Influence of growth stage on sensitivity to helminthosporol toxin of Bipolaris sorokiniana of barley (Hordeum vulgare)*

*Short note Based on Ph D thesis of the first author submitted to Banaras Hindu University, Varanasi in 2010. 1 Scientist (e mail: [email protected]), Division of Plant Pathology, IARI, New Delhi; 2 Professor (e mail: [email protected]), Department of Mycology and Plant Pathology; 3 Professor (e mail: [email protected]), Department of Genetics and Plant Breeding; 4 Professor (e mail: [email protected]), Department of Genetics and Plant Breeding and Senior Wheat Breeder (e mail: [email protected]), CIMMYT South Asia, Regional Office, Singh Durbar Plaza Marg, Kathmandu, Nepal Bipolaris sorokiniana (Sacc.) Shoemaker (syn. Helminthosporium sativum teleomorph: Cochliobolus sativus), a hemibiotrophic phytopathogenic fungus, is a wellknown for inciting spot blotch disease in barley and wheat. Among a variety of symptoms induced by this pathogen on all parts of the plant, foliar Spot blotch caused by B. sorokiniana has emerged as one the major biotic stresses hampering the commercial production of barley (Ghazvini and Tekauz 2007). The disease is found all over the world wherever the barley is grown. It is a serious pathogen of wheat and barley in North America (Mathre 1997), South America and several countries of Asia (Joshi et al. 2007). Ludwig (1957) demonstrated that B. sorokiniana (Helminthosporium sativum ) produces a toxin that is essential for the development of the diseases in the attacked plants. Numerous compounds have been isolated from culture filtrates of B. sorokiniana and highest phytotoxic activity has been associated with the metabolites of sesquiterpene nature (De Mayo et al. 1961; Carlson et al. 1991). The nonselective phytotoxin produced by B. sorokiniana is capable of membrane disruption, inhibition of mitochondrial electron transport and oxidative phosphorylation, general degeneration of nuclei (Aggarwal et al. 2011) and induce chlorosis (Ludwig 1957). Among the various toxins produced by B. sorokiniana helminthosporol is the major sesquiterpene metabolite when grown in broth culture and is responsible for the disease (Carlson et al. 1991). The helminthosporol toxin produces similar symptoms as it was obtained with nonpurified filtered fungus culture on test plant (Stoessl 1981). Toxin has been utilized for screening of barley (Bashyal et al. 2009, Carlson et al. 1991). However, effect of helminthosporol at different growth stages of barley is not known yet. Fourteen diverse genotypes reported for variable disease severity were obtained from the Directorate of Wheat Research Karnal, Haryana (Chand et al. 2008). The experiment was carried out during 2008–09 in the Agricultural Research Farm, Banaras Hindu University,Varanasi. The crop was raised in November with row to row and plant to plant distance of 25 cm and 5 cm respectively. Agronomic practices recommended for normal fertility (60N:40P:30K) under irrigated conditions were followed. Crude toxin was obtained by following the method of De Mayo et al. 1961. Isolate of B. sorokiniana (29 B) was grown in Minimal Medium (Leach et al. 1982) for eight days. Mycelium of B. sorokiniana was filtered with Whatman paper 36 to obtain the cell free culture filtrate. Culture filtrate was adjusted to pH 2 and activated charcoal was added and stirred and kept for 30 min. The charcoal with the absorbed toxin was separated from the liquid in a centrifuge and suspended in ethanol. After filtering on a Buchner Funnel the charcoal cake was suspended in chloroform, stirred and filtered. Process was repeated three times. The chloroform extracts were combined and the solvent removed in vaccuo to yield crude extract. Barley plants of growth stage 33 (third node detectable), 60 (beginning of flowering) and 75 (medium milk) of (Zadoks et al. 1974) were selected for the infiltration of toxin. Individual penultimate leaf of each plant was infiltered with 100 μl (1: 10 dilution) of crude toxin using a 1 ml plastic syringe (Mercado et al. 2006). Each penultimate leaf was infiltered in the centre and water soaked area was marked with non-toxic felt pen at each end of water soaking point. Observations were taken for appearance of necrotic symptom