Early assessment of fungal and oomycete pathogens in greenhouse irrigation water using Oxford nanopore amplicon sequencing.

Water-borne plant pathogenic fungi and oomycetes are a major threat in greenhouse production systems. Early detection and quantification of these pathogens would enable us to ascertain both economic and biological thresholds required for a timely treatment, thus improving effective disease management. Here, we used Oxford nanopore MinION amplicon sequencing to analyze microbial communities in irrigation water collected from greenhouses used for growing tomato, cucumber and Aeschynanthus sp. Fungal and oomycete communities were characterized using primers that amplify the full internal transcribed spacer (ITS) region. To assess the sensitivity of the MinION sequencing, we spiked serially diluted mock DNA into the DNA isolated from greenhouse water samples prior to library preparation. Relative abundances of fungal and oomycete reads were distinct in the greenhouse irrigation water samples and in water samples from setups with tomato that was inoculated with Fusarium oxysporum. Sequence reads derived from fungal and oomycete mock communities were proportionate in the respective serial dilution samples, thus confirming the suitability of MinION amplicon sequencing for environmental monitoring. By using spike-ins as standards to test the reliability of quantification using the MinION, we found that the detection of spike-ins was highly affected by the background quantities of fungal or oomycete DNA in the sample. We observed that spike-ins having shorter length (538bp) produced reads across most of our dilutions compared to the longer spikes (>790bp). Moreover, the sequence reads were uneven with respect to dilution series and were least retrievable in the background samples having the highest DNA concentration, suggesting a narrow dynamic range of performance. We suggest continuous benchmarking of the MinION sequencing to improve quantitative metabarcoding efforts for rapid plant disease diagnostic and monitoring in the future.

What is new in FungiDB: a web-based bioinformatics platform for omics-scale data analysis for fungal and oomycete species.

FungiDB (https://fungidb.org) serves as a valuable online resource that seamlessly integrates genomic and related large-scale data for a wide range of fungal and oomycete species. As an integral part of the VEuPathDB Bioinformatics Resource Center (https://veupathdb.org), FungiDB continually integrates both published and unpublished data addressing various aspects of fungal biology. Established in early 2011, the database has evolved to support 674 datasets. The datasets include over 300 genomes spanning various taxa (e.g. Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Mucoromycota, as well as Albuginales, Peronosporales, Pythiales, and Saprolegniales). In addition to genomic assemblies and annotation, over 300 extra datasets encompassing diverse information, such as expression and variation data, are also available. The resource also provides an intuitive web-based interface, facilitating comprehensive approaches to data mining and visualization. Users can test their hypotheses and navigate through omics-scale datasets using a built-in search strategy system. Moreover, FungiDB offers capabilities for private data analysis via the integrated VEuPathDB Galaxy platform. FungiDB also permits genome improvements by capturing expert knowledge through the User Comments system and the Apollo genome annotation editor for structural and functional gene curation. FungiDB facilitates data exploration and analysis and contributes to advancing research efforts by capturing expert knowledge for fungal and oomycete species.

Morphology, phylogeny, and host range of the novel early-diverging oomycete Sirolpidium dinoletiferum sp. nov. parasitizing marine dinoflagellates.

Oomycetes are fungus-like heterotrophic organisms with a broad environmental distribution, including marine, freshwater, and terrestrial habitats. They function as saprotrophs that use the remains of other organisms or as parasites of a variety of eukaryotes, including protists, diatoms, dinoflagellates, macroalgae, plants, fungi, animals, and even other oomycetes. Among the protist hosts, the taxonomy, morphology, and phylogenetic positions of the oomycete parasitoids of diatoms have been well studied; however, this information concerning the oomycete parasitoids of dinoflagellates is poorly understood. During intensive sampling along the east and west coasts of Korea in May and October 2019, a new species of oomycetes was discovered and two strains of the new parasitoid were successfully established in cultures. The new oomycete parasitoid penetrated the dinoflagellate host cell and developed to form a sporangium, which was very similar to the perkinsozoan parasitoids that infect marine dinoflagellates. The most distinctive morphological feature of the new parasitoid was a central large vacuole forming several long discharge tubes. The molecular phylogenetic tree inferred based on the small subunit (SSU) ribosomal DNA (rDNA) revealed that the new parasitoid forms a distinct branch unrelated to other described species belonging to early-diverging oomycetes. It clustered with species belonging to the genus Sirolpidium with strong support values in the cytochrome c oxidase subunit 2 (cox2) tree. Cross-infection experiments showed that infections by the new parasitoid occurred in only six genera belonging to dinoflagellates among the protists tested in this study. Based on the morphological and molecular data obtained in this study, we propose to introduce a new species, Sirolpidium dinoletiferum sp. nov., for this novel parasitoid, conservatively within the genus Sirolpidium.

Effect of Cucurbit Host, Production Region, and Season on the Population Structure of Pseudoperonospora cubensis in Florida.

Pseudoperonospora cubensis, the causal agent of Cucurbit downy mildew (CDM), is one of the most important diseases affecting cucurbit production in the United States. This disease is especially damaging to Florida production areas, as the state is a top producer of many cucurbit species. In addition, winter production in central and south Florida likely serves as a likely source of P. cubensis inoculum for spring and summer cucurbit production throughout the eastern United States, where CDM is unable to overwinter in the absence of a living host. Over 2 years (2017 and 2018) and four seasons (spring 2017, spring 2018, fall 2017, and fall 2018), 274 P. cubensis isolates were collected from cucurbit hosts at production sites in south, central, and north Florida. The isolates were analyzed with 10 simple sequence repeat (SSR) markers to establish population structure and genetic diversity and further assigned to a clade based on a qPCR assay. Results of population structure and genetic diversity analyses differentiated isolates based on cucurbit host and clade (1 or 2). Of the isolates assigned to clade by qPCR, butternut squash, watermelon, and zucchini were dominated by clade 1 isolates, whereas cucumber isolates were split 34 and 59% between clades 1 and 2, respectively. Clade assignments agreed with isolate clustering observed within discriminant analysis of principal components (DAPC) based on SSR markers, although watermelon isolates formed a group distinct from the other clade 1 isolates. For seasonal collections from cucumber at each location, isolates were typically skewed to one clade or the other and varied across locations and seasons within each year of the study. This variable population structure of cucumber isolates could have consequences for regional disease management. This is the first study to characterize P. cubensis populations in Florida and evaluate the effect of cucurbit host and clade-type on isolate diversity and population structure, with implications for CDM management in Florida and other United States cucurbit production areas.

Advances in understanding grapevine downy mildew: From pathogen infection to disease management.

Plasmopara viticola is geographically widespread in grapevine-growing regions. Grapevine downy mildew disease, caused by this biotrophic pathogen, leads to considerable yield losses in viticulture annually. Because of the great significance of grapevine production and wine quality, research on this disease has been widely performed since its emergence in the 19th century. Here, we review and discuss recent understanding of this pathogen from multiple aspects, including its infection cycle, disease symptoms, genome decoding, effector biology, and management and control strategies. We highlight the identification and characterization of effector proteins with their biological roles in host-pathogen interaction, with a focus on sustainable control methods against P. viticola, especially the use of biocontrol agents and environmentally friendly compounds.

Comparative Analysis of Transcriptomics and Metabolomics Reveals Defense Mechanisms in Melon Cultivars against Pseudoperonospora cubensis Infection.

Melon (Cucumis melo L.) represents an agriculturally significant horticultural crop that is widely grown for its flavorful fruits. Downy mildew (DM), a pervasive foliar disease, poses a significant threat to global melon production. Although several quantitative trait loci related to DM resistance have been identified, the comprehensive genetic underpinnings of this resistance remain largely uncharted. In this study, we utilized integrative transcriptomics and metabolomics approaches to identify potential resistance-associated genes and delineate the strategies involved in the defense against DM in two melon cultivars: the resistant 'PI442177' ('K10-1') and the susceptible 'Huangdanzi' ('K10-9'), post-P. cubensis infection. Even in the absence of the pathogen, there were distinctive differentially expressed genes (DEGs) between 'K10-1' and 'K10-9'. When P. cubensis was infected, certain genes, including flavin-containing monooxygenase (FMO), receptor-like protein kinase FERONIA (FER), and the HD-ZIP transcription factor member, AtHB7, displayed pronounced expression differences between the cultivars. Notably, our data suggest that following P. cubensis infection, both cultivars suppressed flavonoid biosynthesis via the down-regulation of associated genes whilst concurrently promoting lignin production. The complex interplay of transcriptomic and metabolic responses elucidated by this study provides foundational insights into melon's defense mechanisms against DM. The robust resilience of 'K10-1' to DM is attributed to the synergistic interaction of its inherent transcriptomic and metabolic reactions.

Comparative Transcriptome Analysis between Resistant and Susceptible Pakchoi Cultivars in Response to Downy Mildew.

Downy mildew caused by the obligate parasite Hyaloperonospora brassicae is a devastating disease for Brassica species. Infection of Hyaloperonospora brassicae often leads to yellow spots on leaves, which significantly impacts quality and yield of pakchoi. In the present study, we conducted a comparative transcriptome between the resistant and susceptible pakchoi cultivars in response to Hyaloperonospora brassicae infection. A total of 1073 disease-resistance-related differentially expressed genes were identified using a Venn diagram. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that these genes were mainly involved in plant-pathogen interaction, plant hormone signal transduction, and other photosynthesis-related metabolic processes. Analysis of the phytohormone content revealed that salicylic acid increased significantly in the resistant material after inoculation with Hyaloperonospora brassicae, whereas the contents of jasmonic acid, abscisic acid, and 1-aminocyclopropane-1-carboxylic acid decreased. Exogenous salicylic acid treatment also significantly upregulated Hyaloperonospora brassicae-induced genes, which further confirmed a crucial role of salicylic acid during pakchoi defense against Hyaloperonospora brassicae. Based on these findings, we suggest that the salicylic-acid-mediated signal transduction contributes to the resistance of pakchoi to downy mildew, and PAL1, ICS1, NPR1, PR1, PR5, WRKY70, WRKY33, CML43, CNGC9, and CDPK15 were involved in this responsive process. Our findings evidently contribute to revealing the molecular mechanism of pakchoi defense against Hyaloperonospora brassicae.

Obligate biotroph downy mildew consistently induces near-identical protective microbiomes in Arabidopsis thaliana.

Hyaloperonospora arabidopsidis (Hpa) is an obligately biotrophic downy mildew that is routinely cultured on Arabidopsis thaliana hosts that harbour complex microbiomes. We hypothesized that the culturing procedure proliferates Hpa-associated microbiota (HAM) in addition to the pathogen and exploited this model system to investigate which microorganisms consistently associate with Hpa. Using amplicon sequencing, we found nine bacterial sequence variants that are shared between at least three out of four Hpa cultures in the Netherlands and Germany and comprise 34% of the phyllosphere community of the infected plants. Whole-genome sequencing showed that representative HAM bacterial isolates from these distinct Hpa cultures are isogenic and that an additional seven published Hpa metagenomes contain numerous sequences of the HAM. Although we showed that HAM benefit from Hpa infection, HAM negatively affect Hpa spore formation. Moreover, we show that pathogen-infected plants can selectively recruit HAM to both their roots and shoots and form a soil-borne infection-associated microbiome that helps resist the pathogen. Understanding the mechanisms by which infection-associated microbiomes are formed might enable breeding of crop varieties that select for protective microbiomes.

Mining of Minor Disease Resistance Genes in V. vinifera Grapes Based on Transcriptome.

Intraspecific recurrent selection in V. vinifera is an effective method for grape breeding with high quality and disease resistance. The core theory of this method is the substitution accumulation of multi-genes with low disease resistance. The discovery of multi-genes for disease resistance in V. vinifera may provide a molecular basis for breeding for disease resistance in V. vinifera. In this study, resistance to downy mildew was identified, and genetic analysis was carried out in the intraspecific crossing population of V. vinifera (Ecolly × Dunkelfelder) to screen immune, highly resistant and disease-resistant plant samples; transcriptome sequencing and differential expression analysis were performed using high-throughput sequencing. The results showed that there were 546 differential genes (194 up-regulated and 352 down-regulated) in the immune group compared to the highly resistant group, and 199 differential genes (50 up-regulated and 149 down-regulated) in the highly resistant group compared to the resistant group, there were 103 differential genes (54 up-regulated and 49 down-regulated) in the immune group compared to the resistant group. KEGG analysis of differentially expressed genes in the immune versus high-resistance group. The pathway is mainly concentrated in phenylpropanoid biosynthesis, starch and sucrose metabolism, MAPK signaling pathway-plant, carotenoid biosyn-thesis and isoquinoline alkaloid biosynthesis. The differential gene functions of immune and resistant, high-resistant and resistant combinations were mainly enriched in plant-pathogen interaction pathway. Through the analysis of disease resistance-related genes in each pathway, the potential minor resistance genes in V. vinifera were mined, and the accumulation of minor resistance genes was analyzed from the molecular level.

Wall-associated kinase BrWAK1 confers resistance to downy mildew in Brassica rapa.

The plant cell wall is the first line of defence against physical damage and pathogen attack. Wall-associated kinase (WAK) has the ability to perceive the changes in the cell wall matrix and transform signals into the cytoplasm, being involved in plant development and the defence response. Downy mildew, caused by Hyaloperonospora brassicae, can result in a massive loss in Chinese cabbage (Brassica rapa L. ssp. pekinensis) production. Herein, we identified a candidate resistant WAK gene, BrWAK1, in a major resistant quantitative trait locus, using a double haploid population derived from resistant inbred line T12-19 and the susceptible line 91-112. The expression of BrWAK1 could be induced by salicylic acid and pathogen inoculation. Expression of BrWAK1 in 91-112 could significantly enhance resistance to the pathogen, while truncating BrWAK1 in T12-19 increased disease susceptibility. Variation in the extracellular galacturonan binding (GUB) domain of BrWAK1 was found to mainly confer resistance to downy mildew in T12-19. Moreover, BrWAK1 was proved to interact with BrBAK1 (brassinosteroid insensitive 1 associated kinase), resulting in the activation of the downstream mitogen-activated protein kinase (MAPK) cascade to trigger the defence response. BrWAK1 is the first identified and thoroughly characterized WAK gene conferring disease resistance in Chinese cabbage, and the plant biomass is not significantly influenced by BrWAK1, which will greatly accelerate Chinese cabbage breeding for downy mildew resistance.

Genome-Enabled Insights into Downy Mildew Biology and Evolution.

Oomycetes that cause downy mildew diseases are highly specialized, obligately biotrophic phytopathogens that can have major impacts on agriculture and natural ecosystems. Deciphering the genome sequence of these organisms provides foundational tools to study and deploy control strategies against downy mildew pathogens (DMPs). The recent telomere-to-telomere genome assembly of the DMP Peronospora effusa revealed high levels of synteny with distantly related DMPs, higher than expected repeat content, and previously undescribed architectures. This provides a road map for generating similar high-quality genome assemblies for other oomycetes. This review discusses biological insights made using this and other assemblies, including ancestral chromosome architecture, modes of sexual and asexual variation, the occurrence of heterokaryosis, candidate gene identification, functional validation, and population dynamics. We also discuss future avenues of research likely to be fruitful in studies of DMPs and highlight resources necessary for advancing our understanding and ability to forecast and control disease outbreaks.

A comparison of transporter gene expression in three species of Peronospora plant pathogens during host infection.

Protein transporters move essential metabolites across membranes in all living organisms. Downy mildew causing plant pathogens are biotrophic oomycetes that transport essential nutrients from their hosts to grow. Little is known about the functions and gene expression levels of membrane transporters produced by downy mildew causing pathogens during infection of their hosts. Approximately 170-190 nonredundant transporter genes were identified in the genomes of Peronospora belbahrii, Peronospora effusa, and Peronospora tabacina, which are specialized pathogens of basil, spinach, and tobacco, respectively. The largest groups of transporter genes in each species belonged to the major facilitator superfamily, mitochondrial carriers (MC), and the drug/metabolite transporter group. Gene expression of putative Peronospora transporters was measured using RNA sequencing data at two time points following inoculation onto leaves of their hosts. There were 16 transporter genes, seven of which were MCs, expressed in each Peronospora species that were among the top 45 most highly expressed transporter genes 5-7 days after inoculation. Gene transcripts encoding the ADP/ATP translocase and the mitochondrial phosphate carrier protein were the most abundant mRNAs detected in each Peronospora species. This study found a number of Peronospora genes that are likely critical for pathogenesis and which might serve as future targets for control of these devastating plant pathogens.

DNA-Barcoding Identification of Plant Pathogens for Disease Diagnostics.

The accurate identification of plant pathogens is a critical step to prevent their spread and attenuate their impact. Among the wide range of methods available, DNA-barcoding, i.e., the identification of an organism through the PCR amplification and sequencing of a single locus, remains one of the most straightforward and accurate plant-pathogen identification techniques that can be used in a generic molecular biology lab. This chapter provides a detailed protocol for the isolation of genomic DNA of fungal and oomycete pathogens from fresh field samples and the amplification and sequencing of the internal transcribed spacer (ITS) locus for DNA-barcoding purpose. Amendments to the protocol are provided to help in resolving issues related to the analysis of complicated samples and to the lack of species resolution that can be encountered with ITS barcodes.

Harnessing CRISPR-Cas for oomycete genome editing.

Oomycetes are a group of microorganisms that include pathogens responsible for devastating diseases in plants and animals worldwide. Despite their importance, the development of genome editing techniques for oomycetes has progressed more slowly than for model microorganisms. Here, we review recent breakthroughs in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technologies that are expanding the genome editing toolbox for oomycetes - from the original Cas9 study to Cas12a editing, ribonucleoprotein (RNP) delivery, and complementation. We also discuss some of the challenges to applying CRISPR-Cas in oomycetes and potential ways to overcome them. Advances in CRISPR-Cas technologies are being used to illuminate the biology of oomycetes, which ultimately can guide the development of tools for managing oomycete diseases.

TaqMan-MGB PCR Method for Rapid Detection of QoI Fungicide Resistance in Chinese Populations of Plasmopara viticola.

Grape downy mildew caused by Plasmopara viticola is one of the most devastating diseases of grapevine worldwide. Quinone outside inhibitor (QoI) fungicides are commonly used for the control of the pathogen in grape fields across China. However, their recurrent use could lead to the emergence of resistance against these compounds. Based on the most common mutation in resistant isolates, a glycine to alanine substitution at amino acid position 143 (G143A) in the cytochrome b protein, a TaqMan-MGB PCR was developed for the rapid detection of resistance to the QoI fungicide azoxystrobin in P. viticola. Specificity and sensitivity of this method showed it could specifically detect the point mutations linked with QoI resistance in P. viticola, and the detection limit was 0.2 pg. It could also quantify the resistance allele even in isolate mixtures containing as little as 5% QoI-resistant P. viticola strains. With this method, a large P. viticola population (n = 2,373) was screened, and QoI-resistant isolates were identified for the first time in China. The average frequencies of the resistant genotype from eight major-grapevine regions were up to 66%. Taken together, the results not only provide a novel tool for the rapid distinction and quantification of the QoI-resistant allele in P. viticola but also provide important references for fungicide selection and application, which will facilitate resistance management of grape downy mildew and improve grape production systems in Chinese vineyards.

High-throughput time series expression profiling of Plasmopara halstedii infecting Helianthus annuus reveals conserved sequence motifs upstream of co-expressed genes.

Downy mildew disease of sunflower, caused by the obligate biotrophic oomycete Plasmopara halstedii, can have significant economic impact on sunflower cultivation. Using high-throughput whole transcriptome sequencing, four developmental phases in 16 time-points of Pl. halstedii infecting Helianthus annuus were investigated. With the aim of identifying potential functional and regulatory motifs upstream of co-expressed genes, time-series derived gene expression profiles were clustered based on their time-course similarity, and their upstream regulatory gene sequences were analyzed here. Several conserved motifs were found upstream of co-expressed genes, which might be involved in binding specific transcription factors. Such motifs were also found associated with virulence related genes, and could be studied on a genetically tractable model to clarify, if these are involved in regulating different stages of pathogenesis.

Genome Resource of Streptomyces atratus PY-1, a Broad-Spectrum Antimicrobial Strain in Particular Antagonistic Against Plasmopara viticola.

Streptomyces atratus PY-1 exhibited promising antimicrobial properties; in particular, it is highly inhibitory to Plasmopara viticola, which causes downy mildew of grape. It is very necessary to carry out systematic and in-depth research on the PY-1 strain for the improvement, application, and promotion of biocontrol agents. The PY-1 genome was fully sequenced and assembled. We present the draft genome sequence of PY-1, with a size of 9, 254, and 781 bp. Preliminary analysis on the PY-1 genome sequence shows that at least 35 gene clusters are involved in the biosynthesis of polyketides, terpenes, and nonribosomally synthesized peptides.