Multiple deletions of candidate effector genes lead to the breakdown of partial grapevine resistance to downy mildew.

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.

Transcriptome analysis of Phytophthora cactorum infecting strawberry identified RXLR effectors that induce cell death when transiently expressed in Nicotiana benthamiana.

Phytophthora cactorum is a plant pathogenic oomycete that causes crown rot in strawberry leading to significant economic losses every year. To invade the host, P. cactorum secretes an arsenal of effectors that can manipulate host physiology and impair its defense system promoting infection. A transcriptome analysis was conducted on a susceptible wild strawberry genotype (Fragaria vesca) 48 hours post inoculation with P. cactorum to identify effectors expressed during the early infection stage. The analysis revealed 4,668 P. cactorum genes expressed during infection of F. vesca. A total of 539 secreted proteins encoded by transcripts were identified, including 120 carbohydrate-active enzymes, 40 RXLRs, 23 proteolytic enzymes, nine elicitins, seven cysteine rich proteins, seven necrosis inducing proteins and 216 hypothetical proteins with unknown function. Twenty of the 40 RXLR effector candidates were transiently expressed in Nicotiana benthamiana using agroinfiltration and five previously unreported RXLR effector genes (Pc741, Pc8318, Pc10890, Pc20813, and Pc22290) triggered cell death when transiently expressed. The identified cell death inducing RXLR effectors showed 31-66% identity to known RXLR effectors in different Phytophthora species having roles in pathogenicity including both activation and suppression of defense response in the host. Furthermore, homology analysis revealed that these cell death inducing RXLR effectors were highly conserved (82 - 100% identity) across 23 different strains of P. cactorum originating from apple or strawberry.

Design, synthesis, and anti-oomycete activity of 3-acyloxymaltol/ethyl maltol derivatives.

Twenty 3-acyloxymaltol/ethyl maltol derivatives (7a-j and 8a-j) were synthesized and evaluated in vitro for their anti-oomycete activity against Phytophthora capsici, respectively. Among all of twenty derivatives, more than half of the compounds 7f, 7h, 8a-h and 8j had anti-oomycete activity higher than the positive control zoxamide (EC50 = 22.23 mg/L), and the EC50 values of 18.66, 20.32, 12.80, 16.18, 10.59, 14.98, 16.80, 10.36, 15.32, 12.64, and 13.59 mg/L, respectively. Especially, compounds 8c and 8f exhibited the best anti-oomycete activity against P. capsici with EC50 values of 10.59 and 10.36 mg/L, respectively. Overall, hydroxyl group of maltol/ethyl maltol is important active modification site.

An old unknown: 40 years of crayfish plague monitoring in Switzerland, the water tower of Europe.

The oomycete Aphanomyces astaci is the causative agent of crayfish plague, a disease threatening susceptible freshwater crayfish species in Europe. To detect its spatiotemporal occurrence in Switzerland, we reviewed (1) the literature regarding occurrence of crayfish plague and North American crayfish carrier species and (2) the necropsy report archive of the Institute for Fish and Wildlife Health (FIWI) from 1968 to 2020. In the past, crayfish plague was diagnosed through several methods: conventional PCR, culture, and histology. When available, we re-evaluated archived Bouin's or formalin-fixed, paraffin-embedded samples collected during necropsies (1991-2020) with a recently published quantitative PCR. Literature research revealed putative reports of crayfish plague in Switzerland between the 1870s and 1910s and the first occurrence of three North American crayfish species between the late 1970s and 1990s. Finally, 54 (28.1%) cases were classified as positive and 9 (4.7%) cases as suspicious. The total number of positive cases increased by 14 (14.7%) after re-evaluation of samples. The earliest diagnosis of crayfish plague was performed in 1980 and the earliest biomolecular confirmation of A. astaci DNA dated 1991. Between 1980-1990, 1991-2000 and 2001-2010 crayfish plague spread from one to two and finally three catchment basins, respectively. Similar to other European countries, crayfish plague has occurred in Switzerland in two waves: the first at the end of the 19th and the second at the end of the 20th century in association with the first occurrence of North American crayfish species. The spread from one catchment basin to another suggests a human-mediated pathogen dispersal.

Genome Resource of Raspberry Root Rot Pathogen Phytophthora gonapodyides.

Phytophthora gonapodyides is a newly reported oomycetes pathogen associated with root rot of red raspberry. We generated high-quality whole genome resource for P. gonapodyides, which was pathogenic on red raspberry. The genome size was 88,717,598 bp with a BUSCO completeness score of 93.9%. This genome resource provides insight on pathogen biology of Phytophthora spp. causing root rot of raspberry. To our best knowledge, this is the first complete genome assembly of plant pathogenic P. gonapodyides.

A MYB-related transcription factor regulates effector gene expression in an oomycete pathogen.

Phytophthora pathogens possess hundreds of effector genes that exhibit diverse expression patterns during infection, yet how the expression of effector genes is precisely regulated remains largely elusive. Previous studies have identified a few potential conserved transcription factor binding sites (TFBSs) in the promoters of Phytophthora effector genes. Here, we report a MYB-related protein, PsMyb37, in Phytophthora sojae, the major causal agent of root and stem rot in soybean. Yeast one-hybrid and electrophoretic mobility shift assays showed that PsMyb37 binds to the TACATGTA motif, the most prevalent TFBS in effector gene promoters. The knockout mutant of PsMyb37 exhibited significantly reduced virulence on soybean and was more sensitive to oxidative stress. Consistently, transcriptome analysis showed that numerous effector genes associated with suppressing plant immunity or scavenging reactive oxygen species were down-regulated in the PsMyb37 knockout mutant during infection compared to the wild-type P. sojae. Several promoters of effector genes were confirmed to drive the expression of luciferase in a reporter assay. These results demonstrate that a MYB-related transcription factor contributes to the expression of effector genes in P. sojae.

Litchi aspartic protease LcAP1 enhances plant resistance via suppressing cell death triggered by the pectate lyase PlPeL8 from Peronophythora litchii.

Plant cell death is regulated in plant-pathogen interactions. While some aspartic proteases (APs) participate in regulating programmed cell death or defense responses, the defense functions of most APs remain largely unknown. Here, we report on a virulence factor, PlPeL8, which is a pectate lyase found in the hemibiotrophic pathogen Peronophythora litchii. Through in vivo and in vitro assays, we confirmed the interaction between PlPeL8 and LcAP1 from litchi, and identified LcAP1 as a positive regulator of plant immunity. PlPeL8 induced cell death associated with NbSOBIR1 and NbMEK2. The 11 conserved residues of PlPeL8 were essential for inducing cell death and enhancing plant susceptibility. Twenty-three LcAPs suppressed cell death induced by PlPeL8 in Nicotiana benthamiana depending on their interaction with PlPeL8. The N-terminus of LcAP1 was required for inhibiting PlPeL8-triggered cell death and susceptibility. Furthermore, PlPeL8 led to higher susceptibility in NbAPs-silenced N. benthamiana than the GUS-control. Our results indicate the crucial roles of LcAP1 and its homologs in enhancing plant resistance via suppression of cell death triggered by PlPeL8, and LcAP1 represents a promising target for engineering disease resistance. Our study provides new insights into the role of plant cell death in the arms race between plants and hemibiotrophic pathogens.

A Burkholderia cenocepacia-like environmental isolate strongly inhibits the plant fungal pathogen Zymoseptoria tritici.

Fungal phytopathogens cause significant reductions in agricultural yields annually, and overusing chemical fungicides for their control leads to environmental pollution and the emergence of resistant pathogens. Exploring natural isolates with strong antagonistic effects against pathogens can improve our understanding of their ecology and develop new treatments for the future. We isolated and characterized a novel bacterial strain associated with the species Burkholderia cenocepacia, termed APO9, which strongly inhibits Zymoseptoria tritici, a commercially important pathogenic fungus causing Septoria tritici blotch in wheat. Additionally, this strain exhibits inhibitory activity against four other phytopathogens. We found that physical contact plays a crucial role for APO9's antagonistic capacity. Genome sequencing of APO9 and biosynthetic gene cluster (BGC) analysis identified nine classes of BGCs and three types of secretion systems (types II, III, and IV), which may be involved in the inhibition of Z. tritici and other pathogens. To identify genes driving APO9's inhibitory activity, we screened a library containing 1,602 transposon mutants and identified five genes whose inactivation reduced inhibition efficiency. One such gene encodes for a diaminopimelate decarboxylase located in a terpenoid biosynthesis gene cluster. Phylogenetic analysis revealed that while some of these genes are also found across the Burkholderia genus, as well as in other Betaproteobacteria, the combination of these genes is unique to the Burkholderia cepacia complex. These findings suggest that the inhibitory capacity of APO9 is complex and not limited to a single mechanism, and may play a role in the interaction between various Burkholderia species and various phytopathogens within diverse plant ecosystems. IMPORTANCE: The detrimental effects of fungal pathogens on crop yields are substantial. The overuse of chemical fungicides contributes not only to environmental pollution but also to the emergence of resistant pathogens. Investigating natural isolates with strong antagonistic effects against pathogens can improve our understanding of their ecology and develop new treatments for the future. We discovered and examined a unique bacterial strain that demonstrates significant inhibitory activity against several phytopathogens. Our research demonstrates that this strain has a wide spectrum of inhibitory actions against plant pathogens, functioning through a complex mechanism. This plays a vital role in the interactions between plant microbiota and phytopathogens.

The Phytophthora parasitica effector AVH195 interacts with ATG8, attenuates host autophagy, and promotes biotrophic infection.

BACKGROUND: Plant pathogens secrete effector proteins into host cells to suppress immune responses and manipulate fundamental cellular processes. One of these processes is autophagy, an essential recycling mechanism in eukaryotic cells that coordinates the turnover of cellular components and contributes to the decision on cell death or survival. RESULTS: We report the characterization of AVH195, an effector from the broad-spectrum oomycete plant pathogen, Phytophthora parasitica. We show that P. parasitica expresses AVH195 during the biotrophic phase of plant infection, i.e., the initial phase in which host cells are maintained alive. In tobacco, the effector prevents the initiation of cell death, which is caused by two pathogen-derived effectors and the proapoptotic BAX protein. AVH195 associates with the plant vacuolar membrane system and interacts with Autophagy-related protein 8 (ATG8) isoforms/paralogs. When expressed in cells from the green alga, Chlamydomonas reinhardtii, the effector delays vacuolar fusion and cargo turnover upon stimulation of autophagy, but does not affect algal viability. In Arabidopsis thaliana, AVH195 delays the turnover of ATG8 from endomembranes and promotes plant susceptibility to P. parasitica and the obligate biotrophic oomycete pathogen Hyaloperonospora arabidopsidis. CONCLUSIONS: Taken together, our observations suggest that AVH195 targets ATG8 to attenuate autophagy and prevent associated host cell death, thereby favoring biotrophy during the early stages of the infection process.

Proteomic analysis revealed that the oomyceticide phosphite exhibits multi-modal action in an oomycete pathosystem.

Phytopathogenic oomycetes constitute some of the most devastating plant pathogens and cause significant crop and horticultural yield and economic losses. The phytopathogen Phytophthora cinnamomi causes dieback disease in native vegetation and several crops. The most commonly used chemical to control P. cinnamomi is the oomyceticide phosphite. Despite its widespread use, the mode of action of phosphite is not well understood and it is unclear whether it targets the pathogen, the host, or both. Resistance to phosphite is emerging in P. cinnamomi isolates and other oomycete phytopathogens. The mode of action of phosphite on phosphite-sensitive and resistant isolates of the pathogen and through a model host was investigated using label-free quantitative proteomics. In vitro treatment of sensitive P. cinnamomi isolates with phosphite hinders growth by interfering with metabolism, signalling and gene expression; traits that are not observed in the resistant isolate. When the model host Lupinus angustifolius was treated with phosphite, proteins associated with photosynthesis, carbon fixation and lipid metabolism in the host were enriched. Increased production of defence-related proteins was also observed in the plant. We hypothesise the multi-modal action of phosphite and present two models constructed using comparative proteomics that demonstrate mechanisms of pathogen and host responses to phosphite. SIGNIFICANCE: Phytophthora cinnamomi is a significant phytopathogenic oomycete that causes root rot (dieback) in a number of horticultural crops and a vast range of native vegetation. Historically, areas infected with phosphite have been treated with the oomyceticide phosphite despite its unknown mode of action. Additionally, overuse of phosphite has driven the emergence of phosphite-resistant isolates of the pathogen. We conducted a comparative proteomic study of a sensitive and resistant isolate of P. cinnamomi in response to treatment with phosphite, and the response of a model host, Lupinus angustifolius, to phosphite and its implications on infection. The present study has allowed for a deeper understanding of the bimodal action of phosphite, suggested potential biochemical factors contributing to chemical resistance in P. cinnamomi, and unveiled possible drivers of phosphite-induced host plant immunity to the pathogen.

Isolation of a Novel Pythium Species, P. thermoculicivorax, and Trichoderma sp. from Natural Enzootic Mosquito Larval Infections.

Only a handful of microbial mosquito larval pathogens have been described to date. Sampling several natural enzootic infections of mosquito larvae in southwestern Florida indicated the presence of microbial pathogens capable of extensive larval mortality. A microscopic analysis of one sample site revealed extensive apparent growth of a Pythium-like microbe on mosquito larvae, with the highest degree of infection observed in the siphon and head regions. Structures consistent with sporangia were seen on infected insects after lactophenol blue staining, and higher-resolution scanning electron microscopy (SEM) micrographs showed sporangia and encysted zoospores targeting the head and siphon regions. The isolate was single-colony purified, and molecular identification targeting the ITS and COX1 loci coupled to phylogenetic reconstruction indicated that the isolate belonged to the Pythium genus but was distinct from its closest characterized species, P. inflatum. Morphological features were characterized, with the isolate showing rapid growth on all mycological media tested and relatively high thermotolerance, capable of robust growth at 37 °C; hence, it was designated P. thermoculicivorax. Sampling from a second series of natural infections of mosquito larvae resulted in the molecular identification of three Trichoderma isolates, one with high similarity to T. strigosum and the other two clustering closely with T. asperellum. These data highlight the occurrence of natural enzootic infections of mosquito larvae, potentially as a resource for the identification of new mosquito pathogens.

Design, synthesis and anti-oomycete activity of 2-acyloxyhinokitiol derivatives.

In order to explore novel natural product-based anti-oomycete agent, ten 2-acyloxyhinokitiol derivatives (5a-j) were designed and synthesised, and structurally confirmed by 1H NMR,13C NMR, HRMS, and melting point. The stereochemical configuration of compound 5f was unambiguously confirmed by single-crystal X-ray diffraction. Furthermore, we evaluated the target compounds 5a-j as anti-oomycete activity against a serious agricultural disease of Phytophthora capsici. Among the ten hinokitiol ester derivatives tested, four compounds 5d, 5g, 5h and 5j had anti-oomycete activity higher than the positive control zoxamide (EC50 = 23.59 mg/L), and the EC50 values of 18.90, 20.62, 13.61 and 21.29 mg/L, respectively. Especially compound 5h exhibited the best anti-oomycete activity against P. capsici with EC50 value of 13.61 mg/L. Overall, the anti-oomycete activities of 2-acyloxyhinokitiol derivatives is higher than that of 2-sulfonyloxyhinokitiol derivatives. The results laid a good foundation for the subsequent synthesis of hinokitiol ester derivatives with significant anti-oomycete activity.

Fitness difference between two synonymous mutations of Phytophthora infestans ATP6 gene.

BACKGROUND: Sequence variation produced by mutation provides the ultimate source of natural selection for species adaptation. Unlike nonsynonymous mutation, synonymous mutations are generally considered to be selectively neutral but accumulating evidence suggests they also contribute to species adaptation by regulating the flow of genetic information and the development of functional traits. In this study, we analysed sequence characteristics of ATP6, a housekeeping gene from 139 Phytophthora infestans isolates, and compared the fitness components including metabolic rate, temperature sensitivity, aggressiveness, and fungicide tolerance among synonymous mutations. RESULTS: We found that the housekeeping gene exhibited low genetic variation and was represented by two major synonymous mutants at similar frequency (0.496 and 0.468, respectively). The two synonymous mutants were generated by a single nucleotide substitution but differed significantly in fitness as well as temperature-mediated spatial distribution and expression. The synonymous mutant ending in AT was more common in cold regions and was more expressed at lower experimental temperature than the synonymous mutant ending in GC and vice versa. CONCLUSION: Our results are consistent with the argument that synonymous mutations can modulate the adaptive evolution of species including pathogens and have important implications for sustainable disease management, especially under climate change.

Mining oomycete proteomes for phosphatome leads to the identification of specific expanded phosphatases in oomycetes.

Phosphatases are important regulators of protein phosphorylation and various cellular processes, and they serve as counterparts to kinases. In this study, our comprehensive analysis of oomycete complete proteomes unveiled the presence of approximately 3833 phosphatases, with most species estimated to have between 100 and 300 putative phosphatases. Further investigation of these phosphatases revealed a significant increase in protein serine/threonine phosphatases (PSP) within oomycetes. In particular, we extensively studied the metallo-dependent protein phosphatase (PPM) within the PSP family in the model oomycete Phytophthora sojae. Our results showed notable differences in the expression patterns of PPMs throughout 10 life stages of P. sojae, indicating their vital roles in various stages of oomycete pathogens. Moreover, we identified 29 PPMs in P. sojae, and eight of them possessed accessory domains in addition to phosphate domains. We investigated the biological function of one PPM protein with an extra PH domain (PPM1); this protein exhibited high expression levels in both asexual developmental and infectious stages. Our analysis confirmed that PPM1 is indeed an active protein phosphatase, and its accessory domain does not affect its phosphatase activity. To delve further into its function, we generated knockout mutants of PPM1 and validated its essential roles in mycelial growth, sporangia and oospore production, as well as infectious stages. To the best of our knowledge, this study provides the first comprehensive inventory of phosphatases in oomycetes and identifies an important phosphatase within the expanded serine/threonine phosphatase group in oomycetes.

Climate acts as an environmental filter to plant pathogens.

Climate shapes the distribution of plant-associated microbes such as mycorrhizal and endophytic fungi. However, the role of climate in plant pathogen community assembly is less understood. Here, we explored the role of climate in the assembly of Phytophthora communities at >250 sites along a latitudinal gradient from Spain to northern Sweden and an altitudinal gradient from the Spanish Pyrenees to lowland areas. Communities were detected by ITS sequencing of river filtrates. Mediation analysis supported the role of climate in the biogeography of Phytophthora and ruled out other environmental factors such as geography or tree diversity. Comparisons of functional and species diversity showed that environmental filtering dominated over competitive exclusion in Europe. Temperature and precipitation acted as environmental filters at different extremes of the gradients. In northern regions, winter temperatures acted as an environmental filter on Phytophthora community assembly, selecting species adapted to survive low minimum temperatures. In southern latitudes, a hot dry climate was the main environmental filter, resulting in communities dominated by drought-tolerant Phytophthora species with thick oospore walls, a high optimum temperature for growth, and a high maximum temperature limit for growth. By taking a community ecology approach, we show that the establishment of Phytophthora plant pathogens in Europe is mainly restricted by cold temperatures.

Evaluating the Efficacy of Hymexazol in Controlling Globisporangium spinosum, the Newly Identified Pathogen Causing Root Rot in Houttuynia cordata.

Houttuynia cordata is a prevalent vegetable in several Asian countries and is commonly used as a traditional Chinese medicinal herb for treating various diseases in China. Unfortunately, its yield and quality are adversely affected by root rot. However, the pathogen responsible for the losses remains unidentified, and effective fungicides for its management have not been thoroughly explored. In this work, we demonstrate the first report of Globisporangium spinosum as the causative agent causing root rot of H. cordata. Moreover, we evaluated the efficacy of hymexazol to manage the disease, which displayed remarkable inhibitory effects against mycelial growth of G. spinosum in vitro, with EC50 values as low as 1.336 µg/ml. Furthermore, hymexazol completely inhibited sporangia in G. spinosum at a concentration of 0.3125 µg/ml. Specifically, we observed that hymexazol was highly efficacious in reducing the incidence of H. cordata root rot caused by G. spinosum in a greenhouse setting. These findings offer a potential management tool for utilization of hymexazol in controlling H. cordata root rot in field production.

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

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

Computed tomographic characteristics of confirmed and presumed noncutaneous pythiosis in 25 dogs.

Pythium insidiosum is an aquatic oomycete that causes granulomatous infection in dogs, most commonly cutaneous and gastrointestinal. Ultrasonographic characteristics of gastrointestinal pythiosis have been described; occasionally, CT is utilized in the clinical setting, and CT features of pythiosis have not been published. The purpose of this retrospective, multicenter, descriptive study is to describe CT characteristics of noncutaneous canine pythiosis. The following CT parameters were recorded: lesion anatomic location, number, shape, margination, size, attenuation pre- and postcontrast, enhancement pattern, lymph nodes affected, other lesions identified, and presence of peritoneal effusion or steatitis. Descriptive statistics demonstrating the frequency of lesion appearances were performed. Twenty-five dogs with noncutaneous pythiosis lesions that underwent CT were included; 19 had primarily gastrointestinal infections, four primarily arterial infections, one intrathoracic and intra-abdominal infection, and one primary pulmonary infection. In dogs with primary gastrointestinal infection, lesions were most common at the ileocolic junction and were most frequently focal, well-defined, moderate to marked circumferential wall thickening that was homogeneous and smoothly marginated precontrast, with moderate heterogeneous contrast enhancement. Most dogs had involvement of multiple gastrointestinal regions. Of four dogs with primary arterial involvement, three had large aneurysmal dilatations of the cranial mesenteric artery with severe mural thickening. All dogs had regional lymphadenopathy, which was variable but generally mild. Nine dogs had peritoneal effusion; six dogs had steatitis. CT features of pythiosis can overlap with neoplasia, but pythiosis should be considered as a differential, especially in young dogs. Findings supported using CT as an adjunct imaging test for increasing clinical suspicion of noncutaneous pythiosis.

Potential anti-Pythium insidiosum therapeutics identified through screening of agricultural fungicides.

Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Clinical manifestations of pythiosis include an eye, blood vessel, skin, or gastrointestinal tract infection. Pythiosis has been increasingly reported worldwide, with an overall mortality rate of 28%. Radical surgery is required to save patients' lives due to the limited efficacy of antimicrobial drugs. Effective medical treatments are urgently needed for pythiosis. This study aims to find anti-P. insidiosum agents by screening 17 agricultural fungicides that inhibit plant-pathogenic oomycetes and validating their efficacy and safety. Cyazofamid outperformed other fungicides as it can potently inhibit genetically diverse P. insidiosum isolates while exhibiting minimal cellular toxicities. The calculated therapeutic scores determined that the concentration of cyazofamid causing significant cellular toxicities was eight times greater than the concentration of the drug effectively inhibiting P. insidiosum. Furthermore, other studies showed that cyazofamid exhibits low-to-moderate toxicities in animals. The mechanism of cyazofamid action is likely the inhibition of cytochrome b, an essential component in ATP synthesis. Molecular docking and dynamic analyses depicted a stable binding of cyazofamid to the Qi site of the P. insidiosum's cytochrome b orthologous protein. In conclusion, our search for an effective anti-P. insidiosum drug indicated that cyazofamid is a promising candidate for treating pythiosis. With its high efficacy and low toxicity, cyazofamid is a potential chemical for treating pythiosis, reducing the need for radical surgeries, and improving recovery rates. Our findings could pave the way for the development of new and effective treatments for pythiosis.IMPORTANCEPythiosis is a severe infection caused by Pythium insidiosum. The disease is prevalent in tropical/subtropical regions. This infectious condition is challenging to treat with antifungal drugs and often requires surgical removal of the infected tissue. Pythiosis can be fatal if not treated promptly. There is a need for a new treatment that effectively inhibits P. insidiosum. This study screened 17 agricultural fungicides that target plant-pathogenic oomycetes and found that cyazofamid was the most potent in inhibiting P. insidiosum. Cyazofamid showed low toxicity to mammalian cells and high affinity to the P. insidiosum's cytochrome b, which is involved in energy production. Cyazofamid could be a promising candidate for the treatment of pythiosis, as it could reduce the need for surgery and improve the survival rate of patients. This study provides valuable insights into the biology and drug susceptibility of P. insidiosum and opens new avenues for developing effective therapies for pythiosis.

Reference Genome Resource for the Citrus Pathogen Phytophthora citrophthora.

Phytophthora citrophthora is an oomycete pathogen that infects citrus. Its occurrence in citrus-growing regions worldwide is considered a major contributor to crop losses. This study presents a high-quality genome resource for P. citrophthora, which was generated using PacBio HiFi long-read high-throughput sequencing technology. We successfully assembled a 48.5 Mb genome containing 16,409 protein-coding genes from high-quality reads. This marks the first complete genome assembly of P. citrophthora, providing a valuable resource to enhance the understanding of pathogenic behaviour and fungicide sensitivity of this destructive citrus pathogen.