Infection of tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus with betasatellites, results in enhanced level of helper virus components and antagonistic interaction between DNA B and betasatellites.

Tomato leaf curl New Delhi virus (ToLCNDV) (Geminiviridae) is an important pathogen that severely affects tomato production. An extensive survey was carried out during 2003-2010 to study the diversity of begomoviruses found in tomato, potato, and cucurbits that showed symptoms of leaf puckering, distortion, curling, vein clearing, and yellow mosaic in various fields in different regions of India. Ten begomovirus isolates were cloned from infected samples and identified as belonging to the species ToLCNDV. A total of 44 % of the samples showed association of betasatellites, with CLCuMuB and LuLDB being the most frequent. The ToLCNDV cloned component DNA A and DNA B were agroinoculated on Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with or without betasatellites, CLCuMuB or LuLDB. The viral genome levels were then monitored by real-time polymerase chain reaction at different time points of disease development. Plants co-inoculated with betasatellites showed enhanced symptom severity in both N. benthamiana and tomato, as well as increases in helper viral DNA A and DNA B levels. The DNA B and betasatellites acted antagonistically to each other, so that the level of DNA B was 16-fold greater in the presence of betasatellites, while accumulation of betasatellites, CLCuMuB and LuLDB, were reduced by 60 % in the presence of DNA B. DNA B-mediated symptoms predominated in CLCuMuB-inoculated plants, whereas betasatellite-mediated leaf abnormalities were prominent in LuLDB-co-inoculated plants. Inoculation with the cloned components will be a good biotechnological tool in resistance breeding program.

Molecular Evidence for the Occurrence of Abutilon mosaic virus, A New World Begomovirus in India.

During an investigation in the year 2010, on the weed reservoir of begomovirus, Abutilon pictum showing bright yellow mosaic symptoms was observed in Udhagamandalam, Tamil Nadu, India. The complete bipartite genome of a begomovirus was cloned and sequenced which revealed association of Abutilon mosaic virus (AbMV). Nicotiana benthamiana plants inoculated biolistically with the concatemers generated through rolling circle amplification of the cloned DNAs were asymptomatic; however three out of nine plants showed presence of viral DNA A. A recombination event in the ORF BC1 with ToLCNDV DNA B (HM989846) was detected. This is the first molecular evidence of AbMV in India.

Infectivity analysis of a blackgram isolate of Mungbean yellow mosaic virus and genetic assortment with MYMIV in selective hosts.

Yellow mosaic disease in grain legumes in Indian subcontinent is caused by two important virus species viz. Mungbean yellow mosaic virus (MYMV) and Mungbean yellow mosaic India virus (MYMIV), belonging to the genus Begomovirus of the family Geminiviridae. The genomic components of a begomovirus causing yellow mosaic disease in blackgram in southern India were cloned and sequenced. Nucleotide sequence comparison of DNA A component shows the virus isolate to be a variant of Mungbean yellow mosaic virus:-(MYMV-[IN:Vam:05]). However, DNA B component of the present virus isolate has greater similarity (92%) to Mungbean yellow mosaic India virus. Agroinoculations of the viral clones produced typical yellow mosaic symptoms in blackgram and mungbean, severe leaf curl and stunting in French bean, similar to blackgram isolate of MYMIV. Blackgram isolates of both the virus species were only mildly infectious on cowpea, produced atypical leaf curl symptoms and not yellow or golden mosaic. In agroinoculations done by exchanging genomic components, symptom expression was seen only in French bean. In cowpea, blackgram and mungbean there was no visible symptoms though viral DNA could be detected by PCR.

Tomato leaf curl Bangalore virus (ToLCBV): infectivity and enhanced pathogenicity with diverse betasatellites.

Genomic components of a begomovirus isolated from tomato plants showing leaf curl and stunting symptoms in farmer's fields at Hessarghatta village near Bangalore, India, were cloned by rolling-circle amplification. The virus was identified as a variant of strain C of the species Tomato leaf curl Bangalore virus and designated as Tomato leaf curl Bangalore virus-C[India:Hessarghatta:2008], ToLCBV-C[IN:Hess:08]. The betasatellite isolated from these samples belongs to the betasatellite species Tomato leaf curl Bangalore betasatellite. ToLCBV-C[IN:Hess:08] induced severe symptoms in Nicotiana benthamiana and Solanum lycopersicum plants when co-inoculated with the cognate betasatellite, Tomato leaf curl Bangalore betasatellite-[India:Hessarghatta:2008], ToLCBB-[IN:Hess:08] and with two other non-cognate betasatellites, Cotton leaf curl Multan betasatellite-[India:SriGanganagar:2002] and Luffa leaf distortion betasatellite-[India:Luffa:2004].

Engineering Resistance Against Mungbean yellow mosaic India virus Using Antisense RNA.

Yellow mosaic disease of cultivated legumes in South-East Asia, is caused by Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) belonging to the genus Begomovirus of the family Geminiviridae. Efforts to engineer resistance against the genus Begomovirus are focused mainly on silencing of complementary-sense virus genes involved in virus replication. Here we have targeted a complementary-sense gene (ACI) encoding Replication initiation Protein (Rep) to develop resistance against soybean isolate of Mungbean yellow mosaic India virus-[India:New Delhi:Soybean 2:1999], a bipartite begomovirus prevalent throughout the Indian subcontinent. We show that the legume host plants co-agroinoculated with infectious constructs of soybean isolate of Mungbean yellow mosaic India virus [India:New Delhi:Soybean 2:1999] along with this antisense Rep gene construct show resistance to the virus.

Cowpea golden mosaic disease in Gujarat is caused by a Mungbean yellow mosaic India virus isolate with a DNA B variant.

It has long been assumed that cowpea golden mosaic disease (CGMD) in southern Asia is caused by a begomovirus distinct from those causing disease in other legumes. The components of a begomovirus causing CGMD in western India were isolated, cloned and sequenced. Analysis of the sequences shows the virus to be an isolate of Mungbean yellow mosaic India virus, but with a distinct DNA B component with greater similarity to components of a second legume-infecting begomovirus occurring in the region, Mungbean yellow mosaic virus. The clones of the virus were readily infectious to cowpea, mungbean, blackgram and French bean by agroinoculation. However, the wild-type isolate was shown to be easily transmissible by whiteflies between cowpea plants but not to blackgram and mugbean, suggesting that the insect vector plays a major role in determining the natural host range of these viruses.

An improved DNA isolation method and PCR protocol for efficient detection of multicomponents of begomovirus in legumes.

A relatively inexpensive protocol for the detection of genomic components of whitefly-transmitted begomoviruses in symptomatic legumes in the field is described. The method involves extraction with a modified CTAB buffer containing beta-mercaptoethanol upto 5% and sodium chloride concentration from 1.4 to 2.0M. Using this method PCR amplifiable DNA could be extracted from mature leaves of legume hosts rich in polyphenols, tannins and polysaccharides. The non-coding region and full-length DNA A, DNA B components of yellow mosaic viruses were consistently amplifiable from 97 samples, out of 136 tested in PCR reaction, employing primers specific for intergenic regions and full-length genome. The system is robust and the protocol is useful for the detection and identification of begomoviruses infecting grain legumes.