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Biologia 66/2: 288—293, 2011 Section Cellular and Molecular Biology DOI: 10.2478/s11756-011-0021-6 The first investigation of the diversity of bacteria associated with Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) Hacer Muratoglu, Zihni Demirbag &KazimSezen* Karadeniz Technical University, Faculty of Arts and Sciences, Department of Biology, 61080 Trabzon, Turkey; e-mail: [email protected] Abstract: Colorado potato beetle, Leptinotarsa decemlineata (Say), is a devastating pest of potatoes in North America and Europe. L. decemlineata has developed resistance to insecticides used for its control. In this study, in order to find a more effective potential biological control agent against L. decemlineata, we investigated its microbiota and tested their insecticidal effects. According to morphological, physiological and biochemical tests as well as 16S rDNA sequences, microbiota was identified as Leclercia adecarboxylata (Ld1), Acinetobacter sp. (Ld2), Acinetobacter sp. (Ld3), Pseudomonas putida (Ld4), Acinetobacter sp. (Ld5) and Acinetobacter haemolyticus (Ld6). The insecticidal activities of isolates at 1.8×109 bacteria/mL dose within five days were 100%, 100%, 35%, 100%, 47% and 100%, respectively, against the L. decemlineata larvae. The results indicate that Leclercia adecarboxylata (Ld1) and Pseudomonas putida (Ld4) isolates may be valuable potential biological control agents for biological control of L. decemlineata. Key words: Leptinotarsa decemlineata; 16S rDNA; microbiota; insecticidal activity; microbial control. Abbreviations: ANOVA, one-way analysis of variance; LSD, least significant difference; PBS, phosphate buffer solution. Introduction used because of marketing concerns and limited num- ber of transgenic varieties available. Also, recombinant Potato is an important crop with ∼4.3 million tons defence molecules in plants may affect parasitoids or of production on 192,000 hectares of growing area predators indirectly (Bouchard et al. 2003). in Turkey. The Colorado potato beetle, Leptinotarsa In general, the control of this pest is accomplished decemlineata (Say), is a pre-eminent pest on solana- by utilizing insecticides. However, during the 1980’s, re- ceous crops worldwide, and many methods have been sistance against many insecticides commonly used for used to control it, including hand-picking, bird pre- the control of L. decemlineata began to develop. This dation, introduction of natural enemies, trapping bor- insect is well-known for its rapid resistance development der sprays, trench traps, propane flames and crop to pesticides (Dickens 2002). Also, insect’s natural en- rotation (Casagrande 1987). Both adults and larvae emies were influenced by pesticides used against the feed on this host, and often cause complete defo- L. decemlineata (Ferro & Boiteau 1993). However, the liation of potato plants attacked, with considerable development of insecticide resistance in target popula- yield losses (50% of the crop in some European and tions and concern about the detrimental effects of these Mediterranean Plant Protection Organization coun- chemicals on non-target arthropods, the environment, tries). In favourable weather and biological circum- and human health have spurred interest in alternative stances, populations are liable to expand dramati- insect control agents. cally; even with 90% egg mortality and varying de- Other microorganisms associated with insects have grees of larval mortality, after 5 years without control, been implicated in altering the population ecology of a population of 1.1×1012 could build up from a sin- their hosts. For example, ice-nucleating bacteria have gle pair of parents. L. decemlineata is also suspected been shown to decrease the overwintering capacity of L. of spreading several potato diseases, including Ralsto- decemlineata (Lee et al. 1994). Such bacteria, even in a nia solanacearum and Clavibacter michiganensis subsp. killed form, have been documented to raise the lowest sepedonicus (http://www.eppo.org/QUARANTINE/ temperature at which the insect will survive in soil. In insects/Leptinotarsa decemlineata/LPTNDE ds.pdf). addition, there may be as yet undiscovered associations Genetically engineered resistant varieties contain- of individual microbes or microbial communities that ing toxin genes from Bacillus thuringiensis tenebrio- can affect the establishment of insects or their ability nis are highly effective, but they are not now being to overwinter. Components of the microbiota of insects * Corresponding author c 2011 Institute of Molecular Biology, Slovak Academy of Sciences Bacteria from Leptinotarsa decemlineata 289 may also affect the ability through antagonism or com- 1978). Both larvae and adults were homogenized in a nu- petition of plant pathogen partners in synergisms with trient broth by using a sterilized glass tissue grinder. Ten insects. insects were used in every homogenisation and all homogeni- Microscopic techniques and physiological together sations were repeated three times on different occasions. In with biochemical tests generally cannot distinguish be- the repeat, bacterial suspensions were prepared in one of the two following ways. In the first way, the suspension was tween different bacteria. Relatedness of species could filtered twice through two layers of cheesecloth to remove be determined with the bacterial taxonomy molecu- debris (Poinar 1978). The suspension was diluted to 10−8 lar techniques. DNA hybridization studies on entire (Christine & Ted 1992). One hundred µL of every diluted genomes or selected genes are the basis for compari- sample were spread on nutrient agar plates and incubated son between species. Similarly, the potential of sequence at 28 ◦C for 48–96 h. Alternatively the suspension was ap- analysis of genes coding for rRNA and of certain pro- plied directly to nutrient agar plates. In the second way, ◦ teins for interfering the evolution of taxa through bil- the suspension was heated at 80 C for 10 min in a water lions has been documented (Woese 1987). The intro- bath to eliminate the non-spore forming bacteria (Thiery & µ duction of PCR amplification of 16S rDNA genes from Frachon 1997). One hundred L of the heat-treated sam- ples were spread directly on nutrient agar plates and incu- bacterial DNA is used to differentiate the bacteria from ◦ bated at 28 C for 48–96 h (Lee et al. 1995). After incuba- each other. tion times, all the plates obtained in both ways were exam- At present a lot of pesticides have been used in ined, bacterial colonies being selected based on colour and Turkey to control L. decemlineata;someofthemare morphology. Pure cultures of these colonies were then pre- endosulfan, deltametrin, chlorpyrifos-ethyl, azinphos, pared; every one of different colonies was coded from Ld1 to promecarb, and cypermethrin. Increasing interest in de- Ld6 and these cultures were identified by various tests, such veloping environmentally safe pest control methods has as utilization of organic compounds, spore formation, NaCl inspired us to study the potential of bacteria for con- tolerance, optimum temperature, catalase test, oxidase test trolling L. decemlineata. Since there is no study on in- and gelatine hydrolysis. The API 20E and API 50CH sys- tems were used only to characterize the bacterial isolate, vestigation of microbiota as biological control agent of because, as pointed out by Behrendt et al. (1999) and Peix L. decemlineata, this insect is very attractive for bi- et al. (2003), the identification of non-clinical isolates is of- ological control studies. Infected insect larvae present ten wrong with these systems. certain symptoms that suggest bacterial infection; they The identification procedure of isolated bacteria was become flaccid, lethargic, and stop eating (Osborn et done according to “Bergey’s Manual of Systematic Bacteri- al. 2002). Various bacterial insect pathogens have been ology 1 and 2” (Krieg & Hol, 1986; Sneath et al. 1986). used successfully in microbial control of insects (Sezen & Demirbag 1999, 2006; Demir et al. 2002; Kati et al. 16S rDNA sequencing 2005; Sezen et al. 2007, 2008). For 16S rDNA sequencing, DNA was extracted as described Here we report on the isolation and identification of previously (Sambrook et al. 1989). Amplification and se- bacteria found in living L. decemlineata.Wetestedthe quencing of the nearly complete of 16S rDNA gene was per- formed according to the conventional methods. The PCR insecticidal activities of these bacterial isolates that are product was cloned into pGEM-T easy vector and then the less affected both plant varieties and the natural ene- 16S rDNA gene sequences were determined with an Ap- mies of L. decemlineata against its larvae as possible bi- plied Biosystems model 373A DNA sequencer, using the ological control agents. The main goal of the study was ABI PRISM cycle-sequencing kit. Sequences consisting of to describe the isolation and further characterization of about 1,400 nucleotides of the 16S rDNA gene of isolates bacteria from the adults of L. decemlineata. Using con- were determined. The sequences obtained were compared ventional tests and sequence analysis of the bacterial with those from GenBank (Benson et al. 2010) using the 16S rRNA gene, we identified six bacterial isolates to at BLAST program (Altschul et al. 1990) and 16S rRNA gene least the genus level. Also, in order to find and identify sequences of Ld1-6 have been deposited in GenBank under the accession numbers HQ132731, HQ132732, HQ659186, a new toxic bacterial isolate against the adults of L. de- GU187010,