Isolated bacteria
In the present study, five non-spore-forming bacteria were isolated from the larvae of M. melolontha. These bacteria were identified as Enterobacter cloacae complex (isolate 1M), Serratia marcescens (isolate 3M), Pseudomonas aeruginosa (isolate 4M), Kocuria kristinae (isolate 5M), and Serratia liquefaciens (isolate 8M) according to the results from VITEK bacterial identification systems and 16S rRNA gene sequence analysis.
Isolate 1M of M. melolontha larvae was identified as E. cloacae. It is a rod-shaped, gram-negative, facultatively anaerobic bacterium. Species of the E. cloacae complex are widely encountered in nature, and they can act as pathogens (Mezzatesta et al. 2012). Sezen et al. (2007) also found Enterobacter sp. from M. melolontha. E. cloacae was observed to be the most common species of the poplar pest, Cryptorhynchus lapathi (Yaman et al. 2017).
Isolate 3M, isolated from M. melolontha larvae, was identified as Serratia marcescens (family Enterobacteriaceae). It is a species of rod-shaped, gram-negative and facultative anaerobic bacteria. S. marcescens is one of the best-known and mostly isolated pathogenic bacterium from insects (Thiery and Frachon 1997; Lauzon et al. 2003; Pineda-Castellanos et al. 2015). Among the non-spore-forming bacterial genera, the genus Serratia includes more effective entomopathogenic species (O'Callaghan et al. 1996) and would possibly be the bio-control agents against some insects (Sezen et al. 2001).
Isolate 4M isolated from M. melolontha larvae was identified as Pseudomonas aeruginosa. It is a bacil, gram-negative bacterium found throughout the environment. It is known as a potential pathogen for various insects and has frequently been isolated from infected insects, e.g., Schistocerca gregaria (Forski) (Orthoptera: Acrididae), Melanopus spp. (Orthoptera: Acrididae) (Bucher and Stephens 1959), Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae) (Lysenko 1963), Stonioxys calcitrans (Linnaeus) (Diptera: Muscidae), Amphimallon solstitialis (Linnaeus) (Coleoptera: Scarabaeidae), and Eurygaster intergriceps (Puton) (Hemiptera: Scutelleridae) (Lipa 1975). This bacterium was isolated from dead larvae of Pieris brassicae (Yaman and Demirbağ 2000). P. aeruginosa had the greatest insecticidal effect (67%) on P. brassicae (Bucher and Stephens 1959; Yaman and Demirbağ 2000). Bucher and Stephens (1959) found P. aeruginosa as the main cause of disease in grasshoppers, under both laboratory and natural conditions. Obtained results agree with the abovementioned references.
Isolate 5M Kocuria kristinae was another bacterium isolated from M. melolontha in this study. It is a gram-positive microorganism. Cockburn et al. (2013) isolated and identified this bacterium from the common bed bug, Cimex lectularius. Yaman and Ertürk (2016) isolated and identified it from Crepidodera aurata (Coleoptera, Chrysomelidae) for the first time, recently.
Isolate 8M Serratia liquefaciens was isolated from M. melolontha larvae. It is a species of gram-negative bacteria and the most prevalent Serratia species in the natural environment. This bacterium was diagnosed in Cydia pomonella (Zimmermann et al. 2013). Muratoğlu et al. (2011) isolated this bacterium from Ips typographus. Katı et al. (2017) found both S. marcescens and S. liquefaciens in Xyleborinus saxesenii. Yaman et al. (2017) isolated S. liquefaciens from Cryptorhynchus lapathi.
Bioassay
Bioassay tests showed that all the isolated bacteria had a pathogenic effect against the three economic insect pests tested with different ratios. E. cloacae had 35% mortality on D. micans larvae, 50% on S. marcescens, 55% on P. aeruginosa, 40% on K. kristinae, and 45% on S. liquefaciens (Fig. 1).
Among the tested bacteria, P. aeruginosa was the most effective isolate causing 69.6% mortality rate in the larvae of T. pityocampa. The other isolates, E. cloacae, S. marcescens, K. kristinae, and S. liquefaciens showed different levels of potential such as 56.7, 60.9, 43.5, and 65.2% mortality rate on T. pityocampa larvae, respectively (Fig. 2).
On the other hand, the bacterial isolates against the 2nd instar larvae of L. dispar was tested. E. cloacae was the most effective isolate causing 84% mortality on the larvae of L. dispar. The other isolates, S. marcescens, P. aeruginosa, K. kristinae, and S. liquefaciens showed different levels of effects such as 47.8, 48, 16, and 36% mortality on L. dispar larvae, respectively (Fig. 3).
Sezen et al. (2001) carried out a number of bioassays with S. marcescens, using larvae and/or adults of Agelastica alni, Balaninus nucum, Curculio elephas, Euproctis chrysorrhoea, Hyphantria cunea, Malocosoma neustria, Neodiprion sertifer, Pieris brassicae, and Y. malinellus. They determined 70 and 48% effect on the larvae and adults of A. alni, respectively; 78% on B. nucum adults, 55% on the larvae of C. elephas, 58% on E. chrysorrhoea, 10% on H. cunea, 78% on M. neustria, 88% on N. sertifer, 100% on P. brassicae, and 92% on Y. malinellus larvae. On the other hand, Lipa and Wiland (1972) found 100% mortality on Agrotis sp., Mathew and Mohamed-Ali (1987) recorded 83.3% on C. cadambae larvae, and Onoviran et al. (1985) recorded 65–90% mortality with S. marcescens on Glossiana spp.