Delu Wang

In recent years, diseases of tea plants ( Camellia sinensis) caused by Lasiodiplodia theobromae have significantly impacted the quality and yield of tea leaves in China and India. However, the pathogenic mechanisms of L. theobromae in tea plants remain unclear. In this study, the relative expression levels of ethylene receptor 2 ( ETR2, historically referred to as ethylene response 2) mRNA was analyzed at different hours postinoculation at the first, second, and third leaves on the tea shoot inoculated with L. theobromae hyphae. The results revealed spatiotemporal differences in CsETR2 expression, suggesting its involvement in disease resistance. Subcellular localization analysis indicated that CsETR2 was localized in the endoplasmic reticulum of leaf cells in Nicotiana benthamiana. Through transient overexpression, antisense oligonucleotide (AsODN) silencing in tea leaves, and transgenic N. benthamiana assays, it was confirmed that CsETR2 positively regulated resistance in N. benthamiana. 3,3′-Diaminobenzidine/nitroblue tetrazolium staining and quantitative analysis of reactive oxygen species (ROS) scavenger genes demonstrated that CsETR2 mediated disease resistance through an ROS-mediated mechanism. As a key component in ethylene (ET) signal transduction, the expression of CsETR2 was positively correlated with genes in the ET transduction pathways under overexpression or silencing conditions. β-Glucuronidase and dual-luciferase assays revealed that PC-5p-1042181_5 inhibited CsETR2 mRNA expression by targeting its mRNA, thereby reducing disease resistance. Further experiments with transient overexpression, AsODN silencing, and transgenic N. benthamiana validated the negative regulatory role of PC-5p-1042181_5. This study uncovered a novel disease resistance mechanism involving ROS–ET interference mediated by the PC-5p-1042181_5- CsETR2 module, offering new insights for breeding disease-resistant tea cultivars.

Osthole exhibits strong inhibitory activity against phytopathogenic fungi; however, its antifungal mechanism remains unclear. This study assessed osthole's inhibitory effects on several phytopathogenic fungi, revealing a half-maximal effective concentration of 73.03 μg/ml against the hyphal growth of Botrytis cinerea. Micromorphological analysis showed that osthole caused abnormalities in the hyphae, including unclear organelle boundaries and organelle dissolution. Integrated transcriptomic and metabolomic assays and correlation analysis indicated that osthole induced differentially expressed genes and differentially abundant metabolites, which were enriched particularly in the pathways of glyoxylate and dicarboxylate metabolism, tyrosine metabolism, glycerophospholipid metabolism, fructose and mannose metabolism, citrate cycle, biosynthesis of unsaturated fatty acids, and ABC transporters. Molecular docking and molecular dynamics simulation assays demonstrated that osthole binds stably to amidase, a key enzyme in energy metabolism, with a relatively lower binding energy of −8.5 kcal/mol compared with osthole's analogs, suggesting that amidase may be a potential target protein in the fungus. A microscale thermophoresis assay indicated that the dissociation constant (Kd) value for osthole binding to amidase was significantly lower compared with that of osthole's analog 7-methoxycoumarin. Overall, this study demonstrates that osthole disrupts energy metabolism, nitrogen metabolism, substance transport, and the metabolism of the hyphal cell wall and cell membrane, potentially targeting the amidase of B. cinerea. These findings highlight the potential of osthole for controlling gray mold.

Tea disease caused by Lasiodiplodia theobromae is an emerging fungal disease that significantly reduces the yield and quality of tea in tea-producing regions owing to the lack of effective control methods. In this study, we evaluated the antifungal activity of azithromycin, a macrolide antibiotic, against L. theobromae. In vitro assays demonstrated strong inhibitory activity, with a half-maximal effective concentration (EC ₅₀ ) of 140.61 μg/ml, and in vivo application resulted in a 54.30% lesion inhibition rate at 800 μg/ml against tea leaf spot. Morphological and ultrastructural analyses using optical and transmission electron microscopy revealed that azithromycin induced pronounced hyphal abnormalities, including cytoplasmic disorganization and membrane disruption. Integrated transcriptomic, proteomic, and metabolomic analyses indicated that azithromycin treatment led to systemic disruptions in L. theobromae, including dysregulation of genes involved in carbohydrate metabolism, protein degradation, glycoprotein and cell wall biosynthesis, ergosterol biosynthesis, mitochondrial function, and stress response pathways. Molecular docking and bioinformatic analyses identified glycoside hydrolase family 35 (GH35), a key protein in carbohydrate metabolism, as the principal target of azithromycin, exhibiting the most favorable binding energy of −7.9 kcal/mol. These findings demonstrate that azithromycin disrupts multiple metabolic and cellular processes in L. theobromae, primarily by targeting GH35, providing a robust multitarget approach for fungal inhibition and sustainable disease control strategies for tea cultivation.

Spinetoram, a widely used insecticide, exhibits previously unexplored antifungal properties. This study provides the first evidence of its broad-spectrum antifungal activity against phytopathogens from tea plants, vegetables, and fruit trees, with efficacy against gray mold, with a half-maximal effective concentration (EC₅₀) value of 40.41 μg/ml. In vivo trials showed a 60.2% reduction in disease on tomato fruits at a spinetoram concentration of 1,600 μg/ml and a 70.7% reduction on tomato leaves at 800 μg/ml. Morphological analyses involving light microscopy, scanning electron microscopy, and transmission electron microscopy revealed concentration-dependent cellular alterations in Botrytis cinerea hyphae following spinetoram exposure (EC₁₀, EC₅₀, and EC₉₀), including swelling, surface distortion, septal disruption at the exposures of 24 or 36 h, and organelle dissolution at elevated concentrations from EC₅₀ to EC₉₀. Transcriptomic profiling identified ribosomal subunit assembly (40S/60S) and ribosome biogenesis as key enriched pathways, suggesting protein synthesis inhibition as the antifungal mechanism. Molecular docking predicted varying binding affinities, ranging from weak to strong, between spinetoram and 41 ribosomal proteins. Notably, 60S ribosomal protein L27 (RPL27) exhibited a lower binding free energy of −11.8 kcal/mol, with a stable binding process confirmed through molecular dynamics simulations. Our findings establish three novel insights: spinetoram's fungicidal activity against multiple plant pathogens, its disruption of fungal cellular integrity through ribosomal targeting, and RPL27 as a potential binding site for protein synthesis inhibition. This dual pesticidal-fungicidal activity positions spinetoram as a promising lead compound for integrated pest/disease management strategies and fungicide development in the future.

Tea foliar disease, caused by the fungal pathogen Pestalotiopsis trachicarpicola, poses a significant threat to tea production and quality in China. The disease, which includes tea leaf spot and gray blight, severely impacts both tea yield and quality, partly due to the lack of effective epidemiological data and control strategies. Fluoxastrobin, a strobilurin fungicide, has shown promise in controlling various fungal diseases. This study evaluated the inhibitory activity of fluoxastrobin against multiple phytopathogenic fungi, revealing a half-maximal effective concentration of 13.64 μg/ml for P. trachicarpicola in vitro and a maximum in vivo curative activity of 81.63% against tea leaf spot caused by P. trachicarpicola. Observations from optical, scanning electron, and transmission electron microscopy revealed that fluoxastrobin induces severe hyphal deformation, including hyphal swelling, malformation, and rough hyphal surfaces. Integrated transcriptomic, metabolomic, and bioinformatic analyses indicated that fluoxastrobin affects the electron transfer process, therefore disturbing the energy metabolism of hyphae. Biochemical assays indicated that fluoxastrobin can inhibit ATP production in hyphae and increase the membrane potential of hyphae, with these effects being dose-dependent. Molecular docking and molecular dynamics simulations demonstrated that fluoxastrobin binds stably to NAD-dependent epimerase/dehydratase, a key enzyme in energy metabolism, with binding energies of −12.9 kcal/mol, suggesting that these may be potential target proteins in the fungus. Overall, this study demonstrates that fluoxastrobin disrupts electron transfer, reduces ATP production, and inhibits hyphal growth, likely by binding NAD-dependent epimerase/dehydratase. It shows potential for controlling tea foliar diseases in the future.

Allicin exhibits strong inhibitory activity against phytopathogenic fungi; however, its antifungal mechanism remains unclear. This study assessed allicin's inhibitory effects on several phytopathogenic fungi, revealing a half-maximal effective concentration of 125.47 μg/ml against the hyphal growth of Botrytis cinerea. Micromorphological analysis showed that allicin caused abnormalities in the hyphae, including unclear organelle boundaries and organelle dissolution. Integrated transcriptomic, proteomic, and metabolomic assays indicated that allicin induced differential gene and protein expression, particularly in the plasma membrane, oxidative stress processes, and energy metabolism pathways. Additionally, differentially expressed metabolites were involved in the inhibition of hyphal growth. Biochemical assays demonstrated that allicin inhibited ATP production and damaged hyphal cell membranes. Molecular docking revealed that allicin could bind to dihydrolipoamide dehydrogenase, an enzyme rich in cysteine residues, with a binding free energy of −6.322 kcal/mol. Through antimicrobial activity testing of allicin analogs and molecular docking analysis, the active groups of allicin and its interaction with dihydrolipoamide dehydrogenase were identified. This study shows that allicin interferes with energy metabolism, impacts cell membrane and wall integrity by targeting cysteine-containing proteins, and inhibits the proliferation of plant-pathogenic fungi. These insights into the antifungal mechanism will provide valuable data for the development and field application of allicin analogs.

Gray mold is an important disease of crops and is widespread, harmful, difficult to control, and prone to developing fungicide resistance. Screening new fungicides is an important step in controlling this disease. Hydroxychloroquine is an anti-inflammatory and antimalarial agent that has shown marked inhibitory activity against many fungi in medicine. This study evaluated the inhibitory activity of hydroxychloroquine against several phytopathogenic fungi, finding a half-maximal effective concentration of 113.82 μg/ml against the hyphal growth of Botrytis cinerea, with significant in vivo curative effects of 92.37 or 78.37% for gray mold on detached tomato leaves or fruits at 10.0 or 200.0 mg/ml, respectively. Ultrastructural studies indicated that hydroxychloroquine induced collapse of hyphae, with a wrinkled surface, unclear organelle boundaries, and organelle disintegration. Transcriptomic assays revealed that hydroxychloroquine could affect the expression of metabolism-related genes. Molecular docking and molecular dynamics analyses indicated that hydroxychloroquine bound to glucose-methanol-choline oxidoreductase, with a low free energy value of −11.4 kcal/mol. Cell membrane permeability assays and hyphal staining confirmed that hydroxychloroquine damaged the cell membrane, causing leakage of hyphal contents and disturbing cell function. Biochemical assays indicated that hydroxychloroquine reduced the concentration of soluble proteins and reducing sugars in the hyphae. Overall, hydroxychloroquine disturbed amino acid metabolism, therefore inhibiting the production of biomacromolecules, damaging the cell membrane, and restraining the growth of hyphae, hence inhibiting gray mold on tomato. This study explored the use of medicine in the development of agricultural fungicides and their application in managing crop diseases, providing valuable background information.

Due to a lack of understanding of the disease epidemiology and comprehensive control measures, tea leaf spot caused by Didymella segeticola has a significant negative impact on tea yield and quality in the tea plantations of Southwest China. Phenazine-1-carboxamide (PCN) is a phenazine compound derived from Pseudomonas species that exhibits antimicrobial activity against various pathogens. However, its inhibitory mechanism is not yet clear. The current study evaluated the inhibitory activity of PCN against various phytopathogenic fungi and found that PCN has inhibitory activity against multiple pathogens, with a half-maximal effective concentration value for D. segeticola of 16.11 μg/ml in vitro and a maximum in-vivo curative activity of 72.28% toward tea leaf spot. Morphological changes in the hyphae after exposure to PCN were observed through microstructure and ultrastructure analysis and indicated that PCN causes abnormalities in the hyphae, such as cytoplasmic coagulation, shortened hyphal inter-septum distances, and unclear boundaries of organelles. Transcriptomic analysis revealed that PCN upregulated the expression of genes related to energy metabolism. PCN significantly reduced the ATP concentration in the hyphae and decreased mitochondrial membrane potential. Molecular docking analysis indicated that PCN binds to one of the candidate target proteins, pyruvate dehydrogenase, with lower free energy of −10.7 kcal/mol. This study indicated that PCN can interfere with energy metabolism, reducing ATP generation and ultimately affecting hyphal growth. Overall, PCN shows potential for future application in the control of tea leaf spot due to its excellent antifungal activity and unique mode of action.

Tea leaf spot caused by Didymella segeticola is a disease that has recently been discovered in the tea plantations of Southwest China and that has a significant negative impact on the yield and quality of tea leaves. Wuyiencin is a nucleotide antimicrobial that is effective against a range of fungal diseases. However, its mode of action is still unclear. The current study found that wuyiencin inhibited the mycelial growth of D. segeticola in vitro. Meanwhile, in vivo experiments confirmed that wuyiencin had a significant curative effect on tea leaf spot. Microscopic observation indicated that it damaged the organelles and nucleus in fungal cells. Reverse transcription quantitative PCR assays revealed that mycelium treated with wuyiencin at the half-maximal effective concentration (EC₅₀) dosage for 1 h exhibited 3.23 times lower expression of threonine dehydratase ( td) gene, which is responsible for producing pyruvate. The wild-type (WT) strain had a 1.77-fold higher pyruvate concentration than that in the td mutant ( P < 0.05). The td mutant was more sensitive than the WT to wuyiencin treatment, with the EC₅₀ value in the td mutant being 30.01 μg/ml compared with 82.34 μg/ml in the WT. Molecular docking demonstrated that wuyiencin bound to td, with a binding energy of −10.47 kcal/mol. Compared with the WT strain, wuyiencin significantly reduced ATP concentration of the td mutant strain at dosages of 80.0 and 160.0 µg/ml. In total, wuyiencin reduced td activity, inhibited pyruvate production, and decreased ATP content in the phytopathogenic fungus, ultimately disturbing the growth of the mycelium.

Tea leaf spot caused by Lasiodiplodia theobromae is a newly discovered fungal disease in southwest China. Due to a lack of knowledge of its epidemiology and control strategies, the disease has a marked impact on tea yield and quality. Pyriofenone is a new fungicide belonging to the aryl phenyl ketone fungicide group, which has shown marked efficacy in controlling various fungal diseases. However, its mechanism of action is not yet understood. This study found that pyriofenone exhibits strong in vitro inhibitory activity against various phytopathogenic fungi. Specifically, it showed strong inhibitory activity against L. theobromae, with a half-maximal effective concentration (EC₅₀) value of 0.428 μg/ml determined by measuring the mycelial growth rate. Morphological observations, using optical, scanning electron, and transmission electron microscopy, revealed that pyriofenone induces morphological abnormalities in L. theobromae hyphae. At lower doses, the hyphae became swollen, the distance between septa decreased, and the hyphal growth rate slowed. At higher doses and longer exposures, the hyphae collapsed. Transcriptomic and bioinformatic analyses indicated that pyriofenone can affect the expression of genes related to membrane transporters. Homology modeling suggested that pyriofenone may bind to a candidate target protein of the major facilitator superfamily transporter, with a free binding energy of −7.1 kcal/mol. This study suggests that pyriofenone may potentially regulate the transport of metabolites in L. theobromae, thus affecting hyphal metabolism and interfering with hyphal growth. Pyriofenone exhibits in vitro inhibitory activity against various tea foliar pathogens and holds promise for future applications to the control of tea foliar diseases.