Molecular Marker Study Of Metalaxyl Resistance Gene Of Phytophthora Capsici

Phytophthora capsici is an important phytopathogenic oomycete with a wide range of hosts and pepper blight is one of the most important diseases in agricultural production.At present,the production is mainly controlled by metalaxyl and its compounding agent.However,due to the single action site of metalaxyl,after long-term use,it is easy to produce resistant strains in the pathogen population in the field,and the problem of pesticide resistance is becoming more and more serious.The molecular mechanism of P.capsici resistance to metalaxyl is still unclear,which makes it difficult to control drug resistance.Therefore,it is especially important to find genes associated with metalaxyl resistance.This dissertation uses SSR and ISSR molecular markers to search for genes related to metalaxyl resistance,providing the necessary experimental basis for the localization and cloning of metalaxyl resistance genes,as well as monitoring the resistance of P.capsici to metalaxyl,and provide a theoretical basis for the resistance governance.The main results obtained are as follows:1.Screening of mutant strains of P.capsici against metalaxylTwo metalaxyl-resistant mutant strains SD1-9 and SH1-7 were obtained by mutagenesis of the susceptible strain of P.capsici tested with metalaxyl.The results showed that the growth rate of the resistant mutant strain was slower than that of the wild-sensitive strain;at the same time,it was found that the hyphae and sporangia of the resistant mutant strains and the wild-sensitive strains were not significantly different in morphology.The hyphae are white,have no septum,and occasionally have a tumor-like or nodular enlargement;they are also near-right-angled branches with contractures at the branches.The shape of the sporangia of the mutant strain and the wild strain is also irregular,and some are nearly spherical,and some are nearly oblong or inverted pear-shaped,and the mastoid is very obvious.By measuring the resistance level of the resistant mutant strain,the results showed that the resistant mutant strain SH1-7 had an EC50 value of 10.0257 ?g/mL and a resistance level of 16.0978 times;the resistant mutant strain SD1-9 had an EC50 value of 26.0629 ?g/mL,which was resistant.The level is 58.5815 times.2.Marking of metalaxyl resisrance gene of Phytophthora capsiciUsing SSR molecular markers,it was found that differential bands were amplified in the P.capsici resistant strains LN3,LN4,LN5,JX1,JX2,JX3 and JX4,and the differential bands were also found in the above seven strains by ISSR molecular markers.The differential bands in the primers UBC873 and UBC864 were recovered and sequenced,blastx found that the differential sequence 1 in NCBI had the highest similarity with the hypothetical protein PC110_g 19195 of P.cactorum,which was 64.58%;the differential sequence 2 has the highest similarity with the hypothetical protein PC110_g 17981 of P.cactorum,which is 81.02%;after the NCBI and JGI alignment,the sequence 3 found no similar genes or proteins at the nucleic acid level or protein level.When designing specific primers for the differential sequence 1,it was found that the amplified bands in LN3,LN4 and LN5 were significantly brighter than the sensitive strains.The differential sequence 1 was found by RT-PCR to identify the P.capsici resistant strains LN3,LN4 and The relevant expression level in LN5 is higher than that of sensitive strain NM1.When the differential sequence 2 was tested for specific primers,the target band was amplified in the sensitive and resistant strains,However,it was confirmed by RT-PCR that the differential sequence 2 was less expressed in the P.capsici resistant strains LN3,LN4 and LN5 than the sensitive strain NM1.When the differential sequence 3 specific primer was verified,the target band was amplified only in the resistant strains LN3,LN4,LN5,JX1,JX2,JX3 and JX4,The expression level of this sequence in the resistant strains LN3,LN4,LN5,JX1,JX2,JX3 and JX4 was indeed higher than that of the sensitive strain NM1 by RT-PCR.3.Comparison of two molecular markers of SSR and ISSR13 primers were selected from 103 pairs of SSR primers,22 strains of P.capsici were amplified.The results showed that the shannon's diversity index of the seven different regions was from large to small:Jiangxi>Shanghai>Liaoning>Jiangsu>Inner Mongolia>Anhui>Shandong Province.Twenty primers were screened from 47 ISSR primers.The amplification results showed that the Shannon's diversity index of the seven regions was from Shandong to Jiangxi>Shanghai>Liaoning>Anhui>Jiangsu>Inner Mongolia.Both molecular marker clustering results showed that there was some correlation between the tested strains and the geographical source when the threshold was 0.76;there was also some correlation between the sensitivity of P.capsici to metalaxyl and the corresponding clustering when the threshold was 0.66.Correlation analysis was performed on the genetic similarity coefficient matrix obtained from the two molecular markers of SSR and ISSR.The correlation coefficient was 0.434,indicating a weak correlation at the significant level of 0.05.