Effects of different propagating patterns on the virus transmission of cherry seedlings

Abstract:【Objective】At present, the cherry seedling market is disorderly, and the situation for virus carrying and spread through cherry seedlings propagated by different methods is unclear. This study investigated the virus carrying status of cherry seedlings by investigating different propagating methods, so as to provide theoretical guidance for the selection of the most suitable propagating methods for cher- ry seedlings, the virus-free cultivation and management of seedlings in the future.【Methods】Diagonal five-point sampling method was used to randomly collect Chinese cherry seedlings, including cherry rootstock cuttings, 30 samples of sweet cherry seedlings, 60 cherry rootstock tissue culture seedlings, and six types of viruses were tested by RT-PCR. One year old shoot with the same thickness was taken at the same location from each plant, and the phloem was placed into a liquid nitrogen tank for freezing, and stored in a -80 °C refrigerator. Total RNA was extracted from the samples, and six types of major cherry viruses were detected by RT-PCR. Total RNA was extracted with a total RNA extraction and purification kit (Bytec), and RNA integrity was detected by 1% agarose gel electrophoresis. The reverse  transcription kit (Bectec) was used to synthesize cDNA. First, the digestion reaction solution was configured on an ice bath, making up 10 μL with gDnase 1 μL, 10 × Buffer 1 μL, total RNA 2 μL, and Rnase-free H2O and heated at 42 °C for 2 min. Then, 1 μL of Stop Buffer was removed and heated at 65 °C for 2 min on the PCR instrument or water bath for reverse transcription. The reverse transcription reaction solution was prepared as follows: Total RNA or poly (A) RNA 0.2-2 μg, Reverse transcriptase MIX 10 μL, FQ-RT primer Mix 1 μL, and H2O to make up 20 μL according to the following components. The reverse transcription reaction was performed on a PCR instrument under the following conditions: either at 50 °C for 15 min or at 72 °C for 2 min. The cDNA obtained can be used directly for sub- sequent PCR and other reactions, or frozen at -20 °C for later use. The first-strand cDNA synthesized by reverse transcription was used as a template for PCR amplification. The reaction program was pre- denatured at 94 °C for 5 min; 94 °C for 50 s and 72 °C for 50 s, totaling for 35 cycles; 72 °C 10 min. Agarose gel electrophoresis of PCR products: After the amplification was completed, 7 μL of the PCR amplification product was mixed with the loading buffer and electrophoresed on a 1.0%-1.5% agarose gel at a voltage of 150-200 V for 20-30 minutes. The sample was stained in ethidium bromide (EB) solu- tion (10 μg·μL-1) for 10-15 minutes, and then observed with the gel imaging system.【Results】The de- tection results of the six viruses were all positive. Sequence analysis proved that six virus fragments had high identity with the nucleotide sequences of viruses registered in GenBank. The cherry seedlings, cherry rootstock cuttings, cherry rootstock tissue culture seedlings, and sweet cherry seedlings random- ly sampled in this experiment had 86.67%, 70.00%, 0% and 76.67% virus-carrying rates. The Chinese cherry seedlings had 86.67% virus infection rate, and the proportion of PNRSV infection alone was 53.33%. The compound infection rate of PNRSV + CGRMV was the highest (6.67%). There were 2 to 3 types of virus infection and the proportions were 6.67% and 3.33%, respectively. The combined infec- tion rates of PNRSV + ACLSV, PNRSV + LChV2, PNRSV + CGRMV + CNRMV were 3.33%; cherry necrotic rust spot virus (CNRMV) and plum necrosis were found in the samples of cuttings of cherry rootstocks. The detection rates of spotted virus (PNRSV) and cherry green ring mottle virus (CGRMV) were 36.67%, 26.67%, and 20.00%, respectively. These three viruses showed higher rates in the field of cherry cuttings. The highest percentage of compound infection was 6.67%, and there were two to three types of compound infection, the proportions were 13.34% and 6.66%, respectively. The compound infection rate of PNRSV + CNRMV + LChV2, PNRSV + CGRMV + CNRMV was 3.33%. The field vi- rus infection rate of sweet cherry seedlings was 76.67%, 95.66% of which were infected by cherry virus A (CVA) alone, and only the combined infection of two viruses, CNRMV + A (CVA), was 3.33%; A total of 60 samples of cherry rootstock tissue culture seedlings from different production areas were not detected with 6 common viruses in cherry.【Conclusion】The field virus-carrying rates of Chinese cherry seedlings and rootstock cuttings were higher, reaching 86.67% and 70.00%, respectively. Chinese cherry seedlings Prunus necrotic spot virus (PNRSV) had a high infection rate and serious damage, and had a very high virus-carrying rate in Chinese cherry seedlings. Cutting propagation methods are more likely to spread and accumulate Cherry green ring mottle virus (CGRMV) and Cherry necrotic rust spot virus (CNRMV). Sweet cherry seeds cannot be recognized as virus-free by default, but were very susceptible to infection and transmission of Cherry virus A (CVA), which was not the best propagating ma- terial for virus-free seedlings.