Establishment and Application of a Multiple PCR Method to Detect Mating Types of Exserohilum turcicum and Bipolaris maydis
DAI YuLi1, GAN Lin1, TENG ZhenYong2, YANG JingMin3, QI YueYue4, SHI NiuNiu1, CHEN FuRu1, YANG XiuJuan,11 Institute of Plant Protection, Fujian Academy of Agricultural Sciences/Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Fuzhou 350013 2 Fujian Seed Management Station, Fuzhou 350001 3 Jianou Municipal Bureau of Agriculture and Rural Affairs, Jianou 353100, Fujian 4 Zhejiang Tianfeng Biological Science Co. Ltd, Jinhua 321000, Zhejiang
Abstract 【Objective】 Northern corn leaf blight (NCLB) and southern corn leaf blight (SCLB), caused by Exserohilum turcicum and Bipolaris maydis, respectively, are the most important foliar fungal diseases affecting maize production. The objective of this study is to establish a multiple PCR method to detect mating types of E. turcicum and B. maydis, and to provide a technical method for the study of mating type distribution in the field and sexual reproduction of E. turcicum and B. maydis. 【Method】 Mating type-specific primers for the two pathogens were designed on the basis of mating type gene sequences of E. turcicum (accession numbers: GU997138 for MAT1-1; GU997137 for MAT1-2) and B. maydis (accession numbers: X68399 for MAT1-1; X68398 for MAT1-2) obtained from GenBank, and the important parameters of primer annealing temperatures, extension times and amplification cycles in the amplification program were optimized using the single factor method. A multiple PCR method was established to detect mating types of E. turcicum and B. maydis, and the sensitivity and specificity of the multiple PCR were also assessed. Meanwhile, the mating types of 129 strains of E. turcicum and 194 strains of B. maydis from field-collections were detected by the multiple PCR to determine the adaptability of the established method.【Result】The expected 816, 132 bp (E. turcicum), and 490, 136 bp (B. maydis) target fragments were amplified specifically using the multiple PCR with mating type-specific primers of StMAT01-2F/R, StMAT02-3F/R, and ChMAT01-3F/R, ChMAT02-2F/R from MAT1-1 and MAT1-2 strains, respectively. A 25 μL PCR reaction system consisted of 12.5 μL 2×Multiplex PCR Mix, 10 pmol each primer, and 100 ng DNA template. The annealing temperatures for E. turcicum and B. maydis were 57.2℃ and 55.0℃, respectively, and the number of amplification cycles was 35. The multiple PCR method could reliably detect mating types of E. turcicum at 0.1 ng genomic DNA for MAT1-1 or 0.01 ng DNA for MAT1-2 from single-spore strains, while the sensitivity of the multiple PCR to detect mating types of B. maydis was 0.1 ng genomic DNA for both MAT1-1 and MAT1-2 from pure culture strains. The method exhibited specificity in differentiating mating types of E. turcicum and B. maydis from their closely-related species, as well as 14 other fungal genera. The results of mating types detection of E. turcicum and B. maydis strains from different geographical origins indicated that the multiple PCR could reliably detect mating types of 129 and 194 strains of E. turcicum and B. maydis, respectively. These results were consistent with the verification results of laboratorial cross assays with random selected strains from different locations.【Conclusion】The established multiple PCR method for mating types detection of E. turcicum and B. maydis in this study was characterized as high sensitivity, specificity and user-friendly control, it could accurately and rapidly detect mating types of E. turcicum and B. maydis. This study provides a reliable technique and approach for the study of distribution and monitoring of mating types in the field and sexual reproduction of E. turcicum and B. maydis. Keywords:Exserohilum turcicum; Bipolaris maydis;mating type;multiple PCR;sensitivity;specificity
PDF (3975KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文 本文引用格式 代玉立, 甘林, 滕振勇, 杨静民, 祁月月, 石妞妞, 陈福如, 杨秀娟. 玉米大斑病菌和小斑病菌交配型多重PCR 检测方法的建立与应用[J]. 中国农业科学, 2020, 53(3): 527-538 doi:10.3864/j.issn.0578-1752.2020.03.006 DAI YuLi, GAN Lin, TENG ZhenYong, YANG JingMin, QI YueYue, SHI NiuNiu, CHEN FuRu, YANG XiuJuan. Establishment and Application of a Multiple PCR Method to Detect Mating Types of Exserohilum turcicum and Bipolaris maydis[J]. Scientia Acricultura Sinica, 2020, 53(3): 527-538 doi:10.3864/j.issn.0578-1752.2020.03.006
从GenBank网站(https://www.ncbi.nlm.nih.gov/)下载玉米大斑病菌交配型MAT1-1和MAT1-2(登录号:GU997138和GU997137)以及玉米小斑病菌交配型MAT1-1和MAT1-2(登录号X68399和X68398)的基因序列[25,26]。利用DNAMAN 5.0软件进行多重序列比对,寻找各交配型基因的保守序列。通过Primer Premier 5.0软件设计交配型多重PCR检测的特异引物,利用Primer-BLAST(https://www.ncbi.nlm.nih.gov/ tools/primer-blast/)在线工具对设计的引物进行特异性检验[28]。引物委托上海英潍捷基生物技术有限公司合成。
红色和蓝色的方框及箭头分别表示玉米大斑病菌(A)和小斑病菌(B)交配型MAT1-1和MAT1-2型特异引物结合位点 Fig. 1Results of multiple sequence alignment of mating type sequences of E. turcicum and B. maydis and the mating type-specific primer binding locations
The red and blue boxes and arrows show the primer binding locations of E. turcicum (A) and B. maydis (B) mating type of MAT1-1 and MAT1-2, respectively
Table 2 表2 表2玉米大斑病菌和小斑病菌交配型多重PCR检测的特异引物 Table 2Specific primers for multiple PCR to detect mating types of E. turcicum and B. maydis
M:2 kb DNA ladder;1—7:(A)玉米大斑病菌MAT1-1和MAT1-2交配型菌株的基因组DNA的量为100、10、1、0.1、0.01、0.001和0.0001 ng Genomic DNA from MAT1-1 and MAT1-2 strains of E. turcicum at amounts of 100, 10, 1, 0.1, 0.01, 0.001 and 0.0001 ng, respectively;(B)玉米小斑病菌MAT1-1和MAT1-2交配型菌株的基因组DNA的量为10、1、0.1、0.01、0.001、0.0001和0.00001 ng Genomic DNA from MAT1-1 and MAT1-2 strains of B. maydis at amounts of 10, 1, 0.1, 0.01, 0.001, 0.0001 and 0.00001 ng, respectively;8:空白对照Reagent only without DNA template Fig. 3Sensitivity test of the multiple PCR to detect mating types of E. turcicum and B. maydis
M:2 kb DNA ladder;1—6:分别为3株MAT1-1和MAT1-2的玉米大斑病菌(A)和小斑病菌(B)菌株Three MAT1-1 and MAT1-2 strains of E. turcicum (A) and B. maydis (B), respectively;7—10:表1所示的4株玉米大斑病菌(A)和小斑病菌(B)近缘种菌株Four strains of closely related species of Exserohilum (A) and Bipolaris (B), respectively, listed in Table 1;11—24:表1所示的14株其他真菌菌株Fourteen strains of other fungal species listed in Table 1 Fig. 4Specificity of the multiple PCR to detect mating types of E. turcicum and B. maydis
Table 3 表3 表3不同地理来源的玉米大斑病菌和小斑病菌交配型的多重PCR和杂交检测 Table 3Multiple PCR and hybridization detection of mating types of E. turcicum and B. maydis strains from different geographic origins
菌株来源 Source of strains
病原菌 Pathogen
菌株数量 Strain numbers a
交配型的多重PCR和杂交检测结果 Results of mating type detection using multiple PCR and cross assay b
MAT1-1
MAT1-2
福建建瓯Jianou, Fujian
玉米大斑病菌E. turcicum
64 (10)
+ (2)
+ (8)
玉米小斑病菌B. maydis
12 (5)
+
+ (5)
福建松溪Songxi, Fujian
玉米大斑病菌E. turcicum
20 (8)
+ (1)
+ (7)
福建屏南Pingnan, Fujian
玉米大斑病菌E. turcicum
15 (5)
+
+ (5)
玉米小斑病菌B. maydis
24 (10)
+ (3)
+ (7)
福建福州Fuzhou, Fujian
玉米小斑病菌B. maydis
20 (8)
+ (2)
+ (6)
福建莆田Putian, Fujian
玉米大斑病菌E. turcicum
1
ND
+
玉米小斑病菌B. maydis
8 (5)
+
+ (5)
福建南靖Nanjing, Fujian
玉米大斑病菌E. turcicum
20 (10)
+ (1)
+ (9)
玉米小斑病菌B. maydis
11 (5)
+
+ (5)
福建龙岩Longyan, Fujian
玉米大斑病菌E. turcicum
8 (5)
+
+ (5)
玉米小斑病菌B. maydis
9 (5)
+ (1)
+ (4)
安徽霍邱Huoqiu, Anhui
玉米小斑病菌B. maydis
26 (8)
+ (1)
+ (7)
海南三亚Sanya, Hainan
玉米大斑病菌E. turcicum
1
ND
+
玉米小斑病菌B. maydis
24 (6)
+ (2)
+ (4)
浙江泰顺Taishun, Zhejiang
玉米小斑病菌B. maydis
27 (8)
+ (3)
+ (5)
广东Guangdong
玉米小斑病菌B. maydis
33 (10)
+ (4)
+ (6)
总计 Total
玉米大斑病菌E. turcicum
129 (38)
+ (4)
+ (34)
玉米小斑病菌B. maydis
194 (70)
+ (16)
+ (54)
a括号内数字表示随机抽取用于杂交实验的菌株数The number in parentheses represents the number of strains which were randomly selected for cross assays;b“+”:多重PCR方法成功检测出所有待测菌株的交配型Mating types of all tested strains were successfully detected using multiple PCR method;小括号内数字表示杂交检测出的菌株数,且与多重PCR检测结果完全一致The number in parentheses indicates the number of strains detected by cross assays, which are completely consistent with the results of multiple PCR detection;ND:未检测No detection
WEEMS JD, BRADLEY CA . Exserohilum turcicum race population distribution in the north central United States Plant Disease, 2018,102(2):292-299. [本文引用: 1]
DAI YL, GANL, RUAN HC, SHI NN, DU YX, LIAOL, WEI ZX, TENG ZY, CHEN FR, YANG XJ . Sensitivity of Cochliobolus heterostrophus to three demethylation inhibitor fungicides, propiconazole, diniconazole and prochloraz, and their efficacy against southern corn leaf blight in Fujian Province, China. European Journal of Plant Pathology, 2018,152(2):447-459. [本文引用: 1]
WANGM, WANG SQ, MAJ, YU CJ, GAO JX, CHENJ . Detection of Cochliobolus heterostrophus races in South China. Journal of Phytopathology, 2017,165(10):681-691. [本文引用: 1]
HUMAN MP, BARNESI, CRAVENM, CRAMPTON BG . Lack of population structure and mixed reproduction modes in Exserohilum turcicum from South Africa. Phytopathology, 2016,106(11):1386-1392. [本文引用: 1]
DAI YL, GANL, RUAN HC, SHI NN, DU YX, CHEN FR, YANG XJ . Genetic diversity and pathogenicity of different propiconazole-sensitive isolates of Bipolaris maydis in Fujian Province. Acta Phytopathologica Sinica, 2019,49(1):64-74. (in Chinese) [本文引用: 2]
ZHAO SW, YANG XL, GUOD . Epidemic causes of Exserochilum turcicum and its integrated management measures. China Plant Protection, 2005,25(3):10-12. (in Chinese) [本文引用: 1]
PEI JY, GAOF, YANG YW, ZHANGS, YANG XD . Preliminary quantitative studies on important epidemic links of Bipolaris maydis Ⅰ. The spores germination penetration, incubation and expand of lesion. Journal of Jilin Agricultural University, 2007,29(1):28-32. (in Chinese) [本文引用: 1]
GUO LY, JIAH, CAO ZY, GU SQ, SUN SQ, DONG JG . Analysis on mating type and parasitic fitness diversity in sexual hybridization offsprings of Setosphaeria turcica. Scientia Agricultura Sinica, 2013,46(19):4058-4065. (in Chinese) [本文引用: 4]
WANG YP, WANG XM, MAQ . Races of Exserohihun turcicum, causal agent of northern leaf blight in China. Journal of Maize Sciences, 2007,15(2):123-126. (in Chinese) [本文引用: 1]
GUO JG, YANG FZ, DUH, JIN SL, ZHANG XR . Structural composition of physiological races and mating types of Setosphaeria turcica in Gansu. Plant Protection, 2018,44(6):85-90. (in Chinese) [本文引用: 2]
CHAO C CT, ELLINGBOE AH . Selection for mating competence in Magnaporthe grisea pathogenic to rice. Canadian Journal of Botany, 1991,69(10):2130-2134. [本文引用: 1]
NOTTEGHEM JL, SILUéD . Distribution of the mating type in Magnaporthe grisea populations pathogenic on rice. Phytopathology, 1992,82(4):421-424. [本文引用: 1]
CHéRIFM, CHILVERS MI, AKAMATSUH, PEEVER TL, KAISER WJ . Cloning of the mating type locus from Ascochyta lentis (teleomorph: Didymella lentis) and development of a multiplex PCR mating assay for Ascochyta species. Current Genetics, 2006,50(3):203-215. [本文引用: 2]
SHI WQ, GONG SJ, ZENG FS, XUE MF, YANG LJ, YU DZ . Sexual reproduction and detection of mating-type of Blumeria graminis f. sp. tritici populations. Acta Phytopathologica Sinica, 2016,46(5):645-652. (in Chinese) [本文引用: 2]
BREWER MT, CADLE-DAVIDSONL, CORTESIP, SPANU PD, MILGROOM MG . Identification and structure of the mating-type locus and development of PCR-based markers for mating type in powdery mildew fungi Fungal Genetics and Biology, 2011,48(7):704-713. [本文引用: 3]
TAKAN JP, CHIPILIJ, MUTHUMEENAKSHIS, TALBOT NJ, MANYASA EO, BANDYOPADHYAYR, SEREY, NUTSUGAH SK, TALHINHASP, HOSSAINM, BROWN AE, SREENIVASAPRASADS . Magnaporthe oryzae populations adapted to finger millet and rice exhibit distinctive patterns of genetic diversity, sexuality and host interaction Molecular Biotechnology, 2012,50(2):145-158. [本文引用: 1]
DE MICCOLISANGELINI R M, ROTOLOC, POLLASTROS, FARETRAF . Molecular analysis of the mating type (MAT1) locus in strains of the heterothallic ascomycete Botrytis cinerea. Plant Pathology, 2016,65(8):1321-1332. [本文引用: 1]
FOSTER SJ, SINGHG, FITT BD L, ASHBYA M. Development of PCR based diagnostic techniques for the two mating types of Pyrenopeziza brassicae (light leaf spot) on winter oilseed rape (Brassica napus ssp. oleifera). Physiological and Molecular Plant Pathology, 1999,55(2):111-119. [本文引用: 1]
LINDE CC, ZALAM, CECCARELLIS, MCDONALD BA . Further evidence for sexual reproduction in Rhynchosporium secalis based on distribution and frequency of mating-type alleles. Fungal Genetics and Biology, 2003,40(2):115-125. [本文引用: 1]
ALLIOUIN, SIAHA, BRINISL, REIGNAULTP, HALAMAP . Mating type distribution provides evidence for sexual reproduction of Mycosphaerella graminicola in Algeria. Canadian Journal of Plant Pathology, 2014,36(4):475-481. [本文引用: 1]
PEARCE TL, SCOTT JB, HAY FS, PETHYBRIDGE SJ . Mating-type gene structure and spatial distribution of Didymella tanaceti in pyrethrum fields. Phytopathology, 2016,106(12):1521-1529. [本文引用: 1]
TURGEON BG, YODER OC . Proposed nomenclature for mating type genes of filamentous ascomycetes Fungal Genetics and Biology, 2000,31(1):1-5. [本文引用: 1]
RAMATHANII, BIRUMAM, MARTINT, DIXELIUSC, OKORIP . Disease severity, incidence and races of Setosphaeria turcica on sorghum in Uganda. European Journal of Plant Pathology, 2011,131(3):383-392. [本文引用: 3]
TURGEON BG, BOHLMANNH, CIUFFETTI LM, CHRISTIANSEN SK, YANGG , SCH?FERW , YODERO C . Cloning and analysis of the mating type genes from Cochliobolus heterostrophus. Molecular Genetics and Genomics, 1993,238(1/2):270-284. [本文引用: 3]
DAI YL, GANL, RUAN HC, SHI NN, DU YX, CHEN FR, YANG XJ . Pathogen identification of small leaf spots on sweet corn plants in Fujian Fujian Journal of Agricultural Sciences, 2017,32(12):1341-1349. (in Chinese) [本文引用: 1]
YEJ, COULOURISG, ZARETSKAYAI, CUTCUTACHEI, ROZENS, MADDEN TL . Primer-blast: A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics, 2012,13:134. [本文引用: 1]
GAO JX, GAO ZG, ZHANG XF, ZHANGS, CHENL . A simple and feasible method for single-spore isolation of Exserohilum turcicum. Microbiology China, 2010,37(10):1548-1550. (in Chinese) [本文引用: 1]
DAI YL, YANG XJ, GANL, CHEN FR, RUAN HC, DU YX, SHI NN, GAO ZM . First report of southern leaf blight caused by Cochliobolus heterostrophus on corn (Zea mays L.) in Fujian Province, China. Plant Disease, 2016,100(8):1781. [本文引用: 1]
OIDES, KRASNOFF SB, GIBSON DM, TURGEON BG . Intracellular siderophores are essential for ascomycete sexual development in heterothallic Cochliobolus heterostrophus and homothallic Gibberella zeae. Eukaryotic Cell, 2007,6(8):1339-1353. [本文引用: 1]
GLASS LN, KULDAU GA . Mating type and vegetative incompatibility in filamentous ascomycetes Annual Review of Phytopathology, 1992,30:201-224. [本文引用: 1]
BIY, HUJ, CUIX, SHAOJ, LUX, MENGQ, LIUX . Sexual reproduction increases the possibility that Phytophthora capsici will develop resistance to dimethomorph in China. Plant Pathology, 2014,63(6):1365-1373. [本文引用: 1]
JINDAL KK, TENUTA AU, WOLDEMARIAMT, ZHU XY, HOOKER DC, REID LM . Occurrence and distribution of physiological races of Exserohilum turcicum in Ontario, Canada. Plant Disease, 2019,103(7):1450-1457. [本文引用: 1]
GAFURA, TANAKAC, OUCHIS, TSUDAM . A PCR-based method for mating type determination in Cochliobolus heterostrophus. Mycoscience, 1997,38(4):455-458. [本文引用: 1]
DAI YL, GANL, RUAN HC, SHI NN, DU YX, CHEN FR, YANG XJ . A PCR method to detect mating types of Cochliobolus heterostrophus. Canadian Journal of Plant Pathology, 2018,40(3):358-367. [本文引用: 3]
LIU RC, CHENG YP, CHAI AL, SHI YX, XIE XW, PATIGULI, LIB J. Establishment and application of a triplex PCR detection system for vegetable soil-borne pathogens Scientia Agricultura Sinica, 2019,52(12):2069-2078. (in Chinese) [本文引用: 1]
LU SW, PLATZ GJ, EDWARDS MC, FRIESEN TL . Mating type locus-specific polymerase chain reaction markers for differentiation of Pyrenophora teres f. teres and P. teres f. maculata, the causal agents of barley net blotch. Phytopathology, 2010,100(12):1298-1306. [本文引用: 1]
SUN SQ, WEN LL, DONG JG . Identification of physiological races and mating type of Exserohilum turcicum. Journal of Maize Sciences, 2005,13(4):112-113, 123. (in Chinese) [本文引用: 1]
WANG LZ, KANG ZY, WU JZ, WU YX, MAO ZC, HE YQ . Identification of physiological races and analysis of mating type of Setosphaeria turcica in Yunnan Province. Journal of Huazhong Agricultural University, 2011,30(2):187-192. (in Chinese) [本文引用: 1]