Molecular Variability of Yellow Mosaic Virus (YMV) of Blackgram in Southern India

that the begomovirus causing YMD of blackgram in southern India is explored to be as strains of MYMV and MYMIV .


INTRODUCTION
Blackgram [Vigna mungo (L.) Hepper] (2n=22) is one of the important pulse crops of the tropics and sub tropics. It is the third major pulse crop after chickpea and pigeonpea cultivated in the Indian sub-continent. India is a major producer of blackgram with 3.06 million tonnes production harvested from area of 5.60 million hectares (Department of Agriculture and Cooperation, GOI, 2018-19). Despite large area under blackgram cultivation in India, the productivity is very low due to many pests (e.g.: pod borers, sucking pests etc.) and diseases (e.g.: Alternaria leaf spot, Cercospora leafspot, powdery mildew, viral diseases etc.). Among all the diseases, YMV is the major problem and has the most damaging effect on the productivity of blackgram crop across the Indian sub-continent with annual estimated yield loss up to 100% under favourable environmental conditions (Varma et al., 1992;Varma and Malathi 2003;Sathees et al., 2019). Nariani (1960) was the first person ever to report the Mungbean Yellow Mosaic Virus (MYMV) disease of mungbean from the fields of IARI, New Delhi India. It is transmitted by whitefly (Bemesia tabaci) in a semi persistent manner. Symptoms are characterized by presence of bright yellow patches on leaves interspersed with green areas, complete yellowing and stunting of the plant. The infected leaves turn necrotic and the diseased plants usually mature later and had relatively few flowers and pods (Nene 1972;Singh et al., 1980;Rathi et al., 2002). As an obligate whiteflyborne pathogen, YMV has an extensive natural host-range that includes mungbean (Vigna radiata), blackgram (V. mungo), pigeon pea (Cajanus cajan), soybean (Gycine max), mothbean (Vigna aconitifolia) and common bean (Phaseolus vulgaris) causing yield loss of about $300 million by upsetting the physiological machinery of the host. The disease in southern Asia is caused by four distinct begomoviruses collectively known as the yellow mosaic viruses (YMVs); Mungbean yellow mosaic virus (MYMV), Mungbean yellow mosaic India virus (MYMIV), Dolichos yellow mosaic virus (DoYMV), Lima bean Yellow Mosaic Virus (LYMV) (Bhagyashree et al., 2017) and Horsegram yellow mosaic virus (HgYMV) (Qazi et al., 2007).
Of these MYMIV and MYMV are most important as it infect large number of legumes in India (Fauquet et al., 2003). MYMIV is more predominant in northern, central and eastern regions of India (Usharani et al., 2004), whereas MYMV occurs in peninsular and southern region of India (Karthikeyan et al., 2004;Girish and Usha 2005;Haq et al., 2011). Polymerase chain reaction with coat protein genespecific primers (RHA-F and AC abut) primers are used to detect MYMV in YMV infected blackgram which specifically amplifies a 900 bp product (Reddy et al., 2015). Viruses of the family Geminiviridae have characteristic twinned quasiicosahedral particles (1830 nm) that encapsulated the circular single-stranded DNA (Stanley et al., 2005). The YMVs are included in the begomovirus genus, being transmitted by the whitefly (Bemisia tabaci) and having bipartite genomes. DNA-A encodes proteins required mainly for encapsidation, replication and transcription regulation, whereas DNA-B for intra-and intercellular protein movement. The DNA-A and DNA-B components of begomoviruses have highly conserved intergenic common region (CR) which comprises stem-loop structure with the loop containing the invariant nano-nucleotide motif (TAATATTAC) that characterizes the origin of viral replication strand. MYMIV and MYMV isolates share only 82% sequence identity between them in the whole of DNA-A component justifying their separation into distinct species. Full length coat protein gene sequences are accepted by International Committee on Taxonomy of Viruses (ICTV) for provisional classification of begomoviruses and it is useful for species/ strain level identification (Fauquet et al., 2008).
Therefore, associations of YMV strains in blackgram was reported in southern India (Karthikeyan et al., 2004;Haq et al., 2011), a study was conducted to investigate and characterize coat protein gene of YMV infecting blackgram and to confirm that Blackgram yellow mosaic virus (BGYMV) from southern Indian states is an isolate or variant of Mungbean yellow mosaic virus (MYMV) or Mungbean Yellow Mosaic India Virus (MYMIV) with the aid of specific CP gene primers.

Selection of locations and sample collection
The experiment was conducted during rabi season of 2019 at Molecular Biology Laboratory, Department of Plant Pathology, Agricultural College, Bapatla, ANGRAU, Andhra Pradesh. Information regarding main blackgram growing locations in southern India was obtained from different Agricultural Research Stations (ARS), State Agricultural Universities (SAU's) and Krishi Vignan Kendra (KVK's) across four states viz, Odisha andhra Pradesh, Karnataka and Telangana of southern India and samples were collected to study the variability. A total of six blackgram samples showing typical yellow mosaic symptoms were collected from six different regions of the southern India and placed in ziplock plastic bags and transported in cold packs to Agricultural college Bapatla, snap-frozen in liquid nitrogen and stored in a -80C freezer until further use. Three samples from Andhra Pradesh i.e., from Tirupati, Bapatla and Lam farm and other three samples each in different states viz., Bengaluru (Karnataka), Bhubaneshwar (Odisha) and Madhira (Telangana) were collected. Samples from healthy plants were collected as controls in all places. The samples were named as Tirupathi isolate, Bapatla isolate, Lam farm isolate, Bengaluru isolate (Karnataka), Bhubaneshwar isolate (Odisha) and Madhira isolate (Telangana).

Total DNA extraction
The total genomic DNA from leaf tissues of healthy (control sample) and YMV-infected blackgram samples were extracted from leaves by the modified CTAB method (Murray and Thompson 1980). Infected plant material (0.5g) was ground in a pre-sterilized pestle and mortar with liquid nitrogen (-196) until a fine powder was obtained and transferred to sterile Eppendorf tube of 1.5 ml. To this, 1ml of pre-heated (65C) extraction buffer (100 mMTris (pH 8.0), 1.4 M NaCl, 50 mM EDTA, 2% CTAB, 1% PVP and 0.1% β-Mercaptoethanl) were added and incubated for 1 hour in water bath at 65C. The extraction mixture in tubes was centrifuged at 10,000 rpm for 10 min at room temperature (27C) and the supernatant was collected into Eppendorf tubes. To this, equal volumes of chloroform and Isoamyl alcohol (24:1) were added and incubated at room temperature for 10-20 min. Then these tubes were centrifuged at 10,000 rpm at 4C for 10 min, separated the supernatant into sterile Eppendorf tube of 1.5 ml and added 0.1 volume of 3M sodium acetate (pH 4.8) and 0.6 volume of ice-cold isopropanol were added and then incubated at -20C for overnight. After incubation, the tubes were taken out and centrifuged at 10,000 rpm at 4C for 20 min to pelletize the DNA. Supernatant was discarded and the DNA pellet was washed three times with 70% DNA alcohol mixture was centrifuged at 5,000 rpm at 4C for 5 min, supernatant

Molecular Variability of Yellow Mosaic Virus (YMV) of Blackgram in Southern India
was discarded, pellet was air dried and dissolved in 50 µl of sterile distilled water. The DNA samples were quantified using Nanodrop spectrophotometer (ND1000, USA).

Polymerase chain reaction and gel electrophoresis
Polymerase chain reaction (PCR) was performed in 25 μl of reaction mixture using 10X PCR reaction buffer, 25 mM of MgCl 2 , 10 mM of each dNTPs, 10 mM of each primer, 5 U/μl of Taq DNA polymerase (Thermo Scientific, Lithuania) and 100 ng of DNA template. The PCR amplification was performed in a Thermocycler machine (Bio-RAD T100, USA). The amplified PCR products were separated on 2% agarose gel in 1X TAE buffer at 110V by using gel electrophoresis. Specific primers used for amplification of YMV and their sequence were presented in (Table 1). The composition of the reaction mixture (25 μl) used in polymerase chain reaction (PCR) for DNA amplification of respective isolate are given in (Table 2). The migration pattern of the DNA fragments in the gel was noted by using gel documentation system (Omni Doc, Cleaver Scientific, UK) in an auto exposure mode.

Nucleotide Sequencing and construction of phylogenetic tree
DNA amplicons were excised from agarose gel, eluted, purified and sequenced by automated DNA Sanger's dideoxy chain-termination sequencing facility (Eurofin Genomics India Pvt. Ltd., Bangalore). Sequence assembling was analysed by using the software programme BioEdit version 7.0.5 (Hall, 1999). Later, the nucleotide homology searches for a partial sequence of the DNA-A segment of YMV isolates were done with the BLASTN sequence analysis of the NCBI. Phylogenetic tree was constructed from aligned sequences by using the neighbour-joining method in MEGA X (Molecular Evolutionary Genetics Analysis) software version 10.0 (Tamura et al., 2007) based on partial DNA-A segment coat protein gene sequences with 30 others known begomovirus causing diseases in pulses and other crops, downloaded from NCBI GenBank database (www.ncbi.org). Robustness of trees was determined by bootstrap sampling of multiple sequence alignment with 500 replications.

Symptoms of yellow mosaic virus disease in blackgram
YMV infected plants were identified based on visual symptoms such as small yellow round spots on early emerged leaves to yellow mosaic patches alternate with green colour leaf lamina on older leaves. Severely infected plants showed reduced leaf size with stunted growth and smaller number of pods showing yellow discolorations (Fig 1).

Phylogenetic analysis of partial DNA-A nucleotide sequences of MYMV and MYMIV
DNA was extracted from the infected blackgram samples

Molecular Variability of Yellow Mosaic Virus (YMV) of Blackgram in Southern India
whereas Tirupati isolate was successfully amplified with MYMV-CP-704 bp primer, indicating the fields in these areas are infected with Mungbean yellow mosaic virus (MYMV) strain (Fig 2). The synthetic primers designed for the partial DNA-A segment coat protein gene of MYMV and MYMIV amplified a specific product of 704 bp and 500 bp respectively (Fig 2). The partial nucleotide sequences of the DNA-A segment of MYMV and MYMIV isolates along with available corresponding DNA-A sequences of yellow mosaic virus's (YMV's) infecting pulses when subjected to the phylogenetic analysis (Fig 3) formed two distinct clusters. The DNA-A sequence of MYMV Tirupati isolate clustered with other available corresponding sequences for DNA-A of MYMV isolates. It also showed more relationship with DNA-A sequence of Horsegram yellow mosaic virus (HYMV) than other YMV's sequences. While DNA-A sequences of MYMIV isolates from Bengaluru, Guntur, Bapatla and Madhira clustered together with isolates Hyderabad, Guntur, Raipur, Sehore, Raichur, Jabalpur, Indore, Karnool and Kadapa. Whereas, isolate, Bhubaneswar clustered along with isolates Tamil Nadu, Bangladesh and Bengal. The coat protein gene is the most highly conserved gene in the family Geminiviridae. It is the sole structural protein of begomoviruses and has been shown to play a determinative role in the transmission of these viruses (Sharma et al., 2005). CP gene has traditionally useful for identification and classification of plant viruses (Mayo and Pringle, 1997). The International Committee on Taxonomy of Viruses (ICTV) accepts the classification of begomoviruses based on CP gene sequences, when full length sequences are not available (Rybicki, 1998). Reddy et al. (2015) observed six isolates causing YMD of blackgram in Andhra Pradesh are closely related to MYMIV (old world gemini viruses) than MYMV (New world gemini viruses). The YMV infecting blackgram and greengram in Telangana and AP is closely related to MYMIV rather than MYMV or other begomoviruses was reported by Banu (2014). Therefore, the present results with partial coat protein gene sequence alignment and construction of phylogenetic tree using CP gene sequences had clearly revealed that YMV infecting the blackgram crop is not an identical strain in southern India.

CONCLUSION
The present study showed that YMV infecting blackgram in different southern Indian regions is not a same strain. Our results showed that isolates collected from Bengalore, Madhira, Odisha, Bapatla and Lam farm were amplified with MYMIV specific primers and represents the MYMIV strain of virus, whereas the isolate collected from Tirupati was amplified with MYMV specific primer and represents the MYMV strain of virus. The partial nucleotide sequences of the DNA-A segment of MYMV and MYMIV isolates along with available corresponding DNA-A sequences of yellow mosaic virus's (YMV's) infecting pulses when subjected to the phylogenetic analysis formed two distinct clusters.