Rapid Molecular Detection Of Three Species Of Pratylenchus

Pratylenchus spp.known commonly as root lesion nematodes,which is an important migrate plant endoparasitic nematode and widely distributed throughout the world,causing serious damage to many crops.Root-knot nematodes,cyst nematodes and root lesion nematodes are recognized as the top three major plant-parasitic nematodes.Sugarcane and corn in South China are usually infected by Pratylenchus zeae,P.parazeae and P.brachyurus,causing serious economic losses.In addition,it was found that P.zeae and P.parazeae infected sugarcane together as mixed populations.It is of challenge for relying on traditional morphology to distinguish them.Therefore,in this study,three rapid molecular detection system,including multiplex PCR system,real-time PCR system and loop-mediated isothermal amplification(LAMP)system,were developed to identify and distinguish the three Pratylenchus species.The main results are listed as follows:1.A multiplex PCR system that can simultaneously detect P.zeae,P.parazeae and P.brachyurus was developed.The system contains the universal primers D3A/D3 B that can detect all nematodes,the specific primers 18S/Praty-R for detecting P.zeae,PpzF/PpzR for detecting P.parazeae and PbF2/Pb R1 for detecting P.brachyurus.This developed multiplex PCR system has high amplification spec ificity,and could detect not only single individual nematode of P.zeae,P.parazeae and P.brachyurus separately,also the mixture DNA extraction of these three Pratylenchus spp.2.Based on rDNA-ITS region,the specific primers q YF2/q YR2 and the probe qYP for detecting P.zeae,and the specific primers qNF/qNR and the probe qNP for detecting P.parazeae were developed.Meantime,based on mt DNA COI region,the specific primers qBF2/q BR1 and the probe q BP for detecting P.brachyurus were developed.These developed real times PCR assays could specific detect individual template of P.zeae,P.parazeae and P.brachyurus,separately.The Ct values are 25.70-29.90,25.35-25.67 and 22.19-22.81 respectively.In addition,it could sensitively detect the DNA template 100-fold diluted from one single nematode DNA extraction.3.Based on the mt DNA COI,the primer pairs for detecting P.zeae,P.parazeae and P.brachyurus were designed in order to develop the LAMP assays.The outer primers and the inner primers for detecting P.zeae were PZF3/PZB3 and PZFIP/PZBIP.The outer primers and the inner primers for detecting P.parazeae were PPF3/PPB3 and PPFIP/PPBIP.The outer primers and the inner primers for detecting P.brachyurus were PBF3/PBB3 and PBFIP/PBBIP.The developed LAMP assay could specific detect P.zeae,P.parazeae and P.brachyurus,separately.Moreover,it could sensitive ly detect the DN A template 10-fold diluted from one single nematode DNA extraction.4.In order to evaluate the application of multiplex PCR,Real-time PCR and LAMP assay as diagnostic tool for detecting P.zeae,P.parazeae and P.brachyurus in fields,76 rhizosphere soil samples from different hosts and six provinces across China were collected.The Real-time PCR systems can successfully dectect all samples;the LAMP can successfully detect all target Pratylenchus nematodes,however false positive amplification appeared in 5 samples;in addition,6 samples containg P.zeae,4 samples containg P.parazeae and one sample containg P.brachyurus were not detected in the Multiplex PCR system,however no false positive amplifications were found.The developed multiplex PCR,real-time PCR and LAMP systems can be used to rapidly dectect P.zeae,P.parazeae and P.brachyurus.Of these systems,real-time PCRs have the best effect in detecting practical samples,the detection rate is up to 100%.These rapid detection tools can be used in quarantine and agriculture departments and can be applied to diagnose the root rot disease caused by these three root lesion nematodes.