Analysis of the complete genome sequence of Cucumber mosaic virus strain K

The complete genome sequence of Cucumber mosaic virus strain K was determined by deep RNA sequencing. The tripartite genome consists of a 3,382-nucleotide (nt) RNA1, a 3,050-nt RNA2, and a 2,218-nt RNA3 segment. Phylogenetic analysis placed RNA1 and RNA2 in subgroup IB. However, RNA3 grouped with subgroup IA isolates, indicating a likely recombination event. © 2018 Moyle et al

Epidemic spread of smut fungi (Quambalaria) by sexual reproduction in a native pathosystem

Quambalaria are fungal pathogens of Corymbia, Eucalyptus and related genera of Myrtaceae. They are smut fungi (Ustilaginomycota) described from structures that resemble conidia and conidiophores. Whether these spore forms have asexual or sexual roles in life cycles of Quambalaria is unknown. An epidemic of Q. pitereka destroyed plantations of Corymbia in New South Wales and Queensland (Australia) in 2008. We sampled 177 individuals from three plantations of C. variegata and used AFLPs to test hypotheses that the epidemic was spread by asexual reproduction and dominated by a single genotype. There was high genotypic diversity across ≥600 AFLP loci in the pathogen populations at each plantation, and evidence of sexual reproduction based on neighbour-net analyses and rejection of linkage disequilibrium. The populations were not structured by host or location. Our data did not support a hypothesis of asexual reproduction but instead that Q. pitereka spreads exclusively by sexual reproduction, similar to life cycles of other smut fungi. Epidemics were exacerbated by monocultures of Corymbia established from seed collected from a single provenance. This study showcases an example of an endemic pathogen, Q. pitereka, with a strictly outbreeding life cycle that has caused epidemics when susceptible hosts were planted in large monoculture plantations

Both Constitutive and Infection‐Responsive Secondary Metabolites Linked to Resistance against Austropuccinia psidii (Myrtle Rust) in Melaleuca quinquenervia

Austropuccinia psidii is a fungal plant pathogen that infects species within the Myrtaceae, causing the disease myrtle rust. Myrtle rust is causing declines in populations within natural and managed ecosystems and is expected to result in species extinctions. Despite this, variation in response to A. psidii exist within some species, from complete susceptibility to resistance that prevents or limits infection by the pathogen. Untargeted metabolomics using Ultra Performance Liquid Chromatography with Ion Mobility followed by analysis using MetaboAnalyst 3.0, was used to ex-plore the chemical defence profiles of resistant, hypersensitive and susceptible phenotypes within Melaleuca quinquenervia during the early stages of A. psidii infection. We were able to identify three separate pools of secondary metabolites: (i) metabolites classified structurally as flavonoids that were naturally higher in the leaves of resistant individuals prior to infection, (ii) organoheterocyclic and carbohydrate‐related metabolites that varied with the level of host resistance post‐infection, and (iii) metabolites from the terpenoid pathways that were responsive to disease progression re-gardless of resistance phenotype suggesting that these play a minimal role in disease resistance during the early stages of colonization of this species. Based on the classes of these secondary me-tabolites, our results provide an improved understanding of key pathways that could be linked more generally to rust resistance with particular application within Melaleuca. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

Transcriptome analysis of Eucalyptus grandis implicates brassinosteroid signaling in defense against myrtle rust (Austropuccinia psidii)

Eucalyptus grandis, in its native Australian range, varies in resistance to Austropuccinia psidii (syn. Puccinia psidii). The biotrophic rust fungus, A. psidii is the causal agent of myrtle rust and poses a serious threat to Australian biodiversity. The pathogen produces yellow pustules of urediniospores on young leaves and shoots, resulting in shoot tip dieback, stunted growth, and death. Dissecting the underlying mechanisms of resistance against this pathogen will contribute to improved breeding and control strategies to mitigate its devastating effects. The aim of this study was to determine the molecular dialogue between E. grandis and A. psidii, using an RNA-sequencing approach. Resistant and susceptible E. grandis seedlings grown from seed collected across its natural range were inoculated with the pandemic biotype of A. psidii. The leaf tissue was harvested at 12-h post inoculation (hpi), 1-day post inoculation (dpi), 2-dpi and 5-dpi and subjected to RNA-sequencing using Illumina 50 bp PE reads to a depth of 40 million reads per sample. Differential gene expression and gene ontology enrichment indicated that the resistant seedlings showed controlled, coordinated responses with a hypersensitive response, while the susceptible seedlings showed no systemic response against myrtle rust. Brassinosteroid signaling was apparent as an enriched term in the resistant interaction at 2-dpi, suggesting an important role of this phytohormone in defense against the pathogen. Brassinosteroid mediated signaling genes were also among the candidate genes within two major disease resistance loci (Puccinia psidii resistance), Ppr3 and Ppr5. While brassinosteroids have been tagged as positive regulators in other plant disease resistance interactions, this is the first report in the Eucalyptus – Austropuccinia psidii interaction. Furthermore, several putative resistance genes, underlying known resistance loci and implicated in the interaction have been identified and highlighted for future functional studies. This study provided further insights into the molecular interactions between E. grandis and A. psidii, contributing to our understanding of this pathosystem.The South African National Research Foundation (NRF) and the Technology Innovation Agency of South Africa.https://www.frontiersin.org/journals/forests-and-global-change#am2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Chromium sequencing: The doors open for genomics of obligate plant pathogens

It is challenging to sequence and assemble genomes of obligate plant pathogens and microorganisms because of limited amounts of DNA, comparatively large genomes and high numbers of repeat regions. We sequenced the 1.2 gigabase genome of an obligate rust fungus, Austropuccinia psidii, the cause of rust on Myrtaceae, with a Chromium 10X library. This technology has mostly been applied for single-cell sequencing in immunological studies of mammals. We compared scaffolds of a genome assembled from the Chromium library with one assembled from combined paired-end and mate-pair libraries, sequenced with Illumina HiSeq. Chromium 10X provided a superior assembly, in terms of number of scaffolds, N50 and number of genes recovered. It required less DNA than other methods and was sequenced and assembled at a lower cost. Chromium sequencing could provide a solution to sequence and assemble genomes of obligate plant pathogens where the amount of available DNA is a limiting factor. © 2018 Future Science. All rights reserved

Sexual reproduction is the null hypothesis for life cycles of rust fungi

Sexual reproduction, mutation, and reassortment of nuclei increase genotypic diversity in rust fungi. Sexual reproduction is inherent to rust fungi, coupled with their coevolved plant hosts in native pathosystems. Rust fungi are hypothesised to exchange nuclei by somatic hybridisation with an outcome of increased genotypic diversity, independent of sexual reproduction. We provide criteria to demonstrate whether somatic exchange has occurred, including knowledge of parental haplotypes and rejection of fertilisation in normal rust life cycles

Fungal Genomics Challenges the Dogma of Name-Based Biosecurity

Microorganisms have inadvertently been spread via the global movement and trade of their substrates, such as animals, plants, and soil. This intercontinental exchange in the current era of globalisation has given rise to significant increases in the distribution of known pests and pathogens. Importantly, it has also resulted in many novel, emerging, infectious diseases. Biosecurity and quarantine, which aim to prevent the establishment of foreign or harmful organisms in a non-native area, are under significant pressure due to the massive increases in travel and trade. Traditionally, quarantine regulations have been implemented based on pathogens that already cause significant disease problems on congener hosts in other parts of the world (e.g., Q-bank, available at http://www.q-bank.eu). Well-known pathogens are described, named, and studied to determine their disease cycle, epidemiology, and impact. Their importance is assessed based on their risk of infection, establishment, and economic or environmental consequences. This then shapes phytosanitary practices.The Tree Protection Co-operative Programme (TPCP), THRIP initiative of the Department of Trade and Industry, Department of Science and Technology (DST)/ National Research Foundation (NRF), and Centre of Excellence in Tree Health Biotechnology (CTHB).http://www.plospathogens.orgam2016Forestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologyPlant Scienc

The life cycle and field epidemiology of Uromycladium acaciae (Pucciniales) on Acacia mearnsii in South Africa

Uromycladium acaciae has damaged plantations of Acacia mearnsii in southern Africa since 2013. Uredinia of a species of Uromycladium have been known on A. mearnsii in South Africa since the 1980s. However, the recent damage is associated with telia and spermogonia. Uredinia and telia were previously treated as conspecific with a phylogenetic species concept. However, uredinia did not form after previous artificial inoculation experiments with teliospores. Controlled studies identified the optimum conditions for basidiospore infection, but the optimum conditions for sporulation and dispersal have not been identified. To investigate the life cycle and field epidemiology of Uromycladium on A. mearnsii, spores were trapped weekly and development of disease symptoms and plant phenology were monitored monthly at three plantations. Telia and spermogonia developed independently from uredinia, and nucleotide polymorphisms between rDNA of uredinia and telia were fixed based on high throughput sequencing and PCRs. All three weather variables measured had a significant effect on teliospore abundance at two of the three sites. Teliospore abundance was greatest during trapping periods when mean relative humidity was high, mean rainfall was 4–5 mm day−1 and mean temperature was 15–16°C. Teliospore counts peaked at the end of summer, potentially the result of epidemic build-up. Results support the hypothesis that despite sharing a most recent common ancestor, uredinia on A. mearnsii in southern Africa are independent to the life cycle of the telial rust, which likely constitutes a new introduction. Furthermore, teliospores of U. acaciae disperse under wet conditions, and the wet season between October and March is the optimal period for wattle rust development.Members of the Tree Protection Co-operative Programme (TPCP), the Department of Science and Technology (DST)/National Research Foundation (NRF) Centre of Excellence in Tree Health Biotechnology (CTHB) and the Wattle Rust Steering Committee funded by the Sector Innovation Fund (SIF) of the Department of Science and Technology (DST). The Genomics Research Institute of the University of Pretoria provided funding for high-throughput sequencing of Uromycladium acaciae. Stuart Fraser acknowledges Postdoctoral Fellowship funding from the University of Pretoria and New Zealand Ministry for Business Innovation and Employment through the Science Strategic Investment Fund.http://wileyonlinelibrary.com/journal/aabhj2022Forestry and Agricultural Biotechnology Institute (FABI)Plant Production and Soil Scienc

Exogenous double-stranded RNA inhibits the infection physiology of rust fungi to reduce symptoms in planta

Rust fungi (Pucciniales) are a diverse group of plant pathogens in natural and agricultural systems. They pose ongoing threats to the diversity of native flora and cause annual crop yield losses. Agricultural rusts are predominantly managed with fungicides and breeding for resistance, but new control strategies are needed on non-agricultural plants and in fragile ecosystems. RNA interference (RNAi) induced by exogenous double-stranded RNA (dsRNA) has promise as a sustainable approach for managing plant-pathogenic fungi, including rust fungi. We investigated the mechanisms and impact of exogenous dsRNA on rust fungi through in vitro and whole-plant assays using two species as models, Austropuccinia psidii (the cause of myrtle rust) and Coleosporium plumeriae (the cause of frangipani rust). In vitro, dsRNA either associates externally or is internalized by urediniospores during the early stages of germination. The impact of dsRNA on rust infection architecture was examined on artificial leaf surfaces. dsRNA targeting predicted essential genes significantly reduced germination and inhibited development of infection structures, namely appressoria and penetration pegs. Exogenous dsRNA sprayed onto 1-year-old trees significantly reduced myrtle rust symptoms. Furthermore, we used comparative genomics to assess the wide-scale amenability of dsRNA to control rust fungi. We sequenced genomes of six species of rust fungi, including three new families (Araucariomyceaceae, Phragmidiaceae, and Skierkaceae) and identified key genes of the RNAi pathway across 15 species in eight families of Pucciniales. Together, these findings indicate that dsRNA targeting essential genes has potential for broad-use management of rust fungi across natural and agricultural systems

Dual RNA-sequencing of Eucalyptus nitens during phytophthora cinnamomi challenge reveals pathogen and host factors influencing compatibility

Damage caused by Phytophthora cinnamomi Rands remains an important concern on forest tree species. The pathogen causes root and collar rot, stem cankers, and dieback of various economically important Eucalyptus spp. In South Africa, susceptible cold tolerant Eucalyptus plantations have been affected by various Phytophthora spp. with P. cinnamomi considered one of the most virulent. The molecular basis of this compatible interaction is poorly understood. In this study, susceptible Eucalyptus nitens plants were stem inoculated with P. cinnamomi and tissue was harvested five days post inoculation. Dual RNA-sequencing, a technique which allows the concurrent detection of both pathogen and host transcripts during infection, was performed. Approximately 1% of the reads mapped to the draft genome of P. cinnamomi while 78% of the reads mapped to the Eucalyptus grandis genome. The highest expressed P. cinnamomi gene in planta was a putative crinkler effector (CRN1). Phylogenetic analysis indicated the high similarity of this P. cinnamomi CRN1 to that of Phytophthora infestans. Some CRN effectors are known to target host nuclei to suppress defense. In the host, over 1400 genes were significantly differentially expressed in comparison to mock inoculated trees, including suites of pathogenesis related (PR) genes. In particular, a PR-9 peroxidase gene with a high similarity to a Carica papaya PR-9 ortholog previously shown to be suppressed upon infection by Phytophthora palmivora was down-regulated two-fold. This PR-9 gene may represent a cross-species effector target during P. cinnamomi infection. This study identified pathogenicity factors, potential manipulation targets, and attempted host defense mechanisms activated by E. nitens that contributed to the susceptible outcome of the interaction.The National Research Foundation of South Africa for the grant no. 76225 and 86936; the Forest Molecular Genetics Program by Mondi and Sappi, the Technology and Human Resources for Industry Program (80118), and Department of Science and Technology, The Eucalyptus Genomics Platform: Tree genomics and biotechnology for wood fibre, bioenergy and biomaterials.http://www.frontiersin.orgam2016Forestry and Agricultural Biotechnology Institute (FABI)GeneticsPlant Scienc