Abstract
The common smut of corn, caused by Ustilago maydis is a troublesome disease of maize. Early and accurate detection of U. maydis is essential for the disease management. In this study, primer set Pep-2 was selected for LAMP (loop-mediated isothermal amplification) from 12 sets of primers targeting three U. maydis effector genes See1, Pit2 and Pep1 according to primer screening. The optimal concentrations of Bst DNA polymerase and Mg2+ as well as inner/outer primer ratio of the LAMP reaction system were screened by combining a single factor experiment and an orthogonal design arrangement. The specificity of this real-time LAMP (RealAmp) assay was confirmed by negative testing for other pathogens. The detection sensitivity of the RealAmp assay was 200 times higher than that of detection through conventional PCR. Results of the RealAmp assay for quantifying the genomic DNA of U. maydis were confirmed by testing with both artificially and naturally infected samples. In addition, the RealAmp reaction could be conducted via an improved tube scanner to implement a “electricity free” assay from template preparation to quantitative detection. The resulting assay could be more convenient for use in the field as a simple, rapid, and effective technique for monitoring U. maydis.
Highlights
Ustilago maydis is the causative agent of common smut of corn, which is considered to be one of the major prevalent diseases of maize plants in China
In order to screen for an optimal target gene for use in the LAMP assay, a total of 12 sets of LAMP primers were designed that targeted three U. maydis effector genes, which included UmPep[1], UmPit[2] and UmSee[1]
For the LAMP primer sets Pep-1, Pep-3, Pit-2, Pit-3, See-2, See-3 and See-4, pseudo-positive amplification was observed, indicating that they are not specific to U. maydis DNA (Fig. 1)
Summary
Ustilago maydis is the causative agent of common smut of corn, which is considered to be one of the major prevalent diseases of maize plants in China. A Taq-man real-time PCR-based assay was developed for the early detection and identification of S. scitamineum using primers specific to the bE gene and a Taqman probe[11] These methods may be specific for detecting U. maydis and other pathogenic fungi which share a similar life cycle or induce symptoms similar to smut disease. Two research groups successfully developed LAMP (loop-mediated isothermal amplification)-based assays for the detection of S. scitamineum in which internal transcribed spacer sequences and the genomic sequence of the pep[1] gene were selected as targets for LAMP primer design, respectively[12,13] These two LAMP protocols provided a specific, sensitive, and rapid test for the detection of S. scitamineum infection in sugarcane. Real-time fluorescence LAMP (RealAmp) is currently employed for quantitative assays of soil-borne pathogens such as Fusarium oxysporum f. sp. cubense and Pythium inflatum[26,27]
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