Oomycete Plant Pathogens Research Articles

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.

Nano-emulsion of three essential oils as the potential eco-friendly control to Phytophthora capsici

Phytophthora capsici is a plant pathogenic oomycete that causes significant damage to many crops, including black peppers, chili, cucurbits, and tomatoes. This pathogen is widespread and can survive in soil and plant debris for several years, making it difficult to control. The aim of the research was to observe the effectiveness of three essential oils as nano-emulsion formulas (NS, NC, NE and NA) against the growth of P. capsici in vitro and in vivo. In vitro testing was carried out using 2 methods, namely the poison food and volatile disc assay. Testing of active ingredients using the GCMS method and measuring active ingredient particles using the DLS method. In vivo testing was carried out on chili seeds to determine their growth and germination. The result indicated that all nano-emulsions at 0.50% inhibited the growth of P. capsici which varied between 33,25% to 88,50%. The NC, NS, and NA formula caused complete mycelial growth inhibition at a concentration of 1.0%. Total inhibition of growth also occurred with volatile compounds produced by NS and NA formula. The NC, NS, NE and NA formulas are not phytotoxicity to chili seeds and their germination. Therefore, three essential oils (NC, NS, NE) and the mixture of them (NA) are potential to be used in managing P. capsici as eco-friendly control and possibly as an alternative to synthetic fungicides.

Oomycete pathogen pectin acetylesterase targets host lipid transfer protein to reduce salicylic acid signaling.

During initial stages of microbial invasion, the extracellular space (apoplast) of plant cells is a vital battleground between plants and pathogens. The oomycete plant pathogens secrete an array of apoplastic carbohydrate active enzymes, which are central molecules for understanding the complex plant-oomycete interactions. Among them, pectin acetylesterase (PAE) plays a critical role in the pathogenesis of plant pathogens including bacteria, fungi, and oomycetes. Here, we demonstrated that Peronophythora litchii (syn. Phytophthora litchii) PlPAE5 suppresses litchi (Litchi chinensis) plant immunity by interacting with litchi lipid transfer protein 1 (LcLTP1). The LcLTP1-binding activity and virulence function of PlPAE5 depend on its PAE domain but not on its PAE activity. The high expression of LcLTP1 enhances plant resistance to oomycete and fungal pathogens, and this disease resistance depends on BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) and Suppressor of BIR1 (SOBIR1) in Nicotiana benthamiana. LcLTP1 activates the plant salicylic acid (SA) signaling pathway, while PlPAE5 subverts the LcLTP1-mediated SA signaling pathway by destabilizing LcLTP1. Conclusively, this study reports a virulence mechanism of oomycete PAE suppressing plant LTP-mediated SA immune signaling and will be instrumental for boosting plant resistance breeding.

A Systematic Review and Quantitative Synthesis of the Efficacy of Quaternary Ammonium Compounds in Disinfesting Non-fungal Plant Pathogens.

This quantitative review and systematic analysis of the effectiveness of quaternary ammonium compounds (QACs) in disinfesting non-fungal plant pathogens in agricultural and horticultural cropping systems is a complementary follow-up to a previous study that evaluated the efficacy of QACs against fungal plant pathogens. In the present study, a meta-analysis involving 67 studies was conducted to assess the overall efficacy of QACs against plant pathogenic bacteria, oomycetes and viruses, and to identify factors associated with observed differences in product efficacy. Across all studies, QACs resulted in a significant (P < 0.0001) reduction in either disease intensity or propagule viability with a mean Hedges' g (g+) of 1.75, indicating that overall QAC treatments were moderately effective against non-fungal pathogens. Significant differences in product efficacy were observed between organism types (P = 0.0001), with QAC interventions resulting in higher efficacy (P = 0.0002) against oomycetes ( g+ = 4.20) than against viruses ( g+ = 1.42) and bacteria ( g+ = 1.07), which were not different (P = 0.2689) from each other. As a result, bacterium and virus types were combined into a composite set (BacVir). QAC intervention against BacVir resulted in significant differences in efficacy within categorical moderator subgroups for genus (P = 0.0133), target material (P = 0.0001) and QAC product generation (P = 0.0281). QAC intervention against oomycetes resulted in significant differences in efficacy only for genus (P < 0.0001). For the BacVir composite, five random effects (RE) meta-regression models were significant (P = 0.05), where models with dose and time, dose and genus, time and genus, dose and target; and time and target accounted for 62%, 61%, 52%, 83% and 88%, respectively, of the variance in true effect sizes (R2) associated with . For oomycetes, three RE meta-regression models were significant (P = 0.05), where models with dose and time; dose and genus, and time and genus accounted for 64%, 86% and 90 %, respectively, of R2 associated with g+. These results show that while QACs are moderately effective against non-fungal plant pathogens, the observed variability in their efficacy due to dose of active ingredient and contact time of these products can be influenced by organism type, genus within organism type, the target being treated, and the generation of QAC products.

Genome sequence and annotation of Periconia digitata a hopeful biocontrol agent of phytopathogenic oomycetes

The Periconia fungal genus belongs to the phylum Ascomycota, order Pleosporales, family Periconiaceae. Periconia are found in many habitats, but little is known about their ecology. Several species from this genus produce bioactive molecules. Periconia digitata extracts were shown to be deadly active against the pine wilt nematode. Furthermore, P. digitata was shown to inhibit the plant pathogenic oomycete Phytophthora parasitica. Because P. digitata has great potential as a biocontrol agent and high quality genomic resources are still lacking in the Periconiaceae family, we generated long-read genomic data for P. digitata. Using PacBio Hifi sequencing technology, we obtained a highly-contiguous genome assembled in 13 chromosomes and totaling ca. 39 Mb. In addition, we produced a reference transcriptome, based on 12 different culture conditions, and proteomic data to support the genome annotation. Besides representing a new reference genome within the Periconiaceae, this work will contribute to our better understanding of the Eukaryotic tree of life and opens new possibilities in terms of biotechnological applications.

Non-canonical two-step biosynthesis of anti-oomycete indole alkaloids in Kickxellales

BackgroundFungi are prolific producers of bioactive small molecules of pharmaceutical or agricultural interest. The secondary metabolism of higher fungi (Dikarya) has been well-investigated which led to > 39,000 described compounds. However, natural product researchers scarcely drew attention to early-diverging fungi (Mucoro- and Zoopagomycota) as they are considered to rarely produce secondary metabolites. Indeed, only 15 compounds have as yet been isolated from the entire phylum of the Zoopagomycota.ResultsHere, we showcase eight species of the order Kickxellales (phylum Zoopagomycota) as potent producers of the indole-3-acetic acid (IAA)-derived compounds lindolins A and B. The compounds are produced both under laboratory conditions and in the natural soil habitat suggesting a specialized ecological function. Indeed, lindolin A is a selective agent against plant-pathogenic oomycetes such as Phytophthora sp. Lindolin biosynthesis was reconstituted in vitro and relies on the activity of two enzymes of dissimilar evolutionary origin: Whilst the IAA–CoA ligase LinA has evolved from fungal 4-coumaryl-CoA synthetases, the subsequently acting IAA-CoA:anthranilate N-indole-3-acetyltransferase LinB is a unique enzyme across all kingdoms of life.ConclusionsThis is the first report on bioactive secondary metabolites in the subphylum Kickxellomycotina and the first evidence for a non-clustered, two-step biosynthetic route of secondary metabolites in early-diverging fungi. Thus, the generally accepted “gene cluster hypothesis” for natural products needs to be reconsidered for early diverging fungi.

Lignin Biosynthesis Gene Expression Is Associated with Age-related Resistance of Winter Squash to Phytophthora capsici

The Oomycete plant pathogen, Phytophthora capsici, causes root, crown, and fruit rot of winter squash (Cucurbita moschata) and limits production. Some C. moschata cultivars develop age-related resistance (ARR), whereby fruit develop resistance to P. capsici 14 to 21 days postpollination (DPP) because of thickened exocarp; however, wounding negates ARR. We uncovered the genetic mechanisms of ARR of two C. moschata cultivars, Chieftain and Dickenson Field, that exhibit ARR at 14 and 21 DPP, respectively, using RNA sequencing. The sequencing was conducted using RNA samples from ‘Chieftain’ and ‘Dickenson Field’ fruit at 7, 10, 14, and 21 DPP. A differential expression and subsequent gene set enrichment analysis revealed an overrepresentation of upregulated genes in functional categories relevant to cell wall structure biosynthesis, cell wall modification/organization, transcription regulation, and metabolic processes. A pathway enrichment analysis detected upregulated genes in cutin, suberin monomer, and phenylpropanoid biosynthetic pathways. A further analysis of the expression profile of genes in those pathways revealed upregulation of genes in monolignol biosynthesis and lignin polymerization in the resistant fruit peel. Our findings suggest a shift in gene expression toward the physical strengthening of the cell wall associated with ARR to P. capsici. These findings provide candidate genes for developing Cucurbita cultivars with resistance to P. capsici and improve fruit rot management in Cucurbita species.

Fusaricidin produced by the rhizobacterium Paenibacillus polymyxa NX20 is involved in the biocontrol of postharvest plant-pathogenic oomycete Phytophthora capsici

Phytophthora capsici is a destructive oomycete pathogen that mainly threatens solanaceous and cucurbitaceous vegetable crops, resulting in severe economic losses annually. Paenibacillus polymyxa, a typical biological control agent (BCA), has been widely used to control plant diseases due to its efficacy and environmentally friendly properties. However, the application of P. polymyxa to control pathogenic oomycete P. capsici during the postharvest period has not been reported. In this study, we found that P. polymyxa NX20 exhibited the best inhibitory effect on the mycelial growth of P. capsici among 160 cultivable isolates, and the lipopeptide fusaricidin was the main factor in its antagonistic action. Fusaricidin exhibited good inhibition activity against P. capsici with an EC50 value of 2.46 mg L−1. Both zoospore production and germination were suppressed by fusaricidin in a dose-dependent manner. According to the microscopy observation and cell membrane permeability assay, fusaricidin was proved to impaired the plasma membrane structure and led to cell death in P. capsici. Additionally, after exposure to fusaricidin, the reactive oxygen species accumulation was induced and DNA/protein synthesis was inhibited in P. capsici. RNA-Seq analysis revealed that fusaricidin treatment could potentially affect plasma membrane integrity, transmembrane transport, energy metabolism, and signal transduction in P. capsici. Furthermore, fusaricidin exhibited good control efficacy against P. capsici on watermelon, pepper, and cherry tomato fruit. Overall, these findings elucidated the anti-oomycete mechanism of fusaricidin and highlighted the potential of P. polymyxa strain NX20 on vegetable protection and preservation.

Isolation of two triterpenoids from Phlomis purpurea, one of them with anti-oomycete activity against Phytophthora cinnamomi, and insights into its biosynthetic pathway.

Phytophthora cinnamomi is an important plant pathogen responsible for dieback diseases in plant genera including Quercus, Fagus, Castanea, Eucalyptus, and Pinus, among others, all over the world. P. cinnamomi infection exerts tremendous ecological and economic losses. Several strategies have been developed to combat this pathogenic oomycete, including the search for novel anti-oomycete compounds. In this work, a Mediterranean vascular plant, Phlomis purpurea, has been screened for secondary bioactivity against this pathogen. The genus Phlomis includes a group of herbaceous plants and shrubs described as producers of many different bioactive compounds, including several triterpenoids. Triterpenoids are well-known molecules synthesized by plants and microorganisms with potent antioxidant, antitumoral, and antimicrobial activities. We have isolated by HPLC-DAD and characterized by HPLC-MS and NMR two nortriterpenoid compounds (phlomispentaol A and phlomispurtetraolone) from the root extracts of P. purpurea. One of them (phlomispentaol A) is active against the plant pathogenic oomycete P. cinnamomi (based on in vitro inhibition bioassays). Based on their chemical structure and their relationship to other plant triterpenoids, oleanolic acid is proposed to be the common precursor for these molecules. The anti-oomycete activity shown by phlomispentaol A represents a promising alternative to counteract the worldwide-scale damage caused to forest ecosystems by this pathogen.

Protein phosphatase 2A: a high-value target of virulence factors

Pathogen-encoded virulence factors perturb host physiology and immune function to promote infection. Reinforcing the concept that protein phosphatase 2A (PP2A) is convergently targeted by virulence factors from diverse pathogens, Li et al. demonstrate that PP2A is coopted by members of a modular family of effector proteins from a plant-pathogenic oomycete.

Divergent sequences of tetraspanins enable plants to specifically recognize microbe-derived extracellular vesicles

Extracellular vesicles (EVs) are important for cell-to-cell communication in animals. EVs also play important roles in plant–microbe interactions, but the underlying mechanisms remain elusive. Here, proteomic analyses of EVs from the soybean (Glycine max) root rot pathogen Phytophthora sojae identify the tetraspanin family proteins PsTET1 and PsTET3, which are recognized by Nicotiana benthamiana to trigger plant immune responses. Both proteins are required for the full virulence of P. sojae. The large extracellular loop (EC2) of PsTET3 is the key region recognized by N. benthamiana and soybean cells in a plant receptor-like kinase NbSERK3a/b dependent manner. TET proteins from oomycete and fungal plant pathogens are recognized by N. benthamiana thus inducing immune responses, whereas plant-derived TET proteins are not due to the sequence divergence of sixteen amino acids at the C-terminal of EC2. This feature allows plants to distinguish self and non-self EVs to trigger active defense responses against pathogenic eukaryotes.

PGPR control Phytophthora capsici in tomato through induced systemic resistance, early hypersensitive response and direct antagonism in a cultivar-specific manner

Plant pathogens cause significant crop losses worldwide and present significant challenges to reliable food systems. The hemibiotroph Phytophthora capsici is ranked within the top 10 most problematic oomycete plant pathogens. P. capsici induces significant damage to plants by causing root rot, stem blight, and fruit rot, leading to decreased crop yields, economic losses, and increased plant susceptibility to secondary infections. The current study tested the hypothesis that plant growth promoting rhizobacteria (PGPR), namely Bacillus velezensis UQ9000N and Pseudomonas azotoformans UQ4510An, have inhibitory effects against various fungi and oomycetes. This study also hypothesised that the plant genotype affects the capability of UQ4510An to control P. capsici infection. Our results revealed that B. velezensis UQ9000N and P. azotoformans UQ4510An inhibited the growth of several plant pathogens by 50% or higher. These bacteria also induced abnormal mycelial morphology of P. capsici and Fusarium oxysporum f. sp. lycopersici. Moreover, P. azotoformans UQ4510An exhibited anti-oomycete activity in vitro and in planta, by reducing symptoms of P. capsici infection in various tomato plant cultivars in a genotype-dependent manner. Some tomato cultivars which presented less improvements of phenotypic parameters from the UQ4510An inoculation in the absence of the pathogen had a more pronounced reduction in symptoms upon P. capsici infection. Furthermore, our results indicated that the main biocontrol mechanism of P. azotoformans UQ4510An against P. capsici is a combination of direct antagonism and induction of induced systemic resistance (ISR) involving a hypersensitive response (HR) in the plant host at early stages of infection.

EffectorO: Motif-Independent Prediction of Effectors in Oomycete Genomes Using Machine Learning and Lineage Specificity.

Oomycete plant pathogens cause a wide variety of diseases, including late blight of potato, sudden oak death, and downy mildews of plants. These pathogens are major contributors to loss in numerous food crops. Oomycetes secrete effector proteins to manipulate their hosts to the advantage of the pathogen. Plants have evolved to recognize effectors, resulting in an evolutionary cycle of defense and counter-defense in plant-microbe interactions. This selective pressure results in highly diverse effector sequences that can be difficult to computationally identify using only sequence similarity. We developed a novel effector prediction tool, EffectorO, that uses two complementary approaches to predict effectors in oomycete pathogen genomes: i) a machine learning-based pipeline that predicts effector probability based on the biochemical properties of the N-terminal amino-acid sequence of a protein and ii) a pipeline based on lineage specificity to find proteins that are unique to one species or genus, a sign of evolutionary divergence due to adaptation to the host. We tested EffectorO on Bremia lactucae, which causes lettuce downy mildew, and Phytophthora infestans, which causes late blight of potato and tomato, and predicted many novel effector candidates while recovering the majority of known effector candidates. EffectorO will be useful for discovering novel families of oomycete effectors without relying on sequence similarity to known effectors. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Characterization of Arthropeptide B, an Antifungal Cyclic Tetrapeptide from Arthrobacter humicola.

Disease suppressive composts are known, yet little information on the potential role of specific microbial antagonist within are available. Arthrobacter humicola isolate M9-1A has been obtained from a compost prepared from marine residues and peat moss. The bacterium is a non-filamentous actinomycete with antagonistic activity against plant pathogenic fungi and oomycetes sharing its ecological niche in agri-food microecosystems. Our objective was to identify and characterize compounds with antifungal activity produced by A. humicola M9-1A. Arthrobacter humicola culture filtrates were tested for antifungal activity in vitro and in vivo and a bioassay-guided approach was used to identify potential chemical determinants of its observed activity against molds. The filtrates reduced the development of lesions of Alternaria rot on tomatoes and the ethyl acetate extract inhibited growth of Alternaria alternata. A compound, arthropeptide B [cyclo-(L-Leu, L-Phe, L-Ala, L-Tyr)], was purified from the ethyl acetate extract of the bacterium. Arthropeptide B is a new chemical structure reported for the first time and has shown antifungal activity against A. alternata spore germination and mycelial growth.

Sequence and phylogenetic analysis of a novel alphaendornavirus, the first virus described from the oomycete plant pathogen Phytophthora heveae

Here, we report the discovery and complete genome sequence of a novel virus, designated as “Phytophthora heveae alphaendornavirus 1” (PhAEV1), from a single isolate of the plant pathogenic oomycete Phytophthora heveae (kingdom Stramenipila) isolated from a tropical evergreen lowland rainforest in northern Vietnam. PhAEV1 was detected by both cellulose affinity chromatography of dsRNA and high-throughput sequencing of total RNA, and its presence and sequence were confirmed by RT-PCR and Sanger sequencing. The PhAEV1 genome, 12,820 nucleotides (nt) in length, was predicted to encode a single large polyprotein with the catalytic core domain of viral (superfamily 1) RNA helicase (HEL, amino acid [aa] positions 1,287-1,531), glycosyltransferase (GT, aa positions ca. 2,800-3,125), and RNA-directed RNA polymerase (RdRp, aa positions 3,875-4,112). PhAEV1 is the most similar to Phytophthora cactorum alphaendornavirus 3, sharing 39.4% and 39.1% nt and aa sequence identity, respectively. In addition to the first 5′-terminal AUG codon, three additional in-frame methionine codons were found in close proximity (nt 14-16, 96-98, and 176-178), suggesting potential additional translation initiation sites. Conserved RdRp motifs (A-E) similar to those detected in related endornaviruses were identified in PhAEV1, as well as in several previously described alphaendornaviruses from other Phytophthora species in which these motifs had not been identified previously. Phylogenetic analysis showed that PhAEV1 clusters with members of the genus Alphaendornavirus in the family Endornaviridae and is basal to two other alphaendornaviruses described from another oomycete, Phytophthora cactorum. PhAEV1 is the first virus reported in P. heveae.

Detection of Phytophthora, Pythium, Globisporangium, Hyaloperonospora, and Plasmopara Species in High-Throughput Sequencing Data by In Silico and In Vitro Analysis Using Microbe Finder (MiFi)

Species identification of plant pathogenic oomycetes based on morphology is challenging because of the limited number of variable characters available. Hence, pure culture isolation followed by internal transcribed spacer sequencing is routinely conducted for diagnostic purposes. E-probe Diagnostic Nucleic acid Analysis (EDNA), a pipeline that couples high-throughput sequencing and metagenomic sequence analysis, is a detection system used to identify targets without relying on often time-consuming serological and molecular approaches. The EDNA pipeline, implemented through the Microbe Finder (MiFi), was used to design short species-specific sequences (e-probes) with sizes from 40 to 80 nucleotides and to detect plant pathogenic oomycetes in metagenomic data. To avoid false positives and increase target specificity, we removed the e-probes with sequences similar to nontarget species in NCBI's nucleotide database. The designed e-probes were tested using in silico simulated metagenomes with different target pathogen and host sequence levels. E-probes of Phytophthora nicotianae were further tested with metagenomes generated using nanopore sequencing. In silico detection with 100% sensitivity was obtained at 0.01 to 1% pathogen read abundances with the three e-probe lengths tested. MiFi also successfully identified Ph. nicotianae in the sequenced metagenomes with nanopore DNA sequencing. Thus, EDNA-MiFi could enable rapid detection (&lt;24 h from sample collection to diagnosis) without requiring post-sequencing processing. EDNA, as implemented in the MiFi web application, is a powerful tool for detecting oomycetes from metagenomic samples. Our study shows the outstanding potential of EDNA-MiFi coupled with nanopore sequencing for plant and possible on-site pathogen diagnosis. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Glucosinolate-extracts from residues of conventional and organic cultivated broccoli leaves (Brassica oleracea var. italica) as potential industrially-scalable efficient biopesticides against fungi, oomycetes and plant parasitic nematodes

This study aimed to standarize a protocol for obtaining a bioactive extract from broccoli (Brassica oleracea var. italica) crop residues, that is suitable for application on an industrial scale and effective in reducing plant disease incidence. For this purpose, the influence of several extraction factors in the glucosinolate (GSL) content was studied with leaves collected from two conventional broccoli fields and two organic broccoli fields. The analysis showed that lyophilization had no influence on the GSL content. Storage of plant material under two different temperatures (− 20ºC and − 80ºC) had no influence on the GSLs content of the extracts. Phytotoxicity of the extracts was studied with six different plant seeds, and also cytotoxicity was determined with human liver cells in vitro. The extracts were phytotoxic at dilutions above 10%, while cell toxicity was low. Extracts concentrations of 0.1%, 1% and 2% were tested in vitro against eight plant pathogenic fungi and two oomycetes in solid and in liquid media. The extracts reduced the growth of several plant pathogenic fungi at 2% dilution by up to 38.37% against Alternaria alternata and up to 46.55% against Sclerotinia sclerotiorum. When combined with myrosinase enzyme the effect of the extracts was enhanced, reaching inhibition values of 67.06% against A. alternata in solid medium and 68.52% against Rhizoctonia solani in liquid medium. In contrast, the same extracts increased the growth of the plant pathogenic oomycetes Pythium ultimum and Phytophthora cactorum. The effect of the same extracts in the free leaving larvae, J2s, of the plant-parasitic nematode (PPN) Meloidogyne javanica was not obvious. Minor significant differences were obtained but with no clear dose-response in nematode mortality, and no inhibition of eggs hatching was observed. These results show the industrial potential of using broccoli residues to obtain extracts with biopesticide activity against plant pathogenic fungi.

Design, Synthesis, and Antifungal/Antioomycete Activity of Thiohydantoin Analogues Containing Spirocyclic Butenolide.

Novel fungicidal agents were designed based on the combination of two privileged scaffolds, thiohydantoin and spirocyclic butenolide, which are widely found in natural products. The synthesized compounds were characterized by 1H NMR, 13C NMR, and high-resolution electrospray ionisation mass spectrometry. The in vitro antioomycete activity evaluation showed that most of the compounds exhibited excellent inhibitory activities against different developmental stages in the life cycle of pathogenic oomycete Phytophthora capsici. Compound 5j could inhibit the mycelial growth, sporangium production, zoospore release, and cystospore germination significantly with EC50 values of 0.38, 0.25, 0.11, and 0.026 μg/mL, respectively. The in vivo antifungal/antioomycete bioassay results revealed that the series of compounds generally showed outstanding control efficacies against the pathogenic oomycete Pseudoperonospora cubensis, and compounds 5j, 5l, 7j, 7k, and 7l possessed broad-spectrum antifungal activities against the test phytopathogens. The in vivo protective and curative efficacies against P. capsici of the representative compound 5j were excellent, which were better than those of azoxystrobin. More prominently, 5j significantly promoted the biomass accumulation of the root system and reinforced the cell wall by callose deposition. The pronounced upregulation of immune response-related genes indicated that the active oomycete inhibitor 5j also functioned as a plant elicitor. Transmission electron microscopy observation and the enzyme activity test demonstrated that the mechanism of action of 5j was to bind to the pivotal protein, complex III on the respiratory chain, which resulted in a shortage of energy supply. Molecular docking results exhibited that compound 5j appropriately matched with the Qo pocket and had no interaction with the most commonly mutated site Gly-142, which may be of significant benefit in Qo fungicide resistance management. Compound 5j showed great advantages and potential in oomycete control, resistance management, and induction of disease resistance. A further investigation of 5j with a unique structure might have direct implications for the creation of novel oomycete inhibitors against plant-pathogenic oomycetes.

RNA silencing proteins and small RNAs in oomycete plant pathogens and biocontrol agents.

Oomycetes cause several damaging diseases of plants and animals, and some species also act as biocontrol agents on insects, fungi, and other oomycetes. RNA silencing is increasingly being shown to play a role in the pathogenicity of Phytophthora species, either through trans-boundary movement of small RNAs (sRNAs) or through expression regulation of infection promoting effectors. To gain a wider understanding of RNA silencing in oomycete species with more diverse hosts, we mined genome assemblies for Dicer-like (DCL), Argonaute (AGO), and RNA dependent RNA polymerase (RDRP) proteins from Phytophthora plurivora, Ph. cactorum, Ph. colocasiae, Pythium oligandrum, Py. periplocum, and Lagenidium giganteum. Moreover, we sequenced small RNAs from the mycelium stage in each of these species. Each of the species possessed a single DCL protein, but they differed in the number and sequence of AGOs and RDRPs. SRNAs of 21nt, 25nt, and 26nt were prevalent in all oomycetes analyzed, but the relative abundance and 5' base preference of these classes differed markedly between genera. Most sRNAs mapped to transposons and other repeats, signifying that the major role for RNA silencing in oomycetes is to limit the expansion of these elements. We also found that sRNAs may act to regulate the expression of duplicated genes. Other sRNAs mapped to several gene families, and this number was higher in Pythium spp., suggesting a role of RNA silencing in regulating gene expression. Genes for most effector classes were the source of sRNAs of variable size, but some gene families showed a preference for specific classes of sRNAs, such as 25/26 nt sRNAs targeting RxLR effector genes in Phytophthora species. Novel miRNA-like RNAs (milRNAs) were discovered in all species, and two were predicted to target transcripts for RxLR effectors in Ph. plurivora and Ph. cactorum, indicating a putative role in regulating infection. Moreover, milRNAs from the biocontrol Pythium species had matches in the predicted transcriptome of Phytophthora infestans and Botrytis cinerea, and L. giganteum milRNAs matched candidate genes in the mosquito Aedes aegypti. This suggests that trans-boundary RNA silencing may have a role in the biocontrol action of these oomycetes.

Uptake of oomycete RXLR effectors into host cells by clathrin-mediated endocytosis.

Filamentous (oomycete and fungal) plant pathogens deliver cytoplasmic effector proteins into host cells to facilitate disease. How RXLR effectors from the potato late blight pathogen Phytophthora infestans enter host cells is unknown. One possible route involves clathrin-mediated endocytosis (CME). Transient silencing of NbCHC, encoding clathrin heavy chain, or the endosome marker gene NbAra6 encoding a Rab GTPase in the model host Nicotiana benthamiana, attenuated P. infestans infection and reduced translocation of RXLR effector fusions from transgenic pathogen strains into host cells. By contrast, silencing PP1c isoforms, susceptibility factors not required for endocytosis, reduced infection but did not attenuate RXLR effector uptake. Endosome enrichment by ultracentrifugation and sucrose gradient fractionation revealed co-localization of RXLR effector Pi04314-RFP with clathrin-coated vesicles. Immunopurification of clathrin- and NbAra6-associated vesicles during infection showed that RXLR effectors Pi04314-RFP and AvrBlb1-RFP, but not apoplastic effector PiSCR74-RFP, were co-immunoprecipitated during infection with pathogen strains secreting these effectors. Tandem mass spectrometry analyses of proteins co-immunoprecipitated with NbAra6-GFP during infection revealed enrichment of host proteins associated with endocytic vesicles alongside multiple pathogen RXLR effectors, but not apoplastic effectors, including PiSCR74, which do not enter host cells. Our data show that the uptake of P. infestans RXLR effectors into plant cells occurs via CME.