New reports of pathogen spectrum associated with bulb rot and their interactions during the development of rot in tulip.

Bulb rot, a highly damaging disease of tulip plants, has hindered their profitable cultivation worldwide. This rot occurs in both field and storage conditions posing significant challenges. While this disease has been attributed to a range of pathogens, previous investigations have solely examined it within the framework of a single-pathogen disease model. Our study took a different approach and identified four pathogens associated with the disease: Fusarium solani, Penicillium chrysogenum, Botrytis tulipae, and Aspergillus niger. The primary objective of our research was to examine the impact of co-infections on the overall virulence dynamics of these pathogens. Through co-inoculation experiments on potato dextrose agar, we delineated three primary interaction patterns: antibiosis, deadlock, and merging. In vitro trials involving individual pathogen inoculations on tulip bulbs revealed that B. tulipae,was the most virulent and induced complete bulb decay. Nonetheless, when these pathogens were simultaneously introduced in various combinations, outcomes ranged from partial bulb decay to elongated rotting periods. This indicated a notable degree of antagonistic behaviour among the pathogens. While synergistic interactions were evident in a few combinations, antagonism overwhelmingly prevailed. The complex interplay of these pathogens during co-infection led to a noticeable change in the overall severity of the disease. This underscores the significance of pathogen-pathogen interactions in the realm of plant pathology, opening new insights for understanding and managing tulip bulb rot.

Exploring Synergistic Effects of Levan and Levan-Metabolizing Bacillaceae in Promoting Growth and Enhancing Immunity of Tomato and Wheat.

Boosting plant immunity by priming agents can lower agrochemical dependency in plant production. Levan and levan-derived oligosaccharides (LOS) act as priming agents against biotic stress in several crops. Additionally, beneficial microbes can promote plant growth and protect against fungal diseases. This study assessed possible synergistic effects caused by levan, LOS and five levan- and LOS-metabolizing Bacillaceae (Bacillus and Priestia) strains in tomato and wheat. Leaf and seed defense priming assays were conducted in non-soil (semi-sterile substrate) and soil-based systems, focusing on tomato-Botrytis cinerea and wheat-Magnaporthe oryzae Triticum (MoT) pathosystems. In the non-soil system, seed defense priming with levan, the strains (especially Bacillus velezensis GA1), or their combination significantly promoted tomato growth and protection against B. cinerea. While no growth stimulatory effects were observed for wheat, disease protective effects were also observed in the wheat-MoT pathosystem. When grown in soil and subjected to leaf defense priming, tomato plants co-applied with levan and the bacterial strains showed increased resistance to B. cinerea compared with plants treated with levan or single strains, and these effects were synergistic in some cases. For seed defense priming in soil, more synergistic effects on disease tolerance were observed in a non-fertilized soil as compared to a fertilized soil, suggesting that potential prebiotic effects of levan are more prominent in poor soils. The potential of using combinations of Bacilliaceae and levan in sustainable agriculture is discussed.

Analysis of Methylesterase Gene Family in Fragaria vesca Unveils Novel Insights into the Role of FvMES2 in Methyl Salicylate-Mediated Resistance against Strawberry Gray Mold.

Methylesterases (MESs) hydrolyze carboxylic ester and are important for plant metabolism and defense. However, the understanding of MES' role in strawberries against pathogens remains limited. This study identified 15 FvMESs with a conserved catalytic triad from the Fragaria vesca genome. Spatiotemporal expression data demonstrated the upregulated expression of FvMESs in roots and developing fruits, suggesting growth involvement. The FvMES promoter regions harbored numerous stress-related cis-acting elements and transcription factors associated with plant defense mechanisms. Moreover, FvMES2 exhibited a significant response to Botrytis cinerea stress and showed a remarkable correlation with the salicylic acid (SA) signaling pathway. Molecular docking showed an efficient binding potential between FvMES2 and methyl salicylate (MeSA). The role of FvMES2 in MeSA demethylation to produce SA was further confirmed through in vitro and in vivo assays. After MeSA was applied, the transient overexpression of FvMES2 in strawberries enhanced their resistance to B. cinerea compared to wild-type plants.

Novel Sulfoximine Derivatives Containing Cyanoguanidine and Nitroguanidine Moieties: Design, Synthesis, and Bioactivities.

A total of 32 novel sulfoximines bearing cyanoguanidine and nitroguanidine moieties were designed and synthesized by a rational molecule design strategy. The bioactivities of the title compounds were evaluated and the results revealed that some of the target compounds possessed excellent antifungal activities against six agricultural fungi, including Sclerotinia sclerotiorum, Fusarium graminearum, Phytophthora capsici, Botrytis cinerea, Rhizoctonia solani, and Pyricularia grisea. Among them, compounds 8e1 and 8e4 exhibited significant efficacy against P. grisea with EC50 values of 2.72 and 2.98 µg/mL, respectively, which were much higher than that of commercial fungicides boscalid (47.95 µg/mL). Interestingly, in vivo assays determined compound 8e1 possessed outstanding activity against S. sclerotiorum with protective and curative effectiveness of 98 and 95.6% at 50 µg/mL, which were comparable to those of boscalid (93.2, 91.9%). The further preliminary mechanism investigation disclosed that compound 8e1 could damage the structure of the cell membrane of S. sclerotiorum, increase its permeability, and suppress its growth. Overall, the findings enhanced that these novel sulfoximine derivatives could be potential lead compounds for the development of new fungicides.

Kiwifruit resistance to gray mold is enhanced by yeast-induced modulation of the endophytic microbiome.

The influence of endophytic microbial community on plant growth and disease resistance is of considerable importance. Prior research indicates that pre-treatment of kiwifruit with the biocontrol yeast Debaryomyces hansenii suppresses gray mold disease induced by Botrytis cinerea. However, the specific underlying mechanisms remain unclear. In this study, Metagenomic sequencing was utilized to analyze the composition of the endophytic microbiome of kiwifruit under three distinct conditions: the healthy state, kiwifruit inoculated with B. cinerea, and kiwifruit treated with D. hansenii prior to inoculation with B. cinerea. Results revealed a dominance of Proteobacteria in all treatment groups, accompanied by a notable increase in the relative abundance of Actinobacteria and Firmicutes. Ascomycota emerged as the major dominant group within the fungal community. Treatment with D. hansenii induced significant alterations in microbial community diversity, specifically enhancing the relative abundance of yeast and exerting an inhibitory effect on B. cinerea. The introduction of D. hansenii also enriched genes associated with energy metabolism and signal transduction, positively influencing the overall structure and function of the microbial community. Our findings highlight the potential of D. hansenii to modulate microbial dynamics, inhibit pathogenic organisms, and positively influence functional attributes of the microbial community.

Enhancing Botrytis disease management in tomato plants: insights from a Pseudomonas putida strain with biocontrol activity.

AIMS: This study explores the biocontrol potential of Pseudomonas putida Z13 against Botrytis cinerea in tomato plants, addressing challenges posed by the pathogen's fungicide resistance. The aims of the study were to investigate the in vitro and in silico biocontrol traits of Z13, identify its plant-colonizing efficacy, evaluate the efficacy of different application strategies against B. cinerea in planta, and assess the capacity of Z13 to trigger induced systemic resistance (ISR) in plants. METHODS AND RESULTS: The in vitro experiments revealed that Z13 inhibits the growth of B. cinerea, produces siderophores, and exhibits swimming and swarming activity. Additionally, the Z13 genome harbors genes that encode compounds triggering ISR, such as pyoverdine and pyrroloquinoline quinone. The in planta experiments demonstrated Z13's efficacy in effectively colonizing the rhizosphere and leaves of tomato plants. Therefore, three application strategies of Z13 were evaluated against B. cinerea: root drenching, foliar spray, and the combination of root drenching and foliar spray. It was demonstrated that the most effective treatment of Z13 against B. cinerea was the combination of root drenching and foliar spray. Transcriptomic analysis showed that Z13 upregulates the expression of the plant defense-related genes PR1 and PIN2 upon B. cinerea inoculation. CONCLUSION: The results of the study demonstrated that Z13 possesses significant biocontrol traits, such as the production of siderophores, resulting in significant plant protection against B. cinerea when applied as a single treatment to the rhizosphere or in combination with leaf spraying. Additionally, it was shown that Z13 root colonization primes plant defenses against the pathogen.

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.

Mno-miR164a and MnNAC100 regulate the resistance of mulberry to Botrytis cinerea.

Although microRNAs (miRNAs) regulate the defense response of a variety of plant species against a variety of pathogenic fungi, the involvement of miRNAs in mulberry's defense against Botrytis cinerea has not yet been documented. In this study, we identified responsive B. cinerea miRNA mno-miR164a in mulberry trees. After infection with B. cinerea, the expression of mno-miR164a was reduced, which was fully correlated with the upregulation of its target gene, MnNAC100, responsible for encoding a transcription factor. By using transient infiltration/VIGS mulberry that overexpressed mno-miR164a or knocked-down MnNAC100, our study revealed a substantial enhancement in mulberry's resistance to B. cinerea when mno-miR164a was overexpressed or MnNAC100 expression was suppressed. This enhancement was accompanied by increased catalase (CAT) activity and reduced malondialdehyde (MDA) content. In addition, mno-miR164a-mediated inhibition of MnNAC100 enhanced the expression of a cluster of defense-related genes in transgenic plants upon exposure to B. cinerea. Meanwhile, MnNAC100 acts as a transcriptional repressor, directly suppressing the expression of MnPDF1.2. Our study indicated that the mno-miR164a-MnNAC100 regulatory module manipulates the defense response of mulberry to B. cinerea infection. This discovery has great potential in breeding of resistant varieties and disease control.

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.

Binding Mode and Molecular Mechanism of the Two-Component Histidine Kinase Bos1 of Botrytis cinerea to Fludioxonil and Iprodione.

Gray mold caused by Botrytis cinerea is among the 10 most serious fungal diseases worldwide. Fludioxonil is widely used to prevent and control gray mold due to its low toxicity and high efficiency; however, resistance caused by long-term use has become increasingly prominent. Therefore, exploring the resistance mechanism of fungicides provides a theoretical basis for delaying the occurrence of diseases and controlling gray mold. In this study, fludioxonil-resistant strains were obtained through indoor drug domestication, and the mutation sites were determined by sequencing. Strains obtained by site-directed mutagenesis were subjected to biological analysis, and the binding modes of fludioxonil and iprodione to Botrytis cinerea Bos1 BcBos1 were predicted by molecular docking. The results showed that F127S, I365S/N, F127S + I365N, and I376M mutations on the Bos1 protein led to a decrease in the binding energy between the drug and BcBos1. The A1259T mutation did not lead to a decrease in the binding energy, which was not the cause of drug resistance. The biological fitness of the fludioxonil- and point mutation-resistant strains decreased, and their growth rate, sporulation rate, and pathogenicity decreased significantly. The glycerol content of the sensitive strains was significantly lower than that of the resistant strains and increased significantly after treatment with 0.1 µg/ml of fludioxonil, whereas that of the resistant strains decreased. The osmotic sensitivity of the resistant strains was significantly lower than that of the sensitive strains. Positive cross-resistance was observed between fludioxonil and iprodione. These results will help to understand the resistance mechanism of fludioxonil in Botrytis cinerea more deeply.

Carboxylesterase and Cytochrome P450 Confer Metabolic Resistance Simultaneously to Azoxystrobin and Some Other Fungicides in Botrytis cinerea.

Plant pathogens have frequently shown multidrug resistance (MDR) in the field, often linked to efflux and sometimes metabolism of fungicides. To investigate the potential role of metabolic resistance in B. cinerea strains showing MDR, the azoxystrobin-sensitive strain B05.10 and -resistant strain Bc242 were treated with azoxystrobin. The degradation half-life of azoxystrobin in Bc242 (9.63 days) was shorter than that in B05.10 (28.88 days). Azoxystrobin acid, identified as a metabolite, exhibited significantly lower inhibition rates on colony and conidia (9.34 and 11.98%, respectively) than azoxystrobin. Bc242 exhibited higher expression levels of 34 cytochrome P450s (P450s) and 11 carboxylesterase genes (CarEs) compared to B05.10 according to RNA-seq analysis. The expression of P450 genes Bcin_02g01260 and Bcin_12g06380, along with the CarEs Bcin_12g06360 in Saccharomyces cerevisiae, resulted in reduced sensitivity to various fungicides, including azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin, iprodione, and carbendazim. Thus, the mechanism of B. cinerea MDR is linked to metabolism mediated by the CarE and P450 genes.

Antifungal efficiency and mechanisms of ethyl ferulate against postharvest pathogens.

Postharvest loss caused by a range of pathogens necessitates exploring novel antifungal compounds that are safe and efficient in managing the pathogens. This study evaluated the antifungal activity of ethyl ferulate (EF) and explored its mechanisms of action against Alternaria alternata, Aspergillus niger, Botrytis cinerea, Penicillium expansum, Penicillium digitatum, Geotrichum candidum and evaluated its potential to inhibit postharvest decay. The results demonstrated that EF exerts potent antifungal activity against a wide board of postharvest pathogens. Results also revealed that its antifungal mechanism is multifaceted: EF may be involved in binding to and disturbing the integrity of the fungal plasma membrane, causing leakage of intracellular content and losing normal morphology and ultrastructure. EF also induced oxidative stress in the pathogen, causing membrane lipid peroxidation and malondialdehyde accumulation. EF inhibited the critical gene expression of the pathogen, affecting its metabolic regulation, antioxidant metabolism, and cell wall degrading enzymes. EF exhibited antifungal inhibitory activity when applied directly into peel wounds or after incorporation with chitosan coating. Due to its wide board and efficient antifungal activity, EF has the potential to provide a promising alternative to manage postharvest decay.

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.

Industrial Distillation Fractions of Garlic Essential Oil, Design, Synthesis, and Antifungal Activity Evaluation of Aliphatic Substituted Trisulfide Derivatives.

Garlic oil has a wide range of biological activities, and its broad-spectrum activity against phytopathogenic fungi still has the potential to be explored. In this study, enzymatic treatment of garlic resulted in an increase of approximately 50 % in the yield of essential oil, a feasible GC-MS analytical program for garlic oil was provided. Vacuum fractionation of the volatile oil and determination of its inhibitory activity against 10 fungi demonstrated that garlic oil has good antifungal activity. The antifungal activity levels were ranked as diallyl trisulfide (S-3)>diallyl disulfide (S-2)>diallyl monosulfide (S-1), with an EC50 value of S-3 against Botrytis cinerea reached 8.16 mg/L. Following the structural modification of compound S-3, a series of derivatives, including compounds S-4~7, were synthesized and screened for their antifungal activity. The findings unequivocally demonstrated that the compound dimethyl trisulfide (S-4) exhibited exceptional antifungal activity. The EC50 of S-4 against Sclerotinia sclerotiorum reached 6.83 mg/L. SEM, In vivo experiments, and changes in mycelial nucleic acids, soluble proteins and soluble sugar leakage further confirmed its antifungal activity. The study indicated that the trisulfide bond structure was the key to good antifungal activity, which can be developed into a new type of green plant-derived fungicide for plant protection.

Antifungal Activity of Novel Indole Derivatives Containing 1,3,4-Thiadiazole.

In this study, 24 indole derivatives containing 1,3,4-thiadiazole were discovered and synthesized. The target compounds' antifungal efficacy against 14 plant pathogenic fungal pathogens was then determined in vitro. With an EC50 value of 2.7 µg/mL, Z2 demonstrated the highest level of bioactivity among them against Botrytis cinerea (B.c.), exceeding the concentrations of the control prescription drugs azoxystrobin (Az) (EC50 = 14.5 µg/mL) and fluopyram (Fl) (EC50 = 10.1 µg/mL). Z2 underwent in vivo testing on blueberry leaves in order to evaluate its usefulness in real-world settings. A reasonable protective effect was obtained with a control effectiveness of 93.0% at 200 µg/mL, which was superior to those of Az (83.0%) and Fl (52.0%). At 200 µg/mL, this chemical had an efficacy of 84.0% in terms of curative efficacy. These figures outperformed those of Az (69.0%) and Fl (48.0%). Scanning electron microscopy (SEM) experiments and light microscopy experiments showed that Z2 altered the integrity of the cell wall and cell membrane of the pathogenic fungus B.c., which led to an increase in the content of malondialdehyde (MDA), cellular leakage, and cellular permeability. Enzyme activity assays and molecular docking studies indicated that Z2 could act as a potential succinate dehydrogenase inhibitor (SDHI). It was hypothesized that Z2 could cause disruption of mycelial cell membranes, which in turn leads to mycelial death. According to the research, indole derivatives containing 1,3,4-thiadiazole were expected to evolve into new fungicides due to their significant antifungal effects on plant fungi.

Microstructural and physicochemical quality maintenance in green bell pepper infected with Botrytis cinerea and treated with thyme essential oil combined with carnauba wax.

Bell pepper presents rapid weight loss and is highly susceptible to gray mold caused by the fungus Botrytis cinerea. The most employed method to control this disease is the application of synthetic fungicides such as thiabendazole (TBZ); however, its continued use causes resistance in fungi as well as environmental problems. For these reasons, natural alternatives arise as a more striking option. Currently, bell pepper fruits are coated with carnauba wax (CW) to prevent weight loss and improve appearance. Moreover, CW can be used as a carrier to incorporate essential oils, and previous studies have shown that thyme essential oil (TEO) is highly effective against B. cinerea. Therefore, this study aimed to evaluate the effect of CW combined with TEO on the development of gray mold and maintenance of microestructural and postharvest quality in bell pepper stored at 13°C. The minimal inhibitory concentration of TEO was 0.5%. TEO and TBZ provoked the leakage of intracellular components. TEO and CW + TEO treatments were equally effective to inhibit the development of gray mold. On the quality parameters, firmness and weight loss were ameliorated with CW and CW + TEO treatments; whereas lightness increased in these treatments. The structural analysis showed that CW + TEO treatment maintained the cell structure reducing the apparition of deformities. The results suggest that CW + TEO treatment could be used as a natural and effective antifungal retarding the appearance of gray mold and maintaining the postharvest quality of bell pepper. PRACTICAL APPLICATION: CW and TEO are classified as generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA). This combination can be employed on the bell pepper packaging system to extend shelf life and oppose gray mold developments. Bell pepper fruits are normally coated with lipid-base coatings such as CW before commercialization; therefore, TEO addition would represent a small investment without any changes on the packaging system infrastructure.

Synthesis, antimicrobial activity, antioxidant activity and molecular docking of novel chitosan derivatives containing glycine Schiff bases as potential succinate dehydrogenase inhibitors.

Succinate dehydrogenase (SDH) is an important inner mitochondrial membrane-bound enzyme involved in redox reactions during the tricarboxylic acid cycle. Therefore, a series of novel chitosan derivatives were designed and synthesized as potential microbicides targeting SDH and precisely characterized by FTIR, 1H NMR and SEM. Their antifungal and antibacterial activities were evaluated against Botrytis cinerea, Fusarium graminearum, Staphylococcus aureus and Escherichia coli. The bioassays revealed that these chitosan derivatives exerted significant antifungal effects, with four of the compounds achieving 100 % inhibition of Fusarium graminearum merely at a concentration of 0.5 mg/mL. Additionally, CSGDCH showed 79.34 % inhibition of Botrytis cinerea at a concentration of 0.1 mg/mL. In vitro antibacterial tests revealed that CSGDCH and CSGDBH have excellent Staphylococcus aureus and Escherichia coli inhibition with MICs of 0.0156 mg/mL and 0.03125 mg/mL, respectively. Molecular docking studies have been carried out to explore the binding energy and binding mode of chitosan and chitosan derivatives with SDH. The analyses indicated that chitosan derivatives targeted the active site of the SDH protein more precisely, disrupting its normal function and ultimately repressing the growth of microbial cells. Furthermore, the chitosan derivatives were also evaluated biologically for antioxidation, and all of these compounds had a greater degree of reducing power, superoxide radical, hydroxyl radical and DPPH-radical scavenging activity than chitosan. This research has the potential for the development of agricultural antimicrobial agents.

Design, selective synthesis and biological activities evaluation of novel thiazol-2-ylbenzamide and thiazole-2-ylbenzimidoyl chloride derivatives.

To promote the development and exploitation of novel antifungal agents, a series of thiazol-2-ylbenzamide derivatives (3A-3V) and thiazole-2-ylbenzimidoyl chloride derivatives (4A-4V) were designed and selective synthesis. The bioassay results showed that most of the target compounds exhibited excellent in vitro antifungal activities against five plant pathogenic fungi (Valsa mali, Sclerotinia scleotiorum, Botrytis cinerea, Rhizoctonia solani and Trichoderma viride). The antifungal effects of compounds 3B (EC50 = 0.72 mg/L) and 4B (EC50 = 0.65 mg/L) against S. scleotiorum were comparable to succinate dehydrogenase inhibitors (SDHIs) thifluzamide (EC50 = 1.08 mg/L) and boscalid (EC50 = 0.78 mg/L). Especially, compounds 3B (EC50 = 0.87 mg/L) and 4B (EC50 = 1.08 mg/L) showed higher activity against R. solani than boscalid (EC50 = 2.25 mg/L). In vivo experiments in rice leaves revealed that compounds 3B (86.8 %) and 4B (85.3 %) exhibited excellent protective activities against R. solani comparable to thifluzamide (88.5 %). Scanning electron microscopy (SEM) results exhibited that compounds 3B and 4B dramatically disrupted the typical structure and morphology of R. solani mycelium. Molecular docking demonstrated that compounds 3B and 4B had significant interactions with succinate dehydrogenase (SDH). Meanwhile, SDH inhibition assay results further proved their potential as SDHIs. In addition, acute oral toxicity tests on A. mellifera L. showed only low toxicity for compounds 3B and 4B to A. mellifera L. populations. These results suggested that these two series of compounds had merit for further investigation as potential low-risk agricultural SDHI fungicides.

Cytokinin oxidase gene CKX5 is modulated in the immunity of Arabidopsis to Botrytis cinerea.

In our previous work, cytokinin (CK) signaling and biosynthesis were found to be modulated during Arabidopsis defense against infection by the necrotrophic pathogen Botrytis cinerea. Notably, the expression level of CYTOKININ OXIDASE/DEHYDROGENASE 5 (CKX5) was significantly induced in B. cinerea-infected leaves and later in distant B. cinerea-untreated leaves of the same plant. To confirm and determine how CKX5 is involved in the response to B. cinerea infection, transcript levels of CKX family genes were analyzed in B. cinerea-inoculated leaves, and only CKX5 was remarkably induced by B. cinerea infection. Furthermore, CKX5-overexpressing Arabidopsis plants were more resistant to B. cinerea than wild-type plants. Transcription factors (TFs) binding to the CKX5 promoter were then screened by yeast one-hybrid assays. Quantitative Real-Time Reverse Transcription PCR (qRT-PCR) analysis further showed that genes encoding TFs, including WRKY40, WRKY33, ERF6, AHL15, AHL17, ANAC003, TCP13 and ANAC019, were also strongly induced in infected leaves, similar to CKX5. Analysis of ERF6-overexpressing plants and ERF6-and AHL15-knockout mutants indicated that ERF6 and AHL15 are involved in plant immunity to B. cinerea. Furthermore, CKX5 upregulation by B. cinerea infection was affected when ERF6 or AHL15 levels were altered. Our work suggests that CKX5 levels are controlled by the plant defense system to defend against attack by the pathogen B. cinerea.

Antifungal Activity and Putative Mechanism of HWY-289, a Semisynthetic Protoberberine Derivative, against Botrytis cinerea.

The emergence of resistant pathogens has increased the demand for alternative fungicides. The use of natural products as chemical scaffolds is a potential method for developing fungicides. HWY-289, a semisynthetic protoberberine derivative, demonstrated broad-spectrum and potent activities against phytopathogenic fungi, particularly Botrytis cinerea (with EC50 values of 1.34 µg/mL). SEM and TEM imaging indicated that HWY-289 altered the morphology of the mycelium and the internal structure of cells. Transcriptomics revealed that it could break down cellular walls through amino acid sugar and nucleotide sugar metabolism. In addition, it substantially decreased chitinase activity and chitin synthase gene (BcCHSV) expression by 53.03 and 82.18% at 1.5 µg/mL, respectively. Moreover, this impacted the permeability and integrity of cell membranes. Finally, HWY-289 also hindered energy metabolism, resulting in a significant reduction of ATP content, ATPase activities, and key enzyme activities in the TCA cycle. Therefore, HWY-289 may be a potential candidate for the development of plant fungicides.