不同繁育方式对樱桃幼苗主要病毒病的影响

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

【目的】调查不同繁育方式获得樱桃苗木的病毒携带情况,为今后樱桃苗木最适繁育方式的选择以及苗木无毒化栽培管理提供理论指导。【方法】采取对角线五点取样法随机采集中国樱桃实生苗、樱桃砧木扦插苗、甜樱桃实生苗的样本各30份,樱桃砧木组培苗60份,采用RT-PCR法对6种病毒进行检测。【结果】6种病毒检测结果均呈阳性,经序列分析证明,6种病毒片段与GenBank中已登录的病毒核苷酸序列有较高一致性。本实验田间随机取样的中国樱桃实生苗、樱桃砧木扦插苗、樱桃砧木组培苗、甜樱桃实生苗的带毒率分别为86.67%、70.00%、0%和76.67%。中国樱桃实生苗样本植株带毒率为86.67%,单独侵染PNRSV的比例较高,为53.33%;樱桃砧木扦插苗的样本中樱桃坏死锈斑病毒(CNRMV)、李属坏死斑病毒(PNRSV)和樱桃绿环斑驳病毒(CGRMV)检出率分别为36.67%、26.67%和20.00%,这3种病毒樱桃扦插苗田间携带率较高;甜樱桃实生苗的田间带毒率为76.67%,其中95.66%为樱桃病毒A(CVA)单独侵染;从两个不同产区所取樱桃砧木组培苗(共60份样品)未检测到樱桃常见6种病毒。【结论】中国樱桃实生苗与砧木扦插苗的田间带毒率均较高,分别为86.67%和70.00%。甜樱桃种子不可默认为无病毒携带,反而极易感染与传播樱桃病毒A(CVA),并非无毒苗最佳繁育材料。

【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 cherry 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℃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 10μL with gDnase 1μL,10×Buffer 1μL,total RNA 2μL,and Rnase-free H2O and heated at 42℃for 2 min.Then,1μL of Stop Buffer was removed and heated at 65℃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 0.2-2μg,Reverse transcriptase MIX 10μL,FQ-RT primer Mix 1μL,and H2O to make up 20μL according to the following components.The reverse transcription reaction was performed on a PCR instrument under the following conditions:either at 50℃for 15 min or at 72℃for 2 min.The cDNA obtained can be used directly for subsequent PCR and other reactions,or frozen at-20℃for later use.The first-strand cDNA synthesized by reverse transcription was used as a template for PCR amplification.The reaction program was predenatured at 94℃for 5 min;94℃for 50 s and 72℃for 50 s,totaling for 35 cycles;72℃10 min.Agarose gel electrophoresis of PCR products:After the amplification was completed,7μL 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)solution(10μg·μL^-1)for 10-15 minutes,and then observed with the gel imaging system.【Results】The detection 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 randomly 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 infection 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 virus 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 material for virus-free seedlings.