(Zanthoxylum Armatum) by Virome Analysis

viruses Article Discovery of Four Novel Viruses Associated with Flower Yellowing Disease of Green Sichuan Pepper (Zanthoxylum armatum) by Virome Analysis 1,2, , 1,2, 1,2 1,2 3 3 Mengji Cao * y , Song Zhang y, Min Li , Yingjie Liu , Peng Dong , Shanrong Li , Mi Kuang 3, Ruhui Li 4 and Yan Zhou 1,2,* 1 National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China 2 Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China 3 Chongqing Agricultural Technology Extension Station, Chongqing 401121, China 4 USDA-ARS, National Germplasm Resources Laboratory, Beltsville, MD 20705, USA * Correspondences: [email protected] (M.C.); [email protected] (Y.Z.) These authors contributed equally to this work. y Received: 17 June 2019; Accepted: 28 July 2019; Published: 31 July 2019 Abstract: An emerging virus-like flower yellowing disease (FYD) of green Sichuan pepper (Zanthoxylum armatum v. novemfolius) has been recently reported. Four new RNA viruses were discovered in the FYD-affected plant by the virome analysis using high-throughput sequencing of transcriptome and small RNAs. The complete genomes were determined, and based on the sequence and phylogenetic analysis, they are considered to be new members of the genera Nepovirus (Secoviridae), Idaeovirus (unassigned), Enamovirus (Luteoviridae), and Nucleorhabdovirus (Rhabdoviridae), respectively. Therefore, the tentative names corresponding to these viruses are green Sichuan pepper-nepovirus (GSPNeV), -idaeovirus (GSPIV), -enamovirus (GSPEV), and -nucleorhabdovirus (GSPNuV). The viral population analysis showed that GSPNeV and GSPIV were dominant in the virome. The small RNA profiles of these viruses are in accordance with the typical virus-plant interaction model for Arabidopsis thaliana. Rapid and sensitive RT-PCR assays were developed for viral detection, and used to access the geographical distributions. The results revealed a correlation between GSPNeV and the FYD. The viruses pose potential threats to the normal production of green Sichuan pepper in the affected areas due to their natural transmission and wide spread in fields. Collectively, our results provide useful information regarding taxonomy, transmission and pathogenicity of the viruses as well as management of the FYD. Keywords: RNA viruses; nepovirus; idaeovirus; enamovirus; nucleorhabdovirus; high-throughput sequencing; transcriptome; small RNA; RNA silencing; RT-PCR 1. Introduction Zanthoxylum is a genus of approximately 225 species of deciduous and ever green shrubs, trees and woody climbers in the family Rutaceae [1]. It is native primarily to tropical and subtropical regions, and some of its species extend to warm temperate zones [2,3]. The genus is economically important and has been used in pharmaceutics, cosmetics, and culinary applications [2–4]. Leaves, bark, fruits and fruit oil are utilized in traditional medicines to treat asthma, common cold, gout, jaundice, diarrhea, pain and stomach upsets [4–6]. Fruits and fruit oil are also extensively used as spice in eastern Asian countries [3,6]. In China, for a long history of cultivation the production of Zanthoxylum is so far worth multi-billion dollars. In particular, green Sichuan pepper (Z. armatum v. novemfolius) is one of the most economically important commercial crops in Chongqing Province, which produces 70% Viruses 2019, 11, 696; doi:10.3390/v11080696 www.mdpi.com/journal/viruses Viruses 2019, 11, 696 2 of 18 of dry fruits of the variety nationwide. For instance, in 2018, approximately 530,000 acres of this variety were planted only in Jiangjin District, with a record high sale value of over half billion dollars. Several pathogens including fungi and phytoplasmas cause serious diseases in cultivated Zanthoxylum spp. [7–11]. However, no virus or viroid has previously been reported to be associated with this crop. Recently,2.3. HTS a virus-like Data Analysis disease, and flowerVirus Identification yellowing disease (FYD), was found on green Sichuan pepper trees in Chongqing.RNA reads Typical obt symptomsained from observed RNA- and of thesRNA FYD-seq include were analyzed internode using shortening, the software leaf curl,CLC pistil abortion,Genomics stamen Workbench yellowing 9.5 and (Qiagen intumescence,, Germany) and. After eventually adaptor trimming fruit dropping. and filtering After of an raw irreversible reads, de and degenerativenovo assembly progress of clean during reads a fewwas years,carried the out diseased using the trees algorithms decline Trinity and die, (RNA thus) or causing Velvet (sRN severeA) yield reduction[20,21 in]. The crop clean production. reads from Initial RNA symptoms-seq were ofmapped the blossoms against the usually Citrus appear sinensis only and onC. aclementina few branches of diseasedreference trees, genomes and more(downloaded and more from branches https://www.citrusgenomedb.org) show the symptoms following to remove the host disease reads progress.and Withthen propagations assembled, of while green sRNA Sichuan clean pepper reads being by seeds trimmed and grafting,from sRNA symptomatic-seq were directly trees wereassembled. randomly Assembled contigs were subjected to a BLASTx and BLASTn search distributed in the fields (Figure1g). It appears that the FYD is naturally transmitted someway from (https://blast.ncbi.nlm.nih.gov/Blast.cgi) of viral (taxid:10239) and viroidal (taxid:12884) sequences source trees to healthy trees. Unfortunately, the etiology of the disease is unknown. in GenBank Databases, respectively. FigureFigure 1. (a ,1.b ,e(a,) Greenb and Sichuane) Green pepperSichuan plant pepper infected plant infected with flower with yellowingflower yellowing disease disease (FYD) (FYD) exhibiting exhibiting yellowing and tumescent stamen, and yellowing and small leaf. (c, d and f) Normal yellowing and tumescent stamen, and yellowing and small leaf. (c,d,f) Normal stamen and leaf of stamen and leaf of healthy plant. (g) The FYD-affected tree (yellow arrow) among asymptomatic healthy plant. (g) The FYD-affected tree (yellow arrow) among asymptomatic trees (red arrows). trees (red arrows). Advances in high-throughput sequencing (HTS) have led to the development of metagenomic 2.4. Determination of Virus Genomes analyses of genomes from a variety of living forms [12]. This technology has also been used to study populationsTo determine of many organisms.the genome sequence One such of examplea novel virus, is viral specific metagenomics primers (Table or S1) the were generation designed of the based on its contig sequences obtained in this study and used in RT-PCR for amplification of virome from a single species, instead of entire microbial communities [13]. The first study of human overlapped viral fragments. One-step RT-PCR was carried out using the PrimeScript One Step virome was initiated as a systematic exploration of viruses infecting humans [14]. This systematic RT-PCR Kit (Takara, Japan). RACE (5' and 3' termini) was performed using the GeneRacer Core Kit approach(Invitrogen, for viruses USA). wasPCR thenamplicons extended were topurified animals using and the plants Gel Extraction [15,16]. TheKit (Biome studyg ofa, plantUSA) and viromes suchthen as that cloned of grapevineusing the pEASY and pepper-T1 Vector provided System (TransGen, significant China). information At least five on viralclones populations, of each PCR and viralamplicon quasispecies were fully [17,18 sequenced]. In principle, (Sanger) HTS, and sequences techniques of all such amplicons as RNA were sequencing assembled (RNA-seq)to generate and smallcomplete RNA sequencing genome sequences (sRNA-seq). yield short sequences of viral RNA or sRNA entity extracted from plants for in silico assembly to generate longer sequences (contigs) that facilitate BLAST-based or BLAST-independent2.5. HTS Data Reanal annotationysis [19]. Viruses 2019, 11, 696 3 of 18 As for this study, the HTS of both RNA- and sRNA-seq coupled with the bioinformatics analysis allowed discovery of four new RNA viruses, tentative members of the genera Nepovirus (subgroup C), Idaeovirus, Enamovirus and Nucleorhabdovirus, in diseased green Sichuan pepper plants. To understand the cause of FYD, a subsequent large-scale field survey, during which many indications pointed to the nepovirus, was performed. The prevalence of viruses and FYD as well as associated symptoms, and the fact which demonstrated that the viruses have the ability to spread through farm operation (grafting), or still unspecified natural transmissions in the open field, could not be neglected so that relevant works are of great significance. 2. Materials and Methods 2.1. Plant Materials In June 2017, leaves and inflorescences were collected from green Sichuan pepper trees with or without symptoms and stored at 80 C. One tree with foliar yellowing and dwarf as well as flower − ◦ yellowing and deformation was used for high-throughput sequencing (HTS) analysis (Figure1a,b,e). An asymptomatic tree was used as a negative control (Figure1c,d,f). 2.2. RNA Preparation and HTS To identify potential viral and viroidal pathogens, total RNA was extracted from both samples using the EASY spin Plus Complex Plant RNA Kit (Aidlab, China) for RNA sequencing (RNA-seq), and total sRNA was extracted from the samples using the EASYspin Plant microRNA Extract kit (Aidlab) for small RNA sequencing (sRNA-seq). For RNA-seq, after depletion of the ribosomal RNA, the libraries were constructed with a TruSeq RNA Sample Prep kit (Illumina, USA) and sequenced using the Illumina HiSeq

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