Detection of Apple chlorotic leaf spot virus in hawthorn and comparison of virus elimination methods

Abstract:【Objective】Virus disease in fruit tree was first reported in 1923 by American scholars. More than 600 kinds of virus diseases have been found in fruit trees around the world by 2003. Apple chlorotic leaf spot virus (ACLSV), as one of the important latent recessive virus in fruit trees, was widely distributed in a variety of plants, including apple, pear, peach, plum and cherry. In 2015, ACLSV was found in hawthorn(Crataegus pinnatifida) in China. The virus can cause a decline in hawthorn trees and the quality of fruits, and even bring devastating disasters in fruit yield. Propagation of the virus-free hawthorn plants by tissue culture is the main method to prevent and cure virus disease, while, it demands a rapid, accurate and specific virus detection method. In this study, we established an optimized RT-PCR detection system for detection of ACLSV in hawthorn plants. At the same time, we studied the technology for ACLSV elimination from hawthorn with tissue culture.【Methods】Young plant leaves and fruits of the accessions of Crataegus spp. in this study were collected from the National Hawthorn Germplasm Repository in Shenyang Agricultural University. Total RNA of hawthorn was extracted from leaves or young fruits by magnetic bead RNA extraction machine produced by Thermo Fisher Scientific. The sample was 100 mg, and the amount of buffer was 30 μL. Agarose gel electrophoresis was applied to analyze RNA quality and integri-ty, and Nano Drop 2000 ultraviolet spectrophotometer was used to test the concentration and purity of RNA. Primers were designed based on the genomic sequence of ACLSV hawthorn isolates. Reverse transcription reactions were performed at 37 ℃ for 30 min, then 85 ℃ for 5 s with Prime Script®RT reagent kit(Ta Ka Ra, Dalian) according to the manufacturer's instructions. PCR reactions were carried out in 20 μL total volumes with reaction mixtures that contained 1 μL c DNA, 1.6 μL of each d NTP(2.5 mmol·L^-1), 2.0μL 10× PCR buffer, 1.0 μL Mg Cl2(25 mmol·L^-1), 0.5 μL of each primer(10 μmol·L^-1), 1 U Taq DNA polymerase(Promega, USA) and dd H2 O to yield a 20 μL final volume. After the RT-PCR reaction, agarose gel electrophoresis was used to test the reaction product. PCR products were gel-extracted and ligated into a p MD18-T vector(Ta Ka Ra, Dalian). Positive clones for each product were sequenced at Beijing Genomics Institute, China. Nucleotide and amino acid identities were compared using the DNAMAN software package(Version 5.2.2.0). In addition, we used‘Qiuhong'and‘Xinbinruanzi'hawthorn as materials to establish the tissue culture plantlets of hawthorn. The annual branch shoots as explants, the best way of sterilization was 70% alcohol(30 s), 0.1% Hg Cl2(8 min), sterile water rinsing over 5 times. The initiation culture medium is SC+GA3(1.0 mg·L^-1)+6-BA(1.0 mg·L^-1)+IAA(0.5 mg·L^-1). The suitable subculture medium is SC+ GA3(2.0 mg·L^-1) + 6-BA(1.0 mg·L-1)+IAA(0.5 mg·L^-1). The proliferation medium is SC + GA3(2.5 mg·L^-1) + ZT(2.0 mg·L^-1) + IAA(0.5 mg·L^-1). Then, thermotherapy and chemistry method were used to eliminate ACLSV from tissue culture plantlets of hawthorn and the efficiencies of virus elimination were compared between thermotherapy and chemistry method. The method of virus elimination by thermotherapy was as below: the plantlets were cultured at temperature of 31 ℃, rising 1 ℃ every day until 37 ℃ for 30 d with illumination intensity 5 000 lx and illumination times 16 h·d^-1. The virus elimination by chemistry method was conducted by culturing the plantlets in proliferation medium supplemented with 20 mg·L^-1 ribavirin for 40 d with illumination intensity 3 000 lx and illumination times 16 h·d^-1.【Results】Based on the transcriptome sequencing data using Illumina technology for the young fruits of hawthorn, the complete genomic sequence of ACLSV isolate SY01(Gen Bank accession No. KM207212)was assembled. According to SY01 genome sequence, forward primer F1 and inverse primer R1 were designed to amplify a 210 bp fragment. With this pair of primer, we could detect ACLSV from some varieties of hawthorn, but sometimes the bands were weaker. Later, the genomic sequence of ACLSV hawthorn isolate SY02(Gen Bank accession No. KU870524) was assembled with transcriptome data of hawthorn leaves. Sequence analysis showed that two hawthorn isolates shared an overall nucleotide identity of92.1%. Based on the nucleotide sequences of SY01 and SY02, three pairs of virus detection primers(F1/R1, F2/R2, and F3/R3) of ACLSV were designed. With these primers, the virus' s specific fragments of the expected size(380 bp, 267 bp, 408 bp) were amplified, respectively. By comparison, the optimal primer pair F3/R3 was selected. The PCR product amplified from two varieties of hawthorn using F3/R3 were gel-extracted, ligated into a p MD18-T vector, and sequenced with the Sanger method. The data of sequencing showed that the fragments were 267 bp in length, and the sequences showed more than 90 % nucleotide identity with the ACLSV sequences in NCBI. The RT-PCR detection system for ACLSV was optimized by comparing the c DNA concentration, the kinds of Taq polymerase and the kinds of organs. An optimized PCR system for detection ACLSV in hawthorns was as below: 1 μL of undiluted c DNA was used for a 20 μL PCR volume; Taq DNA polymerase of Promega was screened out; and young leaves were used as the material. Eighty hawthorn varieties were analyzed, and ACLSV was found in 18 varieties. ACLSV was eliminated from hawthorn plantlets by both thermotherapy and chemistry treatment with ribavirin. The rate of virus-free plantlets was 100% after thermotherapy or the treatment of thermotherapy combinedwith chemotherapy, while the rates of virus-free plantlets rate were 88.9% for‘Xinbinruanzi'and 57.1%for‘Qiuhong'after chemotherapy.【Conclusion】We screened out the fittest primer and established a optimized RT-PCR system for detection ACLSV in hawthorns. Thermotherapy was the most effective method to eliminate ACLSV form hawthorn tissue culture plantlets.