Prevalence and Molecular Characterization Coat Protein Gene of Tobacco streak virus Causing Peanut Stem Necrosis Disease in Coastal and Rayalaseema Districts of Andhra Pradesh, South-India

Groundnut (Arachis hypogaea L.) is an annual oil seed legume crop, widely grown in tropical and semi-arid tropical regions of the world. It commonly cultivated for edible oil, vegetable protein, fodder, medicinal and cosmetic products. It is also used to improve soil fertility by fixing atmospheric nitrogen. Groundnut is grown in an area of 26.4 M. ha with a total production of 46.1 M.M.T (million metric ton) (USDA, 2020). China and India are the world’s largest groundnut producers, accounting for 38 percent and 11 per cent, respectively. India ranked second in production with 5.5 M.M.T of shell groundnuts (USDA 2020).  In India, Gujarat ranked first in groundnut production, followed by Rajasthan, Andhra Pradesh and Karnataka (APEDA 2018-19). However, the groundnut productivity was very low in India as compared to world groundnut productivity, Major constraint for not attaining higher productivity of groundnut was due to biotic (viral and fungal diseases), abiotic conditions and rain fed nature of crop.
 
Among the viral diseases infecting groundnut, peanut stem necrosis disease (PSND) caused by Tobacco streak virus (TSV) is a major constraint for groundnut productionin south Indian states. Thrips species serves as vectors for TSV and carry the infected pollens from weed hosts to crop plants. TSV cannot spread by seed from infected plants to healthy groundnut plants under Indian conditions (Vemana and Jain, 2010). Parthenium hysterophorus weed was a non-symptomatic host and considered as principal source of TSV-infected pollen leading to disease epidemics in nearby crops (Prasada Rao et al., 2003). TSV on groundnut was first reported in Ananthapuram district of Andhra Pradesh (A.P.), India, TSV caused an epidemic on groundnut crop in 2000 resulting in a loss estimated to the tune of Rs.300 crore (Reddy et al., 2002). Since then, the occurrence of PSND has frequently been reported in A.P (2005-14.9%, 2006-21.0%, 2007-11.4%, 2011-7.9%, 2013-4.4%) (Anonymous 2005, 2006, 2007, 2011 and 2013). Later, its occurrence was detected in the adjacent groundnut growing states of Karnataka and Tamil Nadu (T.N). TSV belongs to the family Bromoviridae and the genus Ilarvirus having quasi isometric shape. TSV genome has RNA 1 (3.5 kb), RNA 2 (2.9 kb) and RNA 3 (2.2 kb) (Fauquet et al., 2005). RNA 1 encodes for RNA dependent RNA polymerase responsible for virus replication. RNA 2 encodes the nonstructural 2a protein necessary for viral replication and an overlapping 2b protein. RNA 3 genome encodes for coat protein (CP) and movement protein (MP). PSND is a serious problem of groundnut cultivation in A.P. However, studies on incidence and spread of TSV confined to Rayalaseema (Anatapuramu, Y.S.R. Kadapa, Kurnool, Chittoor) districts of A.P.

Hence, present study was taken up for the first time to identify the incidence and spread of TSV on groundnut in coastal and Rayalaseema districts A.P. Further, molecular characterization of coat protein (CP) was made for the test isolate by comparing with other TSV isolates originated from different hosts and locations.

Glasshouse facility and molecular laboratory studies were carried out at Agricultural Research Station, Kadiri, All India Coordinated Research project on Groundnut (AICRP-G) centre, A.P.
 
Survey
 
Roving survey was conducted in major groundnut growing districts of A.P during kharif 2017-18 to know per cent disease incidence of PSND. TSV infected groundnut plant samples were collected from different locations of A.P. (Table 1). Randomly five spots selected in each field by walking across the field and counted the number of TSV suspected plants vs total number plants per square meter area. Later, the percent disease incidence was calculated using following formula:
 

 
Diseased leaf samples from all surveyed locations were stored in plastic bags and brought to the laboratory. The suspected TSV samples exhibiting characteristic symptoms such as small necrotic rings on leaf, severe necrosis on leaf lamina, petioles and plants with peg necrosis, were collected and tested by direct antigen coating enzyme linked immunosorbent assay (DAC-ELISA) using TSV specific antisera (Clark and Joseph, 1984) supplied by International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).
 
RT-PCR
 
The TSV positive samples confirmed by DAC-ELISA were once again tested by reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from the collected leaf samples using RNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA). First strand cDNA was synthesized using revert aid first strand cDNA synthesis kit (Thermo fisher) according to manufacturer’s instructions. Synthesized cDNA was subjected to PCR using primer pair TSV1 (forward) (5'-ATGAATACTTTGATCCAAGGTCCN-3') and TSV2 (5'-TCAGTCTTGATTCACCAGGAAAN-3') (reverse) specific to CP gene of TSV. The PCR amplifications were carried out in thermal cycler (Eppendorf master cycler gradient). The reaction mixture contained 2 μl of cDNA template (50-60 ng), 1 μl each of 10ìM forward and reverse primers, 5 μl of 10 X PCR buffer, 3 μl 25 mM MgCl₂,1 μl of 10 mM dNTPs,0.5 μl of Taq DNA polymerase (5U/µl) and added RNase free water to make up final volume up to 50 μl. All the above reaction components were procured from Thermo scientific company. PCR profile consists of initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 45sec, annealing at 50°C for 45 sec and extension for 1 min at 72°C. Final extension was allowed for 30 min at 72°C (Daliyamol et al., 2019). Subsequently PCR product was run on 1 per cent agarose gel for 1 hour at 50 volts and product of ~700 bp was amplified in all the samples (Fig 2).
 
Maintenance of TSV culture
 
The representative groundnut TSV isolate collected from Vetapalem in Prakasam district A.P. (TSV-GN-VPIND) was selected for further study by maintaining on cowpea (Vigna unguiculata cv. PUSA KOMAL) by following standard sap inoculation method in glass house at 25±2°C. Five to eight seeds were sown in each plastic pot (4" diameter) allowed to germinate. Seven day old seedlings with uniform growth were selected for inoculation. Infected as well as healthy tissues from field samples were macerated separately using sterilized chilled pestle and motor in 0.1M phosphate buffer (pH 7.2, 1:1 w/v) containing 0.1% mercaptoethanol as an extraction buffer (EB). The sap was kept on ice till the inoculation was completed. The collected sap was rub-inoculated on surface of young leaves of 7-day-old cowpea seedlings previously dusted with celite (diatomaceous earth). The excess inoculum was washed off with distilled water after three min and kept in the insect-proof glass house. Inoculations were preferably carried out in the evening. The inoculated seedlings were observed for symptom development. Typical symptoms of necrotic lesions observedon cowpea after three days’ post inoculation (PDI) and subsequently TSV culture mass multiplied by transferring single lesion to fresh cowpea seedlings. Similar symptoms were observed on healthy groundnut seedlings upon re-inoculation using above TSV culture and same was used for further molecular study.
 
Molecular characterization of CP gene of TSV
 
The TSV-Vetapalem (TSV-GN-INDVP) groundnut isolate from A.P. was characterized on the basis of coat protein (CP) gene to confirm its molecular identity with already reported TSV isolates. The extracted total RNA from artificially inoculated cowpea leaf was used as template for RT-PCR amplification of CP using primer pair TSV1 and TSV2. In RT-PCR, amplicon of ~ 700 bp was amplified and it was gel-purifed (Gene JET, Fermentas, India) and sent for sequencing facility at Bangalore (Eurofins Genomics Bangalore India). Low quality nucleotide sequences of coat protein (CP) gene of TSV-GN-INDVP trimmed both ends in chromas (version 2.6.6). Trimmed sequences assembled using bioedit (version 7.0.5.3). Assembled sequence of partial CP gene (443bp) first evaluated using BLASTN program from the NCBI website (www.ncbi.nlm.nih.gov) to know the identity and homology with other reported TSV isolates. The partial sequence obtained was deposited in the GenBank. For further analysis, reference sequences of CP gene sequences of TSV available in database were downloaded from NCBI website (Table 2).  Cucumber mosaic virus (CMV) was used as an out-group member. Partial CP gene of TSV was compared with thirty reported TSV isolates from different parts of the world infecting oilseeds, vegetables, pulses, ornamentals and weed host. All the sequences were aligned using MUSCLE algorithm of MEGAX (Version 10.1.1). The Phylogenetic tree for CP gene was constructed using aligned sequences with 1000 bootstrap replicates following neighbor-joining phylogeny of MEGAX (Sudhir et al., 2018).

Roving survey
 
Extensive roving survey was conducted during kharif 2017-18 to record the incidence of PSND in groundnut crop from 12 districts of Andhra Pradesh (Table 1). TSV incidence was observed in 8 out of 12 districts surveyed. PSND incidence was observed in all Rayalaseema districts (Anathapuramu, YSR Kadapa, Kurnool and Chittoor) of A.P. In coastal A.P, only 4 districts (Krishna, Guntur, Sri Pottisriramulu Nellore and Prakasam) showed PSND incidence and no incidence were observed in the remaining districts (Srikakulam, Vizianagaram, Visakhapatnam and West Godavari). During survey typical symptoms of TSV were observed on groundnut crop viz., severe necrosis on leaf lamina, necrotic streaks on petioles stem and peg necrosis, necrotic spots on pods and stunted growth with axillary shoot proliferation (Fig 1). Maximum TSV incidence was recorded from Bathalapalli (22.2%) and minimum incidence was in Mulakalacheruvu (4.1%). Thirty-eight out of seventy-two ground nut samples collected from different locations during survey in A.P were found positive by DAC-ELISA. The absorbance values (A405 nm;1 h) ranged from 0.09 -2.24 (Table 1). During survey predominant weed host observed around the surveyed fields were mentioned in the Table 1. Positively tested DAC-ELISA samples once again confirmed by RT-PCR assay and got amplification using coat protein (CP) gene specific primers (Fig 2).




 
In India, TSV was first recorded on sunflower in Karnataka in the year 1997 (Annual Progress Report 1997; Singh et al., 1997) Later, incidence of TSV was first observed on groundnut in 2000 (Reddy et al., 2002) at Ananthapuramu district of A.P and since then it has been frequently recorded in A.P. on groundnut (2005-14.9%, 2006-21.0%, 2007-11.4%, 2011-7.9%, 2013-4.4%) (Anonymous 2005, 2006, 2007, 2011 and 2013). Subsequently, TSV expands its host range by infecting 16 different crop species, 4 ornamentals and 11 weed hosts (Krishna Reddy et al., 2017). Recently Sunil Kumar et al., 2018 surveyed in Ananthapuramu and Kurnool districts for TSV infection in agricultural and horticultural crops in A.P and recorded 9-28 per cent in groundnut, 6-18 per cent in sunflower and 5-22 per cent in cucumber.
 
In the present study, the PSND incidence was observed whenever alternative weed host Parthenium hysterophorus exists around the field in most surveyed places (Table 1). Parthenium is widely distributed and is a symptom less carrier of TSV. It plays a major role in perpetuation and spread of the disease by supplying infected pollen as TSV is pollen borne (Prasada Rao et al., 2003). Screening technique was developed to screen groundnut genotypes using parthenium infector border growing around the screening plot in advance to groundnut crop that result in high disease pressure of PSND (90.0%) (Vemana et al., 2016). Kanchan et al., 1980 also suggested that large quantities of parthenium pollen are air borne and could move significant distances. In some areas disease incidence is low because farmers have acquired knowledge on the spread of disease and removed Parthenium well advance to groundnut crop.
 
TSV symptomatology in glasshouse
 
The representative TSV-GN-INDVP groundnut isolate was sap inoculated on seven-day old cowpea seedlings in glasshouse and expressed typical symptoms of TSV within the 3-5 days DPI. Symptoms appeared on leaves include viz., chlorotic rings, venal necrosis, necrotic rings, necrotic spots on systemic leaves (Fig 3). Systemic infection was observed on young leaves after seven DPI.
 


Molecular characterization of CP gene of TSV-INDVP
 
Total RNA isolated from TSV-GN-INDVP groundnut isolate maintained on cowpea and got amplification of ~700 bp by RT-PCR (Fig 4). Amplified product sequenced bidirectional and obtained sequence deposited in the GenBank (Accession No MW014340). The partial CP of TSV-GN-INDVP groundnut isolate shared wide range of nucleotide identities (80.72-98.62 %) with isolates reported globally. Present study isolate shared 97.97-98.51% nucleotide identities with groundnut isolates and 97.51–98.62% nucleotide identities with other crop TSV isolates from India. Among Indian isolates, TSV-CA-INDTPT (KC683810) showed the highest nucleotide identity (98.62%) with present study TSV-GN-INDVP groundnut isolate. On the other side, isolate TSV-GN-INDVP shared relatively less nucleotide identity with other country isolates (80.72-88.84%). Among other country isolates TSV-FB-SUD (TSV-Vicia Faba) (AM933669.) shared highest nucleotide identity (88.84%) with isolate TSV-GN-INDVP. The nucleotide-based phylogenetic analysis showed all Indian TSV isolates clustered together irrespective of crop and location which indicated that the CP gene of Indian TSV isolates is highly conserved. Earlier studies also showed that the CP gene of TSV isolates occurring in India is highly conserved (Bhat et al., 2002, Krishna reddy et al., 2003, Jain et al., 2005, Daliyamol et al., 2019). On the contrary, all other country’s TSV isolates clustered together. Isolate TSV-GN-INDVP closely clustered with TSV-CO-INDCO (TSV-Cotton) isolate from Coimbatore. All the TSV isolates clustered separately to that of the CMV isolate as it is out group member (Fig 5).

All previous survey reports for TSV confined to Rayalaseema districts (Anathapuramu, Y.S.R. Kadapa, Kurnool, Chittoor) of A.P and there is no information about incidence of PSND in coastal Andhra Pradesh. The present study recorded PSND incidence for the first time from four coastal A.P. districts (Krishna, Guntur, Sri Pottisriramulu Nellore, Prakasham) with confirmation using DAC-ELISA and RT-PCR. The molecular characterization of TSV-GN-INDVP groundnut isolate revealed that TSV population in Indian subcontinent is highly conserved irrespective of hosts and locations based on CP gene sequences. The genetic information of TSV-GN-INDVP groundnut isolate from A.P can be used in the development of coat protein mediated resistance program in groundnut due to its highly conserved nature over the years. Overall the results on survey indicated incidence of PSND identified in coastal A.P. and control measures have to be taken to avoid epidemic occurrence on groundnut in future.

I was highly indebted to the Acharya N.G. Ranga Agricultural University, Guntur for the financial support for my PhD work carried out at Agricultural Research Station, Kadiri.