Insecticidal activity of isolated bacteria from Hyphantria cunea ( Drury ) ( Lepidoptera : Arctiidae ) Hyphantria cunea ( Drury ) ( Lepidoptera : Arctiidae ) ’ den izole edilen bakterilerin insektisidal aktivitesi

Amerikan Beyaz Kelebegi, Hyphantria cunea (Drury) sayisiz konukcu bitkisi olan polifag bir zararlidir. Bu calismada, H. cunea ’nin bakteriyal florasi zararliya karsi mikrobiyal kontrol ajani olabilecek yeni organizmalari identifiye etmek icin arastirildi. H. cunea ’dan alti bakteri izole edilerek saflastirildi. Bakteriyal izolatlarin morfolojik, biyokimyasal, diger fenotipik ozellikleri ( API 20E, API 50 CH, API Staph ve API Coryne) belirlendi. Ilave olarak, 16S rRNA gen sekans analizi de yapildi. Yapilan calismalar sonucunda izolatlar; Lysinibacillus sphaericus (Abk1), Bacillus amyloliquefaciens (Abk2), Staphylococcus sciuri (Abk4), Kocuria palustris (Abk6), Arthrobacter arilaitensis (Abk7) ve Microbacterium oxydans (Abk8) olarak tanimlandi. Butun bakteriler H. cunea ’nin ucuncu-dorduncu gomlek larvalarina karsi oniki gun boyunca test edildi. En yuksek insektisidal aktivite %30 ile L. sphaericus (Abk1)’dan elde edildi (p<0.05). Bu sonuclar, L. sphaericus (Abk1)’u n, H. cunea ’nin mikrobiyal mucadelesinde dikkate alinabilecegini gostermistir. Gelecekte, L. sphaericus (Abk1)’un patojenite zenginlestirme stratejileri (diger entomopatojelerle veya insektisitlerle kombine edilmesi gibi) kullanilarak H. cunea uzerindeki etkinliginin artirilmasi yonunde calismalar yapilacaktir.


INTRODUCTION
The fall webworm, Hyphantria cunea (Drury) is a pest polyphagous Lepidoptera feed on the number of host plants from wide ranges of forest and fruit trees to several agricultural crops.Native to North America, H. cunea spread of trees different areas in Europe and Asia.
Several some studies have been performed to determine the different enthomopathogens in H. cunea (Yamanaka et al. 1985;Yaman et al. 2002;Albayrak Iskender et al. 2012).However, H. cunea is a very harmful pest species around the world, sustainable method for controlling of this pest still have not been.In the current study, we investigated bacterial flora of H. cunea to identify new candidate organisms as a possible microbial control agent against the pest.

Collection of insects
H. cunea larvae were collected from Georgia, in April-May 2012.The collected larvae carefully were placed in sterile plastic boxes and immediately were transported to the laboratory.

Isolation of bacteria
The larvae were separated to dead and live, surface sterilized in 70% alcohol and then washed three times with sterile distilled water and homogenized in nutrient broth media by using a glass tissue grinder.Suspensions were diluted and 0.1ml suspension was plated on nutrient agar.Plates were incubated at 30ºC for 2-3 days.After the incubation period, the plates were examined and bacterial colonies were selected.The colonies determined were purified by subculture on the plates.

Identification of bacterial isolates
Some morphological (cell morphology, endospore formation and mobility) and biochemical properties (gram reaction, oxidase, catalase and reduction of nitrate) of bacterial isolates were examined using standard protocols (Harley and Prescott 2002).The commercially available API 20E, API 50 CH, API Staph kits and API Coryne kit (bioMe´rieux) also was used to determine some other phenotypic properties according to the manufacturer's instructions.

16S rRNA gene sequencing
Bacterial colonies were inoculated into nutrient broth and incubated approximately 18 h at 30 0 C in order to extract DNA from bacterial isolates.At the end of incubation time, the bacterial cells were collected by centrifuging from the culture medium.Then, genomic DNA was isolated with Genomic DNA Purification Kit (Promega, Germany) in accordance with the manufacturer's recommendations.16S rRNA gene sequences of the bacterial isolates were performed with the following universal primers: UNI16S-L: 5'-ATT CTA GAG TTT GAT CAT GGC TCA -3' as forward and UNI16S-R: 5'ATG GTA CCG TGT GAC GGG CGG TGT GTA-3' as reverse.PCR conditions were adjusted according to the instructions of Weisburg et al. (1991).The amplified 16S rRNA gene product was sent to RefGen Biotechnology Laboratory (Ankara, Turkey) for sequencing.The sequences obtained were used for identification of the isolates and phylogenetic analyses.

Phylogenetic relationship of the bacterial isolates
The sequences obtained were used to perform BLAST searches using the NCBI GenBank database to confirm isolate identification Altschul et al. (1990).Evolutionary relationships of the eleven bacterial isolates were evaluated.Cluster analyses of the sequences were performed using BioEdit (version7.09)with Clustal W followed by neighbor joining analysis on aligned sequences performed with MEGA 6.0 software (Tamura et al. 2013).Reliability of dendograms was tested by bootstrap analysis with 1000 replicates using MEGA 6.0.

The insecticidal effects of bacterial isolates
Third-fourth instar larvae of H. cunea were used for the insecticidal assay of bacterial isolates.All of the bacterial isolates were tested as overnight cultures after removing the growth medium.Bacterial isolates were incubated for 18 hours (72 h for sporulation) at 30ºC in the nutrient broth medium.After incubation, bacterial cells were centrifuged at 3000 rpm for 10 min (Ben-Dov et al. 1995).The pellet was resuspended by adding sterile PBS.The optical density of the cells was adjusted to 1.89 at OD (optical density)600 (Moar et al. 1995).Fresh mulberry leaves were inoculated by dipping them into the bacterial suspensions and placed in a sterile plastic box (150ml).The control group was treated with sterile PBS.Ten thirdfourth instar larvae were placed into each box and fresh mulberry leaves was provided in each day for twelwe days.These boxes were incubated at 26±2ºC and 60% RH during 12 h L: 12 h D photoperiod.Mortality was recorded twelwe days later.At least thirty larvae were assayed for each isolate.All experiments were repeated three times.
Mortalities were corrected according to Abbott's formula (Abbott 1925).The data were subjected to ANOVA and subsequently to LSD multiple comparison test to compare isolates against the control group and to determine differences among isolates using SPSS 15.0 statistical software.
All isolates were catalase positive.Oxidase and reduction of nitrate tests varied depending on the isolate.Some morphological and biochemical characteristics of bacterial isolates are summarized in Table 1.Phylogenetic tree was constructed by using Neighbor Joining method (Figure 1).The similarities between isolates were ranged between 98%-99% compared to other species.
+ positive, -negative, ±: weak positive --+ positive, -negative, ±: weak positive 16S rRNA gene sequence analysis results of isolates are given in Table 6.The 16S rRNA partial gene sequences generated in this study have been deposited with the GenBank database under the accession numbers KF704369, KF704370, KU200945, KF704371, KU200946 and KU200947, respectively.All these bacteria were tested against third-fourth instar larvae of H. cunea.The highest insecticidal activity 30% was obtained from L. sphaericus (Abk1) (p<0.05),whereas insecticidal activity was not obtained from K. palustris (Abk6) within twelwe days.Insecticidal activity of the remaining isolates (Abk2, Abk4 and Abk7 the same, Abk8) were as follows; 16.6%, 13.3% and 10%, respectively.The insecticidal activity of the isolates on H. cunea adults are shown in Figure 2.

DISCUSSION
In the current study, we determined the culturable bacterial flora of H. cunea to identify new candidate organisms as a possible biocontrol agent against pest.Bacteria that belong to Lysinibacillus sphaericus (Abk1), Bacillus amyloliquefaciens (Abk2), Staphylococcus sciuri (Abk4), Kocuria palustris (Abk6) and Microbacterium oxydans (Abk8) with insects has previously been demonstrated (Leroy et al. 2011, Tranchida et al. 2011, Gupta et al. 2012, Huang et al. 2012, Chandel et al. 2013, Ozsahin et al. 2014).However, to our knowledge, this is the first documentation of Arthrobacter arilaitensis (Abk7) from any insects.Isolate Abk1, L. sphaericus, is a naturally occurring, mesophilic, soil bacterium, toxic to mosquito larvae.L. sphaericus was isolated from dead larvae of Culex pipiens and Palomena prasina in the previous studies and investigated on the pathogenicity of the isolated hosts.(Tranchida et al. 2011, Ozsahin et al. 2014) The insecticidal property of this organism is due to two proteins produced during sporulation.These proteins are the binary toxins, which accumulate as crystal inclusions and mosquitocidal toxins (Mtx proteins) are produced during vegetative growth of the bacteria.The bacteria are not harmful to humans and other animals, L. sphaericus is an ideal insecticide.L. sphaericus (Abk1), was found to be causing the highest pathogenicity rates (30%) within twelwe days in larvae of H. cunea.Isolate Abk2, B. amyloliquefaciens is an important source of alpha-amylase and protease for industrial applications.This bacterium was isolated from the gut of house flies, Musca domestica, in the previous studies (Gupta et al. 2012).Geetha et al. were reported for the first time mosquitocidal activity of B. amyloliquefaciens.But, we found that it has 16.6% insecticidal effect on larvae of H. cunea.

Fig. 1 .
Fig. 1.Neighbor-joining tree of bacterial isolates from H. cunea and their closely related 12 bacterial species.The dendrogram was constructed by MEGA 6.0 software based on the partial sequences of the 16S rRNA gene.Bootstrap values shown next to nodes are based on 1000 replicates.The scale on the bottom of the dendrogram shows the degree.API test results are listed in Tables 2, 3, 4 and 5, respectively.

Table 2 .
The results of API 20E test system of bacteria isolated from H. cunea

Table 3 .
The results of API 50CH test system of Abk1 and Abk2 isolates

Table 4 .
The API STAPH test results of Abk4 and Abk6 isolates

Table 5 .
The API Coryne test results of Abk7 and Abk8 isolates

Table 6 .
Conclusion identification and GenBank Accession numbers of bacterial isolates according to the partial 16S rRNA gene sequence.