An internet-based bioinformatics toolkit for plant biosecurity diagnosis and surveillance of viruses and viroids.

BACKGROUND: Detection and preventing entry of exotic viruses and viroids at the border is critical for protecting plant industries trade worldwide. Existing post entry quarantine screening protocols rely on time-consuming biological indicators and/or molecular assays that require knowledge of infecting viral pathogens. Plants have developed the ability to recognise and respond to viral infections through Dicer-like enzymes that cleave viral sequences into specific small RNA products. Many studies reported the use of a broad range of small RNAs encompassing the product sizes of several Dicer enzymes involved in distinct biological pathways. Here we optimise the assembly of viral sequences by using specific small RNA subsets. RESULTS: We sequenced the small RNA fractions of 21 plants held at quarantine glasshouse facilities in Australia and New Zealand. Benchmarking of several de novo assembler tools yielded SPAdes using a kmer of 19 to produce the best assembly outcomes. We also found that de novo assembly using 21-25 nt small RNAs can result in chimeric assemblies of viral sequences and plant host sequences. Such non-specific assemblies can be resolved by using 21-22 nt or 24 nt small RNAs subsets. Among the 21 selected samples, we identified contigs with sequence similarity to 18 viruses and 3 viroids in 13 samples. Most of the viruses were assembled using only 21-22 nt long virus-derived siRNAs (viRNAs), except for one Citrus endogenous pararetrovirus that was more efficiently assembled using 24 nt long viRNAs. All three viroids found in this study were fully assembled using either 21-22 nt or 24 nt viRNAs. Optimised analysis workflows were customised within the Yabi web-based analytical environment. We present a fully automated viral surveillance and diagnosis web-based bioinformatics toolkit that provides a flexible, user-friendly, robust and scalable interface for the discovery and diagnosis of viral pathogens. CONCLUSIONS: We have implemented an automated viral surveillance and diagnosis (VSD) bioinformatics toolkit that produces improved viruses and viroid sequence assemblies. The VSD toolkit provides several optimised and reusable workflows applicable to distinct viral pathogens. We envisage that this resource will facilitate the surveillance and diagnosis viral pathogens in plants, insects and invertebrates.

Phytophthora niederhauserii sp. nov., a polyphagous species associated with ornamentals, fruit trees and native plants in 13 countries.

A non-papillate, heterothallic Phytophthora species first isolated in 2001 and subsequently from symptomatic roots, crowns and stems of 33 plant species in 25 unrelated botanical families from 13 countries is formally described here as a new species. Symptoms on various hosts included crown and stem rot, chlorosis, wilting, leaf blight, cankers and gumming. This species was isolated from Australia, Hungary, Israel, Italy, Japan, the Netherlands, Norway, South Africa, Spain, Taiwan, Turkey, the United Kingdom and United States in association with shrubs and herbaceous ornamentals grown mainly in greenhouses. The most prevalent hosts are English ivy (Hedera helix) and Cistus (Cistus salvifolius). The association of the species with acorn banksia (Banksia prionotes) plants in natural ecosystems in Australia, in affected vineyards (Vitis vinifera) in South Africa and almond (Prunus dulcis) trees in Spain and Turkey in addition to infection of shrubs and herbaceous ornamentals in a broad range of unrelated families are a sign of a wide ecological adaptation of the species and its potential threat to agricultural and natural ecosystems. The morphology of the persistent non-papillate ellipsoid sporangia, unique toruloid lobate hyphal swellings and amphigynous antheridia does not match any of the described species. Phylogenetic analysis based on sequences of the ITS rDNA, EF-1α, and ß-tub supported that this organism is a hitherto unknown species. It is closely related to species in ITS clade 7b with the most closely related species being P. sojae. The name Phytophthora niederhauserii has been used in previous studies without the formal description of the holotype. This name is validated in this manuscript with the formal description of Phytophthora niederhauserii Z.G. Abad et J.A. Abad, sp. nov. The name is coined to honor Dr John S. Niederhauser, a notable plant pathologist and the 1990 World Food Prize laureate.

Phytophthora bisheria sp. nov., a new species identified in isolates from the Rosaceous raspberry, rose and strawberry in three continents.

A homothallic semipapillate slow growing Phytophthora species associated with root rot of strawberries from greenhouse-grown plants in North Carolina, USA, root rot of roses in the Netherlands, and root rot of raspberry in Knoxfield, Australia, was identified. The main character of this organism is the production of paragynous antheridia with broad attachment to the oogonial wall. The morphology of the pathogen does not match that of any of the more than 85 described Phytophthora species. Phylogenetic analysis based on sequences of the internal transcribed spacer rDNA region (ITS1-5.8S-ITS2) of this taxon and those from other Phytophthora species from GenBank supports the conclusion that this organism is an unreported new species. In the phylogenetic tree with other reported Phytophthora species at the GenBank, the new species is more closely related to others in ITS clade 2 comprising semipapillate taxa including P. botryosa, P. citrophthora, P. colocasiae, P. meadii, P. citricola, P. inflata, P.tropicalis, P. capsici, Phytophthora sp. 'glovera' and P. multivesiculata. The most closely related species is P. multivesiculata isolated from Cymbidium orchid in the Netherlands. In this paper we describe the morphological characteristics and the phylogenetic relationships that support the description of this taxon as a new species Phytophthora bisheria sp. nov.

Molecular Identification of Oidium neolycopersici as the Causal Agent of the Recent Tomato Powdery Mildew Epidemics in North America.

A previous morphological study of Oidium anamorphs responsible for the recent tomato (Lycopersicon esculentum) powdery mildew outbreaks worldwide suggested that, despite controversial data in the literature, the North American epidemics were caused solely by a newly erected species, O. neolycopersici. We report here the first molecular evidence that the North American anamorphs do belong to O. neolycopersici. The internal transcribed spacer sequences of the North American anamorphs of this study were identical with those of three Japanese and four European specimens of O. neolycopersici. A morphological study confirmed that all the North American Oidium anamorphs included in this study produced conidia singly, similar to O. neolycopersici. These fungi were readily distinguished from O. lycopersici, which produces conidia in chains and is known to infect tomato only in Australia. The phylogenetic analysis showed that O. neolycopersici is a distinct powdery mildew species, and it is neither identical nor closely related to any known polyphagous species of the Erysiphaceae. Apparently, it was introduced into the United States and Canada only in the 1990s, but its origin is still unknown.

The molecular phylogeny of the white blister rust genus Pustula reveals a case of underestimated biodiversity with several undescribed species on ornamentals and crop plants.

Despite their economic importance, the knowledge of the biodiversity of many plant pathogens is still fragmentary. In this study we show that this is true also for the white blister rust genus Pustula that is parasitic on several genera in the asterids, including sunflower and the gentian, Eustoma. It is revealed that several distinct species exist in Pustula, suggesting that species are mostly host genus specific. No geographic patterns were observed in the occurrence of Pustula, the host range of which includes the Araliaceae, Asteraceae, Gentianaceae, and Goodeniaceae. Evidence points to these becoming hosts as a result of jumps from the Asteraceae, with subsequent host-specific adaptation and speciation. Among the undescribed species are pathogens of economic importance, e.g. the white blister rusts of sunflower, or with still restricted geographical ranges, e.g. Pustula centaurii, which could potentially spread with international seed trade, if no quarantine restrictions are implemented.

Neoerysiphe kerribeeensis sp. nov. (Ascomycota: Erysiphales), a new species of Neoerysiphe on native and introduced species of Senecio (Asteraceae) in Australia.

Anamorphic powdery mildew fungi on introduced taxa of Senecio and Pericallis × hybrida in Australia have previously been identified as Neoerysiphe cumminsiana on the basis of a combination of Euoidium-type conidiophores and lobed mycelial and germ tube appressoria. But, two specimens with chasmothecia on the indigenous Senecio glossanthus did not agree with published descriptions of N. cumminsiana. The teleomorph of the S. glossanthus mildew differed from that of N. cumminsiana in the morphology of its peridial cells, the pigmentation of its appendages, and the morphology and pigmentation of some secondary hyphae. Ribosomal DNA ITS sequences from the two S. glossanthus mildew specimens and five other specimens of Senecio mildews from south-eastern Australia demonstrated that all Australian Senecio mildews are conspecific and distinct from the northern hemisphere Senecio mildew (N. cumminsiana) and from other Neoerysiphe taxa. Based on morphological characters and rDNA sequence data, the Australian Senecio mildew is described as a new species, Neoerysiphe kerribeeensis. This is the first native teleomorphic powdery mildew described from Australia.

Phylogeny and taxonomy of the oak powdery mildew Erysiphe alphitoides sensu lato.

Phylogenetic analyses of Erysiphe alphitoides s. lat. using sequences of the rDNA ITS region and the D1/D2 domains of the 28S rDNA revealed a complex consisting of several genetically and morphologically distinguished taxa, including the already described Erysiphe alphitoides s. str. and E. hypophylla. The ascomata (chasmothecia) of E. hypophylla are morphologically very similar to those of E. alphitoides, but the two species are easily distinguishable by their symptoms, as well as the shape and size of the conidia. The fungus on Quercus phillyraeoides, distributed in warmer regions in southern Japan, is genetically clearly separated from E. alphitoides s. str., and morphologically characterized by having chasmothecia with appendages consistently shorter than the chasmothecial diameter. This fungus, named Erysiphe quercicola in this paper, is also able to infect some other oak species, and it is genetically identical with anamorphs on some tropical trees of other host genera. Collections of E. alphitoides s. lat. on Quercus acutissima and Q. variabilis, both belonging to Quercus sect. Cerris, are genetically distinct from E. alphitoides s. str., E. hypophylla and E. quercicola. They form two genetically and morphologically differentiated groups. The hypophyllous taxon on Q. acutissima and Q. variabilis, named Erysiphe hypogena in this paper, is characterized by forming distinctive persistent hypophyllous mycelial patches, causing necrotic discolouration of the host tissue. The epiphyllous taxon on these hosts, for which the name E. epigena is proposed, differs in having epiphyllous mycelium, smaller chasmothecia with fewer appendages, and does not cause leaf discolouration.

Genetic variation within Podosphaera tridactyla reveals a paraphyletic species complex with biological specialization towards specific Prunus subgenera.

Podosphaera tridactyla (Ascomycota: Erysiphales) is a morphologically variable species occurring on Prunus s. lat. In order to assess the genetic variation within this species, the rDNA ITS region was amplified from 29 specimens from a range of Prunus species collected in Australia, Switzerland, and Korea. RFLP analysis of the PCR products revealed six groups, and a comparison of sequences from representatives of these groups revealed three clades: Clade I contained all specimens from Prunus subgen. Prunus; Clade 2 specimens from a variety of Prunus subgenera, except subgen. Prunus, and Podosphaera longiseta was closely allied to this clade; and Clade 3 contained two specimens, from Japan and Korea. Phylogenetic analysis comparing P. tridactyla with of a range of Podosphaera species suggests that P. tridactyla is paraphyletic.

Two new closely related species of Pseudocercospora on unrelated host families from south-eastern Australia.

A species of Pseudocercospora causing foliar lesions on Hibbertia aspera (Dilleniaceae) is morphologically indistingushable from a species of Pseudocercospora causing foliar lesions on Platylobium formosum (Fabaceae) from the same locality. In order to assess the degree to which these fungi were related, we sequenced the ribosomal DNA ITS region of cultures derived from single conidial isolates. Cultures were obtained from four specimens of each respective host. The ITS sequences for cultures derived from the same host were identical, with the exception of one isolate that had a 4 bp insert, while the Pseudocercospora species from Hibbertia consistently differed at three bases from the species isolated from Platylobium. These data suggest that two distinct species of Pseudocercospora are represented, and that they probably result from a recent evolutionary divergence. P. platylobii and P. hibbertiae-asperae spp. nov. are described and illustrated.