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Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 μg and 200 μg.

Introduction Fungal diseases are responsible for considerable yield losses in sugarcane crop. About 160 fungi have been reported to infect sugarcane, while seven diseases with unknown etiologies have been recognized and documented (Rott, 2000). Fungal diseases reduce cane yield by up to 31% (Jayashree et al., 2010). Among them, the most common disease is red rot, which is also known as a "cancer" of sugarcane (Khan et al., 2011), and is caused by the fungus Colletotrichum falcatum Went. Red rot severely affects the quantity and quality of the cane, with about a 29% reduction in cane weight and 30% less sugar recovery (Hussnain and Afghan, 2006). Red rot is one of the major, oldest, broadly distributed, and documented diseases of sugarcane in many countries, including Pakistan, the United States, Bangladesh, Taiwan, and Thailand (Viswanathan and Samiyappan, 2002; Viswanathan, 2012). It infects the sugar cane stalk at both the initial and mature stages of growth, causing discoloration, while the pathogen-produced invertase enzyme causes sucrose inversion in addition to drying of the cane stalks. Thus, overall, the vegetative growth of the plant halts (Sharma and Tamta, 2015). Various methods are adopted for red rot disease management, including chemical, biological, breeding, and tissue culture methods. All of these have certain limitations and disadvantages. For instance, chemical methods increase the cost of production and contaminate the environment as well, while biological control applied through plant growth-promoting bacteria (Malathi and Viswanathan, 2013) does not show consistent results in the field. Similarly, in tissue culture, the selection of fungus-resistant cells (Mohanraj et al., 2003; Sengar et al., 2009) is most often associated with somaclonal variations. Red rot-resistant varieties can be produced by breeding methods (Agnihotri, 1996), but breeding is laborious and time-consuming. To overcome all these limitations, genetic modification is the best available option for management of red rot disease in sugarcane crops. Specific genes can be introduced into the genome of sugarcane to create resistance against a particular pathogen (Enríquez-Obregón et al., 1998).

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