Isolation and identification of bacterial strains with insecticidal activities from Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae)

Several pathogenic bacteria strains were isolated from dead larvae of theweevil, Rhynchophorus ferrugineus Oliver, collected on palm trees in Taiwan, China. Strains of pure cultures were temporarily named TW-1, TW-2, TW-3, TW-4, TW-5, TW-6, TW-7, TW-8, TW-9, TW-10, TW-11, TW-12, TW-13 and TW-14. These strains were identified by synthetically using colony morphological characters, physiological and biochemical tests and molecular biological analysis. Our results showed that these 14 isolated strains belonged to four genera and five species. The expected approximate 1400-bp bands were obtained through 16S rDNA universal primer amplification. The sequencing results obtained from a commercial sequencing company were initially compared with a corresponding database from NCBI using BLAST, and a phylogenetic tree was constructed using mega 5.1 software. The phylogenetic results indicated that these isolated strains had more than 99% homology with type strains. To further confirm these strains, numerous physiological and biochemical indexes were confirmed. TW-1, TW-2, TW-3, TW-9, TW-12, TW-13 and TW-14 were identified as Serratia marcescens; TW-4, TW-5, TW-8 and TW-10 as Staphylococcus sciuri; TW-6 as Klebsiella pneumonia subsp. pneumonia; TW-7 as Proteus vulgaris and TW-11 as Proteus mirabilis. The preliminary single-dose bioassay on fourth instar larvae showed that S.marcescens had a higher virulence at a density of 1.89 OD600 (56.37% mortality) than the other strains. The corrected mortalities of the other strains were all less than 50%. The lowest toxicities were found for P.vulgaris and P.mirabilis where the corrected mortalities were only 28.18% and 25.57%, respectively. LC50 of S.marcescens was 1.2x10(7)CFU/ml inoculums. Our results indicated that S.marcescens from facultative cultivable bacterial flora isolated from R.ferrugineus could potentially be used as a microbial control agent for this widespread pest.