Research on Apple Virus in China

Detailed research on apple virus initiated in China from 1978. The species of main apple viruses have been identified and the characteristics of these viruses are discussed in this paper. These viruses include Apple mosaic virus, Apple scar skin viroid, Apple green crinkle virus, Apple chlorotic leaf spot virus, Apple stem pitting virus and Apple stem grooving virus. The main detection technologies are also described in this paper, including indicator plant, electron microscopy, serology method and molecular biological techniques.

A Study of the Distribution of Apple stem pitting virus in Tissues of Pear Tree Using In Situ Hybridization and In Situ RT-PCR

To understand the distribution of Apple stem pitting virus （ASPV） in tissues of pear tree and provide directly theorical and technical support for shoot tips detoxication, leaves and shoot tips of Korla pear were used as the materials, cDNA probe for ASPV was synthesized through RT-PCR reaction system using the unradioactive digoxigenin-labeled probe, and the specificity and sensitivity of probe were verified by blot hybridization method. Paraffin slice for in situ PCR and in situ hybridization was made and the location and distribution of ASPV RNA were detected in paraffin slices using in situ reverse transcription polymerase chain reaction, and the important factors which influenced the experiment results were optimized. ASPV mainly distributed in palisade tissue of mesophyll cells, external cortex of the tip, and the corresponding newborn vascular bundles. 20 min was the suitable digestive time for proteinase K. For the better amplication, RT reaction system should be above 0.2 U μL^-1 and 0.4 mmol L^-1 for RNasin and dNTPs respectively, 0.1-1.3 U μ L^-1 SuperScript Ⅱ, 0.6- 0.8 μmol L^-1 primer concentration, and above 0.5 U 100 μL^-1 LA Taq DNA polymerase. The suitable annealing temperature in PCR reaction was 60℃ with 35 cycles. The apical meristem of 0.25 mm was the region of virus-free.

Apple stem grooving virus is associated with leaf yellow mottle mosaic disease on Citrus grandis cv. Huangjinmiyou in China

Although it is usually latent on citrus, apple, and pear, apple stem grooving virus(ASGV) poses a great risk to many sensitive cultivars. Since severe leaf yellow mottle mosaic(LYMM) symptoms have been observed on Huangjinmiyou(HJY) pummelos(Citrus grandis cv. Huangjinmiyou), a commercial variety that is widely cultivated in South China, high throughput sequencing(HTS) was used to find potential pathogens and only three divergent ASGV variants were identified. The three ASGV variants shared 81.03–82.34% genome-wide pairwise identities with each other, and were separately closest to other ASGV variants from different hosts and/or geographical regions, as indicated by viral phylogenies. However, these new variants may have developed from viral interstrain interactions, based on the results of recombination analysis. A large-scale survey using reverse transcription-PCR(RT-PCR) protocols designed for the three ASGV variants revealed a high incidence(92.7–100%) of ASGV in symptomatic HJY trees from 11 major citrusproducing regions in China. None of ASGV were detected in asymptomatic trees. Temperature treatments applied to the symptomatic HJY plants showed that ASGV is sensitive to high temperatures(30–35°C), at which not only the plants recovered, but also the viruses were not detected by RT-PCR, while at low temperatures(20–24°C), both the symptoms and viruses remained detectable. These data show that ASGV is associated with the LYMM disease prevalent on HJY in China, and this is the significant basis especially of taking appropriate measures timely to manage the disease.

Studies on cDNA Probe Detection Technology for Apple Stem Pitting Virus in Kuala Pear

Using Kuala pear leaves and cortexes as materials, the total RNA was extracted using two methods and these two methods were compared. The most suited methods for Kuala pear were screened; and biotin-labeled cDNA probe was synthesized using RT-PCR. The main factors that affected the sensitivity of hybridization were studied. Studies indicated that the highest sensitivity was obtained under the following conditions： probe concentration 400 ng mL^-1, formamide concentration 45%, temperature 42℃, hybridization time 6 hours. The best hybridization results were obtained when the nitrocellulose membrane purchased from Gelman was used. Better blocking of hybridization was obtained using Tween 20 compared with albumin in bovine. The detection of the total RNA using different tissues and different extraction methods was compared. This study indicates that the total RNA of fresh leaf, old leaf, cortexes, and frozen leaf showed signs of hybridization using the two extraction methods.