Bacteria associated with the cuticle protect Phyllophaga larvae against infection with Metarhizium pingshaense but not against Beauveria pseudobassiana

Abstract

Beauveria pseudobassiana S.A. Rehner & Humber (Hypocreales) and Metarhizium pingshaense Q.T. Chen & H.L. Guo (Hypocreales) are fungal pathogens that infect Phyllophaga larvae in the field. Previous laboratory bioassays showed less than 30% mortality in larvae inoculated with conidia of these pathogens, indicating strong resistance to fungal infection. However, injecting M. pingshaense blastospores directly into the larvae's hemocoel caused 100% mortality, suggesting external barriers may limit infection. As some insect-associated bacteria can provide protection against invading pathogens, we hypothesized that bacteria associated with Phyllophaga larvae might offer such protection. We isolated bacteria from the cuticle of Phyllophaga larvae, identified morphotypes based on colony morphology and determined their antagonistic activity against B. pseudobassiana and M. pingshaense. Selected bacterial morphotypes were identified using partial 16S rRNA gene sequencing. Based on antagonism results, eight isolates were selected and their effect on the germination of conidia from both fungal species was evaluated. Finally, the mortality of antibiotic-treated and untreated Phyllophaga polyphylla Bates (Coleoptera: Scarabaeidae) larvae was evaluated after inoculation with B. pseudobassiana and M. pingshaense. Of the 90 bacterial isolates obtained, all but two exhibited antagonistic activity against M. pingshaense, and 10 showed antagonism against B. pseudobassiana. Molecular analysis of selected morphotypes (62 isolates) revealed that the two most common genera were Acinetobacter (Moraxellales: Moraxellaceae) with 22 isolates and Serratia (Enterobacterales: Yersiniaceae) with 12 isolates. The remaining isolates were distributed among the families Flavobacteriaceae, Comamonadaceae, Enterobacteriaceae, Weeksellaceae and Pseudomonadaceae. Conidial germination of B. pseudobassiana was reduced to below 20% by most isolates, and only two allowed over 80% germination. For M. pingshaense, germination was suppressed to between 6% and 56% in most cases; only one isolate allowed 98% germination. When exposed to M. pingshaense, antibiotic-treated larvae showed 68% mortality, whereas untreated larvae had only 26% mortality. In contrast, mortality from B. pseudobassiana was statistically similar between treated (23%) and untreated (7%) groups. Overall, our results suggest that bacteria can provide protection against M. pingshaense but not B. pseudobassiana.