Study On Distribution And Elimination Of Apple Latent Virus In Seed Of Malus Robusta Rehd

Apple virus diseases are severely harmful to growth, yield and fruit quality of appletrees, and there is no effective chemical for controlling apple virus disease up to now. Theresult is once the trees are infected by the virus, it’s almost impossible to eliminate thevirus. So propagation of virus-free seedling becomes one of effective measures to controlapple virus diseases. Seeding on dwarfing interstocks and standard rootstocks have beenbecame a trend in china, which are both grafted on seedlings. Malus robusta Rehd. iswidely used as rootstock of apple, because of its well developed root, strong adaptabilityand saline tolerance. It is consistently thought that the seedling is free of virus for a longtime, but the symptoms caused by virus were observed on them. Our research goal is todetermine whether seeds of Malus robusta Rehd. were infected by apple latent virus andscreen the efficient elimination method. The multiplex RT-PCR system was used to detectapple latent virus in the seeds of Malus robusta Rehd., and evaluate the effect of fourtreatments(Hot water, NaOH, KMnO4, Na3PO4) on the virus elimination and distribution ofapple latent virus in this study. The main results are as follows:1. Fruits and seeds of Malus robusta Rehd. were used as material to evaluate twomethods for isolating total RNA. The results showed that the method based on the use ofRNAiso Plus and RNAiso-mate for Plant Tissue can obtain high-quality total RNA frombranch bark, seeds and seed embryos of Malus robusta Rehd., but could not successfullyextracted from peel and flesh. The application of total RNA plant method to the isolation ofpeel and flesh of Malus robusta Rehd.was successful. The RNA can be used to RT-PCR.2. The seed of Malus robusta Rehd. which was collected randomly, were tested bytwo-step multiplex RT-PCR. Results showed that seeds were infected by ASGV andACLSV but free of ASPV. The overall of apple latent virus infection rate was33%, but thecoinfecton rate was only4%, the infection rates of ASGV and ACLSV were28%and9%respectively.3. Fruits of Malus robusta Rehd., collected from mature trees that infected by apple latentvirus, was used as material to detect the regularity of infection of apple latent virus. Two-step multiplex RT-PCR system was used to detect apple latent virus in the seeds, seedembryos, peel and flesh of Malus robusta Rehd.. Results showed that, the infection rates ofthree kinds of apple latent virus were different significantly, in different developmentperiods of the fruit. Peel, flesh and seeds at the stage of fruit cell rapid elongation were overall infected by ASGV and ACLSV, but the infection rate of ASPV was53.33%,13.33%and88.23%respectively. Peel and flesh of mature fruits were all infected byASGV, ACLSV and ASPV, but the infection rate of ASGV, ACLSV and ASPV in matureseeds without washing, were69.23%,53.25%and42.86respectively. Seed embryos werealso infected by ASGV and ACLSV, but free of ASPV, and the infection rates were25%and8.34%respectively.4. The seeds collected from infected trees fruit were washed with clear water, and wastested for three kinds of apple latent virus. Results showed that the infection of ASGV andACLSV were64%and26%respectively. No ASPV was detected.5. Physical and Chemical treatments can reduce seed viral disease. The seed of Malusrobusta Rehd. which was collected randomly, were tested by two-step multiplex RT-PCR.The effects of four treatments including hot water and2%sodium hydroxide solution,1%potassium permanganate solution and10%trisodium phosphate solution on viruselimination were different. After seeds had been soaked for30minutes, germination testwas carried too. Results showed that the infection rates of seeds of Malus robusta Rehd.were reduced to varying degrees. The elimination rate of ASGV varied with treatments.The elimination efficiency of seeds treated by1%potassium permanganate solution and10%trisodium phosphate solution were42%, high virus elimination efficiency at82%wasachieved by the treatment with2%sodium hydroxide solution. But the treatment with hotwater had no effect on ASGV. Physical and Chemical treatments affect germination rateand germination energy of seeds, and treatment with2%sodium hydroxide solution hadstrong effect on that of seed.6. In order to get the best combinations of concentration and time, different treatmentswas applied to screen. Seeds were soaked with1%and2%sodium hydroxide soaking for10min,20min,30min,40min and60min respectively. Results showed that1%sodiumhydroxide had little effect on elimination of apple latent virus. But the eliminationefficiency of2%sodium hydroxide varied with different combinations. When seeds weresoaked for20min,30min and40min with2%sodium hydroxide, the virus eliminationefficiency were70%,64%and70%respectively. And the seed had no significantly effecton germination.Seeds collected from mature trees infected by apple latent virus were used as materialto screen better combinations. Soak seeds for20min、30min and40min with1.5%sodiumhydroxide, and30min with2%sodium hydroxide. Results showed that the eliminationefficiency of1.5%sodium hydroxide soaking seeds for40minutes was equal to2%sodium hydroxide soaking seeds for30minutes. So the optimized concentration were1.5%sodium hydroxide soaking seeds for40minutes or2%sodium hydroxide soaking seeds for20minutes.