The antagonistic activity of Streptomyces spiroverticillatus (No. HS1) against of poplar canker pathogen Botryosphaeria dothidea.

BACKGROUND: Poplar canker caused by Botryosphaeria dothidea is one of the most severe plant disease of poplars worldwide. In our study, we aimed to investigate the modes of antagonism by fermentation broth supernatant (FBS) of Streptomyces spiroverticillatus HS1 against B. dothidea. RESULTS: In vitro, the strain and FBS of S. spiroverticillatus HS1 significantly inhibited mycelial growth and biomass accumulation, and also disrupted the mycelium morphology of B. dothidea. On the 3rd day after treatment, the inhibition rates of colony growth and dry weight were 80.72% and 52.53%, respectively. In addition, FBS treatment damaged the plasma membrane of B. dothidea based on increased electrical conductivity in the culture medium, and malondialdehyde content of B. dothidea mycelia. Notably, the analysis of key enzymes in glycolysis pathway showed that the activity of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK), Ca2+Mg2+-ATPase were significantly increased after FBS treatment. But the glucose contents were significantly reduced, and pyruvate contents were significantly increased in B. dothidea after treatment with FBS. CONCLUSIONS: The inhibitory mechanism of S. spiroverticillatus HS1 against B. dothidea was a complex process, which was associated with multiple levels of mycelial growth, cell membrane structure, material and energy metabolism. The FBS of S. spiroverticillatus HS1 could provide an alternative approach to biological control strategies against B. dothidea.

Biocontrol of rusted root rot in Panax ginseng by a combination of extracts from Bacillus amyloliquefaciens YY8 crude protein and Enterobacteriaceae YY115 ethyl acetate.

BACKGROUND: Rusted root rot is one of the most common root diseases in Panax ginseng, and Cylindrocarpon destructans is one of the main pathogenic fungus. The objective of this study was to screen and explore the extracts of biocontrol bacteria isolated from ginseng rhizosphere soil against Cylindrocarpon destructans. RESULTS: Bacterial strains Bacillus amyloliquefaciens YY8 and Enterobacteriacea YY115 were isolated and found to exhibit in vitro antifungal activity against C. destructans. A combination of crude protein extract from B. amyloliquefaciens YY8 and ethyl acetate extract from Enterobacteriacea YY115 in a 6:4 ratio exhibited the strongest antifungal activity against C. destructans. Measurements of electrical conductivity, protein content, and nucleic acid content in suspension cultures of C. destructans treated with a mixture extracts indicated that the extracts disrupted the cell membranes of rusted root rot mycelia, resulting in the leakage of electrolytes, proteins, and nucleic acids from the cells, and ultimately inhibiting the growth of C. destructans. The combined extracts suppressed the infection of ginseng roots discs by C. destructans effectively. CONCLUSION: The extracts obtained from the two bacterial strains effectively inhibited C. destructans in P. ginseng. It can provide scientific basis for the development of new biological control pesticides, reduce the use of chemical pesticides, and promote the sustainable development of agriculture.

The occurrence and mechanism of field resistance to boscalid and pyraclostrobin in Stemphylium solani, the causal agent of tomato gray leaf spot in China.

The destructive disease gray leaf spot, caused by Stemphylium solani, is prevalent in tomato plants in China. A variety of fungicides have been extensively used for controlling the disease, with a particular focus on succinate dehydrogenase inhibitors (SDHIs) and quinone outside inhibitors (QoIs). However, there was a lack of information regarding the resistance of S. solani to boscalid (SDHI) and pyraclostrobin (QoI) in China. In this study, the sensitivity of S. solani to boscalid and pyraclostrobin was monitored. The EC50 values for boscalid ranged from 0.02 to 3.0 µg∙mL-1, with an average value of 0.62 µg∙mL-1, while the EC50 values for pyraclostrobin ranged from 0.21 to 14.71 µg∙mL-1, with an average value of 6.03 µg∙mL-1. Based on these findings, the frequencies of observed resistance were as follows: 36.7% for boscalid and 50% for pyraclostrobin; while the resistance frequency to both boscalid and pyraclostrobin in S. solani was 19.4%. The mutation associated with boscalid resistance in S. solani within tomato fields was identified as SdhB-H277Y, while the mutation related to pyraclostrobin resistance was found in cytochrome b, specifically Cytb-G143A. The resistant mutants displayed diminished fitness in terms of mycelial growth, yet their pathogenicity exhibited no significant disparities. To delay the development of resistance, it is advisable to employ a rotation strategy using alternative fungicides with different modes of action or mix with fungicides with multi-site-contact activity for disease management.

The Preliminary Analysis of Flavonoids in the Petals of Rhododendron delavayi, Rhododendron agastum and Rhododendron irroratum Infected with Neopestalotiopsis clavispora.

The petal blight disease of alpine Rhododendron severely impacts the ornamental and economic values of Rhododendron. Plant secondary metabolites play a crucial role in resisting pathogenic fungi, yet research on metabolites in alpine Rhododendron petals that confer resistance to pathogenic fungi is limited. In the present study, the secondary metabolites in Rhododendron delavayi, R. agastum, and R. irroratum petals with anti-pathogenic activity were screened through disease index analysis, metabolomic detection, the mycelial growth rate, and metabolite spraying experiments. Disease index analysis revealed that R. delavayi petals exhibited the strongest disease resistance, while R. agastum showed the weakest, both under natural and experimental conditions. UHPLC-QTOF-MS/MS analysis identified 355 and 274 putative metabolites in positive and negative ion modes, respectively. The further antifungal analysis of differentially accumulated baicalein, diosmetin, and naringenin showed their half-inhibitory concentrations (IC50) against Neopestalotiopsis clavispora to be 5000 mg/L, 5000 mg/L, and 1000 mg/L, respectively. Spraying exogenous baicalein, diosmetin, and naringenin significantly alleviated petal blight disease caused by N. clavispora infection in alpine Rhododendron petals, with the inhibition rates exceeding 64%. This study suggests that the screened baicalein, diosmetin, and naringenin, particularly naringenin, can be recommended as inhibitory agents for preventing and controlling petal blight disease in alpine Rhododendron.

Biochemical mechanism of chlorine dioxide fumigation in inhibiting Ceratocystis fimbriata and black rot in postharvest sweetpotato.

The inhibitory properties and underlying mechanism of chlorine dioxide (ClO2) fumigation on the pathogen Ceratocystis fimbriata (C. fimbriata) and resultant sweetpotato black rot were investigated in vitro and in vivo. Results revealed that the ClO2 fumigation effectively inhibited fungal growth and induced obvious morphological variation of C. fimbriata mycelia. Furthermore, the mycelial membrane suffered damage, as evidenced by a significant increase in malondialdehyde content and the leakage of protein and nucleic acid from mycelia cells, accompanied by a marked decrease in ergosterol content. Additionally, ClO2 fumigation caused spores cell membrane damage, a notable decrease in spore viability, and induced cell apoptosis as indicated by reductions in spore germination rate, two fluorescence staining observations, and flow cytometry analysis. Moreover, the decay diameter of sweetpotato black rot lesions decreased significantly after ClO2 fumigation, and the growth of C. fimbriata was also inhibited. These findings present a novel and effective technology for inhibiting the progression of sweetpotato black rot.

Carbon-Dots-Mediated Improvement of Antimicrobial Activity of Natural Products.

The development of new microbicidal compounds has become a top priority due to the emergence and spread of drug-resistant pathogenic microbes. In this study, blue-emitting and positively charged carbon dots (CDs), called Du-CDs, were fabricated for the first time utilizing the natural product extract of endophyte Diaporthe unshiuensis YSP3 as raw material through a one-step solvothermal method, which possessed varied functional groups including amino, carboxyl, hydroxyl, and sulfite groups. Interestingly, Du-CDs exhibited notably enhanced antimicrobial activities toward both bacteria and fungi as compared to the natural product extract of YSP3, with low minimum inhibitory concentrations. Moreover, Du-CDs significantly inhibited the formation of biofilms. Du-CDs bound with the microbial cell surface via electronic interaction or hydrophobic interaction entered the microbial cells and were distributed fully inside the cells. Du-CDs caused cell membrane damage and/or cell division cycle interruption, resulting in microbial cell death. Moreover, Du-CDs exhibited an improved antimicrobial effect and accelerated wound healing ability with good biocompatibility in the mouse model. Overall, we demonstrate that the formation of CDs from fungal natural products presents a promising and potential means to develop novel antimicrobial agents with great fluorescence, improved microbiocidal effect and wound healing capacity, and good biosafety for combating microbial infections.

Discovery of a Hybrid Molecule with Phytotoxic Activity by Genome Mining, Heterologous Expression, and OSMAC Strategy.

Genome mining in association with the OSMAC (one strain, many compounds) approach provides a feasible strategy to extend the chemical diversity and novelty of natural products. In this study, we identified the biosynthetic gene cluster (BGC) of restricticin, a promising antifungal agent featuring a reactive primary amine, from the fungus Aspergillus sclerotiorum LZDX-33-4 by genome mining. Combining heterologous expression and the OSMAC strategy resulted in the production of a new hybrid product (1), along with N-acetyl-restricticin (2) and restricticinol (3). The structure of 1 was determined by spectroscopic data, including optical rotation and electronic circular dichroism (ECD) calculations, for configurational assignment. Compound 1 represents a fusion of restricticin and phytotoxic cichorin. The biosynthetic pathway of 1 was proposed, in which the condensation of a primary amine of restricticin with a precursor of cichorine was postulated. Compound 1 at 5 mM concentration inhibited the growth of the shoots and roots of Lolium perenne, Festuca arundinacea, and Lactuca sativa with inhibitory rates of 71.3 and 88.7% for L. perenne, 79.4 and 73.0% for F. arundinacea, and 58.2 and 52.9% for L. sativa. In addition, compound 1 at 25 µg/mL showed moderate antifungal activity against Fusarium fujikuroi and Trichoderma harzianum with inhibition rates of 22.6 and 31.6%, respectively. These results suggest that heterologous expression in conjunction with the OSMAC approach provides a promising strategy to extend the metabolite novelty due to the incorporation of endogenous metabolites from the host strain with exogenous compounds, leading to the production of more complex compounds and the acquisition of new physiological functions.

Discovery of Benzothiazol-2-ylthiophenylpyrazole-4-carboxamides as Novel Succinate Dehydrogenase Inhibitors.

Succinate dehydrogenase (SDH) has been considered an ideal target for discovering fungicides. To develop novel SDH inhibitors, in this work, 31 novel benzothiazol-2-ylthiophenylpyrazole-4-carboxamides were designed and synthesized using active fragment exchange and a link approach as promising SDH inhibitors. The findings from the tests on antifungal activity indicated that most of the synthesized compounds displayed remarkable inhibition against the fungi tested. Compound Ig N-(2-(((5-chlorobenzo[d]thiazol-2-yl)thio)methyl)phenyl)-3-(difluoromethyl)-1-methyl-1H-yrazole-4-carboxamide, with EC50 values against four kinds of fungi tested below 10 µg/mL and against Cercospora arachidicola even below 2 µg/mL, showed superior antifungal activity than that of commercial fungicide thifluzamide, and specifically compounds Ig and Im were found to show preventative potency of 90.6% and 81.3% against Rhizoctonia solani Kühn, respectively, similar to the positive fungicide thifluzamide. The molecular simulation studies suggested that hydrophobic interactions were the main driving forces between ligands and SDH. Encouragingly, we found that compound Ig can effectively promote the wheat seedlings and the growth of Arabidopsis thaliana. Our further studies indicated that compound Ig could stimulate nitrate reductase activity in planta and increase the biomass of plants.

Stereoselective Bioactivity and Action Mechanism of the Fungicide Isopyrazam.

Understanding the stereoselective bioactivity of chiral pesticides is crucial for accurately evaluating their effectiveness and optimizing their use. Isopyrazam, a widely used chiral SDHI fungicide, has been studied for its antifungal activity only at the racemic level. Therefore, to clarify the highly bioactive isomers, the stereoselective bioactivity of isopyrazam isomers against four typical phytopathogens was studied for the first time. The bioactivity ranking of the isomers was trans-1S,4R,9R-(+)-isopyrazam > cis-1R,4S,9R-(+)-isopyrazam > trans-1R,4S,9S-(-)-isopyrazam > cis-1S,4R,9S-(-)-isopyrazam. SDH activity was assessed by molecular docking simulation and actual detection to confirm the reasons for stereoselective bioactivity. The results suggest that the stereoselective bioactivity of isopyrazam is largely dependent on the differential binding ability of each isomer to the SDH ubiquitin-binding site, located within a cavity formed by the iron-sulfur subunit, the cytochrome b560 subunit, and the cytochrome b small subunit. Moreover, to reveal the molecular mechanism of isopyrazam stereoselectively affecting mycelial growth, the contents of succinic acid, fumaric acid, and ATP were measured. Furthermore, by measuring exospore polysaccharides and oxalic acid content, it was determined that 1S,4R,9R-(+)- and 1R,4S,9R-(+)-isopyrazam more strongly inhibited the ability of Sclerotinia sclerotiorum to infect plants. The findings provided essential data for the development of high-efficiency isopyrazam fungicides and offered a methodological reference for analyzing the enantioselective activity mechanism of SDHI fungicides.

Rhamnolipids and fengycins interact differently with biomimetic lipid membrane models of Botrytis cinerea and Sclerotinia sclerotiorum: Lipidomics profiles and biophysical studies.

Rhamnolipids (RLs) and Fengycins (FGs) are biosurfactants with very promising antifungal properties proposed to reduce the use of synthetic pesticides in crops. They are amphiphilic molecules, both known to target the plasma membrane. They act differently on Botrytis cinerea and Sclerotinia sclerotiorum, two close Sclerotiniaceae phytopathogenic fungi. RLs are more efficient at permeabilizing S. sclerotiorum, and FGs are more efficient at permeabilizing B. cinerea mycelial cells. To study the link between the lipid membrane composition and the activity of RLs and FGs, we analyzed the lipid profiles of B. cinerea and S. sclerotiorum. We determined that unsaturated or saturated C18 and saturated C16 fatty acids are predominant in both fungi. We also showed that phosphatidylethanolamine (PE), phosphatidic acid (PA), and phosphatidylcholine (PC) are the main phospholipids (in this order) in both fungi, with more PA and less PC in S. sclerotiorum. The results were used to build biomimetic lipid membrane models of B. cinerea and S. sclerotiorum for all-atom molecular dynamic simulations and solid-state NMR experiments to more deeply study the interactions between RLs or FGs with different compositions of lipid bilayers. Distinctive effects are exerted by both compounds. RLs completely insert in all the studied model membranes with a fluidification effect. FGs tend to form aggregates out of the bilayer and insert individually more easily into the models representative of B. cinerea than those of S. sclerotiorum, with a higher fluidification effect. These results provide new insights into the lipid composition of closely related fungi and its impact on the mode of action of very promising membranotropic antifungal molecules for agricultural applications.

Hybrid de novo whole genome assembly of lipopeptide producing novel Bacillus thuringiensis strain NBAIR BtAr exhibiting antagonistic activity against Sclerotium rolfsii.

Bacillus thuringiensis Berliner is recognized as a predominant bioinsecticide but its antifungal potential has been relatively underexplored. A novel B. thuringiensis strain NBAIR BtAr was isolated and morphologically characterized using light and scanning electron microscopy, revealing presence of bipyramidal, cuboidal, and spherical parasporal crystals. The crude form of lipopeptides was extracted from NBAIR BtAr and assessed for its antagonistic activity in vitro, and demonstrated 100 % inhibition of Sclerotium rolfsii Sacc. at a minimum inhibitory concentration of 50 µL of the crude lipopeptide extract per mL of potato dextrose agar. To identify the antagonistic genes responsible, we performed whole genome sequencing of NBAIR BtAr, revealing the presence of circular chromosome of 5,379,913 bp and 175,362 bp plasmid with 36.06 % guanine-cytosine content and 5814 protein-coding sequences. Average nucleotide identity and whole genome phylogenetic analysis delineated the NBAIR BtAr strain as konkukian serovar. Gene ontology analysis revealed associations of 1474, 1323, and 1833 genes with biological processes, molecular function, and cellular components, respectively. Antibiotics & secondary metabolite analysis shell analysis of the whole genome yielded secondary metabolites biosynthetic gene clusters with 100 %, 85 %, 40 %, and 35 % similarity for petrobactin, bacillibactin, fengycin, and paenilamicin, respectively. Also, novel biosynthetic gene clusters, along with antimicrobial genes, including zwittermicin A, chitinase, and phenazines, were identified. Moreover, the presence of eight bacteriophage sequences, 18 genomic islands, insertion sequences, and one CRISPR region indicated prior occurrences of genetic exchange and thus improved competitive fitness of the strain. Overall, the whole genome sequence of NBAIR BtAr is presented, with its taxonomic classification and critical genetic attributes that contribute to its strong antagonistic activity against S. rolfsii.

Antifungal Effect of Bauhinia variegata Lectin (BvL) on Bipolaris oryzae.

The search for less harmful, ecologically efficient, more specific, and natural alternatives for the control of pathogens is essential. Bauhinia variegata lectin (BvL) is a protein that has numerous biological activities, including antifungal. The present study examines the potential in vitro of B. variegata lectin against the fungus Bipolaris oryzae, responsible for agricultural losses in southern Brazil, due to damage to rice fields during seed germination. Bioassays to assess the inhibition potential of BvL were performed, including fungal growth, spore formation, and germination, in concentrations of 0, 25, 50, and 100 µg mL-1. Only the concentration of 100 µg mL-1 successfully inhibited mycelial growth and spore germination, while in spore formation, all treatments inhibited sporulation. In addition, fluorescence microscopy analysis demonstrated the ability of lectin to bind to the fungus and the lack of detection in the presence of lactose, suggesting its interaction with the fungal cell wall structures. This study highlights the potential of B. variegata seed lectin to control mycelial growth, sporulation, and germination of the phytopathogenic fungus B. oryzae, posing as a new biotechnological possibility for biological control.

Discovery, Optimization, and Biological Evaluation of Novel Pyrazol-5-yl-phenoxybenzamide Derivatives as Potent Succinate Dehydrogenase Inhibitors.

The diphenyl ether molecular pharmacophore has played a significant role in the development of fungicidal compounds. In this study, a variety of pyrazol-5-yl-phenoxybenzamide derivatives were synthesized and evaluated for their potential to act as succinate dehydrogenase inhibitors (SDHIs). The bioassay results indicate certain compounds to display a remarkable and broad-spectrum in their antifungal activities. Notably, compound 12x exhibited significant in vitro activities against Valsa mali, Gaeumannomyces graminis, and Botrytis cinerea, with EC50 values of 0.52, 1.46, and 3.42 mg/L, respectively. These values were lower or comparable to those of Fluxapyroxad (EC50 = 12.5, 1.93, and 8.33 mg/L, respectively). Additionally, compound 12x showed promising antifungal activities against Sclerotinia sclerotiorum (EC50 = 0.82 mg/L) and Rhizoctonia solani (EC50 = 1.86 mg/L), albeit lower than Fluxapyroxad (EC50 = 0.23 and 0.62 mg/L). Further in vivo experiments demonstrated compound 12x to possess effective protective antifungal activities against V. mali and S. sclerotiorum at a concentration of 100 mg/L, with inhibition rates of 66.7 and 89.3%, respectively. In comparison, Fluxapyroxad showed inhibition rates of 29.2 and 96.4% against V. mali and S. sclerotiorum, respectively. Molecular docking analysis revealed that compound 12x interacts with SDH through hydrogen bonding, π-cation, and π-π interactions, providing insights into the probable mechanism of action. Furthermore, compound 12x exhibited greater binding energy and SDH enzyme inhibitory activity than Fluxapyroxad (ΔGcal = -46.8 kcal/mol, IC50 = 1.22 mg/L, compared to ΔGcal = -41.1 kcal/mol, IC50 = 8.32 mg/L). Collectively, our results suggest that compound 12x could serve as a promising fungicidal lead compound for the development of more potent SDHIs for crop protection.

Degradation-Risk-Inspired Optimization of the Antifungal Oxazolinyl Aniline Lead by a Fusion of Triazole with Nicotinamide.

The discovery of readily available and easily modifiable new models is a crucial and practical solution for agrochemical innovation. Antifungal function-oriented fusion of triazole with the prevalidated lead (R)-LE001 affords a novel framework with a broad and enhanced antifungal spectrum. Characterized by the easy accessibility and adjustability of [1,2,4]triazolo[4,3-a]pyridine, modular fine-tuning provided a set of unprecedented leads (e.g., Z23, Z25, Z26, etc.) with superior antifungal potentials than the positive control boscalid. Candidate Z23 exhibited a more promising antifungal activity against Sclerotinia sclerotiorum, Botrytis cinerea, and Phytophthora capsici with EC50 values of 0.7, 0.6, and 0.5 µM, respectively. This candidate could effectively control boscalid-resistant B. cinerea strains and also exhibit good vivo efficacy in controlling gray mold. Noteworthily, both the SDH-inhibition and the efficiency against Oomycete P. capsici are quite distinct from that of the positive control boscalid. A molecular docking simulation also differentiates Z23 from boscalid. These findings highlight the potential of [1,2,4]triazolo[4,3-a]pyridine amide as a novel antifungal model.

Discovery of Novel Acethydrazide-Containing Flavonol Derivatives as Potential Antifungal Agents.

In this study, a series of novel hydrazide-containing flavonol derivatives was designed, synthesized, and evaluated for antifungal activity. In the in vitro antifungal assay, most of the target compounds exhibited potent antifungal activity against seven tested phytopathogenic fungi. In particular, compound C32 showed the best antifungal activity against Rhizoctonia solani (EC50 = 0.170 µg/mL), outperforming carbendazim (EC50 = 0.360 µg/mL) and boscalid (EC50 = 1.36 µg/mL). Compound C24 exhibited excellent antifungal activity against Valsa mali, Botrytis cinerea, and Alternaria alternata with EC50 values of 0.590, 0.870, and 1.71 µg/mL, respectively. The in vivo experiments revealed that compounds C32 and C24 were potential novel agricultural antifungals. 3D quantitative structure-activity relationship (3D-QSAR) models were used to analyze the structure-activity relationships of these compounds. The analysis results indicated that introducing appropriate electronegative groups at position 4 of a benzene ring could effectively improve the anti-R. solani activity. In the antifungal mechanism study, scanning electron microscopy and transmission electron microscopy analyses revealed that C32 disrupted the normal growth of hyphae by affecting the structural integrity of the cell membrane and cellular respiration. Furthermore, compound C32 exhibited potent succinate dehydrogenase (SDH) inhibitory activity (IC50 = 8.42 µM), surpassing that of the SDH fungicide boscalid (IC50 = 15.6 µM). The molecular dynamics simulations and docking experiments suggested that compound C32 can occupy the active site and form strong interactions with the key residues of SDH. Our findings have great potential for aiding future research on plant disease control in agriculture.

Design, Synthesis, and Biological Activity of Silicon-Containing Carboxamide Fungicides.

Bioisosteric silicon replacement has proven to be a valuable strategy in the design of bioactive molecules for crop protection and drug development. Twenty-one novel carboxamides possessing a silicon-containing biphenyl moiety were synthesized and tested for their antifungal activity and succinate dehydrogenase (SDH) enzymatic inhibitory activity. Among these novel succinate dehydrogenase inhibitors (SDHIs), compounds 3a, 3e, 4l, and 4o possessing appropriate clog P and topological polar surface area values showed excellent inhibitory effects against Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, and Fusarium graminearum at 10 mg/L in vitro, and the EC50 values of 4l and 4o were 0.52 and 0.16 mg/L against R. solani and 0.066 and 0.054 mg/L against S. sclerotiorum, respectively, which were superior to those of Boscalid. Moreover, compound 3a demonstrated superior SDH enzymatic inhibitory activity (IC50 = 8.70 mg/L), exhibiting 2.54-fold the potency of Boscalid (IC50 = 22.09 mg/L). Docking results and scanning electron microscope experiments revealed similar mode of action between compound 3a and Boscalid. The new silicon-containing carboxamide 3a is a promising SDHI candidate that deserves further investigation.

Effectiveness of Fludioxonil, a New-Generation Reduced-Risk Fungicide, Against Brown Rot Pathogens.

Brown rot, caused by Monilinia species, is a destructive disease of pome and stone fruits that can lead to significant losses in production. Disease management is mainly based on fungicide applications during the growing season. Fludioxonil, a "new-generation reduced-risk fungicide", is one of the most important fungicide used. The objectives of the present study were to compare and determine the toxicity of fludioxonil to selected M. laxa, M. fructigena and M. fructicola isolates, to test its effectiveness in detached fruits and to assess its effectiveness under practical control conditions. A total of 27 isolates (10 isolates of M. laxa, 8 of M. fructigena and 9 of M. fructicola) were tested for sensitivity to fludioxonil in vitro. Isolates from each species exhibited a homogeneous response to the fungicide, while differences among the different species were determined. Based on calculated resistance factors (RF), the examined isolates were classified into two categories: sensitive and moderately resistant. In vivo testing of the effectiveness of the label concentration of fludioxonil on detached fruit did not reveal differences between isolates classified into different sensitivity categories; fludioxonil used at the label concentration (0.1%) inhibited decay development 93.5 to 100%, regardless of the isolate category. Field trials revealed the very high efficacy of fludioxonil in preventing brown rot on fruits, ranging from 92.2 to 100 for peach, 90.7 to 97.3 for plum and 84.9 to 91.9% for sour cherry. In conclusion, fludioxonil was highly effective according to in vitro sensitivity tests and when used under practical field conditions for brown rot control.

Novel mandelic acid derivatives containing piperazinyls as potential candidate fungicides against Monilinia fructicola: Design, synthesis and mechanism study.

Brown rot of stone fruit, a disease caused by the ascomycete fungus Monilinia fructicola, has caused significant losses to the agricultural industry. In order to explore and discover potential fungicides against M. fructicola, thirty-one novel mandelic acid derivatives containing piperazine moieties were designed and synthesized based on the amide skeleton. Among them, target compound Z31 exhibited obvious in vitro antifungal activity with the EC50 value of 11.8 mg/L, and significant effects for the postharvest pears (79.4 % protective activity and 70.5 % curative activity) at a concentration of 200 mg/L. Antifungal activity for the target compounds was found to be significantly improved by the large steric hindrance of the R1 groups and the electronegative of the piperazines in the molecular structure, according to a three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis. Further mechanism studies have demonstrated that the compound Z31 can disrupt cell membrane integrity, resulting in increased membrane permeability, release of intracellular electrolytes, and affect the normal growth of hyphae. Additional, morphological study also indicated that Z31 may disrupt the integrity of the membrane by inducing generate excess endogenous reactive oxygen species (ROS) and resulting in the peroxidation of cellular lipids, which was further verified by the detection of malondialdehyde (MDA) content. These studies have provided the basis for the creation of novel fungicides to prevent brown rot in stone fruits.

Identification of plant based potential antifungal compounds against BMK-1 protein of Bipolaris oryzae using molecular docking approach.

Rice brown spot is an important disease of rice worldwide that inflicts substantial yield losses. The antimicrobial potential of methanol, acetone and dimethyl sulfoxide (DMSO) extracts of different medicinal plants, viz., Syzygium aromaticum, Saussurea costus, Acorus calamus, Bergenia ciliate, Geranium pratense, Mentha longifolia, Inula racemosa, Podophyllum hexandrum, Heracleum candicans and Picrorhiza kurroa, against the brown spot pathogen Bipolaris oryzae in vitro was evaluated via mycelial growth inhibition and spore germination inhibition assays. Among the plant extracts tested, 100% mycelial inhibition was observed for the methanol extract of Syzygium aromaticum at all three concentrations (2000 ppm, 3000 ppm and 4000 ppm), followed by the methanol extract of Inula racemosa (90.33%) at 4000 ppm. A maximum conidial germination inhibition of 83.54% was exhibited by the Heracleum candicans leaf extract. Phytochemical profiling of Syzygium aromaticum and Inula racemosa through liquid chromatography and mass spectrometry (HR-LCMS) revealed the presence of several compounds, such as eugenol, ursolic acid, quercetin, chlorogenic acid, and noscapine. A molecular docking approach was used to identify key inhibitory molecules against B. oryzae. Among the compounds detected in S. aromaticum and Inula racemosa, ursolic acid and noscapine were found to have the greatest binding affinity for the Big Mitogen Activated Protein Kinase (BMK-1) enzyme present in B. oryzae. In conclusion, S. aromaticum and Inula racemosa are potent compounds that could serve as lead compounds for drug discovery in the future.

Determination of the amino linkage of the D-Dab residue of the cyclic lipo-octapeptide occidiofungins A-D and the antifungal activity of their analogues.

Recently, the demand for new antifungal drugs has increased due to the presence of antimicrobial resistant bacteria and their side effects. Occidiofungins (Ocfs) are cyclic lipo-octapeptides that possess unusual amino acids and potent antifungal activities. However, the chemical structure of the 2,4-diamino butyric acid (Dab) residue in the backbone of Ocfs has not been clarified thus far. Therefore, we conducted a structural analysis of the tripeptides around the Dab residue in Ocfs using 1H-NMR spectroscopy. We determined that the D-Dab residue in the peptide backbone of Ocfs has an α-amino linkage. Additionally, we found that Ocf A (5) and Bk-1119 have the same chemical structure. Moreover, the analogue possessing D-αDab (13) showed potent antifungal activity against A. oryzae.