Mehlika ALPER, Hatice GÜNEŞ, Arzu TATLIPINAR, Bekir ÇÖL

Distribution, occurrence of cry genes, and lepidopteran toxicity of native Bacillus thuringiensis isolated from fig tree environments in Aydın Province

Bacillus thuringiensis (Bt) has a significant impact on biological pest control because of the insecticidal activity through its parasporal inclusion bodies (crystal proteins). Fig is an economically important plant in Turkey; agricultural pests result in a considerable economic loss in fig quality and cultivation. The aim of this work was to isolate, characterize, and determine the lepidopteran toxicity of Bt obtained from fig groves in Aydın Province. A total of 606 colonies (out of 1167) obtained from 380 samples were identified as Bt based on parasporal crystal formation. The highest Bt index of 0.60 was observed in the Kuyucak region. A total of 288 Bt isolates were characterized in terms of cry gene content by PCR analysis. It was found that the cry1 plus cry2 genotype was the most abundant (40%) in our collection. Bioactivity tests indicated that 6 isolates exhibited high mortalities against Cadra cautella and 3 isolates were found to exhibit high toxicity against Carpophilus hemipterus. Moreover, 13 Bt isolates exhibiting toxic activity against fig pests were further characterized based on specific cry gene content, protein profiles, and PCR-RFLP analysis. Among cry1 genes, the cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, and cry1Ea genes were the most frequent (100%). Protein profiles of isolates toxic to C. cautella were different from those of isolates toxic to C. hemipterus. PCR-RFLP analysis indicated that toxic isolates differed from the reference strain with respect to cry1 type gene. Finally, it was concluded that Bt strains isolated from fig groves showed high level of toxicity against fig pests. These strains can serve as potential biopesticides for the control of C. cautella in the region as well as alternative biopesticides in the case of pesticide resistance in insects.

Keywords:

Bacillus thuringiensis, bioactivity, cry gene, fig groves,

PDF

___

Abbott WS (1925). A method of computing the effectiveness of an insecticide. J Econ Entomol 18: 265–267.
Akşit T, Özsemerci F, Çakmak İ (2003). Aydın ilinde incir ağaçlarında saptanan zararlı türler. Türk Entomol Derg 27: 181–189 (in Turkish).
Apaydin O, Yenidünya AF, Harsa S, Günes H (2005). Isolation and characterization of Bacillus thuringiensis strains from different grain habitats in Turkey. World J Microbiol Biotechnol 21: 285–292.
Ausebel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1994). Current Protocols in Molecular Biology 3. New York, NY, USA: John Wiley & Sons.
Bel Y, Granero F, Alberola TM, Martinez-Sebastian MJ, Ferre J (1997). Distribution, frequency, and diversity of Bacillus thuringiensis in olive tree environments in Spain. Syst Appl Microbiol 20: 652–658.
Ben-Dov E, Wang Q, Zaritsky A, Manasherob R, Barak Z, Schneider B, Khamraev A, Baizhanov M, Glupov V, Margalith Y (1999). Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains. Appl Environ Microbiol 65: 3714–3716.
Ben-Dov E, Zaritsky A, Dahan E, Barak Z, Sinal R, Manasherob R, Khamraev A, Troitskaya E, Dubitsky A, Berezina N et al. (1997). Extend screening by PCR for seven cry-group genes from field-collected strains of Bacillus thuringiensis. Appl Environ Microbiol 63: 4883–4890.
Bravo A, Sarabia S, Lopez L, Ontiveros H, Abarca C, Ortiz A, Ortiz M, Lina L, Villalobos FJ, Peña G et al. (1998). Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Appl Environ Microbiol 64: 4965–4972.
Bravo A, Likitvivatanavong S, Gill SS, Soberόn M (2011). Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem Mol Biol 41: 423–431.
Brizzard BL, Whiteley HR (1988). Nucleotide sequence of an additional crystal protein gene cloned from Bacillus thuringiensis subsp. thuringiensis. Nucleic Acids Res 16: 2723– 2724.
Ceron J, Covarrubias L, Quintero R, Ortiz A, Ortiz M, Aranda E, Lina L, Bravo A (1994). PCR analysis of the cryI insecticidal crystal family genes from Bacillus thuringiensis strain collection. Appl Environ Microbiol 60: 353–356.
Ceron J, Ortiz A, Quintero R, Güereca L, Bravo A (1995). Specific PCR primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain collection. Appl Environ Microbiol 61: 3826–3831.
Chak KF, Chao DC, Tseng MY, Kao SS, Tuan SJ, Feng TY (1994). Determination and distribution of cry type genes of Bacillus thuringiensis isolates from Taiwan. Appl Environ Microbiol 60: 2415–2420.
Chambers JA, Jelen A, Gilbert MP, Jany CS, Johnson TB, Grawn- Burke C (1991). Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai. J Bacteriol 173: 3966–3976.
Chilcutt CF, Tabashnik BE (1997). Independent and combined effects of Bacillus thuringiensis and parasitoid Cotesia plutella (Hymenoptera: Broconidae) on susceptible and resistant diamondback month (Lepidoptera: Plutellidae). J Econ Entomol 90: 397–403.
Cinar C, Apaydın O, Yenidunya AF, Harsa S, Gunes H (2008). Isolation and characterization of Bacillus thuringiensis strains from olive-related habitats in Turkey. J Appl Microbiol 104: 515–525.
Feitelson JS, Payne J, Kim L (1992). Bacillus thuringiensis: insects and beyond. Bio/Technology 10: 271–275.
Gao M, Li R, Dai S, Wu Y, Yi D (2008). Diversity of Bacillus thuringiensis strains from soil in China and their pesticidal activities. Biol Control 44: 380–388.
González JM, Dulmage HT, Carlton BC (1981). Correlation between specific plasmids and δ-endotoxin production in Bacillus thuringiensis. Plasmid 5: 351–365.
Höfte H, Whiteley HR (1989). Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53: 242–255.
Hoy MA (1998). Myths, models and mitigation of resistance to pesticides. Phil Trans R Soc Lond B 353: 1787–1795.
Hubert J, Kudlíková-Křížková I, Stejskal V (2008). Effect of MON 810 Bt transgenic maize diet on stored-product moths (Lepidoptera: Pyralidae). Crop Prot 27: 489–496.
Jara S, Madvell P, Orduz S (2006). Diversity of Bt strains in the maize and bean phylloplane and their respective soils in Colombia. J Appl Microbiol 101: 117–124.
Kuo WS, Chak KF (1996). Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA. Appl Environ Microbiol 62: 1369–1377.
Laemmli UK (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685.
Magana C, Hernandez-Crespo P, Ortego F, Castanera P (2007). Resistance to malathion in field populations of Ceratitis capitata. J Econ Entomol 100: 1836–1843.
Martin PAW, Travers RS (1989). Worldwide abundance and distribution of Bacillus thuringiensis isolates. Appl Environ Microbiol 55: 2437–2442.
Martínez C, Ibarra JE, Caballero P (2005). Association analysis between serotype, cry gene content, and toxicity to Helicoverpa armigera larvae among Bacillus thuringiensis isolates native to Spain. J Invertebr Pathol 90: 91–97.
Nazarian A, Jahangiri R, Jouzai GH, Seifinejad A, Soheilivand S, Bagheri O, Keshavarzi M, Alamisaeid K (2009). Coleopteran- specific and putative novel cry genes in Iranian native Bacillus thuringiensis collection. J Invertebr Pathol 102: 101–109.
Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62: 775–806.
Seifinejad A, Salehi Jouzani GR, Hosseinzadeh A, Abdmishani C (2008). Characterization of Lepidoptera-active cry and vip genes in Iranian Bacillus thuringiensis strain collection. Biol Control 44: 216–226.
Smith R, Couche G (1991). The phylloplane as a source of Bacillus thuringiensis variants. Appl Environ Microbiol 57: 311–315.
Thammasittirong A, Attathom T (2008). PCR-based method for the detection of cry genes in local isolates of Bacillus thuringiensis from Thailand. J Invertebr Pathol 98: 121–126.
Travers RS, Martin PAW, Reichelderfer CF (1987). Selective process for efficient isolation of soil Bacillus spp. Appl Environ Microbiol 53: 1263–1266.
Uribe D, Martinez W, Ceron, J (2003). Distribution and diversity of cry genes in native strains of Bacillus thuringiensis obtained from different ecosystems from Colombia. J Invertebr Pathol 82: 119–127.
Vidal-Quist JC, Castañera P, González-Cabrera J (2009). Diversity of Bacillus thuringiensis strains isolated from citrus orchards in Spain and evaluation of their insecticidal activity against Ceratitis capitata. J Microbiol Biotechnol 19: 749–759.
Yu H, Zhang J, Huang D, Gao J, Song F (2006). Characterization of Bacillus thuringiensis strain Bt185 toxic to the Asian cockchafer: Holothrichia parallela. Curr Microbiol 53: 13–17.