Occurrence of Bemisia tabaci Asia 1 in Association with BYVMV in Okra

Bhendi Yellow Vein Mosaic Virus (BYVMV) incidence caused by white of the The present investigation was carried out in three major okra-growing districts of Dharmapuri Dindugul on whitefly incidence and occurence. A field survey on these districts revealed that the mean whitefly population of 1.82 per plant was observed while the incidence of BYVMV in the Coimbatore district was 13 per cent. The least mean population was observed in the Dharmapuri district with a mean of 0.48 whiteflies per plant and BYVMV incidence of 15.75 %. In order whitefly collected from 8 locations of Tamil Nadu, and mitochondrial cytochrome subunit results that the whitefly belongs to Asia I genotype. Thus, the present study confirmed the presence of Asia 1 genotype in B. tabaci throughout Tamil Nadu okra growing regions.


INTRODUCTION
Okra, Abelmoschus esculentus L. (Moench), (Family: Malvaceae) is widely grown in tropical and sub-tropical regions of the world. It is an important vegetable component in the human diet due to its dietary fibers and is rich in magnesium folate, antioxidants, potassium, vitamins C, K1 and A (Hughes, 2008). The whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is the most devastating insect pest in agricultural crops worldwide (Brown et al., 1995, de Barro et al., 2000. It was first collected and described as Aleyrodes tabaci (Gennadius) from tobacco, Nicotiana tabacum L., in Greece in 1889. It was subsequently renamed (Russell, 1957) as B. tabaci and found across the globe in United States, Africa, Middle East, the Orient, Russia, China, Southeast Asia, and South America (Brown et al., 1995). Its geographical diversity and broad host range gave rise to several common names associated with host plants such as sweet potato whitefly, cotton whitefly, etc. Different populations of B. tabaci are morphologically undefined but display distinctive biological, physiological, and genetic variation, and thus are deemed a cryptic species complex (Boykin et al., 2007de Barro et al., 2011;Dinsdale et al., 2010;Tay et al., 2012). The B. tabaci complex consists of cryptic species that need to be separated and distinguished. As these cryptic species are morphologically indistinguishable, various molecular markers have been utilized such as RAPD PCR (Gawel andBartlett, 1993, de Barro andDriver, 1997), AFLP (Cervera et al., 2000), mitochondrial cytochrome oxidase gene subunit I (mtCOI) (Frohlich et al., 1999, Brown et al. 2000 and the ribosomal ITS1 nucleotide sequence (de Barro et al., 2000). The most widely accepted method is differentiation on the basis of nucleotide sequence of mtCOI. Using mtCOI-based Bayesian phylogenetic analysis, Dinsdale et al. (2010) and de Barro et al. (2011) proposed a speciation framework keeping 3.5% pairwise divergence as threshold. Based on these criteria, recently 42 putative species and 12 major genetic groups have been separated at global level (Kanakala and Ghanim, 2019). Differentiation of cryptic species on the basis of mating behavior, insecticide resistance, oviposition and transmission characteristics was examined. In the present study, the genetic affiliation of B. tabaci populations used in mtCOI analysis (de Barro et al., 2011) and incidence of B.tabaci and BYVMV infestations occurring on bhendi in Tamil Nadu, India were studied.

MATERIAL AND METHODS
The field survey was conducted in three major okra growing districts of Tamil Nadu viz., Dharmapuri, Coimbatore and Dindigul and the incidence of whitefly and Bhendi Yellow Vein Mosaic Virus (BYVMV) were randomly observed in five different locations of each district during 2018 to 2019. Adults of B. tabaci were counted on three leaves per bhendi plant, one from top, middle and bottom from ten randomly selected plants per field leaving the border rows . Population count was taken from early morning hours and expressed as number per plants. The location of sample collection and genotypic details are given in table.1. The total number of plants and number of plants infected with BYVMV were calculated from fifty randomly selected plants at the flowering stage leaving the outer two rows on all the four sides in each field and expressed as per cent disease incidence (Venkataravanappa et al., 2012).

Number of YVMV infected plants Per cent incidence of YVMV
Total number of plants observed The experiment was replicated three times. The incidence of BYVMV was observed based on characteristic symptoms viz., various levels of chlorosis, yellowing of veins and veinlets, smaller leaves, fewer and smaller fruits and stunting. Adults of B. tabaci were collected from distinct locations in three districts using hand held aspirator for genetic identification. At each location, individual insect samples were collected from okra in separate 1.5 mL Eppendorf tubes containing 70% ethanol and stored in a freezer at -80 0 C, until used.

DNA isolation
Genomic DNA was isolated from individual adult whitefly using Hot SHOT method according to Montero-pau et al. (2008). Individual insect sample was homogenized with 50 μL of alkaline lysis buffer (125 μL of NaOH, 20 μL of Na2EDTA (pH 8) and 50 mL of ddH2O) and transferred to Eppendorf tubes and incubated at 95°C for 30 min in the water bath and allowed to cool down at 4 0 C. Then 50 μL of neutralizing solution was added (315 mg of Tris-HCL and 50 mL of ddH2O). The substances were spun and vortexed for 5 s and stored at -20 °C for further analysis.

mtCOI subunit I amplification and sequence analysis
The genomic DNA of whitefly samples collected from 8 locations were confirmed for the presence of mtCOI gene using LCO 1490 forward primer 5' GGTCAACAAATCATAAAGATATTGG 3' and HCO 2198 reverse primer 5' TAAACTTCAGGGTGACCAAAAAATCA 3' (Folmer et al., 1994). The PCR reaction mix consisted of 5μL of template DNA (approximately 50 ng), 10.5 μL of sterile distilled water, 2.5 μL of dNTPs, 2.5 μL of PCR buffer, 1.0 μL of MgCl2, 1.5 μL of each forward and reverse primer, 0.5 μL of Taq polymerase. PCR was performed with initial denaturation at 94 °C for 2 min, followed by 35 cycles each consisting of denaturation for 1 min at 94 °C, annealing for 1 min at 52 °C with extension for 1 min at 72 °C, followed by final extension for 10 min at 72 °C. The PCR products were eluted and sequenced in Agrigenome Labs Pvt. Ltd., Cochin, Kerala. mtCOI gene sequence corresponding to 34 different genetic groups of B. tabaci were downloaded from the National Center for Biotechnology Information (NCBI) GenBank (https://www.ncbi.nlm.nih.gov/Blast.cgi). Sequence alignment was performed employing MUSCLE implemented in Seaview (Thompson et al., 1994). The tree was generated by neighbour joining method employing MEGA 7 software (Saitou and Nei, 1987). Genetic divergence was calculated employing MEGA 7 using ClustalW (Kumar et al., 2016). The mtCOI DNA sequences generated in the study were submitted to NCBI database.

PCR amplification and sequenced
The genotype of the whitefly population collected from okra leaf sample in eight distinct locations was genetically identified based on mtCOI universal primer. Among the eight populations tested for the presence of mtCOI gene, all the tested isolates had amplicon of 700 bp. Sequence details of all the isolates showed 99% similarity to B.tabaci Asia I and the divergence being less than 3.5%, the threshold value kept for demarcation of the species (de Barro et al., 2011). These PCR amplified sequences were submitted to NCBI and accession number was obtained. (Table 2).

Phylogenetic analysis
Based on the phylogenetic tree constructed (Fig. 2) with the accession numbers viz., MT011400 Coimbatore, MT011401 Dindigul, MT011402 Coimbatore, MT011403 Coimbatore, MT011404 Coimbatore, MT011405 Dharmapuri, MT011406 Dindigul, and MN911178 of Dindigul district were clustered with B.tabaci Asia I From the above findings, it is concluded that all okra fields show the presence of homogenous population of whitefly genotypes.

CONCLUSION
The survey and analyses performed in the study provided a detailed information on the various species of whitefly population in Coimbatore, Dharmapuri and Dindigul region of Tamil Nadu. The study revealed the specimens collected from okra belong to Asia 1 genetic group and its highly prevalent genetic group in the region. Hashmi et al. (2016) reported that Asia II-1 genetic groups were widely distributed across the Indian Agricultural Research Institute, New Delhi. Interestingly, our study provided evidence for the presence of Asia 1 in okra throughout Tamil Nadu. The present survey and the data from GenBank showed that Asia I genetic group was the most prevalent genetic group in these regions of Tamil Nadu.  Volume xxx | Issue xxxx | 2