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

Abstract

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.

Deciphering core microbiota in rhizosphere soil and roots of healthy and Rhizoctonia solani-infected potato plants from various locations

“…The potato's ability to adapt to various climates has made it crucial for both economy and global food security (Xia et al, 2017;Ajayi-Oyetunde and Bradley, 2018;Iradukunda et al, 2022;Shuang et al, 2022). However, its continuous cultivation has led to an increase in black scurf disease, caused by Rhizoctonia solani, affecting potato yields worldwide (Lankau et al, 2020;Zhang X.-Y.…”

Revisión del estado actual de las investigaciones sobre Rhizoctonia solani Kühn

“…En cuanto a esto, [40] aplicó ensayos basados en pcr múltiple para la detección rápida del ag-3 pt, por lo que se diseñaron cebadores específicos y se optimizó la temperatura de alineamiento y la concentración de los cebadores para mejorar la técnica. Los ensayos demostraron que la pcr múltiple, bajo esas condiciones, resultó ser altamente sensible y específica para los genes diana, incluso a bajas concentraciones de adn.…”

“…Los ensayos demostraron que la pcr múltiple, bajo esas condiciones, resultó ser altamente sensible y específica para los genes diana, incluso a bajas concentraciones de adn. Así mismo, esta prueba detectó exitosamente a R. solani ag-3 presente en muestras de suelo y tubérculos de papa, por lo que se concluyó que la pcr múltiple puede ser utilizada para identificar a R. solani ag-3 en muestras de suelo y papa contaminadas con este patógeno [40]. La técnica de qPCR fue aplicada también por [30] para amplificar aislamientos de R. solani y R. solani ag1-ib, este último identificado como el principal ag causante de la pudrición del tallo de la lechuga en la región de Montérégie (Quebec).…”

Unveiling the Genetic Tapestry: Exploring Rhizoctonia solani AG-3 Anastomosis Groups in Potato Crops across Borders

“…Although several different R. solani AGs have been linked to potatoes, AG3-PT is the primary causal agent of black scurf and stem canker [ 8 ]. Variable pathogenicity and disease types have been linked to AG2-1, AG2-2, AG2-2IIIB, AG4, and AG5, whereby AG2-1 predominantly relates to stem canker, exhibiting diverse genetic and virulence features, while the three homogeneous groups (HGI, HGII, and HGIII) within AG4 primarily induce canker symptoms and, to a lesser extent, black scurf; additionally, AG1, AG7, AG8, and AG9 are among the other AGs occasionally documented to infect potatoes [ 7 , 9 ]. Different anastomosis groups (AGs) of Rhizoctonia solani have been associated with specific symptoms in potatoes, with AG8 primarily affecting the roots and AG7 spreading to the stalks, stolons, and tubers, whereas sporadic reports of AG1-1B, AG6, AG10, AG11, AG12, and AG13 on potatoes also exist, but the symptoms caused by many of these AGs remain unconfirmed [ 7 ].…”