Establishment and Application of a Multiplex PCR Assay for the Rapid Detection of Rhizoctonia solani Anastomosis Group (AG)-3PT, the Pathogen Causing Potato Black Scurf and Stem Canker.

Rhizoctonia solani anastomosis group 3 (AG-3) is the main causative agent of the soil-borne disease known as potato black scurf, which poses a huge threat to potato production. Rapid and accurate identification of R. solani AG-3 isolates in soil and potato seed tubers prior to planting is essential for good production. In this study, a multiplex PCR assay was established for the detection of R. solani AG-3. Two pairs of target-specific primers were designed from sequences for endopolygalacturonase and pyridoxine biosynthesis genes downloaded from GenBank. The main factors influencing PCR amplification, such as annealing temperature and primer concentration, were optimized. Results show that the proposed multiplex PCR assay is highly sensitive and specific for the target genes in the pathogen even when the DNA concentration is reduced to 20 fg/µL. The resulting calibration plot shows a linear relationship between electrophoretic band peaks and genomic DNA concentration (R2 = 0.98). The primer specificity was confirmed by applying them to other R. solani AG groups and plant pathogen species on which no amplicons were produced. Using the primers, we successfully detected small amounts of R. solani AG-3 present in soil and potato tuber samples. Taken together, the detection assay developed in this study has high sensitivity, strong specificity, and accuracy and can be used to detect and identify soil and potato seed tubers infected with Rhizoctonia solani AG-3.

A gold nanoparticle-based lateral flow biosensor for sensitive visual detection of the potato late blight pathogen, Phytophthora infestans.

Phytophthora infestans, the causal agent of late blight in potatoes and tomatoes, is the most important and ongoing pathogenic threat to agricultural production worldwide. Rapid and early identification of P. infestans is an essential prerequisite for countering the further spread of infection. In this study, a novel method for visual detection of P. infestans has been developed by integrating universal primer mediated asymmetric PCR with gold nanoparticle (AuNP)-based lateral flow biosensor. We employed asymmetric PCR to generate large amounts of single-stranded DNA (ssDNA) by amplifying a region of P. infestans-specific repetitive DNA sequence. The ssDNA products were then applied to the lateral flow biosensor to perform a visual detection using sandwich-type hybridization assays. In the presence of target DNA, sandwich-type hybridization reactions among the AuNP-probe, target DNA and capture probe were performed on the test line of the biosensor, and then a characteristic red band was produced for the accumulation of AuNPs. Quantitative analysis obtained by recording the optical intensity of the red band demonstrated that this biosensor could detect as little as 0.1 pg µL-1 genomic DNA. Furthermore, the specificity of the biosensor was confirmed by detecting three other Phytophthora species and two pathogenic fungi. We believe this method has potential application in early prediction of potato late blight disease and instigation of management actions to reduce the risk of epidemic development.