Oomycete Plant Pathogens Research Articles

Synthesis and Antifungal Activity of Norbornene Carboxamide/sulfonamide Derivatives as Potential Fungicides Targeting Laccase.

To explore more potential fungicides with new scaffolds, thirty-seven norbornene carboxamide/sulfonamide derivatives were designed, synthesized, and assayed for inhibitory activity against six plant pathogenic fungi and oomycetes. The preliminary antifungal assay suggested that the title derivatives showed moderate to good antifungal activity against six plant pathogens. Especially, compound 6 e presented excellent in vitro antifungal activity against Sclerotinia sclerotiorum (EC50=0.71 mg/L), which was substantially stronger than pydiflumetofen. In vivo antifungal assay indicated 6 e displayed prominent protective and curative effects on rape leaves infected by S. sclerotiorum. The preliminary mechanism research displayed that 6 e could damage the surface morphology and inhibit the sclerotia formation of S. sclerotiorum. In addition, the in vitro enzyme inhibition bioassay indicated that 6 e displayed pronounced laccase inhibition activity (IC50=0.63 μM), much stronger than positive control cysteine. Molecular docking elucidated the binding modes between 6 e and laccase. The bioassay results and mechanism investigation demonstrated that this class of norbornene carboxamide/sulfonamide derivatives could be promising laccase inhibitors for novel fungicide development.

Viable protoplast isolation, organelle visualization and transformation of the globally distributed plant pathogen Phytophthora cinnamomi

Phytophthora cinnamomi is an oomycete plant pathogen with a host range of almost 5000 plant species worldwide and therefore poses a serious threat to biodiversity. Omics technology has provided significant progress in our understanding of oomycete biology, however, transformation studies of Phytophthora for gene functionalisation are still in their infancy. Only a limited number of Phytophthora species have been successfully transformed and gene edited to elucidate the role of particular genes. There is a need to escalate our efforts to understand molecular processes, gene regulation and infection mechanisms of the pathogen to enable us to develop new disease management strategies. The primary obstacle hindering the advancement of transformation studies in Phytophthora is their challenging and unique nature, coupled with our limited comprehension of why they remain such an intractable system to work with. In this study, we have identified some of the key factors associated with the recalcitrant nature of P. cinnamomi. We have incorporated fluorescence microscopy and flow cytometry along with the organelle-specific dyes, fluorescein diacetate, Hoechst 33342 and MitoTracker™ Red CMXRos, to assess P. cinnamomi-derived protoplast populations. This approach has also provided valuable insights into the broader cell biology of Phytophthora. Furthermore, we have optimized the crucial steps that allow transformation of P. cinnamomi and have generated transformed isolates that express a cyan fluorescent protein, with a transformation efficiency of 19.5%. We therefore provide a platform for these methodologies to be applied for the transformation of other Phytophthora species and pave the way for future gene functionalisation studies.

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

BackgroundPlant 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.ResultsWe 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.ConclusionsTaken 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.

The Effects of Antofine on the Morphological and Physiological Characteristics of Phytophthora capsici.

Phytophthora capsici is an important plant pathogenic oomycete that causes great losses to vegetable production around the world. Antofine is an important alkaloid isolated from Cynanchum komarovii Al. Iljinski and exhibits significant antifungal activity. In this study, the effect of antofine on the mycelial growth, morphology, and physiological characteristics of P. capsici was investigated using colorimetry. Meanwhile, the activity of mitochondrial respiratory chain complexes of P. capsici was evaluated following treatment with a 30% effective concentration (EC30), as well as EC50 and EC70, of antofine for 0, 12, 24, and 48 h. The results showed that antofine had a significant inhibitory effect against P. capsici, with an EC50 of 5.0795 μg/mL. After treatment with antofine at EC50 and EC70, the mycelia were rough, less full, and had obvious depression; they had an irregular protrusion structure; and they had serious wrinkles. In P. capsici, oxalic acid and exopolysaccharide contents decreased significantly, while cell membrane permeability and glycerol content increased when treated with antofine. Reactive oxygen species (ROS) entered a burst state in P. capsici after incubation with antofine for 3 h, and fluorescence intensity was 2.43 times higher than that of the control. The activities of the mitochondrial respiratory chain complex II, III, I + III, II + III, V, and citrate synthase in P. capsici were significantly inhibited following treatment with antofine (EC50 and EC70) for 48 h compared to the control. This study revealed that antofine is likely to affect the pathways related to the energy metabolism of P. capsici and thus affect the activity of respiratory chain complexes. These results increase our understanding of the action mechanism of antofine against P. capsici.

Specific Detection of Phytophthora parasitica by Recombinase Polymerase Amplification Assays Based on a Unique Multicopy Genomic Sequence.

Phytophthora parasitica is a highly destructive oomycete plant pathogen that is capable of infecting a wide range of hosts including many agricultural cash crops, fruit trees, and ornamental garden plants. One of the most important diseases caused by P. parasitica worldwide is black shank of tobacco. Rapid, sensitive, and specific pathogen detection is crucial for early rapid diagnosis, which can facilitate effective disease management. In this study, we used a genomics approach to identify repeated sequences in the genome of P. parasitica by genome sequence alignment and identified a 203-bp P. parasitica-specific sequence, PpM34, that is present in 31 to 60 copies in the genome. The P. parasitica genome specificity of PpM34 was supported by PCR amplification of 24 genetically diverse strains of P. parasitica, 32 strains representing 12 other Phytophthora species, one Pythium species, six fungal species, and three bacterial species, all of which are plant pathogens. Our PCR and real-time PCR assays showed that the PpM34 sequence was highly sensitive in specifically detecting P. parasitica. Finally, we developed a PpM34-based high-efficiency recombinase polymerase amplification assay, which allowed us to specifically detect as little as 1 pg of P. parasitica total DNA from both pure cultures and infected Nicotiana benthamiana at 39°C using a fluorometric thermal cycler. The sensitivity, specificity, convenience, and rapidity of this assay represent a major improvement for early diagnosis of P. parasitica infection.

Decoding the Arsenal: Protist Effectors and Their Impact on Photosynthetic Hosts.

Interactions between various microbial pathogens including viruses, bacteria, fungi, oomycetes, and their plant hosts have traditionally been the focus of phytopathology. In recent years, a significant and growing interest in the study of eukaryotic microorganisms not classified among fungi or oomycetes has emerged. Many of these protists establish complex interactions with photosynthetic hosts, and understanding these interactions is crucial in understanding the dynamics of these parasites within traditional and emerging types of farming, including marine aquaculture. Many phytopathogenic protists are biotrophs with complex polyphasic life cycles, which makes them difficult or impossible to culture, a fact reflected in a wide gap in the availability of comprehensive genomic data when compared to fungal and oomycete plant pathogens. Furthermore, our ability to use available genomic resources for these protists is limited by the broad taxonomic distance that these organisms span, which makes comparisons with other genomic datasets difficult. The current rapid progress in genomics and computational tools for the prediction of protein functions and interactions is revolutionizing the landscape in plant pathology. This is also opening novel possibilities, specifically for a deeper understanding of protist effectors. Tools like AlphaFold2 enable structure-based function prediction of effector candidates with divergent protein sequences. In turn, this allows us to ask better biological questions and, coupled with innovative experimental strategies, will lead into a new era of effector research, especially for protists, to expand our knowledge on these elusive pathogens and their interactions with photosynthetic hosts. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome characterization of a novel narnavirus infecting the plant-pathogenic fungus Ustilaginoidea virens.

Mycoviruses are viruses that infect fungi and oomycetes. They are widespread in all major groups of plant-pathogenic fungi and oomycetes. To date, only the full genome of dsRNA mycoviruses and the contigs of positive-sense single-stranded RNA (+ssRNA) mycoviruses have been reported in Ustilaginoidea virens, which is the notorious causal agent of rice false smut (RFS). Here, we report the molecular characterization of a novel +ssRNA mycovirus, Ustilaginoidea virens narnavirus 4 (UvNV4), isolated from U. virens strain Uv418. UvNV4 has a genome of 3,131 nucleotides (nt) and possesses an open reading frame (ORF) predicted to encode an RNA-dependent RNA polymerase (RdRp) of 1,017 amino acids (aa) sequence with a molecular mass of 116.6 kDa. BLASTp analysis revealed that the RdRp showed 50.34% aa sequence identity to that of the previously described Zhangzhou Narna tick virus 1. Phylogenetic analysis indicated that UvNV4 is closely related to members of the family Narnaviridae. Taken together, these results clearly demonstrate that UvNV4 is a novel +ssRNA virus infecting U. virens.

Sensitivity of 40 Pythium species to the new fungicide Picarbutrazox

Picarbutrazox is the active ingredient of Vayantis®, a novel seed treatment fungicide developed by Syngenta and discovered by Nippon Soda. Vayantis® received registration from the US EPA in 2021. Picarbutrazox is a systemic fungicide and belongs to the tetrazolyloximes chemical group of fungicides, and its mode of action is still unknown (FRAC group U17). Picarbutrazox targets oomycete plant pathogens and controls diseases caused by Pythium, Phytophthora, and downy mildews. Picarbutrazox is in a new mode-of-action group with no known cross-resistance to other oomycete compounds. The sensitivity to Picarbutrazox of 528 isolates belonging to 40 different Pythium species was determined in vitro. Pythium isolates were collected from infected plants including corn, soybean, cotton, and other crops from 17 different states. Pythium isolates were also obtained from collections kept at universities. Isolates were identified at the species level using morphological and molecular tools. Mycelial growth inhibition was quantified using the following Picarbutrazox concentrations: 0, 0.00001, 0.0001, 0.001, 0.01, and 0.1 mg/L. All the species of Pythium tested were sensitive to Picarbutrazox. The sensitivity distribution (EC50 values) across all the Pythium species tested had a mean sensitivity of 0.000376 mg ai/L. The Picarbutrazox sensitivity of 40 species of Pythium species has been documented, and results demonstrate the high in vitro intrinsic activity of this fungicide to control Pythium species. The high potency of Picarbutrazox to control Pythium diseases in corn, soybeans, cotton, and other crops has also been demonstrated under field conditions when used as a seed treatment. Picarbutrazox, with its novel mode of action, will contribute to the management of potential fungicide resistance issues developed by other fungicide products controlling Pythium, including mefenoxam and ethaboxam.

Early assessment of fungal and oomycete pathogens in greenhouse irrigation water using Oxford nanopore amplicon sequencing.

Water-borne plant pathogenic fungi and oomycetes are a major threat in greenhouse production systems. Early detection and quantification of these pathogens would enable us to ascertain both economic and biological thresholds required for a timely treatment, thus improving effective disease management. Here, we used Oxford nanopore MinION amplicon sequencing to analyze microbial communities in irrigation water collected from greenhouses used for growing tomato, cucumber and Aeschynanthus sp. Fungal and oomycete communities were characterized using primers that amplify the full internal transcribed spacer (ITS) region. To assess the sensitivity of the MinION sequencing, we spiked serially diluted mock DNA into the DNA isolated from greenhouse water samples prior to library preparation. Relative abundances of fungal and oomycete reads were distinct in the greenhouse irrigation water samples and in water samples from setups with tomato that was inoculated with Fusarium oxysporum. Sequence reads derived from fungal and oomycete mock communities were proportionate in the respective serial dilution samples, thus confirming the suitability of MinION amplicon sequencing for environmental monitoring. By using spike-ins as standards to test the reliability of quantification using the MinION, we found that the detection of spike-ins was highly affected by the background quantities of fungal or oomycete DNA in the sample. We observed that spike-ins having shorter length (538bp) produced reads across most of our dilutions compared to the longer spikes (>790bp). Moreover, the sequence reads were uneven with respect to dilution series and were least retrievable in the background samples having the highest DNA concentration, suggesting a narrow dynamic range of performance. We suggest continuous benchmarking of the MinION sequencing to improve quantitative metabarcoding efforts for rapid plant disease diagnostic and monitoring in the future.

Characteristics of fluopicolide-resistance mutants in Phytophthora nicotianae, the pathogen causing black shank disease in tobacco

Black shank, a devastating disease in tobacco production worldwide, is caused by the oomycete plant pathogen Phytophthora nicotianae. Fluopicolide is a pyridinylmethyl-benzamides fungicide with a unique mechanism of action and has been widely used for controlling a variety of oomycetes such as Plasmopara viticola, Phytophthora infestans, Pseudoperonospora cubensis, P. nicotianae and Bremia lactucae. However, the fluopicolide-resistance risk and molecular basis in P. nicotianae have not been reported. In this study, the sensitivity profile of 141 P. nicotianae strains to fluopicolide was determined, with a mean median effective concentration (EC50) value of 0.12 ± 0.06μg/mL. Five stable fluopicolide-resistant mutants of P. nicotianae were obtained by fungicide adaptation, and the compound fitness index of these resistant mutants were lower than that of their parental isolates. Additionally, cross-resistance tests indicated that the sensitivity of fluopicolide did not correlate with other oomycete fungicides, apart from fluopimomide. DNA sequencing revealed two point mutations, G765E and N769Y, in the PpVHA-a protein in the fluopicolide-resistant mutants. Transformation and expression of PpVHA-a genes carrying G765E and N769Y in the sensitive wild-type isolate confirmed that it was responsible for fluopicolide resistance. These results suggest that P. nicotianae has a low to medium resistance risk to fluopicolide in laboratory and that point mutations, G765E and N769Y, in PpVHA-a are associated with the observed fluopicolide resistance.

Autophagy-related protein PlATG2 regulates the vegetative growth, sporangial cleavage, autophagosome formation, and pathogenicity of peronophythora litchii

ABSTRACT Autophagy is an intracellular degradation process that is important for the development and pathogenicity of phytopathogenic fungi and for the defence response of plants. However, the molecular mechanisms underlying autophagy in the pathogenicity of the plant pathogenic oomycete Peronophythora litchii, the causal agent of litchi downy blight, have not been well characterized. In this study, the autophagy-related protein ATG2 homolog, PlATG2, was identified and characterized using a CRISPR/Cas9-mediated gene replacement strategy in P. litchii. A monodansylcadaverine (MDC) staining assay indicated that deletion of PlATG2 abolished autophagosome formation. Infection assays demonstrated that ΔPlatg2 mutants showed significantly impaired pathogenicity in litchi leaves and fruits. Further studies have revealed that PlATG2 participates in radial growth and asexual/sexual development of P. litchii. Moreover, zoospore release and cytoplasmic cleavage of sporangia were considerably lower in the ΔPlatg2 mutants than in the wild-type strain by FM4–64 staining. Taken together, our results revealed that PlATG2 plays a pivotal role in vegetative growth, sporangia and oospore production, zoospore release, sporangial cleavage, and plant infection of P. litchii. This study advances our understanding of the pathogenicity mechanisms of the phytopathogenic oomycete P. litchii and is conducive to the development of effective control strategies.

PsAF5 functions as an essential adapter for PsPHB2-mediated mitophagy under ROS stress in Phytophthora sojae

Host-derived reactive oxygen species (ROS) are an important defense means to protect against pathogens. Although mitochondria are the main intracellular targets of ROS, how pathogens regulate mitochondrial physiology in response to oxidative stress remains elusive. Prohibitin 2 (PHB2) is an inner mitochondrial membrane (IMM) protein, recognized as a mitophagy receptor in animals and fungi. Here, we find that an ANK and FYVE domain-containing protein PsAF5, is an adapter of PsPHB2, interacting with PsATG8 under ROS stress. Unlike animal PHB2 that can recruit ATG8 directly to mitochondria, PsPHB2 in Phytophthora sojae cannot recruit PsATG8 to stressed mitochondria without PsAF5. PsAF5 deletion impairs mitophagy under ROS stress and increases the pathogen’s sensitivity to H2O2, resulting in the attenuation of P. sojae virulence. This discovery of a PsPHB2-PsATG8 adapter (PsAF5) in plant-pathogenic oomycetes reveals that mitophagy induction by IMM proteins is conserved in eukaryotes, but with differences in the details of ATG8 recruitment.

Biocontrol potential of endophytic Pseudomonas strain IALR1619 against two Pythium species in cucumber and hydroponic lettuce.

The use of fungicides to manage disease has led to multiple environmental externalities, including resistance development, pollution, and non-target mortality. Growers have limited options as legacy chemistry is withdrawn from the market. Moreover, fungicides are generally labeled for traditional soil-based production, and not for liquid culture systems. Biocontrol agents for disease management are a more sustainable and environmentally friendly alternative to conventional agroprotectants. Pythium ultimum is a soil borne oomycete plant pathogen with a broad taxonomic host range exceeding 300 plants. Cucumber seedlings exposed to P. ultimum 1 day after a protective inoculation with bacterial endophyte accession IALR1619 (Pseudomonas sp.) recorded 59% survival; with the control assessed at 18%. When the pathogen was added 5 days post endophyte inoculation, 74% of the seedlings treated survived, compared to 36% of the control, indicating a longer-term effect of IALR1619. Under hydroponic conditions, IALR1619 treated leaf type lettuce cv. 'Cristabel' and Romaine cv. 'Red Rosie' showed 29% and 42% higher shoot fresh weight compared to their controls, respectively. Similar results with less growth decline were observed for a repeat experiment with IALR1619. Additionally, an experiment on hydroponic lettuce in pots with perlite was carried out with a mixture of P. ultimum and P. dissotocum after IALR1619 inoculation. The endophyte treated 'Cristabel' showed fresh weight gain, but the second cultivar 'Pensacola' yielded no increase. In summary, the endophyte IALR1619 provided short term as well as medium-term protection against Pythium blight in cucumber seedlings and may be used as an alternative to conventional fungicides in a greenhouse setting. This study also demonstrated the potential of ALR1619 as a biocontrol agent against Pythium blight in hydroponic lettuce.

Oomycetes as damping‐off pathogens and functional plant traits associated with the susceptibility of west Australian native plant species

AbstractDamping‐off oomycete and fungal plant pathogens reduce the germination, emergence, and survival of seedlings. In agricultural systems, this poses a significant problem as it may reduce productivity; in contrast, the impact is more subtle in natural ecosystems and may be essential for maintaining the structure and diversity of plant communities. The oomycete genus Phytophthora is frequently detected in natural plant communities causing disease in mature plants, but it is rarely assessed as a damping‐off pathogen. A pathogenicity experiment was established with five oomycete treatments, three putatively native Phytophthora species, the invasive P. cinnamomi, and Pythium irregulare, to determine the damping‐off host range in selected plant species from a hyper‐diverse Mediterranean‐type ecosystem. Additionally, plant and seed traits were analysed to determine if they were correlated with susceptibility to pre‐emergent damping‐off. Phytophthora arenaria was the only putatively native generalist damping‐off pathogen, causing 12 of 19 plant species to experience significantly reduced seedling emergence or survival relative to the control treatment. Pythium irregulare and the introduced P. cinnamomi caused significant damping‐off for 12 and 13 plant species, respectively. Plant species that were slowest to germinate and emerge were the most susceptible to pre‐emergence damping‐off caused by P. arenaria. These results suggest native and invasive Phytophthora may substantially influence the structure and diversity of natural plant communities through damping‐off. While mature plant species are the most common focus in Phytophthora research, our study indicates damping‐off should be considered to determine the full impact of these soil‐borne plant pathogens.

A circular economy approach: A new formulation based on a lemon peel medium activated with lactobacilli for sustainable control of post-harvest fungal rots in fresh citrus fruit

The aim of the study was to develop an effective bio-formulation for preventing post-harvest fungal rots in fresh citrus fruit through a multi-step process that includes isolation and characterization of lactic acid bacteria (LABs) from citrus fruit peel, selection of LAB strains with high antifungal activity, chemical characterization of cell free supernatants (CFS), formulation of a lemon peel powdery-based medium (LM) activated with LAB fermentates, chemical characterization of fermented LM, and evaluation of the efficacy of the new bio-formulation against blue mold. Thirteen LABs were recovered from the rind of citrus fruit and identified by the peptide mass fingerprinting method. The antifungal activity of LABs, isolated from citrus fruit, as well as cell free supernatants (CFSs), obtained from their liquid cultures, was tested against diverse plant pathogenic fungi and oomycetes, using the dual-culture overlay and the diffusion agar assays, respectively. Two isolates designated as N3B2 and M2B2, both identified as Lactiplantibacillus plantarum, were selected among the others for their relevant antifungal activity. Both isolates showed a broad spectrum antagonistic activity comprising also major postharvest pathogens of citrus fruit, such as Penicillium digitatum and P. italicum, the causal agents of green and blue moulds, respectively, Alternaria alternata, Colletotrichum gloeosporioides, C. karsti, Phytophthora citrophthora and Ph. nicotianae. The N3B2 and M2B2 isolates were used as starter cultures to ferment a lemon peel powder medium enriched with a nutrient water solution (LM). The two formulations obtained by fermenting LM with the N3B2 and M2B2 isolates showed a strong in vitro inhibitory activity on the same broad range of pathogens used in the preliminary screening of LABs. Moreover, the two LM-based formulations reduced the severity of blue mold infection and inhibited the sporulation of P. italicum on artificially inoculated lemon fruit. The chemical analysis of LM-based bioformulations revealed their antifungal activity was very probably due to the LAB-derived acidic components, including lactic, acetic, DL-3-phenyllactic, 3-4-dihydroxyhydrocinnamic, salicylic acid vanillic acids. These innovative LM-based bioformulations activated with LAB-derived antifungal compounds will be exploited as edible and biodegradable fruit coatings to prolong the shelf-life and prevent post-harvest rots of fresh citrus fruit.

Antimicrobial mechanisms of g-C3 N4 @ZnO against oomycetes Phytophthora capsici: from its metabolism, membrane structures and growth.

Phytophthora capsici, a refractory and model oomycete plant pathogen, especially threatens multiple vegetable crops. A limited number of chemical pesticides play a vital role in controlling oomycete plant diseases. However, this approach often leads to excessive use of chemical agent, exacerbates environmental issues and more and more drug-resistant strains of oomycete. Therefore, it is imperative to devise innovative solutions that can effectively address the infection of oomycete while maintaining high levels of environmental sustainability and low toxicity. In this study, g-C3 N4 @ZnO heterostructure was synthesized and characterized. The g-C3 N4 @ZnO showed higher toxicity on Phytophthora capsici than graphitic carbon nitride (g-C3 N4 ) nanosheets and zinc oxide (ZnO) nanoparticles in vitro and in vivo. Except the hyphal growth of Phytophthora capsici, their germination rate of spores, sporangium formation and number of spores were all suppressed by g-C3 N4 @ZnO heterostructure. Furthermore, we found that this g-C3 N4 @ZnO heterostructure has higher photocatalytic activity under visible light, which potentially enhanced the reactive oxygen species (ROS) mediated stress on Phytophthora capsici. Ultrastructural morphology, global changes of gene expression and weighted gene co-expression network analysis all supported that the anti-oomycete activity of g-C3 N4 @ZnO was manifested in the destruction of membrane system and inhibition of multiple metabolisms of Phytophthora capsici under visible irradiation, which also could be attributed to the ROS and zinc ion (Zn2+ ) mediated stress. This works offers a novel oomycete disease management strategy by using g-C3 N4 @ZnO, which were attributed to the ROS stress, destruction of membrane system and inhibition of multiple metabolisms. © 2023 Society of Chemical Industry.

Staurosporine-producing Streptomyces sp. strain 11 × 1 cell-free culture filtrates control diseases caused by the oomycete plant pathogens Pythium myriotylum and Phytophthora sojae

ABSTRACT Oomycetes are devastating soil-borne plant pathogens, and cause numerous losses on diverse crops worldwide. In this study, our aim was to identify actinomycetes with antimicrobial activity towards oomycetes from the rhizosphere of soybean. Strong growth inhibition of P. myriotylum was found in dual culture with a strain called 11 × 1, and also from using the 11 × 1 cell-free culture filtrate and EtOAc extract. The P. myriotylum growth-inhibiting activity of the 11 × 1 culture filtrates was stable at high temperatures, and acidic and alkaline conditions. The 11 × 1 strain was identified as a Streptomyces strain with the highest 16S rDNA sequence identity to S. scabiei, and phylogenomic analysis also showed clustering with S. scabiei strains. Applying the 11 × 1 culture filtrates to ginger and soybean leaves inhibited the necrosis caused by P. myriotylum and P. sojae, respectively. Pot assay experiments showed a strong control effect of the 11 × 1 culture filtrate against P. myriotylum-caused soft-rot disease of ginger, and against P. sojae caused root-rot of soybean. Analysis of the biosynthetic gene clusters from the genome of 11 × 1 predicted the production of staurosporine, and staurosporine was subsequently identified from LC-MS analysis of the 11 × 1 extract. The concentrations of staurosporine quantified in the 11 × 1 extracts were in the range of the staurosporine minimum inhibitory concentrations for P. myriotylum, supporting the contribution of staurosporine to the biocontrol effect of 11 × 1. The use of 11 × 1 culture filtrate to control both ginger and soybean diseases could be beneficial in an intercropping system of ginger and the leguminous nitrogen-fixing soybean.

Active substances of myxobacteria against plant diseases and their action mechanisms.

Myxobacteria have a complex life cycle and unique social behavior, and obtain nutrients by preying on bacteria and fungi in soil. Chitinase, β-1,3 glucanase and β-1,6 glucanase produced by myxobacteria can degrade the glycosidic bond of cell wall of some plant pathogenic fungi, resulting in a perforated structure in the cell wall. In addition, isooctanol produced by myxobacteria can lead to the accumulation of intracellular reactive oxygen species in some pathogenic fungi and induce cell apoptosis. Myxobacteria can also perforate the cell wall of some plant pathogenic oomycetes by β-1,3 glucanase, reduce the content of intracellular soluble protein and protective enzyme activity, affect the permeability of oomycete cell membrane, and aggravate the oxidative damage of pathogen cells. Small molecule compounds such as diisobutyl phthalate and myxovirescin produced by myxobacteria can inhibit the formation of biofilm and lipoprotein of bacteria, and cystobactamids can inhibit the activity of DNA gyrase, thus changing the permeability of bacterial cell membrane. Myxobacteria, as a new natural compound resource bank, can control plant pathogenic fungi, oomycetes and bacteria by producing carbohydrate active enzymes and small molecular compounds, so it has great potential in plant disease control.

Autophagy-related protein PlAtg3 participates in vegetative growth, sporangial cleavage, autophagy and pathogenicity of Peronophythora litchii

Litchi downy blight, caused by the plant pathogenic oomycete Peronophythora litchii, is one of the most devastating diseases on litchi and resulted in huge economic losses. Autophagy plays an essential role in the development and pathogenicity of the filamentous fungi. However, the function of autophagy in oomycetes remain elusive. Here, an autophagy-related protein Atg3 homolog PlAtg3 was identified and characterized in P. litchii. The absence of PlATG3 through the CRISPR/Cas9 gene replacement strategy compromised vegetative growth and sexual/asexual development. Cytological analyses revealed that the deletion of PlATG3 impaired autophagosome formation with monodansylcadaverine (MDC) staining and significantly disrupted zoospore release due to defects of sporangial cleavage with FM4-64 staining. Atg8 is considered to be an autophagy marker protein in various species. Western blot analysis indicated that PlAtg3 is involved in degradation of PlAtg8-PE. Interestingly, PlAtg3 was unable to interact with PlAtg8 in yeast two hybrid (Y2H) assays, possibly due to the absence of the Atg8 family interacting motif (AIM) in PlAtg3. Furthermore, pathogenicity assays revealed that the deletion of PlATG3 considerably reduced the virulence of P. litchii. Taken together, our data reveal that PlAtg3 plays an important role in radial growth, asexual/sexual development, sporangial cleavage and zoospore release, autophagosome formation, and pathogenicity in P. litchii. This study contributes to a better understanding of the pathogenicity mechanisms of P. litchii and provides insights for the development of more effective strategies to control oomycete diseases.

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.