Molecular characterisation of a novel sadwavirus infecting cattleya orchids in Australia.

The complete genome sequence of a novel sadwavirus infecting cattleya orchids in South East Queensland is described. Isometric virions of c. 27 nm diameter were observed in sap extracts viewed under a transmission electron microscope, and the genome sequence of this virus was determined by high-throughput sequencing. The viral genome consists of two RNA components, 5,910 and 4,435 nucleotides (nt) in length, each encoding a long polyprotein, with predicted cleavage sites at H/Y, E/G, Q/S, and Q/G for the RNA1 and T/G for the RNA2 translation products, respectively. RNA2 has an additional small ORF of 684 nt near the 3' untranslated region. Phylogenetic analysis based on an amino acid sequence alignment of the Pro-Pol region suggested that this virus is most closely related to pineapple secovirus A, a member of the subgenus Cholivirus, but warrants classification as a member of a new species because it exhibited no more than 64% amino acid identity in pairwise sequence comparisons. Because of the prominent purple ringspots that were observed on the leaves of some of the plants, we propose the name "cattleya purple ringspot virus" for this virus (suggested species name: "Sadwavirus cattleyacola").

Endogenous Caulimovirids: Fossils, Zombies, and Living in Plant Genomes.

The Caulimoviridae is a family of double-stranded DNA viruses that infect plants. The genomes of most vascular plants contain endogenous caulimovirids (ECVs), a class of repetitive DNA elements that is abundant in some plant genomes, resulting from the integration of viral DNA in the chromosomes of germline cells during episodes of infection that have sometimes occurred millions of years ago. In this review, we reflect on 25 years of research on ECVs that has shown that members of the Caulimoviridae have occupied an unprecedented range of ecological niches over time and shed light on their diversity and macroevolution. We highlight gaps in knowledge and prospects of future research fueled by increased access to plant genome sequence data and new tools for genome annotation for addressing the extent, impact, and role of ECVs on plant biology and the origin and evolutionary trajectories of the Caulimoviridae.

Surveillance for Avocado Sunblotch Viroid Utilizing the European Honey Bee (Apis mellifera).

Avocado is one of the world's fastest growing tropical fruit industries, and the pathogen avocado sunblotch viroid (ASBVd) is a major threat to both production and access to international export markets. ASBVd is seed transmissible, with infection possible via either the male (pollen) or female gametes. Surveillance for ASBVd across commercial orchards is a major logistical task, particularly when aiming to meet the stringent standards of evidence required for a declaration of pest freedom. As with many fruit crops, insect pollination is important for high avocado yields, and honey bee (Apis mellifera) hives are typically moved into orchards for paid pollination services. Exploiting the foraging behavior of honey bees can provide a complementary strategy to traditional surveillance methods. High-throughput sequencing (HTS) of bee samples for plant viruses shows promise, but this surveillance method has not yet been tested for viroids or in a targeted plant biosecurity context. Here, we tested samples of bees and pollen collected from pollination hives in two ASBVd orchard locations, one in Australia, where only four trees in a block were known to be infected, and a second in South Africa, where the estimated incidence of infection was 10%. Using real-time RT-PCR and HTS (total RNA-seq and small RNA-seq), we demonstrated that ASBVd can be confidently detected in bees and pollen samples from hives within 100 m of infected trees. The potential for using this approach in ASBVd surveillance for improved orchard management and supporting market access is discussed.

CAULIFINDER: a pipeline for the automated detection and annotation of caulimovirid endogenous viral elements in plant genomes.

Plant, animal and protist genomes often contain endogenous viral elements (EVEs), which correspond to partial and sometimes entire viral genomes that have been captured in the genome of their host organism through a variety of integration mechanisms. While the number of sequenced eukaryotic genomes is rapidly increasing, the annotation and characterization of EVEs remains largely overlooked. EVEs that derive from members of the family Caulimoviridae are widespread across tracheophyte plants, and sometimes they occur in very high copy numbers. However, existing programs for annotating repetitive DNA elements in plant genomes are poor at identifying and then classifying these EVEs. Other than accurately annotating plant genomes, there is intrinsic value in a tool that could identify caulimovirid EVEs as they testify to recent or ancient host-virus interactions and provide valuable insights into virus evolution. In response to this research need, we have developed CAULIFINDER, an automated and sensitive annotation software package. CAULIFINDER consists of two complementary workflows, one to reconstruct, annotate and group caulimovirid EVEs in a given plant genome and the second to classify these genetic elements into officially recognized or tentative genera in the Caulimoviridae. We have benchmarked the CAULIFINDER package using the Vitis vinifera reference genome, which contains a rich assortment of caulimovirid EVEs that have previously been characterized using manual methods. The CAULIFINDER package is distributed in the form of a Docker image.

Genome sequence of pineapple secovirus B, a second sadwavirus reported infecting Ananas comosus.

The complete genome sequence of pineapple secovirus B (PSV-B), a new virus infecting pineapple (Ananas comosus) on the island of Oahu, Hawaii, was determined by high-throughput sequencing (HTS). The genome comprises two RNAs that are 5,956 and 3,808 nt long, excluding the 3'-end poly-A tails, both coding for a single large polyprotein. The RNA1 polyprotein contains five conserved domains associated with replication, while the RNA2 polyprotein is cleaved into the movement protein and coat protein. PSV-B is representative of a new species in the subgenus Cholivirus (genus Sadwavirus; family Secoviridae), as the level of amino acid sequence identity to recognized members of this subgenus in the Pro-Pol and coat protein regions is below currently valid species demarcation thresholds.

Untangling the taxonomy of dahlia mosaic virus.

In this brief note, we review the taxonomic history of dahlia mosaic virus (DMV) and related viruses. DMV is the only officially recognized caulimovirus known to infect dahlia (Dahlia variabilis) plants, although this virus appears to be relatively rare as a pathogen compared to a more recently described but unclassified caulimovirus called dahlia common mosaic virus (DCMV). We have undertaken a new set of analyses to test the hypothesis that DCMV represents a new caulimovirus species whose members infect dahlia, but we ultimately reject this hypothesis. A probable sequencing error was identified in the reference genome sequence of DMV, and consequently, we recommend that an alternative virus isolate be nominated as the exemplar for this species. In accordance with the new binomial nomenclatural system, it is proposed that the virus species be called "Caulimovirus dahliae".

Bermuda grass latent virus in Australia: genome sequence, sequence variation, and new hosts.

Bermuda grass latent virus (BGLV; genus Panicovirus) is identified for the first time in Australia and in only the second country after the USA. A full-length genome sequence was obtained, which has 97% nucleotide sequence identity to that of the species exemplar isolate. Surveys for BGLV, utilising a newly designed universal panicovirus RT-PCR assay for diagnosis, demonstrated widespread infection by this virus in a broad variety of Bermuda grass cultivars (Cynodon dactylon and C. dactylon × C. transvaalensis) grown in both New South Wales and Queensland. The virus was also detected in Rhodes grass (Chloris gayana) and Kikuyu grass (Cenchrus clandestinus), which are both important pasture grasses in subtropical Australia, and the latter is also grown as turf. Furthermore, the Rhodes grass plant, which had strong mosaic symptoms, was also infected with sugarcane mosaic virus, warranting further investigations as to whether synergistic interactions occur between these two viruses.

Viruses Infecting Greenhood Orchids (Pterostylidinae) in Eastern Australia.

The Australasian biogeographic realm is a major centre of diversity for orchids, with every subfamily of the Orchidaceae represented and high levels of endemism at the species rank. It is hypothesised that there is a commensurate diversity of viruses infecting this group of plants. In this study, we have utilised high-throughput sequencing to survey for viruses infecting greenhood orchids (Pterostylidinae) in New South Wales and the Australian Capital Territory. The main aim of this study was to characterise Pterostylis blotch virus (PtBV), a previously reported but uncharacterised virus that had been tentatively classified in the genus Orthotospovirus. This classification was confirmed by genome sequencing, and phylogenetic analyses suggested that PtBV is representative of a new species that is possibly indigenous to Australia as it does not belong to either the American or Eurasian clades of orthotospoviruses. Apart from PtBV, putative new viruses in the genera Alphaendornavirus, Amalgavirus, Polerovirus and Totivirus were discovered, and complete genome sequences were obtained for each virus. It is concluded that the polerovirus is likely an example of an introduced virus infecting a native plant species in its natural habitat, as this virus is probably vectored by an aphid, and Australia has a depauperate native aphid fauna that does not include any species that are host-adapted to orchids.

Adaptation of a filter paper method for RNA template preparation for the detection of avocado sunblotch viroid by reverse transcription qPCR.

An easy, rapid and inexpensive method of preparing RNA template for a reverse transcription qPCR assay for avocado sunblotch viroid (ASBVd) is described. This method depends on the principle of reversible binding of viroid RNA to filter paper under different concentrations of monovalent cation. Lysis buffers containing either sodium chloride or lithium chloride were compared, and 1.5 M lithium chloride was shown to be optimal for the adsorption of the viroid RNA to the filter paper. The extraction method was validated using field samples and equivalent yields of viroid RNA were obtained using this method and either a commercial RNA extraction kit or a dsRNA chromatography method. The filter paper method of RNA extraction is ideally suited for the large-scale surveillance for ASBVd.

Development of a one-step RT-qPCR detection assay for the newly described citrus viroid VII.

An apscaviroid, tentatively named citrus viroid VII (CVd-VII), was recently discovered in citrus in Australia. A diagnostic assay using real-time reverse transcription polymerase chain reaction was developed and validated to detect the viroid in citrus plants. The assay showed a high level of sensitivity, reliably detecting 2000 plasmid copies per reaction, while down to 20 plasmid copies per reaction were occasionally detected. The assay showed high specificity, producing no false positives or cross-reactivity with a range of other citrus graft-transmissible pathogens, including viroids, viruses and bacteria. The real-time assay was also found to be more sensitive than the available end-point reverse transcription polymerase chain reaction assay by a factor of 100,000 and could be a useful tool for the rapid detection of CVd-VII in diagnostic and research environments.

Characterisation of novel endogenous geminiviral elements in macadamia.

BACKGROUND: The presence of geminivirus sequences in a preliminary analysis of sRNA sequences from the leaves of macadamia trees with abnormal vertical growth (AVG) syndrome was investigated. RESULTS: A locus of endogenous geminiviral elements (EGE) in the macadamia genome was analysed, and the sequences revealed a high level of deletions and/or partial integrations, thus rendering the EGE transcriptionally inactive. The replication defective EGE in the macadamia genome indicates its inability to be the source of new viral infections and thus cause AVG or any other disease in macadamia. The EGE sequences were detected in two edible Macadamia species that constitute commercial cultivars and the wild germplasm of edible and inedible species of Macadamia. This strongly suggests that the integration preceded speciation of the genus Macadamia. A draft genome of a locus of EGE in Macadamia was developed. The findings of this study provide evidence to suggest the endogenization of the geminiviral sequences in the macadamia genome and the ancestral relationship of EGE with Macadamia in the Proteaceae family. Random mutations accumulating in the EGE inform that the sequence is evolving. CONCLUSIONS: The EGE in Macadamia is inactive and thus not a direct cause of any diseases or syndromes including AVG in macadamia. The insertion of the EGE in the macadamia genome preceded speciation of the genus Macadamia.

Genome sequence and geographic distribution of a new nepovirus infecting Stenotaphrum secundatum in Australia.

The genome sequence of a new subgroup C nepovirus from Stenotaphrum secundatum in Australia is described. This virus, tentatively named Stenotaphrum nepovirus (SteNV), was present in separate plants as a mixed infection with either sugarcane mosaic virus or Panicum mosaic virus. The virus genome was divided between two RNA segments, 7,824 and 7,104 nucleotides (nt) in length, which each encode a single long polyprotein with putative 3C-like cysteine protease sites of the type H/G, H/S or L/S. The 3' untranslated region of RNA2, at 2,155 nt, is the longest observed for any subgroup C nepovirus. Phylogenetic analyses using protease-polymerase and coat protein amino acid alignments suggest that SteNV is most closely related to cherry leaf roll virus. Using a newly developed RT-PCR assay, this virus was detected at multiple localities in New South Wales, Queensland and Western Australia, and in a second host species, Digitaria didactyla. No consistent association between virus infection and symptoms could be established. The economic importance, pathogenicity and transmission of this novel virus species warrant further investigation.

Insertion of Badnaviral DNA in the Late Blight Resistance Gene (R1a) of Brinjal Eggplant (Solanum melongena).

Endogenous viral elements (EVEs) are widespread in plant genomes. They result from the random integration of viral sequences into host plant genomes by horizontal DNA transfer and have the potential to alter host gene expression. We performed a large-scale search for co-transcripts including caulimovirid and plant sequences in 1,678 plant and 230 algal species and characterized 50 co-transcripts in 45 distinct plant species belonging to lycophytes, ferns, gymnosperms and angiosperms. We found that insertion of badnavirus EVEs along with Ty-1 copia mobile elements occurred into a late blight resistance gene (R1) of brinjal eggplant (Solanum melongena) and wild relatives in genus Solanum and disrupted R1 orthologs. EVEs of two previously unreported badnaviruses were identified in the genome of S. melongena, whereas EVEs from an additional novel badnavirus were identified in the genome of S. aethiopicum, the cultivated scarlet eggplant. Insertion of these viruses in the ancestral lineages of the direct wild relatives of the eggplant would have occurred during the last 3 Myr, further supporting the distinctiveness of the group of the eggplant within the giant genus Solanum.

Complete genome sequence of aucuba ringspot virus.

A new badnavirus, aucuba ringspot virus (AuRV), was identified in plants of Aucuba japonica showing mild mosaic, vein banding, and yellow ringspot symptoms on the leaves. The complete nucleotide sequence of the AuRV genome was determined and found to be 9,092 nt in length, and the virus was found to have a genome organization typical of members of the genus Badnavirus. ORF3 was predicted to encode a polyprotein containing conserved movement protein, coat protein, aspartic protease, reverse transcriptase (RT), and RNase H domains. Phylogenetic analysis suggested that this virus is most closely related to codonopsis vein clearing virus but belongs to a distinct species, based on only 69.6% nucleotide sequence identity within the part of ORF 3 encoding the RT and RNase H domains. The vector of AuRV is unknown, but based on phylogenetic relationships, it is predicted to be a type of aphid.

Discovery of Single Nucleotide Polymorphisms for Resistance to Abnormal Vertical Growth in Macadamia.

Abnormal vertical growth (AVG) syndrome is a serious threat to the Australian macadamia industry as it decreases the yield of nuts by as much as 70% per annum. A lack of information on the cause of AVG has hindered the development of an effective disease management strategy. Discovery of genetic markers associated with disease resistance can be used as tool for rapid selection of elite cultivars, hence helps in efficient disease management. Differences in field susceptibility of macadamia cultivars provide an opportunity for discovery of genetic markers that are associated with host resistance. REML mixed model analysis was performed to estimate the AVG rating of 51 cultivars from multiple origins using phenotypic data from 359 trees planted in four sites. Most of the Hawaiian cultivars were found as susceptible, while selections from the Australian macadamia industry breeding program were predominantly resistant. All the cultivars were genotyped for 13,221 DArTseq-based single nucleotide polymorphism (SNP) markers. A bulked sample analysis was performed using 20 genotypes each at the extremes of AVG phenotypic ratings. Ten SNP markers were predicted to be associated with AVG resistance and two arbitrarily selected SNP markers were validated using PCR and Sanger sequencing. Our findings suggest that AVG resistance in the commercial cultivars may be derived from the genomic introgression of Macadamia tetraphylla through interspecific hybridization. The results may support marker-assisted selection for macadamia germplasm with AVG resistance.

Identification of putative viroplasms within banana cells infected by banana streak MY virus.

The badnavirus replication cycle is poorly understood and most knowledge is based on extrapolations from model viruses such as Cauliflower mosaic virus (CaMV). However, in contrast to CaMV, badnaviruses are thought not to produce viroplasms and therefore it has been a mystery as to where virion assembly occurs. In this study, ultrathin sections of a banana leaf infected with a badnavirus, banana streak MY virus (BSMYV), were examined by transmission electron microscopy. Electron-dense inclusion bodies (EDIBs) were sporadically distributed in parenchymatous tissues of the leaf, most commonly in the palisade and spongy mesophyll cells. These EDIBs had a characteristic structure, comprising an electron-dense core, a single, encircling lacuna and an outer ring of electron-dense material. However, much less frequently, EDIBs with two or three lacunae were observed. In the outer ring, densely packed virions were visible with a shape and size consistent with that expected for badnaviruses. Immunogold labelling was done with primary antibodies that detected the N-terminus of the capsid protein and strong labelling of the outer ring but not the central core or lacuna was observed. It is concluded that the EDIBs that were observed are equivalent in function to the viroplasms of CaMV, although obviously different in composition as there is not a paralogue of the transactivation/viroplasm protein in the badnavirus genome. It is postulated that production of a viroplasm could be a conserved characteristic of all members of the Caulimoviridae.

ICTV Virus Taxonomy Profile: Caulimoviridae.

Caulimoviridae is a family of non-enveloped reverse-transcribing plant viruses with non-covalently closed circular dsDNA genomes of 7.1-9.8 kbp in the order Ortervirales. They infect a wide range of monocots and dicots. Some viruses cause economically important diseases of tropical and subtropical crops. Transmission occurs through insect vectors (aphids, mealybugs, leafhoppers, lace bugs) and grafting. Activation of infectious endogenous viral elements occurs in Musa balbisiana, Petunia hybrida and Nicotiana edwardsonii. However, most endogenous caulimovirids are not infectious. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Caulimoviridae, which is available at ictv.global/report/caulimoviridae.

Spatiotemporal Spread of Abnormal Vertical Growth of Macadamia in Australia Informs Epidemiology.

Australian macadamia production is threatened by a disorder known as abnormal vertical growth (AVG), for which the etiology is unknown. AVG is characterized by vigorous upright growth and reduced lateral branching, flowering, and nut set that results in over 70% yield loss annually. Six commercial macadamia orchards were surveyed in 2012 and again in 2018 to examine spatiotemporal dynamics of the epidemic. Data were subjected to point-pattern and geostatistical analyses. AVG incidence in all orchards showed a better fit to the beta-binomial distribution than the binomial distribution. AVG incidence in the different orchards varied between 5 and 47% in 2012, and 13 and 55% in 2018 and the rate of spread was slow, averaging at about 2% increase in disease incidence per annum. Spatial patterns of AVG were highly aggregated on both survey years and spread was mainly between neighboring trees in a row or trees that were opposite to each other in different rows. Semivariograms showed large range values (approximately 15 to 120), indicating aggregation of AVG-affected trees beyond quadrat levels. Furthermore, clusters of disease were mainly at the edge of the orchard on the first survey date and the disease progressed toward the center of the orchard over time. It is concluded that AVG is caused by an infectious agent, and based on patterns of spread, we hypothesize that spread is facilitated by root grafting or root-to-root contact. Furthermore, a vascular-limited pathogen could be involved that modulates plant hormone production.

Three novel Ambrosia Fusarium Clade species producing clavate macroconidia known (F. floridanum and F. obliquiseptatum) or predicted (F. tuaranense) to be farmed by Euwallacea spp. (Coleoptera: Scolytinae) on woody hosts.

The Ambrosia Fusarium Clade (AFC) comprises at least 16 genealogically exclusive species-level lineages within clade 3 of the Fusarium solani species complex (FSSC). These fungi are either known or predicted to be farmed by Asian Euwallacea ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) in the tribe Xyleborini as a source of nutrition. To date, only 4 of the 16 AFC lineages have been described formally. In the absence of Latin binomials, an ad hoc nomenclature was developed to distinguish the 16 species lineages as AF-1 to AF-16. Herein, Fusarium species AF-3, AF-5, and AF-7 were formally described as F. floridanum, F. tuaranense, and F. obliquiseptatum, respectively. Fusarium floridanum farmed by E. interjectus on box elder (Acer negundo) in Gainesville, Florida, was distinguished morphologically by the production of sporodochial conidia that were highly variable in size and shape together with greenish-pigmented chlamydospores. Fusarium tuaranense was isolated from a beetle-damaged Para rubber tree (Hevea brasiliense) in North Borneo, Malaysia, and was diagnosed by production of the smallest sporodochial conidia of any species within the AFC. Lastly, F. obliquiseptatum was farmed by an unnamed ambrosia beetle designated Euwallacea sp. 3 (E. fornicatus species complex) on avocado (Persea americana) in Queensland, Australia. It uniquely produces some clavate sporodochial conidia with oblique septa. Maximum likelihood analysis of a multilocus data set resolved these three novel AFC taxa as phylogenetically distinct species based on genealogical concordance. Particularly where introduced into exotic environments, these exotic mutualists pose a serious threat to the avocado industry, native forests, and urban landscapes in diverse regions throughout the world.

Questions surrounding the taxonomic validity of the species Garlic mite-borne filamentous virus (genus Allexivirus).

Garlic mite-borne filamentous virus is one of the oldest recognized allexivirus species but, paradoxically, one with the least well studied member viruses. In this paper, we review the history of this taxon and highlight problems in designating a holotype (exemplar isolate). Analyses are presented that suggest that GarMbFV is conspecific with Garlic virus A, and therefore the former taxon should be abolished.