Developing a qPCR assay for the quantification of Calonectria ilicicola in soil of soybean field

Abstract

Effective quantification of Calonectria ilicicola in naturally infested soils is critical for evaluating and validating control measures for managing red crown rot disease in soybean. Counting of microsclerotia has been used to quantify this pathogen, but the method is laborious and time-consuming to process large numbers of soil samples using wet sieving. To overcome these issues, we developed and tested a qPCR method to detect and quantify C. ilicicola in naturally infested soil of soybean fields. The primer pairs and probe, designed based on the intergenic spacer region of the ribosomal DNA, showed high specificity with a single melting curve peak in DNA samples from nine C. ilicicola isolates. No amplification product was detected with 2 isolates of other Calonectria species and 12 isolates of common soil-borne fungi. There was a significant positive correlation between the amount of C. ilicicola DNA and the number of C. ilicicola microsclerotia. The qPCR method is a promising tool to quantify C. ilicicola in soil.

Soybean Hypocotyls Prevent Calonectria ilicicola Invasion by Multi-Layered Defenses

“…Real-time PCR was run on a Thermal Cycler Dice TP800 system (Takara Bio Inc., Otsu, Japan) using SYBR premix Ex Taq mixture (Takara) with cycles of 95°C for 5 s, 55°C for 20 s, and 72°C for 20 s. Three technical replicates were used for each biological replicate sample. The PCR primers used were as follows: (1) primers targeting the intergenic spacer region of the C. ilicicola rDNA, CiIGSF (forward) = 5′-TCCATTGCCTCTATTTATCCTGC-3′, and CiIGSR (reverse) = 5′-GCGTAAAGATTTTCCAACCCG-3′ ( Ochi and Kuroda, 2021 ); (2) primers for soybean β-Actin gene (Gm-β-Actin; Glyma.15G050200), Gm-β-ActinF (forward) = 5′-GAGCTATGAATTGCCTGATGG-3′, and Gm-β-ActinR (reverse) = 5′-CGTTTCATGAATTCCAGTAGC-3′ ( Sugano et al, 2013 ). Relative fungal growth was expressed as C. ilicicola rDNA amplification folds relative to the host actin gene amplification ( Jiang et al, 2020 ).…”

Soybean red crown rot: Current knowledge and future challenges

“…Recently, several DNA‐based techniques, including PCR and loop‐mediated isothermal amplification (LAMP)‐based methods have been developed for the detection of multiple soilborne soybean pathogens including C . ilicicola (Jiang et al, 2020; Ochi & Kuroda, 2020; Rocha et al, 2022; Ye et al, 2020). These molecular techniques can address some deficiencies and limitations of the visual symptom‐based assay with more specific and sensitive diagnoses of the causal agents.…”

“…The application of the real‐time PCR method provides a sensitive and highly specific quantification of C . ilicicola (Jiang et al, 2020; Ochi & Kuroda, 2020). This method is much more time‐consuming and costly than the visual rating methods described above.…”