DNA metabarcoding reveals high relative abundance of trunk disease fungi in grapevines from Marlborough, New Zealand.

Grapevine trunk diseases (GTDs) are a threat to grape production worldwide, with a diverse collection of fungal species implicated in disease onset. Due to the long-term and complex nature of GTDs, simultaneous detection of multiple microbial species can enhance understanding of disease development. We used DNA metabarcoding of ribosomal internal transcribed spacer 1 (ITS1) sequences, supported by specific PCR and microbial isolation, to establish the presence of trunk pathogens across 11 vineyards (11-26 years old) over three years in Marlborough, the largest wine producing region in New Zealand. Using a reference database of trunk pathogen sequences, species previously associated with GTD, such as Cadophora luteo-olivacea, Diplodia seriata, Diplodia mutila, Neofusicoccum australe, and Seimatosporium vitis, were identified as highly represented across the vineyard region. The well-known pathogens Phaeomoniella chlamydospora and Eutypa lata had especially high relative abundance across the dataset, with P. chlamydospora reads present between 22 and 84% (average 52%) across the vineyards. Screening of sequences against broader, publicly available databases revealed further fungal species within families and orders known to contain pathogens, many of which appeared to be endemic to New Zealand. The presence of several wood-rotting basidiomycetes (mostly Hymenochaetales) was detected for the first time in the Marlborough vineyard region, notably, the native Inonotus nothofagii which was present at 1-2% relative abundance in two vineyards.

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.

Biowaste Mixtures Affecting the Growth and Elemental Composition of Italian Ryegrass ().

Biosolids (sewage sludge) can be beneficially applied to degraded lands to improve soil quality. Plants grown on biosolids-amended soils have distinct concentrations of macronutrients and trace elements, which can be beneficial or present a risk to humans and ecosystems. Potentially, biosolids could be blended with other biowastes, such as sawdust, to reduce the risks posed by rebuilding soils using biosolids alone. We sought to determine the effect of mixing biosolids and sawdust on the macronutrient and trace element concentration of ryegrass over a 5-mo period. was grown in a low fertility soil, typical for marginal farm areas, that was amended with biosolids (1250 kg N ha), biosolids + sawdust (0.5:1) and urea (200 kg N ha), as well as a control. Biosolids increased the growth of from 2.93 to 4.14 t ha. This increase was offset by blending the biosolids with sawdust (3.00 t ha). Urea application increased growth to 4.93 t ha. The biowaste treatments increased N, P, Cu, Mn, and Zn relative to the control, which may be beneficial for grazing animals. Although biowaste application caused elevated Cd concentrations (0.15-0.24 mg kg) five- to eightfold higher than control and urea treatments, these were below levels that are likely to result in unacceptable concentrations in animal tissues. Mixing biosolids with sawdust reduced Cd uptake while still resulting in increased micronutrient concentrations (P, S, Mn, Zn, Cu) in plants. There were significant changes in the elemental uptake during the experiment, which was attributed to the decomposition of the sawdust.

Cross-kingdom host shifts of phytomyxid parasites.

BACKGROUND: Phytomyxids (plasmodiophorids and phagomyxids) are cosmopolitan, obligate biotrophic protist parasites of plants, diatoms, oomycetes and brown algae. Plasmodiophorids are best known as pathogens or vectors for viruses of arable crops (e.g. clubroot in brassicas, powdery potato scab, and rhizomania in sugar beet). Some phytomyxid parasites are of considerable economic and ecologic importance globally, and their hosts include important species in marine and terrestrial environments. However most phytomyxid diversity remains uncharacterised and knowledge of their relationships with host taxa is very fragmentary. RESULTS: Our molecular and morphological analyses of phytomyxid isolates-including for the first time oomycete and sea-grass parasites-demonstrate two cross-kingdom host shifts between closely related parasite species: between angiosperms and oomycetes, and from diatoms/brown algae to angiosperms. Switching between such phylogenetically distant hosts is generally unknown in host-dependent eukaryote parasites. We reveal novel plasmodiophorid lineages in soils, suggesting a much higher diversity than previously known, and also present the most comprehensive phytomyxid phylogeny to date. CONCLUSION: Such large-scale host shifts between closely related obligate biotrophic eukaryote parasites is to our knowledge unique to phytomyxids. Phytomyxids may readily adapt to a wide diversity of new hosts because they have retained the ability to covertly infect alternative hosts. A high cryptic diversity and ubiquitous distribution in agricultural and natural habitats implies that in a changing environment phytomyxids could threaten the productivity of key species in marine and terrestrial environments alike via host shift speciation.

Insect phylogeny structures the bacterial communities in the microbiome of psyllids (Hemiptera: Psylloidea) in Aotearoa New Zealand.

The bacterial microbiome of psyllids has been studied for decades, with a strong focus on the primary and secondary endosymbionts capable of providing essential amino acids for the insects' diet and therefore playing a key role in the insects' ability to radiate on novel plant hosts. Here, we combine metabarcoding analysis of the bacterial communities hosted by psyllids with a multi-gene phylogenetic analysis of the insect hosts to determine what factors influence the bacterial diversity of the psyllids' microbiomes, especially in the context of the dispersal and evolutionary radiation of these insects in Aotearoa New Zealand. Using multi-gene phylogenetics with COI, 18S and EF-1α sequences from 102 psyllid species, we confirmed for the first time monophyly for all the six genera of native/endemic Aotearoa New Zealand psyllids, with indications that they derive from at least six dispersal events to the country. This also revealed that, after its ancestral arrival, the genus Powellia has radiated onto a larger and more diverse range of plants than either Psylla or Ctenarytaina, which is uncommon amongst monophyletic psyllids globally. DNA metabarcoding of the bacterial 16S gene here represents the largest dataset analysed to date from psyllids, including 246 individuals from 73 species. This provides novel evidence that bacterial diversity across psyllid species is strongly associated with psyllid phylogenetic structure, and to a lesser degree to their host plant association and geographic distribution. Furthermore, while the strongest co-phylogenetic signals were derived from the primary and secondary symbionts, a signal of phylosymbiosis was still retained among the remaining taxa of the bacterial microbiome, suggesting potential vertical transmission of bacterial lineages previously unknown to have symbiotic roles.

Grapevines escaping trunk diseases in New Zealand vineyards have a distinct microbiome structure.

Grapevine trunk diseases (GTDs) are a substantial challenge to viticulture, especially with a lack of available control measures. The lack of approved fungicides necessitates the exploration of alternative controls. One promising approach is the investigation of disease escape plants, which remain healthy under high disease pressure, likely due to their microbiome function. This study explored the microbiome of grapevines with the disease escape phenotype. DNA metabarcoding of the ribosomal internal transcribed spacer 1 (ITS1) and 16S ribosomal RNA gene was applied to trunk tissues of GTD escape and adjacent diseased vines. Our findings showed that the GTD escape vines had a significantly different microbiome compared with diseased vines. The GTD escape vines consistently harbored a higher relative abundance of the bacterial taxa Pseudomonas and Hymenobacter. Among fungi, Aureobasidium and Rhodotorula were differentially associated with GTD escape vines, while the GTD pathogen, Eutypa, was associated with the diseased vines. This is the first report of the link between the GTD escape phenotype and the grapevine microbiome.

Evolution of Rhizaria: new insights from phylogenomic analysis of uncultivated protists.

BACKGROUND: Recent phylogenomic analyses have revolutionized our view of eukaryote evolution by revealing unexpected relationships between and within the eukaryotic supergroups. However, for several groups of uncultivable protists, only the ribosomal RNA genes and a handful of proteins are available, often leading to unresolved evolutionary relationships. A striking example concerns the supergroup Rhizaria, which comprises several groups of uncultivable free-living protists such as radiolarians, foraminiferans and gromiids, as well as the parasitic plasmodiophorids and haplosporids. Thus far, the relationships within this supergroup have been inferred almost exclusively from rRNA, actin, and polyubiquitin genes, and remain poorly resolved. To address this, we have generated large Expressed Sequence Tag (EST) datasets for 5 species of Rhizaria belonging to 3 important groups: Acantharea (Astrolonche sp., Phyllostaurus sp.), Phytomyxea (Spongospora subterranea, Plasmodiophora brassicae) and Gromiida (Gromia sphaerica). RESULTS: 167 genes were selected for phylogenetic analyses based on the representation of at least one rhizarian species for each gene. Concatenation of these genes produced a supermatrix composed of 36,735 amino acid positions, including 10 rhizarians, 9 stramenopiles, and 9 alveolates. Phylogenomic analyses of this large dataset revealed a strongly supported clade grouping Foraminifera and Acantharea. The position of this clade within Rhizaria was sensitive to the method employed and the taxon sampling: Maximum Likelihood (ML) and Bayesian analyses using empirical model of evolution favoured an early divergence, whereas the CAT model and ML analyses with fast-evolving sites or the foraminiferan species Reticulomyxa filosa removed suggested a derived position, closely related to Gromia and Phytomyxea. In contrast to what has been previously reported, our analyses also uncovered the presence of the rhizarian-specific polyubiquitin insertion in Acantharea. Finally, this work reveals another possible rhizarian signature in the 60S ribosomal protein L10a. CONCLUSIONS: Our study provides new insights into the evolution of Rhizaria based on phylogenomic analyses of ESTs from three groups of previously under-sampled protists. It was enabled through the application of a recently developed method of transcriptome analysis, requiring very small amount of starting material. Our study illustrates the potential of this method to elucidate the early evolution of eukaryotes by providing large amount of data for uncultivable free-living and parasitic protists.

Rapid increases in soil pH solubilise organic matter, dramatically increase denitrification potential and strongly stimulate microorganisms from the Firmicutes phylum.

Rapid and transient changes in pH frequently occur in soil, impacting dissolved organic matter (DOM) and other chemical attributes such as redox and oxygen conditions. Although we have detailed knowledge on microbial adaptation to long-term pH changes, little is known about the response of soil microbial communities to rapid pH change, nor how excess DOM might affect key aspects of microbial N processing. We used potassium hydroxide (KOH) to induce a range of soil pH changes likely to be observed after livestock urine or urea fertilizer application to soil. We also focus on nitrate reductive processes by incubating microcosms under anaerobic conditions for up to 48 h. Soil pH was elevated from 4.7 to 6.7, 8.3 or 8.8, and up to 240-fold higher DOM was mobilized by KOH compared to the controls. This increased microbial metabolism but there was no correlation between DOM concentrations and CO2 respiration nor N-metabolism rates. Microbial communities became dominated by Firmicutes bacteria within 16 h, while few changes were observed in the fungal communities. Changes in N-biogeochemistry were rapid and denitrification enzyme activity (DEA) increased up to 25-fold with the highest rates occurring in microcosms at pH 8.3 that had been incubated for 24-hour prior to measuring DEA. Nitrous oxide reductase was inactive in the pH 4.7 controls but at pH 8.3 the reduction rates exceeded 3,000 ng N2-N g-1 h-1 in the presence of native DOM. Evidence for dissimilatory nitrate reduction to ammonium and/or organic matter mineralisation was observed with ammonium increasing to concentrations up to 10 times the original native soil concentrations while significant concentrations of nitrate were utilised. Pure isolates from the microcosms were dominated by Bacillus spp. and exhibited varying nitrate reductive potential.

Not in your usual Top 10: protists that infect plants and algae.

Fungi, nematodes and oomycetes belong to the most prominent eukaryotic plant pathogenic organisms. Unicellular organisms from other eukaryotic lineages, commonly addressed as protists, also infect plants. This review provides an introduction to plant pathogenic protists, including algae infecting oomycetes, and their current state of research.

Intron-rich gene structure in the intracellular plant parasite Plasmodiophora brassicae.

Plasmodiophora brassicae, a pathogen of Brassicaceae plants, is grouped within the eukaryotic supergroup, the Rhizaria. Although a large diversity of protists is found in the Rhizaria, genomes of organisms within the group have barely been examined. In this study, we identified DNA sequences spanning or flanking 24 P. brassicae genes, eventually sequencing close to 44 kb of genomic DNA. Evidence from this preliminary genome survey suggested that splicing is an important feature of P. brassicae gene expression; the P. brassicae genes were rich in spliceosomal introns and two mini-exons of less than 20 bp were identified. Consensus splice sites and branch-point sequences in P. brassicae introns were similar to those found in other eukaryotes. Examination of the promoter and transcription start sites of genes indicated that P. brassicae transcription is likely to begin from initiator elements rather than TATA-box containing promoters. Where neighbouring genes were confirmed, intergenic distances were short, ranging from 44 to 470 bp, but a number of larger DNA fragments containing no obvious genes were also sequenced.

Elongation Factor-1α Accurately Reconstructs Relationships Amongst Psyllid Families (Hemiptera: Psylloidea), with Possible Diagnostic Implications.

The superfamily Psylloidea (Hemiptera: Sternorrhyncha) lacks a robust multigene phylogeny. This impedes our understanding of the evolution of this group of insects and, consequently, an accurate identification of individuals, of their plant host associations, and their roles as vectors of economically important plant pathogens. The conserved nuclear gene elongation factor-1 alpha (EF-1α) has been valuable as a higher-level phylogenetic marker in insects and it has also been widely used to investigate the evolution of intron/exon structure. To explore evolutionary relationships among Psylloidea, polymerase chain reaction amplification and nucleotide sequencing of a 250-bp EF-1α gene fragment was applied to psyllids belonging to five different families. Introns were detected in three individuals belonging to two families. The nine genera belonging to the family Aphalaridae all lacked introns, highlighting the possibility of using intron presence/absence as a diagnostic tool at a family level. When paired with cytochrome oxidase I gene sequences, the 250 bp EF-1α sequence appeared to be a very promising higher-level phylogenetic marker for psyllids.

Identification of genes from the obligate intracellular plant pathogen, Plasmodiophora brassicae.

Plasmodiophora brassicae is an intracellular pathogen that infects plants in the Brassicaceae family. Although an important pathogen group, information on the genomic makeup of the plasmodiophorids is almost completely lacking. We performed suppression subtractive hybridization (SSH) between RNA from P. brassicae-infected and uninfected Arabidopsis tissue, then screened 232 clones from the resulting SSH library. In addition, we used an oligo-capping procedure to screen 305 full-length cDNA clones from the infected tissue. A total of 76 new P. brassicae gene sequences were identified, the majority of which were extended to full length at the 5' end by the use of RACE amplification. Many of the unisequences were predicted to contain signal peptides for ER translocation. Although we located few sequences in total, these markedly increase available data from the plasmodiophorids, and provide new opportunities to examine plasmodiophorid biology. Our study also points towards the best methods for future plasmodiophorid gene discovery.

Mitochondrial genome sequence of the potato powdery scab pathogen Spongospora subterranea.

Spongospora subterranea is a soil-borne obligate parasite responsible for potato powdery scab disease. S. subterranea is a member of the order Plasmodiophorida, a protist taxa that is related to Cercozoa and Foraminifera but the fine details of these relationships remain unresolved. Currently there is only one available complete mtDNA sequence of a cercozoan, Bigelowiella natans. In this work, the mitochondrial sequence of a S. subterranea isolate infecting an Andean variety of S. tuberosum ssp. andigena (Diacol-Capiro) is presented. The mtDNA codes for 16 proteins of the respiratory chain, 11 ribosomal proteins, 3 ribosomal RNAs, 24 tRNAs, a RNA processing RNaseP, a RNA-directed polymerase, and two proteins of unknown function. This is the first report of a mtDNA genome sequence from a plasmodiophorid and will be useful in clarifying the phylogenetic relationship of this group to other members in the supergroup Rhizaria once more mtDNA sequences are available.

A novel methyltransferase from the intracellular pathogen Plasmodiophora brassicae methylates salicylic acid.

The obligate biotrophic pathogen Plasmodiophora brassicae causes clubroot disease in Arabidopsis thaliana, which is characterized by large root galls. Salicylic acid (SA) production is a defence response in plants, and its methyl ester is involved in systemic signalling. Plasmodiophora brassicae seems to suppress plant defence reactions, but information on how this is achieved is scarce. Here, we profile the changes in SA metabolism during Arabidopsis clubroot disease. The accumulation of SA and the emission of methylated SA (methyl salicylate, MeSA) were observed in P. brassicae-infected Arabidopsis 28 days after inoculation. There is evidence that MeSA is transported from infected roots to the upper plant. Analysis of the mutant Atbsmt1, deficient in the methylation of SA, indicated that the Arabidopsis SA methyltransferase was not responsible for alterations in clubroot symptoms. We found that P. brassicae possesses a methyltransferase (PbBSMT) with homology to plant methyltransferases. The PbBSMT gene is maximally transcribed when SA production is highest. By heterologous expression and enzymatic analyses, we showed that PbBSMT can methylate SA, benzoic and anthranilic acids.

Genomics of biotrophic, plant-infecting plasmodiophorids using in vitro dual cultures.

The plasmodiophorids are a phylogenetically distinct group of parasitic protists that infect plants and stramenopiles, causing several important agricultural diseases. Because of the obligate intracellular part of their lifecycle, none of the plasmodiophorids has been axenically cultured. Further, the molecular biology of the plasmodiophorids is poorly understood because pure cultures are not available from any species. We report on an in-vitro dual culture system of the plasmodiophorids Plasmodiophora brassicae and Spongospora subterranea with their respective plant hosts, Brassica rapa and Solanum tuberosum. We show that these plasmodiophorids are capable of initiating and maintaining stable, long-term plant cell callus cultures in the absence of exogenous plant growth regulators. We show that callus cultures harbouring S. subterranea provide an excellent starting material for gene discovery from this organism by constructing a pilot-scale DNA library. Bioinformatic analysis of the sequences established that almost all of the DNA clones from this library were from S. subterranea rather than the plant host. The Spongospora genome was found to be rich in retrotransposable elements, and Spongospora protein-coding genes were shown to contain introns. The sequence of a near full-length non-LTR retrotransposon was obtained, the first transposable element reported from a cercozoan protist.

The liverwort Marchantia foliacea forms a specialized symbiosis with arbuscular mycorrhizal fungi in the genus Glomus.

Microscopic evidence suggests that fungi forming endosymbioses with liverworts in the Marchantiales are arbuscular mycorrhizal (AM) fungi from the Glomeromycota. Polymerase chain reaction amplification of ribosomal sequences confirmed that endophytes of the New Zealand liverwort, Marchantia foliacea, were members of the genus Glomus. Endophytes from two Glomus rDNA phylotypes were repeatedly isolated from geographically separated liverwort samples. Multiple phylotypes were present in the same liverwort patch. The colonizing Glomus species exhibited substantial internal transcribed spacer sequence variation within phylotypes. This work suggests that certain liverwort species may serve as a model for studying DNA sequence variation in colonizing AM phylotypes and specificity in AM-host relationships.