Assessment of oral virulence against Spodoptera litura, acquired by a previously non-pathogenic Metarhizium anisopliae isolate, following integration of a midgut-specific insecticidal toxin

Abstract

All entomopathogenic fungi infect insects by direct penetration through the cuticle rather than per os through the gut. Genetic transformation can confer fungi with per os virulence. However, unless the recipient isolate is nonpathogenic to the target insect, mortality caused by a transgenic isolate cannot be attributed solely to oral virulence due to the potential for some simultaneous cuticular infection. Here, a Metarhizium anisopliae wild-type isolate (MaWT) nonpathogenic to Spodoptera litura was genetically engineered to provide a transformed isolate (MaVipT31) expressing the insect midgut-specific toxin Vip3Aa1. Toxin expression was confirmed in MaVipT31 hyphae and conidia using Western blotting. Mortality, leaf consumption and body weight of S. litura larvae (instars I–IV) exposed to a range of concentrations of MaWT conidia were not significantly different to controls although the number of conidia ingested by surviving larvae during the bioassay ranged from 2.3×105 (instar I) to 8.1×106 (instar IV). In contrast, consumption of MaVipT31 conidia caused high mortalities, reduced leaf consumption rates and decreased body weights in all instars evaluated, demonstrating that oral virulence had been acquired by MaVipT31. Larval mortalities were much more dependent on the number of MaVipT31 conidia ingested than the duration of time spent feeding on conidia-treated leaves (r2: 0.83–0.94 for instars I–IV). LC50 and LT50 trends for MaVipT31 estimated by time-concentration-mortality modeling analyses differed greatly amongst the instars. For 50% kill to be achieved, instar I larvae required 3, 4 and 5days feeding on the leaves bearing 103, 28 and 8conidia/mm2 respectively; instar IV larvae required 6, 7 and 8days feeding on leaves bearing 1760, 730 and 410conidia/mm2 respectively. Our results provide a deeper insight into the high oral virulence acquired by an engineered isolate and highlight its great potential for biological control.