Species Identification Of The Rootlesion Nematodes In Tobacco AndConstruction Of RPA RapidDetection Technique Systemfor Three ImportantPratvlenchus Nematodes

Pratylenchus spp.also referred to as root-lesion nematodes,is widely distributed in the world.Among them,11 species,such as P.coffeae,P.scribneri and P.thornei,are recognized as important plant-parasitic nematodes.The crop root-rot nematode disease caused by this kind of nematode has become an important problem in crop production in China because of its strong colonization,adaptability,many host species and wide geographical distribution.The damage caused by Pratylenchus elegans to plant roots can lead to dwarf,stunted,chlorotic and uneven growth of plant shoots,resulting in a significant reduction in crop yield and a greater threat to agricultural safety production.Pratylenchus is often neglected due to its small size and hidden parasitic.The traditional identification of Pratylenchus is based on morphological identification and combined with molecular identification.This process requires professional technicians,sophisticated equipment and a large number of nematode samples,which is difficult to popularize.Therefore,it is very important to establish a rapid detection method for root-lesion nematodes for early diagnosis of pathogens and timely prevention and control measures.Identification of the species,and determination of the pathogenicity of different populations of the isolated rhizosphere root-lesion nematode based on the isolation of the isolated species in this study.In view of the backward status of molecular detection technology of Pratylenchus,the Basic-RPA rapid molecular detection system of P.coffeae,P.scribneri and P.thornei was constructed,and the LFD-RPA visual detection of P.coffeae was realized on the basis of Basic-RPA.The main research contents are as follows:1.Species identification and determination of pathogenicity of different populations of root-lesion nematode in tobacco: The research,the nematode population from the rhizosphere of tobacco-infected plants in Weifang,Shandong Province was identified as P.coffeae by combining morphological and molecular biology methods.Five different populations of P.coffeae were inoculated into N.benthamiana by pot method.The results showed that the Rf values of the five P.coffeae populations in the tobacco rhizosphere were all greater than 1.According to the host plant determination standard proposed,N.benthamiana was determined to be the suitable host for the five populations of P.coffeae.Combined with root weight per plant and nematode reproduction factor(Rf),the root weight per plant of N.benthamiana treated with SD-YC-1 population was 1.8 g,which was lower than that of other populations.The Rf was 4.2±0.7,which was higher than that of other populations,indicating that the pathogenicity of SD-YC-1 to N.benthamiana was stronger than that of the other four populations.2.Establishment of Basic-RPA rapid molecular detection system for three important Pratylenchus species: RDNA-ITS gene was selected as the target sequence by multiple gene sequence alignment,and RPA primers for rapid detection of three important Pratylenchus species were designed.Using these primers,RPA rapid detection systems for the detection of three Pratylenchus nematodes were established.The results of sensitivity determination showed that the minimum detection limit of Basic-RPA technology was 10-2ng/μl,which was equivalent to the sensitivity of PCR technology,but the reaction time was greatly shortened.The specificity test results showed that the specific primers of the three Pratylenchus nematodes were designed to specifically amplify the DNA of the target nematodes,and the closely related species and other pathogenic nematodes could not be detected,and only the target gene was specifically amplified in the mixed sample DNA.The system has strong sensitivity and high specificity,which provides technical support for the rapid diagnosis of plant root-lesion nematode diseases.3.LFD-RPA rapid visual detection technology of P.coffeae: Based on the screening of Basic-RPA,the specific primers were designed and the primers and probes were modified.A technical system for rapid visual detection of P.coffeae was established.The results of sensitivity determination showed that the minimum detection limit of LFD-RPA technology was 10-2ng/μl,which was equivalent to the sensitivity of PCR technology and Basic-RPA,but the reaction time was greatly shortened and the detection results could be interpreted on site.The specificity test results showed that the designed specific primers and probes specifically amplified the DNA of P.coffeae,and the similar species and other pathogenic nematodes could not be detected.This method provides a new technical means for the rapid visual detection of P.coffeae.