Detection of Mycotoxins Producing of Fusarium Species Using Morphological and Molecular Characteristics in Corn

Document Type : Research Paper

Authors

1 Former MSc Student of Plant Pathology, Department of Plant Protection, Kashmar University of Science and Culture, Iran.

2 Assistant Professor Department of Plant Protection, Agricultural and Natural Resources Research and Education Center of Khorasan-Razavi Province, Mashhad, Iran.

3 Instructor, Department of Plant Protection, Agricultural and Natural Resources Research and Education Center of Khorasan-Razavi Province, Mashhad, Iran.

4 3Professor, Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.

Abstract

Abstract
Fusarium, a phytopathogenic fungus in fields of corn, not only reduces the quality and quantity of crops but also causes food contamination and endangers people  ̓s health by producing some mycotoxins such as Fumonisin (FUM). In the present study, samples were collected from fungal disease symptoms- showing corn from different livestock and poultry storage in Khorasan-Razavi province. Having placed the corn on PDA and WA, isolates belonging to Fusarium genus were isolated and purified. As a result, a total of 40 isolates were obtained among which three species were identified as Fusarium verticillioides, F. graminearum and F. proliferatum with isolated frequencies of 34, 4 and 2, respectively. To support these results,  DNA was extracted from the isolates from corn seeds and the fungal ribosomal DNA was amplified using PCR with species- specific primers. In addition, using PCR with the specific primers (FUM1 F/R) 15 isolates of F. verticillioides, as the prevalent species were examined for fumonisin production. The results indicated that all of the examined F. verticllioides isolates gave the expected fragment with  183 bp in size. Therefore, this study demonstrated that F. verticllioides isolates genetically have a potential for fumonisin-production. It also showed that the PCR-based technique can be used for the identification, distinction and detection of the Fusarium species.
 

Keywords


آشناگر ل، رضایی س، درویش­نیا م، و سیاهپوش س، 1389. شناسایی عوامل پوسیدگی فوزاریومی بلال در استان لرستان. صفحه 1 خلاصه مقاله های نوزدهمین کنگره­ی گیاهپزشکی ایران، تهران، ایران.
پرچمیان م، رهجو و، زمانی م، پیرنیا م، و ذهابی ع، 1389. جداسازی و شناسایی جدایه­های قارچ عامل پوسیدگی فوزاریومی خوشه ذرت. صفحه 62 خلاصه مقاله های نوزدهمین کنگره­ی گیاهپزشکی ایران، موسسه تحثقیقات گیاهپزشکی ایران، تهران، ایران.
داوودی ع، 1381. معرفی قارچ عامل پوسیدگی طوقه ذرت در قزوین. صفحه 82 خلاصه مقاله های پانزدهمین کنگره­ی گیاهپزشکی ایران، کرمانشاه، ایران.
داوودی ع، و مهریان ف، 1383. شناسایی عوامل بیماریزای قارچی خوشه و دانه ذرت در منطقه قزوین. صفحه 115 خلاصه مقاله های شانزدهمین کنگره­ی گیاهپزشکی ایران، تبریز، ایران.
ربیعی مطلق ا، فلاحتی رستگار م، روحانی ح، جعفرپور ب و جهان بخش و، 1389. بررسی فوزاریوم­های بذرزاد پیاز دراستان های خراسان شمالی و رضوی. نشریه حفاظت گیاهان، جلد 24، شماره 2. صفحه­های 137 تا 144.
زمانی م، 1383. بررسی پوسیدگی ساقه ذرت و ارزیابی تعدادی از ترکیبات با استفاده از آلودگی مصنوعی نسبت به گونه غالب. صفحه 114 خلاصه مقاله های شانزدهمین کنگره­ی گیاهپزشکی ایران، تبریز، ایران.
عظیمی ص، 1390. بررسی پوسیدگی فوزاریومی بلال ذرت در استان خوزستان. مجله تحقیقات غلات، جلد1، شماره 1. صفحه­های 75 تا 83.
کاظمی ه، و مومنی ح، 1395. گزارش قارچ Fusarium graminearum و فرم جنسی آن از ذرت در منطقه دشت مغان. جلد 1- صفحه 148 خلاصه مقاله­های بیست و­دومین کنگره گیاهپزشکی ایران، پردیس کشاورزی و منابع طبیعی دانشگاه تهران، کرج.
محمدی ع، فرخی­نژاد ر، و میناسیان و، 1381. تنوع ژنتیکی در جدایه­های Fusarium moniliformeجداسازی شده از نیشکر و بذور ذرت با استفاده از گروههای سازگار رویشی و تعیین ارتباط گروهها با بیماریزایی. صفحه 135 خلاصه مقاله های پانزدهمین کنگره­ی گیاهپزشکی ایران، کرمانشاه، ایران.
مهریان ف، 1370. معرفی عامل بیماریزای خوشه ذرت در استان­های مازندران و گیلان. صفحه 289 خلاصه مقاله های چهاردهمین کنگره­ی گیاهپزشکی ایران، اصفهان، ایران.
نادرپور م، 1383. مایکوفلور بذر ذرت هیبرید رقم سینگل کراس 704 دشت مغان. صفحه 120 خلاصه مقاله های شانزدهمین کنگره­ی گیاهپزشکی ایران، تبریز، ایران.
Abd-El Fatah SI, Naguib MM, El-Hossiny EN, Sultan YY, Abodalam TH and Yli-Mattila T, 2015. Molecular versus Morphological Identification of Fusarium spp. isolated from Egyptian corn. Research Journal of Pharmaceutical, Biological and Chemical 6(4): 1813-1822.
Acuna A, Maria CL, Maria CDeG and Gonzalo JD, 2005. Prevalence of Fusarium species of the Liseola section on selected Colombian animal feedstuffs and their ability to produce fumonisins. Mycopathologia 160(1):63-66.
Bluhm BM, Cousin MA and Woloshuk CP, 2004. Multiplex real-time PCR detection of fumonisin producing and trichothecene producing groups of Fusarium species. Journal of Food Protection 67(3): 536–543.
Burgess LW, Summerell BA, Bullock P and Backhouse D, 1994. Laboratory Manual for Fusarium Research. Department of Crop Science. 3rd ed. University of Sydney.
Duan C, Qin Z, Yang Zh, Li W, Sun S, Zhu Z and W X, 2016. Identification of Pathogenic Fusarium spp. Causing Maize Ear Rot and Potential Mycotoxin Production in China. Toxins 8(6): 1-17.
Ghiasian SA, Kord-Bacheh P, Rezayat SM, Maghsood AH and Taherkhani H, 2004. Mycoflora of Iranian Maize Harvested in the Main Production Areas in 2000. Mycopathologia 158(1): 113-121.
Gonzalez-Jaen MT, Mirete S, Patino B, Lopez-Errasquin E and Covadonga V, 2004. Genetic markers for the analysis of variability and for production of specific diagnostic sequences in fumonisin-producing strains of Fusarium verticillioides. European Journal of Plant Pathology 110(5): 525–532.
Halstensen AS, 2008. Species-specific fungal DNA in airborne dust as surrogate for occupational mycotoxin exposure. International Journal of Molecular Science 9(12): 2543-2558.
Hendricks K, 1999. Fumonisins and neural tube defects in south Texas. Epidemiology 10(2): 198-200.
Hollingsworth ML, Andra Clark ALEX, Forrest LL, Richardson J, Pennington R, Long DG and Hollingsworth PM, 2009. Selecting barcoding loci for plants: evaluation of seven candidate loci with species‐level sampling in three divergent groups of land plants. Molecular Ecology Resources 9(2): 439-457.
Jimenez M, Rodrıguez S, Mateo JJ, Gil JV, Mateo R, 2000. Characterization of Gibberella fujikuroi complex isolates by fumonisin B1 and B2 analysis and by RAPD and restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA. Systematic and Applied Microbiology 23(4):546-55.
Jurado M, Marín P, Callejas C, Moretti A, Vázquez C and González‐Jaén MT, 2010. Genetic variability and Fumonisin production by Fusarium proliferatum. Food Microbiology 27(1): 50–57.
Jurado M,Vázquez C, Patiño B and González-Jaén MT, 2005. PCR detection assays for the trichothecene-producing species Fusarium graminearum, Fusarium culmorum, Fusarium poae, Fusarium equiseti and Fusarium sporotrichioides. Systematic and Applied Microbiology 28(6):562–568.
Leslie JF and Summerell BA, 2006. The Fusarium Laboratory Manual. Blackwell.
Marasas WFO, Riley RT, Hendricks KA, Stevens VL, Sadler TW, Waes JG-v, Missmer SA, Cabrera J, Torres O, Gelderblom WCA, Allegood J, Martinez C, Maddox J, Miller JD, Starr L, Sullards MC, Roman AV, Voss KA, Wang E, Jr AHM, 2004. Fumonisins disrupt sphingolipid metabolism, folate transport, and neural tube development in embryo culture and in vivo: A potential risk factor for human neural tube defects among populations consuming fumonisin-contaminated maize. Journal of Nutrition 134(4): 711-716.
Mule G, Susca A, Stea G and Moretti A, 2004. Specific detection of the toxigenic species Fusarium proliferatum and F. oxysporum from asparagus plants using primers based on Calmodulin gene sequences. Federation of European Microbiological Societies Microbiology Letters 230(2): 235-240.
Munkvold GP, 2003. Epidemiology of Fusarium diseases and their mycotoxins in maize ears. European Journal of Plant Pathology 109(7): 705-713.
Nelson PE, Toussoun TA and Marasas WFO, 1983. Fusarium species an illustrated manual for identification. Pennsylvania State University Press.
O’Donnell K, Cigelnik E, Nirenberg HI, 1998. Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycology 90(3):465-93.
Rocha LO, Nakai VK, Braghini R, Reis TA, Kobashigawa E, Corrêa B, 2009. Mycoflora and co-occurrence of fumonisins and aflatoxins in freshly harvested corn in different regions of Brazil. International Journal of Molecular Sciences 10(11): 5090–5103.
Russell R, Paterson M, 2006. Identification and quantification of mycotoxigenic fungi by PCR. Process Biochemistry 41(7):1467-1474.
Schultz J and Wolf M, 2009. ITS2 sequence structure analysis in phylogenetics: a how to manual for molecular systematics. Molecular Phylogenetics and Evolution 52(2): 520-523.
Sreenivasa MY, Dass RS, Raj APC and Janardhana GR, 2008. PCR method for the detection of genus Fusarium and fumonisin-producing isolates from freshly harvested sorghum grains grown In Karnataka, India. Journaloffood safety28(2): 247-236.

Turner NW, Subrahmanyam S and Piletsky SA, 2009. Analytical methods for determination of mycotoxins: A review. Analytica Chimica Acta 632(2):168-80.

Visentin I, Tamietti G, Valentino D, Portis E, Karlovsky P, Moretti A, Cardinale F, 2009. The ITS region as a taxonomic discriminator between Fusarium verticillioides and Fusarium proliferatum. Mycological Research 113(10): 1137-1145.
White T J, Bruns T, Lee S and Taylor J, 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315–322 In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds.), PCR Protocols : A guide to Methods and Applications Academic Press, New York, USA.
Yazeed HAE, Hassan A, Moghaieb REA, Hamed M, and Refai M, 2011. Molecular detection of fumonisin-producing Fusarium species in animal feeds using polymerase chain reaction (PCR). Journal of Applied Sciences Research 7(4): 420–427.
Yli-Mattila T, Paavanen-Huhtala S, Parikka P, Konstantinova P, and Gagkaeva TY, 2004. Molecular and morphological diversity of Fusarium species in Finland and north-western Russia. European Journal of Plant Pathology 110(5): 573-585.
Zamani, M., Alizadeh, A. and Choukan, R. 2000. Evaluation of resistance of selected corn lines to Fusarium moniliforme ear rot. Seed and Plant 15(4): 331-342.
Zare R and Ershad D, 1997. Fusarium species isolated from cereals in Gorgan area. Iranian Journal of Plant Pathology 33(1/2): 1-14.