苹果锤头类病毒(AHVd,Pelamoviloid,Avsunviroidae)是感染苹果的五种病毒之一。自确认其类病毒性质以来,除澳大利亚外,已在所有大陆上进行了鉴定(DiSerio等人。2018年;CABI和EPPO2022)。在苹果树中发现了AHVd,显示出叶子马赛克,环斑和死回(Hamdi等人。,2021)。苹果(MalusdomesticaBorkh。),其野生亲戚传统上生长在黑山。年产7767吨,216公顷,它是该国第二重要的果树(仅次于李子)(Anonymous2022)。在2020-2022年的调查中,29棵表现出病毒样症状的苹果树(如马赛克,坏死)在整个黑山取样,包括八个城市的16个地点(波德戈里察,Danilovgrad,Niksic,Mojkovac,BijeloPolje,Berane,Pljevlja和Savnik)。使用mirVanamiRNA分离试剂盒(Ambion,LifeTechnologies),并汇集成三个批量样本。每块包含9至10个样品。使用离子总RNA-Seq试剂盒v2构建sRNA的文库,并根据制造商的说明书使用XpressRNA-Seq条形码1-16试剂盒(IonTorrent)进行条形码化。在Illumina平台(NovogeneEurope)上进行小RNA文库测序,在三个文库中产生9.9、9.8和1860万个读段。使用CLCGenomicsWorkbench软件,使用“DemultiplexReads”工具将读取解复用为池。在线程序VirusDetect(Zheng等人。2017)用于病毒/类病毒检测和鉴定。除了已知感染苹果的病毒(苹果茎切槽病毒,苹果茎点蚀病毒,苹果花叶病毒),在所有三个块中都鉴定了映射到AHVd的重叠群,从而能够进行完整的AHVd基因组重建。为了验证AHVd的存在,使用AHVdPG13f/PG12r引物通过逆转录聚合酶链反应(RT-PCR)测试了所有29个苹果样品(Messmer等人。2017)。在来自本体1的三个样品(30/21、32/21和38/21)和来自本体2的两个样品(47/21和55/21)中获得AHVd扩增子,而来自本体3的所有样品潜在地由于病原体的低滴度或引物的3'末端的核苷酸错配而测试为阴性。对来自主体1的三个扩增子进行Sanger测序,并从其中两个(30/21和32/21)获得超过200nts的部分AHVd基因组(GenBankacc。不。OQ863319和OR020603)。此外,通过将Sanger序列映射到来自VirusDetect的重叠群上,在GeneiousPrime中组装了三个完整的AHVd基因组,并命名为30/21、32/21和38/21(acc。不。分别为PP133245、-46和-47)。所有三个基因组都表现出保守的锤头图案(Messmer等人。2017)。在BLASTn分析中,来自黑山的分离株30/21与来自捷克的分离株SA-36(ON564299)具有最高的nt同一性(98.8%),而32/21和38/21显示出最高的身份(95.4%和92.3%),来自加拿大的SD17_2-3(MK188691)和来自捷克的JF2(ON564298),分别。据我们所知,这是在黑山共和国首次报告的AHVd感染家蚕。AHVd阳性样品30/21和32/21来自Niksic的至少20年历史的苹果树,而38/21来自Mojkovac区的一棵40岁的树,这表明这种类病毒长期以来一直存在于这个国家的不同地区。黑山的AHVd发现应在该国的任何植物检疫法规和pome水果认证计划中予以考虑。
Apple hammerhead viroid (AHVd, Pelamoviroid, Avsunviroidae) is one of the five
viroids infecting apples. It has been identified on all continents except Australia since its viroid nature was confirmed (DiSerio et al. 2018; CABI and EPPO 2022). AHVd has been found in apple trees showing leaf mosaic, ringspot and dieback (Hamdi et al., 2021). Apple (Malus domestica Borkh.) and its wild relatives are traditionally grown in Montenegro. With an annual production of 7767 tons on 216 ha, it is the second most important fruit tree (after plum) in the country (Anonymous 2022). In a 2020-2022 survey, 29 apple trees exhibiting virus-like symptoms (e.g. mosaic, necrosis) were sampled throughout Montenegro, including 16 locations in eight municipalities (Podgorica, Danilovgrad, Niksic, Mojkovac, Bijelo Polje, Berane, Pljevlja and Savnik). Small RNAs were isolated using the mirVana miRNA Isolation Kit (Ambion, Life Technologies) and pooled into three bulk samples. Each bulk contained 9 to 10 samples. Libraries of sRNAs were constructed using the Ion Total RNA-Seq Kit v2 and barcoded using the Xpress RNA-Seq Barcode 1-16 Kit (Ion Torrent) according to the manufacturer\'s instructions. Small RNA library sequencing was performed on Illumina platform (Novogene Europe) yielding 9.9, 9.8 and 18.6 million reads in the three libraries. The CLC Genomics Workbench software was used to demultiplex the reads into pools using the \'Demultiplex Reads\' tool. The online program VirusDetect (Zheng et al. 2017) was used for virus/viroid detection and identification. Besides viruses known to infect apple (apple stem grooving virus, apple stem pitting virus, apple mosaic virus), contigs mapping to AHVd were identified in all three bulks enabling full AHVd genomes reconstruction. To verify AHVd presence, all 29 apple samples were tested by reverse transcription-polymerase chain reaction (RT-PCR) using the AHVd PG13f/PG12r primers (Messmer et al. 2017). AHVd amplicons were obtained in three samples (30/21, 32/21 and 38/21) from bulk 1 and two samples (47/21 and 55/21) from bulk 2, while all samples from bulk 3 tested negative potentially due to the low titer of the pathogen or nucleotide mismatches at the 3\' end of the primers. The three amplicons from bulk 1 were Sanger sequenced and partial AHVd genomes over 200 nts were obtained from two of them (30/21 and 32/21) (GenBank acc. nos. OQ863319 and OR020603). Furthermore, three full consensus AHVd genomes were assembled in Geneious Prime by mapping Sanger sequences onto contigs from Virus Detect and named 30/21, 32/21 and 38/21 (acc. nos. PP133245, -46, and -47, respectively). All three genomes exhibited conserved hammerhead motifs (Messmer et al. 2017). In BLASTn analysis, the isolate 30/21 from Montenegro shared the highest nt identity (98.8%) with the isolate SA-36 (ON564299) from Czechia, while 32/21 and 38/21 showed the highest identities (95.4% and 92.3%) with isolates SD17_2-3 (MK188691) from Canada and JF2 (ON564298) from Czechia, respectively. To the best of our knowledge, this is the first report of AHVd infecting Malus domestica in Montenegro. The AHVd-positive samples 30/21 and 32/21 originated from at least two-decade-old apple trees from Niksic, whilst 38/21 came from a 40-year-old tree from Mojkovac district, suggesting that this viroid has long been present in different parts of the country. The AHVd discovery in Montenegro should be considered in any phytosanitary regulations and pome fruit certification program in the country.