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Rapid and sensitive detection of Curvularia lunata associated with maize leaf spot based on its Clg2p gene using semi-nested PCR
被引：16
|作者：
Hou, J. M.
[1
]
Ma, B. C.
[1
]
Zuo, Y. H.
[1
]
Guo, L. L.
[1
]
Gao, S. G.
[2
]
Wang, Y. Y.
[1
]
Liu, T.
[1
,3
]
机构：
[1] Heilongjiang Bayi Agr Univ, Inst Plant Pathol & Appl Microbiol, Daqing 163319, Heilongjiang, Peoples R China
[2] Shanghai Jiao Tong Univ, Sch Agr & Biol, Minist Agr, Key Lab Urban Agr South, Shanghai 200030, Peoples R China
[3] Northwest A&F Univ, State Key Lab Crop Stress Biol Arid Areas, Yangling, Shaanxi, Peoples R China
基金：
国家教育部博士点专项基金资助;
关键词：
Curvularia lunata;
detection;
diagnosis;
infected;
specific primer;
PHYTOPHTHORA-NICOTIANAE;
SOIL SAMPLES;
IDENTIFICATION;
AMPLIFICATION;
PRIMERS;
D O I：
10.1111/lam.12040
中图分类号：
Q81 [生物工程学（生物技术）];
Q93 [微生物学];
学科分类号：
071005 ;
0836 ;
090102 ;
100705 ;
摘要：
Curvularia lunata (Wakker) Boed, the causative agent of Curvularia leaf spot in maize, was determined according to conidiophore and conidium morphology in a previous study. In the current study, a sensitive polymerase chain reaction assay was developed for the detection of C.lunata. Two specific forward (ClgD1/ClgD2) and one reverse primers (ClgD3) were designed based on a Ras-related (Clg2p) gene. Eight C.lunata isolates that represent different virulent strains in maize, six other Curvulariaspp., and 22 fungal plant pathogens were used to test the specificity of the primers. PCR amplification using ClgD1/ClgD3 as the first-round primers resulted in an 870-bp band from the C.lunata isolates. The detection sensitivity using ClgD1/ClgD3 was 100pg of genomic DNA. In the second round of PCR, a 1:50 dilution of the first-round PCR products was used as a template with the ClgD2/ClgD3 primer pair, which increased the detection sensitivity to 1fg. This semi-nested PCR procedure could also be used to detect C.lunata from infected maize leaves. The proposed PCR-based assay may be used for diagnosing and monitoring maize Curvularia leaf spot. Significance and Impact of the Study The semi-nested PCR assay may provide researchers and laboratory technologists a tool to rapidly detect C. lunata, which causes maize Curvularia leaf spot, compared with histological examination.
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页码：245 / 250
页数：6
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