Developing a qPCR assay for the quantification of Calonectria ilicicola in soil of soybean field
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ORIGINAL ARTICLE
Developing a qPCR assay for the quantification of Calonectria ilicicola in soil of soybean field Sunao Ochi 1,2 & Tomohisa Kuroda 3,4 Received: 23 April 2020 / Accepted: 8 September 2020 # Sociedade Brasileira de Fitopatologia 2020
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. Keywords Molecular quantification . Real-time PCR . Red crown rot . Intergenic spacer region
Introduction Calonectria ilicicola is the main causal agent of red crown rot of soybean in Japan (Akamatsu et al. 2020) and black rot of peanut in the USA (Brenneman and Butts 2003). Because C. ilicicola is soil-borne, accurate measures or estimations of the populations in naturally infested soils are critical for assessing pathogen survival as well as understanding the relationship between inoculum density and plant disease incidence under field conditions. In Japan, the damage caused by red crown rot is particularly important in soybean continuous
* Sunao Ochi [email protected] 1
Research Center for Agricultural Information Technology, NARO, 2-1-9 Kannondai, Tsukuba, Ibaraki 305-0856, Japan
2
Central Region Agricultural Research Center, NARO, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8666, Japan
3
Niigata Agricultural Research Institute, Horticultural Research Center, 177, Mano, Seirochou, Kitakanbaragun, Niigata 957-0111, Japan
4
Niigata Agricultural Research Institute, Crop Research Center, 857, Nagakuramachi, Nagaoka, Niigata 940-0826, Japan
cropping systems. Yield loss can reach 50% when disease incidence is maximal in the field (Berner et al. 1988). The mean weight of total harvested soybean seeds per plant and the mean weight per 100 soybean seeds may decrease by 10% for severely diseased plants as compared with healthy plants (Akamatsu et al. 2020). Thus far, disease epidemiology is poorly understood, especially the seasonal dynamics of the soil-borne pathogen population. Part of the reason is the lack of a simple and effective method for quantifying the inoculum of C. ilicicola in the soil and in consequence to evaluate the efficacy o
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