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Detection and differentiation of Fusarium oxysporum f. sp. lycopersici race 1 using loop‐mediated isothermal amplification with three primer sets
Author(s) -
Ayukawa Y.,
Komatsu K.,
Kashiwa T.,
Akai K.,
Yamada M.,
Teraoka T.,
Arie T.
Publication year - 2016
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.12597
Subject(s) - loop mediated isothermal amplification , primer (cosmetics) , biology , fusarium wilt , fusarium oxysporum f.sp. lycopersici , fusarium oxysporum , gene , dna , polymerase chain reaction , dna extraction , microbiology and biotechnology , genetics , chemistry , organic chemistry
Fusarium oxysporum f. sp. lycopersici ( Fol ) causes tomato wilt. Based on the difference in pathogenicity towards tomato cultivars, Fol is classified into three races. In this study, a rapid method is developed for the detection and discrimination of Fol race 1 using a loop‐mediated isothermal amplification ( LAMP ) assay with two primer sets targeting a region of the nucleotide sequence of the SIX 4 gene specific for race 1 and a primer set targeting the SIX 5 gene, conserved in all known Fol isolates. Upon LAMP reaction, amplification using all three primer sets was observed only when DNA of Fol race 1 was used as a template, and not when DNA of other Fol races or other fungal species was used. This method could detect 300 fg of Fol race 1 DNA , a 100‐fold higher sensitivity than that obtained by conventional PCR . The method can also detect DNA extracted from soil artificially infested with Fol race 1. It is now possible to detect Fol race 1 in colonies and infected tomato stems without DNA isolation. This method is a rapid and simple tool for discrimination of Fol race 1. Significance and Impact of the Study This study developed a loop‐mediated isothermal amplification ( LAMP ) assay for detection and differentiation of Fusarium oxysporum f. sp. lycopersici ( Fol ) race 1 by using three primer sets targeting for the SIX 4 and SIX 5 genes. These genes are present together only in Fol race 1. This method can detect Fol race 1 in infected tomato stems without DNA extraction, affording an efficient diagnosis of Fusarium wilt on tomatoes in the field.