3个苹果新品种病毒病的发生状况及ASSVd序列分析

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

【目的】近年来,苹果病毒病的发生日趋严重,对产业的健康发展造成了严重影响,但是其在苹果新品种瑞阳、瑞雪、瑞香红上的发病状况尚不明确。【方法】在2021—2022年采用RT-PCR方法对白水地区198份新品种样品进行病毒病检测,并对感染苹果锈果类病毒(apple scar skin viroid,ASSVd)植株的果实症状进行了调查和基因克隆、测序。【结果】检测结果显示白水地区新品种潜隐性病毒的感病率显著高于非潜隐性病毒,其中苹果褪绿叶斑病毒(apple chlorotic leaf spot virus,ACLSV)的检出率最高,达到了91.72%,其次是苹果茎沟病毒(apple stem grooving virus,ASGV)和苹果茎痘病毒(apple stem pitting virus,ASPV),检出率分别达到了77.16%、37.13%,苹果坏死花叶病毒(apple necrotic mosaic virus,ApNMV)的检出率为30.31%,苹果锈果类病毒和苹果凹果类病毒(apple dimple fruit viroid,ADFVd)的检出率接近10%;通过对新品种携带病毒类型分析发现,新品种瑞阳整体的检出率低于瑞雪和瑞香红,且73.17%植株病毒复合侵染种类以2~3种为主,而瑞雪、瑞香红57.55%、69.39%的植株以3~4种为主;3个新品种感染苹果锈果类病毒后,瑞阳主要表现为“花脸型”,瑞雪、瑞香红表现为“花脸型”“果面凹凸不平”2种症状;通过对3个新品种ASSVd分离物进行测序、分析,发现新品种瑞阳分离物与伊朗苹果分离物(KM213397.1)同属一个分支,新品种瑞雪、瑞香红分离物序列与韩国、加拿大苹果分离物(AF421195.1、X71599.1)同属一个分支。【结论】检测结果表明,白水地区3个新品种苹果褪绿叶斑病毒、苹果茎沟病毒感病率较高,非潜隐性病毒的感病率相对较低,且新品种瑞阳对病毒的敏感性较低;新品种瑞雪、瑞香红苹果锈果类病毒分离物属于同一分支,而它们拥有共同的亲本(克氏粉红×秦富1号),这从寄主亲缘关系远近的角度表明ASSVd侵染存在寄主特异性。

【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 cultivars 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.