Development and evaluation of an RNase H-dependent PCR (rhPCR) and an rh-quantitative PCR (rhqPCR) system for detection of a new potato pathogen-Ilyonectria pseudodestructans

Abstract

Ilyonectria pseudodestructans is a plant pathogen causing root rot on fruit trees such as grapevine and apple. Recently, it was reported to be a pathogen of potato. The increasing risk of this pathogen on plants 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 a RNA nucleotide at the site where a single-nucleotide polymorphism between I. pseudodestructans and strains of other Ilyonectria species 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 base pairs (bp). In the specificity test, by alignment via the Blastn tool, the RNA nucleotide bases on both the forward and reverse primers were identical to the corresponding genomic site of 16 of 17 (94.1%) database-available I. pseudodestructans strains, and different to 43 of 44 (97.7%) database-available strains of other Ilyonectria species. When the rhPCR/rhqPCR protocols were applied on 11 I. pseudodestructans strains and 46 other strains of different species of plant pathogens, all the I. pseudodestructans generated positive reactions but all 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/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. This is the first report of a probe-based rhqPCR to be applied on plant disease diagnosis; in addition, this is also the first rapid molecular protocol to detect I. pseudodestructans, and therefore, the new rhPCR/rhqPCR methods are recommended to plant disease diagnostic labs for their routine work.