Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay.

Xiefeng Yao,Jinghua Xu,Runsheng Ren,Guang Liu,Xingping Yang,Pingfang Li,Man Zhang

doi:10.3389/fmicb.2016.01372

Xiefeng Yao, Jinghua Xu + Show 5 more

Open Access

https://doi.org/10.3389/fmicb.2016.01372

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Abstract

Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL-1 of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics.

Highlights

Gummy stem blight (GSB) is a highly prevalent disease on cucurbit crops worldwide (Keinath, 2011; Li et al, 2015)
As loop-mediated amplification (LAMP) assays have a high amplification efficiency, the LAMP method developed in this study showed a high sensitivity at 0.1 fg μL−1 of D. bryoniae genomic DNA, which was 1000-fold higher than that of a conventional polymerase chain reaction (PCR) (Figure 6)
Compared with reported LAMP assays and conventional PCR, the LAMP assay reported here is more advantageous owing to its sensitivity and efficiency, and is robust enough to be used in latently infected crop testing applications in the field. These results indicate that the LAMP assay established in this study can be used for early detection of the disease as the detection limit is low, and the larger range for field use will significantly increase the efficiency of gummy stem blight (GSB) diagnosis and management

Summary

Introduction

Gummy stem blight (GSB) is a highly prevalent disease on cucurbit crops (e.g., cantaloupe, muskmelon, watermelon, and cucumber) worldwide (Keinath, 2011; Li et al, 2015). D. bryoniae can be spread by a low level of contaminated seeds (Somai et al, 2002; Sudisha et al, 2006; Keinath, 2011). Cultural practices and fungicides play an important role in GSB management (Finger et al, 2014), they require diagnosis of the pathogen during early stages of disease development in cucurbit crop production. Early diagnosis, sensitive and rapid detection of D. bryoniae is very important to limit the spread of GSB in plants being moved in greenhouses or in the field

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