The Titer of Citrus Yellow Vein Clearing Virus is Positively Associated with the Severity of Symptoms in Infected Citrus Seedlings

Citrus yellow vein clearing virus is a new member of the genus Mandarivirus in the family Alphaflexiviridae. Citrus yellow vein clearing virus (CYVCV) is the causal agent of citrus yellow vein clearing disease and is widely distributed in Pakistan, India, Turkey, and China. CYVCV is transmitted from citrus to citrus by Dialeurodes citri, grafting, and contaminated knife blades, threatening citrus production. In this study, four infectious full-length cDNA clones of CYVCV (namely AY112, AY132, AY212, and AY221) derived from CYVCV isolate AY were obtained through yeast homologous recombination and inoculated to ‘Eureka’ lemon (Citrus limon Burm. f.) by Agrobacterium-mediated vacuum infiltration. Pathogenicity analysis indicated that the clones AY212 and AY221 caused more severe symptoms than AY112 and AY132. Northern blot and quantitative reverse transcription PCR (qRT-PCR) analyses showed that the titers of virulent clones (AY212 and AY221) were significantly higher than those of attenuated clones (AY112 and AY132) in the infected ‘Eureka’ lemon (Citrus limon Burm. f.) seedlings. Subsequent comparative studies of viral infectivity, accumulation, and symptoms induced by AY221 in nine citrus cultivars indicated that (i) the infectivity of AY221 varied from 25% to 100% among different cultivars; (ii) ‘Oota’ ponkan (C. reticulata L.) showed the lowest infection rate with mild symptoms, which might be a useful resource for CYVCY-resistance genes; (iii) CYVCV titer was positively associated with the symptom development in infected citrus seedlings. In general, this report revealed the biological properties of CYVCV, thus laying a foundation for further investigation of pathogenic mechanisms in this virus.

A Simulation of the Use of High Throughput Sequencing as Pre-Screening Assay to Enhance the Surveillance of Citrus Viruses and Viroids in the EPPO Region

Citrus are affected by many viruses and viroids, some globally widespread and some restricted to particular countries or areas. In this study, we simulated the use of high throughput sequencing (HTS) and the bioinformatic analysis of small interfering RNAs (siRNA) as a pre-screening method to guide bioindexing and molecular detection to enhance the surveillance survey of some key or emerging citrus viruses, such as non-European citrus tristeza virus isolates (non-EU CTV), citrus tatter leaf virus, citrus leprosis virus, citrus yellow mosaic virus, and citrus bark cracking viroid, present in the EPPO lists, and the citrus yellow vein clearing virus. The HTS’s ability to detect other citrus viroids was also evaluated. The results demonstrate that HTS provides a comprehensive phytosanitary status of citrus samples either in single and multiple infections of viruses and viroids. It also provides effective information on citrus tristeza virus mixed infections despite not being able to identify the non-EU variants of the virus. Bioindexing checks each single virus infection but does not differentiate viroids on the Etrog citron indicator and is time-consuming. Molecular assays are valuable as confirmation tests of viruses and viroids but many pairs of primers are needed for a full screening and new or non-target pathogens remain undetected . In addition, the genomes of two isolates of the citrus yellow vein clearing virus and the citrus tatter leaf virus, detected in a sample from China, are described.

First Report of Apricot vein clearing-associated virus Infecting flowering apricot (Prunus mume) in the United States

Apricot vein clearing-associated virus is the type species of genus Prunevirus, family Betaflexiviridae. The virus was first discovered from an Italian apricot tree (Prunus armeniaca) showing leaf vein clearing and mottling symptoms (Elbeaino et al. 2014). Since then, apricot vein clearing-associated virus (AVCaV) has been reported in symptomatic and asymptomatic plants from other countries (Marais et al. 2015; Kinoti et al. 2017; Kubaa et al. 2014). In 2018, a domestic selection of a flowering apricot (P. mume cv. Peggy Clarke) (PC01) with no discernible foliar virus-like symptoms was received for inclusion in the Foundation Plant Services (UC-Davis) collection. The plant originated from a private Prunus collection located in California. Total nucleic acids (TNA) were isolated from PC01 leaves using MagMax Plant RNA Isolation Kit (Thermo Fisher Scientific). The TNA were analyzed for a panel of 15 Prunus-infecting viruses by reverse-transcription quantitative PCR (RT-qPCR) (Diaz-Lara et al. 2020). In addition, to screen for sap-transmissible viruses, young leaves of PC01 were homogenized in inoculation buffer and were rubbed onto leaves of herbaceous indicator plants, Chenopodium amaranticolor, C. quinoa, Cucumis sativus, and Nicotiana clevelandii (Rowhani et al. 2005). The source PC01 tested negative for the 15 screened viruses. Interestingly, vein clearing symptoms were observed on leaves of C. quinoa and C. amaranticolor plants (Figure S1). These results suggested the presence of a mechanically transmissible virus in PC01. To determine the identity of mechanically transmissible viral agent, symptomatic C. quinoa and PC01 plant were advanced for high throughput sequencing analysis. Aliquots of TNA from PC01 and C. quinoa were rRNA-depleted and used for cDNA library preparation with TruSeq Stranded Total RNA kit (Illumina). The raw reads were trimmed, de novo assembled, and subsequently were annotated using tBLASTx algorithm (Al Rwahnih et al. 2018). A total of 47,261,138 and 8,812,296 single-end reads were obtained from cDNA libraries of PC01 and C. quinoa, respectively. The de novo assembly generated near-complete contigs resembling AVCaV genome ) from both PC01 and C. quinoa, which were 99.8% identical at the nucleotide level. The longest contig (8,342 nucleotides, 73.5x coverage depth) obtained from PC01 was further completed using SMARTer RACE 5’/3’ kit (Takara Bio). The complete genome sequence of AVCaV-PC01 is 8,364 nucleotides long (GenBank: MK170158). The full-length virus genome was compared with GenBank database using BLASTn, which the best hit corresponded to KY132099 with 98% identity. Additionally, AVCaV infection was confirmed in both PC01 selection and the symptomatic C. quinoa by RT-PCR as previously described (Marais et al. 2015). Lastly, symptomatic leaves of C. quinoa were used in leaf dip method to visualize virus particles by transmission electron microscope. As a result, flexuous rod-shaped virions were observed from leaf dips of symptomatic C. quinoa plants (Figure S2). Therefore, our results represent the first report of AVCaV in California, USA. Furthermore, mechanical transmission of an AVCaV isolate infecting flowering apricot to herbaceous hosts was confirmed. Field surveys and biological studies are underway to determine the prevalence of AVCaV in commercial orchards and assess its effect on tree performance.

Grapevine vein clearing virus is prevalent and genetically variable in grape aphid (Aphis illinoisensis Shimer) populations

Grapevine vein clearing virus (GVCV) causes severe stunting and death of cultivated grapevines and is prevalent in native Vitis spp. and Ampelopsis cordata in the Midwest region of the USA. GVCV can be transmitted from wild A. cordata to Vitis spp. by grape aphid (Aphis illinoisensis) under greenhouse conditions, but its prevalence, genetic composition and genome number in native grape aphids are unknown. In this study, we collected grape aphids from native Vitaceae across the state of Missouri in 2018 and 2019, and conducted diagnostic, genetic and quantitative analyses. GVCV was detected in 91 (87%) of the 105 randomly sampled communities on 71 Vitaceae plants. It was present in 211 (40%) of 525 single grape aphids. Diverse GVCV variants from aphids were present on both GVCV-negative and -positive plants. Identical GVCV variants were found in grape aphids sampled from wild and cultivated Vitaceae, indicating that viruliferous aphids likely migrate and disperse GVCV variants among wild and cultivated Vitaceae. In addition, we found that the number of GVCV genomes varies largely in the stylet and body of individual aphids. Our study provides a snapshot of GVCV epidemics and genetic structure in its mobile vector and sessile hosts. This presents a good model for studying epidemiology, ecology and evolution of a plant virus.