Exploration of Amylases Producing Competency of Helicoverpa armigera Gut Bacterial Strain, Bacillus subtilis RTSBA6 6.00

The Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is a polyphagous insect pest of agriculturally important crops. The alkaline gut of this insect pest possesses diverse bacterial communities which may assist in digestive physiology. As part our investigations of understanding the role of gut bacterial communities in insect gut, here amylase producing competency of earlier identified H. armigera gut bacterial strain, i.e., Bacillus subtilis RTSBA6 6.00 is reported. Initial screening for amylase activity was assessed by starch agar plate. Upon 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis amylase zymography, bacterial culture supernatant produced seven amylase bands on the gel. The observed molecular weights of amylases were 191.2 KDa, 158.0 KDa, 131.7 KDa, 54.0 KDa, 31.3 KDa, 67.2 KDa, and 44.6 KDa, respectively. Considerable amylase activity was observed in neutral to alkaline pH with optimum at pH 6.8. The optimal activity temperature of amylases was found to be 50°C, and the activity decreased dramatically at temperatures above 75°C

Proteinase inhibitors of pigeonpea cv. BSMR 736: Characterization and bioefficacy against Helicoverpa armigera

Pigeonpea is an agriculturally important leguminous crop with high vulnerability to insect pest attack specifically, Helicoverpa armigera. The proteinase inhibitors (PIs) mediated host plant resistance against insect pests is a promising sustainable agricultural research practice. The current study was carried out to perceive biochemical characterization of proteinase inhibitors named PPTI in the pigeonpea (cv. BSMR 736). The purification of PPTI from crude protein seed extract was achieved by acetone precipitation, N-LP-IEF, and trypsin affinity chromatography. It was found to inhibit bovine trypsin and HaGPs in vitro. The optimal conditions for inhibition were pH 8 and temperature 40ºC. The PPTI showed four isoinhibitors bands on native, non-reducing and reducing SDS-PAGE in the range of 26.7–19.3 KDa. Upon resolution on two-dimensional gel electrophoresis (2-DE), PPTI produced nine pI variant spots having isoelectric point (pI) 6.6, 6.6, 6.3, 6.1, 5.9, 5.8, 5.7, 5.6 and 5.6. An artificial diet containing PPTI reduced the H. armigera larval weight about 69%, with 25% mortality. For eco-friendly sustainable agricultural practices, natural compounds like PPTI could be expressed in transgenic crops to prevent the invasion of H. armigera in pigeonpea

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

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, TRFL