A Genomic Resource for the Strawberry Powdery Mildew Pathogen Podosphaera aphanis.

Powdery mildew is one of the most economically destructive diseases in protected strawberry production. Here we present the first genome assembly for Podosphaera aphanis, the causal agent of powdery mildew on strawberry. This obligate-biotrophic fungal pathogen was sampled from a naturally occurring outbreak on Fragaria × ananassa 'Malling Centenary' plants grown under cover in the United Kingdom. Assembled reads resolved a 55.6 Mb genome, composed of 12,357 contigs whose annotation led to prediction of 17,239 genes encoding 17,328 proteins. The genome is highly-complete, with 97.5% of conserved single-copy Ascomycete genes shown to be present. This annotated P. aphanis genome provides a molecular resource for further investigation into host-pathogen interactions in the strawberry powdery mildew pathosystem.

Impact of Future Elevated Carbon Dioxide on C3 Plant Resistance to Biotic Stresses.

Before the end of the century, atmospheric carbon dioxide levels are predicted to increase to approximately 900 ppm. This will dramatically affect plant physiology and influence environmental interactions and, in particular, plant resistance to biotic stresses. This review is a broad survey of the current research on the effects of elevated CO2 (eCO2) on phytohormone-mediated resistance of C3 agricultural crops and related model species to pathogens and insect herbivores. In general, while plants grown in eCO2 often have increased constitutive and induced salicylic acid levels and suppressed induced jasmonate levels, there are exceptions that implicate other environmental factors, such as light and nitrogen fertilization in modulating these responses. Therefore, this review sets the stage for future studies to delve into understanding the mechanistic basis behind how eCO2 will affect plant defensive phytohormone signaling pathways under future predicted environmental conditions that could threaten global food security to inform the best agricultural management practices.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Leucanthemum vulgare in Korea.

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.

First Report of Coleosporium clematidis Causing Rust Disease on Clematis patens in Korea.

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.

First Report of Puccinia modiolae Causing Rust Disease on Malva verticillata in Korea.

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.

Genome-wide analysis of Claviceps paspali: insights into the secretome of the main species causing ergot disease in Paspalum spp.

BACKGROUND: The phytopatogen Claviceps paspali is the causal agent of Ergot disease in Paspalum spp., which includes highly productive forage grasses such as P. dilatatum. This disease impacts dairy and beef production by affecting seed quality and producing mycotoxins that can affect performance in feeding animals. The molecular basis of pathogenicity of C. paspali remains unknown, which makes it more difficult to find solutions for this problem. Secreted proteins are related to fungi virulence and can manipulate plant immunity acting on different subcellular localizations. Therefore, identifying and characterizing secreted proteins in phytopathogenic fungi will provide a better understanding of how they overcome host defense and cause disease. The aim of this work is to analyze the whole genome sequences of three C. paspali isolates to obtain a comparative genome characterization based on possible secreted proteins and pathogenicity factors present in their genome. In planta RNA-seq analysis at an early stage of the interaction of C. paspali with P. dilatatum stigmas was also conducted in order to determine possible secreted proteins expressed in the infection process. RESULTS: C. paspali isolates had compact genomes and secretome which accounted for 4.6-4.9% of the predicted proteomes. More than 50% of the predicted secretome had no homology to known proteins. RNA-Seq revealed that three protein-coding genes predicted as secreted have mayor expression changes during 1 dpi vs 4 dpi. Also, three of the first 10 highly expressed genes in both time points were predicted as effector-like. CAZyme-like proteins were found in the predicted secretome and the most abundant family could be associated to pectine degradation. Based on this, pectine could be a main component affected by the cell wall degrading enzymes of C. paspali. CONCLUSIONS: Based on predictions from DNA sequence and RNA-seq, unique probable secreted proteins and probable pathogenicity factors were identified in C. paspali isolates. This information opens new avenues in the study of the biology of this fungus and how it modulates the interaction with its host. Knowledge of the diversity of the secretome and putative pathogenicity genes should facilitate future research in disease management of Claviceps spp.

Dual RNA-Seq analysis of Medicago truncatula and the pea powdery mildew Erysiphe pisi uncovers distinct host transcriptional signatures during incompatible and compatible interactions and pathogen effector candidates.

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.

Transcriptomic Profiling of Acute Cold Stress-Induced Disease Resistance (SIDR) Genes and Pathways in the Grapevine Powdery Mildew Pathosystem.

Temperatures from 2 to 8°C transiently induce quantitative resistance to powdery mildew in several host species (cold stress-induced disease resistance [SIDR]). Although cold SIDR events occur in vineyards worldwide an average of 14 to 21 times after budbreak of grapevine and can significantly delay grapevine powdery mildew (Erysiphe necator) epidemics, its molecular basis was poorly understood. We characterized the biology underlying the Vitis vinifera cold SIDR phenotype-which peaks at 24 h post-cold (hpc) treatment and results in a 22 to 28% reduction in spore penetration success-through highly replicated (n = 8 to 10) RNA sequencing experiments. This phenotype was accompanied by a sweeping transcriptional downregulation of photosynthesis-associated pathways whereas starch and sugar metabolism pathways remained largely unaffected, suggesting a transient imbalance in host metabolism and a suboptimal target for pathogen establishment. Twenty-six cold-responsive genes peaked in their differential expression at the 24-hpc time point. Finally, a subset of genes associated with nutrient and amino acid transport accounted for four of the eight most downregulated transcripts, including two nodulin 1A gene precursors, a nodulin MtN21 precursor, and a Dynein light chain 1 motor protein precursor. Reduced transport could exacerbate localized nutrient sinks that would again be transiently suboptimal for pathogen growth. This study links the transient cold SIDR phenotype to underlying transcriptional changes and provides an experimental framework and library of candidate genes to further explore cold SIDR in several systems, with an ultimate goal of identifying novel breeding or management targets for reduced disease.

Metatranscriptomic dynamics after Verticillium dahliae infection and root damage in Olea europaea.

BACKGROUND: The olive tree is of particular economic interest in the Mediterranean basin. Researchers have conducted several studies on one of the most devastating disorders affecting this tree, the Verticillium wilt, which causes substantial economic losses in numerous areas. We analyzed metatranscriptomic samples taken from a previous study conducted on leaves and roots of Olea europaea that were infected with Verticillium dahliae. In addition, we also analyzed mechanically damaged roots. The aim of our approach is to describe the dynamics of the root microbiome after severe perturbations. RESULTS: Our results not only describe the dynamics of the microbial community associated with the disturbance, but also show the high complexity of these systems and explain how this can lead to a conflicting assignment of the various types of parasitism observed in a specific organism. CONCLUSIONS: Our findings indicate that this infection, although led by Verticillium, is driven not by a single species, but by a polymicrobial consortium that also includes natural endophytes of the olive tree. This community contains both biotrophic and necrotrophic organisms that alternate and live together during the infection. In addition, opportunistic organisms appear that take profit not from plant tissues, but from new emerging populations of microorganisms. Therefore, this system can be described as a complex biological system composed of different interacting communities. Notably, our work has important considerations when it comes to classifying the type of parasitism of a given species.

Network analysis exposes core functions in major lifestyles of fungal and oomycete plant pathogens.

BACKGROUND: Genomic studies demonstrate that components of virulence mechanisms in filamentous eukaryotic pathogens (FEPs, fungi and oomycetes) of plants are often highly conserved, or found in gene families that include secreted hydrolytic enzymes (e.g., cellulases and proteases) and secondary metabolites (e.g., toxins), central to the pathogenicity process. However, very few large-scale genomic comparisons have utilized complete proteomes from dozens of FEPs to reveal lifestyle-associated virulence mechanisms. Providing a powerful means for exploration, and the discovery of trends in large-scale datasets, network analysis has been used to identify core functions of the primordial cyanobacteria, and ancient evolutionary signatures in oxidoreductases. RESULTS: We used a sequence-similarity network to study components of virulence mechanisms of major pathogenic lifestyles (necrotroph (ic), N; biotroph (ic), B; hemibiotroph (ic), H) in complete pan-proteomes of 65 FEPs and 17 saprobes. Our comparative analysis highlights approximately 190 core functions found in 70% of the genomes of these pathogenic lifestyles. Core functions were found mainly in: transport (in H, N, B cores); carbohydrate metabolism, secondary metabolite synthesis, and protease (H and N cores); nucleic acid metabolism and signal transduction (B core); and amino acid metabolism (H core). Taken together, the necrotrophic core contains functions such as cell wall-associated degrading enzymes, toxin metabolism, and transport, which are likely to support their lifestyle of killing prior to feeding. The biotrophic stealth growth on living tissues is potentially controlled by a core of regulatory functions, such as: small G-protein family of GTPases, RNA modification, and cryptochrome-based light sensing. Regulatory mechanisms found in the hemibiotrophic core contain light- and CO2-sensing functions that could mediate important roles of this group, such as transition between lifestyles. CONCLUSIONS: The selected set of enriched core functions identified in our work can facilitate future studies aimed at controlling FEPs. One interesting example would be to facilitate the identification of the pathogenic potential of samples analyzed by metagenomics. Finally, our analysis offers potential evolutionary scenarios, suggesting that an early-branching saprobe (identified in previous studies) has probably evolved a necrotrophic lifestyle as illustrated by the highest number of shared gene families between saprobes and necrotrophs.

The biotroph Agrobacterium tumefaciens thrives in tumors by exploiting a wide spectrum of plant host metabolites.

Agrobacterium tumefaciens is a niche-constructing biotroph that exploits host plant metabolites. We combined metabolomics, transposon-sequencing (Tn-seq), transcriptomics, and reverse genetics to characterize A. tumefaciens pathways involved in the exploitation of resources from the Solanum lycopersicum host plant. Metabolomics of healthy stems and plant tumors revealed the common (e.g. sucrose, glutamate) and enriched (e.g. opines, γ-aminobutyric acid (GABA), γ-hydroxybutyric acid (GHB), pyruvate) metabolites that A. tumefaciens could use as nutrients. Tn-seq and transcriptomics pinpointed the genes that are crucial and/or upregulated when the pathogen grew on either sucrose (pgi, kdgA, pycA, cisY) or GHB (blcAB, pckA, eno, gpsA) as a carbon source. While sucrose assimilation involved the Entner-Doudoroff and tricarboxylic acid (TCA) pathways, GHB degradation required the blc genes, TCA cycle, and gluconeogenesis. The tumor-enriched metabolite pyruvate is at the node connecting these pathways. Using reverse genetics, we showed that the blc, pckA, and pycA loci were important for aggressiveness (tumor weight), proliferation (bacterial charge), and/or fitness (competition between the constructed mutants and wild-type) of A. tumefaciens in plant tumors. This work highlighted how a biotroph mobilizes its central metabolism for exploiting a wide diversity of resources in a plant host. It further shows the complementarity of functional genome-wide scans by transcriptomics and Tn-seq to decipher the lifestyle of a plant pathogen.

Salicylic acid-dependent immunity contributes to resistance against Rhizoctonia solani, a necrotrophic fungal agent of sheath blight, in rice and Brachypodium distachyon.

Rhizoctonia solani is a soil-borne fungus causing sheath blight. In consistent with its necrotrophic life style, no rice cultivars fully resistant to R. solani are known, and agrochemical plant defense activators used for rice blast, which upregulate a phytohormonal salicylic acid (SA)-dependent pathway, are ineffective towards this pathogen. As a result of the unavailability of genetics, the infection process of R. solani remains unclear. We used the model monocotyledonous plants Brachypodium distachyon and rice, and evaluated the effects of phytohormone-induced resistance to R. solani by pharmacological, genetic and microscopic approaches to understand fungal pathogenicity. Pretreatment with SA, but not with plant defense activators used in agriculture, can unexpectedly induce sheath blight resistance in plants. SA treatment inhibits the advancement of R. solani to the point in the infection process in which fungal biomass shows remarkable expansion and specific infection machinery is developed. The involvement of SA in R. solani resistance is demonstrated by SA-deficient NahG transgenic rice and the sheath blight-resistant B. distachyon accessions, Bd3-1 and Gaz-4, which activate SA-dependent signaling on inoculation. Our findings suggest a hemi-biotrophic nature of R. solani, which can be targeted by SA-dependent plant immunity. Furthermore, B. distachyon provides a genetic resource that can confer disease resistance against R. solani to plants.

The compact genome of the plant pathogen Plasmodiophora brassicae is adapted to intracellular interactions with host Brassica spp.

BACKGROUND: The protist Plasmodiophora brassicae is a soil-borne pathogen of cruciferous species and the causal agent of clubroot disease of Brassicas including agriculturally important crops such as canola/rapeseed (Brassica napus). P. brassicae has remained an enigmatic plant pathogen and is a rare example of an obligate biotroph that resides entirely inside the host plant cell. The pathogen is the cause of severe yield losses and can render infested fields unsuitable for Brassica crop growth due to the persistence of resting spores in the soil for up to 20 years. RESULTS: To provide insight into the biology of the pathogen and its interaction with its primary host B. napus, we produced a draft genome of P. brassicae pathotypes 3 and 6 (Pb3 and Pb6) that differ in their host range. Pb3 is highly virulent on B. napus (but also infects other Brassica species) while Pb6 infects only vegetable Brassica crops. Both the Pb3 and Pb6 genomes are highly compact, each with a total size of 24.2 Mb, and contain less than 2 % repetitive DNA. Clustering of genome-wide single nucleotide polymorphisms (SNP) of Pb3, Pb6 and three additional re-sequenced pathotypes (Pb2, Pb5 and Pb8) shows a high degree of correlation of cluster grouping with host range. The Pb3 genome features significant reduction of intergenic space with multiple examples of overlapping untranslated regions (UTRs). Dependency on the host for essential nutrients is evident from the loss of genes for the biosynthesis of thiamine and some amino acids and the presence of a wide range of transport proteins, including some unique to P. brassicae. The annotated genes of Pb3 include those with a potential role in the regulation of the plant growth hormones cytokinin and auxin. The expression profile of Pb3 genes, including putative effectors, during infection and their potential role in manipulation of host defence is discussed. CONCLUSION: The P. brassicae genome sequence reveals a compact genome, a dependency of the pathogen on its host for some essential nutrients and a potential role in the regulation of host plant cytokinin and auxin. Genome annotation supported by RNA sequencing reveals significant reduction in intergenic space which, in addition to low repeat content, has likely contributed to the P. brassicae compact genome.

Antifungal Activity of Phyllospheric Bacteria Isolated from Coffea arabica against Hemileia vastatrix.

Peru is one of the leading countries that produce and export specialty coffees, favorably positioned in the international markets for its physical and organoleptic cup qualities. In recent years, yellow coffee rust caused by the phytopathogenic fungus Hemileia vastatrix stands out as one of the main phytosanitary diseases that affect coffee culture yields. Many studies have demonstrated bacteria antagonistic activity against a number of phytopathogen fungi. In this context, the aim of this work was to select and characterize phyllospheric bacteria isolated from Coffea arabica with antagonistic features against coffee rust to obtain biocontrollers. For that purpose, a total of 82 phyllospheric bacteria were isolated from two coffee leaf rust-susceptible varieties, typica and caturra roja, and one tolerant variety, catimor. Of all the isolates, 15% were endophytic and 85% were epiphytes. Among all the isolates, 14 were capable of inhibiting the mycelial radial growth of Mycena citricolor, and Colletotrichum sp. 16S rRNA gene sequence-based analysis showed that 9 isolates were related to Achromobacter insuavis, 2 were related to Luteibacter anthropi and 1 was related to Rodococcus ceridiohylli, Achromobacter marplatensis and Pseudomonas parafulva. A total of 7 representative bacteria of each group were selected based on their antagonistic activity and tested in germination inhibition assays of coffee rust uredinospores. The CRRFLT7 and TRFLT8 isolates showed a high inhibition percentage of urediniospores germination (81% and 82%, respectively), similar to that obtained with the chemical control (91%). An experimental field assay showed a good performance of both strains against rust damage too, making them a promising alternative for coffee leaf rust biocontrol.

Genetic loci associated with tissue-specific resistance to powdery mildew in octoploid strawberry (Fragaria × ananassa).

Powdery mildew is one of the most problematic diseases in strawberry production. To date, few commercial strawberry cultivars are deemed to have complete resistance and as such, an extensive spray programme must be implemented to control the pathogen. Here, a large-scale field experiment was used to determine the powdery mildew resistance status of leaf and fruit tissues across a diverse panel of strawberry genotypes. This phenotypic data was used to identify Quantitative Trait Nucleotides (QTN) associated with tissue-specific powdery mildew resistance. In total, six stable QTN were found to be associated with foliar resistance, with one QTN on chromosome 7D associated with a 61% increase in resistance. In contrast to the foliage results, there were no QTN associated with fruit disease resistance and there was a high level of resistance observed on strawberry fruit, with no genetic correlation observed between fruit and foliar symptoms, indicating a tissue-specific response. Beyond the identification of genetic loci, we also demonstrate that genomic selection can lead to rapid gains in foliar resistance across genotypes, with the potential to capture >50% of the genetic foliage resistance present in the population. To date, breeding of robust powdery mildew resistance in strawberry has been impeded by the quantitative nature of natural resistance and a lack of knowledge relating to the genetic control of the trait. These results address this shortfall, through providing the community with a wealth of information that could be utilized for genomic informed breeding, implementation of which could deliver a natural resistance strategy for combatting powdery mildew.

A new and effective method to induce infection of Phyllachora maydis into corn for tar spot studies in controlled environments.

BACKGROUND: Tar spot of corn is a significant and spreading disease in the continental U.S. and Canada caused by the obligate biotrophic fungus Phyllachora maydis. As of 2023, tar spot had been reported in 18 U.S. states and one Canadian Province. The symptoms of tar spot include chlorotic flecking followed by the formation of black stromata where conidia and ascospores are produced. Advancements in research and management for tar spot have been limited by a need for a reliable method to inoculate plants to enable the study of the disease. The goal of this study was to develop a reliable method to induce tar spot in controlled conditions. RESULTS: We induced infection of corn by P. maydis in 100% of inoculated plants with a new inoculation method. This method includes the use of vacuum-collection tools to extract ascospores from field-infected corn leaves, application of spores to leaves, and induction of the disease in the dark at high humidity and moderate temperatures. Infection and disease development were consistently achieved in four independent experiments on different corn hybrids and under different environmental conditions in a greenhouse and growth chamber. Disease induction was impacted by the source and storage conditions of spores, as tar spot was not induced with ascospores from leaves stored dry at 25 ºC for 5 months but was induced using ascospores from infected leaves stored at -20 ºC for 5 months. The time from inoculation to stromata formation was 10 to 12 days and ascospores were present 19 days after inoculation throughout our experiments. In addition to providing techniques that enable in-vitro experimentation, our research also provides fundamental insights into the conditions that favor tar spot epidemics. CONCLUSIONS: We developed a method to reliably inoculate corn with P. maydis. The method was validated by multiple independent experiments in which infection was induced in 100% of the plants, demonstrating its consistency in controlled conditions. This new method facilitates research on tar spot and provides opportunities to study the biology of P. maydis, the epidemiology of tar spot, and for identifying host resistance.

Dose-Dependent Genetic Resistance to Azole Fungicides Found in the Apple Scab Pathogen.

The evolution of azole resistance in fungal pathogens presents a major challenge in both crop production and human health. Apple orchards across the world are faced with the emergence of azole fungicide resistance in the apple scab pathogen Venturia inaequalis. Target site point mutations observed in this fungus to date cannot fully explain the reduction in sensitivity to azole fungicides. Here, polygenic resistance to tebuconazole was studied across a population of V. inaequalis. Genotyping by sequencing allowed Quantitative Trait Loci (QTLs) mapping to identify the genetic components controlling this fungicide resistance. Dose-dependent genetic resistance was identified, with distinct genetic components contributing to fungicide resistance at different exposure levels. A QTL within linkage group seven explained 65% of the variation in the effective dose required to reduce growth by 50% (ED50). This locus was also involved in resistance at lower fungicide doses (ED10). A second QTL in linkage group one was associated with dose-dependent resistance, explaining 34% of variation at low fungicide doses (ED10), but did not contribute to resistance at higher doses (ED50 and ED90). Within QTL regions, non-synonymous mutations were observed in several ATP-Binding Cassette and Major Facilitator SuperFamily transporter genes. These findings provide insight into the mechanisms of fungicide resistance that have evolved in horticultural pathogens. Identification of resistance gene candidates supports the development of molecular diagnostics to inform management practices.

Molecular plant immunity against biotrophic, hemibiotrophic, and necrotrophic fungi.

Pathogenic fungi use diverse infection strategies to obtain nutrients from plants. Biotrophic fungi feed only on living plant tissue, whereas necrotrophic fungi kill host cells to extract nutrients. To prevent disease, plants need to distinguish between pathogens with different life cycles, as a successful defense against a biotroph, which often involves programmed cell-death around the site of infection, is not an appropriate response to some necrotrophs. Plants utilize a vast collection of extracellular and intracellular receptors to detect the signatures of pathogen attack. In turn, pathogens are under strong selection to mask or avoid certain receptor responses while enhancing or manipulating other receptor responses to promote virulence. In this review, we focus on the plant receptors involved in resistance responses to fungal pathogens and highlight, with examples, how the infection strategy of fungal pathogens can determine if recognition responses are effective at preventing disease.

Communication is key: extracellular vesicles as mediators of infection and defence during host-microbe interactions in animals and plants.

Extracellular vesicles (EVs) are now understood to be ubiquitous mediators of cellular communication. In this review, we suggest that EVs have evolved into a highly regulated system of communication with complex functions including export of wastes, toxins and nutrients, targeted delivery of immune effectors and vectors of RNA silencing. Eukaryotic EVs come in different shapes and sizes and have been classified according to their biogenesis and size distributions. Small EVs (or exosomes) are released through fusion of endosome-derived multivesicular bodies with the plasma membrane. Medium EVs (or microvesicles) bud off the plasma membrane as a form of exocytosis. Finally, large EVs (or apoptotic bodies) are produced as a result of the apoptotic process. This review considers EV secretion and uptake in four eukaryotic kingdoms, three of which produce cell walls. The impacts cell walls have on EVs in plants and fungi are discussed, as are roles of fungal EVs in virulence. Contributions of plant EVs to development and innate immunity are presented. Compelling cases are sporophytic self-incompatibility and cellular invasion by haustorium-forming filamentous pathogens. The involvement of EVs in all of these eukaryotic processes is reconciled considering their evolutionary history.