Mungbean Yellow Mosaic Virus in the AVRDC Mungbean ...

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164 International Consultation coordination by AVRDC of a biannual information bulletin/ newsletter on all aspects of MYMV research and breeding ▪ regular regional meetings to evaluate progress of MYMV research Multilocational Testing of MYMV resistant Materials The most agronomically promising IMMYMV-resistant mungbean materials developed by the different national agricultural research systems in South Asia and by AVRDC have been collected and multiplied. These materials now undergo multilocational testing in India, Pakistan, Nepal, Sri Lanka, and Bangladesh in the so-called 'hot spots,' where the disease is endemic. Preliminary results of this multilocational testing are reported elsewhere. Development of Molecular Markers for MYMV Resistance A cooperative project with the University of Minnesota is in progress to develop restriction fragment length polymorphism (RFLP) markers to locate MYMV resistance genes in mungbean germplasm. Progress on this project is reported elsewhere. Development of MYMV specific DNA Probes The various recommendations drawn up at the international workshop on MYMV, particularly on the host range and geographic distribution of MYMV, and on the variability of MYMV and its relationship with other geminiviruses affecting legumes, clearly showed the need for improved diagnostic tools for the detection of MYMV. Because of the difficulty in isolating and purifying the virus, polyclonal antisera are either not available or, if available, are nonspecific (Honda and Ikegami 1986, Ikegami and Shimizu 1988, Shimizu et al. 1987, Varma et al. 1992). Similarly, monoclonal antibodies (MABS) produced against specific geminiviruses, are known to cross-react with other geminiviruses (Macintosh et al. 1992, Swanson et al. 1992). Virus-specific DNA probes are, therefore, considered the most effective for the exact diagnosis of geminiviruses. A DNA specific probe has been produced against the geminivirus which has caused yellow mosaic of mungbean in Thailand (Chiemsombat 1992, Morinaga et al. 1990). This virus is, however, clearly distinct from the one affecting mungbean in the Indian subcontinent because of its transmissibility by sap (Honda et al. 1983, Honda and Ikegami 1986). The virus only occurred sporadically in Thailand in 1977-81 (Thongmeearkom et al. 1981) but after 1981 its presence in the country was never confirmed (Chiemsombat 1992). So far it is not known whether the Indian MYMV is related to or different from the Thailand MYMV.
Workshop on Mungbean 165 AVRDC, in cooperation with the University of Wisconsin, Madison, USA has developed a DNA probe against an isolate of MYMV from India. Mungbean leaf samples were collected in the spring of 1993 in Southern India from plants showing symptoms typical of MYMV infection. DNA was extracted from air-dried leaf samples by using a modification of the method described by Dellaporta et al 1983. DNA was amplified by polymerase chain reaction (PCR). Three primer pairs were used: two general primer pairs for whitefly-transmitted geminiviruses, i.e., one pair for the top and one pair for the bottom half of the DNA-A component and another primer pair specifically designed from the top half of the DNA-B sequence of the Thailand MYMV. PCRderived DNA fragments and the plasmid pBluescript II KS (+) were digested with PST 1 restriction enzyme. The DNA-A component was split into three DNA-A fragments by Pst 1. DNA and the plasmid pBluescript II KS (+) were litigated and transformed into competent E. coll. Selection for inserted plasmids was on TY agarose medium, containing IPTG, X-gal, ampicillin, and tetracycline. Four MYMV-specific clones, pIM 5, pIM 13, pIM 20, and pIM 9 were selected and identified by DNA sequencing. The first three clones contained fragments of the DNA-A, whereas pIM 9 contains a fragment of the DNA-B component of MYMV. It was shown that MYMV from India had 95% sequence homology with the MYMV from Thailand. However, restriction enzyme digests made from PCR fragments of geminivirus-infected Croton sparsiflorns and Acalypha indica showed that each contained a distinct geminivirus which, however, was not MYMV. These two hosts have been implied as other natural hosts of MYMV. The cloned DNA-A and DNA-B were used as templates to synthesize MYMVspecific DNA probes by labeling with digoxigenin-11-dUTP (Boehringer Mannheim). These will be used for determining levels of resistance, and for epidemiological studies, such as host range, geographic distribution, and presence of strains of MYMV. Positive signals have been obtained from yellow mosaic diseased mungbean, cowpea, and soybean from various regions in India and also from mungbean in Pakistan. With its commitment to intensify work on MYMV, AVRDC hopes to coordinate efforts of the NARS in the Indian subcontinent to develop high-yielding mungbean lines with stable MYMV resistance which will lead to increased acreage and sustainable production of this crop in the region and elsewhere in Asia. References Ahmad, M. 1975. Screening of mungbean (Vigna radiata) and urdbean (V. inungo) germplasm for resistance to yellow mosaic virus. Journal of Agricultural Research v.13(1):349-354. AVRDC. 1979. Mungbean. Asian Vegetable Research and Development Center, Progress report, 1978:71-91, 120-123, 141-143.
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