Analyses of virus/viroid communities in nectarine trees by next-generation sequencing and insight into viral synergisms implication in host disease symptoms

www.nature.com/scientificreports OPEN Received: 4 February 2019 Accepted: 9 August 2019 Published: xx xx xxxx Analyses of virus/viroid communities in nectarine trees by next-generation sequencing and insight into viral synergisms implication in host disease symptoms Yunxiao Xu1, Shifang Li1,2, Chengyong Na3, Lijuan Yang1 & Meiguang Lu 1 We analyzed virus and viroid communities in five individual trees of two nectarine cultivars with different disease phenotypes using next-generation sequencing technology. Different viral communities were found in different cultivars and individual trees. A total of eight viruses and one viroid in five families were identified in a single tree. To our knowledge, this is the first report showing that the most-frequently identified viral and viroid species co-infect a single individual peach tree, and is also the first report of peach virus D infecting Prunus in China. Combining analyses of genetic variation and sRNA data for co-infecting viruses/viroid in individual trees revealed for the first time that viral synergisms involving a few virus genera in the Betaflexiviridae, Closteroviridae, and Luteoviridae families play a role in determining disease symptoms. Evolutionary analysis of one of the most dominant peach pathogens, peach latent mosaic viroid (PLMVd), shows that the PLMVd sequences recovered from symptomatic and asymptomatic nectarine leaves did not all cluster together, and intra-isolate divergent sequence variants co-infected individual trees. Our study provides insight into the role that mixed viral/viroid communities infecting nectarine play in host symptom development, and will be important in further studies of epidemiological features of host-pathogen interactions. Peach is one of the most widely grown fruit crops in China, and nectarine (Prunus persica cv. nectarina) is an important cultivar of peach. Viruses and viroids can cause significant negative effects on fruit quality and yield in peaches and nectarines. Previous reports have shown that apple chlorotic leaf spot virus (ACLSV), plum pox virus (PPV), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), apple mosaic virus (ApMV), plum bark necrosis stem pitting-associated virus (PBNSPaV), peach latent mosaic viroid (PLMVd), and hop stunt viroid (HSVd) are the major pathogens that infect these trees1. Yu et al. (2013) performed a large-scale field survey of the major viruses and viroids that infect peach trees in China using RT-PCR and ELISA, and the results showed that only ACLSV, PNRSV, cherry green ring mottle virus (CGRMV), apricot pseudo-chlorotic leaf spot virus (APCLSV), PLMVd, and HSVd were detected2. However, next-generation sequencing (NGS) approaches have opened new avenues in recent years for the identification of viruses and viroids (including novel pathogens), and this technology is well suited for large-scale pathogen surveys, not only because it can increase the speed at which a wide range of known pathogens are detected, but also because it can be used to detect newly-emerging or potential pathogens for which other diagnostic tools are not yet available3–6. Several NGS-based strategies have been developed to overcome problems with traditional approaches, and these have resulted in the identification of known and novel viruses in peach. These include two novel luteoviruses, nectarine stem-pitting-associated virus 1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. 2Environment and Plant Protection Institute of Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, China. 3The Agricultural Development and Service Center of WFD, Liaoning, China. Correspondence and requests for materials should be addressed to M.L. (email: ) Scientific Reports | (2019) 9:12261 | https://doi.org/10.1038/s41598-019-48714-z 1 www.nature.com/scientificreports www.nature.com/scientificreports/ Sample Origin Cultivar Leaf and fruit symptoms T01 Greenhouse 1 Youtao 1233 Leaf bleaching, asymptomatic fruit T02 Greenhouse 1 Youtao 1233 Leaf bleaching, fruit pitting T03 Greenhouse 2 Zhongyou 4 Leaf and fruit asymptomatic T04 Greenhouse 2 Zhongyou 4 Leaves asymptomatic, fruits dimpled T05 Greenhouse 2 Zhongyou 4 Leaves with chlorotic mottling, asymptomatic fruit Table 1. Nectarine tissue samples used for NGS analyses of small RNAs. (NSPaV)5,7,8 and peach-associated luteovirus (PaLV)9; two marafiviruses, nectarine virus M (NeVM)5 and peach virus D (PeVD)10; a novel fabavirus in the family Secoviridae, peach leaf pitting-associated virus (PLPaV)11, and three very similar members of the genus Foveavirus, asian prunus virus 1 (APV1), APV2, and APV312. In addition, complex mixed infections have been found among fruit tree-infecting viruses2,5,12,13. Thus, the potential contribution of each single virus infection to the symptoms observed cannot easily be associated with a disease in the infected Prunus trees. In fact, many horticultural plants that are routinely clonally propagated are reservoirs of a large variety of viruses and viroids. The importance of the virome in mammalian biology, and the emerging concept of virome-host interactions and their relationship to host genetics was first described by Virgin (2014)14. The virome of the microbiome interactions with the host, especially in mammalian biology, has recently become a hot research topic that relies on bioinformatic tools and NGS technology14–18. However, only a limited number of studies have revealed viral communities or viromes in peach17. In this study, we used NGS technology to study the viral communities in nectarine trees with different disease phenotypes. We identified both known and novel viruses and viroids, and performed comparative analyses of the potential contribution of the pathogens to disease symptoms. Our results will extend the range and kinds of virus and viroid species that infect peach trees, and provide insight into the viral synergisms and the agents that might be associated with disease symptoms in nectarine. Results Virus and viroid accumulation and pathogen communities within individual nectarine trees. In the five nectarine tree samples, T01 and T02 were collected in greenhouse #1 from the same nectarine cultivar ‘Youtao 1233’ (10 year-old trees), while T03, T04, and T05 were collected in greenhouse #2 from nectarine cultivar ‘Zhongyou 4’ (5 year-old trees). The five samples came from trees that showed different leaf and fruit symptoms (Table 1, Fig. 1). To perform comparative analyses of the different symptoms observed in the nectarine trees, we used NGS of the sRNAs extracted from the five samples to obtain a complete survey of the virus and viroid communities infecting each tree. The Illumina reads obtained from sequencing the five cDNA libraries, which were prepared using RNA extracted from the scion parts of the grafted trees, (...truncated)