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Abstract

Cucumber mosaic virus (CMV) causing mosaic and chlorosis of banana in Marthwada region was characterized on the basis of biological, serological and RT-PCR amplification of Coat protein. Mechanical inoculation tests, the virus was found to infect members of chenopodiaceae, Cucurbitaceae, Fabaceae, and Solanaceae. Partially purified virus preparations revealed the presence of isometric particles of 28nm in the electron microscopy. Insect vector transmission of CMV has indicated that Aphis gossypii was more efficient than A. craccivora in transmitting CMV in a non persistent manner. Using DAS ELISA CMV could be efficiently detected in different banana sample which are both symptomatic and symptom less plants. This would help in development of diagnostic tool for identifying and certifying planting material to check spread of disease.

Keywords: Banana, Chlorosis disease, Cucumber mosaic virus, ELISA detection, RT-PCR amplification

Introduction

Banana is an important fruit crop grown throughout the country. Banana crop is vulnerable to many diseases; among these viral diseases are the major production constraints. Infectious chlorosis disease of banana was first reported in Australia (Magee, 1940) and later in South and Central America, the Caribbean, India and the Philippines (Bouhida and Lockhart, 1990, Joshi and Joshi, 1974, Mali and Deshpande, 1976, Palukaitis et al., 1992). Banana chlorosis or heart rot disease has been reported to be caused by cucumber mosaic virus (CMV) (Magee, 1940; Singh et al., 1995, Kiranmai et al., 1998).

Cucumber mosaic virus (CMV), a positive sense RNA plant virus with a tripartite genome, is the type member of the genus Cucumovirus. CMV has a world -wide distribution and exists as a variety of isolates that differ in host range and pathogenicity (Palukaitis et al., 1992). CMV causes great losses in vegetables, ornamentals and fruits, and it is destructive due to its rapid spread by more than 60 aphid species in the field (Palukaitis and Garcia-Arenal, 2003). Transmission through planting material is also significant in some crop and weed hosts (Hsu et al., 2000). A number of CMV isolates have been described previously and classified into two subgroups, I and II, according to serological relationships, peptide mapping of the coat protein (CP), nucleic acid hybridization and nucleotide sequence identity (Palukaitis et al., 1992). More recently, phylogenetic analysis of a number of CMV isolates led to a further subdivision of subgroup I into subgroups IA and IB (Roossinck, 2002; Roossinck et al., 1999).

In India occurrence of CMV has been reported from many hosts. However, only limited reports are available on biological and molecular characteristics of these isolates (Madhubala et al., 2005, Verma et al., 2005,2006). CMV causes chlorosis, mosaic and heart rot in banana and has been found in most banana growing areas of the world. Infectious chlorosis disease of banana oftenconfused with zinc deficiency and or with the symptoms of Banana streak virus. Therefore the present investigations wereundertaken to identify the causal virus prevailing in Marthwada region based on transmission, serological testing and RT-PCR amplification.

Materials and Methods

Collection and maintenance of virus culture: Survey was conducted in Parbhani and Nanded districts of major banana growing areas of Marathwada region for the incidence of virus disease. Viral incidence was recorded based on symptoms observed on plants The symptomatic samples collected were tested by DAC-ELISA using CMV antisera and the samples which are positive to CMV were mechanically inoculated to Cucumber and the virus cultures were maintained for further studies.

Mechanical transmission and host range of virus</p>

Fort transmission and host range studies, mechanical inoculations were carried out by extracting infected leaf tissues in 0.1 M phosphate buffer, pH 7.0 containing 1.0% sodium sulphite (1:2W/V). Crude sap was inoculated mechanically on a number of test plant species of five plants each and inoculated plants were observed for one month for the appearance of symptoms. The plant species which are tested for host range are Cajanus cajan, Capsicum annuum, Chenopodium amaranticolor, Cicer arietinum, Cucumis sativus, Cucurbita pepo, Glycine max, Lycopersicon esculentum, Nicotiana glutinosa, N. tabacum, Phaseolus vulgaris, Vigna mungo, Vigna radiata, and Vigna unguiculata. Symptoms were recorded and checked for the presence of virus by backinoculation on to C. amaranticolor and by ELISA.

Aphid transmission of the virus</p>

For aphid transmission tests, Aphis gossypii was used with a preacquisition starvation period of 2 h, acquisition access period of 2 min and an inoculation period of 1 to 2 h on 8 plants of Nicotiana tabacum. The inoculated plants were observed for symptom development and presence of virus is confirmed by back inoculation on C. amaranticolor.

Virus Purification

The virus was propagated on N. glutinosa plants by mechanical inoculation. Leaves of N. glutinosa harvested three weeks after inoculation was used for purification. Purification was also done from virus infected banana samples by the method described by Tomlinson et al., (1973) with some modification. The inoculated leaves (100 g) were harvested after 4-5 days of virus inoculation and ground in 0.1 M citrate buffer, pH 6.5 in the ratio 1: 3 (w/v). Differential centrifugation was followed for purification of the virus.

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