v V f.” x .. “T v! ’z‘r . x. ,r, 5... .u ,. w—gézv ‘1. .u f. v 19%“ Q‘IM - ~r $1 “ ' .4 u. ‘ , .‘ , - ~ we: '3?” 1.. "‘ f"- w " ‘ r" ‘11:};- V‘ '3. )Mr’t, 41‘72'Ew 0 $35: .1“ We llll’llllllllllllllllllllll l l 3 1293 01024 0053 ,2 C H +3) This is to certify that the thesis entitled STUDIES ON BEAN MILD MOSAIC VIRUS, A NEW VIRUS OF BEAN IN MICHIGAN presented by PAULINA S. SEPULVEDA has been accepted towards fulfillment of the requirements for MASTER PLANT PATHOLOGY degree in WV) My \Qdajor professor Date 191”)!” v v 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LlBRARY Mlchlgan State University PLACE N RETURN BOX to remove this chockout from yourpcord. TO AVOID FINES Mum on or baton data duo. DATE DUE DATE DUE DATE DUE WM! STUDIES ON BEAN MILD MOSAIC VIRUS, A NEW VIRUS OP BEAN IN MICHIGAN by PAULINA S.SEPULVEDA A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1989 ABSTRACT STUDIES ON BEAN MILD MOSAIC VIRUS, A NEW VIRUS OF BEAN IN MICHIGAN by PAULINA s . SEPULVEDA Bean mild mosaic virus (BMMV), a spherical RNA virus of 28 nm diameter, was found for the first time affecting beans (Rngggglug yulgaris L.) in Michigan. The virus was iidentified by the immunosorbent electron microscopy (ISEM), gel double-diffusion and double antibody sandwich ELISA (DAS-ELISA) tests. Infected plants showed mosaic, mottling, vein banding, curling of the leaves,or no 'symptoms at all. The virus has very narrow host range which includes, soybean (glycine mg; L. (Merr), pea (gigum sativum L.) and bean (Phaseglus vulgaris L.). All eighteen bean cultivars inoculated in this study were susceptible. Bean mild mosaic virus was seed transmitted in three bean cultivars with a range of 3.3 to 5.0%. Seed infection was also demonstrated in Michigan grown bean seed lots. Several bean cultivars exhibited more severe symptoms when BMMV and bean yellow mosaic virus (BYMV) were inoculated together compared with single infection by either viruses. This is the first report of BMMV affecting beans in Michigan and the United States. To my parents for their love and support 111 ACKNOWLEDGEMENTS I wish to express my sincere appreciation to my major professor, Dr. Alfred Saettler for being so helpful and supportive throughout the course of this study. He created a very enjoyable atmosphere for working and study. Special thanks are also extended to Dr. Donald Ramsdell and Jerri Gillet for their friendship and technical assistance with the serology aspects of this study. I also thank the other members of my graduate committee Drs. James Kelly and Karen Klomparens for their helpful suggestions during the preparation of this manuscript. Special thanks to Joe Clayton who accompanied me on the disease survey in 1989. Thanks also to Instituto de Investigaciones Agropecuarias (INIA) (CHILE) for the financial support during my study leave. Finally I would like to thank Wendy Whitford, Jill Pendergrass, Barry Stein, Rodrigo Hoyos, and all those friends who directly or indirectly helped me complete my studies. iv TABLE OF CONTENTS PAGE LIST OF TABLES ..................................... Vii LIST OF FIGURES .................................... viii INTRODUCTION ....................................... 1 LITERATURE REVIEW Identification of plant viruses ............... 3 Bean mild mosaic virus (BMMV) ................. 5 Seed transmission of plant viruses ............ 8 Mixed infection by plant viruses .............. 10 MATERIALS AND METHODS Identification of bean mild mosaic virus (BMMV) as a contaminant virus in bean yellow mosaic virus (BYMV) studies Mechanical transmission .................. 12 Electron microscopy ...................... 13 Serology ................................. 13 Immunosorbent electron microscopy (ISEM) . 14 Mechanical inoculation and host range .......... 15 Double antibody sandwich ELISA (DAS-ELISA) 16 Seed transmission of BMMV ...................... 18 Detection of seed infection by BMMV in Michigan grown bean seeds ............................. 18 Distribution of BMMV and BYMV in Michigan dry bean fields O...OOOOOOOOOOOOOOOOOOOOOOOCOOOOOO 19 Synergistic effect of BMMV and BYMV on several bean cultivars 000.00.00.00...OOOOOOOOOOOOOOOO 20 RESULTS Identification of bean mild mosaic virus (BMMV) as a contaminant virus in bean yellow mosaic virus (BYMV) studies Mechanical transmission .................. 25 PAGE Electron microscopy ...................... 25 serOIOgy OOOOOOOOOOOOOOOOOOOOOOOOOOOIOOOOO 25 Immunosorbent electron microscopy (ISEM) . 32 Mechanical inoculation and host range .......... 32 Seed transmission of BMMV ...................... 38 Detection of seed infection by BMMV in Michigan grown bean seeds ............................. 41 Distribution of BMMV and BYMV in Michigan dry bean fields 0.0...OOOOOOOOOOOOOOOOOOOOOOO0.... 41 Synergistic effect of BMMV and BYMV on several bean cultivars 0.00....OOOOOOOOOOOCOOOOOOOOOOO 43 DISCUSSION ........ OOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOOOO 50 LIST OF REFERENCES .0...OOOOOOOOOOOOOOOOOOOOOOOOOO... 56 vi TABLE LIST OF TABLES Field survey for BMMV and BYMV in Bean Fields in Central Michigan, 1988 and 1989 ........ Symptoms in different species inoculated with W-MiCh 0..OOOOOOOOOOOOOOOOOOOOOO00...... Seed transmission of BMMV-Mich. in three bean cultivars OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOCOO Transmission of BMMV in lots of Michigan grown bean seed ........................... Distribution of BMMV and BYMV in field grown bean plants in three counties in central Michigan, 1988 and 1989 ........... Symptomatology and detection by OAS-ELISA of BMMV and BYMV in plants of different bean cultivars inoculated either with one or the other or both viruses ..................... vii PAGE 21 35 4O 42 44 48 LIST OF FIGURES FIGURE PAGE 1 Vein banding caused by BMMV-Mich. in bean cultivar Domino, 10 - 15 days after inocu1ation .0.00000000000000000000000...O 26 2 Mottling caused by BMMV-Mich. in bean cultivar Domino, 10 - 15 days after inoculation O...OOOOOOOOOOOOOOO0.00.0.0... 27 3 Transmission electron micrograph of Bean mild mosaic virus-Mich. virus particles, negatively stained with 2% ammonium molybdate. Bar represents 50 nm ......... 28 4 Distribution of particle size of BMMV-Mich. in bean IO...00.0.0000...OOOOOOIOOOOOOOOOOO 30 5 Typical reactions of BMMV-Mich. with homologous antiserum at a 1:4 dilution in agar double-diffusion tests. Wells a : healthy Domino bean; b : positive control, bean inoculated with BMMV; c : Domino bean inoculated with BMMV-Mich................. 31 6 Immunosorbent electron microscopy of BMMV-Mich. virus particles negatively stained with 2% ammonium molybdate. A : decorated and B : non decorated particles. Bar represents 100 nm ......... 33 7 Mild mosaic symptoms caused by BMMV-Mich. in Corsoy soybean, 10 - 15 days after inoculation ............................... 37 8 Moderate mosaic and curling of the leaves caused by BMMV-Mich. in Domino bean, 10 - 14 days after inoculation ................. 39 9 Moderate mosaic and mottling in Black Turtle Soup bean caused by BMMV-Mich., 10 — 14 days after inoculation .................... 45 viii PAGE 10 Local necrosis in Orfeo INIA bean doubly infected with BYMV (C-20 isolate) and BMMV-Mich., 7 - 10 after inoculation ...... 46 11 Epinasty caused by BYMV (C-20 isolate) in Domino bean, 7 days after inoculation ..... 47 ix INTRODUCTION Common bean (Baggeglug yglggrig L.) is an important food crop in many parts of the world, and is widely cultivated as a protein source. Beans are particularly important as a food crop in eastern Africa, Latin America and some Asian regions. Numerous diseases caused by different pathogens can affect bean crops and reduce yields. Virus diseases are among the most important, affecting beans worldwide. At least 70 different viruses have been reported to infect beans (Morales, 1986). Some of these viruses are of economic importance because of a direct influence on yield, others are considered as potentially damaging pathogens (Morales, 1986). Bean common mosaic virus (BCMV) and bean yellow mosaic virus (BYMV) are included among the most economically important viruses worldwide. Other viruses have been reported to be found in mixed infections with these or other bean viruses (Waterworth, 1981). Bean mild mosaic virus (BMMV) has been reported in bean from Central and South America and causes mild mosaic symptoms; BMMV can also produce a synergistic effect and cause severe mosaic symptoms when occurred in combination with other viruses, such as bean curly dwarf 1 2 mosaic virus or cowpea mosaic virus (Waterworth et a1, 1977). In mixed infections, BMMV could easily be overlooked because of the mild symptoms it usually produces (Waterworth et a1, 1977). Since BMMV is a highly stable 'virus producing mild foliar symptoms and is seed transmitted, it could easily become distributed over most bean producing regions of the world. The objectives of the present study were to determine the identity of a contaminant virus in greenhouse bean plantings, determine its host range, seed transmissibility, distribution in Michigan bean production areas and determine the possible synergistic interactions with bean yellow mosaic virus in different bean cultivars. LITERATURE REVIEW Identification of plant viruggs Various procedures are useful for identification of plant viruses, and include such things as variation in symptoms, host range, methods of transmission, physical properties, interactions with other viruses, serological tests, electron microscopy and chemical composition. Each of these procedures provides information to determine the identity of a specific virus (Walkey, 1985). Host range studies reveal the infection capacity of the virus. Some viruses, including BYMV, have very large host ranges which may include several species and genera; other viruses have a very narrow host range, and are usually restricted to certain species. Serological methods have been used extensively in viral characterization and determining relationships among viruses. The great value of antigen-antibody reactions is due to the specificity of reaction: an antibody will combine only with an antigen which contains groupings of amino acid sequences similar to those causing antibody formation in an animal (Ball, 1974). The introduction of agar diffusion techniques, immunosorbent electron microscopy (ISEM) (Derrick, 1973) and enzyme linked immunosorbent assay (ELISA) (Clark and 3 4 Adams, 1977) has allowed routine testing for a number of viruses. Frequently a combination of these three techniques are used for virus identification (Ball, 1974). Agar diffusion techniques allow visual determination of the virus as a precipitation line. There are two kinds of diffusion techniques, single and double, in which one or both reactants diffuse through a liquid phase in the gel (Crowle, 1973). The agar-gel double diffusion test (Ouchterlony test) was extensively used in the past, but has been replaced by more precise tests which require very small volumes of antiserum. Nonetheless, Ouchterlony tests are still used. because of easy set up (Crowle, 1973) and because a visible antibody-antigen reaction is obtained (Van Regenmortel, 1982). The recently developed ELISA is extremely sensitive and is increasingly used to detect viruses that occur in low concentrations (Clark and Adams, 1977). A virus- specific conjugate of antibody enzyme is used to indicate virus presence by the appearance of coloration of a substrate added at the end of the test. The enzyme substrate is hydrolyzed and a bright yellow color indicates a positive reaction. Frequently, a spectrophotometer is used to quantify color as absorbance at wavelength 405 nm (Clark, 1981). The double antibody sandwich ELISA (DAS- ELISA), as developed by Clark and Adams (1977) has proven to be an economical, quick and sensitive assay that requires nominal basic laboratory skills. Enzyme linked immunosorbent assay (ELISA) has been used successfully by plant pathologists for diagnosis of virus diseases of perennial and vegetatively propagated crops such as trees and ornamentals; the test is also used to detect virus in seed (Clark, 1981; Clark and Bar-Joseph, 1984). Immunosorbent electron microscopy (ISEM) permits visualization of virus particles which are "trapped" by the antiserum. The use of ISEM can increase by 2 - 10,000 times the number of virus particles observed per grid as compared with other techniques not using antiserum as a trapping agent. The virus is visualized by its morphology in the electron microscope. Some advantages of ISEM are that a very small amount of antiserum is used, and results are rapidly obtained. Some of the disadvantages are that ISEM involves costly equipment, is labor intensive, requires skills with the electron microscope, and is not suitable for handling a large number of samples (Milne and Lesemann, 1984). Bean_mild_m9sais_xiru§ (BMMV) Bean mild mosaic virus was first reported in 1977 by Waterworth et a1 as occurring in a mixed infection with curly dwarf mosaic virus in plants of several bean cultivars (Enaseglug vulgaris L.) obtained from El Salvador, Central America. The virus was isolated from bean plants showing symptoms resembling those caused by bean common mosaic virus or bean golden mosaic virus. Plants also exhibited mosaic, leaf rugosity, curling and extreme dwarfing. The virus has recently been reported to induce mild mosaic symptoms in beans in Colombia (Jayasinghe, 1982). Waterworth et a1 (1977) and Jayasinghe (1982) indicate that BMMV has a very narrow host range which includes mainly legume species. Symptoms in bean (Phaseglus yulggrig L.) can vary from symptomless infection to barely discernible mild mosaic. Slight vein banding or roughening of the leaf surface has also been observed. The virus is easily transmitted mechanically and usually 100% of infection is achieved. Chrysomelid beetles mainly Diabmtiea sp-. gramme spa 3211191312; sp. and EXDQDQIQQIQLIQA sp. are efficient vectors of BMMV (Hobbs, 1981; Jayasinghe, 1982). No transmission has been demonstrated by other insects such as leafhopper, aphids and mites (Jayasinghe, 1982). Seed transmission has been found to occur in a range of 1 - 4% depending on the bean cultivar (Jayasinghe, 1982). Bean mild mosaic virus is composed of spherical particles containing RNA, 28 nm in diameter, sedimenting as a single component. It is a very stable virus in sap with a thermal inactivation point of 84°C, dilution end-point around 10"8 and a longevity in yitrg of 6 weeks at 22°C (Waterworth, 1981). Waterworth (1981) also stated that BMMV is not serologically related to 35 other spherical particle viruses, including 10 viruses usually associated with legumes. The virus is also extremely infectious in legumes and spreads rapidly among beans in the greenhouse, often without inciting symptoms. Virus transmission was readily demonstrated by root contact, when healthy plants were grown in "infested soil" from which most of the roots of infected plants were not removed or when healthy plants were grown in "non-cleansed" infested pots (Hampton and Hancock, 1981). Bean mild mosaic virus can occur in mixed infections with other viruses, such as bean curly dwarf mosaic virus, bean common mosaic virus, peanut stunt virus, cowpea mosaic virus and bean rugose mosaic virus (Waterworth, 1977: Hampton and Hancock, 1981). Little is known about the economic loss in beans due to infection with BMMV. Though infected plants grew normally under greenhouse conditions, and produced healthy- looking pods, flowering and pod formation were delayed about a week (Jayasinghe, 1982). Serology has been a reliable and easy method for detecting BMMV in the field (Jayasinghe, 1982). According to Morales (1986), BMMV is likely to be widely distributed throughout the bean-producing regions of the world because of its high infectivity, efficient vector transmission and seed transmissibility. WW Seed transmission is one of the most important means of inoculum dispersal for many plant pathogens. Seed dispersal also has the disadvantage of allowing pathogen survival for long periods, as well as allowing spread of the pathogen from one location to another. The intimate association between host and virus in the seed gives a high opportunity for infection to occur at early stages of plant development. Only a few plant viruses have been reported to be seedborne in beans, namely bean common mosaic virus (BCMV), southern bean mosaic virus (SBMV) cucumber mosaic virus (CMV) and bean mild mosaic virus (BMMV) (Morales, 1986). There are two means of seed transmission of viruses, in or on the seed coat, and in the embryo of the seed, the later commonly referred to "true seed transmission" (Walkey, 1985). Most viruses are found in the embryo. Bean common mosaic virus (BCMV) is a good example of true seed transmission. It has been detected internally in cotyledons and embryos but not from seed coats on beans (Ekpo and Saettler, 1974). Other viruses such as SBMV are transmitted both on the seed coat, as a contaminant, and internally in the embryo (Uyemoto and Grogan, 1977). The incidence of seed infection with viruses varies greatly with the virus and with the host species infected. Up to 100% of transmission has been found in seeds of individual soybean plants infected with tobacco ring spot virus (Baker and Smith, 1966). Seed transmission of BCMV can vary between 10 to 83% (Phatak, 1974). Other viruses such as BMMV have been shown to be transmitted in ranges of 1.2 to 3.6% in different gnaggglgg yglggzig L. cultivars. This percentage of seed transmission was considered low compared with other beetle transmitted virus in legumes, like cowpea mosaic virus which has 10% seed transmission in cowpea yigna unguigulata L.(Jayasinghe, 1982). Percentage of seed transmission is influenced by stage of plant development at the time of infection, differences in strain and cultivars and environmental conditions. The rate of seed transmission may also be influenced by the presence of other viruses in mixed infection (Allen, 1983). Kuhn and Dawson (1973) found that the incidence of seed- borne southern bean mosaic virus (SBMV) was 20% in the presence of COWpea chlorotic mottle virus (CCMV) as compared with only 12% in single viral infections. Studies with BCMV showed different levels of seed infection depending on age of host at inoculation; the highest incidence of seed transmission occurred when plants were infected at early stages (Schippers, 1963). Morales and Castano (1987) found maximum seed transmission of BCMV in plants inoculated at the primary leaf stage. Little or no infection was found on plants infected after blossoming. Even though the optimum stage of plant development for 10 seed infection by most viruses is prior to flowering, not all seeds of infected plants carry the virus. This is probably due to the fact that not all mega- and microspores are invaded by the virus (Shippers, 1963; Crowley, 1957). The irregular distribution of infected seeds within a pod has also been mentioned by several researchers. Nelson (1932), suggested that viruses are restricted to certain tissues in the plant. Seed transmission is one of the most important means of survival for some viruses, especially those with a narrow host range such as BCMV and BMMV. It'llfililll's Mixed plant virus infections are common in nature (Uyemoto gt g1, 1981). Many crops can be infected by more than one virus at the same time, as pointed out by Rochow (1972) who reported three different viruses simultaneously infecting sugar beet. Competition between mixtures of virus strains has been observed in plant protoplasts infected with variants of cowpea chlorotic mottle virus and raspberry ringspot virus (Carr and Kim, 1983). Plant diseases caused by double infection with two unrelated viruses may cause more severe and different symptoms than the symptoms caused by each individual virus. The virus concentrations may also be significantly altered compared to those in singly infected plants (Kassanis, 11 1963). Variations of virus host range and breakdown of vector specificity can also occur as a result of mixed virus infections. Several newly discovered plant diseases have been found to be caused by virus mixtures (Clark gt gt, 1980; Uyemoto gt g1, 1981; Khan and Demski, 1982). Bean yellow mosaic virus has been found together in beans infected with cowpea mosaic virus (CPMV) or cowpea severe mosaic virus (CPSMV) (Carr and Kim, 1983). Another example of double infection is found in soybean affected by soybean mosaic virus (SoyMV) and bean pod mottle virus (BPMV). Foliage distortion, chlorotic mottling, stunting, misshapen fruit, and necrosis have been observed in soybean fields doubly infected with both viruses; only mild mosaic symptoms are found in plants infected with the individual viruses (Lee and Ross, 1972). MATERIALS AND METHODS Idggtifiggtign of bean mild mosgig viggg (BMMV) gs g o t ' n vi 5 ' n ow o a' ' 5 st 'es. A host range study, to differentiate several BYMV strains included glyging mgx L.(Merr), Lgng gulingtig Medik., Nicotiang giutinosg L., gt tgsticg L., gt b um L., mm a is L., mm L., 171.331: misuleta L., Irifelium incl—Matti L., m mange L. and yigig tgbg L. During this study some bean and pea cultivars previously reported resistant to BYMV (severe strain) (Herrera and Sepulveda, 1986) developed mosaic symptoms. At the same time control plants inoculated with just buffer showed curling of the leaves, mosaic and mottling symptoms. These plants, as well as the resistant cultivars, were used to isolate and identify the contaminant virus. Mechanical transmission Control (non-inoculated) plants of Domino bean cultivar showing mosaic symptoms were used to isolate the virus. Leaf sap was prepared from these plants by grinding leaves showing symptoms 1:10 (w/v) in a cold mortar and pestle with 0.01 M phosphate buffer, pH 7.0. The primary leaves of 7-day-old bean plants were rub-inoculated with 12 13 the sap solution using a sterile foam rubber sponge. The leaves were dusted with 300 mesh carborundum prior to inoculation. Plants were then maintained under greenhouse conditions with a temperature range of 25° to 35°C. Electron microscopy A negative staining procedure was used to examine plant tissue for virus particles with transmission electron microscopy. Leaf sap dips of bean leaves exhibiting mild mosaic symptoms were prepared by chopping a 1 cm square of infected leaf in a 30 ul drop of 2% ammonium molybdate on a glass slide. A drop of the suspension was placed on carbon-coated Parlodion-filmed grids for 1 minute. Grids were then blotted with filter paper and examined in a Philips 201 transmission electron microscope operated at 60 RV. Serology Identity of the virus was determined using gel double diffusion tests in agarose gel containing of 0.8% (w/v) agarose with 0.1% (w/v) NaN3 and 0.85% (w/v) NaCl. Antisera against the following viruses were used in these tests: bean mild mosaic virus (BMMV), bean pod mottle virus (BPMV), black gram mottle virus (BCMV), cowpea chlorotic mottle virus (CCMV), cowpea mosaic virus (CPMV), cowpea severe mosaic virus (CPSMV) and southern bean mosaic virus (SBMV). Antisera to BMMV and SBMV were kindly provided by Dr. Francisco Morales, CIAT (Colombia) and the remainder by 14 Dr. Rose Gergerich, University of Arkansas. The different antisera were diluted 1:2 and 1:4 (v/v) in 0.85% NaCl solution (normal saline). Virus-containing sap was obtained from non-inoculated Domino bean plants showing mild mosaic symptoms. One gram of infected leaves was ground with a sterile mortar and pestle in 10 ml of 0.01 M phosphate buffer and aliquots placed in the different wells. Positive controls consisted of sap from infected leaves with the known virus pathogen, while negative controls consisted of sap from healthy bean leaves. Immunosorbent electron microscopy (ISEM) Antiserum for BMMV was obtained from Dr. Francisco Morales (CIAT). A further modification of the Derrick technique (1973) as modified by Milne and Luisoni (1977) and Haufler and Fulbright (1983) was used for ISEM. Freshly prepared carbon-coated Parlodion-filmed 300 mesh grids were floated on 30 ul of a 1:500 dilution of BMMV- antiserum in 0.06 M NaZHPO4 - KH2P04 buffer at pH 7.0 (ISEM buffer) to coat grids with specific antiserum. Drops of antiserum were placed on parafilm placed in a petri dish containing moistened filter paper. Grids were incubated at 37°C for 3 hrs, then rinsed twice for 10 min in ISEM buffer. Grids were then briefly blotted with filter paper and floated in 30 ul drops of plant extract obtained by grinding 0.5 - 1 gr of infected bean leaves in 3 - 5 m1 of ISEM buffer. Grids were incubated overnight at 4°C. After 15 incubation grids were briefly blotted with filter paper and negatively stained with 2% ammonium molybdate, pH 7.0. To enhance visualization of virus particles, most grids were coated a second time (decorated) with antiserum. For decoration, grids incubated overnight were blotted with filter paper and then floated on a 30 ul drop of a 1:500 dilution of antiserum for 1 - 3 hrs at 4°C. Grids were then blotted with filter paper and negatively stained with 2% ammonium molybdate. All grids were examined in a Philips 201 transmission electron microscope (TEM) operated at 60 KV. Mgcngnicgi inocglation gng hgst zangg Virus inoculum was prepared by grinding systemically infected Ehgsggigs yglggtig L. ‘Black Turtle Soup’ leaves 1:10 (w/v) with cold 0.01 M phosphate buffer, pH 7.0 in a cold sterilized mortar. Sap was rub inoculated with sterile foam rubber sponges onto leaves previously dusted with 300 mesh carborundum powder. Control plants were inoculated with buffer instead of sap. The host range included the following species: Chenopodium amaranticoio; Coste & Reyn, Chenopodium ggiggg Willd., Glycine mg; (L.) Merr cultivars Corsoy, Hobbit and Hodgson 78, Gomphzgng glgtggg L., Lgng cgiinatis Medik. cultivars Araucana INIA and Tekoa, Nicotiana glutinosa L., N. rustica L., N. tgbgggm L. cultivar Xanthi, Phasgglus iunatgs L. cultivar 16 Henderson Bush, Eisum ggtiyum L. cultivars Alaska, Burpee Sugar Snap and Early Perfection, Ititgiigm igggtngtgm L., Irifelium eretense L., Elsie fees L. cultivar Minor and yiggg gaggiggigtg L. cultivar Blackeye. The following Ehagggiug xuiggtig L. cultivars were also used: Amanda, Black Turtle Soup, Blanco INIA, Blue Lake Stringless, common cranberry bean (cultivar not known), Domino, Great Northern 31, Great Northern 123, Isabella, Michelite 62, Montcalm, Olathe, Orfeo INIA, Pinto UI 114, Redkloud, Stringless Green Refugee, Top Crop and Tortola INIA. All plants were grown from seeds in 15 cm diameter sterile clay pots with greenhouse soil. With small seeded plants such as clover or Niggtigng sp., the seeds were sown in a nursery bed and the seedlings were transplanted into pots when plants were 2 cm in height. A minimum of 10 plants were inoculated for each species or cultivar and 5 plants were used as controls. The plants were kept for 15 - 20 days under greenhouse conditions with temperature range of 25° - 35°C. The presence of the virus was confirmed by use of double antibody sandwich ELISA (DAS-ELISA). Double antibody sandwich ELISA (DAS-ELISA). The DAS-ELISA procedure described by Clark and Adams (1977) was followed. All ELISA solutions or plant sap were added to the ELISA plates at 200 ul/well and then sealed in plastic bags during incubation to prevent evaporation. Purified IgG, diluted to a concentration of 1 ug/ml 17 (with 0.05 M sodium carbonate buffer at pH 9.6, coating buffer), was added to a Immulon R 1, 96-well flat bottom polystyrene ELISA plates (Dynatech Laboratories, Inc, Chantilly, VA 22021). Plates were incubated at 37°C for 4 hr and then rinsed three times in phosphate-buffered saline (0.02 M phosphate buffer plus 0.15 M NaCl at pH 7.4 containing 0.05% Tween 20 (PBS-Tween)). Bean leaf extracts were prepared by grinding leaf tissue 1:10 (w/v) with an electric tissue grinder (Tissumizer, Tekmar R Model No. SDT 1810) in extraction buffer. Extracts were added to the plates in duplicate and plates incubated overnight at 4°C. The plates were again rinsed with PBS-Tween as above except several rinses were used as needed to completely remove all traces of plant material from the wells. Enzyme conjugated IgG was diluted 1:800 with PBS-Tween containing 2% (w/v) polyvinylpyrrolidone (PVP) and 0.2 % (w/v) egg albumin (extraction buffer), and added to the ELISA plates. After 4 hr incubation at 37°C, the plates were rinsed 3 times in PBS-Tween as above. The substrate p-nitrophenyl phosphate was diluted to 1 mg/ml in 0.97% (v/v) diethanolamine substrate buffer, pH 9.8, and added to the plate. The reaction was allowed to develop at room temperature for 15 to 35 min until negative controls (background) started to increase in absorbance. All plates were read in a Dynatech R mini-reader MR 590 for absorbance at 405 nm. Microelisa A sample was scored positive if the absorbance reading was twice that of the healthy controls. 18 5 'ss' n o B To determine whether BMMV-Mich. is seed transmitted, 7-day-old bean plants of cultivars Orfeo-INIA, Black Turtle Soup and Domino were mechanically inoculated on the primary leaves with BMMV-Mich. Plants were grown to maturity in the greenhouse and seeds harvested. Periodic pesticide applications were used to control mites, white flies, thrips and powdery mildew. The seeds from each cultivar were harvested and planted individually in 10 cm diameter sterile clay pots with greenhouse soil. Plants were observed for a period of 5 weeks after which symptoms were recorded and serology tests (OAS-ELISA) were conducted. 0‘ ‘ o 0 Sue ' ‘ '01 ' 9 Fiche ' w 10:! 0 0.11 Thirty lots of navy and black bean seeds were obtained from the Michigan Department of Agriculture and used to determine the presence of BMMV in seed. One hundred seeds of each lot were planted into vermiculite contained in sterile aluminum trays and kept under greenhouse conditions for 5 weeks. Evaluation of seed transmission was made on the basis of symptomatology and electron microscopy (EM). For electron microscopy observations, a bulk sample of leaves from the plants of each seed lot was used to 19 prepared leaf dips which were negatively stained with 2% ammonium molybdate. Grids were observed in a Philips 201 transmission electron microscope at 60 KV. To analyze the amount of seed transmission of BMMV, one seed lot was selected at random from those showing virus particles as determined from the EM study. One hundred seeds were planted in individual 10 cm diameter sterile clay pots containing greenhouse soil. Plants were kept under greenhouse conditions for 4 weeks, after which virus was assayed in individual plants by DAS-ELISA. 0 s 'b tono MINad : ,1 4 'on a. an; f’ (1 During the summer of 1988, a field survey was conducted in dry bean fields in the area of Munger, Saginaw County, and Tuscola county,Michigan. Plant samples were obtained from several bean cultivars located in separate fields. Samples were collected at random from plants showing mosaic symptoms, mottling, stunting. In 1989 a second survey was performed in bean fields from 4 counties (Clinton, Saginaw, Tuscola and Gratiot). Samples were obtained at random and consisted of 5 - 20 leaves from individual plants at each site: leaves with and without symptoms were collected. A total of 30 samples were collected for the first year and 500 the second year. Individual samples were analyzed by OAS-ELISA to detect the presence of BMMV and BYMV, using the methodology 20 described previously. Table 1 summarizes the sites sampled in 1988 and 1989. er ' t e f c o B ' seve a ean cultivazs Plants of bean cultivar Black Turtle Soup, individualy infected with either BYMV (C-20 isolate) or BMMV-Mich. were used as sources of inoculum. Inoculum was prepared by grinding leaves systemically infected with either viruses~ in 0.01 M phosphate buffer, pH 7.0 (1:10 w/v) in a sterile chilled mortar. Possible synergism was studied by inoculating plants with a mixture of both viruses compared with each individual virus. Such inoculum was prepared by grinding equal amounts of infected leaves obtained from plants infected with either viruses in a concentration 1:10 (w/v) in 0.01 M phosphate buffer, pH 7.0 in a sterile chilled mortar. Sap containing the individual viruses or the mixture was rub-inoculated with a sterile foam rubber sponge onto the primary leaves of 7-day-old bean seedlings. The leaves were previously dusted with 300 mesh carborundum. Ten plants for each bean cultivar were inoculated with the different viruses or the mixture and five plants for each cultivar were inoculated only with buffer as controls. All plants were kept under greenhouse conditions for 15 - 20 days with a temperature range of 25° - 35°C. Sixteen of the eighteen bean cultivars mentioned in 21 m was muuoflm : = mm\ma\m m ea add Ho cacao = = mm\md\m m 8:.mos «nommz = : mm\ma\m ea c: cooaumoam = = mm\ma\m oH pos.moa ooommH : = mm\ma\m m was eooemz g = mm\ma\w OH mOE HOHMHuOm uOHm SOHMOmOM Sm: 3MCflDmm mw\md\w .flm HOHCOU on m: synoncmuo xummm\.om smacsx = mm\an\s .om souumsw>wq cm a >>mz \.om panama = am\an\s ma o> anymocmuo = = am\am\s .om as m: s>az upszmox.om Eocene coucflao mm\an\> m moa.m «nouns .om muumaoz\mnau= naoomss mm\~a\m m pos.moa suumncnuo .om Hammmzm : mm\~a\m oH uos.moa >>mz .om ummmm>xmnauz : ww\-\m m was >>az .om ouumfloz aaoomss mm\~a\m H MOE EHOQCOHU : 8 mm\NN\m a hoe aaouusoz uon suuommom am: 3ocfimmm mm\~a\m magi: H a s>nz \.om muumHoz «accuse mm\~a\m m >.uo& mocwa « mcfiommnm uon nouommom sz 3mcwomm mm\~a\m moamaou uo>wuaao vcwamamm no no no Honasz uaoumahm onus comm QOAHMOOA aucaoo mama .mmma can mama .cmmsnofiz Hmuucmo cw madman :mmm cw >=>m can >z=m you >o>usm cause H manna 22 o N mOE Hana—Hm . Um CHOOCMJ \.om oemuo uoHumuo mm\m\m ow ms umsoHusmz Hmuz\.om soon = mm\m\m 0H 03 UCMHUflZ : : mm\m\m oH a «sound = = mm\m\m oH m: muuon = = mm\m\m oH mos umsoHusm: = = mmxmxm oH as Hoosmm o¢u=\.om umcumo = mm\m\m m oa.mH ooHomz = = mm\m\m oH moa.m: Hanan = = mm\m\m oH mos «moemz menz\.om Hocumo = mm\m\m cu m: s>mz ummm men: mHoomse mm\m\m m nos «nommz uon anaconda om: amcham mm\mH\m on was omuo .om meson ooom = mm\mH\o om mc.ss HmaoHusm: .um meson omov = mm\mH\m om unoHcs m as onto \aucmm oHoco = mmme\m oH moa.mc Hmcsm asoucmHucH = amme\m on was umumummm .um nusosuuom = mm\mH\m om moa.mc umumummm .nm uschs\Hmu: = mm\mH\m H am onto .om som=\om uHoz = mmmexm om m: omuo .om uanns «Hoomas mmmexm m was monmmz = = mmme\m m moa onwaoo uon nuuoomom am: 3mcfivmm mm\mH\m moamaom um>HuHsu onwamaou no no mo Monasz maoumahm maha comm cowumooa mussou oumo u.ucoo " H mHnms vcfisoaaoa u x can mfimouoaso :Ho> u u> Hoammoa oum>om u Em “maoumsxm o: u m: «woodwofluoo ucmwuuzs u o: «onwauuos n HOE “GammOE u moa Howmmos UHHE u as “cowummcon mama n ma “hummcfiam u 0 « 23 mam .3909 oH mos Hammad : = mm\m\m 0H mos ocflaoo uon nuuoumom sz zonammm mm\m\m on m: umuaummm .nm mHHH>>chmum = mm\m\m .om >oscfix cu m: umumummm \.om umHoz mHoomse mm\m\m OH m2 .HO3OHH%ME .. : mw\m\m 0H s mmoemz = = mm\m\m oH mos mom H z = = mo\m\m oH m: mmeemm = = mm\m\m oH m: omuo = = mm\m\m oH m: mooomx = = mm\m\m oH mos onmz xumHm .om odmuo uoHumuo am\m\m moamemm um>fluasu onwamsmu Ho Ho no Honszz maoumshm onus comm cowuouoq xussou mama o.ucoo " H mHnms 24 the host range study, were used in this experiment to inoculate them with the different viruses or the mixture. Plants were observed up to 20 days after inoculation. Symptoms considered as a susceptible reaction included any degree of mosaic, leaf curling, stunting, epinasty or necrosis. All test plants were assayed for the presence of both viruses by DAS-ELISA. RESULTS Idgntification o: bean miid mosgic virus (BMMV) as a ontam' v'rus b a e ow as 'c vi 5 BYMV gtudigs. Mechanical transmission. A virus was successfully transmitted by mechanical inoculation to plants of the Domino bean cultivar. At 10 days after inoculation inoculated plants showed mild mosaic symptoms, mottling, vein banding or mild curling of the leaves (Figure 1 and 2). Electron microscopy Grids were observed at 45,000 and 70,000 X magnification and a large number of spherical particles which measured approximately 28 nm in diameter were found in leaf dips from infected Domino plants (Figure 3). ‘ The size distribution of 100 virus particles is shown on a histogram, and the modal size for the particles was 28.5 nm (Figure 4). Serology A positive reaction, consisting of a precipitin line, was only found between the BMMV-antiserum and the sap obtained from Domino infected plants (Figure 5). The clearest precipitin lines were observed at 1:4 dilution of 25 26 Figure 1 : Vein banding caused by BMMV-Mich. in bean cultivar Domino, 10 - 15 days after inoculation. 27 Figure 2 : Mottling caused by BMMV-Mich. in bean cultivar Domino, 10 - 15 days after inoculation. 28 Figure 3 : Transmission electron micrograph of bean mild mosaic virus-Mich. virus particles, negatively stained with 2% ammonium molybdate. Bar represents 50 nm. 30 .sessions.”a.H.as”:332223.333”3223232233.“. 22232222.”.sagas”: S d — d d d - 1 ‘ d .— d 0 O 0 O O 6 5 4 3 2 mm_o_toa Co 83:52 particle size (nrn) f BMMV in lZ€ 0 Figure 4. Distribution of particle s bean 31 Figure 5 : Typical reactions of BMMV-Mich with homologous antiserum at a 1:4 dilution in agar double- diffusion tests. Wells a : healthy Domino bean; b : positive control, bean inoculated with BMMV, c : Domino bean inoculated with BMMV-Mich. 32 the antiserum. No reactions were observed in tests with sap from healthy leaves. When sap containing BMMV-Mich. was placed in adjacent wells with sap from BMMV infected bean leaves, there was fusion of precipitating lines without spur formation, indicating that the two antigens were closely related if not identical. Immunosorbent electron microscopy (ISEM). The confirmation of BMMV as the causal agent of the symptoms observed on bean was by ISEM. A large number of particles were trapped by BMMV-antiserum. Decorated particles were easily and rapidly detected. Figure 6 shows a comparison between decorated and non-decorated particles of the virus. A distinctive dark halo is found in decorated particles, which corresponds to an additional amount of staining as a result of the virus-antiserum reaction. e 'ca ’ a o d st . The reactions of the different species to BMMV-Mich. are shown in Table 2. The results indicate that the virus has a very narrow host range. All bean cultivars were susceptible to the virus with a wide range of symptoms. Among other species, BMMV infected the three soybean cultivars (gigging mg; (L.) Merr) and the Alaska pea cultivar (Eignn ggtiynn L.). The symptoms in soybeans and beans included mosaic and curling of the leaves (Figures 7 33 Figure 6 : Immunosorbent electron microscopy of BMMV- Mich. virus particles negatively stained with 2% ammonium molybdate. A : decorated: B : non decorated particles. Bar represents 100 nm. 34 35 Table 2 : Symptoms in different species inoculated with BMMV-Mich. SPECIE COMMON NAME SYMPTOMS Qheaeeedism amarantiseler Caste & Reyn~ ' Qheaenedisu guinea Willd. - gigging max L. Merr soybean Corsoy mos, mot Hobbit mos, mot Hodgson 78 mos, mot Seunhrena slsbeaa L. - Lgng gnlingtig Medik. lentil Araucana INIA - Tekoa - Nisetiana.zlu£in2§a L. - EIQQIIEBQ rustica L. - Niggtigng tgtggnn L. tobacco - Enasgglus lnngtng L. lima bean Henderson Bush - Elena satires L. Pea Alaska latent Burpee Sugar Snap - Early Perfection - Itijgiinn inggtngtnn L. crimson clover - Itifiglinn ntatengg L. red clover - yigig {gtg L. faba bean Minor - Vigng unguiculata L. cOWpea Blackeye - 36 Table 2 : cont'd. SPECIE COMMON NAME SYMPTOMS W missile L. common bean Amanda mm Black Turtle Soup mod mos,vb Blanco INIA mod mos,cur1 Blue Lake Stringless mm common cranberry bean mod mos Domino mod mos,curl,vb Great Northern 31 mod mos,vb Great Northern 123 mm Isabella mm Michelite 62 mod mos,vb Montcalm mm Olathe mod mos,ln Orfeo INIA mod mos,mot,curl Pinto UI 114 mod mos Redkloud mm Stringless Green Refugee mm,curl Top Crop mm Tortola INIA mod mos * curl - curling of leaves, latent - no symptoms, however virus was detected by DAS-ELISA, 1n - local necrosis, mm - mild mosaic, mod mos - moderate mosaic, mos - mosaic, mot - mottling, vb - vein banding, - - no symptoms, and absence of the virus confirmed by DAS-ELISA. 37 Figure 7 : Mild mosaic symptoms caused by BMMV-Mich. in Corsoy soybean, 10 - 15 days after inoculation. 38 and 8). Ten to fourteen days after inoculation, the first trifoliolate of bean plants usually showed a mild vein yellowing or vein banding that later developed into mild to moderate chlorotic mosaic. The symptoms were usually slightly more severe in the second trifoliolate. As the plant matured, symptom severity was reduced in some cultivars and only a very mild mosaic was visible, as in the case of Isabella, Montcalm and Blue Lake Stringless cultivars. In other cultivars (Domino, Black Turtle Soup and Orfeo INIA) mosaic symptoms remained moderate to severe. Virus presence was confirmed by DAS-ELISA in tissue taken from all bean cultivars plus soybeans and Alaska pea. No symptoms were observed in Alaska pea even though the virus was present in the tissue: this was considered a latent infection. S d s o 0 Healthy looking pods and seeds were collected from inoculated bean plants and seed transmission of BMMV-Mich was demonstrated in all three cultivars. After 5 weeks very mild mosaic or no symptoms were observed in plants of the different cultivars, but the virus was detected by DAS-ELISA in all of the cultivars. Table 3 summarizes the percentage of seed transmission found in the different cultivars which ranged from 3.3% in Black Turtle Soup to 5.0% in Orfeo INIA. 39 Figure 8 : Moderate mosaic and curling of the leaves caused by BMMV-Mich. in Domino bean, 10 - 15 days after inoculation. 40 Honucou conconCHICOG no some ounau can» Manon: moz 05Ho> uuccnuomno can: o>nunmom oonoonmcou whoa madmamm m¢.H u some unm.H u xca H¢¢.H u one .osmmnu nonconcn non msHo> musmnnomng we. u some “mo. u xoa “No. u one .Honucoo couoowcnlcoc Hon mmsHm> ousmnnomn< .muonumnsm no cannonsocn ans mH Henna Sam «a no coon monac> ousmnuomno «ea momma pmuoswauom no Manes: on» Bonn pmuoasoaou no: conuunamscuu comm no va ommucounom «« «mHHmumao an omuomumc mauH> 4 Ho.H om.H NH. oo.m m cm on «HzH omnho om. hm. ea. hw.m m mad ova OGnEOQ om. mm.a ma. cm.m m om OOH mzom .8 xOMHm «we coo: .xcz .sn: « omuuoncn monam> oucmnuomno «scanmwna mucoam omuocnanoo poucoam nuaao mnc>nuaau coon omnnu an .20n=|>:zm no sonmmnamccnu 000m H m manna 41 I-t- ., -, ‘--- '1 e i- w? =I T Tchio. - own bean §.e_6Q§ Seed infection and seed transmission of BMMV were found in 11 of the 30 bean seed lots tested. Only mild mosaic or yellowing were observed in some plants of specific seed lots after 5 weeks under greenhouse conditions. These symptoms were not always correlated with the presence of virus particles as demonstrated by electron microscopy. The number of virus particles found in leaf samples from different seed lots varied from only a few to many (Table 4).‘ Table 4 shows that seed transmission was ~ found in 9 of 20 navy bean seed lots and 2 of 10 black bean seed lots. A 2% incidence of seed transmission was found in navy bean seed lot 921011, a seed lot chosen at random from those showing virus particles in leaf dips. The presence of the virus in these plants was confirmed by a positive OAS-ELISA test, even though plants showed no symptoms under greenhouse conditions even after 5 weeks. 8 ' 'o o B nd YMV 'c an d b an 'e ds Bean yellow mosaic virus was detected in all 30 samples collected in 1988, but BMMV was not detected in the same samples. The following season, 1989, both viruses were found in field grown bean plants but the incidence was very low. From 508 samples collected during 1989, BYMV was 42 Table 4 : Transmission of BMMV in lots of Michigan grown bean seed Seed lot Seed type / BMMV particles* Symptoms** cultivar 921011 navy few 921360 black/Midnight Black - 921361 navy/Seafarer few 921362 navy/Fleetwood - 921363 navy - 921368 navy - 921369 navy/Bunsi - 921375 black/Black T Soup - 921378 black/T-39 - 921380 navy - 921388 black/Midnight Black many 921389 black/Midnight Black few 921393 navy/B-155 - 921395 navy/C-20 - 921557 navy - 921610 navy few 921718 navy/Aurora few 922142 navy/Seafarer few 922144 navy/C-zo - 922176 navy few 922185 navy/Seafarer - 922186 navy - 922210 black/Black T Soup - 922313 navy - 922314 navy/Seafarer few 922417 navy few 922433 black - 922434 navy few 922446 navy - 922459 navy - virus particles observed in leaf dips, few = less than 10 particles/field: many = more than 10 particles/field. ** mm a mild mosaic, - = no symptoms. 43 found in seven while BMMV was only present in five samples. Table 5 sumarizes the distribution of viruses among the counties and indicates that both viruses were never present together in the same plant, but they were found in the same field as in the case of a navy bean lot in Clinton county, (Dewitt Rd./Livingston Rd.) (Table 1). Also tested for the presence of BMMV and BYMV were twenty samples of weeds collected in 1988 and 1989, in and around virus infected bean fields. Included among the samples were plants identified as: buckhorn Plantain (Blantagg lanssolata). common milkweed (Asslseias sxriasa). lambsquarter (Qngngngginn ginnn), Pennsylvania smartweed (Polygonum pensxlxanisum). prostrate pigweed (Amaranthus glitgiggg). All samples tested negative for the presence of virus. S e 's ' B nd v sultixars After 7 days, plants inoculated with either BMMV-Mich. or BYMV (C-20 isolate), or both exhibited different degrees of mosaic, epinasty, mottling, leaf necrosis and/or stunting (Figures 9, 10 and 11). Analysis by OAS-ELISA showed that cultivars Amanda, Blanco INIA, Great Northern 31 and Tortola INIA were immune to BYMV when infected alone but not to BMMV (Table 6). Some synergistic effects were observed in plants of Amanda and Great Northern 31 cultivars inoculated with both 44 Honucoo couooncH -co: no some monzu can» noswnn onus mosHm> mosonnomnm coca o>HuHmoa oonooncoo onoa moHaEmm mo.H I come .OH.H I sue .oo.H I :Ha .>=wm um¢.H I come .o¢.H I xms .¢¢.H I :H5 .>22m HdmmH "on. I some .mo.H I xma .om. I CHE .>z>m "mmmH NoammHu pouoonsn non moSHo> oocmnnomnw no. I come .oc. I xma .59. I :Ha ”no. I coca .eo. I xma .oo. I cHa ”mmaH ”Honusoo pouooncnucos non moaHm> oocmnnomom hHo>Huoommon .>=Mm can >22m non oumnumnam no conuoodosH :Ha on no mH nouns admo¢< um omen mosHm> oosmnnomom as on» no oosomona * mmOH mm. Hm. wH. m - noSOthwz oHoomSH mm. mm. mm. - H %>uc mHoomSH ow. mm. mH. H - o~-o uHoomde on. an. HH. - H s>uc amaHmmm ow. «N. NH. H - onnon smanwom ma. aw. mm. H - «momwz amchmm mH. mH. «H. mH. mH. SH. H H h>mc couCHHu mmoH mm. mm. mH. N - onond< «Havana nu. m¢.H SN. m - hnnuncmno oHoomfie an. mo.H HN. m - h>oc «HoomzH on. mm. mm. H - annooflmno smanwmm cs. oh. Hm. m - moaHH manooonn 3msnwcm mm. mm. mm. H - aHmousoz accchm mon some .xma .sna some .xma .sHa >zwm >zzn NuMm Nana mmmmflmmmmm nm>HUH50 no as moaHu> coconnomom z>m can >z=m no sonusnnnumHa m oHomH 45 Figure 9 : Moderate mosaic and mottling in Black Turtle Soup bean caused by BMMV-Mich., 10 - 15 days after inoculation. 46 Figure 10 : Local necrosis in Orfeo INIA bean inoculated with BMMV-Mich. and BYMV (C-20 isolate), 10 - 15 days after inoculation. \f" 47 Figure 11 : Epinasty caused by BYMV (C-20 isolate) in Domino bean, 7 - 10 days after inoculation. 48 Table 6 : Symptomatology and detection by DAS-ELISA of BMMV and BYMV in plants of different bean cultivars inoculated either with one or the other or both viruses. Cultivar Blants inoculgtgd with BMMV BYMV BMMV + BYMV * ** Amanda + ns - ns + + mot,mos Black T. Soup + vb,mos + se,s,mos + + se,s,mos Blanca INIA + mot,vb - ns + - mm Blue L. Stringless + ns + e,mm,s + + s,mm common cranberry bean + mm + mos + + mos Domino + vb,mos + se,mos,s + + s,se Gr. Northern 31 + mm - ns + + mot,mos Gr. Northern 123 + mm + ns + + mm Isabella + ns + ns + + mm Michelite 62 + mos,vb + s,mos,mot + + s,e,mos Montcalm + mos + mos + + mos Orfeo INIA + mos + sm,e + + sm,s,ln Pinto UI 114 + vb,mos + mot + + mos,mot,e S. G. Refugee + mm + e,s,mos + + e,mos,ln Top Crop + mm,vb + mm + + s,e,1n Tortola INIA + mm - ns + + mos * + - presence of virus detected by DAS-ELISA - - virus not detected by DAS-ELISA Absorbance values for DAS-ELISA for both viruses ranged from .60 to 1.44; non-infected tissue ranged from .03 to .09;infected tissue ranged from .98 to 1.45. ** e - epinasty; ln - local necosis; m - mos; mm - mild mosaic; mot - mottling; ns — no symptoms; 3 - stunting; se - severe epinasty; sm - severe mosaic; vb - vein banding. 49 viruses. Mottling and mosaic symptoms were observed in doubly infected plants while singly infected plants showed no symptoms. Both viruses were recovered by OAS-ELISA from doubly infected plants while only BMMV was recovered from single infections. Cultivar Amanda showed no symptoms, however. Expression of symptoms was more severe in doubly infected plants compared with single infection for some cultivars (Orfeo INIA, Stringless Green Refugee and Top Crop) (Table 6). No symptoms were observed on Isabella cultivar up to 15 days after single inoculations, however both viruses were detected on leaves by OAS-ELISA. DISCUSSION BMMV was confirmed as the causal agent of the symptoms observed in non-inoculated control plants and resistant bean cultivars by the use of ISEM, gel double diffusion and DAS-ELISA tests. These techniques have been successfully used in the identification of several different viruses (Milne and Luisoni, 1975; Milne and Lesemann, 1978: Roberts and Harrison, 1979; Lesemann, 1982: Haufler and Fulbright, 1983). Further studies revealed the presence of BMMV in some of the BYMV isolates. This was determined by the use of leaf dips from plants infected with several isolates of BYMV. The different morphology of the virus particles allowed an easy identification of both viruses. The BYMV is a flexous rod that measures approximately 750 nm long and~15 nm wide. This virus belongs to the potyvirus group which includes a number of different viruses (B05, 1970). BYMV is considered one of the most important viruses in bean production and has a worldwide distribution (Morales, 1986, Schwartz and Galvez, 1980). Bean mild mosaic virus was readily transmitted by mechanical inoculation to bean plants, transmission to 100% of inoculated plants was obtained in most inoculations. Similar results were observed by Jayasinghe (1982) in the 50 51 transmission of BMMV-CIAT. The virus particle size of BMMV—Mich. was found to be around 28 nm in diameter, which corresponds well to the size mentioned by Waterworth gt g1 (1977): BMMV-CIAT particles were slightly larger having an average size of 32.2 nm in diameter (Jayasinghe, 1982). No relationship was found between BMMV-Mich. and other antisera tested, which supports the results of authors who were unable to place BMMV into any of the five serogroups of the comoviruses (Waterworth gt Q1, 1977: Jayasinghe, 1982). Waterworth (1981) also mentioned that BMMV is not serologically related to ten other spherical viruses that are usually associated with legumes. Moreover, Morales and Gamez (1989) indicated that even though BMMV has similar morphology and physicochemical properties to other viruses, it has not yet been included into any of the recognized plant virus group. Bean mild mosaic virus host range was found to be very narrow among different species. However, all bean cultivars inoculated were found to be susceptible to the virus even though symptoms were difficult to see in some plants. No local lesion host or resistant cultivar was found. These results agree with those of Jayasinghe (1982). In contrast to the results found by Waterworth gt g1 (1977), BMMV-Mich. infected Alaska pea (Eignn ggtiynm L.), causing a latent infection detected only by DAS-ELISA. 52 BMMV-Mich., did not infect Qngngngginn gningg Willd. and anpnzgng glgbggg L. Experiments conducted by Jayasinghe (1982) at CIAT (Colombia) demonstrated that BMMV-CIAT had a host range similar to the one found in this study, even though he did not inoculate pea. The differences in host ranges between the present study and that of Waterworth gt g1 (1977) could be explained by different environmental conditions according to Jaysinghe (1982). Seed transmission of BMMV-Mich. was higher than that. reported by Jayasinghe (1982) for BMMV-CIAT. BMMV-Mich. was seed transmitted in a range of 3 - 5% in different bean cultivars. This percentage could be considered low by other authors who mentioned 10% seed transmission for other beetle transmitted viruses (Shepherd, 1964). Seed infection was found in bean seed lots grown in Michigan and 2% of seed transmission was achieved in one of these seed lots. This is the first report of seed infection by BMMV under natural conditions in Michigan and United States. The virus has been reported infecting beans in Central and South America (Waterworth gt gi, 1977: Schwartz and Galvez, 1980: Morales, 1986) and as a contaminant virus in greenhouse studies in Corvallis, Oregon (Hampton and Hancock, 1981). The fact that BMMV was found in field grown seed lots suggests that the virus is well distributed among the bean producing areas of Michigan. The percentage of seed transmission from field infected seed was lower than that 53 obtained under experimental conditions, but even a low incidence of transmission could allow establishment of primary inoculum sources in the field. Moreover, the Mexican bean beetle, an efficient vector, is commonly found in many Michigan bean fields. In addition there also exists the possibility that BMMV could survive in infected crop debris from one season to the next (Hampton and Hancock, 1981), which could constitute another source of inoculum. Economic losses due to BMMV have not been determined; however the presence of the virus in the seed could represent a potential problem under Michigan field conditions. In addition, the inability for easy detection of seed transmitted virus infection due to the very mild mosaic or absence of symptoms in some cultivars, could be a problem in attempting to eliminate the primary inoculum. Of concern is, the synergistic interactions between BMMV and other viruses which could represent a cause for economic losses in bean fields (Waterworth gt g1, 1977; Morales, 1986) Seed transmission of BMMV could constitute a real problem in efforts to breed for disease resistance at Michigan State University. This is specially true because the symptoms in bean plants could be confused with those associated with BYMV or BCMV. The fact that the limited screening of bean cultivars for reaction to BMMV showed no resistance could also further complicate breeding efforts 54 if BMMV is a contaminant of BYMV or BCMV inoculum sources. (Dr. James Kelly, personal communication). Results obtained in this study confirmed the presence of BMMV in bean fields in Michigan. The virus was found in bean samples collected from three different counties in 1989. BMMV was not present in any sample collected in 1988 possibly because of the small number of sites sampled. The results indicate that BMMV is most likely to be found in navy bean fields. However, in light of the seed transmission studies and infectivity of the virus in other cultivars, the virus could easily be found in any other cultivars, so further studies are necessary to adequately address this question. Bean yellow mosaic virus was present in all samples in 1988 but only in few of 1989. This situation could be due to a sampling effect or to the dry conditions in 1988 which may have favored greater numbers of aphid vectors and therefore greater disease spread. Climatic conditions were more humid and temperate in 1989. The fact that BMMV and BYMV were never found together in the same plant in the field in this study, does not rule out the possibility that both viruses could affect bean plants under field conditions. The evidence that BMMV was present in some BYMV isolates is possible support for this. Bean mild mosaic virus has been mentioned before as causing severe mosaic symptoms in mixed infections (Waterworth gt gi, 1977). This was confirmed in this study 55 when several bean cultivars showed more severe symptoms when BYMV (C-20 isolate) and BMMV were inoculated together. Examples of such synergistic effects were cultivars Amanda and Great Northern 31 cultivars inoculated with both viruses. Bean yellow mosaic virus was able to infect these bean cultivars in the presence of BMMV but not when BYMV (C-20 isolate) was inoculated alone to the same cultivars. Other more severe reactions were also observed in some cultivars when both viruses were together in comparison with single infection, as observed in Orfeo INIA and Pinto UI 114. The real economic importance of BMMV for beans under Michigan conditions could be an interesting topic to study in the next few years, considering the seed transmission, synergistic effects and vector-virus relationship of this new pathogen. LIST 0? REFERENCES LIST OF REFERENCES Allen, D.J. 1983. The Pathology of Tropical Food Legumes. Disease Resistance in Crop Improvement. John Wiley and Sons Ltd. New York. 413pp. Baker, K.F. and S.H. Smith. 1966. Dynamic of seed transmission of plant pathogens. Ann. Rev. 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