Rational modification of melatonin for broad-spectrum antifungal agents discovery.

Natural products, known for their environmental safety, are regarded as a significant basis for the modification and advancement of fungicides. Melatonin, as a low-cost natural indole, exhibits diverse biological functions, including antifungal activity. However, its potential as an antifungal agent has not been fully explored. In this study, a series of melatonin derivatives targeting the mitogen-activated protein kinase (Mps1) protein of fungal pathogens were synthesized based on properties of melatonin, among which the trifluoromethyl-substituted derivative Mt-23 exhibited antifungal activity against seven plant pathogenic fungi, and effectively reduced the severity of crop diseases, including rice blast, Fusarium head blight of wheat and gray mold of tomato. In particular, its EC50 (5.4 µM) against the rice blast fungus Magnaporthe oryzae is only one-fourth that of isoprothiolane (22 µM), a commercial fungicide. Comparative analyzes revealed that Mt-23 simultaneously targets the conserved protein kinase Mps1 and lipid protein Cap20. Surface plasmon resonance assays showed that Mt-23 directly binds to Mps1 and Cap20. In this study, we provide a strategy for developing antifungal agents by modifying melatonin, and the resultant melatonin derivative Mt-23 is a commercially valuable, eco-friendly and broad-spectrum antifungal agent to combat crop disease.

Novel Alkaloids from Aspergillus fumigatus VDL36, an Endophytic Fungus Associated with Vaccinium dunalianum.

Eleven alkaloids (1-11) including seven new ones, 1-7, were isolated from the solid fermentation of Aspergillus fumigatus VDL36, an endophytic fungus isolated from the leaves of Vaccinium dunalianum Wight (Ericaceae), a perennial evergreen shrub distributed across the Southwest regions of China, Myanmar, and Vietnam. Their structures were elucidated on the basis of extensive spectroscopic methods. The isolates were evaluated for in vitro antifungal activities against five phytopathogenic fungi (Fusarium oxysporum, Coriolus versicolor, Fusarium solani, Botrytis cinerea, Fusarium graminearum). As a result, the new compounds fumigaclavine I (1), 13-ethoxycyclotryprostatin A (5), 13-dehydroxycyclotryprostatin A (6), and 12ß-hydroxy-13-oxofumitremorgin C (7) exhibited antifungal activities with MIC values of 7.8-62.5 µg/mL which were comparable to the two positive controls ketoconazole (MIC = 7.8-31.25 µg/mL) and carbendazim (MIC = 1.95-7.8 µg/mL). Furthermore, compounds 1 and 5 demonstrated potent protective and curative effects against the tomato gray mold in vivo. Preliminary structure-activity relationships of the tested indole diketopiperazine alkaloids indicate that the introduction of a substituent group at position C-13 enhances their biological activities.

Biological effects of a copper-based fungicide on the fruit fly, Drosophila melanogaster.

The increased consumption of pesticides can have a negative environmental impact by increasing the essential metals to toxic levels. Bordasul® is a commonly used fungicide in Brazil and it is composed of 20% Cu, 10% sulfur, and 3.0% calcium. The study of fungicides in vivo in non-target model organisms can predict their environmental impact more broadly. The Drosophila melanogaster is a unique model due to its ease of handling and maintenance. Here, the potential toxicity of Bordasul® was investigated by assessing the development, survival, and behavior of exposed flies. Exposure to Bordasul® impaired the development (p < 0.01) and caused a significant reduction in memory retention (p < 0.05) and locomotor ability (p < 0.001). Fungicides are needed to assure the world's food demand; however, Bordasul® was highly toxic to D. melanogaster. Therefore, Bordasul® may be potentially toxic to non-target invertebrates and new environmentally-safe biofertilizers have to be developed to preserve the biota.

Potential fungicidal and antiaflatoxigenic effects of cinnamon essential oils on Aspergillus flavus inhabiting the stored wheat grains.

Wheat is one of the essential crops for the human and animal nutrition, however, contamination with aflatoxigenic fungi, due to the improper storage conditions and high humidity, was the main global threats. So, preventing the growth of aflatoxigenic fungi in stored wheat grains, by using different essential oils was the main objective of this work. Aspergillus flavus EFBL-MU12 PP087400, EFBL-MU23 PP087401 and EFBL-MU36 PP087403 isolates were the most potent aflatoxins producers inhabiting wheat grains. The effect of storage conditions of wheat grains "humidity, temperature, incubation period, and pH" on growth of A. flavus, was assessed by the response surface methodology using Plackett-Burman design and FCCD. The highest yield of aflatoxins EFBL-MU12 B1 and B2 by A. flavus grown on wheat grains were 145.3 and 7.6 µg/kg, respectively, at incubation temperature 35°C, 16% moisture contents, initial pH 5.0, and incubated for 14 days. The tested oils had a powerful antifungal activity for the growth and aflatoxins production by A. flavus in a concentration-dependent manner. Among these oils, cinnamon oil had the highest fungicidal activity for A. flavus at 0.125%, with about 85-90 % reduction to the aflatoxins B1 and B2, conidial pigmentation and chitin contents on wheat grains. From the SEM analysis, cinnamon oils had the most deleterious effect on A. flavus with morphological aberrations to the conidial heads, vegetative mycelia, alteration in conidiophores identity, hyphae shrank, and winding. To emphasize the effect of the essential oils on the aflatoxins producing potency of A. flavus, the molecular expression of the aflatoxins biosynthetic genes was estimated by RT-qPCR. The molecular expression of nor-1, afLR, pKsA and afLJ genes was suppressed by 94-96%, due to cinnamon oil at 0.062% compared to the control. Conclusively, from the results, cinnamon oils followed by the peppermint oils displayed the most fungicidal activity for the growth and aflatoxins production by A. flavus grown on wheat grains.

Enhanced white rot control in garlic bulbil using organic-inorganic hybrid materials as coating membranes.

Organic-inorganic hybrid materials have a range of applications due to their unique properties. Their application in agriculture brings alternatives for the controlled release of nutrients in the soil, the seed coating, the transport of herbicides, and the treatment of plant diseases. The present study aimed to investigate the use of fungicides incorporated into hybrid membranes formed by synthetic hectorite (LAPONITE®) and polymers in the pre-treatment of garlic bulbils exposed to the pathogen Stromatinia cepivora, which causes white rot. The coatings were selected by a germination test, based on the bulbil sprouting index, and by a mycelial growth inhibition test, based on the percentage of mycelial growth inhibition. The chosen membranes were used to coat the bulbils for bioassays conducted in a biochemical oxygen demand incubator at 17 °C. The coated bulbils were planted in soil samples containing three different densities of Stromatinia cepivora: 0.1 g, 1.0 g, and 10 g of sclerotium per L of soil. Membranes containing 2% carboxymethyl cellulose and 2% LAPONITE® incorporated with (i) the fungicide tebuconazole (36 mg L-1) and (ii) the combination of the actives tebuconazole (36 mg L-1) and triadimenol (62 mg L-1) showed the total rate of sprouting and null indices of incidence of symptoms and mortality in its repetitions. The hybrid membranes were characterized employing several techniques, including X-ray diffraction, infrared and Raman spectroscopy, thermogravimetric analysis and differential scanning calorimetry coupled to mass spectrometry, and optical microscopy. Characterization data confirmed the presence of fungicides incorporated into the membranes. Some concentrations of fungicides were low enough not to be detected in all analyses performed, although they guaranteed a protective character to the bulbils about the fungus S. cepivora present in the soil, with a possibility of antifungal pre-treatment with a potential reduction in the concentration used.

Synthesis, Characterization, and Antifungal Activity of Benzimidazole-Grafted Chitosan against Aspergillus flavus.

Aspergillus flavus contamination in agriculture and food industries poses threats to human health, leading to a requirement of a safe and effective method to control fungal contamination. Chitosan-based nitrogen-containing derivatives have attracted much attention due to their safety and enhanced antimicrobial applications. Herein, a new benzimidazole-grafted chitosan (BAC) was synthesized by linking the chitosan (CS) with a simple benzimidazole compound, 2-benzimidazolepropionic acid (BA). The characterization of BAC was confirmed by Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance spectroscopy (1H and 13C NMR). Then, the efficiency of BAC against A. flavus ACCC 32656 was investigated in terms of spore germination, mycelial growth, and aflatoxin production. BAC showed a much better antifungal effect than CS and BA. The minimum inhibitory concentration (MIC) value was 1.25 mg/mL for BAC, while the highest solubility of CS (16.0 mg/mL) or BA (4.0 mg/mL) could not completely inhibit the growth of A. flavus. Furthermore, results showed that BAC inhibited spore germination and elongation by affecting ergosterol biosynthesis and the cell membrane integrity, leading to the permeabilization of the plasma membrane and leakage of intracellular content. The production of aflatoxin was also inhibited when treated with BAC. These findings indicate that benzimidazole-derived natural CS has the potential to be used as an ideal antifungal agent for food preservation.

Risk assessment and molecular mechanism of Sclerotium rolfsii resistance to boscalid.

Boscalid, a widely used SDHI fungicide, has been employed in plant disease control for over two decades. However, there is currently no available information regarding its antifungal activity against Sclerotium rolfsii and the potential risk of resistance development in this pathogen. In this study, we evaluated the sensitivity of 100 S. rolfsii strains collected from five different regions in China during 2018-2019 to boscalid using mycelial growth inhibition method and assessed the risk of resistance development. The EC50 values for boscalid ranged from 0.2994 µg/mL to 1.0766 µg/mL against the tested strains, with an average EC50 value of 0.7052 ± 0.1473 µg/mL. Notably, a single peak sensitivity baseline was curved, indicating the absence of any detected resistant strains. Furtherly, 10 randomly selected strains of S. rolfsii were subjected to chemical taming to evaluate its resistance risk to boscalid, resulting in the successful generation of six stable and inheritable resistant mutants. These mutants exhibited significantly reduced mycelial growth, sclerotia production, and virulence compared to their respective parental strains. Cross-resistance tests revealed a correlation between boscalid and flutolanil, benzovindiflupyr, pydiflumetofen, fluindapyr, and thifluzamide; however, no cross-resistance was observed between boscalid and azoxystrobin. Thus, we conclude that the development risk of resistance in S. rolfsii to boscalid is low. Boscalid can be used as an alternative fungicide for controlling peanut sclerotium blight when combined with other fungicides that have different mechanisms of action. Finally, the target genes SDHB, SDHC, and SDHD in S. rolfsii were initially identified, cloned and sequenced to elucidate the mechanism of S. rolfsii resistance to boscalid. Two mutation genotypes were found in the mutants: SDHD-D111H and SDHD-H121Y. The mutants carrying SDHD-H121Y exhibited moderate resistance, while the mutants with SDHD-D111H showed low resistance. These findings contribute to our comprehensive understanding of molecular mechanisms underlying plant pathogens resistance to SDHI fungicides.

Modes of action and potential as a peptide-based biofungicide of a plant defensin MtDef4.

Due to rapidly emerging resistance to single-site fungicides in fungal pathogens of plants, there is a burgeoning need for safe and multisite fungicides. Plant antifungal peptides with multisite modes of action (MoA) have potential as bioinspired fungicides. Medicago truncatula defensin MtDef4 was previously reported to exhibit potent antifungal activity against fungal pathogens. Its MoA involves plasma membrane disruption and binding to intracellular targets. However, specific biochemical processes inhibited by this defensin and causing cell death have not been determined. Here, we show that MtDef4 exhibited potent antifungal activity against Botrytis cinerea. It induced severe plasma membrane and organelle irregularities in the germlings of this pathogen. It bound to fungal ribosomes and inhibited protein translation in vitro. A MtDef4 variant lacking antifungal activity exhibited greatly reduced protein translation inhibitory activity. A cation-tolerant MtDef4 variant was generated that bound to ß-glucan of the fungal cell wall with higher affinity than MtDef4. It also conferred a greater reduction in the grey mould disease symptoms than MtDef4 when applied exogenously on Nicotiana benthamiana plants, tomato fruits and rose petals. Our findings revealed inhibition of protein synthesis as a likely target of MtDef4 and the potential of its cation-tolerant variant as a peptide-based fungicide.

Soil microbial community fragmentation reveals indirect effects of fungicide exposure mediated by biotic interactions between microorganisms.

Fungicides are used worldwide to improve crop yields, but they can affect non-target soil microorganisms which are essential for ecosystem functioning. Microorganisms form complex communities characterized by a myriad of interspecies interactions, yet it remains unclear to what extent non-target microorganisms are indirectly affected by fungicides through biotic interactions with sensitive taxa. To quantify such indirect effects, we fragmented a soil microbial community by filtration to alter biotic interactions and compared the effect of the fungicide hymexazol between fractions in soil microcosms. We postulated that OTUs which are indirectly affected would exhibit a different response to the fungicide across the fragmented communities. We found that hymexazol primarily affected bacterial and fungal communities through indirect effects, which were responsible for more than 75% of the shifts in relative abundance of the dominant microbial OTUs after exposure to an agronomic dose of hymexazol. However, these indirect effects decreased for the bacterial community when hymexazol doses increased. Our results also suggest that N-cycling processes such as ammonia oxidation can be impacted indirectly by fungicide application. This work sheds light on the indirect impact of fungicide exposure on soil microorganisms through biotic interactions, which underscores the need for higher-tier risk assessment. ENVIRONMENTAL IMPLICATION: In this study, we used a novel approach based on the fragmentation of the soil microbial community to determine to which extent fungicide application could indirectly affect fungi and bacteria through biotic interactions. To assess off-target effects of fungicide on soil microorganisms, we selected hymexazol, which is used worldwide to control a variety of fungal plant pathogens, and exposed arable soil to the recommended field rate, as well as to higher rates. Our findings show that at least 75% of hymexazol-impacted microbial OTUs were indirectly affected, therefore emphasizing the importance of tiered risk assessment.

Pan-azole- and multi-fungicide-resistant Aspergillus fumigatus is widespread in the United States.

Aspergillus fumigatus is an important global fungal pathogen of humans. Azole drugs are among the most effective treatments for A. fumigatus infection. Azoles are also widely used in agriculture as fungicides against fungal pathogens of crops. Azole-resistant A. fumigatus has been increasing in Europe and Asia for two decades where clinical resistance is thought to be driven by agricultural use of azole fungicides. The most prevalent mechanisms of azole resistance in A. fumigatus are tandem repeats (TR) in the cyp51A promoter coupled with mutations in the coding region which result in resistance to multiple azole drugs (pan-azole resistance). Azole-resistant A. fumigatus has been isolated from patients in the United States (U.S.), but little is known about its environmental distribution. To better understand the distribution of azole-resistant A. fumigatus in the U.S., we collected isolates from agricultural sites in eight states and tested 202 isolates for sensitivity to azoles. We found azole-resistant A. fumigatus in agricultural environments in seven states showing that it is widespread in the U.S. We sequenced environmental isolates representing the range of U.S. sample sites and compared them with publicly available environmental worldwide isolates in phylogenetic, principal component, and ADMIXTURE analyses. We found worldwide isolates fell into three clades, and TR-based pan-azole resistance was largely in a single clade that was strongly associated with resistance to multiple agricultural fungicides. We also found high levels of gene flow indicating recombination between clades highlighting the potential for azole-resistance to continue spreading in the U.S.IMPORTANCEAspergillus fumigatus is a fungal pathogen of humans that causes over 250,000 invasive infections each year. It is found in soils, plant debris, and compost. Azoles are the first line of defense antifungal drugs against A. fumigatus. Azoles are also used as agricultural fungicides to combat other fungi that attack plants. Azole-resistant A. fumigatus has been a problem in Europe and Asia for 20 years and has recently been reported in patients in the United States (U.S.). Until this study, we did not know much about azole-resistant A. fumigatus in agricultural settings in the U.S. In this study, we isolated azole-resistant A. fumigatus from multiple states and compared it to isolates from around the world. We show that A. fumigatus which is resistant to azoles and to other strictly agricultural fungicides is widespread in the U.S.

Preventing multi-resistance: New insights for managing fungal adaptation.

Sustainable crop protection is vital for food security, yet it is under threat due to the adaptation of a diverse and evolving pathogen population. Resistance can be managed by maximising the diversity of selection pressure through dose variation and the spatial and temporal combination of active ingredients. This study explores the interplay between operational drivers for maximising the sustainability of management strategies in relation to the resistance status of fungal populations. We applied an experimental evolution approach to three artificial populations of Zymoseptoria tritici, an economically significant wheat pathogen, each differing in initial resistance status. Our findings reveal that diversified selection pressure curtails the selection of resistance in naïve populations and those with low frequencies of single resistance. Increasing the number of modes of action most effectively delays resistance development, surpassing the increase in the number of fungicides, fungicide choice based on resistance risk, and temporal variation in fungicide exposure. However, this approach favours generalism in the evolved populations. The prior presence of multiple resistant isolates and their subsequent selection in populations override the effects of diversity in management strategies, thereby invalidating any universal ranking. Therefore, the initial resistance composition must be specifically considered in sustainable resistance management to address real-world field situations.

Antifungal Activity of Ribosome-Inactivating Proteins.

The control of crop diseases caused by fungi remains a major problem and there is a need to find effective fungicides that are environmentally friendly. Plants are an excellent source for this purpose because they have developed defense mechanisms to cope with fungal infections. Among the plant proteins that play a role in defense are ribosome-inactivating proteins (RIPs), enzymes obtained mainly from angiosperms that, in addition to inactivating ribosomes, have been studied as antiviral, fungicidal, and insecticidal proteins. In this review, we summarize and discuss the potential use of RIPs (and other proteins with similar activity) as antifungal agents, with special emphasis on RIP/fungus specificity, possible mechanisms of antifungal action, and the use of RIP genes to obtain fungus-resistant transgenic plants. It also highlights the fact that these proteins also have antiviral and insecticidal activity, which makes them very versatile tools for crop protection.

Comparative transcriptome analysis provides insights into the resistance regulation mechanism and inhibitory effect of fungicide phenamacril in Fusarium asiaticum.

Fusarium asiaticum is a destructive phytopathogenic fungus that causes Fusarium head blight of wheat (FHB), leading to serious yield and economic losses to cereal crops worldwide. Our previous studies indicated that target-site mutations (K216R/E, S217P/L, or E420K/G/D) of Type I myosin FaMyo5 conferred high resistance to phenamacril. Here, we first constructed one sensitive strain H1S and three point mutation resistant strains HA, HC and H1R. Then we conducted comparative transcriptome analysis of these F. asiaticum strains after 1 and 10 µg·mL-1 phenamacril treatment. Results indicated that 2135 genes were differentially expressed (DEGs) among the sensitive and resistant strains. The DEGs encoding ammonium transporter MEP1/MEP2, nitrate reductase, copper amine oxidase 1, 4-aminobutyrate aminotransferase, amino-acid permease inda1, succinate-semialdehyde dehydrogenase, 2, 3-dihydroxybenzoic acid decarboxylase, etc., were significantly up-regulated in all the phenamacril-resistant strains. Compared to the control group, a total of 1778 and 2097 DEGs were identified in these strains after 1 and 10 µg·mL-1 phenamacril treatment, respectively. These DEGs involved in 4-aminobutyrate aminotransferase, chitin synthase 1, multiprotein-bridging factor 1, transcriptional regulatory protein pro-1, amino-acid permease inda1, ATP-dependent RNA helicase DED1, acetyl-coenzyme A synthetase, sarcoplasmic/endoplasmic reticulum calcium ATPase 2, etc., showed significantly down-regulated expression in phenamacril-sensitive strain but not in resistant strains after phenamacril treatment. In addition, cyanide hydratase, mating-type protein MAT-1, putative purine nucleoside permease, plasma membrane protein yro2, etc., showed significantly co-down-regulated expression in all the strains after phenamacril treatment. Taken together, This study provides deep insights into the resistance regulation mechanism and the inhibitory effect of fungicide phenamacril and these new annotated proteins or enzymes are worth for the discovery of new fungicide targets.

Resistance mechanism of Phomopsis longicolla to fludioxonil is associated with modifications in PlOS1, PlOS4 and PlOS5.

Phomopsis longicolla, a causal agent of soybean root rot, stem blight, seed decay, pod and stem canker, which seriously affects the yield and quality of soybean production worldwide. The phenylpyrrole fungicide fludioxonil exhibits a broad spectrum and high activity against phytopathogenic fungi. In this study, the baseline sensitivity of 100 P. longicolla isolates collected from the main soybean production areas of China to fludioxonil were determined. The result showed that the EC50 values of all the P. longicolla isolates ranged from 0.013 to 0.035 µg/ml. Furthermore, 12 fludioxonil-resistance (FluR) mutants of P. longicolla were generated from 6 fludioxonil-sensitive (FluS) isolates. and the resistance factors (RF) of 12 FluR mutants were >3500. Sequence alignment showed that multiple mutation types were found in PlOS1, PlOS4 or/and PlOS5 of FluR mutants. All the FluR mutants exhibited fitness penalty in mycelial growth, conidiation, virulence and osmo-adaptation. Under fludioxonil or NaCl treatment condition, the glycerol accumulation was significantly increased in FluS isolates, but was slightly increased in FluR mutants, and the phosphorylation level of most FluR mutants was significantly decreased when compared to the FluS isolates. Additionally, positive cross-resistance was observed between fludioxonil and procymidone but not fludioxonil and pydiflumetofen, pyraclostrobin or fluazinam. This is first reported that the baseline sensitivity of P. longicolla to fludioxonil, as well as the biological and molecular characterizations of P. longicolla FluR mutants to fludioxonil. These results can provide scientific directions for controlling soybean diseases caused by P. longicolla using fludioxonil.

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.

Sensitivity analysis and point mutations in BcSDHB confer cyclobutrifluram resistance in Botrytis cinerea from China.

Botrytis cinerea is one of the most destructive pathogens worldwide. It can damage over 200 crops, resulting in significant yield and quality losses. Cyclobutrifluram, a new generation of succinate dehydrogenase inhibitors, exhibits excellent inhibitory activity against B. cinerea. However, the baseline sensitivity and resistance of B. cinerea to cyclobutrifluram remains poorly understood. This study was designed to monitor the sensitivity frequency distribution, assess the resistance risk, and clarify the resistance mechanism of B. cinerea to cyclobutrifluram. The baseline sensitivity of B. cinerea isolates to cyclobutrifluram was 0.89 µg/mL. Cyclobutrifluram-resistant B. cinerea populations are present in the field. Six resistant B. cinerea isolates investigated in this study possessed enhanced compound fitness index compared to the sensitive isolates according to mycelial growth, mycelial dry weight, conidiation, conidial germination rate, and pathogenicity. Cyclobutrifluram exhibited no cross-resistance with tebuconazole, fludioxonil, cyprodinil, or iprodione. Sequence alignment revealed that BcSDHB from cyclobutrifluram-resistant B. cinerea isolates had three single substitutions (P225F, N230I, or H272R). Molecular docking verified that these mutations in BcSDHB conferred cyclobutrifluram resistance in B. cinerea. In conclusion, the resistance risk of B. cinerea to cyclobutrifluram is high, and the point mutations in BcSDHB (P225F, N230I, or H272R) confer cyclobutrifluram resistance in B. cinerea. This study provided important insights into cyclobutrifluram resistance in B. cinerea and offered valuable information for monitoring and managing cyclobutrifluram resistance in the future.

Study on the Design, Synthesis, Bioactivity and Translocation of the Conjugates of Phenazine-1-carboxylic Acid and N-Phenyl Alanine Ester.

The natural pesticide phenazine-1-carboxylic acid (PCA) is known to lack phloem mobility, whereas Metalaxyl is a representative phloem systemic fungicide. In order to endow PCA with phloem mobility and also enhance its antifungal activity, thirty-two phenazine-1-carboxylic acid-N-phenylalanine esters conjugates were designed and synthesized by conjugating PCA with the active structure N-acylalanine methyl ester of Metalaxyl. All target compounds were characterized by 1H NMR, 13C NMR and HRMS. The antifungal evaluation results revealed that several target compounds exhibited moderate to potent antifungal activities against Sclerotinia sclerotiorum, Bipolaris sorokiniana, Phytophthora parasitica, Phytophthora citrophthora. In particular, compound F7 displayed excellent antifungal activity against S. sclerotiorum with an EC50 value of 6.57 µg/mL, which was superior to that of Metalaxyl. Phloem mobility study in castor bean system indicated good phloem mobility for the target compounds F1-F16. Particularly, compound F2 exhibited excellent phloem mobility; the content of compound F2 in the phloem sap of castor bean was 19.12 µmol/L, which was six times higher than Metalaxyl (3.56 µmol/L). The phloem mobility tests under different pH culture solutions verified the phloem translocation of compounds related to the "ion trap" effect. The distribution of the compound F2 in tobacco plants further suggested its ambimobility in the phloem, exhibiting directional accumulation towards the apical growth point and the root. These results provide valuable insights for developing phloem mobility fungicides mediated by exogenous compounds.

Diallyl Trisulfide Acts as a Soil Disinfestation Against the Ilyonectria destructans through Inducing the Burst of Reactive Oxygen Species.

Soil-borne diseases represent an impediment to the sustainable development of agriculture. A soil-borne disease caused by Ilyonectria destructans severely impacts Panax species, and soil disinfestation has proven to be an effective management approach. Here, diallyl trisulfide (DATS), derived from garlic, exhibited pronounced inhibitory effects on the growth of I. destructans in vitro tests and contributed to the alleviation of soil-borne diseases in the field. A comprehensive analysis demonstrated that DATS inhibits the growth of I. destructans by activating detoxifying enzymes, such as GSTs, disrupting the equilibrium of redox reactions. A series of antioxidant amino acids were suppressed by DATS. Particularly noteworthy is the substantial depletion of glutathione by DATS, resulting in the accumulation of ROS, ultimately culminating in the inhibition of I. destructans growth. Briefly, DATS could effectively suppress soil-borne diseases by inhibiting pathogen growth through the activation of ROS, and it holds promise as a potential environmentally friendly soil disinfestation.

Novel Pyrazole-4-Carboxamide Derivatives Containing Oxime Ether Group as Potential SDHIs to Control Rhizoctonia solani.

In the search for novel succinate dehydrogenase inhibitor (SDHI) fungicides to control Rhizoctonia solani, thirty-five novel pyrazole-4-carboxamides bearing either an oxime ether or an oxime ester group were designed and prepared based on the strategy of molecular hybridization, and their antifungal activities against five plant pathogenic fungi were also investigated. The results indicated that the majority of the compounds containing oxime ether demonstrated outstanding in vitro antifungal activity against R. solani, and some compounds also displayed pronounced antifungal activities against Sclerotinia sclerotiorum and Botrytis cinerea. Particularly, compound 5e exhibited the most promising antifungal activity against R. solani with an EC50 value of 0.039 µg/mL, which was about 20-fold better than that of boscalid (EC50 = 0.799 µg/mL) and 4-fold more potent than fluxapyroxad (EC50 = 0.131 µg/mL). Moreover, the results of the detached leaf assay showed that compound 5e could suppress the growth of R. solani in rice leaves with significant protective efficacies (86.8%) at 100 µg/mL, superior to boscalid (68.1%) and fluxapyroxad (80.6%), indicating promising application prospects. In addition, the succinate dehydrogenase (SDH) enzymatic inhibition assay revealed that compound 5e generated remarkable SDH inhibition (IC50 = 2.04 µM), which was obviously more potent than those of boscalid (IC50 = 7.92 µM) and fluxapyroxad (IC50 = 6.15 µM). Furthermore, SEM analysis showed that compound 5e caused a remarkable disruption to the characteristic structure and morphology of R. solani hyphae, resulting in significant damage. The molecular docking analysis demonstrated that compound 5e could fit into the identical binding pocket of SDH through hydrogen bond interactions as well as fluxapyroxad, indicating that they had a similar antifungal mechanism. The density functional theory and electrostatic potential calculations provided useful information regarding electron distribution and electron transfer, which contributed to understanding the structural features and antifungal mechanism of the lead compound. These findings suggested that compound 5e could be a promising candidate for SDHI fungicides to control R. solani, warranting further investigation.

Targeting of Antifungal Metabolites from Grapevine Byproducts by UPLC-HRMS/MS Approaches Using Bioactivity-Based Molecular Networking.

This study focuses on countering Fusarium graminearum, a harmful fungal pathogen impacting cereal crops and human health through mycotoxin production. These mycotoxins, categorized as type B trichothecenes, pose significant health risks. Research explores natural alternatives to synthetic fungicides, particularly investigating phenolics in grapevine byproducts. Thirteen eco-extracts from five French grape varieties (Merlot, Cabernet Sauvignon, Sauvignon blanc, Tannat, and Artaban) exhibited substantial antifungal properties, with ten extracts displaying remarkable effects. Extracts from grapevine stems and roots notably reduced fungal growth by over 91% after five days. Through UHPLC-HRMS/MS analysis and metabolomics, the study identified potent antifungal compounds such as ampelopsin A and cyphostemmin B, among other oligomeric stilbenes. Interestingly, this approach showed that flavan-3-ols have been identified as markers for extracts that induce fungal growth. Root extracts from rootstocks, rich in oligostilbenes, demonstrated the highest antifungal activity. This research underscores grapevine byproducts' potential both as a sustainable approach to control F. graminearum and mycotoxin contamination in cereal crops and the presence of different metabolites from the cultivars of grapevine, suggesting different activities.