Studies On Molecular Detection Technology For Quarantine Pathogenic Fungi Of Imported Apple

Apple diseases caused by quarantine pathogenic fungi of imported apple are very serious and difficult to control,and they are likely to spread via international fruit trade.Once these quarantine pathogenic fungi invade China,the world’s largest apple producer,they will become a great threat to our fruit industry.Therefore,it is neccesary to strengthen the import quarantine efforts so as to prevent the spread of such harmful organisms.The continued development of reliable diagnostic tools for accurate and sensitive detection technology remains a priority to achieve this goal.In recent years,the causal agents(Neofabraea spp.)of bull’s-eye rot on apple have been intercepted by China quarantine department for many times.To date,there has no systematically introduction of this disease and the genus Neofabraea in China.There are many important plant pathogens in the genus Neofabraea,but due to their similar morphology and physiology,species in this genus have been revised on many occasions,which have caused confusion to some extent.With the thriving of molecular biology,a variety of molecular techniques have been applied to the detection and identification of plant pathogenic fungi.The purpose of this study is to develop rapid detection techniques for the quarantine pathogenic fungi of imported apple fruits.The main research achievements are listed as follows:(1)Screening of potential DNA barcoding markers for Neofabraea.Seven DNA barcode candidate gene sequences of β-tubulin,EF-1α,GPDH,GS,ITS,LSU and SSU in Neofabraea were investigated with the amplification efficiency,sequencing success rate and species distinguishing ability.The potential as Neofabraea DNA barcode of EF-1α regions was confirmed.Barcoding Gap analysis shows EF-1α region is even more excellent in species identify than β-tubulin.A pair of general primers(NS1/NS4)was designed for the effective amplification of EF-1α region.(2)Construction of recombinant plasmids containing EF-1α region of quarantine fungi on apple.Partial sequence of the EF-1α coding region(about 500 bp)of nine important quarantine apple pathogenic fungi(Neofabraea malicorticis,N.perennans,N.kienholzii,N.vagabunda,Monilinia fructicola,Botryosphaeria stevensii,Sphaeropsis pyriputrescens,Phialophora malorum,Venturia inaequalis)were cloned and inserted into pGEM-T vectors respectively to construct recombinant plasmid as pGE-Nm,pGE-Np,pGE-Nv,pGE-Nk,pGE-Mf,pGE-Bs,pGE-Sp,pGE-Pm and pGE-Vi,which could be used as positive standard for molecular detection of the above fungi based on EF-1α region.(3)Development of a PLP-RCA method for the rapid detection of Neofabraea species associated with bull’s-eye rot on apple.A rolling circle amplification method with padlock probes that targets on EF-1α regions was developed for rapid detection of apple bull’s eye rot pathogens(Neofabraea malicorticis,N.perennans,N.kienholzii,N.vagabunda).Based on the analysis of the EF-1α sequence alignment,a pair of general RCA primers(RCAf / RCAr)and four species-specific padlock probes(PLP-Nm,PLP-Np,PLP-Nk and PLP-Nv)were designed in this study.(4)Development of a real-time fluorescence PCR method for the rapid detection of Neofabraea species associated with bull’s-eye rot on apple.A real-time fluorescence PCR method with TaqMan probes that targets on EF-1α regions was developed for rapid detection of apple bull’s eye rot pathogens(Neofabraea malicorticis,N.perennans,N.kienholzii,N.vagabunda).A pair of general PCR primers(Neo F/Neo R)and four species-specific TaqMan probes(MAL-P,PER-P,KIE-P,VAG-P)were designed for simultaneous detection of all the above species in one test.(5)Development of a visual DNA chip for the rapid detection of quarantine fungi on apple.Based on the EF-1α region sequence of nine important quarantine pathogenic fungi(Neofabraea malicorticis,N.perennans,N.kienholzii,N.vagabunda,Monilinia fructicola,Botryosphaeria stevensii,Sphaeropsis pyriputrescens,Phialophora malorum,Venturia inaequalis)of apple fruits,a pair of universal primers(Apl F/Apl R)and nine species-specific chip detection probes(Nm-P,Np-P,Nk-P,Nv-P,Mf-P,Bs-P,Sp-P,Pm-P,Vi-P)were designed and a visual DNA chip was constructed for high-throughput detection of all the above fungi.In this study,several molecular detection technologies of important apple quarantine pathogens were constructed,giving full play the advantages of each technology,which could be useful for monitoring apple pathogenic fungi in import quarantine,preventing the invasion of dangerous organisms and favouring the rapid clearance of goods.