Molecular Characterisation Of Rotylenchulus Reniformis Parasitizing Garden Plants And Study On Molecular Detection Of This Nematode

Rotylenchulus reniformis is a semi-endoparasitic nematode with a wide range of host plants and is mainly distributed in tropical and subtropical regions.In recent years,with the increase of exports and imports of garden plants,the problem of nematode disease causing by R.reniformis becomes more and more prominent.In this study,molecular characterisation of R.reniformis from garden plants in China was analyzed,based on which two rapid molecular detection system,including real-time PCR system and loop-mediated isothermal amplification system,were developed to detect R.reniformis.The main results are summarized as follows:1.Ribosomal DNA?rDNA?Internal Transcribed Spacer?ITS?and D2D3 expansion segment of twenty-one R.reniformis populations from different geographical location in China were sequenced and analyzed.Similar to those reported previously,there are also two types of rDNA gene of R.reniformis in China,i.e.typeA and typeB.Of these,the length of typeA ITS sequences was 996-1104bp with 94.2%-100%similarity,and the length of typeB ITS sequences was 1143-1149bp with 93.1%-100%similarity.In addition,the length of typeA D2D3 sequences was 780-786bp with 95.3%-100%similarity and the length of typeB D2D3 sequences was 780-781bp with 95.4%-98.5%similarity.Moreover,there were also two types of rDNA sequences in those clones from the same nematode.2.The mitochondrial DNA?mtDNA?COI gene of twenty-one R.reniformis populations from different geographical location in China were sequenced and analyzed.The results showed the mtDNA-COI region was highly conserved and the sequence similarity was 99.3%-100%.3.Based on ITS,D2D3 and COI sequences,the phylogenetic relationship among R.reniformis and other closely related species was analyzed by using MrBayes.The phylogeny showed ITS and D2D3 sequences of R.reniformis both divided into two types,i.e.typeA and typeB sequence.But all sequences were positioned in one clade inferred from the mtDNA phylogenetic tree.4.Based on D2D3 sequences,we designed R.reniformis-specific primers Rr-28SF/Rr-28SR₂ and developed a real-time PCR assay for detecting R.reniformis.Twenty-one R.reniformis populations from different geographical location in China can be detected using this real-time PCR system,but other nematodes can not be detected,showing the real-time PCR system is specific to R.reniformis.In addition,the real-time PCR system can amplify the DNA template 1000-fold diluted from one single nematode DNA extraction,which was 100 times more sensitive than the conventional PCR.And the threshold cycle values?Ct? are 23.19±0.19 to 29.66±0.07.These showed that the real-time PCR system can detect R.reniformis sensitively and can be applied to detect the low number of R.reniformis in practice.5.Based on D2D3 sequences,we designed R.reniformis-specific primers,including the outer primers Rr-F3/Rr-B3 and the inner primers Rr-FIP/Rr-BIP,to develop a LAMP assay for the detection of R.reniformis.The LAMP detected R.reniformis only by using SYBRGreen I dye,digestion experiments and the sequencing of amplification products,which showed the LAMP is specific to R.reniformis.In addition,the LAMP can amplify the DNA template 100-fold diluted from one single nematode DNA extraction,which was10 times more sensitive than the conventional PCR.This study developed two rapid molecular detection systems for detecting R.reniformis,which provided rapid detection methods of R.reniformis for the import and export quarantine departments and is useful for the diagnosis of reniform nematode disease in garden plants.Meantime,this study characterized the molecular character of R.reniformis from garden plants in China,providing more important information for further understanding of the intra-specific variation of R.reniformis.