Isolation of an Isometric Virus Causing Sunflower Necrosis Disease in India.

Abstract

Sunflower necrosis disease (SND) is becoming a potential threat to sunflower (Helianthus annuus L.) cultivation in the Indian subcontinent. The disease was first recorded in parts of Karnataka state in 1997. Since then the disease has become increasingly important in Andhra Pradesh, Karnataka, Maharashtra, and Tamil Nadu, the four major sunflower-growing states of India, and is a limiting factor in sunflower production; up to 80% of the plants of some open pollinated and hybrids were affected during the 1999 survey in sunflower growing areas. Field symptoms of the disease include extensive necrosis of leaf lamina, petiole, stem and floral calyx and severe stunting with malformation of flowering head when plants are infected early. The association of a tospovirus, antigenically related to groundnut bud necrosis (GBNV) and watermelon silver mottle (WSMV) viruses, with the disease has been reported (1). However, the etiology of the disease remains unaddressed. In this study a sap-transmissible isometric virus was transferred to cowpea (cvs. Pusa Komal and C152) inciting localized chlorotic and necrotic lesions and systemic veinal necrosis. Electron-microscopic studies of leaf-dip preparations from field samples revealed two types of particles (isometric measuring 25 to 28 nm in diameter and flexuous rods with a length of about 600 nm). The sap-inoculated cowpea and sunflower contained only the isometric particles. Some preparations also showed the presence of tubules containing virus particles. The presence of flexuous particles in field samples could be due to mixed infection as the mosaic disease, known to be caused by a flexuous virus, was common in the sunflower fields surveyed in the present investigations. Extracts from the field collected samples or sap-inoculated plants did not react with antisera to cucumber mosaic (CMV) or potato Y (PVY) viruses in direct antigen-coated (DAC)-ELISA and immunosorbent electron microscopy tests. The isometric virus isolated from sunflower was purified from sap-inoculated cowpea plants by differential and sucrose density-gradient centrifugations. The virus was sap transmitted back to sunflower (cv. Morden), which developed symptoms identical to those observed under field conditions. Disease symptoms were also reproduced on sunflower upon mechanical inoculation with the purified virus. Polyclonal antiserum raised in rabbits using purified virus preparations, detected the virus from field and glasshouse collected sunflower plants in DAC-ELISA tests. This will help in epidemiological studies and breeding for disease resistance. The particle size and structure and the presence of tubule containing virus particles in plant extracts suggest that the virus belongs to ILAR group. An ILAR virus is reported to infect sunflower (2), but details of its natural occurrence are not known. This is the first report on the etiology of the sunflower necrosis disease in India. Further studies are in progress.