Detection and variation analysis of three types of apple latent viruses in Malus pumila ‘Saiwaihong’ in Tongliao

Abstract:【Objective】The aim of this study was to determine the incidence and genetic diversity of three types of latent apple viruses, including apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV) and apple stem grooving virus (ASGV) in Saiwaihong apple (Malus pumila) in Tongliao. 【Methods】Fresh twigs of Saiwaihong were randomly collected from 96 trees in 8 different counties in Kailu. Total RNA was extracted from the leaves of the samples using TaKaRa MiniBEST Plant RNA Extraction Kit and served as templates for cDNA synthesis using PrimeScriptⅡ 1st Strand cDNA Syn-thesis Kit. The genes of the three viruses were amplified by RT- PCR, in which the primers were designed based on the conserved parts of the gene sequences published in NCBI GenBank. The amplified fragments were purified and cloned onto pTOPO- TA cloning vectors, and E. coli JM109 competent cells were transformed. Positive clones were sequenced at Beijing Genomics Institute. The sequences were analyzed and assembled using Vector NTI Advance 11, and the nucleotide sequence identity was determined using BLASTn program at NCBI website. Phylogeny analysis was performed based on the sequences using MEGA-X software.【Results】The incidence of the viruses in Saiwaihong in Tongliao was 30.2% (29/96) for ACLSV, 12.5% (12/96) for ASPV and 22.0% (21/96) for ASGV. The triple mixed infection was 4.2% . The double mixed infection was 4.2% for ACLSV and ASPV, 13.5% for ACLSV and ASGV, and 3.1% for ASPV and ASGV. The nucleotide sequence identities among the 29 ACLSV isolates from Saiwaihong were 78.1%-99.9%, while the identities were 69.1%-93.6% with additional 15 isolates published in NCBI. ACLSV isolate Kailu7-10 (GenBank accession No. ON001713) had the highest nucleotide sequence identity of 93.6% with isolate XC-HF (MF678819), and the lowest nucleotide sequence identity of 69.1% was seen between ACLSV Kailu1-5 (ON001689) and the isolate Ta Tao 5 from American peach (EU223295). The nucleotide sequence identities of the 12 ASPV isolates from Saiwaihong were 83.6%-99.7%, and the identities were 75.4%-91.5% with additional 15 ASPV isolates published in NCBI. ASPV Kailu 7-12 (OL953382) and isolate Aurora-1 (HE963831) shared the highest identity of 91.5%. ASPV isolate Kailu7-6 (OL953380) and isolate PR1 (EU095327) shared the lowest nucleotide sequence identity of 75.4%. The nucleotide sequence identities among the 21 ASGV isolates from Saiwaihong were 96.4%-100.0%, and the identities were 89.9%-98.9% with additional 38 ASGV isolates published in NCBI. Both the ASGV isolate Kailu7-5 (OL960751) and isolate Kailu7- 10 (OL960752) had the highest identity of 98.9% with the Canadian isolate 13TF163B (MZ126539). The ASGV isolate Kailu1- 7 (OL960738) had the lowest identity of 89.9% with the isolate PG (MK427058). Phylogenetic trees were reconstructed based on the gene sequences of each virus. On the tree for ACLSV, the 44 ACLSV isolates were clustered into 7 major branches, marked as JB, MO-5, Balatonl, P863, 1, B6 and Ta Tao 5. The 15 isolates from Saiwaihong (ON001687- 89, ON001692, ON001700-04, ON001706-08, ON001713-15) were clustered into JB group together with the isolate SY03 from hawthorn (KU870525), isolate XC- HF from apple (MF678819), and pear isolates KMS (KC935954), YH (KC935955) and JB (KC935956). The isolate Kailu3-12 (ON001699) and the strain Balaton1 (X99752) were clustered together into the Balatonl group. A subclade 1 was formed by isolate Kailu5-11 (ON001705) and isolate Kailu6-9 (ON001709). The other ACLSV isolates from Saiwaihong (ON001690-691 and ON001693-712) were clustered together with isolate QD-13 (KJ522693) and isolate MS (KC847061) was clustered into group B6. The 27 ASPV isolates were clustered into 6 groups, marked as group Ⅰ-Ⅳ. The ASPV isolates from Saiwaihong (OL953372- 75, OL953379 and OL953381- 83) were clustered into the group Ⅰ together with the Canadian apple isolate Aurora- 1 (HE963831). Some isolates from Saiwaihong (OL953379 and OL953380), with the German apple isolate PM8 (KF319056), isolate Palampur (FR694186) and isolate IF38 (AB045371), were clustered into group Ⅳ. The other isolates from Saiwaihong (OL953378 and OL953377) formed a distinct subclade. The 61 ASGV isolates were clustered into 8 main clades, and the 21 isolates from Saiwaihong were clustered together and formed a distinct subclade.【Conclusion】Based on the results above, we concluded that ACLSV, ASPV and ASGV all can infect Saiwaihong, and the incidence was high in Tongliao while ACLSV had the highest incidence of above 30%. The isolates occurred in Saiwaihong showed closer intraspecific relationship to each other in comparison with isolates from other hosts ac-cording to the phylogenetic trees. This work, therefore, provided basic knowledge about the viral diseases on Saiwaihong in Tongliao, and was significant for making prevention and control policies during the sustainable and healthy development of the industry