Rosa roxburghii Tratt. is an economically important fruit crop whose production is severely constrained by top rot disease caused by Colletotrichum fructicola. Although protein elicitors such as Harpin Ea represent a promising sustainable alternative to chemical fungicides, their mode-of-action in this crop is largely unknown. Foliar application of 7.5 mg L-1 Harpin Ea protein under field conditions significantly reduced top rot incidence, with >75% control efficacy over consecutive growing seasons, and increased fruit yield by >9% without negatively affecting quality, despite negligible direct antifungal activity in vitro (3.39-8.25% inhibition). The elicitor enhanced antioxidant enzyme activities, elevated levels of defense-related compounds and improved photosynthetic performance. Structural analyses show that Harpin Ea protein reinforced the fruit epidermis and cell walls, increasing key structural components (cellulose, lignin, carbohydrates) by 102-200%. Integrated transcriptomic and metabolomic profiling demonstrates that Harpin Ea coordinately upregulated the phenylpropanoid-flavonoid biosynthetic pathway, enhancing the expression of genes encoding key enzymes such as phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL) and chalcone synthase (CHS), and promoting the accumulation of critical defense-related metabolites including p-cinnamic acid, caffeic acid, phenylalanine, naringenin, quercetin and hesperidin. Harpin Ea-induced resistance in R. roxburghii is mediated by a synergistic enhancement of physical barriers and systemic activation of phenylpropanoid-flavonoid metabolism. This work provides the first mechanistic evidence linking this dual-layer defense to Harpin Ea-induced disease resistance and establishes a molecular basis for the sustainable application of protein elicitors in fruit crop protection. © 2026 Society of Chemical Industry.

Oil tea (Camellia oleifera) anthracnose caused by Colletotrichum spp. seriously endangers the oil tea forestry. However, there is a lack of suitable targets for the development of efficient and environmentally friendly fungicides and precise prevention of oil tea anthracnose. In this study, 20 highly expressed Colletotrichum fructicola candidate effector proteins (CfCEPs) were predicted as potential targets. Yeast secretion test showed that these 20 CfCEPs have secretory activity, indicating that they may be potential targets. Transient overexpression assay showed that 20 CfCEPs suppressed plant immunity, but only four of them (CfCEP1, CfCEP2, CfCEP7, and CfCEP8) directly promoted pathogen infection. Gene deletion showed that these four CfCEPs directly regulate pathogenicity, rather than indirectly regulating pathogenicity by regulating growth and reproduction, indicating that they are suitable potential targets. Subcellular localization analysis revealed that these four CfCEPs mainly function in the plasma membrane and cytoplasm. qRT-PCR analysis indicated that these four CfCEPs were upregulated during the infection phase. Finally, multiple sequence alignment, phylogenetic analysis, and conserved motif analysis suggested that these four CfCEPs were conserved in Colletotrichum genus. In summary, this study provided four potential targets for the development of novel fungicides and theoretical guidance for the precise and efficient prevention of oil tea anthracnose. © 2026 Society of Chemical Industry.

Carya illinoinensis (pecan) is an important fruit and woody oil tree species widely cultivated in China, but its production is severely affected by Colletotrichum fructicola. Transcriptome analysis indicated that the differentially expressed genes (DEGs) of pecan were significantly enriched in terms associated with disease resistance (e.g., defense response, protein autophosphorylation, and abscisic acid (ABA)-activated signaling) and pathways (e.g., plant-pathogen interaction, MAPK signaling, flavonoid biosynthesis). A key gene, named CiCP12, was screened and identified, which enhanced the resistance of Nicotiana benthamiana to C. fructicola infection by upregulating defense-related genes and having a higher basal antioxidant level. Conversely, silencing CiCP12 in pecan reduced resistance to C. fructicola infection by suppressing the expression of multiple defense-related genes. CiCP12 could also enhance broad-spectrum disease resistance against various pathogens in N. benthamiana. This study identified a key disease-resistance-related gene and provided insights into pecan defense mechanisms against C. fructicola.

MdWRKY61 enhances jasmonic acid signal transduction by inhibiting the expression of MdJAZ1, a negative regulator in the jasmonic acid signaling pathway, thereby increasing apple resistance to Colletotrichum fructicola. Glomerella leaf spot (GLS), a fungal disease caused by Colletotrichum species, poses a significant threat to the global apple industry. The WRKY transcription factor family plays a crucial role in plant defense responses; however, their specific functions and the mechanisms underlying resistance to GLS remain poorly understood. In this study, MdWRKY61 overexpression lines and wild-type (WT) plants were inoculated with Colletotrichum fructicola to assess GLS resistance. At 4 days post-inoculation, overexpression lines exhibited a 45.4% ~ 50.0% lower disease incidence and a 22.0 ~ 29.3 reduction in disease index compared to WT. Physiological assays showed decreased hydrogen peroxide and superoxide anion levels, alongside elevated superoxide dismutase and peroxidase activities in overexpression lines. These findings indicate that overexpression of MdWRKY61 enhances resistance to C. fructicola in apple and alleviates oxidative damage induced by pathogen infection. Hormone analyses revealed that the overexpression of MdWRKY61 enhanced the accumulation of jasmonic acid (JA), and in vitro tests confirmed that JA treatment significantly suppressed C. fructicola mycelial growth. Molecular biological experiments revealed that MdWRKY61 directly binds to the W-box element (TTGACT) in the promoter region of MdJAZ1, a negative regulator in the JA signaling pathway, thereby inhibiting its transcriptional expression. Collectively, these findings suggest that MdWRKY61 enhances JA signal transduction by downregulating MdJAZ1, thereby increasing apple resistance to GLS. These results highlight MdWRKY61 as a promising candidate for genetic engineering to develop novel disease-resistant apple cultivars.

ClWRKY75 regulates lignin biosynthesis to confer Colletotrichum fructicola resistance in watermelon.

(1) Reverse transcription quantitative real-time PCR (RT-qPCR) requires reliable reference genes for accurate data normalization; however, optimal reference genes for the economically and ecologically valuable timber species Phoebe zhennan remain uncharacterized; (2) Here, we selected nine candidate reference genes derived from full-length transcriptome sequencing to evaluate their expression stability across abiotic (drought) and biotic (Colletotrichum fructicola infection) stresses. Transcript abundance was analyzed via RT-qPCR using four distinct algorithms (Delta Ct, geNorm, NormFinder, and BestKeeper), with RefFinder used to reconcile analytical discrepancies and generate a definitive consensus ranking; (3) Our analysis showed that expression stability is highly context-dependent: CYP20-1 and HSP70-1 were the most stable reference genes under drought stress, whereas Actin-101 and Actin constituted the optimal pair under disease stress. For cross-condition assessments, Actin-101 and β-Tubulin served as the most reliable baseline combination. Subsequent empirical validation quantifying stress-responsive transcripts demonstrated a significant positive correlation between RT-qPCR relative expression and corresponding RNA-seq data (drought: R = 0.80; disease: R = 0.76); (4) This study identifies and validates the first set of reference genes for P. zhennan, providing a foundation for accurate gene expression analysis in this species, which is crucial for understanding its response to environmental stresses.

Genome-wide identification of the P-loop NTPase gene family in Pyrus betulifolia and functional characterization of PbePloopNTP21 in resistance to black spot disease.

Peach scab, caused by the fungus Venturia carpophila, is a major threat to peach production world-wide. Despite its economic impact, the molecular mechanisms driving V. carpophila pathogenicity are poorly understood. In this study, we functionally characterized VcAIP1, a novel effector protein secreted by V.carpophila, and elucidated its roles in promoting infection. More specifically, we showed that the heterologous expression of VcAIP1 in peach leaves significantly increases susceptibility to V. carpophila and other pathogenic fungi, namely Colletotrichum fructicola, Lasiodiplodia theobromae, Monilinia fructicola, and Botrytis cinerea. We also showed that VcAIP1 binds to the peach enzyme PpADC, a rate-limiting component in polyamine biosynthesis, and suppresses its activity. This interaction reduced peach hydrogen peroxide accumulation, thereby enhancing fungal virulence. Finally, we showed that overexpression of PpADC enhances peach resistance to V. carpophila and the other tested pathogens, while silencing of this gene increases susceptibility. Importantly, exogenous application of polyamines, metabolites synthesized via PpADC, substantially improved peach resistance to multiple pathogens, offering a sustainable strategy for disease management. Collectively, our findings revealed VcAIP1 as a fungal virulence factor that subverts peach defenses by disrupting polyamine biosynthesis and highlight polyamine-based interventions as promising tools for eco-friendly peach disease control.

Persea americana (avocado) is a fruit rich in nutrients; however, its industry is facing major threats from pathogen infection. Here, we clearly identified Colletotrichum fructicola as the pathogen causing avocado diseases in Pu'er City, Yunnan Province. However, the biological characteristics, genetic transformation system, and early cell cycle regulation of this pathogen remained unclear. In this study, C. fructicola exhibited a maximum growth rate on complete medium (CM), with the conidial yield reaching 2 × 105 conidia/mL after 24 h in liquid CM. Conidia of C. fructicola had nearly fully germinated at 4 h post-inoculation (hpi), with the appressorium formation rate exceeding 95% at 12 hpi. We also established a PEG-CaCl2-mediated genetic transformation system. The GFP-tagged transformants showed no significant differences in core biological function from the wild type. Using eGFP labeling, we visually elucidated the early cell cycle regulation of C. fructicola. Furthermore, cell cycle inhibitor assays demonstrated that C. fructicola conidial germination is independent of nuclear division and relies on cytoskeletal modulation, whereas appressorium formation and mycelial expansion require functional cell cycle regulation. This is probably the first study to systematically elucidate the cell cycle regulatory characteristics of C. fructicola isolated from avocado, and to successfully develop its genetic transformation system. These results provide important theoretical and technical support for the formulation of integrated control strategies against C. fructicola, as well as facilitating the sustainable development of the avocado industry.

Antifungal activity and mechanism of thymol against Colletotrichum fructicola that causes litchi anthracnose.

The aroma quality of grape fruit is a crucial trait for table grapes, yet its relationship with plant disease resistance remains unclear. Using 'Shine Muscat' grapes as material, this study employed HS-SPME-GC-MS combined with odor activity value (OAV) and PLS-DA analysis to investigate the regulatory effects of different rootstocks and GA3/MeJA treatments on volatile aroma compounds. Linalool and α-terpineol were selected as representative compounds for antibacterial experiments and gene expression analysis of terpenoid synthesis. Results indicate that the Lot rootstock and 15.25 mg·L-1 GA3 treatment significantly promoted the accumulation of terpenoid aroma compounds. Linalool exhibited significant inhibitory effects on the mycelial growth of Colletotrichum fructicola and induced upregulation of DXS, TPS56, and TPS gene expression. This study reveals a potential link between aroma metabolism and defense responses, providing a theoretical basis for synergistic optimization of grape aroma quality improvement and disease-resistant cultivation.

Colletotrichum fructicola is the predominant pathogenic agent responsible for anthracnose in Camellia oleifera. RGS2 is a GTPase-activating protein that negatively regulates G-protein signaling by inactivating Gα subunits. In this study, we characterized the ortholog of CfRGS2 in C. fructicola to explore its pathogenic roles. Seven canonical RGS genes were identified through BLASTp and keyword searches. Conserved domains and subcellular localizations were predicted bioinformatically. A CfRGS2 knockout mutant was generated via overlap-PCR and PEG-mediated transformation, verified by PCR, and complemented by reintroducing the wild-type gene. Phenotypic characterization showed that the growth rates of mutants ΔCfrgs2-1 and ΔCfrgs2-2 were significantly reduced compared with those of the wild-type and complemented strains. On both PDA and minimal medium, the mutant strains exhibited significantly smaller colony diameters of 3.3 cm and 3.1 cm, respectively, relative to the control strains. Moreover, conidiation in the mutants was only 4% of that in the wild-type and complemented strains, and appressorium formation was reduced to 6%, with statistical analyses confirming high significance. Under cell wall stress induced by 400 μg/mL Congo red, the growth inhibition rates of ΔCfrgs2-1 and ΔCfrgs2-2 were 44% and 48%, respectively, significantly higher than those of the control strains. Pathogenicity assays demonstrated that the mutants failed to induce lesions on unwounded leaves and caused 47% and 30% smaller lesion areas on wounded apple fruits, respectively. In summary, C. fructicola possesses seven canonical RGS proteins that regulate G-protein signaling, among which CfRgs2 is implicated in growth, conidiation, the stress response to cell wall perturbation, and virulence.

Study on the immune defense response of Epimedium sagittatum induced by Colletotrichum fructicola and its biological control

ABSTRACTPostharvest brown spot (Lasidiplodia theobromae) and crown rot (caused by a pathogen complex) are the most important postharvest issues in Bangladesh. Research information regarding banana postharvest disease control is limited in Bangladesh. Thus, this research aimed to identify the causal agent, and tested certain botanical extracts for their antifungal properties to reduce banana postharvest infections. These two symptoms of local banana provided 37 fungal isolates through standard isolation method. Later, L. theobromae, Colletotrichum fructicola, and Fusarium equiseti were identified morphologically and molecularly. Lasidiplodia theobromae (Lt1) was selected for further disease control studies as this pathogen causes both crown rot and brown spot simultaneously. For the antifungal efficacy tests, several botanical extracts (from Aloe vera, garlic bulb, onion bulb, and moringa leaf t) were chosen based on their local availability and previous research findings (on other fruit). Among the botanicals, Aloe vera + Garlic (0.78 cm), Aloe vera (3.08 cm), and Garlic (0.53 cm) successfully reduced the fungal growth compared with the untreated control (4.78 cm) in vitro. Among these botanicals, Aloe vera (250 mL/L) + Garlic (50 mL/L) successfully reduced both brown spot and crown rot in planta at 11 days after treatment. This botanical combination did not have an adverse effect on the physiochemical properties of banana such as weight, color, firmness, and TSS. This study suggests Aloe vera and garlic as nonchemical green fruit coat to reduce the postharvest fruit diseases and ensure consumer safety.

Prochloraz (Pro) is a broad-spectrum fungicide that is effective against various plant pathogens, including Colletotrichum fructicola, the causal agent of strawberry anthracnose. However, its limited absorption restricts its effectiveness. In this study, zeolitic imidazolate framework-8 (ZIF-8) was utilized as a nanocarrier and combined with the fungicide prochloraz (Pro) to develop an effective delivery system (Pro@ZIF-8), improving the fungicidal activity of prochloraz. Characterization confirmed that Pro@ZIF-8 possesses a distinct dodecahedral crystal structure with an average particle size of 139.6 nm. The system exhibited pH-sensitive release, with 89.8% of the prochloraz released within 24 h at pH 5.0, while remaining stable at pH 7.4. Antifungal assays demonstrated that Pro@ZIF-8 had an EC50 of 0.0150 mg L-1 against C. fructicola, which was significantly lower than that of free prochloraz (0.0429 mg L-1). Pro@ZIF-8 effectively delayed disease lesion development on strawberry leaves and fruits inoculated with C. fructicola. Under storage conditions, Pro@ZIF-8 prevented pathogen infection and kept the fruit fresh for an extended period. Additionally, by tracking fluorescein isothiocyanate (FITC)-labeled ZIF-8 after foliar and root applications, it was confirmed that it was systemically transported within strawberry plants. These findings highlight the potential of Pro@ZIF-8 as a smart fungicide delivery system, offering a promising strategy for improving disease management in strawberries. © 2026 Society of Chemical Industry.

Apples and pears, as important economic fruit crops, are frequently threatened by various diseases, including Valsa canker. Given the numerous advantages of disease resistance breeding, the identification of key resistance genes is particularly important. This study aimed to identify the "Duli-G03" (Pyrus betulifolia) resistance gene PbeZFP3 and clarify its regulatory mechanism in disease resistance via JA/SA pathways, providing a theoretical basis for resistant breeding. In this study, we identified a C2H2-type transcription factor, PbeZFP3, in the Valsa canker-resistant rootstock "Duli-G03". Expression analysis revealed that PbeZFP3 is induced by both Valsa pyri (Vp) and Vp metabolites (VpM). Transient expression in pear and apple fruits and stable expression in suspension cells confirmed that PbeZFP3 positively regulates Valsa canker resistance. Meanwhile, PbeZFP3 not only enhances the resistance of "Duli-G03" cells to Botrytis cinerea infection, but may also act as a negative regulator against Colletotrichum fructicola. The overexpression of PbeZFP3 in "Duli-G03" significantly upregulated the expression of genes related to jasmonic acid (JA) and salicylic acid (SA) signaling. These findings demonstrate that the jasmonic acid (JA) and salicylic acid (SA) signaling pathways are involved in the enhanced Valsa canker resistance conferred by PbeZFP3 overexpression. A deeper understanding of this host resistance mechanism will provide theoretical support for breeding strategies aimed at developing disease-resistant fruit trees.

Scrophularia ningpoensis is a perennial herb widely cultivated in southern China for its medicinal properties, including the treatment of fever, constipation, swelling, rheumatism and inflammation (Guo et al. 2023). In June 2022, leaf anthracnose was observed on S. ningpoensis plants that had been cultivated in the experimental field for three months. The relative humidity was 65% in Jinhua, Zhejiang Province, China (29.12°N, 119.64°E). The affected area covered approximately 200 m2 with a disease incidence of 65%. Symptoms initially manifested as circular or irregular dark brown spots, ranging from 0.25 to 3 mm in diameter, which progressively enlarged and coalesced into necrotic lesions with perforations. Six symptomatic leaves tissues (5×5 mm) were surface-sterilized using 75% alcohol for 30 s, followed by disinfection with 0.1% mercuric chloride solution for 30 s. Samples were then rinsed three times with sterile water (1 minute each) and transferred onto potato dextrose agar (PDA) plates for incubation at 25℃ in the dark. Fungal isolates (SNPF01-06) were obtained via hyphal-tip isolation. Colonies on PDA were circular, initially producing white, cottony mycelium that later developed a gray center with a white peripheral margin. Conidia were oval, unicellular, hyaline, smooth-walled, measuring 9.1-17.3×3.4-7.6 µm (mean 13.3×5.8 µm, n=50). The morphological characteristics of isolate SNPF05 were consistent with those of Colletotrichum spp. (Weir et al. 2012). Molecular identification involved amplification of five gene loci: internal transcribed spacer (ITS), actin (ACT), chitin synthase 1 (CHS-1), glyceradehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin (TUB2), using primers ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GDF/GDR and T1/Bt2b, respectively (Weir et al. 2012). The obtained sequences were deposited in GenBank under accession numbers PQ687468(ITS), PQ763649 (ACT), PQ763650 (CHS-1), PQ763648 (GAPDH), and PQ763651 (TUB2). BLASTn analysis revealed 99%-100% identity with the C. fructicola type strain ICMP:18581 across all loci: ITS (JX010165; 548/549 bp), ACT (FJ907426; 218/219 bp), CHS-1 (JX009866; 285/287 bp), GAPDH (JX010033; 246/247 bp), and TUB2 (JX010405; 699/699 bp). A maximum-likelihood phylogenetic tree was constructed from the concatenated sequence dataset using MEGA 11; isolate SNPF05 clustered with Colletotrichum fructicola. For pathogenicity testing, six healthy S. ningpoensis plants were selected; three were inoculated via foliar spray with a spore suspension (1×10⁶ conidia/mL) of SNPF05, while three control plants were sprayed with sterile water. All plants were maintained at 25°C under 12-hour light/dark cycles while with 65% humidity. Characteristic leaf blight symptoms appeared on inoculated plants after six days, mimicking field observations. No symptoms developed on control plants. The pathogen was reisolated from symptomatic tissues and identified morphologically and via sequence analysis of ITS, ACT, CHS-1, GAPDH, and TUB2, fulfilling Koch’s postulates. This study represents the first record of C. fructicola causing leaf anthracnose on S. ningpoensis in China. Accurate identification of the pathogen will provide a basis for future prevention and control of leaf anthracnose on S. ningpoensis.

Apple is an important economic species, but it suffers from biotic stress during its growth and development. Fungi and pests are two types of biotic stress that have significant destructive effects on apple. Members of the leucine-rich repeat receptor-like kinase (LRR-RLK) family play a key role in regulating plant responses to biotic stress. In this study, overexpressing MdLRR-RLK1 enhanced apple resistance to Colletotrichum fructicola and aphids by promoting the expression of resistance genes such as WRKYs, PRs and JA-pathway genes, as well as increasing the content of antioxidant enzymes and secondary metabolites. Additionally, MdLRR-RLK1 could interact with MdGRP1-LIKE in vivo and in vitro, and MdLRR-RLK1 could phosphorylate MdGRP1-LIKE in vitro. Overexpressing MdGRP1-LIKE enhanced apple resistance to C. fructicola by increasing the expression of resistance genes such as WRKYs and PRs and the content of antioxidant enzymes. However, overexpressing MdGRP1-LIKE did not enhance the apple resistance to aphids. These findings reveal the mechanism by which the MdLRR-RLK1-MdGRP1-LIKE module regulates apple resistance to C. fructicola stress.

The rice sheath rot pathogens Sarocladium attenuatum and Sarocladium oryzae share a conserved biosynthetic gene cluster (sarc), which encodes colocalized highly reducing polyketide synthases (HR-PKSs) and type III polyketide synthases (T3PKSs). Heterologous expression of the sarc cluster in the Aspergillus nidulans strain LO8030 led to the production of six previously unreported alkylresorcinols, sarocladones A-D (1-4) and H-I (5-6), along with three putative artifacts arising from 4, sarocladones E-G (4a-4c). Biological screening revealed that 1 and 4c both exhibit mild cytotoxicity against murine NS-1 myeloma cells, with IC50 values of 13 μM and 9 μM, respectively. In addition, 1, 3, 6 and 7 displayed antiphagocytotic activity against THP1 macrophages. Subsequent bioinformatic analysis identified a homologous biosynthetic gene cluster (col) in the genome of the fungal plant pathogen Colletotrichum fructicola. To expand the structural diversity of alkylresorcinols, we employed a gene-mixing strategy, coexpressing the HR-PKS gene colA with the T3PKS gene sarcB in A. nidulans. This resulted in the production of two new sarocladone analogues, collecladones A (7) and B (8), lacking the C-2-C-3 double bond present in the sarocladones. These findings establish HR-PKS-T3PKS collaboration as an underexplored source of fungal chemical diversity.

Camellia oleifera anthracnose, primarily caused by Colletotrichum fructicola, leads to significant economic losses. As a sustainable alternative to chemical pesticides, this study investigated the biocontrol efficacy of Bacillus tequilensis DZY 6715 against this disease and elucidated the difference by different application methods: foliar spray and root irrigation. Results indicated that DZY 6715 application significantly suppressed disease incidence and lesion expansion, with root irrigation proving superior to foliar spray, achieving a biocontrol efficacy of 53.62 %. This significantly stronger protection resulted from a multi-layered induced resistance more effectively triggered by root irrigation compared to foliar spray. It systemically enhanced physical barriers by triggering stomatal closure, increasing wax layer thickness, and optimizing leaf anatomy. Root irrigation more potently activated the phenylpropanoid pathway, leading to elevated activities of phenylalanine ammonia-lyase (PAL), cinnamic acid-4-hydroxylase (C4H), 4-coumarate: coenzyme A ligase (4CL), cinnamyl alcohol dehydrogenase (CAD), polyphenol oxidase (PPO) and peroxidase (POD) and the accumulation of lignin, cellulose, and hemicellulose. Additionally, root irrigation reconfigured the phyllosphere microbiome to a greater extent, enhancing microbial community stability and network complexity, and suppressing pathogens.