Establishment Of Main Virus Detection And Seedling Detoxification System Of Passion Fruit In Guizhou

Passion fruit（Passiflora caerulea L.）is an important tropical fruit in southern China.In recent years,the planting area of passion fruit has been increasing,however,the decline of fruit quality and yield caused by virus disease restricts the healthy development of the passion fruit industry.So it is urgent to establish a rapid virus detection method and an effective seedling virus-free system.In this paper,the passion fruit collected from Zhenning County,Zhenfeng County,Luodian County and Guanling County of Guizhou Province were used as materials,to proceed virus identification,RT-PCR detection,and virus-free system,and finally to establish a simple and easy culture system for virus-free shoot tip of passion fruit.The results can provide a certain theoretical basis for the production of passion fruit virus-free seedlings.The main results are as follows:1.The the virus species of passion fruit leaves suspected of virus infection in four main production areas were identified by high-throughput sequencing technology and bioinformatics analysis.Among them,EAPV（East Asian Passiflora virus）is the highest,accounting for 63.65%of all virus sequences;followed by PLV（Passiflora latent virus）,accounting for 34.72%;finally,Te MV（Telosma mosaic virus）,accounting for 1.59%.By sequencing and analyzing,the nucleotide sequence identity of Te MV,EAPV and PLV Guizhou isolates were 87.6%～99.7%,85.2%～99.8%and 93.0%～95.4%consistent with the reported ones.2.Virus detection of 105 samples was carried out by RT-PCR method and a multiplex RT-PCR reaction system was established.The detection rate of Te MV was100%in the four major producing areas;the average detection rate of EAPV and PLV in Zhenning County,Zhenfeng County and Luodian County was 80%;the detection rate of PLV in Guanling County was as low as 20%,while EAPV was not detected.The optimized multiplex RT-PCR reaction system was as follow:the upstream and downstream primers（10μmol/L）was 0.5μL,and the optimal annealing temperature was 50℃.3.The disinfection method and culture medium formula were optimized in detoxification culture system.The best disinfection method was 75%ethanol disinfection for 30 s,followed by 0.1%Hg Cl₂solution disinfection for 15 min,which reduced the pollution was as low as 9.0%,and the survival rate was up to 85%.The best induction medium formula was MS+1.0 mg/L 6-BA+0.1 mg/L NAA+30 g/L sucrose+7 g/L agar powder,p H=5.9,and the induction rate was up to 89%.The best proliferation medium formula was MS+1.5 mg/L 6-BA+0.1 mg/L IBA+30g/L sucrose+7 g/L agar powder,p H=5.9,with a proliferation coefficient up to 9.4.The best rooting medium formula was 1/2 MS+0.1 mg/L IBA+30 g/L sucrose+7g/L agar powder,p H=5.9,and the roots grew robustly,and the induction rate reached90%.4.The detoxification effect was evaluated on the detoxification tissue culture seedlings of passion fruit shoot tips.The RT-PCR method was used to detect the virus in 9 tissue culture seedlings,which could remove 8 strains of EAPV and 4 tissue culture seedlings of Te MV,but failed to remove PLV.