Conserved nature of Helicoverpa armigera gut bacterial flora on different host plants and in vitro interactions with PI proteins advocates role in host digestive physiology

Abstract

Helicoverpa armigera is anxious insect pest of agricultural crops. Array of defensive molecules in host plants and extensive use of chemical insecticides are unable to cease the attack incidences. Gut bacterial communities are found to contribute in various physiological activities in most of the arthropods. In the current study the bacterial communities were isolated from gut of H. armigera feeding on three host plants (Pigeonpea, Chickpea and Cotton) by culture dependent and culture independent methods. Predominant bacterial communities were identified by terminal restriction fragment length polymorphism (TRFLP). Three dominant phylotypes namely proteobacteria, actinobacteria and firmicutes were identified by TRFLP and found to conserve on different host plant selected. Five Bacillus species namely Bacillus sp. JR14, Bacillus sp. YP1, Bacillus safensis CG1, Bacillus subtillis KAVK2 and Bacillus megaterium 47N were purified by culture dependent method and identified by 16S rRNA sequencing. Among all identified Bacillus, Bacillus sp. YP1 strain was found to be potent protease producer as assisted by dot-blot assay and in vitro solution assays. The in vitro interactions of these proteases with host plant PIs were studied by reverse zymography and gel X-ray contact print (GXCP) analysis. Reduction in activity of PIs and degradation pattern of PI bands on gels in presence of trypsin and protease extract of Bacillus sp. YP1 indicates inactivation of PIs. Thus, conserved nature and in vitro response to PI proteins advocates role of gut bacterial flora in H. armigera digestive physiology. Keywords: H. armigera, Gut bacterial proteases, Host plant defense, PIs, TRFLP