RESEARCH REPORT
FIRST REPORT OF Mungbean yellow mosaic India virus INFECTING COWPEA IN INDONESIA
Sari Nurulita Sri Hendrastuti Hidayat
DEPARTEMENT OF PLANT PROTECTION FACULTY OF AGRICULTURE
IPB UNIVERSITY
2023
LEMBAR PENGESAHAN LAPORAN SINGKAT PENELITIAN
Title : First Report of MYMIV on Cowpea in Indonesia Authors : Sari Nurulita, Sri Hendrastuti Hidayat
Funding : AUSAID Funded Economic Cooperation Work Program of the ASEAN–Australia–New Zealand Free Trade Agreement and
Department of Plant Protection, Faculty of Agriculture, IPB University Date and Location : April 2012, Central Java
Bogor, 31 January 2023
Act of Dean Head of Department of Plant Protection
Faculty of Agriculture
Prof Dr Ir Suryo Wiyono, MAgr Dr Ir Ali Nurmansyah, MSi
NIP 196902121992031003 NIP 196302121990021001
Abstract
Yellow mosaic disease has been reported on legume for the first time in 2008 in Java. In 2012, yellow mosaic disease was found infecting cowpea (Vigna unguiculata) in Central Java.
Molecular detection was done using degenerate primers for begomovirus. Cloning strategy was conducted to cover full targeted region (partial replicase gene and partial coat protein).
Sequence analysis showed begomovirus sequence from cowpea shared 93.2% – 94.6% identity with Mungbean yellow mosaic India virus.
Key words: begomovirus, cowpea, MYMIV, yellow mosaic disease
Begomovirus infection has been reported to cause yellow leaf curl disease on tobacco (Hidayat et al. 2006), chilli pepper (Hidayat et al. 2006), and tomato plants (Aidawati et al.
2005) in West Java and Central Java. Bright yellow, yellow with green spots, and green mosaic as characteristic of Begomovirus symptom (Fig 1) was observed in cowpea plants (Vigna unguiculata) during field survey to legumes growing areas in West Java, and Central Java in 2012. This type of symptoms was very similar with previous report on infection of Mungbean yellow mosaic virus, a member of Begomovirus, on yard long bean in India, Pakistan, and Thailand (Qazi et al. 2007; Ilyas et al. 2010; Naimuddin et al. 2011a). Recently, Nurulita et al.
(2015) reported similar disease on yard long bean in Java, Indonesia.
Figure 1. Yellow mosaic symptom associated with Begomovirus infection on cowpea in Central Java, Indonesia
To determine the identity of Begomovirus associated with yellow mosaic disease of cowpea, total nucleic acid was extracted from leaf samples by a method described by Doyle and Doyle (1987). Amplification of viral DNA was proceeded using universal primers for Geminivirus, pAL1v1978 and pAR1c715 (Rojas et al. 1993). Amplification was performed using incubation regime of 94C for 60 s once, followed by 35 cycles of 94C for 60 s, 50C for 60 s, 72C for 60 s then a final incubation of 7 min at 72C. The amplified DNA fragment was evaluated by electrophoresis with DNA marker using a 1.0% agarose gel. Specific viral DNA fragment of ca. 1600 bp was successfully amplified from symptomatic samples originated from Magelang, Central Java. The amplicon was purified using QIAquick PCR Purification Kit (Qiagen Inc., Valencia, California, US) then cloned into TOPO TA Cloning Kit for Sequencing (InvitrogenTM – Life Technologies Corp., Carlsbad, California, US). One clone, p61b, was selected and sent to Australian Genome Research Facility Ltd., Australia for nucleotide sequencing. The 1360 bp consensus sequences of Magelang isolate was used for further analysis.
5’- CATCCCTAGG CATATTTGAA GTCGTTTTTG TATCGGTGTA CACCGATTGC TTCTCTACCC CCCTATCGGT GTATCGGTGT ACTATATATA CTAGAGCTAT TAAAAGCCCA TAGGGGCACT CAGCTATAAT ATTACCTGAG TGCCCCGCGA CCGGTGTATT GGGGTTGCTT TAACTTTAAT GCTTTTT – 3’
Figure 2. Nucleotide sequences of the intergenic region of Begomovirus infecting cowpea in Central Java derived from p61b showing TATA sequences (red bold letters), repeat sequences (underlined letters), and the stem-loop region (blue bold letters).
Sequence analysis using BLAST program (National Center for Biotechnology Information, http://www.blast.ncbi.nlm.nih.gov) indicated that the virus isolate causing yellow mosaic disease on cowpea has the highest homology (99.9%) with MYMIV infecting yard long bean in Brebes (Central java) and Purwakarta (West Java). High sequence homology (93.2%
– 94.6%) was also observed with MYMIV isolates from India, Pakistan, Nepal, and Bangladesh, but the homology is low with MYMV isolate from India (Table 1). Further analysis of the nucleotide sequences of p61b revealed specific characteristics of geminivirus common region, i.e. TATA Box, repetitive sequences, and stem loop regions (Fig 2).
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