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Ilyonectria pseudodestructans, a plant pathogen that is known to cause root rot on fruit trees such as grapevine and apple, has recently been reported to also cause tuber decay in potato. The increasing risk of this pathogen on various horticultural crops makes it essential to develop a rapid and accurate detection method. In this study, an RNase H-dependent PCR (rhPCR) protocol and a modified probe-based rh-quantitative PCR (rhqPCR) protocol for I. pseudodestructans detection were developed. Both the forward and reverse primers for rhPCR and rhqPCR carry an RNA nucleotide at the site where a single-nucleotide polymorphism between I. pseudodestructans and strains of other Ilyonectria spp. is located, and the rhqPCR also contains a fluorescent-labeled target-specific probe. The primers were designed based on the sequence of the histone H3 gene and could amplify a DNA fragment of 73 bp. In the specificity test, by alignment via the BLASTn tool, the RNA nucleotide bases on both the forward and the reverse primers were identical to the corresponding genomic site of 16 of 17 (94.1%) database-available I. pseudodestructans strains, and different from 43 of 44 (97.7%) database-available strains of other Ilyonectria spp. When the rhPCR and rhqPCR protocols were applied on 11 I. pseudodestructans strains and 46 other strains of different species of plant pathogens, all of the I. pseudodestructans strains generated positive reactions whereas all of the other strains were negative, which indicated an excellent specificity of the primers. In the sensitivity test, the lowest DNA template amount for a positive reaction using the rhPCR and rhqPCR methods was 2 pg for I. pseudodestructans genomic DNA. When testing the rhqPCR method on gBlock, the lowest number of molecules for a positive reaction was six. These results indicated a high sensitivity of the protocol for I. pseudodestructans detection. To our knowledge, this is the first report of a probe-based rhqPCR to be applied to plant disease diagnosis; in addition, this is also the first rapid molecular protocol to detect I. pseudodestructans. The new rhPCR and rhqPCR methods have a potential to be applied by plant disease diagnostic labs for their routine work.

The planting of clubroot resistant (CR) canola (Brassica napus) is the most effective method to manage clubroot. Since 2013, many Plasmodiophora brassicae isolates capable of overcoming resistance have been detected, often in mixtures with avirulent isolates. To improve understanding of the effect of low concentrations of virulent isolates on host resistance, three CR canola cultivars (45H29, L135C, and L241C) were inoculated with pairs of isolates representing virulent/avirulent pathotypes (2*/2, 3*/3, and 5*/5) collected after or before the introduction of CR canola, respectively. Seven-day-old seedlings of each cultivar were incubated for 2 days in low concentrations (1 × 103 spores/ml) of the virulent isolates, followed by a second inoculation with a high concentration (1 × 107 spores/ml) of the avirulent isolates. Positive controls comprised seedlings inoculated with low concentrations of the virulent isolates followed by high concentrations of the virulent isolates (PC1) or only with high concentrations of virulent isolates (PC2). Negative controls comprised seedlings inoculated only with high concentrations of the avirulent isolates (NC1) or only with low concentrations of the virulent isolates (NC2). Clubroot severity was significantly higher in all nine experimental treatments (low virulent plus high avirulent) than in the negative control NC1 (high avirulent) but was lower in the experimental treatments than in the positive controls (PC1 and PC2). Low concentrations of virulent isolates alone (NC2) caused moderate clubroot. Disease severity correlated well with P. brassicae biomass in canola as determined by quantitative PCR analysis 28 to 35 days after inoculation. This study revealed that low concentrations of virulent isolates compromised canola resistance for infection by avirulent isolates.