Study On Molecular Detection Of Cryphonectria Parasitica

Chestnut blight,also known as chestnut canker,caused by Cryphonectria parasitica,is a destructive disease of branches on chestnut.In recent years,the occurrence of this disease is on the rise,has become the important limiting factors to the healthy development of the Chinese chestnut production.It has seriously affected the quality of trees and fruit production in China and has been listed in one of the forest pest and quarantine object.In this study,we took work on the molecular detection of C.parasitica,as the traditional methods of chestnut blight disease early symptoms are difficult to observe,and the problem of pathogen identification difficult for C.parasiticaIn this study,C.parasitica collected from different regions and other strains as the research material.According to the nucleotide sequence of internal transcribed spacer regions(ITS)of the ribosomal gene of Cryphonectria,a couple of primers CP1/CP2 were developed,then establish the ITS-PCR detection technology of C.parasitica.Meanwhile,by random amplification polymorphism technology,A a specific RAPD fragment of C.parasitica from all the strains tested was identified.After recycling and purification,the specific fragment was cloned and sequenced.The sequence was used to design two specific primers SC1/SC2 and converted RAPD marker to SCAR marker successfully.Through the primer combination and PCR conditions optimization,a duplex PCR method to detect to C.parasitica had been established.The main results are as follows:1,According to the sequence analysis of the ITS of C.parasitica,a pair of specific primers CPI(5 ’-CGGCCCACTAAACTCTTTGT-3 ’)/CP2(5 ’-ATTTCAGGGCCTACCGTTTT-3 ’)was synthesized by the aid of software Primer premier 6.0.The results of PCR showed that a 285 bp specific band was only amplified from C.parasitica,but no other strains.2.Orthogonal design was involved to establish the optimum RAPD system of C.parasitica and the optimal RAPD reaction system was finally obtained as:40ng DNA template,1 U Taq polymerase(5 U/μL),2.0 μL Mg2+(25 mmol/μL),2.5 μL dNTP(25 mmol/μL),1.0 μL primer(10 mmol/μL),2.5 μL10×PCR buffer were contained in 25 μL reaction solution.The PCR amplification program was pre-denaturing at 94℃ 4 min;denaturing at 94℃ 30 s,annealing at 37℃ 40 s,extension at 72℃ 70 s,for 40 cycles;at last extension at 72 ℃ 10 min.3.By using the optimized RAPD reaction system can filter out the specific amplification of C.parasitica random primer S160(5’-AACGGTGACC-3’)and specific RAPD fragment.The specific RAPD fragment of C.parasitic.was purified and inserted into pMD(?)19-T Vector that transformed into E.coli and cloned and sequenced.The results show that primer S160 generated a polymorphic pattern displaying a 389 bp DNA fragment specific for C.parasitica.4.Based on the sequence of RAPD marker,the specific SCAR primers SCI(5 ’-AACGGTGACCCTTCTGGGGC-3 ’)/SC2(5 ’-AACGGTGACCGCAAAGGACTC-3 ’)were designed by the aid of the software Primer 6.0 and the specific primer pairs amplified a single unique band of 389 bp from all C.parasitica isolates and there was no amplification from another species tested.These results clearly demonstrate that the specific fragment was successfully converted to SCAR marker by using SC1/SC2.5.A duplex PCR method,combing primers CP1/CP2 and SC1/SC2,was used to detected C.parasitica after the amplification conditions were orthogonal optimization.The results showed that a 285 bp and 389 bp band were only amplified from the strains of C.parasitica,but not in other strains and negative control.The detection limit with the duplex PCR was 300 fg/μL of DNA which could be detected in a 25 μl PCR reaction.6.The results of detection of plant showed that C.parasitica could be specifically detected by duplex PCR assay from diseased plant tissues collected from filed and artificial inoculation branches.This duplex PCR assay is a convenient and efficient tool for detection of C.parasitica and has great significance for the prevention and control of chestnut blight.