been known to transmit the virus from diseased to healthy plants in a non-persistent manner and they have been primarily responsible for the natural epidemics of BYMV. The most noted species are Aphis fabae, Acyrthosiphon pisum, Macrosiphum euphorbie, and Myzus 22 persicae. Some strains of BYMV are not easily transmitted by aphids while others lose transmissibility during storage or maintenance by mechanical inoculation (7, 59). Transmission through seed in Phaseolus vulgaris L. has not been reported but small percentages have been observed in alternate hosts like Vicia faba, white sweet clover, and in both yellow and white lupine. Highest transmission occurs when young plants become infected (7, 71). BYMV infects both leguminous and non-leguminous plants and its wide host range includes the following species: Phaseolus vulgaris L., Cicer arietinum L., Vicia faba, Glycine max, Phaseolus lunatus, Pisum sativum, Melilotus alba, Vigna species, Chenopodium quinoa, Chenopodium amaranticolor, Gladiolus species, Nicotiana tabacum, and Medicago sativa (6, 58, 71). 2.2. Symptomatology Reaction of host plants varies according to genotype, time of infection, strain of the virus and environmentali conditions. Infected bean plants may exhibit systemic mosaic, systemic necrosis or local necrosis. Systemic mosaic symptoms initially appear as small chlorotic spots which gradually enlarge and coalesce to produce general chlorosis on affected leaves. Young leaves become brittle, glossy, concave on upper leaf 23 surface, and may be malformed. Yellow and green mottling becomes more intense on leaves as they age. Infection causes shortened internodes, proliferation of branches and plant stunting and may delay maturity (71). Systemic necrosis symptoms appear as a purplish coloration at the base of the lower leaves, which may be accompanied by veinal, stem and petiole necrosis, top necrosis at the terminal growing point, or plant death. Local necrotic lesions appear as reddish-brown spots on infected leaves and pods leading to malformation depending on the specific virus strain (59, 71). 2.3. BYMV Strains A number of BYMV strains that commonly infected Gladiolus sp., Melilotus alba, Trifolium pratense, and Trifolium incarnatum are transmitted to beans by aphids. Pierce (45) first described the Type strain of the virus on beans and this was later described in detail by Zaumeyer and Wade (72). The Type strain causes typical systemic mosaic symptoms in susceptible plants. McWhorter and Boyle (40) reported a necrotic or the X-strain of the virus on Blue Lake beans in Oregon. McWhorter (39) reported nine additional strains isolated from beans, gladiolus, alfalfa and peas in Oregon and Washington. He distinguished these strains on the basis of symptomatology, host preference and cytological evidence 24 in the leaf cells of Vicia faba. The isolate from beans, cultivar Blue Lake, was later called the Y-strain. Grogan and Walker (24) reported on a pod-distorting strain of the virus from Idaho Refugee varieties of beans. Bridgemon and Walker (9) isolated the type strain and the pod- distorting strain from naturally infected gladiolus. Frey (23) recorded the identification of the Virus in New Zealand on gladioli that was transmissible to beans. Houston and Oswald (33) isolated two strains of the virus from ladino clover. Conover (14) isolated a virus from soybean that he regarded as a strain of the Type virus. Hagedorn (27) and Hagedorn and Walker (28) described four isolates of BYMV, one of which appeared distinct from the Type strain. Zaumeyer and Fisher (68) described a new necrotic lesion producing strain of the virus. Thomas and Zaumeyer (64) reported a severe yellow mosaic strain which produced local lesions on tobacco. Frandsen [quoted by Zaumeyer and Thomas (71)] described a strain he designated as Marmor Manifestum n. nom. It produced necrosis on Kentucky Wonder beans and yellow mosaic symptoms on many other varieties. Van der Want described a strain which produced top necrosis in many bean varieties. . 808 (7) described the 825 strain of BYMV isolated from a dwarf French bean variety ‘Bataaf’. In most varieties the strain produced epinasty of inoculated 25 primary leaves and chlorotic or necrotic local lesions followed by systemic mottle or mosaic, leaf curling or malformation and often by stunting and mottle and malformation of pods. Bos, Kowalska and Maat (8) classified nine BYMV isolates into three strain groups based on host ranges, symptoms and bean and pea varietal reactions. Group I, consisting of isolates #212 from pea, Li from yellow lupine and 825 from French bean, represented the Bean Mosaic Virus strain. This strain caused systemic green mosaic symptoms in most pea, broad bean and also Chenopodium amaricolor species. Group IIa, consisting of isolates 8198 and 8204 from pea and Kow28 from red clover, represented the Pea Yellow Mosaic strain. This strain caused yellow mosaic in pea and broad bean and mild symptoms only in more BYMV-sensitive P; vulgaris. Group IIb, consisting of isolates 8197, 8199 and E221 from .pea, represented the Pea Necrosis strain. This strain caused characteristic and usually severe whole plant necrosis in pea and broad bean and a hypersensitive reaction in most 3; vulgaris cultivars. Herrera and Sepulveda (32) identified a distinct BYMV strain, Orfeo-INIA, in Chile from cv. Orfeo which had previously been immune to the severe strain prevalent in that region. Orfeo was developed from a cross between cultivar GN-31, resistant to a severe strain of BYMV, with the susceptible local cultivar, Negro Argel (13). The 26 distinction between the two BYMV strains was based on symptomatology, transmission, physical properties, morphology and size of the virus particles. The Orfeo- INIA strain caused necrosis in susceptible Orfeo while the severe strain caused severe systemic mosaic symptoms in susceptible cultivars. Hart et a1. (31) collected an isolate of BYMV, coded ‘C', on P; vulgaris cv. C-20 in Saginaw and Tuscola counties in the State of Michigan which was positively identified to be BYMV on the basis of host ranges and serology by Mink at Washington State University (36). 2.4. Genetics of Resistance to BYMV Resistance to BYMV in P; vulgaris is conditioned by different genes that are virus strain-specific. Baggett (2) reported two or three recessive genes with modifiers from P; coccineus conferring resistance to two severe pod- distorting strains (Y and X) of BYMV in crosses involving susceptible O.S.C.22 Blue Lake (3. vulgaris) and resistant Accession 2014. Baggett and Frazier (5) obtained similar results with the same strains in crosses involving two-P; vulgaris cultivars: GN-31 (resistant) and O.S.C.21 Blue Lake (susceptible). GN-31 however, was found to be susceptible to a necrotic strain, Orfeo-INIA, isolated in Chile (32). Schroeder and Provvidenti (56) reported a single dominant gene, By, in cv. Red Kidney conferring 27 resistance to the PV-2 strain in a Red Kidney x Black Turtle Soup cross. Dickson and Natti (17) reported a single dominant gene, By-2, derived from P; coccineus, conferring resistance to several BYMV strains. Provvidenti and Schroeder (49) found GN 1140 resistant to 82 isolates of the severe strain (BYMV-S) and pea virus 2 (PV-2) and found that resistance was conditioned by a single recessive gene, by-3. The severe strain of BYMV has since been renamed clover yellow vein virus and the by-3 gene has also been renamed cyv (48). Tu (66) reported a similar gene in cultivar Kentwood conditioning resistance to the severe and necrotic strains of BYMV. Tatchell and Baggett (63) found that the inheritance to two strains of BYMV (Type and severe strains) in GN-3l was conditioned by two different genetic factors: resistance to the type strain was conditioned by three recessive genes while two recessive genes conditioned resistance to the severe strain, suggesting independent inheritance. 3. A Common Gene for Resistance to More than one Pathogen Cook (15) reported that resistance to tobacco etch virus and potato virus Y was conferred by a single _ recessive gene, eya, from cultivars P11 and S. C. 46252, both belonging to Capsicum annum species. The F1 progeny produced in crosses involving the two resistant and four 28 susceptible cultivars were susceptible to both viruses. The four susceptible cultivars were California Wonder, Florida Grant, Improved Worldbeat, and Yolo Wonder. The F2 segregation ratio in all four populations to both viruses was 3 susceptible to l resistant (3S:1R) indicating single recessive gene resistance. F2 progeny resulting from crosses between the two resistant cultivars were all resistant to both viruses and no recombinants were observed. He referred to this condition as "spurious pleiotropism", implying at least two different effects emanating from a single gene and resulting in the same type of resistance mechanism. Schroeder and Provvidenti (57) reported a single recessive gene, mo, conditioning resistance to both bean yellow mosaic virus and water melon virus 2 in gisgm sativum. Inoculations of F1, F2, and F3 populations from crosses between BYMV-resistant Bonneville (mo mo) and BYMV-susceptible Ranger (Mo M0) were made. F1 plants (Mo mo) were completely susceptible to both viruses and the Fé populations segregated in the ratio of 3 susceptible to l resistant (38:1R). A common gene for resistance to both viruses was substantiated by the homozygous resistant reactions of F3 plants from families of plants selected as genetically mo mo on the basis of their reactions in the F generation to one or the other virus. 2 29 Kyle et al. (38) studied the genetic relationship among five dominant genetic factors, I, ch, Cam, H53 and st, conditioning hypersensitive resistance to five pathogens belonging to the potyvirus group of plant viruses: been common mosaic virus (BCMV), black eye cowpea mosaic virus (BlCMV), cowpea aphid-borne mosaic virus (CabMV), soybean mosaic virus (SMV) and water melon mosaic virus-2 (WMV-Z) respectively. Evaluation of F3 families from a BT-2 (ii) x BT-l (II) cross and other diverse types of bean lines revealed that all hypersensitive BCMV-resistant lines developed a similar response to the other four viruses at 35°C. In addition, no BCMV-susceptible lines were hypersensitive to any of the other viruses. Furthermore, to establish whether resistance was both dominant and associated with the I locus of Corbett Refugee (a BCMV-resistant variety developed from a spontaneous mutant at the I locus), they made a cross between Corbett Refugee and BT-l (II) and test-crossed the F1 progeny to 8T-2 (ii). Progeny were inoculated with each of the five viruses at 35°C and results showed that all plants developed hypersensitivity. Since the only factor(s) for resistance to these viruses in BT-l are at or closely linked to the I locus, they concluded that either a tight linkage between the five- genetic factors exists, or the I locus determined similar reactions to all five viruses. 30 They also demonstrated that been lines that carry the combination of dominant I gene and homozygous recessive genes at the bc-u and bc-l, bc-2 or bc-3 loci, showed altered or suppressed expression of the I allele with the five viruses. Based on this reaction, they postulated that if these are distinct factors conditioning hypersensitivity to the five viruses, they (factors) are tightly linked, phenotypically indistinguishable and subject to extremely similar epistatic interactions. III. MATERIALS AND METHODS 1. Virus Strains seen The NL3 strain of BCMV was used in this study. It is a necrosis-inducing strain independent of temperature and induces systemic necrosis (top necrosis) in varieties carrying'the unprotected I gene at both normal (20-25°C) and high (30°C) temperatures. It produCes typical systemic mosaic symptoms in certain susceptible genotypes with the recessive 1 gene. In this study it was used to distinguish between II and II bc-3 genotypes because of its necrosis inducing characteristics in II genotypes. On detached leaves of test plants, the NL3 strain induced two reaction types: 1) materials carrying only the dominant resistance (II) were hypersensitive and developed fpin-point" local lesions, 2) materials carrying both the dominant resistance and recessive resistance (IIbc-3) genes gave an immune response, developing no symptoms at all. B. BYMV The ‘C’ isolate of BYMV collected on C-20 cultivar was used. It induces typical systemic mosaic symptoms in 31 32 susceptible plants with yellow and severe leaf mottling (Fig. 3). In other cultivars like Orfeo and Fleetwood it induces a necrotic reaction without the typical top necrosis and plant death of BCMV. Fifty MSU elite breeding lines were screened for reaction to the NL3 strain of BCMV using the necrosis test and the ‘C’ isolate of BYMV using intact plants. This was to confirm the observations made earlier by researchers at Michigan State University, that materials carrying both the dominant I and recessive bc-3 genes were resistant both to the NL-3 strain and to the ‘C' isolate. Four plants of each test line were used for the confirmation test. Two breeding lines and one variety were selected to initiate the crossing program for the genetic study. 2. Bean Varieties and Breeding Lines Seeds of two breeding lines and one variety used in this study were obtained from the bean program at Michigan State University. C-20 and N84004, both white seeded navy beans, carry the dominant I gene while the been breeding line, 885009, carries both the I gene and the recessive bc-3 gene. The pedigrees of the three parental lines are shown in Table 5. The I gene in C-20 and N84004 came from NEP-II anc C-20 respectively. The bc-3 gene in 885009 is from IVT 7214, a selection from a USDA introduction line PI 181954 made by Drijfhout (18), and the I gene came from the commercial bean variety Midnight. The initial crosses 33 Fig. 3. Systemic mosaic symptoms induced on beans breeding line N84004 (genotype = II) by the ‘C' isolate of BYMV. 34 were made at the International Center for Tropical Agriculture (CIAT) between BAT 338, IVT 7233 and IVT 7214. F seeds resulting from this cross were obtained and used 4 in the bean program at Michigan State University where final crosses were made with Midnight. Table 5. The pedigrees of the three parental lnes used in the study. - Lip; Seed color Genotype Pedigree C-20 White II Jamapa/NEP-2//X80018 N84004 White II C-20/N80038 885009 Black IIbc-3bc-3 BAT338//IVT7233/ IVT7214/3/Midnight 3. Inoculum Preparation And Inoculation Seeds of cultivar Sutter Pink, infected with the NL3 strain, and frozen, desiccated leaf tissue of cultivar Aurora infected with the ‘C’ isolate of BYMV were obtained from Dr. A. W. Saettler at Michigan State University. The BCMV infected seeds were planted to produce plants infected with the NL-3 strain. Infected leaves from cultivar Sutter Pink were used for the NL3 inoculum preparation. Similarly, infected leaves from cultivar Aurora were used to prepare the BYMV inoculum. In the case of BCMV, the inoculum was prepared by triturating the infected leaf tissue in 0.01M phosphate buffer, pH 7.2, at the approximate ratio of lg of infected tissue per 5ml of buffer (1:5) (42). For BYMV a similar 35 procedure was used but a ratio of 1:10 was used. All materials used in the inoculations (mortars, pestles and buffer) were sterilized by autoclaving for 30-35 minutes prior to use. Inoculations were performed by dipping the rough surface of the pestle in the inoculum and then rubbing the upper surface of the expanded primary leaves which had been previously dusted lightly with 320 mesh carborundum. When making inoculations with a different strain/isolate, hands were washed between each inoculation to avoid contamination. 4. Virus Propagation and Maintenance of Strains The NL3 strain was propagated in cultivars Sutter Pink and Sanilac. These two cultivars carry the recessive allele of the I gene and produce systemic mosaic symptoms 'when infected with NL3. Infected seeds of these cultivars were harvested and stored for future use. The ‘C' isolate was propagated on cultivars Aurora and Bunsi. The two cultivars carry the dominant I gene (II) but produce systemic mosaic symptoms when infected with isolate ‘C’ of BYMV. The two virus materials were usually maintained in different greenhouses to prevent contamination by either contact of plants or possible virus transmission by insect pests. 36 5. Necrosis Test The necrosis test for BCMV was used in the genetic analysis to distinguish plants with the unprotected I gene from those carrying the I gene protected by the recessive bc-3 gene (II bc-3bc-3) (Figs. 4 and 5). The test allowed the determination of the plant genotype and then permit the observation of the whole plant’s reaction to the ‘C’ isolate of BYMV. These segregating individuals under study were developed from crosses between I gene and the I bc-3 gene parent. At the fully expanded primary leaf-stage, one primary and BCP leaf from each of the F1, F2, BCP BCP 1' 2 3 populations and parental lines was sap-inoculated with NL3 strain and then detached within 30 minutes after inoculation. The plants and leaves were numbered to distinguish them. The detached leaves were placed in plastic boxes which were then covered with plastic tops and maintained in the incubator at a temperature of 28°C. A sheet of moist filter paper was placed between each layer of the detached leaves to prevent them from drying up. After three or four days, the leaves from plants with the unprotected dominant I gene showed "pin-point" local lesions and vein necrosis, whereas the leaves from plants with the protected I gene (Ibo-3) showed no reaction (Fig. 5). ~The second primary leaf from each of the generations I was inoculated with the BYMV isolate ‘C and left intact on 37 Fig. 4. Pin-point local lesions and vein necrosis induced on the detached leaf of N84004 (genotype = II) inoculated with the NL3 strain of BCMV. 38 Fig. 5. Immune response induced on the breeding line 885009 (genotype = II bc-3bc-3). 39 the plants. Throughout the study, both the II and II bc- 3bc-3 genotypes were used as controls. 6. Development of Necrosis Test for BCMV A preliminary study was undertaken to determine the time within which the necrotic strain of BCMV (NL3) would infect the detached inoculated leaf of the II genotypes.. Leaves needed to develop local pin-point lesions sufficiently after being detached in order to detect the I gene but without systemically infecting the whole plant. This was done to protect the intact plant from developing systemic necrosis symptoms typical of BCMV since the same plant was to be used in the test against the ‘C’ isolate of BYMV. Plants of each of the two parental lines carrying the dominant I gene (hypersensitive to BCMV strain NL3) and susceptible to the BYMV isolate ‘C', and one parental line carrying both the I dominant gene and the recessive bc-3 gene (immune to NL3 and resistant to BYMV isolate ‘C’) were used in this study. A Randomized Complete Block Design was used with six sets of time treatments (0, 6, 12, 18, 24, 30 hours) as blocks. Four plants of each parental line were used in each block. At the fully expanded primary leaf-stage, one plant from each line in each block had one of its leaves inoculated with NL3 and detached immediately after inoculation (0 hours), and then at six-hour intervals up to 30 hours. Both the detached 40 leaves and the intact plants were observed for development of systemic necrosis symptoms (Table 7). 7. Crossing Techniques The techniques used in making crosses were as described by Drijfhout (l8) and Morales (42). The standard of an unopened flower bud was lifted up with a pair of tweezers. Then the left wing was pressed downwards, forcing the stigma out of the keel, remaining in this position if the wing had been pressed down far enough. The stigma was then rubbed with the desired pollen, using a pollen-covered stigma from an open flower of a male parent. Emasculation was not used because it is not only slow but also reduces the chances of success since the removal damages the flower structure. Crosses were made in spring and summer of 1988 and involved one 'cultivar and two MSU advanced elite breeding lines. The cultivar C-20 (P2) and one elite breeding line N84004 (P3), carry the dominant I gene and were used as female parents. The other breeding line, 885009 (P1), possesses both the dominant I and the recessive bc-3 genes and was the pollen donor. The incorporation of the recessive gene into I gene materials confers resistance to all strains of BCMV (18). The F1, F2 and backcross generations were grown and screened in the greenhouse during the summer and fall of 1988. 41 and BCP Seeds BCPZ' 3 8. Production of F1, F2, BCPl, Since BYMV is not transmitted through seed or pollen, F1 plants were used for several purposes: 1) confirming the dominant I gene resistance by necrosis test, 2) determination of resistance or susceptibility to BYMV, 3) production of F2 seeds, 4) backcrossing to both parents. BCP1 populations were produced by backcrossing the F1 plants to the resistant parent (II bc-3bc-3), BCP2 and BCP3 plants by backcrossing to the susceptible C-20 and N84004 (II) respectively. 9. Third Generation (F3) Tests As a third supplementary test, the F3 progeny of randomly selected F2 plants from the two populations in each of the three phenotypic classes, i.e., TN/S; TN/R; I/S; and all F2 plants from the I/R class were tested with the same strain and isolate of the viruses as used for the F2 plants. The test was conducted to confirm the reactions observed in the F2 generation. For this test, F3 seeds were harvested in the greenhouse from all F3 plants that set pods and matured. Five plants from each of the three classes, i.e, TN/S; I/S and TN/R, were randomly selected and ten F3 seeds from each plant were planted. All plants from the I/R class were harvested since the numbers were small and all their F3 seeds were tested. At ten days after planting, the 42 necrosis test was performed with the NL3 strain of BCMV and the intact plants were inoculated with the ‘C’ isolate of BYMV. The detached leaves and the intact plants were examined as described previously and the phenotypic reaction for each individual was determined. 10. Comparison between Genotypes Carrying the bc-3 Recessive Gene and Other Known Sources of Resistance to BYMV. Provvidenti and Schroeder (49) found that resistance in GN 1140 to the severe (BYMV-S) and pea virus 2 (PV-2) strains of BYMV was conditioned by a single recessive gene, by-3 (cyv). Tu (66) reported a similar gene in cultivar Kentwood conferring resistance to the severe and necrotic (BYMV-N) strains of BYMV. Baggett and Frazier (5) reported two or three recessive genes from cultivar GN-31 conferring resistance to the X and Y pod-distorting strains of BYMV. The three cultivars were screened against the ‘C' isolate of BYMV. If they proved resistant, they would be used in the comparative study to determine whether the gene(s) they carry for resistance to the ‘C’ isolate would be related to any of the gene(s) in genotypes carrying the bc-3 gene. Pure line seeds of GN 1140 were obtained from the University of Nebraska, and those of Kentwood and GN-31 were obtained from seed storage at Michigan State 43 University. Four plants of each cultivar were screened against the ‘C’ isolate for possible inclusion in the breeding program. Twenty plants of each of the susceptible lines, C-20 and N84004, and a resistant elite breeding line, 885009, were used as checks (Table 8). IV. RESULTS . 1. Confirmation Tests Table 6 shows the reaction of the forty nine breeding lines and cultivars screened against the NL3 strain of BCMV and the ‘C’ isolate of BYMV. Lines carrying only the dominant I gene and those carrying both the I gene and the recessive bc-22 gene from cultivar Orfeo developed "pin- point" local lesions on detached leaves inoculated with the NL3 strain and systemic mosaic in the growing plants I inoculated with the ‘C isolate. The bc-22 recessive gene protects the plant carrying the dominant I gene against the necrotic strains of BCMV, permitting local lesions only in the inoculated leaves since the virus does not systemically infect the whole plant. The breeding lines carrying both the dominant I gene and the recessive bc-3 gene originating from the breeding line IVT 7214, a selection from a USDA plant introduction line PI 181954, had immune responses to the NL3 strain and also were resistant to the ‘C’ isolate. These results confirmed the earlier observations that the- dominant I gene lines protected by the recessive bc-3 gene had immunity to the NL3 strain of BCMV and exhibited an associated resistance to the ‘C’ isolate of BYMV. 44 45 TABLE 6. Reaction of the forty nine breeding lines to the NL3 strain of BCMV based on the necrosis test and the 'C' isolate of BYMV inoculated on the growing plants. Necrotic reaction to Breeding Abbreviated BCMV BYMV Line Pedigree Genotypes (NL3) ('C') B76002 N2 / BTS II LL 3 c-2o PIJ/N2//X80018 II LL 3 885002 N81017*2/ORFED IIbc-22bc-22 LL 8 385003 MID/lN81017/ORFED " LL 5 884004 MID/N81017/ORFED " LL 5 385005 MID/N81017/ORFED " LL 3 885006 BAT/IVT 7214/lure IIbc-3bc-3 - R 385007 BAT/IVT 7214//MID " - R 385008 BAT/IVT 7214//MID " - R 385009 BAT/IVT 7214/lMID " - R 885010 BAT/IVT 7214//MID " - R 385011 BAT/IVT 7214//MID " - R 385012 BAT/IVT 7214//MID " - R N84004 C-20/N80038 II LL 3 N85090 C-20//N81095/ORFED IIbc-Zzbc-ZZ LL 5 N85091 C-20//N81095/ORFED " LL s N85092 c-20//N81095/0RFED " LL 3 N85093 N79034/ORFED " LL 5 N85094 N79034/ORFED " LL 8 N85095 N79034/ORFED " LL 8 N85096 N81017/ORFED " LL 3 N85097 N81017/ORFED " LL 8 N85098 N81017/ORFED " LL 3 N85099 N81017/ORFED " LL 3 N85100 N81017/ORFED " LL 3 N85101 N81017/ORFED " LL 3 N85102 N81017/ORFED " LL 8 N85103 N81017/ORFED " LL 5 N85104 N81017/ORFED " LL 5 N85105 N81017/ORFED " LL 8 N85106 N81017/ORFED " LL ‘5 N85107 N81017/ORFED . " LL 3 N85108 N81017/ORFED " LL S N85109 BAT/IVT 7214//N79023 IIbc-3bc-3 - R TABLE 6 (Cont'd) Breeding Abbreviated Line N85110 N85111 N85112 N85113 N85114 N85115 N85116 N85117 N85118 N85119 N85120 N85121 N85122 N85123 N85124 N85125 Pedigree BAT/IVT BAT/IVT BAT/IVT BAT/IVT BAT/IVT BAT/IVT BAT/IVT BAT/IVT BAT/IVT BAT/IVT BAT/IVT BAT/IVT 46 Genotypes 7214//N79023 7214//N79023 7214//N79023 7214//N79023 7214//N79023 7214//N81064 7214//N81064 7214//N81064 7214/lN81064 7214//MID 7214/lure 7214//MID C-20//VAT/IVT 7214 IIbc-3bc-3 N79023//8AT/IVT 7214 " N79023//BAT/IVT 7214 " N79023//BAT/IVT 7214 " Necrotic reaction to BCMV (NL3) BYMV ('C' wwwwwwwwwwwwwwww B76002 C-ZO B85009 LL R S BAT MID N2 BTS PIJ II bc-22 bc-3 (+/S) (+/S) Check Check Check (-/R) Local lesions. No local lesions. Resistant. Susceptible. BAT 338 Midnight NEP II Black Turtle Soup Pijao Dominant I gene recessive gene recessive gene 47 2. Development of the Necrosis Test for BCMV Table 7 shows the results of six sets of six-hour time intervals used to develop the necrosis test. The test uses the detached leaf technique to differentiate genotypes carrying the unprotected dominant I gene from those protected by the recessive bc-3 gene. A fully expanded primary leaf of each plant genotype is inoculated with a necrosis inducing strain of BCMV and then detached from the plant. Observations are taken on the detached leaf. The genotypes carrying the dominant I gene and those carrying 2 bc- both the I gene and the recessive bc-22 gene (II bc-2 22) develop local lesions on the detached leaf. However, the II bc-22 bc-22 genotypes, despite developing the local lesions on the detached leaves, do not ultimately develop systemic necrosis in the growing plants because the recessive bc-22 bc-22 gene prevents the expression of hypersensitivity in II genotypes. On the other hand, no local lesions are observed on the detached leaves from genotypes carrying either the recessive 1 gene or the combination of the dominant I gene and the recessive bc-3 gene. In this study the necrosis test achieved the dual purpose of determining the reaction of the segregating individuals to BCMV based on the detached leaf technique while permitting a determination of the reaction of the same individuals to the ‘C’ isolate of BYMV using the intact 48 TABLE 7. Plant systemic infection by the NL3 strain of BCMV in relation to time between inoculation and leaf detaching in the necrosis test. C-ZO N84004 885009 Time (hrs) Detached Intact Detached Intact Detached Intact O + - + - - - 6 + - + - - - 12 + +,Sn + +,Sn - - 18 + +,Sn + +,Sn - - 24 + +,Sn + +,Sn - - 30 + +,Sn + +,Sn - - * = Time in hours between leaf inoculation and removal from plant. + = "pin-point" local lesions on the detached leaves. +,Sn = "pin-point" local lesions followed by systemic necrosis on the growing plants. - = No local lesions observed on the detached leaves, no systemic necrosis on the intact plants. 49 plants. Plants of the susceptible checks (c-ZO and N84004) whose primary leaves were inoculated with the NL3 necrotic strain and detached soon after inoculation (0 hours) did not develop systemic necrosis. Similar observations were obtained on plants whose leaves were detached after six hours. Removal of inoculated leaves between twelve and thirty hours after inoculation produced systemic necrosis in the intact plants and resulted in. plant death. However, plants of the resistant check (B85009), did not develop systemic necrosis regardless of time intervals at which the necrosis test was performed. This was expected since the 885009 breeding line carries the dominant I gene protected by the recessive bc-3 gene. There was, however, importantly no minimal time of attachment to the plant needed by the leaf in cultivars C- 20 and N84004 after inoculation to permit development of necrosis symptoms on the detached leaf. These results provided a sufficiently reasonable time frame in which the segregating F2 individuals could be genotypically differentiated by using the NL3 necrotic strain of BCMV, leaving the plants virus-free to be available for screening against the ‘C' isolate of BYMV. A time interval of up to six hours was sufficient to . inoculate and detach the leaves with no systemic necrosis developing in the test plants. 50 3. Comparative Studies between Genotypes Carrying the bc-3 Recessive Gene and Those Known to Carry Other Resistance Factors to BYMV Table 8 shows the reaction to the ‘C' isolate of BYMV on cultivars GN 31, known to carry recessive resistance genes, and GN 1140 and Kentwood, both known to carry the by-3 (cyv) recessive gene (49, 66). 885009 was the resistant check and C-20 and N84004 were susceptible checks. GN 31, GN 1140 and Kentwood were all susceptible to the ‘C’ isolate. These results suggested that the gene (s) in 885009 conferring resistance to the ‘C’ isolate are different from those known to exist in GN 31, GN 1140 and Kentwood. These data suggest that the resistance afforded by 885009 is therefore at a different locus than the by-3 gene unless it carries a resistance allele at the by-3 locus. 4. Reaction of Parental and Segregating Individuals to BCMV (NL3) and BYMV ( C ) Parental, F F and backcross plants were rated for 1’ 2 reaction to BCMV based on the production of local lesions in the necrosis test or the expression of the immune response to the NL3 strain on detached leaves. Genotypes exhibiting local lesions were susceptible and carried the unprotected hypersensitive resistance I gene. Genotypes with no local lesions carried the dominant I gene protected by the recessive bc-3 gene. 51 Reaction to §. 0 40 4O 40 40 40 TABLE 8. Reaction to the 'C' isolate of BYMV on three check cultivars and three cultivars with known recessive gene resistance to other BYMV strains. Cultivar/ breeding Plants the line Gene(s) Pathogen Source Screened isolate R 885009(*) ? BYMV-'C' MSU 20 20 GN 31 2/3 x, Y (5) 4o 0 ON 1140 by-3 BYMV—S, (45) 40 0 (cyv) PV-2 Kentwood by-3 BYMV-S, (62) 40 0 (cyv) BYMV-N c-20 (#) - - MSU 20 0 N84004(#) - - MSU 20 0 = Resistant S = Susceptible * = Resistant check w as II + = Unknown genes = Undetermined Susceptible checks - No resistance gene(s) 52 Rating for reaction to BYMV was based on a resistant (R) or susceptible (S) reaction of the whole plant. Resistant plants did not exhibit BYMV-induced symptoms while susceptible ones had typical leaf curling, stunting and yellow systemic mosaic symptoms. Reaction of Parental Plants Plants of each parental breeding line were uniform in their reactions to both BCMV and BYMV. All plants of breeding line 885009 were immune to the NL3 necrotic strain of BCMV; plants of line N84004 and cultivar C-20 were susceptible exhibiting the top necrosis reaction. Uniformity to BYMV reaction in 885009 was dependent on certain temperature conditions. Moderate temperature (l8-20°C) produced a resistant response and those above 20°C produced a moderately susceptible response, and the virus was recovered in back-inoculations to the susceptible checks. Both C-20 and N84004 were susceptible to BYMV at temperatures below and above 20°C. Unpublished results from Dr. Saettler’s laboratory at Michigan State university (personal communication) indicate that the expression of susceptibility to the ‘C’ isolate in breeding line 885009 is due to contamination of the BYMV inoculum source with bean mild mosaic virus (BMMV). The discovery of this contaminant could have somewhat affected the results obtained from evaluating the reaction of the 53 segregating individuals to the ‘C’ isolated of BYMV. The susceptible category could have been inflated by individuals which otherwise would have been classified as resistant. Reaction of F1 Plants All F1 plants from the crosses exhibited the hypersensitive or top necrosis reaction induced by the NL3 necrotic strain of BCMV and were susceptible to the ‘C' isolate of BYMV, suggesting recessive resistance to both BCMV and BYMV. Reaction of F2 Plants Tables 9 and 10 show the segregation patterns for reaction to BCMV and BYMV in the F2 progeny of the 885009 x C-20 cross. Table 11 and 12 show similar F2 segregation patterns in the 885009 x N84004 cross. A satisfactory fit of a 3:1 ratio of tOp necrosis to immune plants for BCMV ' was observed in the F2 progeny of both crosses. This ratio indicates that immunity to BCMV was controlled by a single recessive gene. A ratio of 3:1 of susceptible to resistant plants for BYMV was observed and this ratio indicates that resistance to BYMV was controlled by a single recessive gene. 54 Table 9. Inheritance of resistance to the NL3 strain of BYMV resulting from the cross of 885009 x C-ZO. Observed Expected No. Ratio 2* Generation tested I TN I TN tested X P 885009, P1 50 50 0 C-20, P2 40 0 40 P1 x P2, F1 17 0 17 P1 x P2, F2 122 39 83 31 92 1:3 2.78 .05 - .10 F1 x P2, BCP2 40 0 40 0 40 I ' Immune TN ' Top Necrosis * - Chi-square test (df ' 1; p = 0.05) for lI:3TN segregation ratio in the F2 individuals. Table 10. Inheritance of resistance to the ‘C’ isolate of 55 BYMV resulting from the cross of 885009 x C-20 Observed Expected "0' Ratio Generation tested R S R 3 tested x2* P B85009, P1 50 50 0 C-20, P2 40 0 40 P1 x P2, F1 17 0 17 P1 x P2, F2 122 22 100 31 92 1:3 2.90 .05 - .10 F1 1 P1, BCP1 33 15 18 17 17 1:1 0.14 .70 - .10 F1 x P2, BCP2 46 0 46 0 46 R - Resistant S - Susceptible ? = Chi-square test (df = l; p = 0.05) for 1R:3S segregation ratio in the F2 individuals. 56 TABLE 11. Inheritance of resistance to the NL3 strain of BCMV in populations resulting from the cross of 885009 x N84004 Observed Expected No. Ratio 2* Generation Tested I TN I} TN. Tested X P 885009, P1 50 50 0 N84004, P3 40 0 40 P1 x P3, F 37 O 37 P1 x P3, P2 470 128 342 118 352 1:3 1.02 .30-.50 F1 X P1, BCP1 23 9 14 12 12 1:1 0.71 .30-.05 F1 X P3, BCP3 20 0 20 0 20 I = Immune TN = Top Necrosis. * = Chi-square test (df = 1; P = 0.05) for 1 I33 TN segregation ratio in the F2 individuals. 57 ratio in the F2 individuals. TABLE 12. ' Inheritance of resistance to the 'C' isolate of BYMV in populations resulting from the cross of 885009 x N84004. Observed Expected No. Ratio 2* Generation Tested R .§ 5 S Tested X P 885009, P1 50 50 O N84004, P3 40 0 40 P1 x P3, F 37 O 37 P1 x P3, F 470 100 370 118 352 1:3 3.47 .05—.10 15‘1 x P1, BCP1 23 10 13 12 12 1:1 0.21 .50-.70 F1 x P2, BCP3 20 0 20 0 20 R = Resistant. S = Susceptible. * = Chi-square test (df== 1; p = 0.05) for 1 R:3 S segregation 58 Reaction of Backcross Progenies All backcross progeny resulting from crossing F1 hybrids to both susceptible parents, C-20 and N84004, were necrotic to the NL3 strain of BCMV. In addition, the same backcross progeny were also susceptible to BYMV. This indicated the absence of the resistance factors to both BCMV and BYMV. However, backcrossing the F1 hybrids to the resistant parent, B85009, produced progeny segregating in a ratio of 1:1 (Top necrosis: Immune) when inoculated with the NL3 strain of BCMV and also a ratio of 1:1 (Resistant:Susceptib1e) when inoculated with the ‘C’ isolate of BYMV. This result indicates the presence of a single recessive gene conferring immunity to BCMV and also a single recessive gene conferring resistance to BYMV. 5. Association between Genetic Factors Controlling Immunity to the NL3 Strain of BCMV and Resistance to the ‘C’ Isolate of BYMV A heterozygote differing in two gene pairs will produce gametes of four types. If the genes are independent, these types will be produced in equal numbers; if linked, the parental classes will appear with a frequency different from the recombinant classes. In this study, if one assumes that a recessive gene, say by-4, confers resistance to the ‘C’ isolate of BYMV and its dominant allele, 8y-4, confers susceptibility then 59 the genotype for both C-20 and N84004 is IIBC-38C-3B-Y4BY- 4. This genotype will exhibit the top necrosis reaction to the NL3 strain of BCMV because of absence of the double recessive form of the bc-3 gene and will also be susceptible to the ‘C’ isolate of BYMV because of the absence of the double recessive form of the by-4 gene. Likewise the 885009 breeding line will have the genotype IIbc-3bc-3by-4by-4 and will be immune to the NL3 strain- and resistant to the ‘C’ isolate of BYMV. Therefore, crosses between either C-20 or N84004 and 885009 will produce an F1 double heterozygote with the genotype IIBC- 3bc-3BY-4by-4 which will exhibit top necrosis to the NL3 strain and susceptibility to the ‘C’ isolate because of the absence of the double recessive forms of both the bc-3 and the by-4 genes. Considering the fact that the I gene is present in both parental classes, it will not segregate in either the parental or the recombinant classes. The genes that will segregate in subsequent generations will be derived from the F heterozygote of the genotype 8C-3bc-38Y-4by-4. 1 Therefore, this heterozygote will produce gametes of four types; BC-3 BY-4; bc-3 8y-4; BC-3 by-4 and bc-3 by-4. If the genes are unlinked, gametes BC-3BY—4 and bc-3by-4 will be produced in equal numbers; if linked, the parental gametes 8C-3By-4 and bc-3by-4 will appear with a frequency 60 different from the recombinant gametes BC-3by-4 and bc- 38Y-4. In the F2 population developed by selfing the F1 double heterozygotes from a 885009 x C-20 cross, 122 individuals were screened for their reaction to the NL3 strain of BCMV by use of the necrosis test and for their reaction to the ‘C’ isolate of BYMV using the intact plants. Of these, 51 individual plants (42%) were top necrotic to the NL3 and susceptible to the ‘C’ isolate (genotypes BC-3-BY-4); 33 plants (27%) were top necrotic to the NL3 strain and resistant to the ‘C’ isolate (genotypes 8C-3-by-4by-4); 20 plants (16%) were immune to the NL3 strain and susceptible to the ‘C’ isolate (genotypes bc-3bc-38Y—4-) and 18 plants (15%) were immune to the NL3 strain and resistant to the ‘C’ isolate (genotype bc-3bc-3by-4by-4). In the 885009 x N84004 cross, 470 F2 individuals were screened against both the NL3 strain and the ‘C’ isolate. Of these, 276 plants (59%) were top necrotic to the NL3 strain and susceptible to the ‘C’ isolate; 67 plants (14%) were top necrotic to the NL3 strain and resistant to the ‘C’ isolate; 95 plants (20%) were immune to the NL3 strain and susceptible to the ‘C’ isolate; and 32 plants (7%) were immune to the NL3 strain and resistant to the ‘C’ isolate (Table 13). 61 N .coeumcanaooou mo ammunoouod o no commouduo mafia: one I m .omoxnaa on one unoauuomme unocnodouoa meanness .maonve>wcoa m on» a“ cause coeuowouwom M\H~ " mxH m “ «\zhm “ m\zam pom Ano.o I d an I mvv uuou onesaalano I «ax >zwm cu ucmumwmom I m >=un ou muecsaaH I H A.u,o >xsm on manaaamumam I m Aquv >zum on mamouooz doa I 29 mo.o Nm.a an ass Has «mm «mm euuuoauo o.m H o.ns ama.o no.ouao.o N8.o~ as was as man man umsummno sauce mm mm as oau oae sooemz u aoommn m on as ch «Na cauo.u sconce mafia: oases a .Nx a\H m\H a\ze m\za coaosuam uoauouoaoo has: nonvoum _ .oz um .>=~n mo ouoaomu (o. ecu cu oocoueamou can >zum mo cannon qu ecu cu meansssa wnwadouunoo muouoom owuoaom ecu noosuop nofiuowoomm< .mH manna 62 These results indicate a weak coupling phase linkage ’in which ratios of F2 offsprings contained too many of the original parental types, TN/S and I/R, (64% for the 885009 x C-20 cross; 66% for the 885009 x N84004 cross) and fewer of the newly recombinant genotypes, TN/R and I/S, (36% for the 885009 x C-20 cross; 34% for the 885009 x N84004 cross). In the absence of a coupling phase linkage parental genotypes would constitute 62% while recombinant types would represent 38% of all genotypes. 6. Test for Independence between Genetic Resistance Factors to BCMV (NL3) and BYMV (‘C’) The chi-square (X2) goodness of fit test was used to establish independence or association/linkage between the gene conferring immunity to BCMV (NL3 strain) and the gene conferring resistance to BYMV (‘C’ isolate). In similar cases, unlinked genes should give four classes of offspring in the ratio 9:3:3:l and linkage will manifest itself by an excess of the first and last classes, if dominant genes came from the same parents (coupling phase), and by an excess of the second and third classes if they came from different parents (repulsion phase). The observed F2 phenotypes in each of the four classes of the two populations in this study were combined and only’ the total numbers were tested against the expected ratio of 9TN/S : 3TN/R : 3I/S : 3I/R for two unlinked recessive 63 genes. The two populations were combined because they were genotypically similar (half-sibs) and they carried the same two recessive genes whose linkage was to be determined. A combined X2 value of 10.62 was obtained which showed a significant deviation from the expected ratio based on random assortment of two recessive genes. This result indicates the existence of linkage between the recessive gene (be—3) that confers immunity to the NL3 necrotic strain of BCMV and the other recessive gene that I confers resistance to the ‘C isolate of BYMV. 7. Genetic Distance Between the Two Linked Genes The map distances are given as recombination frequencies expressed as percentages and represent relationships based entirely on genetic crossover data. These units are expressed either as morgan, l morgan being equal to 1 percent recombination, or as map units. In this study, the product ratio method of Fisher and Balmukand (22) was used to calculate the linkage value of the two genes and the tables of Stevens (62) were used to convert the product ratio to percentage recombination as a measure of linkage. A combined linkage value of 45.6 +/- 3.0 map units between the two genes was obtained which indicates that the two genes are loosely linked (Table 13). 64 8. Reaction of the F3 Families Table 14 and 15 respectively show the reaction of the F families from the four phenotypic classes of the 885009 3 x C-20 and the 885009 x N84004 crosses to the NL3 strain of BCMV and the ‘C’ isolate of BYMV. Families from most plants in the TN/S class showed segregation for immunity and top necrosis upon inoculation with the NL3 strain. They also showed segregation for resistance and susceptibility to inoculation by the ‘C’ isolate. This result indicates that the selected F2 individuals from which the F3 families were derived were heterozygous for the bc—3 gene. It also indicates that the same F2 individuals were heterozygous for the other gene conferring resistance to the ‘C’ isolate. Families from F individuals selected from the I/S class were all immune 2 to the NL3 strain but most segregated for resistance and susceptibility to the ‘C’ isolate. This indicates the presence of the double recessive form of the bc-3. It also indicates that F2 individuals were heterozygous for the other gene. Families from the TN/R class showed segregation for immunity and top necrosis upon inoculation by the NL3 strain but were all resistant to the ‘C’ isolate. This respectively indicates the absence of the double recessive form of the bc-3 gene and the presence of the double recessive form of the new gene. Finally, 65 TABLE 14. Reaction to the NL3 strain of BCMV and the 'C' isolate of BYMV in the F families derived from F2 selections of the 885009 X C-20 cross. Reaction Reaction to the NL3 to the 'C' F No. of F strain of isolate of 2 3 BCMV BYMV Phenotypic Selections Individuals Class Made Screened I IN 3 § TN/S 88M301-01 8 6 2 2 6 88M304-12 10 5 5 4 6 88M309—01 10 7 3 6 4 88M310-03 10 6 4 2 8 88M311-03 9 6 3 3 6 TN/R 88M302-03 10 4 6 10 0 88M303-05 10 3 7 10 0 88M303-08 10 10 0 10 0 88M306-07 10 6 4 10 0 88M309-03 10 3 7 10 0 I/S 88M303-01 8 8 0 3 5 88M304-14 10 10 0 3 7 88M308-03 10 10 0 3 7 88M310—06 10 10 0 0 10 88M311-09 9 9 0 2 7 I/R 88M302-06 10 10 0 10 0 88M305-05 3 3 0 3 0 88M308-05 9 9 0 9 0 88M310-02 9 9 0 9 0 88M311-07 3 3 0 3 0 TN/S = Top necrotic to the NL3 strain of BCMV/Susceptible to the 'C' isolate of BYMV TN/R = Top necrotic to the NL3 strain/Resistant to the 'C' isolate. I/S = Immune to the NL3 strain/Susceptible to the 'C' isolate. I/R = Immune to the NL3 strain/Resistant to the 'C' isolate. 66 TABLE 15. ‘ Reaction to the NL3 strain of BCMV and the 'C' isolate of BYMV in the F families derived from F2 selections of the 885009 X N84004 cross. Reaction ReaCtion to the NL3 to the 'C' F No. of F strain of isolate of 2 3 BCMV BYMV Phenotypic Selections Individuals Class Made Screened I II. R S TN/X 88M313-05 10 9 1 2 8 88M324-11 10 0 10 l 9 88M330-12 10 0 10 l 9 88M335-05 10 2 8 8? 2 88M341-06 9 5 4 3 6 TN/R 88M313-06 9 5 4 9 0 88M314-09 9 0 9 9 0 88M315-14 10 l 9 10 0 88M338-13 7 6 l 7 0 88M345-10 9 3 6 9 0 I/S 88M317-01 10 10 0 2 8 88M327-01 ..10 10 0 2 8 88M327-04 7 7 0 0 7 88M341-05 10 10 0 3 7 88M342-14 10 10 0 1 9 I/R 88M312-05 4 4 0 4 O 88M315-13 5 5 0 5 0 88M317-07 9 9 0 9 0 88M319-04 2 2 0 2 0 88M319-08 8 8 0 8 O 88M321-03 8 8 0 8 0 88M321-05 9 9 0 9 0 88M323-04 10 10 0 10 0 88M324-10 5 5 0 5 0 88M325-08 4 4 0 4 0 88M326-04 3 3 0 3 0 88M326-12 2 2 0 2 0 88M327-05 3 3 0 3 ‘0 88M331-ll 5 5 0 5 0 88M336-05 9 9 0 9 0 88M339-12 10 10 0 10 0 88M346-07 7 7 0 7 0 88M346-08 8 8 0 8 0 88M346-09 5 5 0 5 0 88M347-03 6 6 0 6 0 TABLE TN/S TN/R I/S I/R 67 15 (cont'd) TOp necrotic to the NL3 strain of BCMV/Susceptible to the 'C' isolate of BYMV. Top necrotic to the NL3 strain/Resistant to the 'C' isolate. Immune to the NL3 strain/Susceptible to the 'C' isolate. Immune to the NL3 strain/Resistant to the 'C' isolate. 68 families from the I/R class were all immune to the NL3 strain and resistant to the ‘C’ isolate. This indicates the presence of the double recessive forms of both the bc- 3 gene and the newly identified gene. V. DISCUSSION. In his extensive studies on BCMV, Drijfhout (l8) determined that bean cultivars carrying both the dominant I gene from Corbett Refugee and the recessive bc-22 gene from cultivar GN-3l developed brownish local necrotic lesions when inoculated with the necrosis inducing strains of BCMV. Similar observations were also made by Morales (42) in his studies using Orfeo, a derivative of GN-3l, as a source of the bc-22 recessive gene. This gene confers additional resistance in genotypes carrying the dominant I gene by producing local lesions only in the infected leaves but the virus does not systemically affect the whole plant. They also observed that cultivars carrying the dominant I gene and the recessive bc-3 gene from a breeding line IVT 7214 were completely immune to all known strains of BCMV at all temperature ranges. In this study, breeding lines carrying the dominant I gene and the recessive bc-22 gene from cultivar Orfeo produced local lesions on the detached leaves in the necrosis test using the NL3 strain of BCMV but the intact plants from which these leaves were taken were systemically infected by the ‘C’ isolate of BYMV (Table 6). The breeding lines carrying the I and bc-3 genes were not only immune to the NL3 strain but were also apparently resistant 69 70 to the ‘C’ isolate at moderate temperatures (l8-20°C). These results suggested that the bc-3 recessive gene could possibly have a dual role in protecting the dominant I gene against infection by specific strains of both BCMV and BYMV. It could also mean that the recessive bc-3 gene was linked to another gene which conferred resistance to the ‘C’ isolate of BYMV. Pleiotropic effects of certain single genes, i.e. a single gene having at least two different effects resulting in the same type of resistance mechanism, have been reported by some workers. Cook (15) reported a single recessive gene in the P 11 and S.C. 46252 strains of pepper (Capsicum 33323 ) conferring resistance to both tobacco etch virus and potato virus Y. Schroeder and Provvidenti (57) reported a single recessive gene in cultivar Bonneville of IIggm sativum L. conferring resistance to BYMV and water melon virus 2. Kyle et a1 (38) reported that the dominant I gene in beans induced hypersensitive resistance to five different virus types; bean common mosaic virus, blackeye conea mosaic virus, cowpea aphid-borne mosaic virus, soybean ‘mosaic virus and water melon mosaic virus. They further reported that dominant I gene lines coupled with homozygous recessive genes at bc-u and bc-l, bc-2, or bc-3 loci showed modified or suppressed necrosis upon infection with each of the same same viruses at 25°C and 35°C. This evidence certainly supports the role of pleiotropic effect of genes which control virus diseases in beans. 71 In the present study, while dominant I gene lines developed top necrosis upon infection with the NL3 strain of BCMV, they also developed systemic mosaic due to infection by the ‘C’ isolate of BYMV even at temperatures as high as 37°C. This shows the lack of the top necrosis pleiotropic effects of the I gene in beans to infection by BCMV (NL3 strain) and BYMV (‘C’ isolate). Furthermore, evaluation of the F2 individuals resulting from crosses between bean cultivars carrying only the dominant I gene and the breeding line having both the dominant I gene and the recessive bc-3 gene deviated from expectation of a pleiotropic effect of a single recessive gene on both BCMV and BYMV. All lines that carried the recessive bc-3 gene were immune to the NL3 \ I strain of BCMV but not all of them were resistant to the C isolate of BYMV. Immunity to BCMV segregated in the expected single recessive gene-effect of l immune to 3 top necrotic (l I : 3 TN). A l : 3 ratio (R : S) was also obtained for individuals segregating for resistance to BYMV (‘C’) indicating single gene resistance. An analysis of independent assortment to determine possible independence or linkage between the recessive genes respectively conferring immunity to BCMV and resistance to BYMV indicated that a coupling phase linkage existed in parent 885009 between the resistance bc-3 recessive gene and another recessive gene that controls resistance to BYMV (Table 13). This finding also ruled out the possibility of pleiotropic effect of the recessive bc-3 gene in beans . 72 conferring resistance to both BCMV and BYMV. The linkage determined between these genes had a distance of 45.6 i 3.0 map units indicating a loose linkage. This loose linkage further precludes the notion that these two recessive genes might be allelic. However, further studies need to be conducted to conclusively answer the question of allelism between the newly identified gene and the by-3 gene that confers resistance to other isolates of BYMV. This coupling phase linkage is of great importance because significant advantages can be realized in exploiting it in breeding individuals that carry the dominant I gene, the recessive bc-3 gene, and the other gene that confers resistance to BYMV. The value of the dominant I gene has long been recognized and extensively used in the control of BCMV in regions where necrosis inducing strains do not exist. In environments where such strains exist, efforts have been made to pyramid into the dominant I gene lines the specific recessive genes that confer resistance or immunity _against infection by the necrotic strains. The present study shows that with an additional recessive gene, lines carrying the dominant I gene are not only able to resist infection by the necrotic strains of BCMV but also capable of resisting infection by the ‘C’ isolate of BYMV. Therefore, the pyramiding of these genes into one genotype can be of great value in environments where both necrotic strains of BCMV and different isolates of BYMV exist. 73 Plant viruses have been known to cause serious losses in yield and quality. Several measures have been attempted to minimize their damage to crops and these include chemical control of vectors that transmit the viruses; crop hygiene; the use of virus free seed; and elimination of alternate hosts. These measures have not only proved to be ineffective but also suffer from problems of cost and plant and animal toxicity. Long-term and effective protection against viruses is possible in those crop species or cultivars where resistance is available and has been successfully utilized in breeding. None of the problems associated with virus control measures listed above have been linked to genetic resistance. The present study adds useful information to the ever expanding use of genes as the ultimate solution in the control of plant viruses in beans. One important tool which was made use of throughout this study was the necrosis test. The test was first used in 1957 by Quantz, a German Scientist (18), and then modified by Drijfhout (18). It is an efficient technique for distinguishing plants carrying the dominant I gene from those carrying its recessive allele. Its importance can be appreciated in bean trade between regions or in germplasm exchanges between been breeding programs. At present, too many bean cultivars with the recessive form of the I gene are maintained in cultivation because they often tolerate prevalent strains, or are only susceptible to strains not prevalent in the bean-growing area thus limiting the damage 74 caused by the virus. Moreover, these cultivars often have valuable characteristics for yield, consumption quality and plant type or are resistant to other diseases. However, keeping cultivars with the recessive I gene in cultivation preserves the virus and sources of infection. Simultaneous production of beans in nearby fields of both ii and II cultivars may easily lead to losses due to systemic necrosis in the latter genotypes when necrotic strain of viruses are present. ‘In the absence of any information on the genotype of the bean cultivars available and where there exists a need to keep separate the recessive (ii) from the dominant (II) genotypes, the necrosis test can be a handy tool in the identification of genotypes. It saves time and space in that information can be obtained within a few days and also a large number of materials can be evaluated using a reasonably small amount of space. Such information is important in making plans for either bean production or research purposes in situations where BCMV is an important consideration. The test was also important in the study for the identification of specific genotypes for genetic studies. Based on their reaction to the NL3 strain of BCMV, the specific genotype was immediately known and could be utilized for other studies. In the absence of this test, it would not have been possible to know the reaction of the II genotypes to the ‘C’ isolate of BYMV because they would have died from systemic infection after inoculation with the NL3 75 strain of BCMV. The test also permits the screening for I gene lines using necrotic strains of BCMV which are most effective and rapid as compared to non-necrotic strains of BCMV . In the comparative studies to determine the similarity or difference between the recessive genes known to confer resistance to specific strains of BYMV cultivars, GN 1140 and Kentwood having recessive resistance to the severe and pea virus 2 strains of BYMV (49,66) were selected. In addition GN-31, with recessive resistance to the X and Y strains of BYMV (17), was also selected. These cultivars were subjected to inoculation by the ‘C’ isolate of BYMV. If these cultivars had been resistant, they would have been used as parents to determine if their resistance genes were identical to the recessive gene in the breeding line B85009. Since the results (Table 8) showed that they were all susceptible, it indicated that the gene conferring resistance to the ‘C’ isolate was not only different from those found in cultivars GN-ll40, Kentwood and GN-3l, but in the absence of tests for allelism, it is not yet known if the new gene is at a separate locus. One way to establish allelism in cultivars that are resistant to a particular isolate of BYMV, is to cross them and observe the reaction of their progenies to both BYMV isolates in which the genes are active. If allelism exists, a different segregation ratio to different BYMV isolates would occur in the F2 in contrast to the ratio exhibited if 76 the genes are at different loci. However, it is possible that cultivars with resistant progenies to a particular disease could carry different but closely linked genes. Bokosi (1986) showed that although the progenies resulting from a cross between Montcalm and 1212D (a Malawian white navy bean) were resistant to the 8A3 isolate of anthracnose, the parents did not carry identical genes because 1212D showed complete immunity to this isolate while Montcalm had some symptoms that were inconsequential. Cook (15) reported an identical gene, eya, in cultivars P 11 and S.C. 46252 of the Capsicum annum species. F2 progenies resulting from crosses between the two resistant cultivars to tobacco etch virus and potato virus Y were also resistant to both viruses. However, no linkage studies were conducted to determine whether or not the two resistant parents had different genes but that they were closely linked. In this study, additional evaluations were carried out in the F3 generation to further elucidate the findings obtained in the F2 populations. The segregations to the NL3 strain of BCMV obtained in the evaluation of most F3 families from the randomly selected F2 individuals in the TN/S and TN/R phenotypic classes from the two populations indicated that the segregating individuals were heterozygous for the recessive bc-3 gene and they had the genotype.II8C- 3bc-3. If these individuals had been homozygous dominant for the dominant bc-3 gene, i.e. IIBC-38C-3, all resulting F families would have shown hypersensitive necrosis 3 77 symptoms upon inoculation by the NL3 strain of BCMV. This is probably the case with the 88M324-11, 88M330-12 and 88M314-09 F2 individuals from Table 13. All F2 genotypes homozygous recessive for the bc-3 gene, i.e. IIbc-3bc-3, showed an immune response to the NL3 strain in F3 generation confirming the presence of fixed double recessive homozygote. However, the sample of ten seeds from each F2 individual may not have been large enough to be representative in giving the expected results. It is possible that some F3 families may not have shown any segregation because of insufficient seed numbers selected. In addition F3 families from a heterozygous F2 individual should have segregated in a 3 top necrosis : l immune ratio with the NL3 strain of BCMV. The fact that many of the F3 progenies deviated from the expected ratio could be due the small sample size used in this study. Although some individual F plants from the TN/S class 3 had the homozygous recessive bc-3 gene as shown by their immune response to the NL3 strain of BCMV, not all were resistant to the ‘C’ isolate of BYMV (Tables 14 and 15). This confirmed the lack of the pleiotropic effects of the recessive bc-3 gene to prevent infection by both BCMV and BYMV. The segregation pattern in the F3 families to inoculation by the ‘C’ isolate of BYMV generally indicates a 3 susceptible to l resistant ratio and confirms the existence of another gene in the heterozygous susceptible 78 F2 individuals. The small samples of up to ten seeds each may again have contributed to the deviation from the expected ratio in some families. The lack of segregation to the ‘C’ isolate in the F3 families from the TN/R phenotypic class confirmed the existence of a new recessive gene and indicated that the gene was fixed as a double recessive homozygote. The individual plants were all resistant to BYMV. The results from F3 families in the I/S phenotypic class confirmed that the recessive bc-3 gene was fixed as demonstrated by the absence of segregation upon inoculation by the NL3 strain of BCMV. They were all immune. The segregation for resistance and susceptibility of most families to inoculation by the ‘C’ isolate revealed that the selfed F2 individuals were heterozygous for the new gene. The families that were all susceptible may have come from a homozygous dominant F2 individual or it may have been that the samples were not large enough to segregate in the expected ratio. The lack of segregation to both BCMV and BYMV on individuals from the I/R class confirmed that both the recessive bc-3 gene and another gene were fixed. The fact that the number of plants expressing an immune response to the NL3 strain of BCMV and occurred at a higher frequency than the recombinant classes suggests that an association exists between the recessive bc-3 gene and a new recessive gene in plants belonging to the I/R class. VI. BREEDING IMPLICATIONS BYMV is a highly variable, ubiquitous and occasionally destructive pathogen of dry beans (30). It has thus far eluded control measures including chemical application on insect vectors that transmit it; use of disease-free seed; and elimination of alternate hosts. These measures have been found to be ineffective, and may have undesirable effects on the environment. Genetic resistance to the virus has been recognized as the only long-term and effective method of control and has not been associated with any of the problems resulting from the use of other control measures. Like most of the other plant diseases, BYMV results from a very close interaction among pathogen, host and environment. Among the most important factors are pathogenic variation, quality and quantity of inoculum, developmental stage, method of inoculation and environmental physical factors. A successful breeding program will, therefore, consider each of these factors. 1. Pathogenic Variation The presence of pathogenic variation in BYMV has been documented by several researchers (45,58,71,72) and the present study makes use of a new pathotype of BYMV isolated 79 80 in Michigan, U.S.A. (31). This pathotype, called the \ I C isolate, was identified to be BYMV based on host ranges and serology (36). It is possible that other pathotypes not yet reported do occur in Michigan or elsewhere. The realization that there is extreme variation in pathogenic potential of this organism is very important and it is recommended that a continuous monitoring system for the occurrence of new different pathotypes should be exercised. This is very important to the development of widely adapted BYMV resistant/tolerant bean cultivars. 2. Method of InoculatioanQuality and Quantitypof Inoculum. The most commonly used method to inoculate plants with BYMV is the rub-inoculation of plant leaves with freshly ground inoculum. This was the method used in this study. Other useful methods include single or multiple-needle pricking techniques done in the greenhouse especially on small leaves. The most appropriate method of inoculation is usually that which is most convenient and appropriate for the circumstances. In addition to methods of inoculation, the quality and quantity of inoculum are very important. The importance of the quality of inoculum is related to pathogenic variation. Quantity of inoculum is also important in that field- ’ tolerance may be over-looked if too high an inoculum concentration is used. On the other hand, susceptible escapes could be regarded as resistant/tolerant if very low 81 inoculum concentrations were used. Therefore, if no previous data exist, it is necessary to carry out preliminary studies in trying to determine and standardize the inoculum concentration. 3. Developmental stagg. The age of the virus tissue to be used for inoculum preparation and also the plant to be inoculated is an important factor to be considered. Morales (42) observed a reduction in infectivity in infected BCMV tissue taken at blossom or beyond (28 days after planting). In this study precautions were taken to use only young BYMV virus tissue to avoid loss of infectivity. Test plants were also inoculated while young as older ones tend to be more tolerant. It is recommended, therefore, that for best results in screening and identification tests, infected tissue should be taken prior to the fourth week after inoculation and the test plants should be within about ten days of age. 4. Methods of Evaluating Disease Reaction. Most methods of assessing disease reactions involve either quantitative or qualitative aspects. In the present study, a qualitative method of resistance (R) and susceptibility (S) to BYMV was employed in evaluating each individual plant of the F2 populations and F3 families where, 82 No symptoms of the virus. 21 II (D II Typical systemic mosaic symptoms. 5. Breeding Strategies. After considering the above factors, a breeding program for resistance to BYMV should therefore emphasize resistance sources and effective methods of incorporation. A. Sources of resistance. In environments where BYMV problems exist, both local and introduced bean germplasm collections should be screened to identify sources of resistance. This screening can either depend on natural sources of the virus under field conditions or can be done under controlled greenhouse conditions. In this study, the 885009 breeding line used in the crossing program was one of the twenty four advanced breeding lines found to be resistant to the ‘C’ isolate of BYMV (Table 8). The pedigrees of all these lines include IVT 7214, a USDA introduction also known to carry a recessive bc-3 gene for resistance to BCMV (18). Thus, possibilities do exist that the IVT 7214 line or its derivatives also carry resistance to other pathotypes of BYMV. Therefore, it is recommended that these lines be screened against. other BYMV isolates with the view of finding additional resistance sources. Through hybridization, resistance could be incorporated from such sources and new local BYMV 83 resistant, agronomically superior varieties could be developed. 8. Breeding systems. Disease resistance is often a highly heritable trait. It may be under the control of a single gene or more genes acting in a complementary fashion. In the present study, resistance to the ‘C’ isolate of BYMV in breeding line 885009 has been identified to be under the control a recessive gene, loosely linked to bc-3 gene. Due to the high heritability of these genes, a useful plant breeding strategy in the development of BYMV resistant bean lines is the adoption of the inbred back cross method. Utilizing 885009 as the donor parent of the recessive resistance genes, resistance can be incorporated into the desired recurrent parent having all the desirable characteristics but only lacking genes for resistance to BYMV. The back- cross F2 plants are then selfed to produce lines similar to the recurrent parent, but carrying more variability from the donor parent, B85009. Selection with these inbred back- cross lines should then be conducted until the resistance factors are fixed and the level of desired homozygosity for other traits is reached. 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