شناسایی مولکولی و تشخیص ژن کدکننده دی اکسی نیوالنول در جدایه های Fusarium graminearum عامل بلایت فوزاریومی سنبله گندم در ایران

نوع مقاله: پژوهشی

نویسندگان

1 اعضای هیت علمی گروه بیماری شناسی گیاهی، دانشکدهکشاورزی و منابع طبیعی دانشگاه آزاد اسلامی تهران واحد علوم و تحقیقات

2 اعضای هیت علمی بخش تحقیقات بیماری های گیاهی، موسسه تحقیقات گیاه پزشکی کشور، تهران

چکیده

بیماری بلایت فوزاریومی سنبله یکی از مهم­ترین بیماری­های قارچی گندم در ایران و سایر نقاط جهان می­باشد. عامل اصلی این بیماری Fusarium graminearum  شناخته شده که گندم را در طی دوره گل­دهی آلوده کرده و نه تنها باعث کاهش محصول می­گردد، بلکه  با تولید زهرابه قارچی، موجب مسمومیت در انسان و دام نیز می شود. در این مطالعه 60 جدایه F. graminearum جداشده از مزارع گندم آلوده از استان­های مختلف ایران مورد بررسی قرار گرفتند. تشخیص مولکولی جدایه­ها بر اساس روش PCR با استفاده از آغازگرهای اختصاصی Fg16F/Fg16R برای این گونه انجام و تعلق کلیه جدایه­ها به گونه F. graminearum مورد تأیید قرار گرفت. در کلیه جدایه­ها یک باند 420 جفت بازی تکثیر شد که در سایر گونه­های نزدیک از جمله F. culmorum مشاهده نگردید. جدایه­ها برای تشخیص ژن کدکننده زهرابه دی اکسی نیوالنول با استفاده از آغازگر اختصاصی Tri13F/Tri13DONR مورد آزمایش قرار گرفتند. تنها در 36 جدایه یک قطعه 228 جفت بازی تکثیر گردید. با توجه به این که روش­های مرسوم بسیار وقت‎گیر و غیراختصاصی هستند، استفاده از آغازگرهای ویژه گونه برای تشخیص سریع کشت­های مشکوک به F. graminearum برای تعیین گونه قارچ عامل بیماری به طور مستقیم دربافت آلوده و تعیین ترکیب زهرابه­های تولیدی آن از اهمیت خاصی برخوردار است.

کلیدواژه‌ها


عنوان مقاله [English]

Molecular identification and detection of gene encoding deoxynivalenol in Fusarium graminearum isolates, the causal agent of fusarium head blight of wheat in Ira

نویسندگان [English]

  • Roya Rezaeian Doloei
  • saeed rezaee 1
  • mansooreh mieabolfathy 2
  • hamidreza zamanizadeh 1
  • mohammad razavi 2
1 department of plant pathology, college of agriculture and natural resources, sciende and research branch, islamic azad university, tehran, iran
2 iranian plant protection research institute
چکیده [English]

Fusarium Head Blight (FHB) is one of the most important fungal diseases of wheat in Iran and throughout the world. Fusarium species infect wheat during the flowering period. In addition to losses of yield, these fungi can also synthesize mycotoxins under suitable environmental conditions, thus threatening animal and human health. In the present study, 60 isolates of F. graminearum collected from infested wheat fields of some provinces of Iran and were characterized morphologically and genetically. Molecular characterization of isolates was performed using an optimized simple and low cost method for isolation of DNA from F. graminearum using polymerase chain reaction with species specific primers. All of the isolates were confirmed as F. graminearum using species specific primers Fg16F/Fg16R through PCR assays. The results indicated that a specific band of 420 bp was amplified, when species specific primers of F. graminearum was used. All of the isolates were molecularly identified as F. graminearum. The primers failed to amplify any band in negative controls including F. culmorum. All of F. graminearum isolates were tested for DON chemotype producing gene by specific Tri13F/Tri13DONR primer by PCR. Our results showed that a specific band of 282 bp was amplified only in 36 isolates (60%). Consequently, using of species specific primers for detection of suspected cultures of F. graminearum in infected tissues and toxin profile of isolates are very interesting when comparing with the morphological trait which are time consuming and non-specific.

کلیدواژه‌ها [English]

  • fusarium head blight (FHB)
  • species specific primer
  • DON
Babadoost M (1995) Occurrence of Fusarium species in seeds of wheats in Azarbayjan-e-Sharqi province and Ardabil, Iran. Iranian Journal of Plant Pathology, 31: 88-100.

Bottalico A, Perrone G (2002) Toxigenic Fusarium species and mycotoxins associated with head blight in small grain cereals in Europe. European Journal of Plant Pathology 198: 611–624.

Brown DW, McCormick SP, Alexander NJ, Proctor RH, Desjardins AE (2002) Inactivation of a cytochrome P-450 is a determinant of trichothecene diversity in Fusarium species. Fungal Genetic Biology 36:224–233.

Burgess LW, Summerell BA, Bullock  S, Gott KP, Backhouse D (1994) Laboratory Manual for Fusarium Research. Third edition. University of Sydney/Royal  Botanic Gardens, Sydney, Australia.

Burlakoti RR, Shaukat A, Secor GA, Neate SM, McMullen MP, and Adhikari TB (2008) Comparative mycotoxin profiles of Gibberella zeae populations from barley, wheat, potatoes, and sugar beets. Applied and Environmental Microbiology, 74(21): 6513-6520

Carter JP, Rezanoor HN, Desjardins AE, Nicholson P (2000) Variation in Fusarium graminearum isolates from Nepal associated with their host of origin. Plant Pathology 49: 452-460.

Carter JP, Rezanoor HN, Holden D, Desjardins AE, Plattner RD, Nicholson P (2002) Variation in pathogenicity associated with the genetic diversity of Fusarium graminearum. European Journal of Plant Pathology 108: 573–583.

Chandler EA, Simpson DR, Thomsett MA, Nicholson P (2003) Development of PCR assays to Tri7 and Tri13 trichothecene biosynthetic genes, and characterisation of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis. Physiological and Molecular Plant Pathology 62: 355-367.

Chung WH, Ishii H, Nishimura K, Ohshima M, Iwama T, Yoshimatsu H (2008) Genetic analysis and PCR based identification of major Fusarium species causing head blight on wheat in Japan. Journal of General Plant Pathology 74: 364-374.

Desjardins AE, Jarosz AM, Platiner RD, Alexander NJ, Brown DW Jurgenson JE (2004) Patterns of Trichothecene production, genetic variability and virulence to wheat of Fusarium graminearum from smallholder farms in Nepal. Journal of Agricultural Food Chemistry 52: 6341-6346.

Forootan A, Ershad D, Dalili  A, Bamdadian T, Gerami GH (1993) Occurrence of head blight of wheat in Mazandaran. Proceeding of the 11th Iranian Plant Protection Congress, Gilan University, Rasht.

Golzar H, (1993) Wheat head blight- etiology, infection and seed transmission. Iranian Journal of Plant pathology 25: 17-22.

Haratian M, Sharifnabi B, Alizadeh A, Safaie N (2006) Detection of genes involved in trichothecene production in Iranian isolates of Fusarium graminearum by PCR. Iranian Journal of Plant Pathology 42: 519-538.

Haratian M, Sharifnabi B, Alizadeh A, Safaie N (2008) PCR analysis of the Tri13 gene to determine the genetic potential of  Fusarium graminearum isolates from Iran to produce Nivalenol and Deoxynivalenol. Mycopathologia 166: 109-116.

Jennings P, Coates ME, Turner JA, Chandler EA, Nicholson P (2004) Determination of deoxynivalenol and nivalenol chemotypes of Fusarium culmorum isolates from England and Wales by PCR assay. Plant Pathology 53: 182– 190

Ji L, Cao K, Hu T, Wang S (2007) Determination of deoxynivalenol and nivalenol chemotypes of Fusarium graminearum isolates from China by PCR assay. Journal of Phytopathology 155: 505-512.

Johanson DD, Wilson WW Diersen M (1995) Quality uncertainty and grain merchandising risk: Vomitoxin in spring wheat. Agric. Econ. Rep. No. 33. North Dakota State University, Fargo.

Langseth A, Bernhoft A, Rundberget T, Kosiak B, Gereis M (1999) Mycotoxin production and cytotoxicity of Fusarium strains isolated from Norwegian cereals. Mycopathologia 144: 103–13.

Lee T, Oh D, Kim H (2001) Identification of deoxynivalenol and nivalenol-producing chemotypes of Gibberella zeae by using PCR. Applied and Environmental Microbiology 67:2966–2972.

Leslie JF, Summerell BA (2006) The fusarium laboratory manual. Ames: Blackwell Publishing, pp:176-179.

Li HP, Wu AB, Zhao CS, Scholten O, Loffler H, Liao YC (2005) Development of a generic PCR detection of deoxynivalenol and nivalenol-chemotypes of Fusarium graminearum. FEMS Microbiology Letter 243:505–511.

Llorens A, Hinojo1 MJ, Mateo R, Medina1 A, Valle-Algarra1 FM, Gonzalez-Jaen MT, Jimenez M (2006) Variability and characterization of mycotoxin-producing Fusarium spp. isolates by PCR-RFLP analysis of the IGS-rDNA region. Antonie van Leeuwenhoek 89: 465–478

McMullen M, Jones R, Gallenberg D (1997) Scab of wheat and barley: a re-emerging disease of devastating impact. Plant Disease 81:1340–1348.

Miller JD, Greenhalgh R, Wang Y Lu M (1991) Trichothecene chemotypes of three Fusarium species, Mycologia 83: 121–130.

Nicholson P, Lees AK, Maurin N, Parry DW, Rezanoor HN (1996) Development of a PCR assay to identify and quantify Microdochium nivale var nivale and Microdochium nivale var. majus in wheat. Physiological and Molecular Plant Pathology 48:257–271.

Nicholson P, Simpson DR, Weston G, Rezanoor HN, Lees AK, Parry DW, Joyce D (1998) Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiological and Molecular Plant Pathology 53: 17– 37.

Nicholson P, Simpson DR, Wilson AH,  Chandler E, Thomsett M (2004) Detection and differentiation of trichothecene and enniatin-producing Fusarium species on small-grain cereals. European Journal of Plant Pathology 110: 503-514.

O’Donnell K, Kistler HC, Tacke BK Casper HH (2000) Gene genealogies reveal global phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum, the fungus causing wheat scab. Proceeding of National Academy of Science USA97: 7905–7910.

Parry DW, Jenkinson P, McLeod L (1995) Fusarium ear blight (scab) in small grains-a review. Plant Pathology 44: 207–238.

Parry DW, Nicholson P (1996) Development of a PCR assay to detect Fusarium poae in wheat. Plant Pathology 45:383–391.

Prodi A, Tonti S, Nipoti P, Pancaldi D, Pis A (2009) Identification of deoxynivalenol and Nivalenol producing chemotypes of F. graminearum isolates from durum wheat in a restricted area of northern Italy. Journal of Plant Pathology 91(3): 727-731

Qu B, Li HP, Zhang JB, Huang T, Carter J, Liao YC, Nicholson P (2008) Comparison of  genetic diversity and pathogenicity of Fusarium head blight pathogens from China and Europe by  SSCP and seedling assays on wheat. Journal of Plant Pathology 57: 642-651

Reader U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Applied Microbiology, 90: 901-908.

Snijders CHA (1990) Fusarium head blight and mycotoxin contamination of wheat, a review. Netherland Journal of Plant Pathology 96: 187-198.

Turner AS, Lees AK, Rezanoor HN, Nicholson P (1998) Refinement of PCR detection of Fusarium avenaceum and evidence from DNA marker for phonetic relatedness to Fusarium tricinetum. Plant Pathology 47: 278-288. 

Waalwijk C, Kastelein P, de Vries I, Kerenyi Z, Van der Lee T, Hesselink T, Kohl J, Kema G (2003) Major changes in Fusarium spp. in wheat in the Netherlands. European Journal of Plant Pathology 10: 743–754.

Wang YZ (1996) Epidemiology and management of wheat scab in China in: Fusarium head scab: Global status and future prospects. Pages: 97-105.

Ward TJ, Bielawski JP, Kistler HC, Sullivan E, O’Donnell K (2002) Ancestral polymorphism and adaptive evolution in the tri-chothecene mycotoxin gene cluster of phytopathogenic Fusarium. Proceeding of National Academy of Science, USA, 99: 9278–9283.

Windels CE (2000) Economic and social impacts of Fusarium head blight: changing farms and rural communities in the northern Great Plains. Phytopathology 90: 17–21.

Yao JB, Lu WZ (2000) Research advances in wheat breeding for scab resistance in China. Jiangsu Journal of Agricultural Science 16:242–248.

Yang L, van der Lee T, Yang X, Yu D, Waalwijk C. (2008) Fusarium populations on Chinese barley show a dramatic gradient in mycotoxin profiles. Phytopathology 98(6): 719-727.

Zamanizadeh HR, Khorsandi H (1995) Fusarium spp. And their mycotoxins in wheat of Mazandaran province. Iranian Journal of Plant Pathology 23: 31-37.

Zhang JB, Li, HP, Dang FJ, Qu B, Xu YB, Zhao CS, Liao YC (2007) Determination of the trichothecene mycotoxin chemotypes and associated geographical distribution and phylogenetic species of the Fusarium graminearum clade from Chinese Mycological Research 111: 967-975.