Zoospore Germination Research Articles

Inhibitory activity and antioomycete mechanism of citral against Phytophthora capsici

The natural terpenoid citral has antifungal activity against multiple fungi, but its bioactivity against oomycetes is unclear. Therefore, this study investigated the antioomycete activity and mechanism of citral against Phytophthora capsici, a highly destructive invasive oomycete. Results showed that citral not only had a great inhibition on the mycelial growth of P. capsici (EC50 = 94.15 mg/L), but also had a significant inhibition on multiple spores, such as sporangia formation, zoospore discharge and zoospore germination. Citral at 4000 mg/L exhibited favorable protective (73.33%) and curative efficacy (55.11%) against pepper Phytophthora blight. Citral significantly damaged the hyphal morphology, disrupted the cell membrane integrity, increased the permeability of cell membrane, and increased the glycerol content in P. capsici. A total of 250 upregulated and 288 downregulated proteins were identified in iTRAQ-based quantitative proteomic analysis. Downregulated proteins were mostly enriched in pathways of ABC transporters, cyanoamino acid metabolism and starch and sucrose metabolism, suggesting an inhibition of citral on transmembrane transporter (e.g., ABC transporters) and pathogenicity (e.g., β-glucosidases) proteins. Upregulated proteins were enriched in biosynthesis of unsaturated fatty acids, pyruvate metabolism and glycolysis/gluconeogenesis, suggesting an activation of citral on energy generation proteins, including acyl-CoA oxidase, D-lactate dehydrogenase, pyruvate kinase, acetyl-CoA synthetase and phosphoenolpyruvate carboxykinase. Biochemical and iTRAQ analysis suggested that cell membrane may be the target of citral in P. capsici.

New Insights into the Anti-Oomycete Mechanisms of a Leaf Extract from Glycyrrhiza glabra Against Pseudoperonospora cubensis.

Cucurbit downy mildew, caused by Pseudoperonospora cubensis, is responsible for high economic losses worldwide in cucumber production. Synthetic pesticides or copper-based products are still important tools to manage the disease. However, the pathogen has developed resistance against common fungicides rather quickly, and there is a need for alternative plant-protecting agents. Glycyrrhiza glabra leaf extract is known for its antifungal activity and was highly effective in former bioassays and semi-commercial trials against downy mildew of cucumber. To elucidate the active ingredients and the mode of action, licorice leaf extract was here fractionated into five fractions (F1 to F5) with a newly developed and optimized separation process via flash chromatography. The crude extract (P1) and fraction F1 inhibited the zoospore release from sporangia, the zoospore germination, and germ tube development of P. cubensis almost completely on two cucumber cultivars, one tolerant and one susceptible to the pathogen. Infestation rates were reduced between 73 and 96%. F1 contained three previously reported antifungal polyphenols: glabranin, pinocembrin, and licoflavanon. Here, we report an additional new compound, naringenin. Furthermore, F5 is found here to show some protective effects against P. cubensis, based on either direct fungicidal or indirect effects via the host plant. The presented results show that licorice leaf extract can serve as an alternative plant protection agent, able to manage P. cubensis infestation on cucumber cultivars with differing levels of susceptibility by interfering with important early stages in the pathogen development.

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.

Enhanced germination and electrotactic behaviour of Phytophthora palmivora zoospores in weak electric fields

Soil-dwelling microorganisms use a variety of chemical and physical signals to navigate their environment. Plant roots produce endogenous electric fields which result in characteristic current profiles. Such electrical signatures are hypothesised to be used by pathogens and symbionts to track and colonise plant roots. The oomycete pathogen Phytophthora palmivora generates motile zoospores which swim towards the positive pole when exposed to an external electric field in vitro. Here, we provide a quantitative characterization of their electrotactic behaviour in 3D. We found that a weak electric field (0.7–1.0 V cm−1) is sufficient to induce an accumulation of zoospore at the positive pole, without affecting their encystment rate. We also show that the same external electric field increases the zoospore germination rate and orients the germ tube’s growth. We conclude that several early stages of the P. palmivora infection cycle are affected by external electric fields. Taken together, our results are compatible with the hypothesis that pathogens use plant endogenous electric fields for host targeting.

Plant secondary metabolite citral interferes with Phytophthora capsici virulence by manipulating the expression of effector genes.

Phytophthora capsici is a notorious pathogen that infects various economically important plants and causes serious threats to agriculture worldwide. Plants deploy a variety of plant secondary metabolites to fend off pathogen attacks, but the molecular mechanisms are largely unknown. In this study, we screened 11 plant secondary metabolites to evaluate their biofumigation effects against P. capsici, and found that citral, carvacrol, and trans-2-decenal exhibited strong antimicrobial effects. Intriguingly, a low concentration of citral was effective in restricting P. capsici infection in Nicotiana benthamiana, but it was unable to inhibit the mycelial growth. A high concentration of citral affected the mycelial growth and morphology, zoospore germination, and cell membrane permeability of P. capsici. Further investigations showed that citral did not induce expression of tested plant immunity-related genes and reactive oxygen species (ROS) production, suggesting that a low concentration of citral could not trigger plant immunity. Moreover, RNA-Seq analysis showed that citral treatment regulated the expression of some P. capsici effector genes such as RxLR genes and P. cactorum-fragaria (PCF)/small cysteine-rich (SCR)74-like genes during the infection process, which was also verified by reverse transcription-quantitative PCR assay. Five candidate effector genes suppressed by citral significantly facilitated P. capsici infection in N. benthamiana or inhibited ROS triggered by flg22, suggesting that they were virulence factors of P. capsici. Together, our results revealed that plant-derived citral exhibited excellent inhibitory efficacy against P. capsici by suppressing vegetative growth and manipulating expression of effector genes, which provides a promising application of citral for controlling Phytophthora blight.

Endophytic Bacterium Lysobacter firmicutimachus Strain 5-7 Is a Promising Biocontrol Agent Against Rice Seedling Disease Caused by Pythium arrhenomanes in Nursery Trays.

Rice root rot disease caused by Pythium spp. is a highly destructive disease in rice nurseries. Biocontrol with endophytic bacteria was developed in this study to control rice seedling diseases. An in planta screening assay revealed that two bacterial endophytes, strains 5-7 and 6-4, displayed strong protection of rice seedlings from attack by Pythium arrhenomanes. Phylogenetic analysis indicated that strain 5-7 is Lysobacter firmicutimachus, while strain 6-4 belongs to the Kitasatospora genus. To quickly evaluate the disease severity of the root system damaged by Pythium spp. in nursery trays, a root surface area measurement assay was developed. By using this measurement, the control efficacy in nursery trays was evaluated, and L. firmicutimachus 5-7 showed promising biocontrol activity against Pythium disease. In a field trial, the two endophytes exhibited significant disease control efficacy on rice brown spot disease caused by Bipolaris oryzae naturally occurring in a commercial nursery field. The two endophytes exhibited multiple enzymatic activities and broad-spectrum antagonistic activities against multiple rice pathogens. The two endophytes colonized the root surface and inside of the root. L. firmicutimachus 5-7 primarily colonized the intercellular space and aerenchyma. Antibiosis is the major mechanism used by strain 5-7 to cause Bipolaris hyphal swelling and inhibit Pythium zoospore germination and sporangium formation, while a hyperparasitism-like phenomenon was found in the interaction of strain 6-4 with Pythium and Bipolaris hyphae. In conclusion, we report the promising biocontrol agent L. firmicutimachus 5-7 and the potential biocontrol agent Kitasatospora sp. 6-4 for disease control of rice seedlings in commercial nursery trays and their possible mechanisms of action.

Evaluation of Fungicides and Fungicide Application Methods to Manage Phytophthora Blight of Pigeonpea

Phytophthora, a blight of pigeonpea caused by Phytophthora cajani, has been significantly increasing in major pigeonpea production regions of India. Limited information on infection with this pathogen and its epidemiology, as well as a lack of adequate resistant cultivars, is hampering the management of Phytophthora blight significantly. Therefore, five fungicides, viz., metiram + dimethomorph, cymoxanil + mancozeb, famoxadone + cymoxanil, mancozeb, and metalaxyl-M + mancozeb, were evaluated against P. cajani under control conditions to control zoospore induction, as well as the infection of zoospores, at the seedling stage. The half-maximal effective concentration (EC50) of fungicides for mycelial inhibition was calculated. The lowest EC50 was recorded in metiram + dimethomorph (0.17 µg/mL), followed by the metalaxyl-M + mancozeb (2.49 µg/mL) and cymoxanil + mancozeb (8.23 µg/mL) fungicides. The formation of the sporangium and zoospores was most significantly affected by metalaxyl-M + mancozeb, followed by metiram + dimethomorph and cymoxanil + mancozeb, in terms of sporangia viability and zoospore germination and encystment. Further, under glasshouse conditions, different fungicide application methods (e.g., seed-treatment; soil-drench; foliar-spray, either singly or in combinations) were evaluated with fungicides on susceptible (ICP 7119) moderately resistant pigeonpea (ICPL 99010, ICPL 20135 and ICPL 99048) cultivars. The seed-treatment + soil-drench, soil-drench + foliar-spray, and soil-drench of fungicide application methods were found to be effective in controlling the Phytophthora blight, at p < 0.001. A combination of the seed-treatment + soil-drench and soil-drench + foliar-spray methods, using metalaxyl-M + mancozeb or metiram + dimethomorph fungicides on moderately resistant cultivars (ICPL 99010), has a synergistic effect on the ability to control the Phytophthora blight at the seedling stage.

Evaluating the potential of environmentally friendly compounds to deactivate different life stages of Phytophthora species

AbstractPhytophthora species are a genus in the oomycete class and are destructive plant pathogens. The ability to inhibit all stages in the life cycle of these aggressive Phytophthora pathogens is essential for the long‐term protection of native trees, plantation forests and horticultural crops that they threaten. Compounds having this ability are currently scarce. In this study, antifungal compounds of natural and synthetic origin were screened against the mycelial, zoospore and oospore life stages of Phytophthora agathidicida (P. agathidicida 18406), P. multivora (P. multivora 20280) and P. cinnamomi (P. cinnamomi 20276, 3910 and 21050). An antifungal lipopeptide showed promising (25–200 μg/mL) antimycelial activity against all the tested Phytophthora species, rapid (c. 30 s) inhibition of P. agathidicida, P. multivora and P. cinnamomi 3910 zoospore motility, inhibition of zoospore germination at 12.5–50 μg/mL and reduced the viability of P. agathidicida and P. multivora oospores at 1000 μg/mL. Polygodial extracted from New Zealand horopito leaves showed more potent (5–50 μg/mL) antimycelial activity and inhibited zoospore motility of P. agathidicida, P. multivora and P. cinnamomi 3910, although at a slightly slower rate; however, it was inactive against oospores. Polygodial and lipopeptide protected kauri and avocado leaves from Phytophthora infection and did not show symptoms of phytotoxicity. Polygodial also inhibited the growth of P. agathidicida in soil. This study demonstrates for the first time the promising antioomycete potential of compounds ranging from small molecule natural products to larger biomolecules such as lipopeptides, which can be developed as compounds to deactivate the different life stages of aggressive Phytophthora species.

Anti-Oomycete Activity and Pepper Root Colonization of Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 against Phytophthora capsici

Previously, Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 from a sequential screening procedure were proven to effectively control Phytophthora blight caused by Phytophthora capsici. In this study, we further investigated the anti-oomycete activities of these strains against mycelial growth, zoospore germination, and germ tube elongation of P. capsici. We also investigated root colonization ability of the bacterial strains in square dishes, including cell motility (swimming and swarming motilities) and biofilm formation. Both strains significantly inhibited mycelial growth in liquid and solid V8 juice media and M9 minimal media, zoospore germination, and germ tube elongation compared with Bacillus vallismortis EXTN-1 (positive biocontrol strain), Sphingomonas aquatilis KU408 (negative biocontrol strain), and MgSO4 solution (untreated control). In diluted (nutrient-deficient) V8 juice broth, the tested strain populations were maintained at >108 cells/ml, simultaneously providing mycelial inhibitory activity. Additionally, these strains colonized pepper roots at a 106 cells/ml concentration for 7 days. The root colonization of the strains was supported by strong swimming and swarming activities, biofilm formation, and chemotactic activity towards exudate components (amino acids, organic acids, and sugars) of pepper roots. Collectively, these results suggest that strains YJR13 and YJR92 can effectively suppress Phytophthora blight of pepper through direct anti-oomycete activities against mycelial growth, zoospore germination and germ tube elongation. Bacterial colonization of pepper roots may be mediated by cell motility and biofilm formation together with chemotaxis to root exudates.

Diketopiperazine cyclo(-l-Leu-l-Phe) with plant elicitation activity and anti-oomycete activity against Plasmopara viticola.

The aim of this study was to contribute to the reduction of synthetic chemical fungicide application in viticulture by using cyclo(-l-Leu-l-Phe) (cLF) produced by Bacillus subtilis KS1, a candidate for biological control agent. cLF is a diketopiperazine and belongs to the family of 2,5-diketopiperazines. KS1 secreted micromolar levels of cLF into culture medium. Micromolar concentrations of cLF structure-dependently decreased by ∼90% both disease severity and lesion density of downy mildew in grapevine cultivated in a growth chamber. Microscopic observation demonstrated that cLF inhibited Plasmopara viticola haustorium formation by 80% but not zoospore germination on leas disks. Interestingly, millimolar concentrations of cLF induced plant defense response, such as expression of genes encoding chitinase and β-1,3-glucanase, in grapevine leaves through the salicylic acid and jasmonate signaling pathways. We demonstrated that cLF was a weapon against P. viticola infection. Further evaluation of cLF in field trials is required to uncover its inherent characteristics.

Una revisión sobre biocontroladores de Phytophthora capsici y su impacto en plantas de Capsicum: Una perspectiva desde el exterior al interior de la planta

Phytophthora capsici is an oomycete that causes various symptoms, such as root, neck, stem, fruit rot, and leaf blight, in different plant species, including the genus Capsicum. One of the tools to counteract this biotic problem, which may be more profitable and respectful to the environment in the long term, is using biocontrollers such as Bacillus, Pseudomonas, Streptomyces (bacteria), and Trichoderma (fungus). It seems that each of these microorganisms has different mechanisms that allow them to inhibit and reduce the growth of P. capsici, negatively affecting the development of sporangia, germination and motility of zoospores, and expansion of germ tube. Although this direct biocontrol action on the phytopathogen correlates with the reduction of symptoms in Capsicum plants or other plant species, this would also involve the activation of defense responses in plants against P. capsici induced by microorganisms. The activity of several enzymes related to the isoflavonoid pathways and reactive oxygen species, as well as the expression of different genes that encode proteins related to pathogenesis and other proteins that can activate the jasmonic acid, salicylic acid, or ethylene signaling pathways. Despite the few existing works related to the biochemical and molecular interaction of Capsicum–P. capsici–biocontroller, in this review, we outlined and elucidated the possible effects and metabolic pathways related to this tripartite pathosystem.

P131 Optimization of MTT assay protocol in Pythium insidiosum zoospores incubated together with neutrophils

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesThe tetrazolium-based MTT assay is widely used to assess cell viability. This colorimetric assay measures cell viability through the enzymatic reduction of MTT, a yellow water-soluble tetrazolium dye, to purple-colored formazan crystals. The zoospores of Pythium insidiosum are the infective form causing pythiosis. There remains a lack of standardized protocol for P. insidiosum zoospore viability assay. Here, we optimized the MTT assay to assess the activity of neutrophils on zoospores.MethodsNeutrophils and zoospores were incubated for 2 h, 4 h, and overnight at 37°C. Neutrophils were lysed in water (pH = 11), MTT was added to each well and incubated at 37°C for 2 h. Then, SDS-HCl solution was added to each well, and absorbance at 570 nm was measured using a microplate reader. The percentage of viable zoospores was then determined.ResultsThe viable zoospores metabolized MTT to insoluble purple formazan product. The zoospores germinated into hyphae when incubated for ˃2 h. Because of the rapid germination of zoospores into hyphae, reliable results would be obtained with an optimum incubation time of 2 h for the zoospores.ConclusionThe simplicity and low cost of the assay make it a valuable tool for zoospore viability studies, with a focus on immunology and immunotherapy.

Biological Control of Oomycete Soilborne Diseases Caused by Phytophthora capsici, Phytophthora infestans, and Phytophthora nicotianae in Solanaceous Crops

Oomycete pathogens that belong to the genus Phytophthora cause devastating diseases in solanaceous crops such as pepper, potato, and tobacco, resulting in crop production losses worldwide. Although the application of fungicides efficiently controls these diseases, it has been shown to trigger negative side effects such as environmental pollution, phytotoxicity, and fungicide resistance in plant pathogens. Therefore, biological control of Phytophthora-induced diseases was proposed as an environmentally sound alternative to conventional chemical control. In this review, progress on biological control of the soilborne oomycete plant pathogens, Phytophthora capsici, Phytophthora infestans, and Phytophthora nicotianae, infecting pepper, potato, and tobacco is described. Bacterial (e.g., Acinetobacter, Bacillus, Chryseobacterium, Paenibacillus, Pseudomonas, and Streptomyces) and fungal (e.g., Trichoderma and arbuscular mycorrhizal fungi) agents, and yeasts (e.g., Aureobasidium, Curvibasidium, and Metschnikowia) have been reported as successful biocontrol agents of Phytophthora pathogens. These microorganisms antagonize Phytophthora spp. via antimicrobial compounds with inhibitory activities against mycelial growth, sporulation, and zoospore germination. They also trigger plant immunity-inducing systemic resistance via several pathways, resulting in enhanced defense responses in their hosts. Along with plant protection, some of the microorganisms promote plant growth, thereby enhancing their beneficial relations with host plants. Although the beneficial effects of the biocontrol microorganisms are acceptable, single applications of antagonistic microorganisms tend to lack consistent efficacy compared with chemical analogues. Therefore, strategies to improve the biocontrol performance of these prominent antagonists are also discussed in this review.

Extracellular self‐DNA plays a role as a damage‐associated molecular pattern (DAMP) delaying zoospore germination rate and inducing stress‐related responses in Phytophthora capsici

AbstractPhytophthora capsici is a highly destructive pathogen of crops. Although chemical pesticides are the most widely used strategy to counter phytopathogens, they have been inefficient to combat P. capsici and have produced significant environmental and health problems. Therefore, sustainable alternatives to control soilborne pathogens, such as the inhibitory effect of self‐extracellular DNA (eDNA), have been proposed. This inhibition phenomenon has been attributed to the action of self‐eDNA as a damage‐associated molecular pattern (DAMP). Here, we describe the effect of self‐eDNA on P. capsici zoospore germination rate, antioxidant enzymes activity and MAPK gene expression. Also, the effect of P. capsici eDNA on the protection of chilli pepper (Capsicum annuum) plants against P. capsici was investigated. The results highlight that P. capsici can sense 2–500 µg/ml self‐eDNA and induce stress‐related responses like SAK1 gene expression, and superoxide dismutase and catalase activities. Moreover, in vitro zoospore germination rate was suppressed with self‐eDNA concentrations ranging from 50 to 500 µg/ml. Interestingly, drench applications of P. capsici eDNA at 60 and 100 µg/ml on chilli pepper plants did not show any protective effect against the phytopathogen, whereas 2 µg/ml of P. capsici eDNA drench application showed a lower percentage of plants with symptoms and lower disease severity. Moreover, phenols and total flavonoids were increased in chilli pepper plants, therefore inducing plant immunity. This study showed that self‐eDNA acts as a DAMP in P. capsici and provides insight into the use of eDNA for the protection of crops of agronomic interest.

Discovery of febrifugine with specific anti-Phytophthora oomycete activity isolated from Dichroa febrifuga Lour

Dichroa febrifuga has been used in traditional Chinese medicine for centuries to treat productive cough and fevers caused by malaria infection, however the antimicrobial activity is still unknown. In this study, the crude alkaloids extract of D. febrifuga leaves showed potent anti-oomycete activity against phytopathogen Phytophthora capsici. Subsequent bioassay-guided fractionation led to identify febrifugine as the main active component. Febrifugine showed specific in vitro anti-oomycete activity against P. capsici, Phytophthora sojae and Phytophthora infestans with EC50 of 1.741, 1.674 and 0.413 μg/mL, respectively, but showed no or weak antifungal activity against twelve test phytopathogenic fungi. Scanning electron microscope and optical microscope results indicated that febrifugine could alter the morphology of P. infestans hyphae, but did not destroy the cell wall. The electrical conductivity and intracellular component leakage experiments suggested the integrity of the cell membrane after the treatment of febrifugine. Moreover, febrifugine possessed inhibitory effects on zoospore release and germination of P. infestans. In vivo bioassay using detached leaves demonstrated that febrifugine had better protective and curing activities than commercialized drug metalaxyl. The application of febrifugine at a dose of 60 μg/mL provided protective efficacies of 93.1% and 91.9%, and curing efficacies of 86.2% and 86.6% for pepper phytophthora blight and potato late blight, respectively. Taken together, this study showed that the crude alkaloids extract of D. febrifuga and its active component febrifugine represent alternatives to control plant diseases caused by oomycetes, and febrifugine is expected to be a new leading structure for the development of oomyceticides.

Fungicidal properties of ginger (Zingiber officinale) essential oils against Phytophthora colocasiae

Recently, plant essential oils (EOs) have attracted special attention in plant disease control and food preservation. Since ancient times, essential oils extracted from plants have exhibited many biological characteristics, especially antimicrobial properties. Recent studies have described the potentials of EOs and derivatives to inhibit the growth and reproduction of microorganisms, mainly in response of overwhelming concerns of consumers about food safety. In the context of returning to nature, with the advancement of science and technology and improved living standards, people have begun to seek solutions for food hygiene without chemical additives. Therefore, biological pesticides and plant-oriented chemicals have received special attention from scientists because they are environmentally friendly and nonhazardous, sustainable, and effective alternatives against many noxious phytopathogens. Present study is intended to appraise the fungicidal properties of ginger EOs to combat leaf blight disease of taro, which threatens global taro production. Farmers often hinge on extremely toxic synthetic fungicides to manage diseases, but the residual effects and resistance of chemicals are unavoidable. The microwave-assisted hydrodistillation method was used for ginger EOs extraction and an FTIR (ATR) spectrometer was used to evaluate their chemical composition and citral was identified as most abundant compound (89.05%) in oil. The pathogen isolated from lesions of diseased taro plants was identified as Phytophthora colocasiae and used as test fungus in the present study. Ginger EO was evaluated in-vitro for antifungal properties against mycelium growth, sporangium production, zoospore germination, leaf, and corm necrosis inhibition. Repeated experiments have shown that the concentration of ginger essential oil (1250 ppm) proved to be the lowest dose to obtain 100% inhibition of fungal growth and spore germination, sporangia formation and leaf necrosis assessment. These results are derived from this fungal species and a hypothesis that involves further research on other plant pathogens to demonstrate the overall potency of essential oils. This study references the easy, economic, and environmental management and control of plant diseases using essential oils and byproducts.

Bioactive compounds in fluid propolis preparations inhibit different life stages of pathogenic oomycetes Aphanomyces astaci and Saprolegnia parasitica

The pathogenic oomycetes Saprolegnia parasitica, causative agent of saprolegniosis in salmonid fish, and Aphanomyces astaci, causative agent of crayfish plague, have a negative impact on freshwater aquaculture. As they are controlled worldwide with chemicals that are harmful to humans and the environment, there is a growing need to replace them with ecologically acceptable alternatives. Propolis has documented antimicrobial activity as well as positive effects on the immune response, growth performance and/or reproductive capacity of fish and crayfish. Therefore, we aimed to investigate the possible inhibition of S. parasitica and A. astaci by fluid propolis formulations. Chemical analysis showed that the propolis formulations were rich in volatile and non-volatile phenolic bioactive components (chrysin was the dominant flavone and pinocembrin the dominant flavanone). Overall, the fluid propolis formulations showed good in vitro inhibition of mycelial growth and zoospores of both pathogens, but we found differences in sensitivity depending on species and life cycle stage. Mycelial growth was more sensitive for A. astaci, with EC50 values (the samples concentrations causing 50% inhibition) up to 8.59 μg/mL, compared to 206.60 μg/mL for S. parasitica. Zoospore motility was more affected in S. parasitica, where MIC (minimum inhibitory concentration) values were up to 61.88 μg/mL, compared to 154.68 μg/mL for A. astaci. Zoospore germination of both pathogens was similarly sensitive to the fluid propolis preparations, with EC50 values of up to 19.52 μg/mL for A. astaci and up to 23.62 μg/mL for S. parasitica. In addition, molecular docking was used to analyze the binding of selected propolis components to oomycete proteins suggested to play a role in pathogenesis. Apigenin, chrysin and pinocembrin were predicted to bind strongly to the endochitinase of A. astaci, which is mainly expressed in the mycelium, and to the thrombospondin of S. parasitica, mainly expressed in the cysts, which is consistent with the results of the in vitro inhibition experiments. Overall, our results suggest that propolis could be used in salmonid and crayfish aquaculture not only as an immunostimulant but also as an antioomycetic agent.

Assessing the effectiveness of oxathiapiprolin toward Phytophthora agathidicida, the causal agent of kauri dieback disease.

Phytophthora species cause disease and devastation of plants in ecological and horticultural settings worldwide. A recently identified species, Phytophthoraagathidicida, infects and ultimately kills the treasured kauri trees (Agathis australis) that are endemic to New Zealand. Currently, there are few options for managing kauri dieback disease. In this study, we sought to assess the efficacy of the oomycide oxathiapiprolin against several life cycle stages of two geographically distinct P. agathidicida isolates. The effective concentration to inhibit 50% of mycelial growth (EC50) was determined to be ∼0.1ng/ml, indicating that P. agathidicida mycelia are more sensitive to oxathiapiprolin than those from most other Phytophthora species that have been studied. Oxathiapiprolin was also highly effective at inhibiting the germination of zoospores (EC50 = 2-9ng/ml for the two isolates) and oospores (complete inhibition at 100ng/ml). In addition, oxathiapiprolin delayed the onset of detached kauri leaf infection in a dose-dependent manner. Collectively, the results presented here highlight the significant potential of oxathiapiprolin as a tool to aid in the control of kauri dieback disease.

4-Ethylphenol, A Volatile Organic Compound Produced by Disease-Resistant Soybean, Is a Potential Botanical Agrochemical Against Oomycetes.

Oomycetes, represented by Phytophthora, are seriously harmful to agricultural production, resulting in a decline in grain quality and agricultural products and causing great economic losses. Integrated management of oomycete diseases is becoming more challenging, and plant derivatives represent effective alternatives to synthetic chemicals as novel crop protection solutions. Biologically active secondary metabolites are rapidly synthesized and released by plants in response to biotic stress caused by herbivores or insects, as well as pathogens. In this study, we identified groups of volatile organic compounds (VOCs) from soybean plants inoculated with Phytophthora sojae, the causal agent of soybean root rot. 4-Ethylphenol was present among the identified VOCs and was induced in the incompatible interaction between the plants and the pathogen. 4-Ethylphenol inhibited the growth of P. sojae and Phytophthora nicotianae and had toxicity to sporangia formation and zoospore germination by destroying the pathogen cell membrane; it had a good control effect on soybean root rot and tobacco black shank in the safe concentration range. Furthermore, 4-Ethylphenol had a potent antifungal activity against three soil-borne phytopathogenic fungi, Rhizoctonia solani, Fusarium graminearum, and Gaeumannomyces graminis var tritici, and four forma specialis of Fusarium oxysporum, which suggest a potential to be an eco-friendly biological control agent.

Isolation and Characterization of Endophyte Bacillus velezensis KOF112 from Grapevine Shoot Xylem as Biological Control Agent for Fungal Diseases.

As the use of chemical fungicides has raised environmental concerns, biological control agents have attracted interest as an alternative to chemical fungicides for plant-disease control. In this study, we attempted to explore biological control agents for three fungal phytopathogens causing downy mildew, gray mold, and ripe rot in grapevines, which are derived from shoot xylem of grapevines. KOF112, which was isolated from the Japanese indigenous wine grape Vitis sp. cv. Koshu, inhibited mycelial growth of Botrytis cinerea, Colletotrichum gloeosporioides, and Phytophthora infestans. The KOF112-inhibited mycelial tips were swollen or ruptured, suggesting that KOF112 produces antifungal substances. Analysis of the 16S rDNA sequence revealed that KOF112 is a strain of Bacillus velezensis. Comparative genome analysis indicated significant differences in the synthesis of non-ribosomal synthesized antimicrobial peptides and polyketides between KOF112 and the antagonistic B. velezensis FZB42. KOF112 showed biocontrol activities against gray mold caused by B. cinerea, anthracnose by C. gloeosporioides, and downy mildew by Plasmopara viticola. In the KOF112–P. viticola interaction, KOF112 inhibited zoospore release from P. viticola zoosporangia but not zoospore germination. In addition, KOF112 drastically upregulated the expression of genes encoding class IV chitinase and β-1,3-glucanase in grape leaves, suggesting that KOF112 also works as a biotic elicitor in grapevine. Because it is considered that endophytic KOF112 can colonize well in and/or on grapevine, KOF112 may contribute to pest-management strategies in viticulture and potentially reduce the frequency of chemical fungicide application.