ISOLATION AND CHARACTERIZATION OF Bacillus ...

© by PSP Volume 11 – No 7. 2002

Fresenius Environmental Bulletin

ISOLATION AND CHARACTERIZATION OF Bacillus thuringiensis ISOLATED FROM HAZELNUT FIELDS IN TURKEY R. Nalcacioglu, M. Yaman, S. Dulger, A. O. Belduz and Z. Demirbag Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University, 61080 Trabzon, TURKEY

SUMMARY In this study seventy-three Bacillus thuringiensis strains were isolated from soil samples collected from eleven hazelnut fields in the north-east coast of Turkey. They were characterized by morphological, biochemical and molecular methods and tested for insecticidal activity to find out the relatively more effective and safe biological control agents against some plant pests, especially hazelnut pests. A total of 40 of the B. thuringiensis strains fit the internally transcribed spacers (ITSs) characterisation of the B. thuringiensis varieties known. Repetitive extragenic palindromic-PCR (rep-PCR) results indicate that there is a significant genomic variation among the B. thuringiensis isolates. Experimental infections were carried out to detect the insecticidal effect of the isolates against three important pests, Hyphantria cunea, Agalastica alni and Neodiprion sertifer from three different orders. Twenty five of the 73 isolates under study showed high insecticidal effect against H. cunea, 10 against A. alni and 25 against N. sertifer and 2 against all the three insects. Four isolates were pathogenic for A. alni and H. cunea.

hazelnut fields in Turkey. Great efforts have been made in the development of biological control agents against these pests. However, until now these techniques have been sporadically applied and need further research. This leads us to consider Bacillus thuringiensis as an ecologically sound microbial control agent, specific for target insects [1]. There has been extensive interest in recent years in collecting, analysing, and screening B. thuringiensis strains isolated from environmental samples. World-wide screening efforts have been based on the possible existence of new strains with new pathogenic spectra or host ranges. A number of different phenotypic and genotypic methods are presently being employed for identification and classification of B.thuringiensis. Generally, DNAbased methods are emerging as the more reliable, simple and inexpensive ways to identify and classify bacteria. In fact, the assignment of genera/species has traditionally been based on DNA-DNA hybridization methods and modern phylogeny,s increasingly based on 16S rRNA sequence analysis. Internally transcribed spacers-PCR (ITSs-PCR) and repetitive extragenic palindromic-PCR (rep-PCR) are techniques based on DNA amplification, which have been found to be extremely reliable, reproducible, rapid and highly discriminatory [2].

KEYWORDS: Bacillus thuringiensis, ITS, repetitive extragenic palindromic-PCR (rep-PCR), insecticidal activity

In this study we report the isolation of B. thuringiensis strains from eleven hazelnut fields during the summer of 1998 in the north-east coast region of Turkey and their characterization with biochemical reactions and molecular (ITS and rep-PCR) methods.

INTRODUCTION With regard to production and export, Turkey is the first among all hazelnut producing countries, but it lies far behind many of them in terms of the amount of the harvested per unit area. A main reason for this is that hazelnut has many agricultural pests that cannot be effectively controlled. Chemicals utilized to control these pests have hazardous effect on the environment. Fortunately, biological control of hazelnut pests is an alternative to the use of chemical pesticides and ecologically very important because of production of honey, milk and fish around

MATERIALS AND METHODS Sample collection

About 10 g of surface soil samples scrapped within 2-5 cm depth with a sterile spatula were collected from hazelnut fields in 11 different areas of north-east coast of Turkey. The samples were placed in sterile plastic bags and stored at 4 ºC.

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Isolation and Identification

Insect bioassays

The acetate selection method described by Hossain et al. [3] was performed to screen soil samples. Collected soil samples were suspended in sterile physiological saline (1:5, w/v), mixed vigorously, and left to stand without any disturbance. A total of 1.5 ml of the clear supernatant was heated at 80 ºC for 5 min and 1 ml out of it was used to grow culture at 31 ºC in nutrient broth containing 0.25 M sodium acetate for 4 h. The whole broth was heated again at 80 ºC for 5 min and after appropriate dilution the culture was grown on 7% sodium chloride nutrient agar plates at 31 ºC for 5 days. Bacterial colonies were picked up and identified as B. thuringiensis by comparison with standard B. thuringiensis strains [Bt Berliner (DSM2046), Bt var. israelensis (DSM-5724), Bt var. kurstaki (DSM-6102)]. Morphological characteristics such as vegetative cells, cell arrangement and spores were examined microscopically. The biochemical characterization was determined according to Logan and Berkeley [4].

Pathogenicity of B. thuringiensis isolates was assessed against larvae of Hyphantria cunea (Lepidoptera; Arctiidae), Neodiprion sertifer (Hymenoptera; Diprionidae), and adults of Agalastica alni (Coleoptera; Crysomelidae). The insects Hyphantria cunea and Agalastica alni for bioassays were collected from the hazelnut fields and Neodiprion sertifer was collected on young pine foliage in Çamburnu - Sürmene. For this purpose, all isolates were grown for 72 h in nutrient broth at 31 ºC on a rotary shaker. After incubation, the density of the bacterial cells was adjusted to 1.89x109/ml at OD600 and 5 ml of the bacterial suspension was centrifuged at 3,000 rpm for 10 min (8). The pellet was resuspended in 5 ml of phosphate buffer solution and used for bioassays (9). In the experimental infections, larvae of Neodiprion sertifer (Hymenoptera; Diprionidae), Hyphantria cunea (Lepidoptera; Arctiidae), and adults of Agalastica alni (Coleoptera; Chrysomelidae) were used. While A. alni and H. cunea were fed with hazelnut leaves, N. sertifer was fed with pine foliage. The diets were dipped in bacterial suspensions and air-dried on filter paper, and then placed in containers for each assay. Larvae and adult insects were placed on the diets. After 48 h, the insects received fresh untreated leaves every 24 h. For the control, 20 insects received leaves dipped in distilled water. All insects were kept at 26 °C and 60 % RH during a 12:12 h photo period (10). The mortality of insect was scored every 24 h and all dead insects were removed immediately. All these bioassays were repeated 3 times. Data were evaluated by using Abbott’s formula (11).

PCR amplification of intergenic 16S-23S rDNA sequences

Template DNA was isolated from 16-18 h cultures in Luria-Bertani broth. DNA extraction was carried out as described by Sambrook et al. [5]. Primers used in this study are: 5’-TGCGGCTGGATCCCCTCCTT-3’ and 5’-CCGGGTTTCCCCATTCGG-3.’ These primers were designed previously from multiple alignments of 16S and 23S genes of diverse bacteria [6]. They are complementary to the conserved regions 4 (positions 1521-1541 for 16S rRNA gene) and 6 (positions 114132 for the 23S rRNA gene) within the rDNA operon and match the recommended positions to detect spacer variation at the species level [7]. DNA templates (5 µg, 10 µl) were amplified in a 100 µl reaction volume that contained 2.5 U Taq DNA polymerase, 1 mM of each primer, 0.2 mM each of the four dNTPs (Promega), 10 mM Tris/HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 and 0.1 mg/ml gelatine. The reaction mixtures were overlaid with 50 µl mineral oil and heated to 95 °C for 2 min prior to amplification. Amplification was carried out in a thermal cycler (Hybraid, PCR Sprint) for 35 cycles. Each amplification cycle was as follows: 1 min at 95 °C (denaturation), 1 min at 55 °C (annealing), and 2 min at 72 °C (extension). Ten µl of the PCR products were electrophoresed in agarose gel (1.5%) containing 0.1 µg/ml ethidium bromide.

RESULTS An extensive study on the isolation and characterisation of B. thuringiensis from hazelnut fields in Turkey is presented. The study of twenty two soil samples collected from eleven fields clearly revealed that B. thuringiensis was ubiquitously distributed in the soil of hazelnut fields in the north east coast of Turkey. According to the acetate selection method, 73 B. thuringiensis isolates were examined for their morphological and biochemical characteristics, and their insecticidal effects were determined. According to the ITS- PCR results, 40 of the B. thuringiensis isolates fit the ITS characterisation of the B. thuringiensis varieties known (Fig.1). Since the results of ITS did not clearly support whether the 33 remaining isolates were B. thuringiensis strains, we only performed rep-PCR fingerprints for the first 40 isolates and observed a significant genomic variation among them (Fig. 2).

rep-PCR genomic fingerprinting

Primers used in repetitive extragenic palindromicPCR (rep-PCR) genomic fingerprinting are REP 1R 5’IIIICgICgICATCIggC-3’ and REP 2I 5’-ICgICTTATCIggCCTAC-3’. The cycling programs and reaction mixture composition (25 µl) were as previously described. Ten microliters of the reaction mixtures were electrophoresed in agarose gel (1.2% ).

The experimental infections carried out to determine the insecticidal activities showed that 38,6% for H. cunea, 21.4% for A. alni, and 34,3% for N. sertifer were pathogenic (Table 1). On the other hand, 2.9% of the isolates

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FIGURE 1 - ITS (internally transcribed spacers) results of B. thuringiensi isolates (Lane M: Molecular weight marker (100 bp); B1: B. thuringiensis Berliner; B2: B. thuringiensis var. israelensis; B3: B. thuringiensis var. Kurstaki; numbers indicate the isolates).

FIGURE 2 - Repetitive extragenic palindromic-PCR (rep-PCR) results of B. thuringiensis (Isolates that have the same ITS results with control strains B1: B. thuringiensis Berliner; B2: B. thuringiensis var.israelensis; B3: B. thuringiensis var. Kurstaki; numbers indicate the isolates).

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TABLE 1 - Bacillus thuringiensis isolates found to be pathogenic against H. cunea, A. alni and N. sertife.r

Insecticidal effect Tested insects Lepidoptera Hyphantria cunea Coleoptera Agalastica alni Hymenoptera Neodiprion sertifer

≥80%

≥60%

≥50%

2, 5, 9, 11, 18, 19, 20, 45, 47, 52, 70, 72 26, 27, 9

6, 10, 23, 28,35, 60, 68 15, 45, 60, 64

22, 26, 38, 53, 62, 29 2, 18, 71

16, 22, 24, 37, 47, 60, 69, 70, 73

1, 19, 21, 35, 45, 46, 50, 64, 67

3, 10, 15, 17, 28, 48, 50

*Bold numbers refer to the isolates which showed 100% insecticidal effect

were pathogenic for all three insects. Of all isolates 3 for A. alni, 9 for N. sertifer and 12 for H. cunea have very high insecticidal activity (>80%). Four isolates for H. cunea and five for N. sertifer caused 100% mortality and 2 isolates insecticidal activity for all three insects. Six isolates were pathogenic for both A. alni and H. cunea.

results. Recently, Forsty and Logan [17] from Northern Victoria Land, Antartica and Lee et al. [18] from Korea have found new serovarieties of B. thuringiensis. Further researches will be directed to determine serovarieties of the Turkish isolates of B. thuringiensis.

Some of the isolates tested for pathogenicity evidenced no pathogenic effects to any of the insects tested. Similarly, Jung et al. [19] recorded that B. thuringiensis subsp.higo BT205 isolated from rice brain has no insecticidal activity against H. cunea. They suggested that this isolate might have novel cry-type genes other than the known cry genes. Additional studies may be needed for these isolates in order to predict the toxicity against insects of other orders.

DISCUSSION Results of biochemical and morphological tests (data not shown) indicate that the isolates had general biochemical characteristics with minor differences similar to those of B. thuringiensis described by Burges [12]. The isolation of B. thuringiensis is valuable because it may help to understand the role of these bacteria in the environment. The ubiquity of B. thuringiensis in soil was also shown by Dulmage and Aizawa [13] and De Lucca et al. [14]. Depending on the ITS results (Fig. 1), we are able to say that the 40 isolates are certainly B. thuringiensis strains. However, studies performed by Daffonchio et al. [15] showed that the 16S-23S ITS of Bacillus cereus are well-preserved in terms of length, in contrast to that of Bacillus licheniformis, for which at least two different ITS fingerprints were observed. Two different ITS-PCR patterns have also been observed for Bacillus subtilis [16]. Thus, it is difficult to underline whether 33 of our isolates are B. thuringiensis or not.

Up to now no extensive study on isolation and characterization of new B. thuringiensis isolates from soil in Turkey has been performed. On the other hand, some studies were performed on insect-originated Bacillus thuringiensis isolates against some hazelnut pests, but they did not reach 100% mortality [20, 21]. Therefore, the fact that 4 of the isolates caused 100% mortality for H. cunea and 5 for N. sertifer, is very promising. The prospects offered by new isolations of B. thuringiensis strains with improved efficacy and specificity may be of benefit in developing alternative control strategies against hazelnut pests, which currently are controlled only by chemical pesticides.

According to the rep-PCR results (Fig. 2), there is also a variation between the control strains (B. thuringiensis Berliner) and two other control strains used. Significant genomic variations among B. thuringiensis isolates leads us to consider that there are many different and possibly new varieties among the 40 B. thuringiensis isolates. Also the variation between control strains, B. thuringiensis Berliner and its two varieties, supports these

These isolates are also candidates for harbouring putative novel cry genes. The identification of putative novel B. thuringiensis strains could be the first step in the sequence for finding novel toxicity, since novel toxins may be toxic for new targets. The isolation and sequencing of novel cry genes should be encouraged once the target insect is identified and more evidence on the potential of novel toxins as biological control agents is available.

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ACKNOWLEDGEMENTS

[15] Daffonchio D, Borin S, Frova G, Manachini P L and Sorlini C 1998 PCR fingerprinting of whole genomes: the spacers between the 16S and 23S rRNA genes and of intergenic tRNA gene regions reveal a different intraspecific genomic variability of Bacillus cereus and Bacillus licheniformis; International Journal of systematic Bacteriology 48 107-116

This study was funded by the Research Council of Karadeniz Technical University (99.111.004.6).

[16] Wunschel D, Fox K F, Black G E and Fox A 1994 Discrimination among B. cereus group, in comparison to B. subtilis, by structural carbohydrate profiles and ribosomal RNA spacer region PCR; Syst appl Microbiol 17 625-635

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Lambert and Peferoen, M. 1992. Insecticidal promise of Bacillus thuringiensis. Bioscience 42, 112-122. Louws F J, Schneider M and de Bruijn F J 1996 in Nucleic Acid Amplification Methods for the Analysis of Environmental samples (ed) Toranzos G (Technomic Publishing Co) pp 63-94

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Hossain M A, Ahmed S and Hoque S 1997 Abundance and distribution of Bacillus thuringiensis in the agricaltural soil of Bangladesh; J. Inver. Pathology 70 221-225

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Logan N A and Berkeley R C W 1984 Identification of Bacillus strains using the API System; J. General Microbioloy 130 1871- 1882

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Sambrook J, Fritsch E F and Maniatis T 1989 Molecular Cloning 2nd ed. Cold Spring Harbor Laboratory Press, New York.

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[17] Forsty G and Logan N A 2000 Isolation of Bacillus thuringiensis from Northern Victoria Land, Antartica; Letters in Applied Microbiology 30 263-266 [18] Lee, H.H., Lee, J. A., Lee, K.Y., Chung, J.., Barjac, H, Charles, J.F., Dumanoir, V.C. and Frachon, E. 1994. New serovars of Bacillus thuringiensis: B. thuringiensis ser. coreanensis (serotype H25), B. thuringiensis ser. leesis (Serotype H33), and B. thuringiensis ser. konkukian (serotype H34); J. Invertebrate Pathol. 63 217-219 [19] Jung Y C, Kim S U, Lecadet M M, Chung Y S and Bok S H 1998 Characterization of a new Bacillus thuringiensis subsp. higo strain isolated from rice bran in Korea; J. Invert. Pathol 71 95-96 [20] Yaman M and Demirbag Z 2000 Isolation, identification and determination of insecticidal activity of two insect-originated Bacillus spp.; Biologia 55 283-287

Normand P, Ponsonnet C, Nesme X, Neyra M and Simonet P 1996 ITS analysis of prokaryotes; in molecular microbial ecology Manual (eds) Akkermans A D L, Van Elsas J D and De Brujin F J (The Netherlands: Kluwer) pp 1-12

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Gürtler V and Stanisich V A 1996 New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region; Microbiology 142 3-16

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Ben-Dov E, Boussiba S, and Zaritsky A 1995 Mosquito Larvicidal Activity of Escherichia coli with Combinations of Genes from Bacillus thuringiensis subsp. Israelensis;. Journal of Bacteriology 177(10) 2581-2587

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Moar W J, Pusztzai-Carey M and Mack T P 1995 Toxity of purified proteins and the HD-1 strain from Bacillus thuringiensis against Lesser Cornstalk Borer (Lepidoptera: Pyralidae); J. Econ. Entomol., 88 606-609

[21] Yaman M, Demirbag Z and Belduz A O 2000 Isolation and insecticidal effects of some bacteria from Euproctis chrysorrhoea L. (Lepidoptera: Lymantriidae); Acta Microbiologica Polonica 49(3-4) 217-224

Received for publication: October 16, 2001 Accepted for publication: June 07, 2002

[10] Dulmage H T, 1981 Insecticidal activity of isolates of Bacillus thuringiensis and their potential for pest control. in Microbial Control of Cest and Plant Diseases (ed) Burges H D (London, Academic Press) pp 193-280

CORRESPONDING AUTHORS

[11] Abbot W S 1925 A method of computing the effectiveness of an insecticide; J. Econ. Entomol. 18 265-267

Zihni Demirbag Department of Biology Faculty of Arts and Sciences Karadeniz Technical University 61080 Trabzon - TURKEY

[12] Burges H D 1981In Microbial Control of Pests and Plant Diseases, 1970 to 1980 Ac ademic Press, London pp 36-42 [13] Dulmage H T and Aizawa K 1982 Distribution of Bacillus thuringiensis in nature in Microbial and viral pesticides (ed.)Kurstak E (Marcel Dekker Inc., New York) pp 209-237

Phone: ++90 462 377 3320 Fax: ++90 462 325 3195 e-mail: [email protected]

[14] DeLucca A J, Simonson J G and Larson A D 1981 Bacillus thuringiensis distribution in soils of the United States; Can. J. Microbiol. 27 865-870

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