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

Identification of Potent Inhibitors of Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) Gut Proteinase from Plant Gum PIs

ABSTRACT We report on the efficacy of proteinase inhibitors (PIs) from six host plants Zea mays, m35 sorghum, Sorghum bicolour, Saccharum spp., Setaria italia and Pennisetum glaucum (L) stem tissue and five non-host Acacia nilotica, Acacia leucophloea, Azardirachta indica, Mangifera indica and Terminalia spp. plant gum extracts PIs, in retarding the growth of Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) larvae, a devastating pest of important crop plants. Enzyme assays and electrophoretic analysis of interaction of C.partellus gut proteinases (CPGPs) with PIs revealed that non-host PIs inhibited CPGPs activity efficiently whereas host PIs were ineffective. In the electrophoretic assay, trypsin inhibitor activity bands were detected in all of the host and non-host plants, but CPGPs inhibitor activity bands were present only in non-host plants. C. partellus larvae reared on a diet containing non-host PIs showed growth retardation, reduction in total and trypsin like proteinase activity. In present study potent PIs identified against C. partellus linsects

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

Role of chloroplastidial proteases in leaf senescence

In this report the effect of hydrogen peroxide (H2O2) on peroxidase (POD) activity during leaf senescence was studied with and without phenylmethylsulfonyl fluoride (PMSF) pre-treatment in detached neem (Azadirachta indica A. juss) leaf chloroplasts. Increased POD activity was detected in natural and H2O2-promoted senescent leaf chloroplasts compared to untreated control mature green leaf chloroplasts. However, under H2O2 POD activity markedly increased at 1 day, and then significantly decreased until 4 days. In the presence of H2O2, PMSF, the induction of POD activity was alleviated at 1 day, whereas reduced after 4 days. In contrast, in the presence of H2O2, cycloheximide (CX), the induction of POD activity was reduced at 1 day, whereas alleviated after 4 days. The was a partial reduction in H2O2-induced POD activity with PMSF and CX, indicating the presence of pre-existing inactive PODs in chloroplasts. We also propose a new role for chloroplastidial proteases as activators of pre-existing inactive PODs during leaf senescence