Identification of Antifungal Substance (Iturin A2) Produced by Bacillus subtilis B47 and Its Effect on Southern Corn Leaf Blight

doi:10.1016/S1671-2927(00)8509

Journal of Integrative Agriculture ›› 2012, Vol. 12 ›› Issue (1): 90-99.DOI: 10.1016/S1671-2927(00)8509

Identification of Antifungal Substance (Iturin A2) Produced by Bacillus subtilis B47 and Its Effect on Southern Corn Leaf Blight

YE Yun-feng, LI Qi-qin, FU Gang, YUAN Gao-qing, MIAO Jian-hua , LIN Wei

1.College of Agriculture, Guangxi University, Nanning 530004, P.R.China
2.Guangxi Botanical Garden of Medicinal Plant, Nanning 530023, P.R.China
3.Microbiology Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, P.R.China

收稿日期:2011-03-01 出版日期:2012-01-01 发布日期:2012-01-13
作者简介:YE Yun-feng, E-mail: yeyunfeng111@126.com
基金资助:
This research was financially supported by the grants from the Department of Science and Technology, Guangxi Zhuang Autonomous Region, China (0991056).

Identification of Antifungal Substance (Iturin A2) Produced by Bacillus subtilis B47 and Its Effect on Southern Corn Leaf Blight

YE Yun-feng, LI Qi-qin, FU Gang, YUAN Gao-qing, MIAO Jian-hua , LIN Wei

1.College of Agriculture, Guangxi University, Nanning 530004, P.R.China
2.Guangxi Botanical Garden of Medicinal Plant, Nanning 530023, P.R.China
3.Microbiology Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, P.R.China

Received:2011-03-01 Online:2012-01-01 Published:2012-01-13
Contact: Correspondence LI Qi-qin, Tel: +86-771-3235674, E-mail: qqli5806@gxu.edu.cn
About author:YE Yun-feng, E-mail: yeyunfeng111@126.com
Supported by:
This research was financially supported by the grants from the Department of Science and Technology, Guangxi Zhuang Autonomous Region, China (0991056).

摘要/Abstract

摘要： Bacillus subtilis B47 is an endophytic bacterium of tomato, and produces substance that strongly inhibits the growth of Bipolaris maydis, the pathogen of southern corn leaf blight (SCLB), as well as several other phytopathogenic fungi. The antifungal substance was purified from the broth culture of the bacterium using acid precipitation, methanol extraction, and three-step chromatography. Based on FT-IR spectrometry, amino acid composition, and MALDI-TOF-MS/MS CID analyses, the antifungal substance was identified as iturin A2, a cyclic lipopeptide antibiotic. To evaluate the efficacy of iturin A2 for control of SCLB, partially purified iturin A2 (75%, w/v) was applied under different conditions. At the concentration of 300 mg kg-1, iturin A2 showed efficacy ranging from 100 to 53.1% under in vitro, in plot and in field conditions. This efficacy was higher than or similar to that of the fungicide chlorothalonil. When the concentration of iturin A2 was increased to 500 mg kg-1, the control efficacy was enhanced to 64.2% in field, which was significantly higher than that of chlorothalonil. These results indicate that iturin A2 has potential for SCLB control and could be a substitute to synthetic fungicides. To our knowledge, this is the first report on using partially purified iturin A for control of SCLB under field conditions.

关键词: identification, Bipolaris maydis, biocontrol

Abstract: Bacillus subtilis B47 is an endophytic bacterium of tomato, and produces substance that strongly inhibits the growth of Bipolaris maydis, the pathogen of southern corn leaf blight (SCLB), as well as several other phytopathogenic fungi. The antifungal substance was purified from the broth culture of the bacterium using acid precipitation, methanol extraction, and three-step chromatography. Based on FT-IR spectrometry, amino acid composition, and MALDI-TOF-MS/MS CID analyses, the antifungal substance was identified as iturin A2, a cyclic lipopeptide antibiotic. To evaluate the efficacy of iturin A2 for control of SCLB, partially purified iturin A2 (75%, w/v) was applied under different conditions. At the concentration of 300 mg kg-1, iturin A2 showed efficacy ranging from 100 to 53.1% under in vitro, in plot and in field conditions. This efficacy was higher than or similar to that of the fungicide chlorothalonil. When the concentration of iturin A2 was increased to 500 mg kg-1, the control efficacy was enhanced to 64.2% in field, which was significantly higher than that of chlorothalonil. These results indicate that iturin A2 has potential for SCLB control and could be a substitute to synthetic fungicides. To our knowledge, this is the first report on using partially purified iturin A for control of SCLB under field conditions.

Key words: identification, Bipolaris maydis, biocontrol

YE Yun-feng, LI Qi-qin, FU Gang, YUAN Gao-qing, MIAO Jian-hua , LIN Wei. Identification of Antifungal Substance (Iturin A2) Produced by Bacillus subtilis B47 and Its Effect on Southern Corn Leaf Blight[J]. Journal of Integrative Agriculture, 2012, 12(1): 90-99.

参考文献

[1]Arun B B, Bose S K. 1964. Biosynthesis of mycobacillin, a new antifungal peptide. Journal of Bacteriology, 87, 1402-1405.

[2]Babasaki K, Takao T, Shimonishi Y, Kurahashi K. 1985. Subtilosin A, a new antibiotic peptide produced by Bacillus subtilis 168: isolation, structural analysis, and biogenesis. Journal of Biological Chemistry, 98, 585- 603.

[3]Balint-Kurti P J, Carson M L. 2006. Analysis of quantitative trait loci for resistance to southern leaf blight in juvenile maize. Phytopathology, 96, 221-225.

[4]Balint-Kurti P J, Krakowsky M D, Jines M P, Robertson L A, Molnár T L, Goodman M M, Holland J B. 2006. Identification of quantitative trait loci for resistance to southern leaf blight and days to anthesis in a maize recombinant inbred line population. Phytopathology, 96, 1067-1071.

[5]Besson F, Peypoux F, Michel G, Delcambe L. 1978. Identification of antibiotics of iturin group in various strains of Bacillus subtilis. Journal of Antibiotics (Tokyo), 31, 284-288.

[6]Besson F, Michel G. 1987. Isolation and characterization of new iturins: iturin D and iturin E. Journal of Antibiotics (Tokyo), 40, 437-442.

[7]Chang W T, Chen M L, Wang S L. 2010. An antifungal chitinase produced by Bacillus subtilis using chitin waste as a carbon source. World Journal of Microbiology and Biotechnology, 26, 945-950.

[8]Chen H, Yuan C, Cai K, Zheng Z, Yu Z. 2008. Purification and identification of iturin A from Bacillus subtilis JA by electrospray ionization mass spectrometry. Acta Microbiologica Sinica, 48, 116-120. (in Chinese)

[9]Delcambe L, Peypoux F, Besson F, Gisnaud M, Michel G. 1977. Structure of iturin and iturin like substances. Biochemical Society Transactions, 5, 1122-1124.

[10]Frick H, Nicholson R L, Hodges T K, Bauman L F. 1976. Influence of Helminthosporium maydis, race T, toxin on potassium uptake in maize roots. Plant Physiology, 57, 171-174.

[11]Eshita S M, Roberto N H, Beale J M, Mamiya B M, Workman R F. 1995. Bacillomycin Lc, a new antibiotic of the iturin group: isolations, structures, and antifungal activities of the congeners. Journal of Antibiotics (Tokyo), 48, 1240-1247.

[12]Fu G, Huang S L, Ye Y F, Wu Y G, Cen Z L, Lin S H. 2010. Characterization of a bacterial biocontrol strain B106 and its efficacy in controlling banana leaf spot and postharvest anthracnose diseases. Biological Control, 55, 1-10.

[13]Gao Z S, Cai H W, Liang G H. 2005. Field assay of seedling and adult-plant resistance to southern leaf blight in maize. Plant Breeding, 124, 356-360.

[14]Gong M, Wang J D, Zhang J, Yang H, Lu X F, Pei Y, Cheng J Q. 2006. Study of the antifungal ability of Bacillus subtilis strain PY-1 in vitro and identification of its antifungal substance (iturin A). Acta Biochimica et Biophysica Sinica (Shanghai), 38, 233-240.

[15]Huang C J, Yang K H, Liu Y H, Lin Y J, Chen C Y. 2010. Suppression of southern corn leaf blight by a plant growth-promoting rhizobacterium Bacillus cereus C1L. Annals of Applied Biology, 157, 45-53.

[16]Isogai A, Takayama S, Murakoshi S, Suzuki A. 1982. Structures of β-amino acids in antibiotics iturin A. Tetrahedron Letters, 23, 3065-3068.

[17]Kajimura Y, Sugiyama M, Kaneda M. 1995. Bacillopeptins, new cyclic lipopeptide antibiotics from Bacillus subtilis FR-2. Journal of Antibiotics (Tokyo), 48, 1095-1103.

[18]Kim J C, Choi G J, Kim H J, Kim H T, Ahn J W, Cho K Y. 2002. Verlamelin, an antifungal compound produced by a Mycoparasite, Acremonium strictum. The Plant Pathology Journal, 18, 102-105.

[19]Kim P I, Ryu J, Kim Y H, Chi Y T. 2010. Production of biosurfactant bipopeptides iturin A, fengycin, and surfactin A from Bacillus subtilis CMB32 for control of Colletotrichum gloeosporioides. Journal of Biotechnology, 20, 138-145.

[20]Klich M A, Arthur K S, Lax A R, Bland J M. 1994. Iturin A: a potential new fungicide for stored grains. Mycopathologia, 127, 123-127.

[21]Klich M A, Lax A R, Bland J M. 1991. Inhibition of some mycotoxigenic fungi by iturin A, a peptidolipid produced by Bacillus subtilis. Mycopathologia, 116, 77-80.

[22]Kugler M, Loeffler W, Rapp C, Kern A, Jung G. 1990. Rhizocticin A, an antifungal phosphono-oligopeptide of Bacillus subtilis ATCC 6633: biological properties. Archives of Microbiology, 153, 276-281.

[23]Lai C Y. 2003. Agricultural Plant Pathology. Science Press, Beijing. (in Chinese) Leclere V, Bechet M, Adam A, Guez J S, Wathelet B, Ongena M, Thonart P, Gancel F, Marlene C I, Jacques P. 2005. Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism’s antagonistic and biocontrol activities. Applied and Environment Microbiology, 71, 4577-4584.

[24]Lim S M, Hooker A L. 1971. Southern corn leaf blight: genetic control of pathogenicity and toxin production in race T and race O of Cochliobolus heterostrophus. Genetics, 69, 115-117.

[25]Li Q Q, Ye Y F, Meng X Y, Peng H W, Lin W, Luo K. 2005. Identification of endophytic bacterium strain B47 and its control effects on tomato bacterial wilt. Chinese Journal of Biological Control, 21, 178-182. (in Chinese)

[26]Lin H Y, Rao Y K, Wu W S, Tzeng Y M. 2007. Ferrous ion enhanced lipopeptide antibiotic iturin A production from Bacillus amyloliquefaciens B128. International Journal of Applied Science and Engineering, 5, 123- 132.

[27]Lu X Y, Li S Z, Li Q S, Kong L X, Liu J, Ma P, Gao S G. 2006. Screening of bacteria as biocontrol agent against corn leaf spot and study on its optimal culture medium. Chinese Journal of Biological Control, 22, 47-53. (in Chinese)

[28]Maget-Dana R, Peypoux F. 1994. Iturins, a special class of pore-forming lipopeptides: biological and physicochemical properties. Toxicology, 87, 151-174.

[29]Mhammedi A, Peypoux F, Besson F, Michel G. 1982. Bacillomycin F, a new antibiotic of iturin group: isolation and characterization. Journal of Antibiotics (Tokyo), 35, 306-311.

[30]Mizumoto S, Hirai M, Shoda M. 2007. Enhanced iturin A production by Bacillus subtilis and its effect on suppression of the plant pathogen Rhizoctonia solani. Applied Microbiology and Biotechnology, 75, 1267- 1274.

[31]Montesinos E. 2003. Development, registration and commercialization of microbial pesticides for plant protection. International Microbiology, 6, 245-252.

[32]Moyne A L, Shelby R, Cleveland T E, Tuzun S. 2001. Bacillomycin D: an iturin with antifungal activity against Aspergillus flavus. Journal of Applied Microbiology, 90, 622-629.

[33]Ongena M, Jacques P, Touré Y, Destain J, Jabrane A, Thonart P. 2005. Involvement of fengycin-type lipopeptides in the multifaceted biocontrol potential of Bacillus subtilis. Applied Microbiology and Biotechnology, 69, 29-38.

[34]Paik S H, Chakicherla A, Hansen J N. 1998. Identification and characterization of the structural and transporter genes for, and the chemical and biological properties of, sublancin 168, a novel lantibiotic produce by Bacillus subtilis 168. Journal of Biological Chemistry, 273, 23134-23142.

[35]Patel P S, Huang S, Fisher S, Pirnik D, Aklonis C, Dean L, Meyers E, Fernandes P, Mayerl F. 1995. Bacillaene, a novel inhibitor of procaryotic protein synthesis produced by Bacillus subtilis: production, taxonomy, isolation, physico-chemical characterization and biological activity. Journal of Antibiotics (Tokyo), 48, 997-1003.

[36]Peypoux F, Guinand M, Michel G, Delcambe L, Das B C, Lederer E. 1978. Structure of iturin A, a peptidolipid antibiotic from Bacillus subtilis. Biochemistry, 17, 3992- 3996.

[37]Peypoux F, Pommier M T, Das B C, Besson F, Delcambe L, Michel G. 1984. Structures of bacillomycin D and bacillomycin L peptidolipid antibiotics from Bacillus subtilis. Journal of Antibiotics (Tokyo), 37, 1600-1604.

[38]Peypoux F, Pommier M T, Marion D, Ptak M, Das B C, Michel G. 1986. Revised structure of mycosubtilin, a peptidolipid antibiotic from Bacillus subtilis. Journal of Antibiotics (Tokyo), 39, 636-641.

[39]Peypoux F, Bonmati J M, Wallach J. 1999. Recent trends in the biochemistry of surfactin. Applied Microbiology and Biotechnology, 51, 553-563.

[40]Raid R N. 1991. Fungicidal control of foliar sweet corn diseases in the presence of high inoculum levels. Proceedings of the Florida State Horticultural Society, 104, 267-270.

[41]Rao Y K, Lin H Y, Wu W S, Tzeng Y M. 2008. Evaluation of HPLC and MEKC methods for the analysis of lipopeptide antibiotic iturin A produced by Bacillus amyloliquefaciens. International Journal of Applied Science and Engineering, 6, 85-96.

[42]Romero D, Vicente A, Rakotoaly R H, Dufour S E, Veening J W, Arrebola E, Cazorla F M, Kuipers O P, Paquot M, Alejandro P G. 2007. The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. American Phytopathological Society, 20, 430-440.

[43]Stover A G, Driks A. 1999. Secretion, localization, and antibacterial activity of Tas A, a Bacillus subtilis poreassociated protein. Journal of Bacteriology, 181, 1664- 1672.

[44]Tenoux I, Besson F, Michel G. 1991. Studies on the antifungal antibiotics: bacillomycin D and bacillomycin D methylester. Microbios, 67, 187-193.

[45]Ullstrup A J. 1972. The impacts of the southern corn leaf blight epidemics of 1970-1971.

[46]Annual Review of Phytopathology, 10, 37-50.

[47]Wang X, Wang S Y, Gao P H. 2008. Evaluation of antagonism of Penicillium TS67 against soybean root rot disease and corn southern leaf blight. Microbiology, 35, 1246- 1250. (in Chinese)

[48]Wang H L, Wen K, Zhao X Y, Wang X D, Li A Y, Hong H Z. 2009. The inhibitory activity of endophytic Bacillus sp. strain CHM1 against plant pathogenic fungi and its plant growth-promoting effect. Crop Protection, 28, 634-639.

[49]Wei J K, Lui K M. 1988. Pathological and physilogical identification of raee C of Bipolaris maydis in China. Phytopathology, 75, 550-554.

[50]Yu G Y, Sinclair J B, Hartman G L, Bertagolli B L. 2002. Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani. Soil Biology & Biochemistry, 34, 955-963.

[51]Yuliar. 2005. Influence of palmitic acid and amino acids addition on iturin A productivity by Bacillus subtilis RB14-CS.

[52]Biodiversitas, 6, 172-174.

[53]Zhang B, Xie C, Yang X. 2008. A novel small antifungal peptide from Bacillus strain B-TL2 isolated from tobacco stems. Peptides, 29, 350-355.

[54]Zimmerman S B, Schwartz C D, Monaghan R L, Pelak B A, Weissberger B, Gilfillan E C, Mochales S, Hernandez S, Currie S A, Tejera E. 1987. Difficidin and oxydifficidin: novel broad spectrum antibacterial antibiotics produced by Bacillus subtilis. I. Production, taxonomy and antibacterial activity. Journal of Antibiotics (Tokyo), 40, 1677-1681.

[55]Zuber P, Nakano M M, Marahiel M A. 1993. Peptide antibiotics. In: Sonenshein A L, Hoch J A, Losick R, eds., Bacillus subtilis and Other Gram-Positive Bacteria. American Society for Microbiology, Washington D.C. pp. 897-916.

Identification of Antifungal Substance (Iturin A2) Produced by Bacillus subtilis B47 and Its Effect on Southern Corn Leaf Blight

Identification of Antifungal Substance (Iturin A2) Produced by Bacillus subtilis B47 and Its Effect on Southern Corn Leaf Blight

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