Small RNA Analyses of a Ceratobasidium Isolate Infected with Three Endornaviruses.

Isolates of three endornavirus species were identified co-infecting an unidentified species of Ceratobasidium, itself identified as a symbiont from within the roots of a wild plant of the terrestrial orchid Pterostylis vittata in Western Australia. Isogenic lines of the fungal isolate lacking all three mycoviruses were derived from the virus-infected isolate. To observe how presence of endornaviruses influenced gene expression in the fungal host, we sequenced fungus-derived small RNA species from the virus-infected and virus-free isogenic lines and compared them. The presence of mycoviruses influenced expression of small RNAs. Of the 3272 fungus-derived small RNA species identified, the expression of 9.1% (300 of 3272) of them were up-regulated, and 0.6% (18 of 3272) were down-regulated in the presence of the viruses. Fourteen novel micro-RNA-like RNAs (Cer-milRNAs) were predicted. Gene target prediction of the differentially expressed Cer-milRNAs was quite ambiguous; however, fungal genes involved in transcriptional regulation, catalysis, molecular binding, and metabolic activities such as gene expression, DNA metabolic processes and regulation activities were differentially expressed in the presence of the mycoviruses.

Spillover of a Tobamovirus from the Australian Indigenous Flora to Invasive Weeds.

The tobamovirus yellow tailflower mild mottle virus (YTMMV) was previously reported in wild plants of Anthocercis species (family Solanaceae) and other solanaceous indigenous species growing in natural habitats in Western Australia. Here, we undertook a survey of two introduced solanaceous weeds, namely Solanum nigrum (black nightshade) and Physalis peruviana (cape gooseberry) in the Perth metropolitan area and surrounds to determine if YTMMV has spread naturally to these species. At a remnant natural bushland site where both solanaceous weeds and indigenous Anthocercis hosts grew adjacent to one another, a proportion of S. nigrum and P. peruviana plants were asymptomatically-infected with YTMMV, confirming spillover had occurred. Populations of S. nigrum also grow as weeds in parts of the city isolated from remnant bushland and indigenous sources of YTMMV, and some of these populations were also infected with YTMMV. Fruit was harvested from virus-infected wild S. nigrum plants and the seed germinated under controlled conditions. Up to 80% of resultant seedlings derived from infected parent plants were infected with YTMMV, confirming that the virus is vertically-transmitted in S. nigrum, and therefore infection appears to be self-sustaining in this species. This is the first report of spillover of YTMMV to exotic weeds, and of vertical transmission of this tobamovirus. We discuss the roles of vertical and horizontal transmission in this spillover event, and its implications for biosecurity.

Fungal endophytes from salt-adapted plants confer salt tolerance and promote growth in wheat (Triticum aestivum L.) at early seedling stage.

With increasing human global population, increased yield under saline conditions is a desirable trait for major food crops. Use of endophytes, isolated from halophytic hosts, seems to be an exciting approach for conferring salt tolerance to a salt-sensitive crop. Therefore, in the current study, fungal endophytes were isolated from halophytic plants' roots and their ability to withstand in vitro salt stress was evaluated. The fungal endophytes could withstand up to 1M NaCl concentrations and this tolerance was independent of their host or tissue source. When inoculated on salt-sensitive wheat seeds/seedlings, several of the endophytes showed a positive impact on germination and biomass-related parameters upon salt stress, both in vitro and under glasshouse conditions. One of the isolates from dicot plants (identified as Microsphaeropsis arundinis) could successfully colonize wheat and promote its growth under salt and no-salt conditions. Amongst the fungal isolates that are known to be natural endophytes of wheat, Chaetomium globosum was the best performing isolate and has previously been reported to be an effective biocontrol agent. Based on the results of our preliminary study, we suggest that these fungal endophytes could prove beneficial for enhancing the salt stress tolerance of wheat crop.

ICTV Virus Taxonomy Profile: Potyviridae 2022.

The family Potyviridae includes plant viruses with single-stranded, positive-sense RNA genomes of 8-11 kb and flexuous filamentous particles 650-950 nm long and 11-20 nm wide. Genera in the family are distinguished by the host range, genomic features and phylogeny of the member viruses. Most genomes are monopartite, but those of members of the genus Bymovirus are bipartite. Some members cause serious disease epidemics in cultivated plants. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Potyviridae, which is available at ictv.global/report/potyviridae.

Differential Symptom Development and Viral RNA Loads in 10 Nicotiana benthamiana Accessions Infected with the Tobamovirus Yellow Tailflower Mild Mottle Virus.

Yellow tailflower mild mottle virus (YTMMV, genus Tobamovirus) was identified from wild plants of solanaceous species in Australia. Nicotiana benthamiana is a species indigenous to the arid north of Australia. N. benthamiana accession RA-4 (the lab type), which has a mutant, functionally defective, RNA-dependent RNA polymerase 1 (Rdr1) gene (Nb-Rdr1m), has played a significant role in plant virology, but little study has been done regarding responses to virus infection by other accessions of N. benthamiana. All wild-collected N. benthamiana accessions used in this study harbored wild-type Rdr1 genes (Nb-Rdr1). We compared symptoms of YTMMV infection and viral RNA load on RA-4 and nine wild-collected accessions of N. benthamiana from mainland Western Australia, an island, and the Northern Territory. After inoculation with YTMMV, RA-4 plants responded with systemic hypersensitivity and all individuals were dead 35 days postinoculation (dpi). Plants of wild-collected accessions exhibited a range of symptoms, from mild to severe, and some, but not all, died in the same period. Quantitative reverse transcription PCR revealed that the Rdr1 mutation was not a predictor of viral RNA load or symptom severity. For example, wild-collected A019412 plants carried more than twice the viral RNA load of RA-4 plants, but symptom expression was moderate. For plants of most accessions, viral RNA load did not increase after 10 dpi. The exception was plants of accession Barrow-1, in which viral RNA load was low until 15 dpi, after which it increased more than 29-fold. This study revealed differential responses by N. benthamiana accessions to infection by an isolate of YTMMV. The Rdr1 gene, whether mutant or wild-type, did not appear to influence viral RNA load or disease expression. Genetic diversity of the 10 N. benthamiana accessions in some cases reflected geographical location, but in other accessions this was not so.

Wisteria Vein Mosaic Virus Detected for the First Time in Iran from an Unknown Host by Analysis of Aphid Vectors.

The development of reverse transcription-polymerase chain reaction using degenerate primers against conserved regions of most potyviral genomes enabled sampling of the potyvirome. However, these assays usually involve sampling potential host plants, but identifying infected plants when they are asymptomatic is challenging, and many plants, especially wild ones, contain inhibitors to DNA amplification. We used an alternative approach which utilized aphid vectors and indicator plants to identify potyviruses capable of infecting common bean (Phaseolus vulgaris). Aphids were collected from a range of asymptomatic leguminous weeds and trees in Iran, and transferred to bean seedlings under controlled conditions. Bean plants were tested serologically for potyvirus infections four-weeks post-inoculation. The serological assay and symptomatology together indicated the presence of one potyvirus, and symptomology alone implied the presence of an unidentified virus. The partial genome of the potyvirus, encompassing the complete coat protein gene, was amplified using generic potyvirus primers. Sequence analysis of the amplicon confirmed the presence of an isolate of Wisteria vein mosaic virus (WVMV), a virus species not previously identified from Western Asia. Phylogenetic analyses of available WVMV sequences categorized them into five groups: East Asian-1 to 3, North American and World. The Iranian isolate clustered with those in the World group. Multiple sequence alignment indicated the presence of some genogroup-specific amino acid substitutions among the isolates studied. Chinese isolates were sister groups of other isolates and showed higher nucleotide distances as compared with the others, suggesting a possible Eastern-Asian origin of WVMV, the main region where Wisteria might have originated.

Challenges to elucidating how endornaviruses influence fungal hosts: Creating mycovirus-free isogenic fungal lines and testing them.

Determining roles of mycoviruses in fungal biology is complicated, especially when fungi are co-infected with multiple viruses. Genetically identical (isogenic) fungal lines that are infected by and not infected by viruses must be created and compared. Here, we study an isolate of Ceratobasidium sp., a fungus isolated from pelotons in roots of a wild terrestrial orchid. The fungal isolate was co-infected with three distinct endornaviruses, isolates of Ceratobasidium endonarvirus B (CbEVB), Ceratobasidium endonarvirus C (CbEVC) and Ceratobasidium endonarvirus D (CbEVD). An experiment to reveal natural distribution of the three mycoviruses within a fungal colony revealed no sectoring; they were all evenly distributed throughout the colony. Hyphal tipping and treatments with one of five antibiotics (kanamycin, streptomycin, cycloheximide, rifampicin and ampicillin) were applied in attempts to 'cure' fungal lines of one, two or three of the viruses present. Surprisingly, the three mycoviruses responded differentially to each curing approach. The isolate of CbEVC was eliminated upon treatment with cycloheximide, but not with kanamycin or streptomycin, whereas the isolate of CbEVD did not respond to cycloheximide. The isolate of CbEVB was eliminated upon all treatments. In some cases, a virus was undetectable by species-specific RT-PCR assay after treatment, but when the fungus was cultured for a period on non-selective medium, the virus was detected again. Effects of mycoviruses on growth characteristics of isogenic fungal lines on two nutrient media were studied. Co-infection by the three viruses reduced mycelial growth rate on both media. In contrast, some fungal lines infected with one or two mycoviruses grew more rapidly than virus-free lines.

Co-Infection with Three Mycoviruses Stimulates Growth of a Monilinia fructicola Isolate on Nutrient Medium, but Does Not Induce Hypervirulence in a Natural Host.

Monilinia fructicola and Monilinia laxa are the most destructive fungal species infecting stone fruit (Prunus species). High-throughput cDNA sequencing of M. laxa and M. fructicola isolates collected from stone fruit orchards revealed that 14% of isolates were infected with one or more of three mycoviruses: Sclerotinia sclerotiorum hypovirus 2 (SsHV2, genus Hypovirus), Fusarium poae virus 1 (FPV1, genus Betapartitivirus), and Botrytis virus F (BVF, genus Mycoflexivirus). Isolate M196 of M. fructicola was co-infected with all three viruses, and this isolate was studied further. Several methods were applied to cure M196 of one or more mycoviruses. Of these treatments, hyphal tip culture either alone or in combination with antibiotic treatment generated isogenic lines free of one or more mycoviruses. When isogenic fungal lines were cultured on nutrient agar medium in vitro, the triple mycovirus-infected parent isolate M196 grew 10% faster than any of the virus-cured isogenic lines. BVF had a slight inhibitory effect on growth, and FPV1 did not influence growth. Surprisingly, after inoculation to fruits of sweet cherry, there were no significance differences in disease progression between isogenic lines, suggesting that these mycoviruses did not influence the virulence of M. fructicola on a natural host.

Novel and divergent viruses associated with Australian orchid-fungus symbioses.

Terrestrial orchids represent a symbiotic union between plants and mycorrhizal fungi. This study describes the occurrence and nature of viruses associated with one population of wild Pterostylis sanguinea orchids, including their fungal symbionts, over two consecutive years. A generic sequencing approach, which combined dsRNA-enrichment from plant and mycelial tissues, random amplification and high throughput shotgun sequencing was used to identify novel viruses. The majority of the virus-like sequences represent partial genomes, and their identification is based solely on de novo assembly of sequencing data. In orchid leaf tissues we found three isolates of a novel totivirus and an unclassified virus; both resemble fungus-infecting viruses. Two isolates of Ceratobasidium sp that were isolated from orchid underground stems contained at least 20 viruses, 16 of which were previously described as alphapartitiviruses and betapartitiviruses. A novel hypovirus and a mitovirus were genetically distant from existing members of the genera and did not readily fit into recognised subgroups.

Host Specificity of Endophytic Mycobiota of Wild Nicotiana Plants from Arid Regions of Northern Australia.

In arid regions of northern Australia, plants survive under water deficit, high temperatures, intense solar radiation and nutrient-impoverished soils. They employ various morpho-physiological and biochemical adaptations including interaction with microbial symbionts. We evaluated identity, host and tissue association with geographical distribution of fungal endophytes isolated from above- and below-ground tissues of plants of three indigenous Australian Nicotiana species. Isolation frequency and α-diversity were significantly higher for root endophyte assemblages than those of stem and leaf tissues. We recorded no differences in endophyte species richness or diversity as a function of sampling location, but did detect differences among different host genotypes and plant tissues. There was a significant pattern of community similarity associated with host genotypes but no consistent pattern of fungal community structuring associated with sampling location and tissue type, regardless of the community similarity measurements used.

The challenges of using high-throughput sequencing to track multiple bipartite mycoviruses of wild orchid-fungus partnerships over consecutive years.

The bipartite alpha- and betapartitiviruses are recorded from a wide range of fungi and plants. Using a combination of dsRNA-enrichment, high-throughput shotgun sequencing and informatics, we report the occurrence of multiple new partitiviruses associated with mycorrhizal Ceratobasidium fungi, themselves symbiotically associated with a small wild population of Pterostylis sanguinea orchids in Australia, over two consecutive years. Twenty-one partial or near-complete sequences representing 16 definitive alpha- and betapartitivirus species, and further possible species, were detected from two fungal isolates. The majority of partitiviruses occurred in fungal isolates from both years. Two of the partitiviruses represent phylogenetically divergent forms of Alphapartitivirus, suggesting that they may have evolved under long geographical isolation there. We address the challenge of pairing the two genomic segments of partitiviruses to identify species when multiple partitiviruses co-infect a single host.

A simple and rapid in vitro test for large-scale screening of fungal endophytes from drought-adapted Australian wild plants for conferring water deprivation tolerance and growth promotion in Nicotiana benthamiana seedlings.

Some fungal endophytes confer novel phenotypes and enhance existing ones in plants, including tolerance to water deprivation stress. A range of fungal endophytes was isolated from wild Nicotiana plants growing in arid parts of northern Australia. These were screened for ability to enhance water deprivation stress tolerance by inoculating seedlings of the model plant N. benthamiana in two in vitro tests. Sixty-eight endophyte isolates were co-cultivated with N. benthamiana seedlings on either damp filter paper or on agar medium before being subjected to water deprivation. Seventeen isolates were selected for further testing under water deprivation conditions in a sand-based test in a glasshouse. Only two fungal isolates, Cladosporium cladosporioides (E-162) and an unknown fungus (E-284), significantly enhanced seedling tolerance to moisture deprivation consistently in both in vitro and sand-based tests. Although a strongly significant correlation was observed between any two screening methods, the result of filter paper test was more strongly reflected (r = 0.757, p < 0.001) in results of the glasshouse test, indicating its relative suitability over the agar-based test. In another experiment, the same 17 isolates carried forward to the sand-based test used in the glasshouse screening test were inoculated to N. benthamiana plants in pots in a nutrient-limiting environment to test their influence on growth promotion. Isolates related to C. cladosporioides, Fusarium equiseti, and Thozetella sp. promoted seedling growth by increasing shoot length and biomass. The fungal isolate E-162 (C. cladosporioides) significantly enhanced moisture deprivation tolerance as well as promoted seedling growth.

Evolution of a wild-plant tobamovirus passaged through an exotic host: Fixation of mutations and increased replication.

Tobamovirus is a group of viruses that have become serious pathogens of crop plants. As part of a study informing risk of wild plant virus spill over to crops, we investigated the capacity of a solanaceous-infecting tobamovirus from an isolated indigenous flora to adapt to new exotic hosts. Yellow tailflower mild mottle virus (YTMMV) (genus Tobamovirus, family Virgaviridae) was isolated from a wild plant of yellow tailflower (Anthocercis littoria, family Solanaceae) and initially passaged through a plant of Nicotiana benthamiana, then one of Nicotiana glutinosa where a single local lesion was used to inoculate a N. benthamiana plant. Sap from this plant was used as starting material for nine serial passages through three plant species. The virus titre was recorded periodically, and 85% of the virus genome was sequenced at each passage for each host. Six polymorphic sites were found in the YTMMV genome across all hosts and passages. At five of these, the alternate alleles became fixed in the viral genome until the end of the experiment. Of these five alleles, one was a non-synonymous mutation (U1499C) that occurred only when the virus replicated in tomato. The mutant isolate harbouring U1499C, designated YTMMV-δ, increased its titre over passages in tomato and outcompeted the wild-type isolate when both were co-inoculated to tomato. That YTMMV-δ had greater reproductive fitness in an exotic host than did the wild type isolate suggests YTMMV evolution is influenced by host changes.

ICTV Virus Taxonomy Profile: Potyviridae.

The Potyviridae is the largest family of RNA plant viruses, members of which have single-stranded, positive-sense RNA genomes and flexuous filamentous particles 680-900 nm long and 11-20 nm wide. There are eight genera, distinguished by the host range, genomic features and phylogeny of the member viruses. Genomes range from 8.2 to 11.3 kb, with an average size of 9.7 kb. Most genomes are monopartite but those of members of the genus Bymovirus are bipartite. Some members cause serious disease epidemics in cultivated plants. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Potyviridae, which is available at www.ictv.global/report/potyviridae.

Quarantine Treatments of Imported Nursery Plants and Exported Cut Flowers by Phosphine Gas (PH3) as Methyl Bromide Alternative.

Quarantine treatments by phosphine (PH3) gas have been performed to replace methyl bromide (MeBr) for export cut flowers and imported nursery plant in Korea. In this preliminary study, two dominant insect pests of cut flowers, Tetranychus urticae Koch and Frankliniella occidentalis Pergande, and the dominant insect pest of nursery plants, Planococcus citri Risso, were used to certify optimum concentration and fumigation time, along with evaluation of phytotoxic damages. To validate the results of preliminary tests, quarantine treatments for export cut flowers was performed in a 58-m3 reefer container. When 14 species of cut flowers were fumigated with 2 g m-3 PH3 for 24 h (Ct product was 30.9 g h m-3) at 5 °C, all pests were effectively controlled and no phytotoxic damage were observed on roses and chrysanthemums. On quarantine trials for imported nursery trees, which was performed at 10 m3 scale covered with a PVC-tarpaulin tent, 2 g m-3 of PH3 for 24 h (Ct product was 30.0 g h m-3) at 15 °C was enough to kill all pests and no damage was observed on seven species of nursery plants. Phosphine gas shows the promise as MeBr alternative to perishable commodities in terms of efficacy to certain quarantine pest and maintenance of its quality as well as being a more environmentally safe fumigant.

Novel Endorna-like viruses, including three with two open reading frames, challenge the membership criteria and taxonomy of the Endornaviridae.

Viruses associated with wild orchids and their mycorrhizal fungi are poorly studied. Using a shotgun sequencing approach, we identified eight novel endornavirus-like genome sequences from isolates of Ceratobasidium fungi isolated from pelotons within root cortical cells of wild indigenous orchid species Microtis media, Pterostylis sanguinea and an undetermined species of Pterostylis in Western Australia. They represent the first endornaviruses to be described from orchid mycorrhizal fungi and from the Australian continent. Five of the novel endornaviruses were detected from one Ceratobasidium isolate collected from one Pterostylis plant. The partial and complete viral replicases shared low (9-30%) identities with one another and with endornaviruses described from elsewhere. Four had genome lengths greater than those of previously described endornaviruses, two resembled ascomycete-infecting endornaviruses, and unlike currently described endornaviruses, three had two open reading frames. The unusual features of these new viruses challenge current taxonomic criteria for membership of the family Endornaviridae.

A novel member of the Tombusviridae from a wild legume, Gompholobium preissii.

As part of an investigation into viruses of wild plants in Australia, a contiguous sequence of 3935 nucleotides was obtained after shotgun sequencing of RNA isolated from an asymptomatic wild legume, Gompholobium preissii. Phylogenetic analysis of the sequence revealed that it most closely resembled that of Trailing lespedeza virus 1 (TLV1), a virus isolated from a wild legume in America. The proposed virus, named Gompholobium virus A, and TLV1 are genetically closest to viruses in the genera Alphacarmovirus and Pelarspovirus, family Tombusviridae, but they share features distinguishing them from both groups.

Differential responses to virus challenge of laboratory and wild accessions of australian species of nicotiana, and comparative analysis of RDR1 gene sequences.

Nicotiana benthamiana is a model plant utilised internationally in plant virology because of its apparent hyper-susceptibility to virus infection. Previously, others showed that all laboratory accessions of N. benthamiana have a very narrow genetic basis, probably originating from a single source. It is unknown if responses to virus infection exhibited by the laboratory accession are typical of the species as a whole. To test this, 23 accessions of N. benthamiana were collected from wild populations and challenged with one to four viruses. Additionally, accessions of 21 other Nicotiana species and subspecies from Australia, one from Peru and one from Namibia were tested for susceptibility to the viruses, and for the presence of a mutated RNA-dependent RNA polymerase I allele (Nb-RDR1m) described previously from a laboratory accession of N. benthamiana. All Australian Nicotiana accessions tested were susceptible to virus infections, although there was symptom variability within and between species. The most striking difference was that plants of a laboratory accession of N. benthamiana (RA-4) exhibited hypersensitivity to Yellow tailflower mild mottle tobamovirus infection and died, whereas plants of wild N. benthamiana accessions responded with non-necrotic symptoms. Plants of certain N. occidentalis accessions also exhibited initial hypersensitivity to Yellow tailflower mild mottle virus resembling that of N. benthamiana RA-4 plants, but later recovered. The mutant Nb-RDR1m allele was identified from N. benthamiana RA-4 but not from any of 51 other Nicotiana accessions, including wild accessions of N. benthamiana, demonstrating that the accession of N. benthamiana used widely in laboratories is unusual.

Catharanthus mosaic virus: A potyvirus from a gymnosperm, Welwitschia mirabilis.

A virus from a symptomatic plant of the gymnosperm Welwitschia mirabilis Hook. growing as an ornamental plant in a domestic garden in Western Australia was inoculated to a plant of Nicotiana benthamiana where it established a systemic infection. The complete genome sequence of 9636 nucleotides was determined using high-throughput and Sanger sequencing technologies. The genome sequence shared greatest identity (83% nucleotides and 91% amino acids) with available partial sequences of catharanthus mosaic virus, indicating that the new isolate belonged to that taxon. Analysis of the phylogeny of the complete virus sequence placed it in a monotypic group in the genus Potyvirus. This is the first record of a virus from W. mirabilis, the first complete genome sequence of catharanthus mosaic virus determined, and the first record from Australia. This finding illustrates the risk to natural and managed systems posed by the international trade in live plants and propagules, which enables viruses to establish in new regions and infect new hosts.