Bacillus subtilis and B. licheniformis Isolated from Heterorhabditis indica Infected Apple Root Borer (Dorysthenes huegelii) Suppresses Nematode Production in Galleria mellonella

Abstract

Heterorhabdits indica successfully controlled apple root borer Dorysthenes huegelii in the orchards, but nematode-infected cadavers revealed the presence of non-symbiotic bacterial B. subtilis and B. licheniformis, and no subsequent generations of H. indica were produced (hampered recycling phenomenon). Intrigued, we tested the effect of the two Bacillus species on symbiotic association of H. indica-Photorhabdus luminescens. One-to-one competitive parallel line in vitro assays were carried out between P. luminescens and the two Bacillus spp., while in vivo H. indica development was studied on the test insect Galleria mellonella which were fed with Bacillus mixed diet, followed by nematode exposure. Where P. luminescens was flanked by either of the two Bacillus species, only B. subtilis significantly suppressed its growth, while in reversed assays both the Bacillus growth was unaffected. Heterorhabditis indica was able to kill Galleria larvae pre-fed with the two Bacillus spp.; these cadavers did not develop the characteristic evenly distributed brick red coloration. Besides P. luminesecns, both Bacillus spp. were found to coexist in these cadavers. Development of hermaphrodites was not affected, but second-generation females, and final nematode progeny was reduced significantly. Monozenic lawns of B. subtilis and B. licheniformis did not support H. indica development. These results show the reduced development of H. indica by the presence of the non-symbiotic bacteria in G. mellonella is likely to affect their ability to recycle in other insect larvae. Reduced recycling caused by non-symbiotic bacteria will reduce the overall long-term pest control benefits and have implications in the development of application strategies using entomopathogenic nematodes (EPNs) as insect control agents.