Research On The Elimination Of Latent Viruses In Apple By Chemotherapy And Thermotherapy

Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus, (ASPV) were three important apple latent viruses, which were worldwide distributed in apple production areas. The three kinds of viruses unusually caused visible symptoms, but could decrease the growth and productivity of infected trees. One of most effective measures for the controlling of these virus diseases was to utilize virus-free seedlings. Thermotherapy and chemotherapy were the most widely used measures for the production of virus-free materials. In this study, the 22 kinds of apple cultivars and rootstocks were used to study the effect of three kinds of chemicals and heat treatment on their growth and virus elimination efficiency by RT-PCR method. The traditional system of virus elimination was further optimized, and the elimination efficiency was improved significantly.The main results were as followed:1 The shoot tips were dark cultured in the medium added with different concentrations of three chemicals i.e. acyclovir, moroxydine and ribavirin under room temperature for forty days, then the 2mm shoot tips were cut and subcultured. The shoot tips after forty days subculture were used to detect the viruses by RT-PCR method. The results showed that the suitable concentration of acyclovir and moroxydine were 60 mg/L and 40 mg/L respectively, which the virus elimination rate of ACLSV And ASGV were both 100% , whereas the virus elimination rate of ASGV were 63% and 62% respectively. The suitable concentration of ribavirin was 10 mg/L, which the virus elimination rate of ACLSV, ASPV and ASGV were both 100%. The virus elimination efficiency of ribavirin, acyclovir and moroxydine were 230.50%, 169.47% and 143.22% respectively.2 The elimination rate of three latent viruses treated by 10 mg/L ribavirin under dark culture, which reached 100%, by whatever heating treatment or room temperature, was higher than that under light culture. For the other difference of multiplication coefficient and differentiation rate of shoot tips, the elimination efficiency under dark culture was higher than that under light culture significantly. The elimination efficiency of heat treatment was significantly higher than that of room temperature culture, whatever under dark culture or light culture.3 The effect of 6-BA, GA3 and NAA on the virus elimination rate were studied by heat treatment under dark culture. The optimum concentration of 6-BA and GA3 were 2.0mg/L and 0.3 mg/L respectively, which virus elimination rate of ACLSV and ASGV were both 100%, and the virus elimination rate of ASGV was both 95%, and the virus elimination efficiency were 273.85% and 109.5% respectively. The optimum concentration of NAA was 0.4mg/L, which virus elimination rate was 90%, the virus elimination efficiency was reduced by 54.2% for the decreased multiplication coefficient.4 The response of different apple cultivars and rootstocks in vitro to heat treatment were different under dark culture. The results showed that the heat tolerence of different apple cultivars and rootstocks were different significantly.The heat tolerence of Orin, Jonagold, Gala, Golden Delicious, Zumi and P22 were stronger than that of Fuji, Tianhong 1, B9, M9, P59, 77-34,Tianhong 2 and Jazz, and that of SH series stocks, Hirosaki and GM256 were the worst. The apple latent virus elimination rate of different apple genotypes under same treatment was different, which of Huimin Fuji Spur, SH17, M9, Hirosaki and GM256 was the highest, followed by 77-34, P59, P22 and Tianhong1, which of Jonagold, SH40, SH38 and Deep Red Fuji was the lowest. Moreover, the difficulty level of elimination was different depending on virus species, the order from easy to difficult was ACLSV, ASPV andASGV.5 The response of 22 kinds of apple cultivars and rootstocks in vitro to 10 mg/L ribavirin treatment were studied. The results showed that the tolerence of different apple cultivars and rootstocks to antiviral agents were different significantly. The tolerence of Gala, Hirosaki, Zumi, B9, P22, P59, 77-34, GM256 and M9 was stronger than that of SH17, Tianhong1, Tianhong2, Huimin Fuji Spur, SH29, SH40, SH38, Orin and Jonagold, followed by Golden Delicious, Deep Red Fuji, SH28 and Jazz. Except for Tianhong 2, Deep Red Fuji, Jazz, SH38 and SH29, the virus elimination rate of the other apple cultivars and rootstocks both reached 100%. The results showed that the 10 mg/L ribavirin treatment was suitable for the most apple cultivars and rootstocks, which indicated its extensive applicability.6 The same series of materials were used to detect the ASGV by PAS-ELISA and RT-PCR method. The results showed that the elimination rate by PAS-ELISA was 85%, but that by RT-PCR was 25%, which indicated that the sensitivity and accuracy of RT-PCR was higher than that of PAS-ELISA.