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Bacillus thuringiensis (Helicoverpa armigera ) Isolation of local isolates of Bacillus thuringiensis from forest soils of and evaluation of their toxicity on larva of Helicoverpa armigera (Lepidoptera: Noctouidae).

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1395 :4/2/ 1394 19/9/ : Bacillus thuringiensis (Bt) . . Bt 24 . LBA . Bt 60 Bt . . ( 56 )6/ . (Helicoverpa armigera ) 5580 . . Bt Helicoverpa armigera Bacillus thuringiensis :

1 [email protected] :

21 ... Bacillus thuringiensis

(Helicoverpa armigera ) . .( Ammouneh et al ., 2011 ) . .( Marzban and Salehi Jouzani, 2005 ) Bacillus thuringiensis ( Bt ) Bt . Bacillaceae .( Keshavarzi, 2008 ) Cry Bacillus sotto ( 1901 ) .( Ishiwata, 1901 ) Sudden- collapsebacillus . Bt (Anagasta kuhniella zell.) .( Hannay and Fitz, 1955 ) Bt 17 Bt Bt . Bt .( Feitelson et al ., 1992 ) . .( Adang, 1991 ) . .( Chilcott and wigley, 1993 ) Bt B. cereus B. thuringiensis Bt .( Dulmage and Aizawa, 1989 ) . (Anear et al ., 1997) .( Marzban and Salehi Jouzani, 2006 ) Bt (Bioassay) (. Marzban, 2002 )

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Bt .

15 200 . . 1 . 4 °C . Bt . (Travers et al ., 1987) Bt 20 30 3 . Bt . (PH= 6/8) 25/0 M . 70 20 . Bt 53 . Bt (LBA 1) 9/0 ( ) . 4 °C

LBA Bt . LBA 53 . 3 100 . ( ) Bt

( ) 10 ×1 10 8 (CFU/ ml) .

1- Luria Bertani Agar

23 ... Bacillus thuringiensis

1 Table 1. Characterization of sample locations from forest soils of Golestan province

Height of sea level (m) Date of collection Name of forest sample location No. of sample Geo. Latitude Geo. Longitude 678 m 55° 28´ 50/1´´E 37° 13´ 3/8´´N 2016/5/30 1395 10/3/ Galikesh forest 1 697 m 55° 20´ 50´´E 37° 13´ 29/9´´N 2016/6/1 1395 /3/12 Kalaleh forest 2 498 m 55° 28´ 25/4´´E 37° 8´ 46/7´´N 2016/6/5 1395 16/3/ Gholitapeh minodasht forest 3 445 m 55° 28´ 32/2´´E 37° 10´ 14/1´´N 2016/6/5 1395 16/3/ Chehehchai minodasht forest 4 319 m 55° 7´ 20/7´´E 36° 59´ 9/7´´N 2016/6/7 1395 18/3/ Tashteh Ramiyan forest 5 309 m 55° 8´ 3/7´´E 36° 59´ 31/5´´N 2016/6/7 1395 18/3/ Dare mola Ramiyan forest 6 104 m 55° 5´12/3´´E 37° 2´ 37/2´´N 2016/6/7 1395 18/3/ Daland forest park 7 120 m 55° 2´ 30/7´´E 36° 58´ 38/7´´N 2016/6/7 1395 18/3/ Khan Bebin forest 8 114 m 55° 1´ 2/8´´E 36° 58´ 41´´N 2016/6/7 1395 18/3/ forest 9 411 m 54° 27´ 50/5´´E 36° 47´ 29/9´´N 2016/6/8 1395 19/3/ Alang Dareh Gorgan forest 10 486 m 54° 28´ 7/3´´E 36° 46´ 15/2´´N 2016/6/8 1395 19/3/ Naharkhoran Gorgan forest 11 471 m 54° 34´48/9´´E 36° 46´ 33/5´´N 2016/6/8 1395 19/3/ Tooskestan Gorgan forest 12 180 m 54° 40´ 3/7´´E 36° 50´ 45/5´´N 2016/6/1 1395 12/3/ Mohamadabad Nodehmalek 13 139 m 54° 40´ 35/4´´E 36° 52´ 54/5´´N 2016/6/1 1395 12/3/ Ghorogh forest 14 408 m 54° 38´ 21/8´´E 36° 54´ 18/5´´N 2016/6/8 1395 19/3/ Gorgan Ziyarat forest park 15 112 m 54° 7´ 38/4´´ E 36° 42´ 35/2´´N 2016/5/31 1395 11/3/ Kordkoy EmamReza forest park 16 150 m 53° 53´ 19/6´´E 36° 41´ 58/9´´N 2016/5/31 1395 11/3/ Bandargaz forest 17 171 m 53° 45´ 14/1´´E 36° 40´ 17/9´´N 2016/5/31 1395 11/3/ Nokandeh forest 18 45 m 53° 50´ 34/9´´E 36° 43´ 4/1´´N 2016/5/31 1395 11/3/ East Livan forest 19 32 m 53° 47´ 21/4´´E 36° 41´ 5/7´´N 2016/5/31 1395 11/3/ West Livan forest 20 695 m 55° 28´ 29/4´´E 37° 18´ 40/7´´N 2016/6/7 1395 18/3/ Maravehtappeh forest 21 398 m 54° 38´ 7/3´´E 36° 42´ 45/2´´N 2016/6/8 1395 19/3/ Gorgan Valeshabad forest 22 578 m 55° 21´ 35/1´´E 37° 27´ 41/9´´N 2016/6/5 1395 16/3/ Minodasht Dozein forest 23 410 m 54° 48´ 7/3´´E 36° 32´ 25/2´´N 2016/6/8 1395 19/3/ Gorgan Taghartapeh forest 24

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(Helicoverpa armigera ) . ) . Teakle and Jensen (1985) 37% ( . . . . 11 18× 5/5× ( 35 17× ) 10% . . . . 27 ± 2 34 . . 8 16 65 ± 5 . ( ) Bt ×6( 10 6 ×3 10 6– ×2 10 7 ×1 10 8) Bt . 3 3 2 . Bt . . . . 1357 . Abbot (1925) M [ %] = [ (t – c) \ (100- c) ] × 100 = M % = t = c . SAS 9.1 .

25 ... Bacillus thuringiensis

24 ( ) 60 . Bt . % 9/0 5 .( 1 ) 8 8 6 34 . 4

Bacillus thuringiensis (Cap) (S) (C) 1 (100 × ) Fig. 1. Crystal (C), Spore (S) and Cap of Bacillus thuringiensis observed under the microscope (Zoom × 100)

Bt 2 Fig. 2. Isolation stages of Bt from soil samples

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LBA Bt 3 Fig. 3. Bacterial colonies grown on LBA medium

4 Fig. 4. Artificial training of host insect and bioassay

Bt . . Bt . Bt . Bt Bacillus thuringiensis Bt . (Travers et al ., 1987) 32 (2014 ) . (Anear et al ., 1997) Bt 144 . (Grayeli et al ., 2014) Bt .

27 ... Bacillus thuringiensis

Bt . (. Martin and Travers, 1989) Bt Bt . . . Bt . (Izadyar et al ., 1998) Bt × Bt Bt ( Bt ) Bt .( 2 ) CFU/ 23 Bt Bt ×2 10 7ml 3.( ) ( a ) 80 Bacillus thuringiensis 2 Table 2. Variance analysis the impact of native isolates of Bt on Helicoverpa armigera compared to control in vitro condition. MS. Significance level (Pr>f) df S.O.V 0.13 2 Replication 3591.93 ** 0.0001 24 Bt 3080.89** 0.0001 3 Concentration 21.71** 0.0001 72 concentration Bt × × Bt 1.11 198 Error 2.33 CV% ( ) **: Significant at 1% level of probability :**

2200 Bt . . (Heliothis armigera ) Bt Bt .( Marzban and Salehi Jouzani, 2006 ) Dipel® Bt 12 . ×6 106 CFU LC50 . Bt .( Izadyar et al ., 2002 ) ×8 106 CFU

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Bacillus thuringiensis 3 Helicoverpa armigera Table 3. Average comparison of mortality percentage and impact of native isolates of Bacillus thuringiensis on Helicoverpa armigera compared to control at in vitro condition. 3×10 6 6×10 6 1×10 7 2×10 7 Bt treatment Bt 3 × 10 6 6 × 10 6 1 × 10 7 2 × 10 7 60 IG 62 GHI 72/1 C 78 a Sample 1 1 58/8 JK 62 GHI 72/1 C 80 a Sample 2 2 62 GHI 65 F 75 B 80 a Sample 3 3 24 V 31 R 37 OP 40 N Sample 4 4 60 IJ 62 GHI 71 CD 75 B Sample 5 5 24 V 31 R 37 OP 40 N Sample 6 6 24 V 31 R 37 OP 40 N Sample 7 7 60 IJ 64 FG 68/8 E 75 B Sample 8 8 24 V 31 R 37 OP 40 N Sample 9 9 24 V 31 R 37 Op 40 N Sample 10 10 50 M 57 K 65 F 72/1 C Sample 11 11 24 V 31 R 37 OP 40 N Sample 12 12 50 M 56/6 K 63/3 FGH 70 DE Sample 13 13 50 M 57 K 58/8 JK 68/8 E Sample 14 14 60 IJ 57 K 61/1 E 70 DE Sample 15 15 60 IJ 65 F 68 E 71 CD Sample 16 16 28 TU 31 R 33 QR 35 PQ Sample 17 17 50 M 54 L 57 K 63/3 FGH Sample 18 18 24 V 26 UV 28 TU 35PQ Sample 19 19 24 V 26 UV 28 TU 35 PQ Sample 20 20 24 V 26 UV 28 TU 33 QR Sample 21 21 24 V 26 UV 28 TU 33 QR Sample 22 22 24 V 26 UV 28 TU 33 QR Sample 23 23 24 V 26 UV 28 TU 33/3 Q Sample 24 24 20 W 33/3 Q 40 N 48/8 M Control ( ) Bt . Ephestia kuheniella zeller. Bt .( Obeidat et al ., 2004 ) 80 55 4050 Tribolium castaneum (. Frouzan et al ., 2012) 80 50 .

29 ... Bacillus thuringiensis

References Abbot, W. S. 1925. A method of computing the effectiveness of an insecticide. Journal of Economic Entomology 18: 265-267. Adang, M. J. 1991. Bacillus thuringiensis insecticidal crystal proteins: gene structure, action and utilization. Pp. 3-24. In: Maramosch, K. (ed.) Biotechnology for biological control of pests and vector. CRC press. Ammouneh, H., Harba, M., Idris, E. and Makee, H. 2011. Isolation and characterization of native Bacillus thuringiensis isolates from Syrian soils and testing of their insecticidal activities against some insect pests. Turkish Journal of Agriculture 35: 421-431. Anear, H. Ahmad, S. and Sirajal, H. 1997. Abundance and Distribution of Bacillus thuringiensis in the agricultural soils of Bangladesh 70: 221-225. Chilcott, C. N. and and Wigley, P. J. 1993. Isolation and Toxicity of Bacillus thuringiensis from soil and insect habitats in New Zealand. Journal of Invertebrate pathology 61: 244-247. Dulmage, H. and Aizawa, K. 1982. Distribution of Bacillus thuringiensis in nature. Pp. 209-237. In: Kurstak, E. (ed.) Microbial and Viral pesticides, Marcel Dekker, Inc., New York. Feitelson, J. S., Payne, J. and Kim, L. 1992. Bacillus thuringiensis : Insects and beyond. Biotechnology 10: 271-275. Frouzan, M., Nuri, H., Rezaei, M. and Hasanzadeh, M. 2012. Extraction of local isolates of Bacillus thuringiensis from vineyard soils in samples and evaluation of its toxicity on larva and adults of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Iranian Journal of Plant Pest Research 1(1): 60-66. Grayeli, F., Mohammadi Sharif, M., Hadizadeh, A. and Babayizad, V. 2014. Identification of genetical structure of native strains of Bacillus thuringiensis isolated from forest soils of Golestan province. Journal of Biotechnology in Agriculture 13(1): 313-321. (In Persian) Hannay, C. L. and Fitz, J. P. 1955. The protein crystals of Bacillus thuringiensis Ber. Canadian Journal of Microbiology 1: 694-710. Ishiwata, S. 1901. On a kind of severe flacherie (Sotto disease). I. Dianihen sanshi kaiho 114, 1(cited in the safety of microbial insecticides, pp. 36-36). Izadyar, S., Amirsadeghi, S. and Rohani, H. 1998. Isolation of native strains Bacillus thuringiensis from Agricultural soil of Northern . Abstract of Sixteenth Iranian Plant Protection Congress P: 198. (In Persian) Izadyar, S., Askari, H. and Talebi Jahromi, KH. 2002. Bioassay of several Iranian isolates of Bacillus thuringiensis on Helicoverpa armigera . Journal of plant pests and diseases 73(1): 30-44. Keshavarzi, M. 2008. Isolation, Identification and differentiation of local Bacillus thuringiensis strains. Journal of Agricultural science & Technology 10: 493-499. Martin, P. A. W. and Travers, R. S. 1989. Worldwide abundance and distribution of Bacillus thuringiensis isolates. Applied Environmental Microbiology 55: 2437-2442. Marzban, R. 2002. Bioassay comparison of several isolates of Bacillus thuringiensis subsp. Kurstaki on Plodia interpunctella Hb. Journal of plant pests and diseases 70(1): 83-90. (In Persian) Marzban, R. and Salehi Jouzani, GH. R. 2006. Distribution of Bacillus thuringiensis in the agricultural soils of Iran. Biotechnology, Agriculture and the food Industry 4: 95-100. Marzban, R. and Salehi Jouzani, GH. R. 2005. Isolation of native strains of Bacillus thuringiensis from agricultural soils of Iran. Journal of New sustainable Agriculture 2: 47-54. (In Persian) Obeidat, M., Hassawi, D. and Ghabeish, I. 2004. Characterization of Bacillus thuringiensis strains from Jordan and their toxicity to the Lepidoptera, Ephestia kuhniella zeller. African Journal of Biotechnology 3: 622-626. Teakle, R. E., Jensen, J. M. 1985. Heliothis armigera . Pp. 313-322. In: Singh, P. and Moor, R. F. (eds.) Handbook of insect rearing. Travers, R. S., Martin, P. A. W. and Reichelderfer, C. F. 1987. Selective process for efficient isolation of soil Bacillus spp . Applied and Environmental Microbiology 53: 1263-1266.

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