| Virus diseases have serious effect on the growth of pear plants and decrease their fruit quality and productivity. Utilizing certified healthy propagation materials is one of the most effective measures for the controlling of virus diseases. Thermotherapy and chemotherapy are the most widely used methods for the production of virus-free plant materials. Many study results have demonstrated that plant viruses could be eliminated by continuous treatment under high temperature or by using antiviral chemicals at a proper concentration. Pear plants are relatively sensitive to high temperature, which can cause a high death rate. Meanwhile, chemotherapy can induce phytotoxicity of host and usually cost long time. In this study, in order to improve the virus elimination efficiency, sand pear Pyrus pyrifolia cv. Jinshui no.2infected with Apple stem grooving virus (ASGV) and Apple chlorolic leaf spot virus (ACLSV) was used as the experimental material, and thermotherapy (35℃) combined with chemotherapy (Ribavirin) was used to eliminate the viruses from in vitro pears. Meanwhile, the population structure of ASGV in in vitro plants of pear was investigated during20sub-culturings and the full genome of an ASGV isolate from sand pear was sequenced. Moreover, the siRNAs derived from ASGV in in vitro sand pears treated by thermotherapy was characterized by high-throughput sequencing, and the titers of six siRNAs were tested during a35d thermotherapy course. Results obtained are as follows:1. In vitro sand pear P. pyrifolia cv.'Huanghua' was used as material. The RT-PCR results showed that ASGV can replicate stably in in vitro sand pear cultures during20passage sub-cultures. The RT-PCR products were cloned and single-strand conformation polymorphism (SSCP) of PCR products of more than20clones from each sub-culturing showed that ASGV persisted as a dominant molecular variant in in vitro cultures of 'Huanghua', and continues sub-culturings had no significant effect on the constitution of ASGV molecular variants.2. The complete genome of an ASGV isolate from in vitro 'Huanghua' was sequenced. The primers used in this study were designed basing on the available sequences from GenBank. The ASGV isolate named ASGV-HH contained6,496nucleotides excluding the poly (A) tail, and consisted of two open reading frames (ORFs), a214kDa polyprotein and a36kDa movement protein, respectively. The full genome of ASGV-HH had identities ranging from79.8%to87.1%with previously reported ASGV isolates ASGV-P-209from apple, CTLV-L and CTLV-Li-23from lily in Japan, ASGV-P from pear in South Korea, CTLV-K and CTLV-LCd-NA-1from citrus in Taiwan, and CTLV-ML from citrus in USA. ASGV-HH had highest identities of86.2%nt and89.9%aa in Rep region with isolate ASGV-P-209. ASGV-HH had highest identities of93.7%and87.6%with isolate CTLV-L in CP and MP, respectively. The phylogenetic tree constructed by amino acid sequences of variable region Ⅱ showed that ASGV-HH was closed to CTLV-L, and separated from isolate ASGV-P in the tree.3. In vitro cultures of P. pyrifolia cv. Jinshui no.2infected by ACLSV and ASGV, were used as materials to evaluate the effects of thermotherapy at35±0.5℃, chemotherapy with ribavirin, and combinations of chemotherapy and thermotherapy on the growth and virus elimination of in vitro pears. Results showed that the15-25μg/mL ribavirin could significantly improve the growth and proliferation of in vitro pears during5-30d treatment periods. The growth of in vitro pears treated by15-25μg/mL ribavirin was increased from2.10cm to2.57-2.61cm, and the proliferation was increased from3.27to4.09-8.82. Shoot tips of0.5-1.0mm long were excised from in vitro plants treated for30d,35d and40d, and viruses in regenerated plants were detected after one or two sub-culturing by RT-PCR. Results showed that the combination of chemotherapy and thermotherapy could enhance the efficiency of virus elimination. The virus elimination efficiency of treatments of15or20μg/mL ribavirin combined with thermotherapy at35±0.5℃for40d were53.8-76.9%for ACLSV and61.5-69.2%for ASGV, which were much higher than50.0-60.0%and41.7-50.0%of virus elimination rates achieved by treatment with15and20μg/mL ribavirin. A high virus eradication efficiency of100%was achieved by chemotherapy of ribavirin at25μg/mL combined with thermotherapy at35℃for40days, followed by culturing of0.5-1.0mm long meristem-tips. Furthermore, the treatment protocol was used for the virus elimination from other three in vitro-cultured pears,'Huanghua','Xuehua','Yuanhuang', and one in vitro-cultured apple 'Chaonan'. The virus elimination rate of100.0%for 'Yuanhuang' and 'Chaonan', and84.6%and64.8%for,'Huanghua' and 'Xuehua' were achieved, respectively.4. Small RNAs in in vitro 'Huanghua' treated by thermotherapy were detected by high-throughput sequencing, and a total of16,372,275unique reads was identified. Sequence alignment to the genome of ASGV-HH revealed3,111reads of ASGV siRNAs (named vsiRNA). The sizes of vsiRNAs ranged from18nt to25nt, and vsiRNAs of21nt and22nt account82.7%of total vsiRNAs. The detected frequency of vsiRNAs was 1-103times. There was a slight bias of vsiRNAs toward the sense strand of ASGV genome, which was55.8%. The titers of six vsiRNAs from six conserved regions of the ASGV genome during thermotherapy were analyzed by Real-Time qRT-PCR. The results showed that the titer of vsiRNAs decreased during the primary period of thermotherapy, and then had a peak in the in vitro cultures treated for25days. The titers of vsiRNAs derived from Met, Het, P-Pro and MP in in vitro pear treated by high temperature for25days was lower than that in untreated pears, whereas, the titer of vsiRNAs of CP and Rdrp in in vitro pear treated by high temperature for25days was higher than that in untreated pears for1.3and2.5times, respectively. During thermotherapy, the titer of ASGV in in vitro cultures decreased, and the band of RT-PCR products in agarose gel was very weak when the treatment was prolonged for25days. It was speculated that high temperature enhanced the RNA silencing.
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