Studies on the Cloning and Expression of Bbchit1 Gene of Beauveria bassiana NCIM 1216

Abstract

Beauveria bassiana is a biocontrol agent which shows entomopathogenecity on insect pests especially the Lepidopterons invading the agriculturally important crops. The mode of infection is through the cuticle by degrading the chitin present on it which is enabled by the exochitinase enzyme of Bbchit1 gene. A good quality genomic DNA was isolated from Beauveria bassiana NCIM 1216 and amplified with specific primers to isolate the gene corresponding to Bbchit1 which codes for the exochitinase enzyme that is responsible for pathogenesis. The Bbchit1 gene of B. bassiana was transformed with the binary plasmid pBANF-bar-pAN-Bbchit1, in which the Bbchit1 gene was placed downstream of the constitutive gpd promoter, which was mediated by A. tumefaciens, and transformants were selected on the basis of herbicide resistance. Fifty herbicide resistant colonies were obtained and analyzed. The exochitinase produced by these transformants was observed maximum on the 7th day of inoculation in both which was 0.09 lmol/ml/min for the purified fraction and 0.06 lmol/ml/min for the crude extract. The chitinolytic activity was observed maximum at pH 5 and at temperature of 40°C. The genetically modified pure form can be used in the production of transgenic plants and in bringing out commercial formulation for the control of Lepidopteran pests.

Insecticidal activity of culture filtrates from liquid medium of Beauveria bassiana isolates from South Sumatra (Indonesia) wetland soil against larvae of Spodoptera litura

“…In this study, the culture filtrate produced from the separation of liquid B. bassiana culture from its spores was toxic and lethal on larvae of test insects. According to Pinnamaneni et al (2010) incubation of test fungi in liquid medium produces exochitinase enzyme and the chitinolytic exochitinase enzyme, able to degrade cuticle insects. Based on the resulting mortality, the culture filtrates of B. bassiana from medium of PDB (pH 6) were more toxic than that of pH 2.5.…”

“…The pH range for chitinase activity of B. bassiana was pH 4-6. More acidic or alkaline nature lowers the activity of culture media (Pinnamaneni et al 2010). In addition, the pH of culture media less than 3 is less ideal for the growth and viability of spores of B. bassiana (Rizkie et al 2017).…”

“…However, there are still species or isolates of the fungi conidia still survive at pH 2 (Rizkie et al 2017). According to Pinnamaneni et al (2010), the minimum pH 4 is generally required by B. bassiana which enables these fungi to produce chitinase enzymes that can kill host insects. The pH range of the culture medium used for growing B. bassiana under acidic is at (4.0), neutral at (7.0), and basic at (11.0) (Qazi 2008).…”

Thermostable chitinase from Cohnella sp. A01: isolation and product optimization

“…A comparable pH optimum has been reported for Bbchit1 of Beauveria bassiana NCIM 1216. 10 This enzyme has it is the best pH in the acidic range and can possibly be used for the control of plant fungal pathogens.…”

“… 3 Most chitin-degrading prokaryotes are the gliding bacteria, pseudomonad, vibrio, enterobacteria, actinomycete, bacilli, and clostridia. 10 Bacterial chitinases have a size range of 20–60 kDa. 11 , 12 Chitinases have potential applications in various areas of biotechnology, biomedicine, agriculture, and nutrition.…”