Rapid detection of Colletotrichum gloeosporioides using a loop-mediated isothermal amplification assay

Abstract

Anthracnose caused by Colletotrichum gloeosporioides is an economic disease that affects soybean production worldwide. This study developed a rapid, sensitive method for the detection of C. gloeosporioides using a loopmediated isothermal amplification (LAMP) assay. By targeting a glutamine synthetase (GS) gene sequence, the GS-Cg-LAMP assay works most efficiently at 64°C and allows the detection of C. gloeosporioides DNA within 70 min based on a color change from orange to yellow-green following the addition of SYBR Green I to the LAMP reaction products. The detection limit was 1 pg μL −1 of genomic DNA per reaction. In specificity tests, the positive reaction (yellowgreen color by the naked eye) was observed only in the presence of C. gloeosporioides, and none of other Colletotrichum spp. or fungal isolates produced a color change. Moreover, the GS-Cg-LAMP assay was successfully used to diagnose anthracnose caused by C. gloeosporioides in diseased soybean samples obtained from the field and detected the pathogen in soybean seeds bought from farmers' markets. Our study provides a simple, sensitive tool for the rapid diagnosis of soybean anthracnose caused by C. gloeosporioides.

Identification and Detection of Botryosphaeria dothidea from Kiwifruit (Actinidia chinensis) in China

“…Approaches to treating and controlling this disease remain limited, and a novel approach to effectively and reliably detecting B. dothidea must be developed. A range of molecular techniques has been designed in recent years to detect plant pathogens including bacteria, viruses, oomycetes, and fungi [ 29 , 30 , 31 , 32 , 33 ]. Several strategies including PCR-based approach, restriction fragment length polymorphism (RFLP), and high-resolution melting have been used to detect isolates.…”

Soybean anthracnose caused by Colletotrichum species: Current status and future prospects

“…Using these molecular techniques, tiny amounts of host samples are sufficient for the detection of Colletotrichum in soybean seeds or other plant tissues. Several PCR‐based strategies are available for these purposes, such as multiplex PCR, loop‐mediated isothermal amplification (LAMP), real‐time or quantitative PCR (qPCR), and droplet digital PCR (ddPCR), among others, using specific primer pairs and sometimes excluding the need for DNA extraction (Ciampi‐Guillardi et al., 2020; Tian et al., 2017; Wang et al., 2017).…”

“…Despite the great potential attributed to the technique, LAMP has not been widely used for detecting Colletotrichum species associated with soybean anthracnose so far. Rapid LAMP diagnostic assays were proposed to detect C. truncatum , targeting the large subunit of RNA polymerase II ( Rpb1 ) coding gene (Tian et al., 2017), and C. gloeosporioides , whose target was a glutamine synthetase ( GS ) gene (Wang et al., 2017) in soybean samples. For C. truncatum the detection limit of the LAMP assay was 100 pg/μl of fungal DNA per reaction, a hundred times greater than the amount detected in the qPCR assay proposed by Tian et al.…”

qPCR-based detection of Colletotrichum truncatum in soybean seeds

“…The primers Colg 1/CT 2 (375 bp) were designed based on the sequence variation in the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA) of Colletotrichum gloeosporioides and C. truncatum (Chen et al 2006). However, ITS sequence is highly conserved in closely related species (Cannon et al 2012;Wang et al 2017). In multilocus analysis, C. truncatum clade occupies a sister position to the combined C. gloeosporioides and Colletotrichum boninense clade; however, in ITS, phylogenetic tree formed C. boninense clade only (Cannon et al 2012).…”