New reports on dixenic associations between the symbionts of entomopathogenic nematodes, Photorhabdus and Xenorhabdus, and non-symbiotic bacteria

By conducting three different methods, we report on the isolation of five novel strains of non-symbiotic bacteria from crushed infective juveniles (IJs) of four species of entomopathogenic nematodes (EPN) including Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema feltiae, and Steinernema glaseri and five bacterial species from hemolymph of insect larvae infected with EPNs. Samples of hemolymph of infected Galleria mellonella L. larvae by EPNs and crushed surface sterilized IJs were bulk streaked onto both MacConkey and NBTA agar. To further ensure diagnoses, extracted DNA from IJs bulk was subjected to PCR by ۱۶S-rRNA bacterial universal primers. Bacteria were identified using biochemical and phylogenetic analysis. Based on ۱۶S-rRNA gene sequence, maximum parsimony, maximum likelihood and neighbour joining phylogenetic analyses were conducted, as well as comparisons of predicted RNA secondary structures. Four species of bacteria were identified including: Stenotrophomonas maltophilia strain IR۱۱ from S. feltiae; S. pavanii strain IR۲۰ from S. glaseri; Acinetobacter junii strain IR۸ from S. carpocapsae; and Alcaligenes faecalis strains IR۱ & IR۱۵ from S. feltiae and H. bacteriophora respectively as non-symbiotic bacteria from IJs and five species probably originated from G. mellonella intestine including Citrobacter gillenii isolate S۳, Enterobacter asburiae isolate S۴, Klebsiella oxytoca isolate S۵, Morganella morganii isolate S۶ and Serratia marcescens isolate S۶.

Keywords:

Stenotrophomonas maltophilia , S. pavanii , Acinetobacter junii , Alcaligenes faecalis , Citrobacter gillenii , Enterobacter asburiae Klebsiella oxytoca , Morganella morganii , Serratia marcescens