Wheat (Triticum spp.) is often coinfected by several soil-borne fungal pathogens, causing serious crop yield and economic losses. Rapid diagnosis of infected fungi is critical for the application of disease control mechanisms. Therefore, we established a multiplex PCR method using specific primers for the simultaneous detection of five common soil-borne fungal pathogens affecting wheat: Rhizoctonia cerealis, Bipolaris sorokiniana, Gaeumannomyces graminis var. tritici, Fusarium pseudograminearum and F. graminearum. The specific primers were designed based on the sequences of several unique genes: ITS (internal transcribed spacer) of R. cerealis, URP-1F (universal rice primer) of B. sorokiniana, β-tubulin of G. graminis var. tritici and TEF1-α (translation elongation factor) of F. pseudograminearum and F. graminearum. During optimization, sensitivity assays showed that the detection limit of multiplex PCR was as low as 0.39 ng of mycelium DNA from a known pool of fungal DNA. To detect pathogens in wheat field, the multiplex PCR method was applied to infected wheat samples collected from wheat fields in the Fengqiu and Taikang counties in Henan Province, China. The R. cerealis and F. graminearum pathogens were identified in samples from Fengqiu, while the R. cerealis, G. graminis var. tritici, B. sorokiniana and F. graminearum pathogens were identified in samples from Taikang. In conclusion, the results indicated that our multiplex PCR method was efficient and specific for the detection of soil-borne pathogens.
Multiplex PCR Detection Soil-borne pathogen
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Funding was provided by Special Fund for Agro-scientific Research in the Public Interest (201503112) and “Five-twelfth” National Science and Technology Support Program of China (2015BAD26B01).
Compliance with ethical standards
We collected the fungus in wheat field where they occurred and no specific permits were required in Henan Province, China. The land used as the collection area is neither privately owned nor protected, and the field studies did not involve endangered or protected species.
Conflict of interest
The authors declared that they have no conflicts of interest to this work.
Aggarwal R, Gupta S, Banerjee S, Singh VB (2011) Development of a SCAR marker for detection of Bipolaris sorokiniana causing spot blotch of wheat. Can J Microbiol 57(11):934–942CrossRefPubMedGoogle Scholar
Aoki T, O’Donnell K (1999) Morphological and molecular characterization of Fusarium pseudograminearum sp. nov., formerly recognized as the Group 1 population of F. graminearum. Mycologia 91(4):597–609CrossRefGoogle Scholar
Asher MJC, Shipton PJ (1981) Biology and control of take-all. Academic Press Inc., Ltd., LondonGoogle Scholar
Benyon FH, Burgess LW, Sharp PJ (2000) Molecular genetics investigations and reclassification of Fusarium species in sections Fusarium and Roseum. Mycol Res 104(10):1164–1174CrossRefGoogle Scholar
Boerema GH, Verhoeven AA (1977) Check-list for scientific names of common parasitic fungi. Series 26: fungi on field crops: cereals and grasses. Neth J Plant Pathol 83(5):165–204CrossRefGoogle Scholar
Bryan GT, Labourdette E, Melton RE, Nicholson P, Daniels MJ, Osbourn AE (1999) DNA polymorphism and host range in the take-all fungus, Gaeumannomyces graminis. Mycol Res 103(3):319–327CrossRefGoogle Scholar
Clarkson JDS, Cook RJ (1983) Effect of sharp eyespot (Rhizoctonia cerealis) on yield loss in winter wheat. Plant Pathol 32(4):421–428CrossRefGoogle Scholar
Coombs JT, Michelsen PP, Franco CM (2004) Evaluation of endophytic actinobacteria as antagonists of Gaeumannomyces graminis var. tritici in wheat. Biol Control 29(3):359–366CrossRefGoogle Scholar
Freeman J, Ward E (2004) Gaeumannomyces graminis, the take all fungus and its relatives. Mol Plant Pathol 5(4):235–252CrossRefPubMedGoogle Scholar
Goodwin PH, Hsiang T, Xue BG, Liu HW (1995) Differentiation of Gaeumannomyces graminis from other turf-grass fungi by amplification with primers from ribosomal internal transcribed spacers. Plant Pathol 44(2):384–391CrossRefGoogle Scholar
Goulds A, Polley RW (1990) Assessment of eyespot and other stem base diseases of winter wheat and winter barley. Mycol Res 94(6):819–822CrossRefGoogle Scholar
Guo YP, Li W, Sun HY, Wang N, Yu HS, Chen HG (2012) Detection and quantification of Rhizoctonia cerealis in soil using real-time PCR. J Gen Plant Pathol 78(4):247–254CrossRefGoogle Scholar
Hamada MS, Yin Y, Ma Z (2011) Sensitivity to iprodione, difenoconazole and fludioxonil of Rhizoctonia cerealis isolates collected from wheat in China. Crop Prot 30(8):1028–1033CrossRefGoogle Scholar
Hameed MA, Rana RM, Ali Z (2012) Identification and characterization of a novel Iraqi isolate of Fusarium pseudograminearum causing crown rot in wheat. Genet Mol Res 11(2):1341–1348CrossRefPubMedGoogle Scholar
Harvey H, Ophel-Keller K (1996) Quantification of Gaeumannomyces graminis var. tritici in infected roots and soil using slot-blot hybridization. Mycol Res 100(8):962–970CrossRefGoogle Scholar
Kumar J, Schäfer P, Hückelhoven R, Langen G, Baltruschat H, Stein E, Nagarajan S, Kogel KH (2002) Bipolaris sorokiniana, a cereal pathogen of global concern: cytological and molecular approaches towards better control. Mol Plant Pathol 3(4):185–195CrossRefPubMedGoogle Scholar
Láday M, Bagi F, MesteRazy A, Szécsi A (2000) Isozyme evidence for two groups of Fusarium graminearum. Mycol Res 104(7):788–793CrossRefGoogle Scholar
Li H, Conner RL, Chen Q, Laroche A, Graf RJ, Kuzyk AD (2004) The transfer and characterization of resistance to common root rot from Thinopyrum ponticum to wheat. Genome 47(1):215–223CrossRefPubMedGoogle Scholar
Li HL, Yuan HX, Fu B, Xing XP, Sun BJ, Tang WH (2012) First report of Fusarium pseudograminearum causing crown rot of wheat in Henan, China. Plant Dis 96(7):1065CrossRefGoogle Scholar
Lipps PE, Herr LJ (1982) Etiology of Rhizoctonia cerealis in sharp eyespot of wheat. Phytopathology 72:1574–1577CrossRefGoogle Scholar
Lu QX, Yang HY, Wang B, Li FF, Shi JR, Song YL, Yang CL (2008) Molecular diagnose of wheat stem soil-borne fungus diseases (in Chinese). J Triticeae Crops 28(3):531–536Google Scholar
Marasas WFO, Rheeder JP, Lamprecht SC, Zeller KA, Leslie JF (2001) Fusarium andiyazi sp. nov., a new species from sorghum. Mycologia 93(6):1203–1210CrossRefGoogle Scholar
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. Physiol Mol Plant Pathol 53(1):17–37CrossRefGoogle Scholar
Nizam S, Verma S, Singh K, Aggarwal R, Srivastava KD, Verma PK (2012) High reliability transformation of the wheat pathogen Bipolaris sorokiniana using Agrobacterium tumefaciens. J Microbiol Methods 88(3):386–392CrossRefPubMedGoogle Scholar
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. Proc Natl Acad Sci 97(14):7905–7910CrossRefPubMedPubMedCentralGoogle Scholar
Poloni A, Pessi IS, Frazzon APG, Van Der Sand ST (2009) Morphology, physiology, and virulence of Bipolaris sorokiniana isolates. Curr Microbiol 59(3):267–273CrossRefPubMedGoogle Scholar
Schilling AG, Moller EM, Geiger HH (1996) Molecular differentiation and diagnosis of the cereal pathogens Fusarium culmorum and F. graminearum. Sydowia 48(1):71–82Google Scholar
Sun BJ, Lei XT, Yuan HX, Xing XP, Li HL (2007) Screening of the fungicides for the chemical control of wheat sharp eyespot (in Chinese). J Triticeae Crops 27(5):914–918Google Scholar
Sun BJ, Yuan HX, Xing XP, Li HL (2008) Control effect of different seed treatments to wheat take-all (in Chinese). Journal of Triticeae Crops 28(4):709–712Google Scholar
Tan MK, Niessen LM (2003) Analysis of rDNA ITS sequences to determine genetic relationships among, and provide a basis for simplified diagnosis of, Fusarium species causing crown rot and head blight of cereals. Mycol Res 107(7):811–821CrossRefPubMedGoogle Scholar
Wang M, Na DC, Ji HT, Zhang DY (2009) A simple and quick method of extracting genomic DNA from wheat leaves. Agric Sci Technol 10(5):34–35, 75Google Scholar
Wang M, Lv BL, Xing XP, Li HL (2011) Composition and virulence variation of the pathogen of wheat sharp eyespot from Henan Province (in Chinese). Acta Phytopathol Sin 41(5):556–560Google Scholar