Molecular characterization, host range and integrated management of bhindi yellow vein mosaic disease

Abstract

Bhindi (Abelmoschus esculentus (L.) Moench) is one of the most important vegetable crops cultivated across the globe. However, its cultivation is often hindered by biotic stresses like incidence of pests and diseases. Among the diseases, yellow vein mosaic disease is one of the major constraints in bhindi cultivation which leads to 100 per cent yield loss especially when infected at an early stage of the crop. In recent years, the evolution of new viral strains is a serious problem especially among Begomoviruses belonging to the family Geminiviridae which has an adverse effect on the host plant resistance. Considering these facts, the present study was undertaken to carry out the molecular characterization of the virus causing bhindi yellow vein mosaic disease (BYVMD), to study the host range and seed transmission of the virus and to develop a sustainable disease management strategy. The project was initiated with purposive sampling survey conducted in elevan different locations of Thrissur district, Kerala. The disease incidence recorded during the survey ranged from 61.20 to 98.16 per cent while the disease severity ranged from 48 to 90 per cent. The predominant symptoms observed on the leaves of infected plants under natural conditions were vein clearing, vein thickening, reduction in leaf area, bleached appearance and marginal necrosis. A novel type of symptom observed during the survey was general yellowing of leaves with severe puckering along the veins and upward curling of leaves. Linear cholorotic striations were observed on the calyx of the flower buds. The immature fruits produced by the infected plants showed linear chlorotic striations, while the mature fruits were bleached in appearance along with reduction in fruit size. The plants infected during the early vegetative stage were extremely stunted. The major symptoms developed under artificial conditions were vein clearing, vein thickening and puckering of leaves.

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Histopathological studies of the infected leaf revealed disruption of parenchymtous cells in the epidermis, disintegration of chloroplast, reduction in number of metaxylem and protoxylem along with abnormality of phloem vessels. The studies on virus transmission confirmed that it is transmitted through grafting and insect vector, Bemisia tabaci. The presence of virus inside the insect body was also confirmed through polymerase chain reaction (PCR) based molecular technique. The studies on seed transmission revealed that BYVMD is not seed-borne. Host range studies revealed that weed species Synedrella nodiflora and Hemidesmus indicus were proved to be hosts of the begomovirus. Molecular detection of the virus causing BYVMD was standardized through PCR, using universal primer specific to the core coat protein gene of Begomovirus which yielded amplicons at expected size of about 550 bp. The amplification was also carried out using primers specific to coat protein gene of bhindi yellow vein mosaic virus (BYVMV) and okra enation leaf curl virus (OELCuV) which yielded amplicons at expected band size of about 770 bp. The molecular characterization of the elevan isolates was carried out through in silico analysis to identify the virus associated with BYVMD and for diversity analysis. The results revealed that all the isolates showed 99-100 per cent nucleotide homology to OELCuV. BLASTp analysis of the isolates also showed 100 per cent identity with coat protein of OELCuV and thus confirming that the virus causing yellow vein mosaic disease in bhindi in Kerala is okra enation leaf curl virus. The identity was further confirmed through DNA barcoding technique and species demarcation analysis. A field experiment was also conducted to develop a disease management package against BYVMD. Among the seven treatments, T5 i.e., integrated management with early seedling protection using insect proof net + yellow sticky trap + seed bio-priming and foliar spray of PGPR mix II + alternate foliar spray of Bougainvillea spectabilis and azadiractin was found to be most effective with

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lowest disease incidence and severity, least whitefly population and maximum yield. It is concluded that yellow vein mosaic disease affecting bhindi cultivation in Kerala is caused by okra enation leaf curl virus, an evolved strain of BYVMV. This virus is transmitted through grafting and the insect vector Bemisia tabaci and not though seeds. The outcome of the study would also facilitate early detection and elimination of sources of infection so as to reduce the spread of the disease. An integrated disease management package was also developed for the benefit of farming community.