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Golovinomyces (Erysiphaceae, Ascomycota) is an obligate plant pathogenic group causing powdery mildew on diverse angiosperm plants, including economically significant crops. Despite advancements in the taxonomy and phylogeny of Golovinomyces species using ribosomal DNA markers (ITS and LSU), several taxonomic issues remain unresolved. Previously, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, which exhibits higher nucleotide variation, has been proposed as an additional marker for powdery mildew species. In this study, we designed a new primer set (GoGPD-F and GoGPD-R) to improve the PCR success and efficiency of the GAPDH gene across various Golovinomyces species and dried herbarium specimens. The primers were successful in amplifying and sequencing the GAPDH gene in sixteen Golovinomyces species, including six species not previously registered in GenBank and two undescribed species. This development is a significant contribution to future research on the identification, taxonomy, and phylogeny of Golovinomyces species, offering a more robust tool for resolving existing taxonomic issues. [ABSTRACT FROM AUTHOR]

Golovinomyces (Erysiphaceae, Ascomycota) is an obligate plant pathogenic group causing powdery mildew on diverse angiosperm plants, including economically significant crops. Despite advancements in the taxonomy and phylogeny of Golovinomyces species using ribosomal DNA markers (ITS and LSU), several taxonomic issues remain unresolved. Previously, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, which exhibits higher nucleotide variation, has been proposed as an additional marker for powdery mildew species. In this study, we designed a new primer set (GoGPD-F and GoGPD-R) to improve the PCR success and efficiency of the GAPDH gene across various Golovinomyces species and dried herbarium specimens. The primers were successful in amplifying and sequencing the GAPDH gene in sixteen Golovinomyces species, including six species not previously registered in GenBank and two undescribed species. This development is a significant contribution to future research on the identification, taxonomy, and phylogeny of Golovinomyces species, offering a more robust tool for resolving existing taxonomic issues.

Virginia creeper (or five-leaved ivy; Parthenocissus quinquefolia) is one of the most popular and widely grown climbers worldwide. In September 2021, Virginia creeper leaves with typical rust symptom were found in an arboretum in Korea, with severe damage. Globally, there is no record of a rust disease on Virginia creeper. Using morphological investigation and molecular phylogenetic inferences, the rust agent was identified as Neophysopella vitis, which is a rust pathogen of other Parthenocissus spp. including Boston ivy (P. tricuspidata). Given that the two ivy plants, Virginia creeper and Boston ivy, have common habitats, especially on buildings and walls, throughout Korea, and that N. vitis is a ubiquitous rust species affecting Boston ivy in Korea, it is speculated that the host range of N. vitis may recently have expanded from Boston ivy to Virginia creeper. The present study reports a globally new rust disease on Virginia creeper, which could be a major threat to the ornamental creeper.

BackgroundDowny mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily.ResultsHere a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic.ConclusionsThe genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.

During 2016 and 2018, Lactuca undulata plants showing symptoms typical of white blister rust were collected in the province of South Khorasan, Iran. Phylogenetic analysis resolved the position of specimens in the genus Pustula. The specimens were characterised by oospore size and ornamentation, and size of secondary sporangia. In addition to morphology, phylogenetic analysis also suggests a new species for the genus distinct from other established Pustula species, which is described here as Pustula lactucae sp. nov. Scanning electron microscope illustrations are also provided. [ABSTRACT FROM AUTHOR]

Pseudoperonospora humuli is an obligate biotrophic oomycete that causes downy mildew, one of the most devastating diseases of cultivated hop, Humulus lupulus. Downy mildew occurs in all production areas of the crop in the Northern Hemisphere and Argentina. The pathogen overwinters in hop crowns and roots, and causes considerable crop loss. Downy mildew is managed by sanitation practices, planting of resistant cultivars, and fungicide applications. However, the scarcity of sources of host resistance and fungicide resistance in pathogen populations complicates disease management. This review summarizes the current knowledge on the symptoms of the disease, life cycle, virulence factors, and management of hop downy mildew, including various forecasting systems available in the world. Additionally, recent developments in genomics and effector discovery, and the future prospects of using such resources in successful disease management are also discussed. Taxonomy: Class: Oomycota; Order: Peronosporales; Family: Peronosporaceae; Genus: Pseudoperonospora; Species: Pseudoperonospora humuli. Disease symptoms: The disease is characterized by systemically infected chlorotic shoots called "spikes". Leaf symptoms and signs include angular chlorotic lesions and profuse sporulation on the abaxial side of the leaf. Under severe disease pressure, dark brown discolouration or lesions are observed on cones. Infected crowns have brown to black streaks when cut open. Cultivars highly susceptible to crown rot may die at this phase of the disease cycle without producing shoots. However, foliar symptoms may not be present on plants with systemically infected root systems. Infection process: Pathogen mycelium overwinters in buds and crowns, and emerges on infected shoots in spring. Profuse sporulation occurs on infected tissues and sporangia are released and dispersed by air currents. Under favourable conditions, sporangia germinate and produce biflagellate zoospores that infect healthy tissue, thus perpetuating the infection cycle. Though oospores are produced in infected tissues, their role in the infection cycle is not defined. Control: Downy mildew on hop is managed by a combination of sanitation practices and timely fungicide applications. Forecasting systems are used to time fungicide applications for successful management of the disease. Useful Websites: https://content.ces.ncsu.edu/hop‐downy‐mildew (North Carolina State University disease factsheet), https://www.canr.msu.edu/resources/michigan‐hop‐management‐guide (Michigan Hop Management Guide), http://uspest.org/risk/models (Oregon State University Integrated Plant Protection Center degree‐day model for hop downy mildew), https://www.usahops.org/cabinet/data/Field‐Guide.pdf (Field Guide for Integrated Pest Management in Hops). [ABSTRACT FROM AUTHOR]

Powdery mildew (PM) is a serious fungal disease of legumes. To gain novel insights into PM pathogenesis and host resistance/susceptibility, we used dual RNA-Seq to simultaneously capture host and pathogen transcriptomes at 1 d post-inoculation of resistant and susceptible Medicago truncatula genotypes with the PM Erysiphe pisi (Ep). Differential expression analysis indicates that R -gene mediated resistance against Ep involves extensive transcriptional reprogramming. Functional enrichment of differentially expressed host genes and in silico analysis of co-regulated promoters suggests that amplification of PTI, activation of the JA/ET signaling network, and regulation of growth-defense balance correlate with resistance. In contrast, processes that favor biotrophy, including suppression of defense signaling and programmed cell death, and weaker cell wall defenses are important susceptibility factors. Lastly, Ep effector candidates and genes with known/putative virulence functions were identified, representing a valuable resource that can be leveraged to improve our understanding of legume-PM interactions. • Dual RNA-Seq was performed to study M. truncatula - E. pisi (Ep) interactions. • R -gene mediated resistance is associated with massive transcriptional reprogramming. • Control of defense-growth balance correlates with effective resistance. • Ep effector candidates exhibit infection-stage specific expression patterns. • An initial resource for the development of powdery mildew resistance in legumes. [ABSTRACT FROM AUTHOR]

Summary: Pea powdery mildew (PM) is an important fungal disease caused by an obligate biotroph, Erysiphe pisi (Ep), which significantly impacts pea production worldwide. The phytopathogen secretes a plethora of effectors, primarily through specialized infection structures termed haustoria, to establish a dynamic relationship with its host. To identify Ep effector candidates, a cDNA library of enriched haustoria from Ep‐infected pea leaves was sequenced. The Ep transcriptome encodes 622 Ep candidate secreted proteins (CSPs), of which 167 were predicted to be candidate secreted effector proteins (CSEPs). Phylogenetic analysis indicates that Ep CSEPs are highly diverse, but, unlike cereal PM CSEPs, exhibit extensive sequence similarity with effectors from other PMs. Quantitative real‐time PCR of a subset of EpCSEP/CSPs revealed that the majority are preferentially expressed in haustoria and exhibit infection stage–specific expression patterns. The functional roles of EpCSEP001, EpCSEP009 and EpCSP083 were probed by host‐induced gene silencing (HIGS) via a double‐stranded (ds) RNA‐mediated RNAi approach. Foliar application of individual EpCSEP/CSP dsRNAs resulted in a marked reduction in PM disease symptoms. These findings were consistent with microscopic and molecular studies, suggesting that these Ep CSEP/CSPs play important roles in pea PM pathogenesis. Homology modelling revealed that EpCSEP001 and EpCSEP009 are analogous to fungal ribonucleases and belong to the RALPH family of effectors. This is the first study to identify and functionally validate candidate effectors from the agriculturally relevant pea PM, and highlights the utility of transcriptomics and HIGS to elucidate the key proteins associated with Ep pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract UV-C irradiation is known to compromise germination of Blumeria graminis conidia and to reduce powdery mildew infestation. However, only scarce information is available on the effects of UV-C irradiation on B. graminis appressorium formation. Applying a Formvar® resin-based in vitro system allowed for analyzing B. graminis germination and appressorium formation in absence of plant defense. UV-C irradiation more strongly affected the differentiation of appressoria than conidial germination. In vivo and in vitro , a single dose of 100 J m−2 UV-C was sufficient to reduce germination to less than 20 % and decrease appressorium formation to values below 5 %. UV-C irradiation negatively affected pustule size and conidiation. White light-mediated photoreactivation was most effective immediately after UV-C irradiation, indicating that a prolonged phase of darkness after UV-C treatment increases the efficacy of B. graminis control. UV-C irradiation increased transcript levels of three putative B. graminis photolyase genes, while mere white light or blue light irradiation did not contribute to the transcriptional up-regulation. Thus, UV-C irradiation effectively controls B. graminis infestation and proliferation by restricting prepenetration processes. Nevertheless, photoreactivation plays an important role in UV-C-based powdery mildew control in crops and hence has to be considered for planning specific irradiation schedules. Graphical abstract Image 1 Highlights • UV-C irradiation compromised appressorium formation of Blumeria graminis conidia. • 100 J m−2 UV-C irradiation completely prevented appressorium formation in vitro. • On host leaves, UV-C irradiation negatively affected pustule size and conidiation. • Three putative B. graminis photolyase genes were up-regulated upon UV-C irradiation. • Irradiation with blue or white light did not induce photolyase gene expression. [ABSTRACT FROM AUTHOR]

Fungi of the Pucciniales order cause rust diseases which, altogether, affect thousands of plant species worldwide and pose a major threat to several crops. How rust effectors-virulence proteins delivered into infected tissues to modulate host functions-contribute to pathogen virulence remains poorly understood. Melampsora larici-populina is a devastating and widespread rust pathogen of poplar, and its genome encodes 1184 identified small secreted proteins that could potentially act as effectors. Here, following specific criteria, we selected 16 candidate effector proteins and characterized their virulence activities and subcellular localizations in the leaf cells of Arabidopsis thaliana. Infection assays using bacterial ( Pseudomonas syringae) and oomycete ( Hyaloperonospora arabidopsidis) pathogens revealed subsets of candidate effectors that enhanced or decreased pathogen leaf colonization. Confocal imaging of green fluorescent protein-tagged candidate effectors constitutively expressed in stable transgenic plants revealed that some protein fusions specifically accumulate in nuclei, chloroplasts, plasmodesmata and punctate cytosolic structures. Altogether, our analysis suggests that rust fungal candidate effectors target distinct cellular components in host cells to promote parasitic growth. [ABSTRACT FROM AUTHOR]

Rust fungi (Pucciniales) are the most speciose and the most complex group of plant pathogens. Historically, rust taxonomy was largely influenced by host and phenotypic characters, which are potentially plastic. Molecular systematic studies suggest that the extant diversity of this group was largely shaped by host jumps and subsequent shifts. However, it has been challenging to reconstruct the evolutionary history for the order, especially at deeper (family-level) nodes. Phylogenomics offer a potentially powerful tool to reconstruct the Pucciniales tree of life, although researchers working at this vanguard still face unprecedented challenges working with nonculturable organisms that possess some of the largest and most repetitive genomes now known in kingdom fungi. In this chapter, we provide an overview of the current status and special challenges of rust genomics, and we highlight how phylogenomics may provide new perspectives and answer long-standing questions regarding the biology of rust fungi. [ABSTRACT FROM AUTHOR]

Current and emerging plant diseases caused by obligate parasitic microbes such as rusts, downy mildews, and powdery mildews threaten worldwide crop production and food safety. These obligate parasites are typically unculturable in the laboratory, posing technical challenges to characterize them at the genetic and genomic level. Here we have developed a data analysis pipeline integrating several bioinformatic software programs. This pipeline facilitates rapid gene discovery and expression analysis of a plant host and its obligate parasite simultaneously by next generation sequencing of mixed host and pathogen RNA (i.e. metatranscriptomics). We applied this pipeline to metatranscriptomic sequencing data of sweet basil (Ocimum basilicum) and its obligate downy mildew parasite Peronospora belbahrii, both lacking a sequenced genome. Even with a single data point, we were able to identify both candidate host defense genes and pathogen virulence genes that are highly expressed during infection. This demonstrates the power of this pipeline for identifying genes important in host-pathogen interactions without prior genomic information for either the plant host or the obligate biotrophic pathogen. The simplicity of this pipeline makes it accessible to researchers with limited computational skills and applicable to metatranscriptomic data analysis in a wide range of plant-obligate-parasite systems.

Plant cells secrete a wide range of proteins in extracellular spaces in response to pathogen attack. The poplar Rust-Induced Secreted Protein (RISP) is a small cationic protein of unknown function that was identified as the most induced gene in poplar leaves during immune responses to the leaf rust pathogen Melampsora larici-populina, an obligate biotrophic parasite. Here, we combined in planta and in vitro molecular biology approaches to tackle the function of RISP. Using a RISP-mCherry fusion transiently expressed in Nicotiana benthamiana leaves, we demonstrated that RISP is secreted into the apoplast. A recombinant RISP specifically binds to M. larici-populina urediniospores and inhibits their germination. It also arrests the growth of the fungus in vitro and on poplar leaves. Interestingly, RISP also triggers poplar cell culture alkalinisation and is cleaved at the C-terminus by a plant-encoded mechanism. Altogether our results indicate that RISP is an antifungal protein that has the ability to trigger cellular responses.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is now established in all major soybean-producing countries. Currently, there is little information about the molecular basis of ASR–soybean interactions, which will be needed to assist future efforts to develop effective resistance. Toward this end, abundance changes of soybean mRNAs were measured over a 7-day ASR infection time course in mock-inoculated and infected leaves of a soybean accession (PI230970) carrying the Rpp2 resistance gene and a susceptible genotype (Embrapa-48). The expression profiles of differentially expressed genes (ASR-infected compared with the mock-inoculated control) revealed a biphasic response to ASR in each genotype. Within the first 12 h after inoculation (hai), which corresponds to fungal germination and penetration of the epidermal cells, differential gene expression changes were evident in both genotypes. mRNA expression of these genes mostly returned to levels found in mock-inoculated plants by 24 hai. In the susceptible genotype, gene expression remained unaffected by rust infection until 96 hai, a time period when rapid fungal growth began. In contrast, gene expression in the resistant genotype diverged from the mock-inoculated control earlier, at 72 h, demonstrating that Rpp2-mediated defenses were initiated prior to this time. These data suggest that ASR initially induces a non-specific response that is transient or is suppressed when early steps in colonization are completed in both soybean genotypes. The race-specific resistance phenotype of Rpp2 is manifested in massive gene expression changes after the initial response prior to the onset of rapid fungal growth that occurs in the susceptible genotype.

Plant cells secrete a wide range of proteins in extracellular spaces in response to pathogen attack. The poplar rust-induced secreted protein (RISP) is a small cationic protein of unknown function that was identified as the most induced gene in poplar leaves during immune responses to the leaf rust pathogen Melampsora larici-populina, an obligate biotrophic parasite. Here, we combined in planta and in vitro molecular biology approaches to tackle the function of RISP. Using a RISP-mCherry fusion transiently expressed in Nicotiana benthamiana leaves, we demonstrated that RISP is secreted into the apoplast. A recombinant RISP specifically binds to M. laricipopulina urediniospores and inhibits their germination. It also arrests the growth of the fungus in vitro and on poplar leaves. Interestingly, RISP also triggers poplar cell culture alkalinisation and is cleaved at the C-terminus by a plant-encoded mechanism. Altogether our results indicate that RISP is an antifungal protein that has the ability to trigger cellular responses. [ABSTRACT FROM AUTHOR]

Plants are susceptible to a limited number of pathogens. Most infections fail due to active defense or absence of compatibility. Many components of the plant's surveillance system and defense arsenal have been identified in the last decades. However, knowledge is limited on compatibility; in particular, the role of plant factors in the infection process. To gain insight into these processes, we have initiated an Arabidopsis thaliana mutant screen for reduced susceptibility to the downy mildew pathogen Hyaloperonospora parasitica. Ethyl methane sulfonate (EMS) mutants were generated in the highly susceptible Arabidopsis line L er eds1-2. Eight downy mildew-resistant (dmr) mutants were analyzed in detail, corresponding to six different loci. Microscopic analysis showed that, in all mutants, H. parasitica growth was severely reduced. Resistance of dmr3, dmr4, and dmr5 was associated with constitutive expression of PR-1. Furthermore, dmr3 and dmr4, but not dmr5, also were resistant to Pseudomonas syringae and Golovinomyces orontii, respectively. However, enhanced activation of plant defense was not observed in dmr1, dmr2, and dmr6. We postulate that, in these susceptibility mutants, cellular processes are disrupted which are required for H. parasitica infection. This interesting new set of mutants provides a basis to elucidate the molecular processes underlying susceptibility to downy mildew in Arabidopsis.

Melampsora larici-populina is a fungal pathogen responsible for foliar rust disease on poplar trees, which causes damage to forest plantations worldwide, particularly in Northern Europe. The reference genome of the isolate 98AG31 was previously sequenced using a whole genome shotgun strategy, revealing a large genome of 101 megabases containing 16,399 predicted genes, which included secreted protein genes representing poplar rust candidate effectors. In the present study, the genomes of 15 isolates collected over the past 20 years throughout the French territory, representing distinct virulence profiles, were characterized by massively parallel sequencing to assess genetic variation in the poplar rust fungus. Comparison to the reference genome revealed striking structural variations. Analysis of coverage and sequencing depth identified large missing regions between isolates related to the mating type loci. More than 611,824 single-nucleotide polymorphism (SNP) positions were uncovered overall, indicating a remarkable level of polymorphism. Based on the accumulation of non-synonymous substitutions in coding sequences and the relative frequencies of synonymous and non-synonymous polymorphisms (i.e. PN/PS), we identify candidate genes that may be involved in fungal pathogenesis. Correlation between non-synonymous SNPs in genes encoding secreted proteins and pathotypes of the studied isolates revealed candidate genes potentially related to virulences 1, 6 and 8 of the poplar rust fungus.

Melampsora larici-populina is a fungal pathogen responsible for foliar rust disease on poplar trees, which causes damage to forest plantations worldwide, particularly in Northern Europe. The reference genome of the isolate 98AG31 was previously sequenced using a whole genome shotgun strategy, revealing a large genome of 101 megabases containing 16,399 predicted genes, which included secreted protein genes representing poplar rust candidate effectors. In the present study, the genomes of 15 isolates collected over the past 20 years throughout the French territory, representing distinct virulence profiles, were characterized by massively parallel sequencing to assess genetic variation in the poplar rust fungus. Comparison to the reference genome revealed striking structural variations. Analysis of coverage and sequencing depth identified large missing regions between isolates related to the mating type loci. More than 611,824 single-nucleotide polymorphism (SNP) positions were uncovered overall, indicating a remarkable level of polymorphism. Based on the accumulation of non-synonymous substitutions in coding sequences and the relative frequencies of synonymous and non-synonymous polymorphisms (i.e., PN/PS), we identify candidate genes that may be involved in fungal pathogenesis. Correlation between non-synonymous SNPs in genes encoding secreted proteins (SPs) and pathotypes of the studied isolates revealed candidate genes potentially related to virulences 1, 6, and 8 of the poplar rust fungus. [ABSTRACT FROM AUTHOR]

Pucciniales are an important group of fungal plant pathogens that cause rust diseases in a diverse group of hosts including ecologically and economically important crops and trees. Rust fungi have intriguing and complex life cycles and are obligate biotrophs. Because of their biological features, these fungi are very difficult to study under laboratory conditions. The recent advances in genomics and transcriptomics have opened great perspectives for making progress in the study of this group of fungi and more particularly to dissect the genetic determinants underlying the host infection process. In this chapter, we provide an overview of the current knowledge on rust genomics and we particularly highlight how next-generation sequencing technologies are moving this field forward, providing new avenues in the understanding of fungal biotrophy. [ABSTRACT FROM AUTHOR]

Phakopsora pachyrhizi, the cause of soybean rust, is an economically important pathogen of soybean in South America. Understanding the pathogenicity of indigenous fungal populations is useful for identifying resistant plant genotypes and targeting effective cultivars against certain populations. Fifty-nine rust populations from Argentina, Brazil, and Paraguay were evaluated for pathogenicity in three cropping seasons, 2007/2008-2009/2010, using 16 soybean differentials. Only two pairs of P. pachyrhizi populations displayed identical pathogenicity profiles, indicating substantial pathogenic variation in the rust populations. Comparative analysis of 59 South American and five Japanese samples revealed that pathogenic differences were not only detected within South America but also distinct between the P. pachyrhizi populations from South America and Japan. In addition, seasonal changes in rust pathogenicity were detected during the sampling period. The differentials containing resistance genes ( Rpp: resistance to P. p achyrhizi) Rpp1, Rpp2, Rpp3, and Rpp4, except for Plant Introduction (PI) 587880A, displayed a resistant reaction to only 1.8-14, 24-28, 22, and 36 % of South American P. pachyrhizi populations, respectively. In contrast, PI 587880A ( Rpp1), Shiranui ( Rpp5), and 3 Rpp-unknown differentials (PI 587855, PI 587905, and PI 594767A) showed a resistant reaction to 78-96 % of all populations. This study demonstrated that P. pachyrhizi populations from South America vary geographically and temporally in pathogenicity and that the known Rpp genes other than Rpp1 in PI 587880A and Rpp5 have been less effective against recent pathogen populations in the countries studied. [ABSTRACT FROM AUTHOR]

The in planta association of the Hyaloperonospora arabidopsidis effector ATR1 with the cognate Arabidopsis thaliana RPP1 immune receptor activates a disease-resistance signaling pathway that inhibits pathogen growth. To define the molecular events specifying effector recognition by RPP1, we determined the crystal structure of ATR1 and assayed in planta the effects of surface polymorphisms that are critical to activating plant immunity. ATR1 adopts an elongated, all-helical, two-domain, seahorse-like structure with an overall architecture unlike any previously described fold. Structural comparisons highlight a tandemly duplicated, five-helix motif in the C-terminal domain that creates a structural framework for rapid diversification. Identification and mapping of critical recognition sites suggest that ATR1 detection by the RPP1 resistance protein is mediated by several distinct protein surfaces that allow the effectors to escape recognition through diverse surface polymorphisms. ATR1 gain-of-recognition mutants demonstrate that multiple amino acid substitutions are necessary for recognition and that surface polymorphisms exert additive effects. These results suggest that ATR1 is a modular repeat protein belonging to an ancient family of oomycete effectors that rapidly evolves to escape host detection and adopt diverse virulence functions. [ABSTRACT FROM AUTHOR]

Abstract: Obligate biotrophic pathogens like the rust fungi are important plant pathogens causing enormous losses on food, forage and biomass crops. The analysis of the molecular details underlying obligate biotrophic host–parasite interactions is mainly hampered by the fact that no system for transformation is available for most obligate biotrophic organisms. Here we report the transient transformation of Uromyces fabae, an obligate biotrophic rust fungus using a biolistic approach. Biolistic bombardment of U. fabae urediospores was used to deliver different color markers (β-glucuronidase (GUS), intron green fluorescent protein (iGFP) and red fluorescent protein (DsRed) and/or a selection marker. Endogenous regulatory elements from U. fabae plasma membrane ATPase (Uf-PMA1) were used to drive expression of the transgenes. In addition to the delivery of color markers, an in planta selection procedure using the fungicide Carboxin was established allowing the propagation of transformants. In addition to mere cytoplasmic expression of the color markers, a nuclear localization signal was fused to DsRed (pRV115-NLS) targeting the fluorescent marker protein to the nuclei. A procedure for the genetic modification of U. fabae was established. The method can be easily adapted for use with other obligate biotrophic fungi. This provides the basis for a more in depth analysis of the molecular principles governing the obligate biotrophic lifestyle. [Copyright &y& Elsevier]

Abstract: White blister rust caused by the obligate biotroph Albugo candida (Albuginaceae; Oomycota) is one of the most notorious and common diseases of Brassicaceae. During the past 5y, A. candida specimens collected from about 30 host genera were phylogenetically and morphologically investigated in several studies. These not only revealed that A. candida s.str. has a broad host range, encompassing a large number of host plants belonging to Brassicales, but also the presence of previously overlooked species of Albugo with hosts in this order. In this study, we examined specimens from Alyssum, Barbarea, and Rorippa, of which many species were commonly recorded as host plants of A. candida but could not be included in previous works due to the paucity of specimens available. It was revealed that Albugo specimens from Alyssum montanum, Barbarea vulgaris, and various Rorippa species, were placed in three phylogenetically distinct clades, but closer to A. candida s.str. than any previously reported species. Oospores were observed from Albugo specimens parasitic to Rorippa and could be distinguished morphologically from A. candida. Therefore, Albugo rorippae sp. nov. is described and illustrated here. In addition, a key of Albugo species described previously from Brassicales is given. The present study reveals that a large number of Albugo species remain still undiscovered, and that species close to A. candida exist. This could help elucidating the basis of the broad host range of A. candida as opposed to the narrow specialisation that is seemingly present in other species of Albugo on the Brassicaceae. [Copyright &y& Elsevier]

To elucidate host processes and components required for the sustained growth and reproduction of the obligate biotrophic fungus Golovinomyces orontii on Arabidopsis thaIiana, laser micro-dissection was used to isolate cells at the site of infection at 5 days postinfection for downstream global Arabidopsis expression profiling. Site-specific profiling increased sensitivity dramatically, allowing us to identify specific host processes, process components, and their putative regulators hidden in previous whole-leaf global expression analyses. For example, 67 transcription factors exhibited altered expression at the powdery mildew (PM) infection site, with subsets of these playing known or inferred roles in photosynthesis, cold/dehydration responses, defense, auxin signaling, and the cell cycle. Using integrated informatics analyses, we constructed putative regulatory networks for a subset of these processes and provided strong support for host cell cycle modulation at the PM infection site. Further experimentation revealed induced host endoreduplication occurred exclusively at the infection site and led us to identify MYB3R4 as a transcriptional regulator of this process. Induced endoreduplication was abrogated in myb3r4 mutants, and G. orontii growth and reproduction were reduced. This suggests that, by increasing gene copy number, localized endoreduplication serves as a mechanism to meet the enhanced metabolic demands imposed by the fungus, which acquires all its nutrients from the plant host. [ABSTRACT FROM AUTHOR]

Plasmopara halstedii, the causal agent of downy mildew of sunflower, is an obligate parasite but viable sporangia and oospores of the pathogen may be found in a quiescent state in seeds of sunflower and therefore may be transported with sunflower seeds in international commercial exchanges. In order to prevent the spread of this pathogen, especially the introduction of potentially new races, an efficient method to analyse sunflower seed samples is required. In this study, a P. halstedii-specific polymerase chain reaction (PCR) test was developed based on the ribosomal large sub unit (LSU) DNA. The forward (PHAL-F) and reverse (PHAL-R) PCR primers were designed from two polymorphic regions of LSU. After screening 22 isolates of P. halstedii corresponding to different races and countries and 32 other oomycete, deuteromycete and ascomycete isolates, the PHAL-F/R primers amplified only a single PCR band of c. 310 bp from P. halstedii. The PHAL-F/R PCR test could detect as little as 3 pg of P. halstedii genomic DNA per 20  µL reaction volume and enabled the direct detection of P. halstedii in 35 g sunflower seed samples without the need for any prior biological baiting step. An internal amplification control (IAC) was developed to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. The test was successfully used on samples of naturally contaminated seeds. These new molecular tools should be of great interest for quarantine seed testing purposes. [ABSTRACT FROM AUTHOR]

In Peronospora parasitica (At) (downy mildew), the genetic determinants of cultivar-specific recognition by Arabidopsis thaliana are the Arabidopsis thaliana-recognised (ATR) avirulence genes. We describe the identification of 10 amplified fragment length polymorphism (AFLP) markers that define a genetic mapping interval for the ATR1Nd avirulence allele, the presence of which is perceived by the RPP1Nd resistance gene. Furthermore, we have constructed a P. parasitica (At) bacterial artificial chromosome (BAC) library comprising over 630 Mb of cloned DNA. We have isolated 16 overlapping clones from the BAC library that form a contig spanning the genetic interval. BAC sequence-derived markers and a total mapping population of 311 F2 individuals were used to refine the ATR1Nd locus to a 1 cM interval that is represented by four BAC clones and spans less than 250 kb of DNA. This work demonstrates that map-based cloning techniques are feasible in this organism and provides the critical foundations for cloning ATR1Nd using such a strategy. [Copyright &y& Elsevier]

Leucanthemum vulgare Lam. (Asteraceae), known as ox-eye daisy, is a perennial herb native to Europe and western Asia (Clements et al. 2004, McDougall et al. 2018). In Korea, this plant was introduced for ornamental purposes but has been naturalized as a widespread invasive species. In June 2015, symptoms of a powdery mildew disease were observed on L. vulgare in a public garden in Goseong (38°14'18"N, 128°32'56"E), Korea. Since then, its findings have continued throughout the country, including Mokpo and Seogwipo (in 2018), Hongcheon and Seoul (in 2020), Boeun, Gunsan, and Namwon (in 2022), where the disease incidence was often higher than 80%. Symptoms first appeared as circular to irregular white powdery patches covering leaves and stems. Affected plants became distorted, eventually losing their aesthetic and ornamental value. A total of sixteen samples were deposited in the herbarium of Korea University (KUS-F), Korea. Microscopic observations showed that hyphal appressoria were nipple-shaped. Conidiophores were cylindrical, 98 to 157 × 9 to 12 μm, and produced 2 to 5 immature conidia in chains with a sinuate outline. Foot cells were cylindrical, straight, and 37 to 65 μm long. Conidia were ellipsoid to barrel-shaped, 23 to 39 × 12 to 19 μm, with a length/width ratio of 1.4 to 2.3 and devoid of fibrosin bodies. Germ tubes were produced in the perihilar position of the conidia. Primary conidia were apically rounded and basally subtruncated. No chasmothecia were found until the plants died in winter. The morphological characteristics were typical for anamorph of the genus Golovinomyces. To identify the fungus, genomic DNA was extracted from the four herbarium specimens (KUS-F 28650, 30839, 31728, and 31787). PCR products were amplified using the primer sets PM10/ITS4 for internal transcribed spacer (ITS) and PM3/TW14 for the large subunit (LSU) of the rDNA (Mori et al. 2000, Bradshaw and Tobin 2020). Sequences obtained in the present study were deposited at GenBank (accession numbers ON834488-91 for ITS and ON834494-7 for LSU). A BLASTn search of the Korean specimens showed 100% identity with reference sequences of G. ambrosiae in GenBank (KX98730, MK452580, and MK452588 for ITS and MF612182, MK452653, and MK452661 for LSU). In phylogenetic trees of a concatenated dataset of the ITS and LSU sequences, the Korean specimens formed a well-supported clade with the reference sequences of G. ambrosiae. Pathogenicity tests were carried out by touching and dusting an infected leaf (KUS-F 31787) onto the upper leaf surface of five healthy plants. Five non-inoculated plants served as controls. After two weeks, all inoculated plants formed white patches on the surface of leaves and stems, whereas the control plants remained symptomless. The fungus on the inoculated plants was identical to that observed on the initially diseased plant, fulfilling Koch's postulates. As a result, the causal agent of the powdery mildew on L. vulgare was confirmed as G. ambrosiae (Schwein.) U. Braun & R.T.A. Cook, based on the current taxonomy and nomenclature of this species by Qiu et al. (2020).. Previously powdery mildew collections on L. vulgare have been reported as Golovinomyces cichoracearum (≡ Erysiphe cichoracearum) s. lat. in Estonia, Finland, Germany, and Switzerland, Golovinomyces biocellatus in Spain, and Podosphaera fusca (probably P. xanthii according to the current taxonomy) in the former Soviet Union (now Russia and adjacent countries) (Farr and Rossman 2022). This study is the first report of powdery mildew disease caused by G. ambrosiae on L. vulgare in Korea. Qiu et al. (2020) confirmed the occurrence of G. ambrosiae on L. maximum, another species of the genus Leucanthemum. As powdery mildew causes damage to the cultivation of L. vulgare by loss of ornamental value, appropriate control measures should be developed.

Malva verticillata (Malvaceae), commonly called Chinese mallow or whorled mallow, is an annual herb native to East Asia and is currently distributed worldwide. In Korea, this plant is cultivated as a leafy vegetable and cooked like spinach or used in soups and also as a medicine material. In March 2022, typical symptoms of rust disease were observed on M. verticillata in a plastic house (37°22'12″ N, 127°34'30" E) in Yeoju, Korea. Yellow or light green round chlorotic spots appeared on the upper surface of infected leaves, while reddish-brown or dark brown rust pustules formed on its lower surface. Infection occurred in 10% of M. verticillata plants surveyed, and disease severity ranged between 30-90%. A representative sample was deposited in the Kunsan National University Herbarium (KSNUH1762). Telia were mostly hypophyllous, reddish-brown to dark brown, round, mostly grouped, and 0.3-0.7 mm in diameter. Teliospores were mostly two-celled, but rarely one or three-celled, yellowish to light brown, fusoid, and 42.9-101 × 10.8- to 18.8 μm (average 72.7 ± 12.3 × 14.2 ± 1.92 μm [mean ± SD]; n = 50), with a smooth, hyaline to yellowish wall of 1.0-2.5 μm thickness. The morphological characteristics were similar to those reported for Puccinia modiolae (Aime and Abbasi 2018; Albu et al. 2019). To confirm the morphological identification, genomic DNA was extracted from the teliospores of an infected leaf. The internal transcribed spacer (ITS) with primers ITS5-u and ITS4rust (Pfunder and Schürch 2001) and the large subunit (LSU) rDNA with primers LRust1R and LRust3 (Beenken et al. 2012) were amplified for sequencing. The resulting sequences were deposited in GenBank with accession numbers ON631218 for ITS and ON631226 for LSU. BLASTn search showed that the Korean sample was identical to the ITS sequences of P. modiolae from Modiola caroliniana (MK458693-MK458697) and the LSU sequences from M. caroliniana, Malva sylvestris, and Alcea rosea (MH742976, MH742977, and MH742978). In the phylogenetic trees of the ITS and LSU sequences, the Korean sample was grouped with the reference sequences of P. modiolae, with the maximum supporting value. For the pathogenicity test, rust-infected leaf discs were placed on the upper or lower surfaces of leaves of three healthy M. verticillata. Three non-inoculated plants served as controls. Inoculated and non-inoculated plants were maintained in a growth chamber at 22°C, a 16/8 h light cycle, and 80% humidity. After three weeks, all inoculated plants developed evident rust symptoms on the upper and lower surfaces of the leaves on which the leaf discs were placed, whereas the control plants remained symptomless. The pathogen present on the inoculated plants was confirmed to be the same pathogen as that observed in the field, fulfilling Koch's postulates. Based on the morphological investigation, sequence analysis, and pathogenicity tests, P. modiolae was identified as the causal agent of rust disease on M. verticillata. To date, this pathogen has been reported on seven Malvaceae plants, including Alcea rosea, Althaea officinalis, Lavatera arborea, Malva parviflora, Malva sylvestris, Modiola caroliniana, and Modiola sp., in North and South America (Farr and Rossman 2022). However, it has not been reported in Asia or Korea. This study is the first report of rust disease on M. verticillata worldwide. Considering its high incidence rate and severe damage, this pathogen is a potential concern for the cultivation of M. verticillata in Korea. This finding could contribute to developing phytosanitary and control treatments for this disease.

Virginia creeper (or five-leaved ivy; Parthenocissus quinquefolia ) is one of the most popular and widely grown climbers worldwide. In September 2021, Virginia creeper leaves with typical rust symptom were found in an arboretum in Korea, with severe damage. Globally, there is no record of a rust disease on Virginia creeper. Using morphological investigation and molecular phylogenetic inferences, the rust agent was identified as Neophysopella vitis , which is a rust pathogen of other Parthenocissus spp. including Boston ivy ( P. tricuspidata ). Given that the two ivy plants, Virginia creeper and Boston ivy, have common habitats, especially on buildings and walls, throughout Korea, and that N. vitis is a ubiquitous rust species affecting Boston ivy in Korea, it is speculated that the host range of N. vitis may recently have expanded from Boston ivy to Virginia creeper. The present study reports a globally new rust disease on Virginia creeper, which could be a major threat to the ornamental creeper., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Korean Society of Mycology.)

Clematis patens (Ranunculaceae), often called big-flower clematis, is a perennial plant native to Northeast Asia, including China, Japan, and Korea. This plant is one of the popular ornamental plants because of its large and colorful flower. In Korea, it is widely cultivated for public and private gardening and medicinal purposes. In September of 2021, symptoms of rust disease were found on C. patens at a public park (ca. 30 ha) in Jeonju (35°52'16"N, 127°03'16"E), Korea, where the disease occurred on 80% of C. patens plants (n = 50) surveyed, and disease severity in each affected plant ranged 60 to 90%. Symptoms appeared as light green, vein-limited chlorotic spots on the upper surface of infected leaves, and yellow or orange rust pustules were formed on the corresponding lower surface of leaves. A representative sample was deposited in the Kunsan National University Herbarium (KSNUH1522). Uredinia were yellow or orange, round to ellipsoidal, mostly scattered, and 0.5-1 mm in diameter. Urediniospores were pale yellow, ellipsoid or ovoid, 23.1 to 34.8 × 14.9 to 24.7 (average 29.3 ± 2.7 × 18.8 ± 2.2 μm [mean ± SD], n = 50) μm with a verrucose and hyaline wall of 1.0-2.0 μm thick. The morphological characteristics were similar to those reported for Coleosporium clematidis (Barclay 1889, Hiratsuka et al. 1992). To confirm morphological identification, genomic DNA was extracted from a representative specimen (KSNUH1522). The internal transcribed spacer (ITS) rDNA with primers ITS5-u and ITS4rust (Pfunder and Schürch 2001) and large subunit (LSU) rDNA with primers LRust1R and LRust3 (Beenken et al. 2012) were amplified for sequencing. Two resulting sequences (Acc. Nos. OM200310 for ITS, OM184262 for LSU) were blasted in GenBank. The ITS sequence of the Korean sample differs at a nucleotide with a sequence of C. clematidis from Clematis sp. (KX386005) but at eight nucleotides with other three sequences of C. clematidis (KX386007, KX386008 and KX386010). The LSU sequence differs at a nucleotide from the sequences of C. clematidis from Clematis sp. (KX386039, KX386040, KX386042). In phylogenetic trees of the ITS and LSU sequences, the Korean isolate formed a well-supported clade with the reference sequences of C. clematidis. For a pathogenicity test, urediniospores (1.25 ×106/ml) were harvested from the infected leaves and inoculated onto three healthy C. patens. Three non-inoculated plants served as controls. Inoculated and non-inoculated plants were kept in a plant growth chamber at 22°C, a 16/8 h of light cycle, 80% humidity. After three weeks, all inoculated plants formed yellow rust pustules on the lower surface of leaves, identical to what was previously observed in the field, whereas the control plants remained symptomless. The same pathogen was confirmed from the symptomatic plants, fulfilling Koch's postulates. Based on morphological characteristics, sequence data and pathogenicity test, the causal agent of rust on Clematis patens was identified as C. clematidis. To our knowledge, this is the first report of rust disease caused by C. clematidis on C. patens in Korea and previously recorded only in Japan (Hiratsuka et al. 1992). Coleosporium clematidis has been reported on about 60 species of Clematis in Asia and Africa but has not been reported in Europe and North America (Farr and Rossman 2022). In Korea, Clematis fusca var. violacea was previously reported as a host plant for the causal pathogen (Cho and Shin 2004). Given the high occurrence and severe damage, this disease could be a potential threat to the cultivation of C. patens.

The PhyloCode is used to classify taxa based on their relation to a most recent common ancestor as recovered from a phylogenetic analysis. We examined the first specimen of Cintractiella ( Ustilaginomycotina ) collected from Australia and determined its systematic relationship to other Fungi . Three ribosomal DNA loci were analysed both with and without constraint to a phylogenomic hypothesis of the Ustilaginomycotina. Cintractiella did not share a most recent common ancestor with other orders of smut fungi. We used the PhyloCode to define the Cintractiellales , a monogeneric order with four species of Cintractiella , including C. scirpodendri sp. nov. on Scirpodendron ghaeri . The Cintractiellales may have shared a most recent common ancestor with the Malasseziomycetes , but are otherwise unresolved at the rank of class., (© 2020 Westerdijk Fungal Biodiversity Institute.)

Rust fungi (Pucciniales) are the most speciose and the most complex group of plant pathogens. Historically, rust taxonomy was largely influenced by host and phenotypic characters, which are potentially plastic. Molecular systematic studies suggest that the extant diversity of this group was largely shaped by host jumps and subsequent shifts. However, it has been challenging to reconstruct the evolutionary history for the order, especially at deeper (family-level) nodes. Phylogenomics offer a potentially powerful tool to reconstruct the Pucciniales tree of life, although researchers working at this vanguard still face unprecedented challenges working with nonculturable organisms that possess some of the largest and most repetitive genomes now known in kingdom fungi. In this chapter, we provide an overview of the current status and special challenges of rust genomics, and we highlight how phylogenomics may provide new perspectives and answer long-standing questions regarding the biology of rust fungi., (© 2017 Elsevier Inc. All rights reserved.)

Current and emerging plant diseases caused by obligate parasitic microbes such as rusts, downy mildews, and powdery mildews threaten worldwide crop production and food safety. These obligate parasites are typically unculturable in the laboratory, posing technical challenges to characterize them at the genetic and genomic level. Here we have developed a data analysis pipeline integrating several bioinformatic software programs. This pipeline facilitates rapid gene discovery and expression analysis of a plant host and its obligate parasite simultaneously by next generation sequencing of mixed host and pathogen RNA (i.e., metatranscriptomics). We applied this pipeline to metatranscriptomic sequencing data of sweet basil (Ocimum basilicum) and its obligate downy mildew parasite Peronospora belbahrii, both lacking a sequenced genome. Even with a single data point, we were able to identify both candidate host defense genes and pathogen virulence genes that are highly expressed during infection. This demonstrates the power of this pipeline for identifying genes important in host-pathogen interactions without prior genomic information for either the plant host or the obligate biotrophic pathogen. The simplicity of this pipeline makes it accessible to researchers with limited computational skills and applicable to metatranscriptomic data analysis in a wide range of plant-obligate-parasite systems.