Manika Sharma

Genetic variation of Alternaria brassicae (Berk.) Sacc., causal agent of dark leaf spot of cauliflower and mustard in India

Cauliflower (Brassica oleracea L. var. botrytis) and mustard [Brassica juncea (L.) Czern] are important cruciferous crops facing serious yield and quality loss in India from dark leaf spot disease caused by Alternaria brassicae (Berk.) Sacc. Genetic variation among 32 pathogenic A. brassicae isolates from both crops was analyzed with random amplified polymorphic DNA and inter-simple sequence repeat primers in which the mean similarity coefficient was found to be 0.73 and 0.84, respectively. Further internal transcribed spacer analysis showed all isolates are 90–100xa0% similar to each other, indicating genetic similarity among different A. brassicae isolates that vary pathogenically.

Heterologous expression and characterization of purified partial endochitinase ( ech-42 ) isolated from Trichoderma harzianum

Chitinase gene from Trichoderma harzianum was cloned and hetrologously over expressed in M15 Escherichia coli. The recombinant protein of 42 kDa from E. coli was purified through Ni-NTA affinity column chromatography. The purified enzyme was active over broad range of pH (2.0 to 8.0) and temperature (10 to 60°C) with the peak activity at pH 5 (0.50 μg/ml) and 20°C with enzyme activity value (0.49 μg/ml). The purified protein fractions were tested for in vitro antifungal activity against different phytopathogens like Fusarium oxysporum f.sp. lycopersici, Sclerotioum rolfsii, Alternaria brassicae and Alternaria brassicicola. Purified endochitinase isolated from T. harzianum caused necrotic lesions, segmentation, branching and hyphal bursting at the concentration of 200 µg ml-1. n n xa0 n n Key words: Antifungal activity, Trichoderma harzianum, Fusarium oxysporum f.sp. lycopersici, Sclerotioum rolfsii, Alternaria brassicae and Alternaria brassicicola.

Cloning and semi-quantitative expression of endochitinase (ech42) gene from Trichoderma spp.

Species of Trichoderma such as T. harzianum , T. viride and T. atroviride are some of the most potent antagonistic fungi, and have been used as plant growth promoters in developed countries. Endochitinase (ech42) gene which is involved in mycoparasitism, was isolated from Trichoderma spp. taken from hot-arid soils of Rajasthan, cloned, sequenced and its expression profiling was carried by reverse transcription-polymerase chain reaction (RT-PCR) technique. The cloned DNA sequence was 1,476 base pairs. Gene encoding endochitinase was ligated in pGEMT cloning vector. The plasmids were transformed in DH5α Escherichia coli competent cells and clones were confirmed through sequencing and restriction analysis. Endochitinase gene expression was then observed for different Trichoderma isolates viz., T. harzianum (T14 and T12) and T. atroviride (T5). Among the three, higher expression of endochitinase was observed in T14 and T12, whereas T5 showed lesser expression with respect to T14 and T12 strain. The Trichoderma chitinase enzyme activity was monitored for all isolates under study. The highest chitinase activity was observed in T14 and T11 viz., 17.21 (1 enzyme μg/ml) and 13.11 enzyme μg/ml, respectively. Keywords: Endochitinase, cloning, expression, Trichoderma atrovirid e, Trichoderma harzianum , Trichoderma viride