Isolation and identification of bacteria from Xylosandrus germanus ( Blandford ) ( Coleoptera : Curculionidae )

Biological control studies have been increasingly performed against agricultural and forest pests. To develop a biological control agent, bacteria was isolated from harmful pests and identified using various tests. Xylosandrus germanus (Blandford, 1894) (Coleoptera: Curculionidae) is a harmful pest in the hazelnut orchards and other fruit-tree cultures. In this study, we identified 16 bacteria isolates from healthy X. germanus collected in hazelnut orchards in Turkey. Isolates were characterized based on morphological, physiological and biochemical properties using the VITEK 2 Identification System and the fatty acid methyl esters (FAME) analysis. In addition, 16S rRNA gene sequencing of bacterial isolates was performed. Associated bacteria were identified as Acinetobacter psychrotolerans (2 strains), Stenotrophomonas maltophilia, Pseudomonas fluorescens (two strains), Staphylococcus sciuri, Staphylococcus warneri, Pantoea agglomerans (two strains), Staphylococcus hominis subsp. hominis, Erwinia billingiae (two strains), Brevibacterium linens, Advenella sp., Pantoea cedenensis and Brevibacterium permense. Several species of these bacteria are used in biological control as an antifungal and insecticidal against agricultural pest. In the future, their biological control properties will be investigated. This is the first study on the bacterial community of X. germanus.


INTRODUCTION
The main purpose of most agricultural studies is to increase the yield of agricultural crops.Although Turkey is first among all hazelnut producing countries (Kılıç 1994), the average yield of hazelnut per unit field is very low.Approximately 150 insect species have been detected in hazelnut orchards.However, only 10-15 of these species result in economic losses (Isık et al., 1987).Ambrosia beetles are an important pest in hazelnuts (Ak et al., 2005a, b, c).
Chemicals used against pest insects have harmful effects on the environment.Intensive use of chemicals leads to resistance in insects, and is also harmful to the environment.Biological pest control is thought to be an alternative method.Biological control provides a safety approach that is less toxic to the environment, credit to its capability of causing disease in insects, it does not harm other animals or plants.Using natural enemies against pest organism has developed the new environmentally friendly methods and microbial pest control strategies have been preferred instead of chemical pesticides worldwide.
Bacteriological studies have been made with the aim of developing biological control agents, especially against other hazelnut pest insects, such as the ambrosia beetles *Corresponding author.E-mail: hatice.kati@giresun.edu.tr.Tel: +90 454 310 1458 Fax: +90 454 310 1477 Xyleborus dispar (Sezen et al., 2007(Sezen et al., , 2008;;Kati et al., 2007).Another closely related beetle, the black stem borer Xylosandrus germanus (Blandford 1894) (Coleoptera: Curculionidae) is also an important hazelnut pest, but its bacterial community is currently unknown.These invasive beetles are native to Asia and were first detected the US in 1932 and introduced to Europe in the 1950`s (Solomon, 1995;Lawrence, 2006).It is polyphagous and attacks a wide variety of host trees (Frank and Sadof, 2011).Bacteria are abundant and diverse on the body surface and within galleries of ambrosia and bark beetles (Hulcr et al., 2012).Here, we aimed to identify the bacterial community of X. germanus for the first time.

Collection of insects and isolation of bacteria
In this study, branches with galleries creating adults of X. germanus in the bark were collected from the hazelnut orchards in Giresun, Turkey, in June and July 2008 and taken to the laboratory.Insects were individually put into sterilized tubes to prevent possible contamination.They were identified by Dr. Kibar Ak (Black Sea Agricultural Research Institute, Samsun, Turkey).Collected adults were surface sterilized with 70% ethanol.The adults were homogenized in a Nutrient broth (NB; containing per liter: 5 g peptone from meat; 3 g meat extract) by using a glass tissue grinder.Then, samples were ten-fold diluted.100 μl of the suspensions were plated on a Nutrient agar (NA; containing per liter: 5 g peptone from meat; 3 g meat extract; 12 g agar-agar).
Plates were incubated at 30°C for 24 or 48 h.Bacteria were selected based on their colours and colony morphologies.Then, pure cultures were prepared and these cultures were identified using various assays.

Phenotypical, physiological, biochemical properties and fatty acid methyl ester analysis of the isolated bacteria
Colony morphologies of the isolates were observed on NA by direct and stereomicroscopic observations of single colonies.Bacteria morphology and motility were examined by light microscopy of native preparations.Gram staining was performed (Claus, 1992).Endospores were observed in light microscopy using negative staining (Elcin, 1995).Temperature, NaCl and pH tolerance values were determined in NB.The VITEK 2 analysis system was used to detect biochemical properties.Fatty acid methyl ester (FAME) analysis of isolates was performed as suggested by Sasser (1990) using the Microbial Identification System (Hewlett-Packard model 5898A, Palo Alto, CA) and using the Tryptic Soy Agar (TSA) database of the Microbial Identification System software package (MIDI; Microbial ID, Inc., Newark, DE).
The PCR was conducted using the following conditions: 5 min at 95°C for initial denaturation, followed by 30 amplification cycles (20 s at 95°C, 45 s at 55°C 1 min at 72°C) and 7 min at 72°C for final primer extension.All PCR products were analysed by 1.3% agarose gel electrophoresis.The resulting gene sequences (length approximately 1,400 bp) were cloned into a pGEM-T easy cloning vector.Sequencing of the cloned products was performed at Macrogen Inc. (Wageningen, Holland).These sequences comparisons were blasted against the GenBank database (Pearson, 1990;Altschul et al., 1990Altschul et al., , 1997)).

G±C analysis of Xg5 isolate
Analysis of the G±C content of the bacterial isolate Xg5 was performed using the DSMZ Identification Service.Its G±C content was determined by HPLC (Cashion et al., 1977;Tamaoka et al., 1984;Mesbah et al., 1989).The DNA was purified on hydroxyapatit according to the procedure of Cashion et al. (1977).

RESULTS
In this study, 16 bacterial isolates from X. germanus were identified using phenotypic, biochemical, physiological, FAME and molecular techniques.According to morphological results, five isolates were Gram-positive, the others were Gram-negative and all isolates were nonsporulating, eight isolates were motile and eight were non-motile.Moreover, the colony colours of two isolates were yellow, that of the other two isolates were orange and the others produced a creamy pigment.Four isolates had the shape of coccobacilli; five isolates were bacilli; seven isolates were cocci (Table 1).
According to pH test results, none of the isolates grow at pH 3 media; and six isolates grow at pH 5.All isolates grew at pH 7. According to heat tolerance test results, all isolates grew at 25 and 30°C, and some isolates grew at 37 and 40°C.According to NaCl tolerance test results, six isolates grow at 2% NaCl media; two isolates grow weakly; the others did not grow (Table 2).Biochemical characteristics of isolates were examined using the VITEK 2 system (Table 3 and 4).In order to identify FAME profiles of the isolates, MIS was used.In this study, according to FAME profiles, all isolates had 9-20 carbons and 46 different fatty acids were detected.Moreover, all the isolates had a C16:0 saturated fatty acid.The FAME profiles of isolates are listed in Table 5.Molecular studies of isolates were performed using 16S rRNA gene sequencing analysis.

DISCUSSION
In order to develop effective biological control agents, it is necessary to identify the bacterial community of insect pests.For this purpose, we aimed to identify the bacterial community of the hazelnut pest X. germanus.In this study, 16 bacteria isolated from X. germanus were identified.
According to FAME analysis and VITEK 2 results, Xg1 and Xg2 isolates were determined as Acinetobacter
Based on FAME analyses, Xg5 and Xg6 isolates were identified as Staphylococcus sp.The Xg5 isolate was identified as S. sciuri, according to FAME analysis and VITEK 2 results.In previous studies, members of the genus Staphylococcus displayed large amounts of the fatty acids anteiso C15:0, C18:0, C20:0 and smaller but significant amounts of the fatty acids iso C15:0, C16:0, iso C17:0 ve anteiso C17:0 fatty acids (Kotilainen et al., 1990;Wieser and Busse, 2000).Our results of the 16S rRNA sequencing identified Xg5 as one of the S. sciuri subspecies: either S. sciuri subsp.carnaticus, S. sciuri subsp.rodentium or S. sciuri subsp.sciuri (Table 7).Thus, G±C analysis of this isolate was performed by DSMZ.We found a G±C content of 32.5% that suggested a new S. sciuri subspecies.
The Xg6 isolate is similar to S. cohnii subsp.cohnii based on FAME analyses.Nevertheless, according to VITEK 2 and 16S rRNA gene sequence analysis results, this isolate resembles Staphylococcus warneri (Table 7).Strains of S. warneri have been shown to grow at 40°C and are susceptiple to novobiocin (Kloos and Schleifer, 1975).These results are consistent with ours.RNA gene restriction polymorphism has been used to differentiate S. pasteuri from S. wameri (Chesneau et al., 1993).Staphylococcus pasteuri should be yellow in VITEK 2 tests, whereas Xg6 appeared to be creamy in our analysis.Therefore, the Xg6 isolate was identified as S. warneri.
Xg7 and Xg15 isolates were identified as Pantoea agglomerans according to VITEK 2. According to FAME analyses results, the Xg7 isolate is similar to P. agglomerans and the Xg15 isolate is similar to Serratia odorifera.16S rRNA gene sequencing identified the Xg15 isolate as Serratia sp. and Xg7 as P. agglomerans (99%).These results were also supported by VITEK 2 analyses.
Xg8 isolate was identified as Staphylococcus hominis subsp.hominis according to FAME analysis and VITEK 2. However, 16S rRNA sequencing indicated that isolate is similar to S. hominis subsp.novobiosepticus.Kloos et al. (1998) reported S. hominis subsp.novobiosepticus is resistant to novobiocin.We found that Xg8 is susceptible to novobiocin in VITEK 2 results and therefore we concluded that Xg8 is S. hominis subsp.hominis (Table 4).
16S rRNA sequencing showed that the isolates belong to the Brevibacteria.Morhopological studies showed that Xg12 and Xg16 isolates are B. linens, B. permense, respectively.Brevibacterium species have been isolated from insect (Katı et al., 2010).
As a result, bacteria isolated from X. germanus were identified in this study.In future, biological control properties of these bacteria will be investigated.In previous studies, several species of Acinetobacter, Stenotrophomonas, Pantoea, Brevibacterium and Pseudomonas bacteria identified in this study exhibited antifungal or insecticidal activities (Selvakumara et al., 2011;Trotel-Aziz et al., 2008;Jankiewicz et al., 2012).

Table 1 .
Morphological characteristics of bacterial isolates of Xylosandrus germanus.

Table 2 .
Physiological characteristics of bacterial isolates of X. germanus.

Table 4 .
Biochemical characteristics of Gram positive bacterial isolates (tested with VITEK 2).

Table 5 .
FAME profiles of bacterial isolates.

Table 7 .
Identity of isolates according to VITEK 2, FAME profiles and 16S rRNA sequencing.