JIA-2019-11

2550 LI Liu et al. Journal of Integrative Agriculture 2019, 18(11): 2549–2560 pumila ‘ Virginia crab’, as well as epinasty and decline of M . domestica ‘Spy227’ (Jelkmann 1994; Brakta et al . 2015). ASPV infection in apple and pear trees occurs frequently in combination with other viruses, including Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple mosaic virus (ApMV). Mixed infection of ACLSV, ASGV andASPV causes significant decreases in the quality and quantity of fruits and even induces tree top working disease (Nemeth 1986; Brakta et al . 2013). There is no insect vector reported for ASPV (Martelli and Jelkmann 1998). ASPV is the type species of the genus Foveavirus , family Betaflexiviridae in the order Tymovirales (Adams et al . 2012). The positive-sense single-stranded RNA (+ssRNA) genome of ASPV is approximately 9 300 nucleotides (nts) in length, consists of 5 open reading frames (ORF1–5), 5´ and 3´ untranslated regions (UTRs), as well as the two short intergenic non-coding regions (IG-NCRs) located between ORF1 and ORF2 and between ORF4 and ORF5. ORF1 encodes a viral replicase polyprotein, ORF2–4 encode triple gene block proteins (TGBps) and ORF5 encodes the viral coat protein (CP) (Jelkmann 1994). Previous studies have revealed the remarkable genetic variability among the ASPV isolates (Wu et al . 2010; Liu et al . 2012; Yeon et al . 2014). Until now, complete genome sequences have been available for 17 ASPV isolates, including 12 isolates (PB66, IF38, PA66, Palampur, YL, YT, PM8, Hannover, GA4, GA2, AKS and XC) from apple grown in different countries and five isolates (PR1, HB-HN1, KL9, KL1 and YLX) from pear grown in China (Liu et al . 2012; Ma et al . 2016). Additionally, a virus identified as apple green crinkle associated virus (AGCaV) was molecularly characterized from Aurora Golden Gala apple showing severe symptoms of green crinkle disease, which was considered as a new virus or a novel strain of ASPV (James et al . 2013). More recently, an isolate AGCaV-CYD involved in a severe disease of quince ( Cydonia oblonga ) was identified and characterized in Italy (Morelli et al . 2017). The two AGCaV isolates have the same genomic structure as that of ASPV isolates, but are molecularly and biologically divergent from the type strain PA66 of ASPV and other ASPV isolates. As one of the most important temperate fruit trees, with the third largest growing area worldwide, pear has a long cultivation history. China is the largest commercial pear fruit producer in the world, and also an important origin center of pears with a long history of pear cultivation (Teng 2011). There are abundant local pear varieties with broad genetic diversities and ecotypes suitable for growing under different ecological conditions in China, but the knowledge on molecular characteristics of ASPV-infecting pear trees is very limited. The pear ‘Chili’ is an old local variety of Pyrus bretschneideri , originated at Laiyang, Chiping and Muping regions of Shandong Province (Teng 2011). Until now, there are still many old ‘Chili’ trees specially grown at these places. To understand the virus infection statues in old ‘Chili’ trees and virus molecular characteristics, we carried out a molecular detection for three well-known viruses ASGV, ASPV and ACLSV in old ‘Chili’ trees and identified an ASPV (ASPV-LYC) with novel molecular characteristics. This study provided the first complete genome sequence of an ASPV isolate from an over 300-year-old pear tree. The serological relationship of ASPV-LYC with other three ASPV isolates was comparatively evaluated. The obtained results provide important information for understanding the molecular evolution of the virus. 2. Materials and methods 2.1. Sample sources In 2013, young leaves were randomly collected from five trees of pear ( P . bretschneideri cv . Chili) grown at Laiyang area, Shandong Province, China. The trees were over 300 years old, without experiencing top-working manipulation, and did not show visible viral disease-like symptom. The leaves from the same pear tree were bulked as one sample and the presence of viruses in each sample was tested by RT-PCR. 2.2. RT-PCR detection Total RNA was extracted from leaves using silica spin column (SSC)-based protocol I (SSC-PI) (Yang et al . 2017). Reverse transcription was performed using Maloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA) and a random primer hexadeoxyribonucleotide mixture pd(N)6 (TaKaRa, Dalian, China) at 37°C for 1.5 h. PCR reaction solution in a final volume of 25 µL contained 2.5 µL of 10× PCR buffer, 2.5 µmol L –1 dNTPs, 0.5 µmol L –1 of each primer, 0.25 U of Taq DNA polymerase (TaKaRa, Dalian, China), and 2 µL of cDNA. PCR reactions were carried out in a Mastercycler (Eppendorf, Hamburg, Germany) using the following conditions: an initial denaturation step at 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 54°C for 30 s, 72°C for 1 min, and a final extension for 10 min at 72°C. The PCR products were separated by electrophoresis on 1.2% agarose gels, stained with ethidium bromide, and visualized under UV light. The primer sets ASPV247-F/ASPV247-R (5´-CAGTAT TGTGCCTTYTAYGCRAAGC-3´/5´-CCATAGAACGGAT GCGGTACATYTG-3´) (Yao et al . 2014) and 370A/370B (5´-ATGTCTGGAACCTCATGCTGCAA-3´/5´-TTGGGAT CAACTTTACTAAAAAGCATAA-3´) (Menzel et al . 2002)