Molecular characterization of pathogenesis involving the GAS 1 gene from Entomopathogenic fungus Lecanicillium lecanii and its virulence against the insect host Diaphorina citri.

The entmopathogenic fungus Lecaniicillium lecanii is a naturally available biological control and it is considered to be one of the best mycoinsecticide agents against the destructive insect pest Diaphorina citri Kuwayama. The present study aimed to extract and characterize the toxic insecticidal protein from L. lecanii and to assess the toxicity level against the Asian citrus psyllid the vector of Huanglongbing disease (HLB), also called citrus greening. Extracts of a toxic substance from submerged batch culture examined by sodium dodecyl sulfate-poly-acrylamide (SDS-PAGE), had a molecular weight of 45 kDa. The most abundant toxic metabolite was subjected to HPLC to purify and identified it by mass spectrometry. Subsequently, metabolite toxicity was tested against D. citri at three different concentrations (1%, 2%, and 3%). The results showed that the highest concentration had a significant maximum mortality at 120 h post application. Furthermore, we investigated the expression of the GAS1 gene which was previously identified to have a role in pathogenicity in in vivo studies in adult insect psyllids. Results of this study indicated that expression of the virulence factor gene was present at three concentrations of the fungal suspension post inoculation. This is the first study to provide this novel approach for the characterization of fungal mediated synthesis of a cuticle degrading soluble protein against the insect D. citri. The present results provide strong information on the in vivo expression of the GAS1 gene involved in fungal virulence pertaining to penetration of the insect cuticle, but not to inhibiting the growth of the host.