World Journal of Microbiology and Biotechnology

, Volume 24, Issue 12, pp 3005–3015 | Cite as

Isolation and identification of bacteria from Thaumetopoea pityocampa Den. and Schiff. (Lep., Thaumetopoeidae) and determination of their biocontrol potential

  • ikbal Agah ince
  • Hatice Katı
  • Hüseyin Yilmaz
  • ismail Demir
  • Zihni DemirbağEmail author
Original Paper


The pine processionary moth Thaumetopoea pityocampa (Den. and Schiff.) is one of the most harmful insect pest for pine species in Mediterranean countries including Turkey. The objective of the present study is to find a more effective and safe biological control agent against T. pityocampa. Thus, we investigated the bacterial flora of the pest insect, collected from the Middle Black Sea Region of Turkey from 2003 to 2004. Based on morphological, physiological, biochemical and molecular methods, 14 different bacterial isolates were determined. The identified bacterial flora of T. pityocampa consisted of bacteria belonging to the Enterobacteriaceae (Tp1), Arthrobacter sp. (Tp2), Staphylococcus spp. (Tp3 and 10), Bacillus subtilis (Tp4), Serratia liquefaciens (Tp5), Bacillus thuringiensis subsp. morrisoni (Tp6 and 14), an acrystalliferous form Bacillus thuringiensis (Tp7), Staphylococcus cohnii (Tp8), Bacillus licheniformis (Tp9), Bacillus pumilus (Tp11), Brevibacterium sp. (Tp12) and Bacillus simplex (Tp13). After analysing the conclusions of conventional and molecular tests Tp1 (Enterobacteriaceae), Tp2 (Arthrobacter sp.) and Tp12 (Brevibacterium sp.) were assigned as novel bacterial species. Isolate Tp5 had a promising insecticidal effect on third instar larvae of T. pityocampa (up to 70% mortality within 10 days).


Thaumetopoea pityocampa Pine processionary moth Bacterial flora 



We are grateful to Michio Ohba and Kumiko Kagoshima (Kyushu University, Japan) for serotyping analysis. This study was supported by T. R. Prime Ministry State Planning Organization (Project Number: 2004.200.200.01)


  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410Google Scholar
  2. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. doi: 10.1093/nar/25.17.3389 CrossRefGoogle Scholar
  3. Aptosoglou SG, Sivropoulou A, Koliais SI (1997) Plasmid patterns of Bacillus thuringiensis strains and isolates. Microbios 91:203–214Google Scholar
  4. Bahar AA, Demirbag Z (2007) Isolation of pathogenic bacteria from Obera linearis (Coleoptera: Cerambycidae). Biologia 62:13–18. doi: 10.2478/s11756-007-0009-4 CrossRefGoogle Scholar
  5. Barry AL, Gavan TL, Badal RE, Telenson MJ (1982) Sensitivity, specificity, and reproducibility of the AutoMicrobic system (with the Enterobacteriaceae-plus biochemical card) for identifying clinical isolates of gram-negative bacilli. J Clin Microbiol 15:582–588Google Scholar
  6. Battisti A, Longo S, Tiberi R, Triggiani O (1998) Results and perspectives in the use of Bacillus thuringiensis Berl. var. kurstaki and other pathogens against Thaumetopoea pityocampa (Den. et Schiff.) in Italy (Lep., Thaumetopoeidae). J Pest Sci 71:72–76Google Scholar
  7. Brosius J, Palmer ML, Kennedy PJ, Noller HF (1978) Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci USA 75:4801–4805. doi: 10.1073/pnas.75.10.4801 CrossRefGoogle Scholar
  8. Bucher GE (1981) Identification of bacteria found in insects. In: Burges HD (ed) Microbial control of pests and plant diseases 1970–1980. Academic, New York, pp 7–33Google Scholar
  9. Cavanagh P, Marsden PD (1969) Bacteria isolated from the gut of some reduviid bugs. Trans R Soc Trop Med Hyg 63:415–416. doi: 10.1016/0035-9203(69)90027-3 CrossRefGoogle Scholar
  10. Claus M (1992) A standardized Gram staining procedure. World J Microbiol Biotechnol 8:451–452. doi: 10.1007/BF01198764 CrossRefGoogle Scholar
  11. Cote JC, Frechette S, Vincent C (1992) Isolation of Bacillus thuringiensis from the tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae). In: 25th Annual meeting of Society for Invertebrate Pathology, Heidelberg, 16–21 AugustGoogle Scholar
  12. Demir I, Sezen K, Demirbag Z (2002) The first study on bacterial flora and biological control agent of Anoplus roboris (Coleoptera: Curculionidae). J Microbiol 40:104–108Google Scholar
  13. Demolin G, Martin JC, Lavanceau P (1993) Controlling secondary insects pest in pine forests. Phytoma 452:13–16Google Scholar
  14. Denneulin JC, Lamy M (1977) Effects of a chitin inhibitor, dimilin 1-(4-chlorophenyl)-3-(2, 6-difluorobenzoyl)urea, on the oenocytes and moulting in the processionary caterpillar (Thaumetopoea pityocampa Schiff.) (Lepidoptera). Ann Endocrinol (Paris) 38:405–406Google Scholar
  15. Er MK, Tunaz H, Gökçe A (2007) Pathogenicity of entomopathogenic fungi to Thaumetopoea pityocampa (Schiff.) (Lepidoptera: Thaumatopoeidae) larvae in laboratory conditions. J Pest Sci 80:235–239. doi: 10.1007/s10340-007-0177-6 CrossRefGoogle Scholar
  16. Erturk O, Yaman M, Aslan I (2008) Effects of four Bacillus ssp. of soil origin on the Colorado potato beetle Leptinotarsa decemlineata (Say). Entomol Res 38:135–138. doi: 10.1111/j.1748-5967.2008.00150.x CrossRefGoogle Scholar
  17. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evol Int J Org Evol 39:783–791. doi: 10.2307/2408678 Google Scholar
  18. Georgevits RP (1979) Comparison of results in the control of Thaumetopoea pityocampa Schiff. with Dimilin, Decis, Bactospeine and Thuricide HP. Anakoinoseis Idrumaton Dasikon Ereun 7:7–34Google Scholar
  19. Georgevits RP (1980) New perspectives for the control of the pine processionary (Thaumetopoea pityocampa Schiff.) with Dimilin. Anakoinoseis Idrumaton Dasikon Ereun 8:7–25Google Scholar
  20. Grimont PAD, Jackson TA, Ageron E, Noonan MJ (1988) Serratia entomophila sp. nov. associated with amber disease in the New Zealand grass grub Costelytra zealandica. Int J Syst Bacteriol 38:1–6Google Scholar
  21. Gumpert J, Schwartz W (1962) Untersuchungenuber die symbiose von tieren mit pilzen und bakterien, X. Die symbiose der triatominen 1. Aufzucht symbiontenhaltiger und symbiontenfreier triatominen und eigenschaften der bei triatominen vorkommenden mikroorganismen. Z Allg Mikrobiol 2:209–302. doi: 10.1002/jobm.3630020306 CrossRefGoogle Scholar
  22. Halperin J (1980) Control of the pine processionary caterpillar (Thaumetopoea wilkinsoni Tams) with diflubenzuron. Phytoparasitica 8:83–91Google Scholar
  23. Ince IA, Demir I, Demirbag Z, Nalcacioglu R (2007) A cytoplasmic polyhedrosis virus isolated from the pine processionary caterpillar, Thaumetopoea pityocampa. J Microbiol Biotechnol 17:632–637Google Scholar
  24. Ishikawa Y, Hayashida T, Ikawa A (1964) On the isolation of Bacillus thuringiensis from silkworm rearing houses in Aichi prefecture. J Sericult Sci 33:480–483Google Scholar
  25. Jackson TA, Pearson JF, O’Callaghan M, Mahanty HK, Willocks M (1992) Pathogen to product development of Serratia entomophila (Enterobacteriaceae) as a commercial biological control agent for the New Zealand grass grub (Costelytra zealandica). In: Jackson TA, Glare R (eds) Use of pathogens in scarab pest management. Intercept, Andover, pp 191–198Google Scholar
  26. Jeyaprakash A, Hoy MA, Allsopp MH (2003) Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences. J Invertebr Pathol 84:96–103. doi: 10.1016/j.jip.2003.08.007 CrossRefGoogle Scholar
  27. Kanat M, Alma MH (2004) Insecticidal effects of essential oils from various plants against larvae of pine processionary moth (Thaumetopoea pityocampa Schiff) (Lepidoptera: Thaumetopoeidae). Pest Manag Sci 60:173–177. doi: 10.1002/ps.802 CrossRefGoogle Scholar
  28. Kashian DR, Dodson SI (2002) Effects of common-use pesticides on developmental and reproductive processes in Daphnia. Toxicol Ind Health 18:225–235. doi: 10.1191/0748233702th146oa CrossRefGoogle Scholar
  29. Katı H, Sezen K, Demirbağ Z, Beldüz AO (2005) Characterization of a Bacillus thuringiensis subsp. Kurstaki strain isolated from Malacosoma neustria L. (Lepidoptera: Lasicompidae). Biologia 60:301–305Google Scholar
  30. Kelly MT, Latimer JM (1980) Comparison of the AutoMicrobic system with API, Enterotube, Micro-ID, Micro-Media systems, and conventional methods for identification of Enterobacteriaceae. J Clin Microbiol 12:659–662Google Scholar
  31. King EG, Bell JV, Martin DF (1975) Control of bacterium Serratia marcescens in an insect-parasite rearing program. J Invertebr Pathol 26:35–40Google Scholar
  32. Klein MG, Kaya HK (1995) Bacillus and Serratia species for scarab control. Mem Inst Oswaldo Cruz Rio J 90:87–95Google Scholar
  33. Konecka E, Kaznowski A, Ziemnicka J, Ziemnicki K (2007) Molecular and phenotypic characterization of Bacillus thuringiensis isolated during epizootics in Cydia pomonella L. J Invertebr Pathol 94:56–63. doi: 10.1016/j.jip.2006.08.008 CrossRefGoogle Scholar
  34. Krieg NR, Holt JG (1986) Gram-negative aerobic rods and cocci. In: Palleroni NJ (ed) Bergey’s manual of systematic bacteriology, vol 1. Williams and Wilkins, Baltimore, pp 140–218Google Scholar
  35. Krieg A, Huger AM, Langenbruch GA (1983) Bacillus thuringiensis var. tenebrionis: a new pathotype effective against larvae of Coleoptera. Z Angew Entomol 96:500–508Google Scholar
  36. Kurstak E (1962) Donnees sur l’epizootie bacterienne naturelle provoguee par un Bacillus du type B. thuringiensis sur Ephestia kuhniella. Entomophaga Mem Hors Ser 2:245–247Google Scholar
  37. Kurt A, Özkan M, Sezen K, Demirbağ Z, Özcengiz G (2005) Cry3Aa11: a new rank of Cry3Aa δ-endotoxin from a local isolate of Bacillus thuringiensis. Biotechnol Lett 27:1117–1121. doi: 10.1007/s10529-005-8460-1 CrossRefGoogle Scholar
  38. Kuzina LV, Peloquin JJ, Vacek DC, Miller TA (2001) Isolation and identification of bacteria associated with adult laboratory Mexican fruit flies, Anastrepha ludens (Diptera: Tephritidae). Curr Microbiol 42:290–294Google Scholar
  39. Lamy M, Pastureaud MH, Ducombs G (1985) Toxicologie-La thaumétopoéine, une protéine urticant de la chenille processionnaire du pin (Thaumetopoea pityocampa Schiff.) (Lépidoptères., Thaumetopoeidea). C R Acad Sci III 301(5):173–176Google Scholar
  40. Lau WW, Jumars PA, Armbrust EV (2002) Genetic diversity of attached bacteria in the hindgut of the deposit-feeding shrimp Neotrypaea (formerly Callianassa) californiensis (Decapoda: Thalassinidae). Microb Ecol 43:455–466. doi: 10.1007/s00248-001-1043-3 CrossRefGoogle Scholar
  41. Li H, Medina F, Vinson SB, Coates CJ (2005) Isolation, characterization, and molecular identification of bacteria from the red imported fire ant (Solenopsis invicta) midgut. J Invertebr Pathol 89:203–209. doi: 10.1016/j.jip.2005.05.008 CrossRefGoogle Scholar
  42. Lipa JJ, Wiland E (1972) Bacteria isolated from cutworms and their infectivity to Agrotis sp. Acta Microbiol Pol 4:127–140Google Scholar
  43. Martinez AJ, Robacker DC, Garcia JA, Esau KL (1994) Laboratory and field olfactory attraction of the Mexican fruit fly (Diptera: Tephritidae) to metabolites of bacterial species. Fla Entomol 77:117–126. doi: 10.2307/3495878 CrossRefGoogle Scholar
  44. Nicholas KB, Nicholas HB, Deerfield DW (1997) GeneDoc: analysis and visualization of genetic variation. EMBnet News 4:1–4Google Scholar
  45. Nishiwaki H, Ito K, Shimomura M, Nakashima K, Matsuda K (2007) Insectisidal bacteria isolated from predatory larvae of the antlion species Myrmeleon bore (Neuroptera: Myrmeleontidae). J Invertebr Pathol 96:80–88. doi: 10.1016/j.jip.2007.02.007 CrossRefGoogle Scholar
  46. Oliveira P, Arnaldo PS, Araújo M, Ginja M, Sousa AP, Almeida O et al (2003) Report of poison in five dogs after contact with Thaumetopoea pityocampa. Rev Port Cienc Vet 98:151–156Google Scholar
  47. Osborn F, Berlioz L, Vitelli-Flores J, Monsalve W, Dorta B, Lemoine VR (2002) Pathogenic effects of bacteria isolated from larvae of Hylesia metabus Crammer (Lepidoptera: Saturniidae). J Invertebr Pathol 80:7–12. doi: 10.1016/S0022-2011(02)00037-X CrossRefGoogle Scholar
  48. Pearson WR (1990) Rapid and sensitive sequence comparison with Fastp and Fasta. Methods Enzymol 183:63–98. doi: 10.1016/0076-6879(90)83007-V CrossRefGoogle Scholar
  49. Rausell C, Martinez-Ramirez AC, Garcia-Robles I, Real MD (1999) The toxicity and physiological effects of Bacillus thuringiensis toxins and formulations on Thaumetopoea pityocampa, the pine processionary caterpillar. Pestic Biochem Physiol 65:44–54. doi: 10.1006/pest.1999.2426 CrossRefGoogle Scholar
  50. Reeves WK, Nayduch D (2002) Pathogenic Bacillus from a larva of the Simulium tuberosum species complex (Diptera: Simuliidae). J Invertebr Pathol 79:126–128. doi: 10.1016/S0022-2011(02)00016-2 CrossRefGoogle Scholar
  51. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425Google Scholar
  52. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  53. Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J et al (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806Google Scholar
  54. Sezen K, Demirbağ Z (1999) Isolation and insecticidal activity of some bacteria from the hazelnut beetle (Balaninus nucum L.). Appl Entomol Zool (Jpn) 34:85–89Google Scholar
  55. Sezen K, Demir I, Demirbağ Z (2001) Identification and insecticidal effects of bacteria isolated from pests of hazelnut. In: Proceedings of the 1st Eurasian Congress on Molecular Biotechnology, Trabzon, Turkey, pp 146–148Google Scholar
  56. Sezen K, Demir I, Kati H, Demirbag Z (2005) Investigations on bacteria as a potential biological control agent of Summer Chafer, Amphimallon solstitiale L. (Coleoptera: Scarabaeidae). J Microbiol 43:463–468Google Scholar
  57. Sezen K, Demir I, Demirbag Z (2007) Identification and pathogenicity of entomopathogenic bacteria from common cockchafer, Melolontha melolontha (Coleoptera: Scarabaeidae). N Z J Crop Hortic Sci 35:79–85Google Scholar
  58. Sezen K, Muratoglu H, Nalcacioglu R, Mert D, Demirbag Z, Kati H (2008) Highly pathogenic Bacillus thuringiensis subsp. tenebrionis from the European shot-hole borer, Xyleborus dispar (Coleoptera: Scolytidae). N Z J Crop Hortic Sci 36:77–84Google Scholar
  59. Sneath PHA, Mair NS, Sharpe ME, Holt JG (1986) Regular, nonsporing gram positive rods. In: Kandler O, Weiss N (eds) Bergey’s manual of systematic bacteriology, vol 2. Williams and Wilkins, Baltimore, pp 1208–1260Google Scholar
  60. Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA gene sequences analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44:846–849CrossRefGoogle Scholar
  61. Steinhaus EA, Marsh GA (1962) Report of diagnosis of diseased insects 1951–1961. Hilgardia 33:349Google Scholar
  62. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599. doi: 10.1093/molbev/msm092 CrossRefGoogle Scholar
  63. Triggiani O, Sidor C (1982) Prove di controllo microbiologico della Processionaria del pino (Thaumetopoea pityocampa SchiV.) nelle pinete pugliesi. Entomologica 17:91–102Google Scholar
  64. Vega JM, Moneo I, Armentia A, Fernandez A, Vega J, de la Fuente R et al (1999) Allergy to the pine processionary caterpillar (Thaumatopoea pityocampa). Clin Exp Allergy 29:1418–1423. doi: 10.1046/j.1365-2222.1999.00626.x CrossRefGoogle Scholar
  65. Vega ML, Vega J, Vega JM, Moneo I, Sanchez E, Miranda A (2003) Cutaneous reactions to pine processionary caterpillar (Thaumetopoea pityocampa) in pediatric population. Pediatr Allergy Immunol 14:482–486. doi: 10.1046/j.0905-6157.2003.00066.x CrossRefGoogle Scholar
  66. Vossen A, Abdulmawjood A, Lammler C, Wei R, Siebert U (2004) Identification and molecular characterization of β-hemolytic streptococci isolated from harbor seals (Phoca vitulina) and grey seals (Halichoerus grypus) of the German North and Baltic seas. J Clin Microbiol 42:469–473. doi: 10.1128/JCM.42.1.469-473.2004 CrossRefGoogle Scholar
  67. Yaman M, Demirbağ Z (2000) Studies of the bacterial flora as a microbial control agent of the large white butterfly Pieris brassicae L. (Lepidoptera: Pieridae). Afr J Entomol 8:144–148Google Scholar
  68. Yaman M, Demirbağ Z, Beldüz AO (1999) Investigations on the bacterial flora as a potential biocontrol agent of chestnut weevil, Curculio elaphas L. (Coleoptera: Curculionidae) in Turkey. Biologia 54:677–681Google Scholar
  69. Yaman M, Demirbag Z, Belduz AO (2000) Isolation and insecticidal effects of some bacteria from Euproctis chrysorrhoea L. (Lepidoptera: Lymanthriidae). Acta Microbiol Pol 49:217–224Google Scholar
  70. Yaman M, Nalcacioglu R, Demirbag Z (2002) Studies on bacterial flora in the population of the fall webworm, Hyphantria cunea Drury, (Lep., Arctiidae). J Appl Entomol 126:470–474. doi: 10.1046/j.1439-0418.2002.00681.x CrossRefGoogle Scholar
  71. Yilmax H, Sezen K, Kati H, Demirbag Z (2006) The first study on the bacterial flora of the European spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae). Biologia 61:679–686. doi: 10.2478/s11756-006-0140-7 CrossRefGoogle Scholar
  72. Yu H, Wang Z, Liu L, Xia Y, Cao Y, Yin Y (2008) Analysis of intestinal microflora in Hepialus gonggaensis (Lepidoptera: Hepialidae). Curr Microbiol 56:391–396. doi: 10.1007/s00284-007-9078-4 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • ikbal Agah ince
    • 1
    • 2
  • Hatice Katı
    • 1
  • Hüseyin Yilmaz
    • 1
    • 2
  • ismail Demir
    • 2
  • Zihni Demirbağ
    • 2
    Email author
  1. 1.Department of Biology, Faculty of Arts and SciencesGiresun UniversityGiresunTurkey
  2. 2.Department of Biology, Faculty of Arts and SciencesKaradeniz Technical UniversityTrabzonTurkey

Personalised recommendations