Identification of a New Badnavirus in the Chinaberry (Melia azedarach) Tree and Establishment of a LAMP-LFD Assay for Its Rapid and Visual Detection.

The Chinaberry tree, a member of the Meliaceae family, is cultivated in China for use in traditional medicines. In 2020, Chinaberry trees with leaf deformation symptoms were found in Hangzhou, Zhejiang province, China. In order to identify possible pathogenic viruses, a symptomatic sample was subjected to deep sequencing of small interfering RNAs. Assembly of the resulting sequences led to the identification of a novel badnavirus, provisionally designated Chinaberry tree badnavirus 1 (ChTBV1). With the recent development of China's seedling industry and increasing online shopping platforms, the risk of tree virus transmission has increased substantially. Therefore, it is important to detect the occurrence of ChTBV1 to ensure the safety of the Chinaberry tree seedling industry. Here, we describe the development and validation of a sensitive and robust method relying on a loop-mediated isothermal amplification (LAMP) assay, targeting a 197 nt region, to detect ChTBV1 from Chinaberry tree leaves. The LAMP assay was also adapted for rapid visualization of results by a lateral flow dipstick chromatographic detection method.

Identification and characterization of a novel virus associated with an eriophyid mite in extracts of fruit trees leaves.

Eriophyid mites are commonly found on the leaf surface of different plant species. In the present study, a novel virus associated with an eriophyid mite species was detected using high-throughput sequencing (HTS) of total RNA from fruit tree leaves, primarily growing under greenhouse conditions. The complete genome sequence was characterized using rapid amplification of cDNA ends followed by Sanger sequencing, revealing a genome of 8885 nucleotides in length. The single positive-stranded RNA genome was predicted to encode typical conserved domains of members of the genus Iflavirus in the family Iflaviridae. Phylogenetic analysis showed this virus to be closely related to the unclassified iflavirus tomato matilda associated virus (TMaV), with a maximum amino acid sequence identity of 59% in the RNA-dependent RNA polymerase domain. This low identity value justifies the recognition of the novel virus as a potential novel iflavirus. In addition to a lack of graft-transmissibility evidence, RT-PCR and HTS detection of this virus in the putative host plants were not consistent through different years and growing seasons, raising the possibility that rather than a plant virus, this was a virus infecting an organism associated with fruit tree leaves. Identification of Tetra pinnatifidae HTS-derived contigs in all fruit tree samples carrying the novel virus suggested this mite as the most likely host of the new virus (p-value < 1e-11), which is tentatively named "eriophyid mite-associated virus" (EMaV). This study highlights the importance of a careful biological study before assigning a new virus to a particular plant host when using metagenomics data.

Zika virus in southeastern Senegal: survival of the vectors and the virus during the dry season.

BACKGROUND: Zika virus (ZIKV, genus Flavivirus, family Flaviviridae) is transmitted mainly by Aedes mosquitoes. This virus has become an emerging concern of global public health with recent epidemics associated to neurological complications in the pacific and America. ZIKV is the most frequently amplified arbovirus in southeastern Senegal. However, this virus and its adult vectors are undetectable during the dry season. The aim of this study was to investigate how ZIKV and its vectors are maintained locally during the dry season. METHODS: Soil, sand, and detritus contained in 1339 potential breeding sites (tree holes, rock holes, fruit husks, discarded containers, used tires) were collected in forest, savannah, barren and village land covers and flooded for eggs hatching. The emerging larvae were reared to adult, identified, and blood fed for F1 production. The F0 and F1 adults were identified and tested for ZIKV by Reverse Transcriptase-Real time Polymerase Chain Reaction. RESULTS: A total of 1016 specimens, including 13 Aedes species, emerged in samples collected in the land covers and breeding sites investigated. Ae. aegypti was the dominant species representing 56.6% of this fauna with a high plasticity. Ae. furcifer and Ae. luteocephalus were found in forest tree holes, Ae. taylori in forest and village tree holes, Ae. vittatus in rock holes. ZIKV was detected from 4 out of the 82 mosquito pools tested. Positive pools included Ae. bromeliae (2 pools), Ae. unilineatus (1 pool), and Ae. vittatus (1 pool), indicating that the virus is maintained in these Aedes eggs during the dry season. CONCLUSION: Our investigation identified breeding sites types and land cover classes where several ZIKV vectors are maintained, and their maintenance rates during the dry season in southeastern Senegal. The maintenance of the virus in these vectors in nature could explain its early amplification at the start of the rainy season in this area.

Virus population structure in the ectomycorrhizal fungi Lactarius rufus and L. tabidus at two forest sites in Southern Finland.

Lactarius fungi belong to the Russulaceae family and have an important ecological role as ectomycorrhizal symbionts of coniferous and deciduous trees. Two Lactarius species, L. tabidus and L. rufus have been shown to harbor bisegmented dsRNA viruses belonging to an unclassified virus group including the mutualistic Curvularia thermal tolerance virus (CThTV). In this study, we characterized the first complete genome sequences of these viruses designated as Lactarius tabidus RNA virus 1 (LtRV1) and Lactarius rufus RNA virus 1 (LrRV1), both of which included two genome segments of 2241 and 2049 bp. We also analyzed spatial distribution and sequence diversity of the viruses in sixty host strains at two forest sites, and showed that the viruses are species-specific at sites where both host species co-occur. We also found that single virus isolates inhabited several different conspecific host strains, and were involved in persistent infections during up to eight years.

A bushel of viruses: Identification of seventeen novel putative viruses by RNA-seq in six apple trees.

Apple decline in Washington state has been increasing in incidence, particularly on Honeycrisp trees grown on G.935 rootstock. In this disease the trees exhibit dieback with necrosis at the graft union and in the rootstock. The cause of this disease remains unknown. To identify viral candidates, RNA-seq was performed on six trees: four trees exhibiting decline and two healthy trees. Across the samples, eight known viruses and Apple hammerhead viroid were detected, however none appear to be specifically associated with the disease. A BLASTx analysis of the RNA-seq data was performed to identify novel viruses that might be associated with apple decline. Seventeen novel putative viruses were detected, including an ilarvirus, two tombus-like viruses, a barna-like virus, a picorna-like virus, three ourmia-like viruses, three partiti-like viruses, and two narna-like viruses. Four additional viruses could not be classified. Three of the viruses appeared to be missing key genes, suggesting they may be dependent upon helper viruses for their function. Others showed a specific tropism, being detected only in the roots or only in the leaves. While, like the known apple viruses, none were consistently associated with diseased trees, it is possible these viruses may have a synergistic effect when co-infecting that could contribute to disease. Or the presence of these viruses may weaken the trees for some other factor that ultimately causes decline. Additional research will be needed to determine how these novel viruses contribute to apple decline.

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

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.

Genomic, Morphological and Biological Traits of the Viruses Infecting Major Fruit Trees.

Banana trees, citrus fruit trees, pome fruit trees, grapevines, mango trees, and stone fruit trees are major fruit trees cultured worldwide and correspond to nearly 90% of the global production of woody fruit trees. In light of the above, the present manuscript summarizes the viruses that infect the major fruit trees, including their taxonomy and morphology, and highlights selected viruses that significantly affect fruit production, including their genomic and biological features. The results showed that a total of 163 viruses, belonging to 45 genera classified into 23 families have been reported to infect the major woody fruit trees. It is clear that there is higher accumulation of viruses in grapevine (80/163) compared to the other fruit trees (each corresponding to less than 35/163), while only one virus species has been reported infecting mango. Most of the viruses (over 70%) infecting woody fruit trees are positive-sense single-stranded RNA (+ssRNA), and the remainder belong to the -ssRNA, ssRNA-RT, dsRNA, ssDNA and dsDNA-RT groups (each corresponding to less than 8%). Most of the viruses are icosahedral or isometric (79/163), and their diameter ranges from 16 to 80 nm with the majority being 25-30 nm. Cross-infection has occurred in a high frequency among pome and stone fruit trees, whereas no or little cross-infection has occurred among banana, citrus and grapevine. The viruses infecting woody fruit trees are mostly transmitted by vegetative propagation, grafting, and root grafting in orchards and are usually vectored by mealybug, soft scale, aphids, mites or thrips. These viruses cause adverse effects in their fruit tree hosts, inducing a wide range of symptoms and significant damage, such as reduced yield, quality, vigor and longevity.

Viromes in Xylariaceae fungi infecting avocado in Spain.

Four isolates of Entoleuca sp., family Xylariaceae, Ascomycota, recovered from avocado rhizosphere in Spain were analyzed for mycoviruses presence. For that, the dsRNAs from the mycelia were extracted and subjected to metagenomics analysis that revealed the presence of eleven viruses putatively belonging to families Partitiviridae, Hypoviridae, Megabirnaviridae, and orders Tymovirales and Bunyavirales, in addition to one ourmia-like virus plus other two unclassified virus species. Moreover, a sequence with 98% nucleotide identity to plant endornavirus Phaseolus vulgaris alphaendornavirus 1 has been identified in the Entoleuca sp. isolates. Concerning the virome composition, the four isolates only differed in the presence of the bunyavirus and the ourmia-like virus, while all other viruses showed common patterns. Specific primers allowed the detection by RT-PCR of these viruses in a collection of Entoleuca sp. and Rosellinia necatrix isolates obtained from roots of avocado trees. Results indicate that intra- and interspecies horizontal virus transmission occur frequently in this pathosystem.

The p33 protein of Citrus tristeza virus affects viral pathogenicity by modulating a host immune response.

Accumulation of reactive oxygen species (ROS) is a general plant basal defense strategy against viruses. In this study, we show that infection by Citrus tristeza virus (CTV) triggered ROS burst in Nicotiana benthamiana and in the natural citrus host, the extent of which was virus-dose dependent. Using Agrobacterium-mediated expression of CTV-encoded proteins in N. benthamiana, we found that p33, a unique viral protein, contributed to the induction of ROS accumulation and programmed cell death. The role of p33 in CTV pathogenicity was assessed based on gene knockout and complementation in N. benthamiana. In the citrus-CTV pathosystem, deletion of the p33 open reading frame in a CTV variant resulted in a significant decrease in ROS production, compared to that of the wild type CTV, which correlated with invasion of the mutant virus into the immature xylem tracheid cells and abnormal differentiation of the vascular system. By contrast, the wild type CTV exhibited phloem-limited distribution with a minor effect on the vasculature. We conclude that the p33 protein is a CTV effector that negatively affects virus pathogenicity and suggest that N. benthamiana recognizes p33 to activate the host immune response to restrict CTV into the phloem tissue and minimize the disease syndrome.

Recent Advances on Detection and Characterization of Fruit Tree Viruses Using High-Throughput Sequencing Technologies.

Perennial crops, such as fruit trees, are infected by many viruses, which are transmitted through vegetative propagation and grafting of infected plant material. Some of these pathogens cause severe crop losses and often reduce the productive life of the orchards. Detection and characterization of these agents in fruit trees is challenging, however, during the last years, the wide application of high-throughput sequencing (HTS) technologies has significantly facilitated this task. In this review, we present recent advances in the discovery, detection, and characterization of fruit tree viruses and virus-like agents accomplished by HTS approaches. A high number of new viruses have been described in the last 5 years, some of them exhibiting novel genomic features that have led to the proposal of the creation of new genera, and the revision of the current virus taxonomy status. Interestingly, several of the newly identified viruses belong to virus genera previously unknown to infect fruit tree species (e.g., Fabavirus, Luteovirus) a fact that challenges our perspective of plant viruses in general. Finally, applied methodologies, including the use of different molecules as templates, as well as advantages and disadvantages and future directions of HTS in fruit tree virology are discussed.

Discovery of Negative-Sense RNA Viruses in Trees Infected with Apple Rubbery Wood Disease by Next-Generation Sequencing.

Apple rubbery wood is a disease of apple found around the world, often associated with Apple flat limb disease, and regulated in many countries. Despite its long history in apple cultivation, the disease's causal agent has remained elusive. In this study, next-generation sequencing (NGS) was used to identify and characterize several related novel viral agents from apple rubbery wood-infected plants, which have been named Apple rubbery wood virus (ARWV) 1 and 2. Additional specimens with apple rubbery wood disease tested positive by polymerase chain reaction with primers designed to ARWV 1 and 2 genomic RNA segments. In an NGS-based screening of over 100 Malus and 100 Prunus specimens from a collection of virus-infected trees, only one Malus specimen was found to be infected with ARWV not known to be infected with the disease, which strongly suggests that ARWV is not commonly found in Malus spp. or other fruit trees. The two viruses are most closely related to members of the order Bunyavirales. Three RNA segments (large, medium, and small) were characterized and the viruses likely represent a new genus under the family Phenuiviridae, with a suggested name of Rubodvirus (Rubbery wood virus).

Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR.

A one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan probes was developed for the simultaneous detection of Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) in total RNA of pome trees extracted with a CTAB method. The sensitivity of the method was established using in vitro synthesized viral transcripts serially diluted in RNA from healthy, virus-tested (negative) pome trees. The three viruses were simultaneously detected up to a 10-4 dilution of total RNA from a naturally triple-infected apple tree prepared in total RNA of healthy apple tissue. The newly developed RT-qPCR assay was at least one hundred times more sensitive than conventional single RT-PCRs. The assay was validated with 36 field samples for which nine triple and 11 double infections were detected. All viruses were detected simultaneously in composite samples at least up to the ratio of 1:150 triple-infected to healthy pear tissue, suggesting the assay has the capacity to examine rapidly a large number of samples in pome tree certification programs and surveys for virus presence.

A rapid silica spin column-based method of RNA extraction from fruit trees for RT-PCR detection of viruses.

Efficient recovery of high quality RNA is very important for successful RT-PCR detection of plant RNA viruses. High levels of polyphenols and polysaccharides in plant tissues can irreversibly bind to and/or co-precipitate with RNA, which influences RNA isolation. In this study, a silica spin column-based RNA isolation method was developed by using commercially available silica columns combined with the application of a tissue lysis solution, and binding and washing buffers with high concentration guanidinium thiocyanate (GuSCN, 50% w/v), which helps remove plant proteins, polysaccharides and polyphenolic compounds. The method was successfully used to extract high quality RNA from citrus (Citrus aurantifolia), grapevine (Vitis vinifera), peach (Prunus persica), pear (Pyrus spp.), taro (Colocosia esculenta) and tobacco (Nicotiana benthamiana) samples. The method was comparable to conventional CTAB method in RNA isolation efficiency, but it was more sample-adaptable and cost-effective than commercial kits. High quality RNA isolated using silica spin column-based method was successfully used for the RT-PCR and/or multiplex RT-PCR amplification of woody fruit tree viruses and a viroid. The study provided a useful tool for the detection and characterization of plant viruses.

Population structure and diversity of citrus tristeza virus (CTV) isolates in Hunan province, China.

Stem-pitting (SP) is the main type of citrus tristeza virus (CTV) that causes severe damage to citrus trees, especially those of sweet orange, in Hunan province, China. Understanding the local CTV population structure should provide clues for effective mild strain cross-protection (MSCP) of the SP strain of CTV. In this study, markers for the p23 gene, multiple molecular markers (MMMs), and sequence analysis of the three silencing suppressor genes (p20, p23 and p25) were employed to analyze the genetic diversity and genotype composition of the CTV population based on 51 CTV-positive samples collected from 14 citrus orchards scattered around six major citrus-growing areas of Hunan. The results indicated that the CTV population structure was extremely complex and that infection was highly mixed. In total, p23 gene markers resulted in six profiles, and MMMs demonstrated 25 profiles. The severe VT and T3 types appeared to be predominantly associated with SP, while the mild T30 and RB types were related to asymptomatic samples. Based on phylogenetic analysis of the amino acid sequences of p20, p23 and p25, 19 representative CTV samples were classified into seven recently established CTV groups and a potentially novel one. A high level of genetic diversity, as well as potential recombination, was revealed among different CTV isolates. Five pure SP severe and two pure mild strains were identified by genotype composition analysis. Taken together, the results update the genetic diversity of CTV in Hunan with the detection of one possible novel strain, and this information might be applicable for the selection of appropriate mild CTV strains for controlling citrus SP disease through cross-protection.

Development of multiplex polymerase chain reaction assay for simultaneous detection of clostero-, badna- and mandari-viruses along with huanglongbing bacterium in citrus trees.

Citrus trees harbor a large number of viral and bacterial pathogens. Citrus yellow vein clearing virus (CYVCV), Indian citrus ringspot virus (ICRSV), Citrus yellow mosaic virus (CYMV), Citrus tristeza virus (CTV) and a bacterium, Candidatus Liberibacter asiaticus (CLa) associated with huanglongbing (HLB) disease, the most prevalent pathogens in citrus orchards of different regions in India and are responsible for debilitating citriculture. For detection of these viral and bacterial pathogens a quick, sensitive and cost effective detection method is required. With this objective a multiplex polymerase chain reaction (mPCR) assay was developed for simultaneous detection of four viruses and a bacterium in citrus. Several sets of primers were designed for each virus based on the retrieved reference sequences from the GenBank. A primer pair published previously was used for greening bacterium. Each pair of primers was evaluated for their sensitivity and differentiation by simplex and mPCR. The constant amplified products were identified on the basis of molecular size in mPCR and were compared with standard PCR. The amplicons were cloned and results were confirmed with sequencing analysis. The mPCR assay was validated using naturally infected field samples for one or more citrus viruses and the huanglongbing bacterium. The mPCR assay developed here will aid in the production of virus free planting materials and rapid indexing for certification of citrus budwood programme.

One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys.

Replacement of a dominant viral pathogen by a fungal pathogen does not alter the collapse of a regional forest insect outbreak.

Natural enemies and environmental factors likely both influence the population cycles of many forest-defoliating insect species. Previous work suggests precipitation influences the spatiotemporal patterns of gypsy moth outbreaks in North America, and it has been hypothesized that precipitation could act indirectly through effects on pathogens. We investigated the potential role of climatic and environmental factors in driving pathogen epizootics and parasitism at 57 sites over an area of ≈72,300 km(2) in four US mid-Atlantic states during the final year (2009) of a gypsy moth outbreak. Prior work has largely reported that the Lymantria dispar nucleopolyhedrovirus (LdNPV) was the principal mortality agent responsible for regional collapses of gypsy moth outbreaks. However, in the gypsy moth outbreak-prone US mid-Atlantic region, the fungal pathogen Entomophaga maimaiga has replaced the virus as the dominant source of mortality in dense host populations. The severity of the gypsy moth population crash, measured as the decline in egg mass densities from 2009 to 2010, tended to increase with the prevalence of E. maimaiga and larval parasitoids, but not LdNPV. A significantly negative spatial association was detected between rates of fungal mortality and parasitism, potentially indicating displacement of parasitoids by E. maimaiga. Fungal, viral, and parasitoid mortality agents differed in their associations with local abiotic and biotic conditions, but precipitation significantly influenced both fungal and viral prevalence. This study provides the first spatially robust evidence of the dominance of E. maimaiga during the collapse of a gypsy moth outbreak and highlights the important role played by microclimatic conditions.

Phage-mediated selection on microbiota of a long-lived host.

It is increasingly apparent that the dynamic microbial communities of long-lived hosts affect their phenotype, including resistance to disease. The host microbiota will change over time due to immigration of new species, interaction with the host immune system, and selection by bacteriophage viruses (phages), but the relative roles of each process are unclear. Previous metagenomic approaches confirm the presence of phages infecting host microbiota, and experimental coevolution of bacteria and phage populations in the laboratory has demonstrated rapid reciprocal change over time. The key challenge is to determine whether phages influence host-associated bacterial communities in nature, in the face of other selection pressures. I use a tree-bacteria-phage system to measure reciprocal changes in phage infectivity and bacterial resistance within microbial communities of tree hosts over one season. An experimental time shift shows that bacterial isolates are most resistant to lytic phages from the prior month and least resistant to those from the future month, providing clear evidence for both phage-mediated selection on bacterial communities and bacterial-mediated selection on phage communities in nature. These reciprocal changes suggest that phages indeed play a key role in shaping the microbiota of their eukaryotic hosts.