Background

New pest management strategies tend to minimize the impact on the environment and non-target organisms (Ruiu et al. 2013). Entomopathogens have relative specificity and lower environmental impact. The successful use of entomopathogens results in an alternative pest management for insect control. Among them, bacteria and their toxins are the most commercially microbial insecticides used successfully in biological control. Entomopathogenic bacteria such as Bacillus thuringiensis are generally known as lower risk pesticides than chemicals. Current goals include searching for entomopathogenic bacteria and determining their potentials (Egami et al. 2009). Entomopathogenic bacteria are commonly isolated from host insects or soil (Thiery and Frachon 1997). Soil plays an important role as a habitat for spore-forming and non-spore-forming entomopathogenic bacteria. While the entomopathogenic bacteria domain has been well represented by spore-forming bacteria, members of Bacillaceae family, non-spore-forming entomopathogenic bacteria have been also documented with insects (Ruiu et al. 2013).

Numerous larvae of Scarabaeidae (Coleoptera) feed on plant materials in the soil, and many of them are known as important plant pests. Scarabs are infected by a number of entomopathogens (Yaman et al. 2016). The European cockchafer, Melolontha melolontha (Linnaeus, 1758) (Coleoptera: Scarabaeidae) causes significant economic damage. It is a soil-inhabiting insect pest at the larval stage and possible target for entomopathogenic bacteria in soil.

In this study, isolation and characterization of non-spore-forming bacteria from M. melolontha larvae were presented and their potentials on three economic insect pests were documented.

Material and methods

Insect samples

Samples of M. melolontha larvae were collected from Ordu province, Turkey, targeting isolate entomopathogenic bacteria. For bioassay tests, three economic insect pests, namely Dendroctonus micans (Yaman et al. 2010), Thaumetopoea pityocampa, and Lymantria dispar, were used. D. micans larvae were obtained from the forest fields near Ordu, Turkey; their cultures were maintained in a laboratory set up by the Turkish Ministry of Forestry in Ordu province. Lymantria dispar larvae were collected from İzmir province, Turkey, brought to the laboratory, and fed on apple leaves. T. pityocampa larvae were collected from pine trees in Sinop province, Turkey, brought to the laboratory, and fed on fresh pine needles.

Bacterial isolation

M. melolontha larvae suspected of bacterial symptoms after a macroscopic examination were used for bacterial isolation (Thiery and Frachon 1997). Before the isolation process, the dead larvae were individually placed into 70% ethanol and gently shaken for 3 min (Yaman et al. 2010). After surface sterilization, considering aseptic conditions samples were washed by a sterilized water. Sterile 1 ml syringes were used to insert into the hemocoel of the insect. Then, 100 μl hemolymph suspension, taken from the hemocoel, was spread on nutrient agar plates. The plates were incubated at 25–36 °C for 24–48 h. After incubation, the plates were examined and bacterial colonies that were similar in terms of colony and color morphology were selected. Different colony types of bacteria were selected and purified on nutrient agar plates by subculturing. Individual colonies were subcultured twice to ensure purity (Kuzina et al. 2001). One of the subcultured colonies was used for identification of species per isolate.

Bacterial strains were preserved for a long-term storage in nutrient broth with 15% glycerol at − 86 °C for further tests at the Department of Molecular Biology and Genetics, Faculty of Arts and Science, Ordu University. The stock culture strains were subcultured onto tryptic soy agar plates to check their purity. All bacterial isolates were initially stained by Gram’s dye for the identification of gram-positive or gram-negative bacteria and tested for some biochemical reactions. A standard bacterial suspension was prepared in 1.8 ml of 0.45% saline, using the VITEK colorimeter for each isolate. The time interval between suspension preparation and card filling was less than 30 min to avoid changes in turbidity. Then, VITEK bacterial identification systems (bioMerieux, Prod. No 21341 and 21342) were used for the identification of the isolated bacteria. Additionally, all isolated bacteria were further identified, using 16S rDNA analysis. For this, pure bacterial suspensions were used for DNA extraction. Equal volumes of bacterial suspension and glass beads were put into an Eppendorf tube and vigorously shaken on the vortex for 1 min at maximum speed. And then, the solutions were incubated by proteinase K at 56 °C for 3 h. After that, nucleic acid extraction was performed by the DNA isolation kit, as according to the manufacturer’s guidelines and Hyliš et al. (2005) with some modifications. To amplify and sequence the bacterial 16S rRNA genes and identify the bacterial isolates, the isolated bacterial DNAs were sent to Sentegen Biotech Company (Ankara, Turkey).

Bioassay tests for potentials of the isolated bacteria

Virulence of the isolated bacteria was tested against the larvae of D. micans, larvae of T. pityocampa, and L. dispar. Bacterial isolates were incubated in nutrient broth medium at 30 °C for 18 h. Bacterial suspensions containing approximately 109 bacteria/ml were used in bioassay according to Ben-Dov et al. (1995). D. micans larvae were fed on spruce barks dipped into the bacterial suspensions (Yaman et al. 2010), T. pityocampa larvae on pine needles, and L. dispar on apple leaves. Each bioassay group was performed by 30 insect larvae at the same laboratory conditions. All tested groups were kept at 24–28 °C and 35–45% RH and 18:6 photoperiod in laboratory conditions. Observations were recorded daily and dead larvae were removed immediately. All bioassays were repeated three times on different days and data was corrected, using Abbott’s formula (Abbott 1925).

Results and discussion

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).

Fig. 1
figure 1

Potential of the isolated bacteria on Dendroctonus micans larvae

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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).

Fig. 2
figure 2

Potential of the isolated bacteria on Thaumetopoea pityocampae larvae

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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).

Fig. 3
figure 3

Potential of the isolated bacteria on Lymantria dispar larvae

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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.

Conclusion

Entomopathogenic bacteria are generally known as lower risk pesticides than chemical pesticides. Five entomopathogenic bacteria were isolated from the forest common pests; D. micans, T. pityocampa, and L. dispar. The results confirmed that the five bacteria originated from M. melolontha larvae and infected the three tested pests. Therefore, the results of this study would be of great interest to propose some effective entomopathogenic bacteria against these important forest pests.