Diverse Phytophthora species, including many important plant pathogens, have been widely detected among surface water irrigation sources. In the past decade, metabarcoding has been used to characterize waterborne Phytophthora populations. Metabarcoding typically involves amplification of portions of the nuclear ribosomal internal transcribed spacer (ITS)1 or ITS2 from Phytophthora species, followed by indexed high throughput sequencing. However, full-length sequences of the entire ITS region are required for resolution of many Phytophthora species. We used metabarcoding with PacBio sequencing of full-length ITS amplicons to analyze populations of Phytophthora in waterways of the Stockton East Water District (SEWD) in the northern San Joaquin Valley of California. This approach yielded species-level resolution of many members of the Phytophthora community. Results were compared to those obtained by using ITS1 or ITS2 regions alone and were found to provide superior species resolution for P. pini, P. capsici, and P. gregata. Samples were collected throughout the 2021 irrigation season from five waterways across the SEWD. Thirty-eight Phytophthora species were detected in the waterways, including tree-crop pathogens P. acerina, P. cactorum, P. pini, P. ×cambivora, P. niederhauserii, P. mediterranea, and P. taxon walnut. These pathogenic species were detected throughout the SEWD during most of the irrigation season. The results demonstrated the utility of full-length ITS amplicon sequencing for identifying Phytophthora species in environmental samples and suggested that some disease risk may be incurred by orchardists irrigating with SEWD water. Additional epidemiological studies will be required to critically evaluate this risk.

In September 2023, thirty declining 30-year-old avocado (Persea americana) trees (\'Hass\' grafted on \'Zutano\' seedlings) were detected in a 1.5-ha orchard in the island of Crete (Chania region). Crown symptoms encompassed wilting and leaf chlorosis, advancing to defoliation and extensive dieback. Tap and feeder roots decayed and brown discoloration of root tissues was evident on heavily infected trees. The disease was severe and widespread, resulting in a 5% mortality rate among 300 trees. The pathogen was isolated with a modified soil baiting technique (Ferguson and Jeffers, 1999). Surface disinfected avocado fruits were immersed in water containing soil samples. Following a period of 2 to 8 days, tissue fragments from the resulting necrotic lesions on the fruit surface were transferred on ΡΑRP medium and subsequently incubated at 20°C (Ferguson and Jeffers, 1999). Three isolates (AV2, AV12 and AV11a) were obtained by transferring single hyphal tips to new Petri dishes containing V8 juice agar. They were grown at 20˚C and used for identification after 10 days. Isolates formed coralloid colonies with abundant clustered spherical hyphal swellings and terminal or intercalary (ratio 1:5) thick-walled chlamydospores measuring 20 to 36 μm (avg 29±0.8 μm) with characteristic thick walls (avg 1.2±0.2 μm). Sporangia, produced in non-sterile soil-extract water, were ovoid to obpyriform, persistent, non-papillate, 32 to 81 μm (avg 56±4.8 μm) long and 20 to 42 μm (avg 31±3.2 μm) wide (n=100). Isolates were heterothallic as they did not produce oospores in single cultures. Based on the morphological traits the isolates were identified as Phytophthora cinnamomi (Erwin and Ribeiro 1996). The internal transcribe spacer region (ITS) including ITS1, 5.8S rDNA region, and ITS2 as well as the cytochrome c oxidase subunit I (coxI) gene of the three representative isolates wereamplified with ITS1/ITS4 and FM83/FM84 primers, respectively (White et al. 1990; Martin and Tooley 2003), and sequenced (GenBank acc. PP506613 to PP506615 and PQ063867 to PQ063869, respectively). BLAST search revealed almost 100% identity with the sequences of P. cinnamomi ex-isotype isolate (KC478663 and KU899315 respectively). Pathogenicity tests using isolate AV2 were conducted following the soil infestation method (Jung et al. 1996) using six-year-old avocado \'Zutano\' seedlings. Six non-inoculated plants treated with vermiculite-multivitamin juice mixture were used as controls. Plants (1 m tall) were grown in pots under greenhouse conditions and watered regularly. Six weeks post inoculation, all inoculated trees showed chlorosis, wilting and root rot, while control plants remained symptomless. Symptoms were similar to those observed in the field and the pathogen was re-isolated and molecularly identified as previously described. This study presents the first documented occurrence of P. cinnamomi, widely regarded as the most destructive avocado pathogen globally, on avocado crops in Greece (Rodger et al. 2019). Additionally, this marks the first recorded presence of this pathogen on the island of Crete, regardless of the host species. The accurate identification of Phytophthora species associated with avocado root rot is essential for implementing an effective disease management strategy, particularly in the selection of appropriate disease-resistant rootstocks.

Oomycete pathogens deliver many effectors to enhance virulence or suppress plant immunity. Plant immune networks are interconnected, in which a few effectors can trigger a strong defense response when recognized by immunity-related proteins. How effectors activate plant defense response remains poorly understood. Here we report Phytophthora capsici effector RxLR23KM can induce plant cell death and plant immunity. RxLR23KM specifically binds to ERD15La, a regulator of abscisic acid and salicylic acid pathway, and the binding intensity depends on the amino acid residues (K93 and M320). NbNAC68, a downstream protein of ERD15La, can stimulate plant immunity that is compromised after binding with ERD15La. Silencing of NbNAC68 substantially prevents the activation of plant defense response. RxLR23KM binds to ERD15La, releasing NbNAC68 to activate plant immunity. These findings highlight a strategy of plant defense response that ERD15La as a central regulator coordinates RxLR23KM to regulate NbNAC68-triggered plant immunity.

Oxathiapiprolin (OXA), which targets the oxysterol-binding protein (OSBP), is an outstanding piperidinyl thiazole isoxazoline (PTI) fungicide that can be used to control oomycetes diseases. In this study, starting from the structure of OXA, a series of novel OSBP inhibitors were designed and synthesized by introducing an indole moiety to replace the pyrazole in OXA. Finally, compound b24 was found to exhibit the highest control effect (82%) against cucumber downy mildew (CDM) in the greenhouse at a very low dosage of 0.069 mg/L, which was comparable to that of OXA (88%). Furthermore, it showed better activity against potato late blight (PLB) than other derivatives of indole. The computational results showed that the R-conformation of b24 should be the dominant conformation binding to PcOSBP. The results of the present work indicate that the 3-fluorine-indole ring is a favorable fragment to increasing the electronic energy when binding with PcOSBP. Furthermore, compound b24 could be used as a lead compound for the discovery of new OSBP inhibitors.

Phytophthora species are oomycetes that have evolved a broad spectrum of biological processes and improved strategies to cope with host and environmental challenges. A growing body of evidence indicates that the high pathogen plasticity is based on epigenetic regulation of gene expression linked to Phytophthora\'s rapid adjustment to endogenous cues and various stresses. As 5mC DNA methylation has not yet been identified in Phytophthora, the reversible processes of acetylation/deacetylation of histone proteins seem to play a pivotal role in the epigenetic control of gene expression in oomycetes. To explore this issue, we review the structure, diversity, and phylogeny of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in six plant-damaging Phytophthora species: P. capsici, P. cinnamomi, P. infestans, P. parasitica, P. ramorum, and P. sojae. To further integrate and improve our understanding of the phylogenetic classification, evolutionary relationship, and functional characteristics, we supplement this review with a comprehensive view of HATs and HDACs using recent genome- and proteome-level databases. Finally, the potential functional role of transcriptional reprogramming mediated by epigenetic changes during Phytophthora species saprophytic and parasitic phases under nitro-oxidative stress is also briefly discussed.

Asparagine (Asn, N)-linked glycosylation is an abundant post-translational modification in which Asn, typically in Nglyco-X-S/T; X ≠ P motifs, are modified with N-glycans. It has essential regulatory roles in multicellular organisms. In this study, we systematically investigate the function of three N-glycosylation motifs (Nglyco-A, Nglyco-D and Nglyco-S) previously identified in Phytophthora sojae, through site-directed mutagenesis and functional assays. In P. sojae expressing glycosylation-dead variants pre-PsDMAP1N70A (Nglyco-A motif) or PsADFN64A (Nglyco-D motif), zoospore release or cyst germination is impaired. In particular, the pre-PsDMAP1N70A mutant reduces DNA methylation levels, and the PsADFN64A mutant disrupts the actin forms, which could explain the decrease in pathogenicity after N-glycosylation is destroyed. Similarly, P. sojae expressing PsNRXN132A (Nglyco-S motif) shows increased sensitivity to H2O2 and heat. Through autophagy or 26S proteasome pathway inhibition assays, we found that unglycosylated pre-PsDMAP1N70A and PsADFN64A are degraded via the 26S proteasome pathway, while the autophagy pathway is responsible for PsNRXN132A clearance. These findings demonstrate that glycosylation of these motifs regulates the stability and function of glycoproteins necessary for P. sojae growth, reproduction and pathogenicity, which expands the scope of known N-glycosylation regulatory functions in oomycetes.

Grapevine downy mildew, caused by the oomycete Plasmopara viticola (P. viticola, Berk. & M. A. Curtis; Berl. & De Toni), is a global threat to Eurasian wine grapes Vitis vinifera. Although resistant grapevine varieties are becoming more accessible, P. viticola populations are rapidly evolving to overcome these resistances. We aimed to uncover avirulence genes related to Rpv3.1-mediated grapevine resistance. We sequenced the genomes and characterized the development of 136 P. viticola strains on resistant and sensitive grapevine cultivars. A genome-wide association study was conducted to identify genomic variations associated with resistant-breaking phenotypes. We identified a genomic region associated with the breakdown of Rpv3.1 grapevine resistance (avrRpv3.1 locus). A diploid-aware reassembly of the P. viticola INRA-Pv221 genome revealed structural variations in this locus, including a 30 kbp deletion. Virulent P. viticola strains displayed multiple deletions on both haplotypes at the avrRpv3.1 locus. These deletions involve two paralog genes coding for proteins with 800-900 amino acids and signal peptides. These proteins exhibited a structure featuring LWY-fold structural modules, common among oomycete effectors. When transiently expressed, these proteins induced cell death in grapevines carrying Rpv3.1 resistance, confirming their avirulence nature. This discovery sheds light on the genetic mechanisms enabling P. viticola to adapt to grapevine resistance, laying a foundation for developing strategies to manage this destructive crop pathogen.

Revealing the effector-host molecular interactions is crucial for understanding the host immunity against Plasmopara viticola and devising innovative disease management strategies. As a pathogenic oomycete causing grapevine downy mildew, Plasmopara viticola employs various effectors to manipulate the defense systems of host plants. One of these P. viticola derived effectors is necrosis- and ethylene-inducing peptide 1 (Nep1) -like protein (PvNLP7), which has been known to elicit cell death and immune responses in plants. However, the underlying molecular mechanisms remain obscure, prompting the focus of this study. Through yeast two-hybrid screening, we have identified the Vitis rotundifolia ADP-ribosylation factor (VrARF1) as a host interactor of PvNLP7. This interaction is corroborated through bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays. Heterologous expression of VrARF1 in Nicotiana benthamiana verifies its accumulation in both the cytoplasm and nucleus, and induction of cell death. Moreover, the VrARF1 gene is strongly induced during early P. viticola infection and upon PvNLP7 transient expression. Overexpression of the VrARF1 gene in grapevine and N. benthamiana enhances resistance to P. viticola and Phytophthora capsici, respectively, via induction of defense related genes PR1 and PR2. Conversely, virus-induced gene silencing (VIGS) of NbARF1 in N. benthamiana, homologous to VrARF1, markedly attenuates PvNLP7-triggered cell death and reduces the expression of four PTI marker genes (PTI5, Acre31, WRKY7 and Cyp71D20) and two defense related genes (PR1 and PR2), rendering plants transiently transformed with PvNLP7 more susceptible to oomycete P. capsici. These findings highlight the role of ARF1 in mediating PvNLP7-induced immunity and indicate its potential as a target for engineering disease-resistant transgenic plants against oomycete pathogens.

While resistant cultivars are valuable in safeguarding crops against diseases, they can be rapidly overcome by pathogens. Numerous strategies have been proposed to delay pathogen adaptation (evolutionary control), while still ensuring effective protection (epidemiological control). For perennial crops, multiple resistance genes can be deployed 1) in the same cultivar (pyramiding strategy), in single-gene-resistant cultivars grown 2) in the same field (mixture strategy) or 3) in different fields (mosaic strategy), or 4) in hybrid strategies that combine the three previous options. In addition, the spatial scale at which resistant cultivars are deployed can affect the plant-pathogens interaction: small fields are thought to reduce pest density and disease transmission. Here we used the spatially-explicit stochastic model landsepi to compare the evolutionary and epidemiological control across spatial scales and deployment strategies relying on two major resistance genes. Our results, broadly focused on resistance to downy mildew of grapevine, show that the evolutionary control provided by the pyramiding strategy is at risk when single-gene-resistant cultivars are concurrently planted in the landscape (hybrid strategies), especially at low mutation probability. Moreover, the effectiveness of pyramiding compared to hybrid strategies is influenced by whether the adapted pathogen pays a fitness cost across all hosts or only for unnecessary virulence, particularly when the fitness cost is high rather than intermediate. Finally, field size did not affect model outputs for a wide range of mutation probabilities and associated fitness costs. The socio-economic policies favoring the adoption of optimal resistant management strategies are discussed.

Filamentous plant pathogens, including fungi and oomycetes, pose significant threats to cultivated crops, impacting agricultural productivity, quality and sustainability. Traditionally, disease control heavily relied on fungicides, but concerns about their negative impacts motivated stakeholders and government agencies to seek alternative solutions. Biocontrol agents (BCAs) have been developed as promising alternatives to minimize fungicide use. However, BCAs often exhibit inconsistent performances, undermining their efficacy as plant protection alternatives. The eukaryotic cell wall of plants and filamentous pathogens contributes significantly to their interaction with the environment and competitors. This highly adaptable and modular carbohydrate armor serves as the primary interface for communication, and the intricate interplay within this compartment is often mediated by carbohydrate-active enzymes (CAZymes) responsible for cell wall degradation and remodeling. These processes play a crucial role in the pathogenesis of plant diseases and contribute significantly to establishing both beneficial and detrimental microbiota. This review explores the interplay between cell wall dynamics and glycan interactions in the phytobiome scenario, providing holistic insights for efficiently exploiting microbial traits potentially involved in plant disease mitigation. Within this framework, the incorporation of glycobiology-related functional traits into the resident phytobiome can significantly enhance the plant\'s resilience to biotic stresses. Therefore, in the rational engineering of future beneficial consortia, it is imperative to recognize and leverage the understanding of cell wall interactions and the role of the glycome as an essential tool for the effective management of plant diseases.