Incidence of virus diseases and ASSVd sequence analysis in three new apple cultivars

Abstract: 【Objective】The incidence of apple virus disease is a key factor limiting the healthy development of apple industry, and it is mainly spread by grafting. In recent years, with the rapid development of the apple industry, dwarf cultivation and high-jointing techniques have been rapidly promoted, which further aggravates the danger of virus diseases. In this study, we investigated the incidence of apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), apple necrotic mosaic virus (ApNMV), apple scar skin viroid (ASSVd), and apple dimple fruit viroid (AFDVd) on the new apple cultivars Ruiyang, Ruixue and Ruixianghong in Baishui county, clarified the current status of virus diseases on the new cultivars, cloned the sequence of ASSVd gene on the new cultivars and clarified their variant types, so as to provide a basis for the sustainable and healthy development of the new apple cultivars.【Methods】In this study, 198 leaf samples of new culti-vars were collected from eight orchards in Baishui county during 2021 to 2022 using a five-point sampling method, carried back to the laboratory at 4 ℃, and tested for ASGV, ASPV, ACLSV, ApNMV, ASSVd and AFDVd by RT-PCR. Samples infected with ASSVd from three new cultivars were cloned and sequenced for ASSVd sequences, and the sequencing results were analyzed using Blast, MEGA7.0, and DNAMAN software.【Results】The main types of viruses infecting the new species in Baishui county were latent viruses, of which ACLSV had the highest detection rate of 91.72%, followed by ASGV and ASPV, with detection rates being 77.16% and 37.13%, respectively. The detection rates of non-latent viruses were relatively low, with ApNMV being 30.31%, and ASSVd and ADFVd being 9.75% and 4.87% , respectively. Among the three new cultivars, the overall detection rate of Ruiyang was lower than that of Ruixue and Ruixianghong, and 73.17% of the plants were infected with 2-3 virus complexes, and 57.55% of the plants of Ruixue and 69.49% of the plants of Ruixianghong were infected with 3- 4 virus complexes. In addition, only 5.97% of the plants of the three new cultivars were infected with a combination of five viruses, and no plants were detected with a combination of six viruses, but the number of plants that did not carry viruses was also relatively small, only accounting for 11.47%. Although the detection rate of ASSVd was low, its damage was serious. The disease-susceptible fruits of all three new cultivars showed“color dappling”, and Ruixue and Ruixianghong also showed“uneven and rough fruit surface”. The internal and external quality of healthy fruit,“color dappling”and“uneven and rough fruit surface”was determined. The average weight per fruit of healthy fruit was 361.83 g, with a longitudinal diameter of 878.13 mm and a transverse diameter of 881.43 mm, while the average weight per fruit of“color dappling”and“uneven and rough fruit surface”was 217.5 g and 234.11 g, longitudinal diameter of 720.06 mm and 729.57 mm, and transverse diameter of 768.39 mm and 776.52 mm, respectively, which were smaller than healthy fruit, resulting in a lower yield. Meanwhile, although there was no significant difference in soluble solids content between the fruits of these two symptoms, the titratable acid content decreased, the solid-acid ratio increased, and the fruit firmness increased to 8.77 kg · cm^-2 and 8.89 kg· cm^-2 , and the firmness of healthy fruits was only 7.87 kg· cm^-2 , which seriously affected the intrinsic quality of the fruits. The ASSVd sequences of three new cultivars infected with ASSVd were cloned and analyzed, and the sequence lengths of ASSVd obtained from Ruiyang, Ruixue and Ruixianghong were 332 nt, 329 nt and 329 nt, respectively and the sequence comparison analysis revealed that the homology with MN598204.1 and MN598215.1 was high, both reaching more than 99%, and the Ruixue and Ruixianghong isolates were similar to MN598204.1 with 99.4% and 98.4% similarity, respectively, which belonged to the same branch with Korean and Canadian apple isolates (AF421195.1 and X71599.1), and Ruiyang isolate with MN598215.1 had higher similarity of 99.4%, and belonged to the same branch with Iranian apple isolate (KM213397.1).【Conclusion】The detection rate of latent viruses of new cultivars in Baishui county was the highest, followed by ACLSV, ASGV and ASPV, and the detection rate of non-latent viruses was relatively low, followed by ApNMV, ASSVd and ADFVd. For the sake of the overall detection rate and the compound infestation status of plants in the three new cultivars, Ruiyang was lower than Ruixue and Ruixianghong, which were less sensitive to viruses, while Ruixue and Ruixianghong were more sensitive. After infection with ASSVd, the internal and external quality of the fruit was greatly affected. ASSVd isolates obtained from susceptible samples of the new cultivars were compared and analyzed to further indicate the existence of host specificity of ASSVd infestation from the perspective of host relatedness.