Apigenin and Apigenin-7-O-β-d-Glucoside Enhance Pepper Defense Against Phytophthora capsici by Inhibiting Pathogen Growth and Reproduction

Apigenin and Apigenin-7-O-β-d-Glucoside Enhance Pepper Defense Against Phytophthora capsici by Inhibiting Pathogen Growth and Reproduction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Apigenin and Apigenin-7-O-β-d-Glucoside Enhance Pepper Defense Against Phytophthora capsici by Inhibiting Pathogen Growth and Reproduction Linying Li, Jiayuxuan Ren, Bingting Lai, Kaidi Zhang, Xiang Qiu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6565949/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Mar, 2026 Read the published version in Phytopathology Research → Version 1 posted You are reading this latest preprint version Abstract Apigenin is a natural plant flavonoid known for its remarkable biological activity. While numerous studies have documented the accumulation of glycosylated and non-glycosylated apigenin in fruits and vegetable crops, their direct and indirect effects on plant resistance remain poorly understood. This study employed non-targeted metabolomics strategies to monitor metabolome-reprogramming events in susceptible, moderately resistant, and highly resistant pepper ( Capsicum annuum) cultivars challenged with Phytophthora capsici . Results revealed a positive correlation between the accumulation of apigenin and apigenin-7-O-β-d-glucoside in pepper seedlings inoculated with P. capsici and the resistance characteristics of the individual pepper cultivars. Further, we demonstrated that both apigenin and apigenin-7-O-β-d-glucoside significantly inhibited the vegetative growth of economically important phytopathogenic oomycetes and fungi, including P. capsici , Phytophthora sojae, Pyricularia oryzae, Fusarium graminearum , and Colletotrichum siamense. These findings highlight the potential applications of glycosylated and non-glycosylated apigenin in developing biopesticides to support the environment-friendly prevention and control of critical crop fungal and oomycete diseases. Plant immunity Apigenin-7-O-β-d-glucoside Apoptosis Pathogen-host interaction Green prevention Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Phytophthora capsici is a destructive soil-borne filamentous fungus-like phytopathogenic oomycete capable of infecting all foliar tissues, including leaves, fruits, and stems/vines, causing diverse diseases such as damping-off, crown rot, foot rot, dieback, phytophthora blight, root rot, fruit rot, and wilting in 78 plant species from 18 families. Hosts of P. capsici include economically significant fruits and vegetables, such as pepper, eggplant, watermelon, cucumber, squash, snap bean, soybean, lima bean, cantaloupe, pumpkin, etc (Hausbeck and Lamour 2004 ; Quesada-Ocampo et al. 2023 ). P. capsici infections account for crop losses valued at $ 1 billion annually (Lamour et al. 2012 ). Environmental conditions, including high temperatures, humidity, rainy weather, and improper irrigation practices accelerate the dissemination, initiation, and development of Phytophthora blight disease caused by P. capsici (Mondal et al. 2020 ). Current control strategies for P. capsici rely heavily on synthetic chemicals and cultural farming practices, including crop rotation, proper spacing, mixed cropping, farm hygiene, enhanced soil drainage, proper tillage, and irrigation (Retes-Manjarrez et al. 2020 ; Wang et al. 2022 ). However, these cultural practices are often less effective than synthetic oomycides (fungicides), such as benzoylamide, metalaxyl, mandipropamid, etc. Chemical control measures, however, tend to increase production costs and negatively impact the environment and human health (Kousik et al. 2011 ; Naqvi et al. 2024 ). Further, prolonged and intensive use of chemicals promotes the development of drug resistance among pathogenic microbes, including field isolates of P. capsici (Tyers and Wright 2019 ; Lee et al. 2023 ; Ishii 2024 ). Attempts to breed resistance in plants against this broad-host pathogen have not yielded durable success due to significant variation in virulence and tissue preference among P. capsici field isolates. Studies have also revealed that P. capsici undergoes frequent genome reshuffling to overcome inbred resistance in host plants (Kamoun et al. 2015 ; Ro et al. 2022 ). Besides external interventions, plants possess and readily deploy genetic and chemical defenses to thwart the initiation of parasitic activities by invading pathogens. The contributions of genetic parameters in host plants, including gene coding for pathogenesis-related (PR) or resistance-associated proteins, such as CaWRKY01-10 and CaWRKY08-4, ascorbate peroxidase, β-1,3-glucanases, aquaporins, late blight resistance protein (CA11g02470), and PR protein (CA04g13070), to either complete or partial resistance of selected fruits and vegetable crops (particularly in pepper cultivars against P. capsici) have attracted considerable research attention (Cheng et al. 2024 ; Shi et al. 2024 ). However, the contributions of plant-specific metabolites (phytochemicals) derived from P. capsici -induced metabolome reprogramming events to the resistance of pepper cultivars against blight pathogens are not fully understood. Classes of plant metabolites reported to accumulate in host tissues or infection sites include terpenes (monoterpenes, sesquiterpenes, diterpenes, and polyterpenes), phenolics (flavonoids, isoflavonoids, tannins, coumarin, etc.), and sulfur-containing compounds (alkaloids), inter alia (Jan et al. 2021 ). Flavonoids constitute a predominant proportion of plant secondary metabolites (Górniak et al. 2019 ). Flavanoids, including apigenin, anthocyanins, cyanidin, caffeic acid, chlorogenic acid, and daidzin, represent essential antioxidant and anti-microbial compounds with significant pharmacological value (Chagas et al. 2022 ). Recent research has revealed that differences in the accumulation of caffeic acid and chlorogenic acid in ripe and unripe pepper fruits during anthrax infection determine pepper’s resistance to anthrax (Baba et al. 2019 ). Although studies have identified apigenin and apigenin-7-O-β-d-glucoside, natural plant flavonoids found in celery ( Apium graveolens ), parsley ( Petroselinum crispum ), and other fruits and vegetables (Williams and Grayer 2004 ; Petrussa et al. 2013 ; Tang et al. 2017 ), with significant anticancer, antioxidant, anti-inflammatory, antibacterial, and antiviral properties (Clavin et al. 2007 ; Benavente-Garcia and Castillo 2008 ; Rossato et al. 2011 ), the inherent contributions of glycosylated and non-glycosylated apigenin to plant defense against phytopathogenic microbes, including P. capsici , are just beginning to attract research attention. Results from comparative metabolome profiling of P. capsici-susceptible , moderately resistant, and resistant pepper cultivars challenged with P. capsici revealed an increasing accumulation of apigenin and apigenin-7-O-β-d-glucoside that correlated with the resistance capabilities of the individual pepper cultivars. Further, we demonstrated that both apigenin and apigenin-7-O-β-d-glucoside significantly inhibited the vegetative growth of economically important phytopathogenic oomycete and fungal species, including P. capsici, Phytophthora sojae, Pyricularia oryzae, Fusarium graminearum , and Colletotrichum siamense. This study provides crucial insights into the antimicrobial efficacy of apigenin and apigenin-7-O-β-d-glucoside, underscoring the potential of glycosylated and non-glycosylated apigenin in developing biopesticides to support the environment-friendly prevention and control of essential plant pathogenic oomycete and fungal pathogens. Results Accumulation of glycosylated and non-glycosylated apigenins in resistant pepper seedlings in response to P. capsici infection To explore the possible connection between the accumulation of apigenin and apigenin-7-O-β-d-glucoside and resistance variations in different pepper cultivars (HNUCB0081, HNUCB0226, and XIAO ZHOU PI) against P. capsici , we analyzed non-targeted metabolomics data from samples collected at different stages of P. capsici -pepper interactions. The results revealed a significant increase in apigenin and apigenin-7-O-β-d-glucoside levels in the completely resistant (XIAO ZHOU PI) and moderately resistant (HNUCB0226) cultivars, compared to the highly susceptible HNUCB0081 cultivar in response to P. capsici infection (Fig. 1 A-B, and Supporting Information, Fig. S1 ). We quantitatively analyzed apigenin and apigenin-7-O-β-d-glucoside levels in inoculated leaf tissues of HNUCB0081, HNUCB0226, and XIAO ZHOU PI seedlings under P. capsici -induced biotic stress using targeted metabolomics. Our analyses revealed a significant increase in intracellular apigenin levels, particularly in the HNUCB0081 and HNUCB0226 cultivars (Fig. 1 C-E). Unexpectedly, the intracellular level of apigenin-7-O-β-d-glucoside in XIAO ZHOU PI before inoculation was significantly higher than in both inoculated and control groups of HNUCB0081 and HNUCB0226. During P. capsici infection, apigenin-7-O-β-d-glucoside levels in XIAO ZHOU PI were significantly higher than in HNUCB0081 and HNUCB0226 under the same conditions (Fig. 1 D-F). Apigenin and Apigenin-7-O-β-d-glucoside inhibit vegetative growth of phytopathogens, including P. capsici We investigated the inhibitory effects of apigenin and apigenin-7-O-β-d-glucoside on P. capsici by assessing the vegetative growth of LT1534 strains on TA agar medium, independently supplemented with different concentrations of apigenin or apigenin-7-O-β-d-glucoside. Compared to the control groups, we observed a significant reduction in the vegetative growth of P. capsici on media containing either compound, with stable IC 50 values of 126.9 µM and 110.6 µM, respectively (Fig. 2 A-B, D-E, and G-H). These results confirm that both apigenin and apigenin-7-O-β-d-glucoside significantly inhibit the vegetative growth of P. capsici . Additionally, we examined the inhibitory effects of a combination of apigenin and apigenin-7-O-β-d-glucoside at a 1:1 molar ratio on TA agar medium. At a concentration of 5 µM or higher inhibited vegetative growth and at 150 µM, almost complete inhibition was observed (Fig. 2 C, F). The IC 50 values calculated for apigenin, apigenin-7-O-β-d-glucoside, and their combination demonstrated that the combined treatment exerted a higher inhibitory effect on P. capsici growth, with an IC 50 of 35.63 µM compared to 126.9 µM and 110.6 µM, for apigenin and apigenin-7-O-β-d-glucoside alone. These findings suggest that the moderate accumulation of these compounds plays a crucial role in enhancing the resistance of pepper cultivars against P. capsici . Further, we evaluated the effects of apigenin, apigenin-7-O-β-d-glucoside, and their combination on the vegetative growth of economically important phytopathogenic fungi and oomycetes, including P. sojae , P. oryzae , C. siamense , and F. graminearum . Exogenous application of each compound showed significant inhibitory efficacy across all species (Supporting Information, Figs. S2-S5). Interestingly, unlike P. capsici , there was no substantial difference in the concentrations required to cause 50% inhibition in the vegetative growth of P. sojae , P. oryzae , C. siamense , and F. graminearum . The IC 50 values for P. capsici were 2–3 times lower than for these other species (Fig. 2 G-I) and Supporting Information, Fig. S2, and S5 G-I). Exogenous apigenins trigger apoptotic-like collapse in vegetative hyphae of P. capsici To gain further insight into the effects of apigenin, apigenin-7-O-β-d-glucoside, and their combination on the morphology and cellular dynamics of vegetative hyphae in P. capsici , LT1534 strains were cultured on TA agar media supplemented with exogenous apigenin (126.9 µM), apigenin-7-O-β-d-glucoside (110.6 µM), a cocktail of apigenin and apigenin-7-O-β-d-glucoside (35.63 µM), DMSO (2% v / v ), and the mock control group, then incubated under optimal growth conditions for 24 h. SEM and TEM analyses revealed that treatment with apigenin and apigenin-7-O-β-d-glucoside induced significant collapse of hyphal cells, compared to DMSO and the control group (Fig. 3 A). TEM analysis further demonstrated dynamic changes, including increased liposome formation, nuclear alterations, mitochondrial disintegration, vacuolar enlargement, and abnormal plasma membrane and cell wall morphology in hyphal cells treated with apigenin (Fig. 3 B), suggesting that the accumulation of glycosylated and non-glycosylated apigenin disrupts vital cellular processes, thereby interfering with the pathophysiological development of P. capsici. Apigenins significantly suppress asexual reproduction in P. capsici Sporangia play a crucial role in the overwintering, reproduction, and dissemination of P. capsici . To investigate the influence of apigenin, apigenin-7-O-β-d-glucoside, or a cocktail of both on sporangium production in P. capsici , we assessed the sporangium production of LT1534 strains grown on TA agar medium supplemented with various concentrations of these compounds. Treatments included apigenin, apigenin-7-O-β-d-glucoside, a cocktail of both compounds, DMSO (2% v/v), and a mock control group. The results showed that apigenin and apigenin-7-O-β-d-glucoside, at concentrations of 100 µM and the cocktail at 25 µM, significantly reduced the sporangium production capabilities of P. capsici (Fig. 4 A-F) and Supporting Information, Fig. S6). We concluded that the accumulation of apigenin and apigenin-7-O-β-d-glucoside in host tissues during pepper-P. capsici interactions also acts to directly compromise the ability of P. capsici to reproduce, thereby limiting its pathogenesis. Apigenins inhibit zoosporogenesis and suppress zoospore germination in P. capsici To investigate the effects of apigenin, apigenin-7-O-β-d-glucoside, and their combination on zoospore release in P. capsici , we used the IC50 value from the growth experiment along with two concentrations (IC 50 ± 10 µM) of each compound to prepare sporangium suspensions. A 30 µL sporangium suspension was inoculated on hydrophobic coverslips, placed in a moist sample box, and incubated at 4°C for 30 and 60 min. Microscopy examinations revealed that compared to 0.2% v/v DMSO and the mock control groups, treatment with apigenin, apigenin-7-O-β-d-glucoside, or their combination significantly suppressed the rate of zoospore release in P. capsici (Fig. 5 A-C). Additionally, we assessed the effects of apigenin and apigenin-7-O-β-d-glucoside on zoospore germination by treating zoospores harvested from LT1534 wild-type strains with various concentrations of each compound and their combination. Microscope examinations of the treated zoospores revealed a substantial reduction in germination efficiency, with the cocktail of apigenin and apigenin-7-O-β-d-glucoside exerting the most significant inhibitory effect on zoospore germination (Fig. 5 D-F). Exogenous apigenin and apigenin-7-O-β-d-glucoside enhance resistance in susceptible pepper cultivars against P. capsici To investigate the effects of apigenin, apigenin-7-O-β-d-glucoside, and their combination on the pathogenicity of P. capsici , we pre-treated P. capsici -susceptible pepper seedlings with apigenin (126.9 µM), apigenin-7-O-β-d-glucoside (110.6 µM), and a combination of both compounds (126.9 µM) and apigenin-7-O-β-d-glucoside (35.63 µM), and incubated the treated seedlings in the dark for 30 min before inoculating the pre-treated leaves with sporangia suspensions (concentration of 3×10⁵ sporangia/mL) prepared from LT1534 strains. Infection assessment results at 72 h post-inoculation (hpi) revealed a significant reduction in lesion expansion in seedlings that were pre-treated. The cocktail of apigenin and apigenin-7-O-β-d-glucoside recorded the highest inhibitory effect on the infection capabilities of P. capsici. Additionally, we confirmed that apigenin, apigenin-7-O-β-d-glucoside, the cocktail, and the 0.2% v/v DMSO treatment had no observable adverse effects on the pepper seedlings (Fig. 6 B). Based on these results, we inferred that the accumulation of apigenin and apigenin-7-O-β-d-glucoside possibly plays a direct role in enhancing pepper immunity against P. capsici. Discussion Apigenin is a naturally occurring flavonoid found in plants. Its two forms are the non-glycosylated (aglycone) form and the glycosylated (glycone) form (Lefort and Blay 2013 ; Tang et al. 2017 ). The biosynthesis of both forms of apigenin has been reported in numerous plants, including parsley, celery, and chamomile, inter alia (Tang et al. 2017 ). However, there are limited reports on the generation and accumulation of apigenin in pepper (Castillejo et al. 2022 ; Islam et al. 2023 ). Here, we demonstrated the generation and accumulation of apigenin and apigenin-7-O-β-d-glucoside in the leaf metabolome of three pepper cultivars. These observations suggest that the regulatory pathways and enzymes involved in apigenin biosynthesis and glycosylation, including flavone synthases (FNS) (Righini et al. 2019 ; Tian et al. 2022 ; Luo et al. 2023 ), Chalcone Isomerase (CHI), chalcone synthase (CHS), 4-coumarate ligase (4-CL), cinnamate-4-hydroxylase (C4H) (Cheng et al. 2018 ), phenylalanine ammonia-lyase (PAL), UDP-glucose-7-O-glucosyltransferase (UGT) (Marín et al. 2017 ; Hou et al. 2023 ), and other cytochrome P450 enzymes (Peng et al. 2017 ), are relatively conserved in plants. Moreover, the accumulation of apigenin in the leaf metabolome of the three pepper cultivars increased in response to P. capsici infection, further underscoring the direct and indirect contributions of apigenin in boosting plant immunity against invading pathogens. This finding is supported by previous research indicating that the biosynthesis and accumulation of apigenin enhance plant immunity against biotic stress by activating multiple plant defense mechanisms, including the generation of defense hormones like salicylic acid and jasmonic acid, and the expression of genes coding for pathogenesis-related (PR) (Falcone Ferreyra et al. 2015 ) Plant flavonoids, including apigenin, generally possess antimicrobial properties against pathogenic bacterial and fungal groups, either by directly inhibiting the growth of pathogenic microbes inside or around the host tissues or indirectly by providing cellular cues that trigger the generation of defense-related compounds and hormones (Nayaka et al. 2014 ). Previous studies confirmed the inhibitory effects of apigenin against diverse human pathogenic bacteria, including Pseudomonas aeruginosa, Salmonella typhimurium, Proteus mirabilis , and Klebsiella pneumoniae (Nayaka et al. 2014 ). We demonstrated that the exogenous application of apigenin, apigenin-7-O-β-d-glucoside, or their combination significantly inhibited the vegetative morphogenesis of economically significant oomycete and fungal species, including P. capsici , P. sojae , P. oryzae , C. siamense , and F . graminearum . These observations are consistent with previous findings regarding the significant inhibitory activities of non-glycosylated apigenin against sporangium formation of P. capsici (Kato et al. 2022 ) and a wide range of soybean pathogens, including Colletotrichum truncatum , Macrophomina phaseolina , Phoma exigua , Phytophthora sojae , Pythium ultimum , Rhizoctonia solani , and Sclerotinia sclerotiorum (Jiang et al. 2012 ). These observations indicated that both non-glycosylated and glycosylated apigenin exhibit broad antimicrobial effects against pathogenic bacteria, oomycetes, and fungi. This partly supports the reasoning that differences in the levels of apigenin and apigenin-7-O-β-d-glucoside between the different pepper cultivars during P. capsici infection possibly account for the variations in resistance among the three pepper cultivars against P. capsici . In Candida albicans, apigenin treatment triggered cell shrinkage and significantly compromised membrane integrity (Lee et al. 2018 ). Apigenin suppresses human carcinogenesis by accelerating apoptosis in cancer cells (Woo et al. 2020 ). Here, we showed that apigenin and apigenin-7-O-β-d-glucoside treatment caused hyphal cells in P. capsici to collapse, triggered the formation of lytic vacuoles, aggregation of lipid bodies, the disintegration of cellular organelles, including nuclear and mitochondrial, and compromised cell wall and membrane integrity. Additionally, we demonstrated that the application of apigenin or apigenin-7-O-β-d-glucoside, either alone or in combination, significantly compromised the survival of zoospores in P. capsici . Accordingly, we speculate that the accumulation of glycosylated and non-glycosylated apigenin in plants disrupts disease development by inducing cell death in the invading pathogen. Future studies should investigate the direct influence of apigenin on the initiation of programmed cell death processes, including apoptosis or autophagy, in phytopathogenic oomycetes and fungi. Apigenin plays a buffering role by regulating the generation and accumulation of cytotoxic compounds, including reactive nitrogen species (RNS) and reactive oxygen species (ROS). For instance, SH-SY5Y neuroblastoma cell lines treated with apigenin were protected from the cytotoxic effects of reactive radicals (Kim et al. 2021 ). Unlike neuroblastoma cells, treating malignant mesothelioma cell lines with apigenin modulates ROS generation and compromises mitochondrial integrity (Lee et al. 2020 ). Further, we showed that pre-treatment of P. capsici -susceptible pepper cultivars with apigenin, apigenin-7-O-β-d-glucoside, and a combination of both pre-enhances the resistance of pepper seedlings against P. capsici without causing physical or cellular injuries in the pre-treated plants. These observations, coupled with previous research findings that implicated apigenin in promoting growth and stress tolerance in rice seedlings (Mekawy et al. 2018 ), suggest that apigenin may modulate different molecular targets in plants than in humans, bacteria, or fungi. In conclusion, these findings provide a solid framework for screening protein targets of glycosylated and non-glycosylated apigenin in phytopathogenic oomycetes and fungi, advancing the development of sustainable apigenin-based strategies for controlling P. capsici and other important fungal pathogens. Conclusions This study demonstrated that the intracellular levels of apigenin and apigenin-7-O-β-d-glucoside correlate with the varying resistance levels of three pepper cultivars to P. capsici . The results suggest that the production of these compounds in response to P. capsic i infection significantly inhibits disease progression by suppressing the reproduction, pathogenic differentiation, and morphological development of P. capsici . Additionally, apigenin and apigenin-7-O-β-d-glucoside also inhibit a wide range of phytopathogenic oomycetes and fungi, including P. sojae , P. oryzae , C. siamense , and F. graminearum . These findings indicate that these compounds could serve as valuable natural agents for sustainable control of oomycete and fungal diseases, opening new opportunities for integrated disease management and the development of pepper cultivars with enhanced resistance. Materials and Methods Test materials and reagents Apigenin (HPLC ≥ 98% purity) was purchased from Hebei Co-create Biotechnology Co., Ltd., Apigenin-7-O-β-d-glucoside (HPLC ≥ 98% purity) from Nanjing Chunqiu Biological Engineering Co., Ltd. and DMSO from Biofroxx, Germany. Test strains and pepper cultivar The wild-type strains of Phytophthora capsici LT1534, Pyricularia oryza e Guy11, Phytophthora sojae P6497, Colletotrichum siamense , and Fusarium graminearum PH-1 were obtained from the strains library of Tropical Plant Disease Control, Microbial Genomics, and Microbial Resource Utilization Laboratory at the Tropical Agriculture and Forestry College of Hainan University. Pepper cultivars HNUCB0081 (highly susceptible) and HNUCB0226 (moderately resistant) were a gift from Professor Zhiwei Wang from the Tropical Agriculture and Forestry College of Hainan University. The completely resistant cultivar, XIAO ZHOU PI, was purchased from the Chunhua Seed Industry Center in Yichun City, Jiangxi Province, P.R. China. Cultivation and preparation of samples from pepper seedlings for targeted metabolomic analysis Leaves from six-week-old susceptible pepper cultivar (HNUCB0081), moderately resistant pepper cultivar (HNUCB0226), and highly resistant pepper cultivar (XIAO ZHOU PI) were inoculated with sporangium suspensions at a concentration of approximately 5×10⁵ sporangia/mL. Forty microliters of the sporangium suspension were pre-incubated in the dark at 25°C for 24 h and then exposed to natural light for 48 h, along with a mock control group treated with autoclaved double-deionized water. The leaves were collected, ground into a powder with liquid nitrogen, and lyophilized for 48 h. Each sample was weighed (30 mg) and mixed with 1 mL of 70% methanol, vortexed at room temperature at 100% power for 20 min, and centrifuged at 12,000 rpm at 4°C for 10 min. The resulting supernatants were pipetted into sterilized centrifuge tubes. One hundred microliters of supernatant were diluted with 900 µL of 70% methanol, vortexed, centrifuged, and 100 µL was used for quantitative metabolic analysis, the data for which data were obtained using Waters ACQUITY UPLC-XEVO TQ-S MS triple quadrupole liquid mass spectrometry instruments at the Metabolomics Research Center, Straits Joint Research Institute, Fujian Agriculture and Forestry University. Assays for assessing the inhibitory effects of apigenin and its glucoside derivatives on the vegetative growth of oomycetes and fungi Vegetative growth assays for P. capsici and P. sojae involved inoculating 4 mm media plugs containing vegetative mycelia from actively growing colonies of P. capsici (LT1534) and P. sojae (P6497) onto tomato juice agar (TA) medium (10% tomato juice, 0.14% CaCO 3 , 1.5% Agar, for P. capsici ) and V8 agar medium (10% V8 juice, 0.14% CaCO 3 , 1.5% Agar, for P. sojae ). These media were supplemented with varying concentrations of apigenin, apigenin-7-O-β-d-glucoside, or a 1:1 combination of both compounds. Cultures were incubated under dark conditions at 25°C for 4 days (for P. capsici ) and 10 days (for P. sojae ), and colony diameters were measured to assess growth. For P. oryzae (Guy11), C. siamense , and F. graminearum (PH-1), 4 mm mycelial plugs were similarly taken from actively growing colonies and inoculated onto a complete medium (CM) for P. oryzae or potato dextrose agar (PDA) for C. siamense and F. graminearum . These media were also supplemented with apigenin, apigenin-7-O-β-d-glucoside, or their 1:1 combination, and incubated at 28°C for 10 days ( P. oryzae ), 7 days ( C. siamense ), and 4 days ( F. graminearum ). The inhibitory effects of apigenin, apigenin-7-O-β-d-glucoside, and their combination on the vegetative growth of P. capsici , P. sojae , P. oryzae , C. siamense , and F. graminearum were evaluated by measuring colony diameters on their respective culture media. Method for evaluating the efficacy of apigenin and its derivatives against P. capsici sporangium formation, encystment, and zoospore release The LT1534 strain was cultured on a TA medium supplemented with different concentrations of apigenin, apigenin-7-O-β-d-glucoside, and a cocktail preparation of both compounds. The treated strains were incubated at 28°C in darkness for 4 days. The plates were further incubated under continuous light for 1–2 days to maximize sporulation. The sporangia were washed from the plates with sterile water, and the final volume of the sporangium suspension was adjusted to 1 mL. The suspension was mixed thoroughly, and 10 µL was placed on a hemocytometer and counted under a light microscope. For spore encystment and zoospore release, the sporangia were washed from the plates with apigenin, apigenin-7-O-β-d-glucoside, or their combination (1:1 mole ratio) and filtered with sterilized Miracloth to generate sporangium suspensions with a concentration of 1×10⁵ sporangia/mL. A 20 µL suspension was inoculated onto hydrophobic coverslips, kept in a moist chamber, and incubated at 4°C for 30 or 60 min. Microscopic assessment of encystment, zoospore release, and survival in response to apigenin, apigenin-7-O-β-d-glucoside, or their combination was performed at 30- and 60 min post-inoculation (mpi) using a light microscope. For zoospore germination, sporangia were washed from culture plates with sterilized water and incubated at 4°C for 30 min to induce encystment and zoospore release. The zoospores were filtered through two layers of Miracloth to obtain zoospore suspensions. The concentration of zoospores in suspensions was adjusted to 1×10⁵zoospores/mL. Zoospores in suspensions were treated with different concentrations of apigenin, apigenin-7-O-β-d-glucoside, and a combination of both, and 20 µL of the suspension was inoculated on hydrophobic coverslips, placed in moist sample boxes, and incubated at room temperature. The influence of different concentrations of apigenin, apigenin-7-O-β-d-glucoside, and their combination on the germination efficiency of zoospores was monitored at 30 and 60 mpi under a light microscope. Assays to evaluate the effects of apigenin on the pathogenicity and virulence of P. capsici Six-week-old pepper seedlings, at the 5–8 leaf stage and susceptible to P. capsici , were pre-sprayed with different concentrations of apigenin, apigenin-7-O-β-d-glucoside, and their combination, and incubated in the dark for 30 min. The pre-treated leaves were inoculated with sporangium suspensions at a final concentration of 3x10⁵ sporangia/mL. For each treatment, 20 µL of sporangia suspension was inoculated on the pre-treated leaves and non-treated control groups. The inoculated seedlings were incubated in a dark growth chamber at 27°C with 80–95% humidity for 24 h and later transferred to a growth chamber with 12h light-dark cycles. Disease severity was assessed by measuring lesion diameters on infected leaves. Scanning electron microscopy and transmission electron microscopy investigation P. capsici LT1534 was inoculated on a TA agar medium containing varying concentrations of apigenin, apigenin-7-O-β-d-glucoside, or a mixture of both. After incubating in the dark at 25°C for 1 day, a mycelium sample was collected, frozen in liquid nitrogen for 10 min at -90°C, and coated with a gold-palladium layer. Images were captured using a Regulus 8100 (HITACHI) scanning electron microscope. For transmission electron microscopy (TEM), P. capsici LT1534 was inoculated into a TA agar medium containing different concentrations of apigenin, apigenin-7-O-β-d-glucoside, or a combination of both. After a day of dark incubation at 25°C, a mycelium sample was fixed in 2.5% glutaraldehyde for 1 h, rinsed thrice with 0.1 M PBS (phosphate buffer saline) for 15 min each, and subsequently fixed in 0.1 M osmium tetroxide for 1–2 h. The sample was rinsed with 0.1 M PBS four times for 15 min each and dehydrated in a graded ethanol series (50%, 70%, 80%, 90%, 95%, 100%) for 15 min at each step. The samples were dehydrated with acetone (2–3 times, 15 min each) before embedding and staining with uranyl acetate and lead citrate. Images were acquired using an H-7650 (HITACHI) transmission electron microscope at the Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China. Statistical analyses All statistical analyses were performed using data from at least three biological experiments, each with 3 to 5 technical replicates. Data were computed using GraphPad Prism 9.5.1 software. Paired t-tests were performed for pairwise comparisons, and non-parametric tests (e.g., Kruskal-Wallis) were used for group comparisons when assumptions for parametric ANOVA were not met. Statistical significance was denoted as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Abbreviations Ap Apigenin Apg Apigenin-7-O-β-d-glucoside CM Complete media CHI Chalcone Isomerase CHS Chalcone Synthase C4H Cinnamate-4-Hydroxylase CW Cellular wall CM Cellular membrane DMSO Dimethyl sulfoxide FNS Flavone synthases hpi H post-inoculation HPLC High High-performance liquid chromatography LV Lytic vacuoles LB Lipid bodies mpi Min post-inoculation MI Mitochondrion N Cellular nuclei PBS Phosphate buffer saline PR Pathogenesis-related PDA Potato dextrose agar PAL Phenylalanine Ammonia-Lyase RNS Reactive nitrogen species ROS Reactive oxygen species SEM Scanning electron microscopy TEM Transmission electron microscopy TA Tomato-juice agar UGT UDP-Glucose-7-O-Glucosyltransferase UPLC Ultra performance liquid chromatography 4-CL 4-Coumarate Ligase Declarations Ethics approval and consent to participate in this study comply with the ethical standards of China, where this research was conducted. Consent for publication All authors have read and consented to the publication of the study. Competing interests The authors declare that they have no competing interests. Supporting information Supporting information may be found in the online version of this article. Funding This work was supported by the Open Fund of the Key Laboratory of Agricultural Microbiome to JB and JN, Hainan University Research Start-up Fund to JN (KYQD(ZR)-21043) and the National Undergraduate Training Program for Innovation and Entrepreneurship research fund to BL (202410589029) Author Contribution JN, JB, LL＋, and JR conceived and designed the experiment. JN, JB, and BL sourced funding for the research. JN, LL＋, JR, BL, XQ, RS and KZ performed the experiments. JN, JB, LL, MS, and WL conducted data and method validation. LL＋, JR, BL, and XQ carried out visualization. LL＋, JR, BL, XQ, WL, and LL drafted the manuscript, while JN, JB, MS, and LL revised the same. All authors contributed to the final manuscript. Acknowledgement We are grateful to the members of JN laboratory for their insightful discussions. Data availability statement The data supporting the findings of this study are available and can be obtained from the corresponding author upon reasonable request. References Baba VY, Constantino LV, Ivamoto ST, Moreira AFP, Madeira TB, Nixdorf SL, et al. Capsicum-Colletotrichum interaction: Identification of resistance sources and quantification of secondary metabolites in unripe and ripe fruits in response to anthracnose infection. Sci Hort. 2019;246:469–77. Benavente-Garcia O, Castillo J. Update on uses and properties of citrus flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory activity. J Agric Food Chem. 2008;56:6185–205. Castillejo N, Martínez-Zamora L, Artés-Hernández F. Postharvest UV radiation enhanced biosynthesis of flavonoids and carotenes in bell peppers. Postharvest Biol Technol. 2022;184:111774. 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Tian S, Yang Y, Wu T, Luo C, Li X, Zhao X, et al. Functional characterization of a flavone synthase that participates in a kumquat flavone metabolon. Front Plant Sci. 2022;13:826780. Tyers M, Wright GD. Drug combinations: a strategy to extend the life of antibiotics in the 21st century. Nat Rev Microbiol. 2019;17:141–55. Wang Z, Gao X, Zhong S, Li Y, Shi M, Zhang B, et al. Host-induced gene silencing of PcCesA3 and PcOSBP1 confers resistance to Phytophthora capsici in Nicotiana benthamiana through NbDCL3 and NbDCL4 processed small interfering RNAs. Int J Biol Macromol. 2022;222:1665–75. Williams CA, Grayer RJ. Anthocyanins and other flavonoids. Nat Prod Rep. 2004;21:539–73. Woo J-S, Choo G-S, Yoo E-S, Kim S-H, Lee J-H, Han S-H, et al. Apigenin induces apoptosis by regulating Akt and MAPK pathways in human melanoma cell A375SM. Mol Med Rep. 2020;22:4877–89. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6565949","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":456365605,"identity":"032c4421-7eea-4bbd-a8e7-4d2d72cae160","order_by":0,"name":"Linying Li","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Linying","middleName":"","lastName":"Li","suffix":""},{"id":456365606,"identity":"011ea29e-9f25-403b-9130-f85c61f3426e","order_by":1,"name":"Jiayuxuan Ren","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Jiayuxuan","middleName":"","lastName":"Ren","suffix":""},{"id":456365607,"identity":"57379766-eda4-4aa3-ab23-84362a3e6312","order_by":2,"name":"Bingting Lai","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Bingting","middleName":"","lastName":"Lai","suffix":""},{"id":456365608,"identity":"602e7132-dd19-4789-99d9-dac02f3f9bf2","order_by":3,"name":"Kaidi Zhang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Kaidi","middleName":"","lastName":"Zhang","suffix":""},{"id":456365610,"identity":"f407e6d0-bb77-4572-8031-4e8a5c36dfd7","order_by":4,"name":"Xiang Qiu","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Qiu","suffix":""},{"id":456365613,"identity":"1f0c42db-90c9-47dd-bce7-c3503586323f","order_by":5,"name":"Lili Lin","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Lin","suffix":""},{"id":456365616,"identity":"3c97dcf8-bd96-4bc1-a35d-571ed2870971","order_by":6,"name":"Ruqi Shi","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Ruqi","middleName":"","lastName":"Shi","suffix":""},{"id":456365618,"identity":"04e97b4d-ce43-4485-a471-04134b9c4ee4","order_by":7,"name":"Wenbo Liu","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Wenbo","middleName":"","lastName":"Liu","suffix":""},{"id":456365620,"identity":"3202e61e-a2c6-454c-a5fe-db1ed52fec1a","order_by":8,"name":"Moosa Sedibe","email":"","orcid":"","institution":"Central University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Moosa","middleName":"","lastName":"Sedibe","suffix":""},{"id":456365621,"identity":"7857dd80-f8a2-4606-99ad-9e4035171a86","order_by":9,"name":"Jiandong Bao","email":"","orcid":"","institution":"Zhejiang Academy of Agricultures Science","correspondingAuthor":false,"prefix":"","firstName":"Jiandong","middleName":"","lastName":"Bao","suffix":""},{"id":456365622,"identity":"883d2784-aab5-439b-b732-eb7ec7d3f8da","order_by":10,"name":"Justice Norvienyeku","email":"data:image/png;base64,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","orcid":"","institution":"Hainan University","correspondingAuthor":true,"prefix":"","firstName":"Justice","middleName":"","lastName":"Norvienyeku","suffix":""}],"badges":[],"createdAt":"2025-04-30 14:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6565949/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6565949/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s42483-026-00414-z","type":"published","date":"2026-03-26T16:09:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82890256,"identity":"216cabd0-01a8-4135-83bf-1c4668360c1d","added_by":"auto","created_at":"2025-05-16 12:08:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3223642,"visible":true,"origin":"","legend":"\u003cp\u003eRelative quantification of apigenin and apigenin-7-O-β-d-glucoside in different pepper cultivars challenged with \u003cem\u003eP. capsici\u003c/em\u003e. (A-B) Comparative abundance of apigenin and apigenin-7-O-β-d-glucoside in susceptible and resistant pepper cultivars at different stages of \u003cem\u003eP. capsici\u003c/em\u003e infection. (C-D)\u003cstrong\u003e \u003c/strong\u003eTargeted metabolomics quantification of apigenin and apigenin-7-O-β-d-glucoside concentrations in the susceptible and resistant pepper cultivars during \u003cem\u003eP. capsici\u003c/em\u003e infection. (E-F)\u003cstrong\u003e \u003c/strong\u003eChromatographic peaks of the accumulation of apigenin and apigenin-7-O-β-d-glucoside detected in the susceptible and resistant pepper cultivars calibrated against HPLC analytical standards as references. *, P ≤ 0.05; **, P ≤ 0.01; ns: no significant. Paired t-test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6565949/v1/2d1f7807853f510fbf0f48bb.png"},{"id":82888141,"identity":"f149dea0-ddab-4d9f-acae-a8561550c462","added_by":"auto","created_at":"2025-05-16 12:00:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9654066,"visible":true,"origin":"","legend":"\u003cp\u003eThe inhibitory effects of apigenins on the vegetative growth of \u003cem\u003eP. capsici\u003c/em\u003e. (A-C) Show the inhibitory efficacy of various concentrations of apigenin, apigenin-7-O-β-d-glucoside, or their combination against the vegetative growth of \u003cem\u003eP. capsici\u003c/em\u003e. (D-F) Histogram represents statistical computation of the inhibitory effects of apigenin, apigenin-7-O-β-d-glucoside, or their combination on the vegetative growth of \u003cem\u003eP. capsici\u003c/em\u003e.\u003cstrong\u003e \u003c/strong\u003e(G-I)\u003cstrong\u003e \u003c/strong\u003eThe line graph shows the efficacy assessment and the effective concentrations (IC\u003csub\u003e50\u003c/sub\u003e) corresponding to 50% inhibition in the vegetative growth of \u003cem\u003eP. capsici\u003c/em\u003e. Abbreviations: Ap: apigenin; Apg: apigenin-7-O-β-d-glucoside. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns: no significant. Non-parametric one-way analysis.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6565949/v1/b2067a6354340200ad4bcbca.png"},{"id":82888155,"identity":"6c9c290b-f36e-4b93-870e-ccc533d1c537","added_by":"auto","created_at":"2025-05-16 12:00:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10042635,"visible":true,"origin":"","legend":"\u003cp\u003eIndividual impacts of apigenins on the morphological and cellular integrity of vegetative structures.\u003cem\u003e \u003c/em\u003e(A-B) Effects of various concentrations of apigenin, apigenin-7-O-β-d-glucoside, or a combination of both on the mycelium and cellular structure of \u003cem\u003eP. capsici.\u003c/em\u003e CW: cellular wall; CM: cellular membrane; LV: lytic vacuoles; LB: lipid bodies; MI: mitochondrion; N, cellular nuclei. Abbreviations: Ap: apigenin; Apg: apigenin-7-O-β-d-glucoside.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6565949/v1/4843fde562a7747fbb9b5dee.png"},{"id":82890255,"identity":"fc31fc7a-da12-44a5-a1e2-b1bc51900df9","added_by":"auto","created_at":"2025-05-16 12:08:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8734901,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of apigenin and apigenin-7-O-β-d-glucoside on sporangium productionin \u003cem\u003eP. capsici\u003c/em\u003e. (A-D) Comparative evaluation of the effects of different concentrations of apigenin and apigenin-7-O-β-d-glucoside on sporangium production.\u003cstrong\u003e \u003c/strong\u003e(E-F) The histogram illustrates the statistical computation of the impacts of these compounds on sporangium production in \u003cem\u003eP. capsici\u003c/em\u003e. Ap: apigenin; Apg: apigenin-7-O-β-d-glucoside. Scale bars: (A-B) 50 μm; (C-D) 100 μm. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns: not significant. Non-parametric one-way analysis.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6565949/v1/eea84b5825df319d9f61468e.png"},{"id":82888192,"identity":"078c1333-dfe7-40c9-88ab-d854f79ec397","added_by":"auto","created_at":"2025-05-16 12:00:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10599090,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of apigenins on the formation, release, survival, and germination of zoospores in \u003cem\u003eP. capsici\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e(A) The impact of various concentrations of apigenin, apigenin-7-O-β-d-glucoside, or their combination on zoospore release and survival. (B-C) Bar graphs depict the statistical effects of these compounds on zoospore release and survival. (D) Influence of apigenin, apigenin-7-O-β-d-glucoside, or their combination on the germination and survival of zoospores. (E-F) The bar graphs present the statistical evaluation of the effects on zoospore germination and survival. Abbreviations: Ap: apigenin; Apg: apigenin-7-O-β-d-glucoside. Scale bars: (A) and\u003cstrong\u003e \u003c/strong\u003e(D) 20 μm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6565949/v1/2cf301dd06c408fb85cd418e.png"},{"id":82888166,"identity":"1cf2f556-9dd7-49aa-99ed-b59a887f4612","added_by":"auto","created_at":"2025-05-16 12:00:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":16166283,"visible":true,"origin":"","legend":"\u003cp\u003eApigenin enhances the resistance of susceptible pepper cultivars against \u003cem\u003eP. capsici\u003c/em\u003e. (A) Pre-inoculation application of exogenous apigenin, apigenin-7-O-β-d-glucoside, and their combination significantly suppressed the development of Phytophthora blight disease. (B) Effects of different concentrations of apigenin, apigenin-7-O-β-d-glucoside, or their combination on pepper leaves. (C)\u003cstrong\u003e \u003c/strong\u003eBar graphs illustrate the statistical effects of these compounds on lesion development. Abbreviations: Ap: apigenin; Apg: apigenin-7-O-β-d-glucoside.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6565949/v1/19ae12a042167cfe74f26022.png"},{"id":105755773,"identity":"58fedf47-c66a-4cf2-8144-1d778d53ee63","added_by":"auto","created_at":"2026-03-30 16:30:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":55666458,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6565949/v1/0c954344-22eb-49cf-87dd-c29077cbc944.pdf"},{"id":82890254,"identity":"e4834781-fbcc-466e-89c2-cdfd572b4637","added_by":"auto","created_at":"2025-05-16 12:08:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1205346,"visible":true,"origin":"","legend":"","description":"","filename":"PRSupplementaryfile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6565949/v1/3a93691cd798de36cb734b95.pdf"},{"id":82890253,"identity":"883fcd2b-60e9-49db-84d9-6d9e802ceb00","added_by":"auto","created_at":"2025-05-16 12:08:52","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1585600,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstractImage1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6565949/v1/decb094c4c00e3d5c6dd7033.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Apigenin and Apigenin-7-O-β-d-Glucoside Enhance Pepper Defense Against Phytophthora capsici by Inhibiting Pathogen Growth and Reproduction","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003ePhytophthora capsici\u003c/em\u003e is a destructive soil-borne filamentous fungus-like phytopathogenic oomycete capable of infecting all foliar tissues, including leaves, fruits, and stems/vines, causing diverse diseases such as damping-off, crown rot, foot rot, dieback, phytophthora blight, root rot, fruit rot, and wilting in 78 plant species from 18 families. Hosts of \u003cem\u003eP. capsici\u003c/em\u003e include economically significant fruits and vegetables, such as pepper, eggplant, watermelon, cucumber, squash, snap bean, soybean, lima bean, cantaloupe, pumpkin, etc (Hausbeck and Lamour \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Quesada-Ocampo et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eP. capsici\u003c/em\u003e infections account for crop losses valued at \u003cspan\u003e$\u003c/span\u003e1\u0026nbsp;billion annually (Lamour et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Environmental conditions, including high temperatures, humidity, rainy weather, and improper irrigation practices accelerate the dissemination, initiation, and development of \u003cem\u003ePhytophthora\u003c/em\u003e blight disease caused by \u003cem\u003eP. capsici\u003c/em\u003e (Mondal et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurrent control strategies for \u003cem\u003eP. capsici\u003c/em\u003e rely heavily on synthetic chemicals and cultural farming practices, including crop rotation, proper spacing, mixed cropping, farm hygiene, enhanced soil drainage, proper tillage, and irrigation (Retes-Manjarrez et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, these cultural practices are often less effective than synthetic oomycides (fungicides), such as benzoylamide, metalaxyl, mandipropamid, etc. Chemical control measures, however, tend to increase production costs and negatively impact the environment and human health (Kousik et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Naqvi et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Further, prolonged and intensive use of chemicals promotes the development of drug resistance among pathogenic microbes, including field isolates of \u003cem\u003eP. capsici\u003c/em\u003e (Tyers and Wright \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ishii \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Attempts to breed resistance in plants against this broad-host pathogen have not yielded durable success due to significant variation in virulence and tissue preference among \u003cem\u003eP. capsici\u003c/em\u003e field isolates. Studies have also revealed that \u003cem\u003eP. capsici\u003c/em\u003e undergoes frequent genome reshuffling to overcome inbred resistance in host plants (Kamoun et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ro et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBesides external interventions, plants possess and readily deploy genetic and chemical defenses to thwart the initiation of parasitic activities by invading pathogens. The contributions of genetic parameters in host plants, including gene coding for pathogenesis-related (PR) or resistance-associated proteins, such as CaWRKY01-10 and CaWRKY08-4, ascorbate peroxidase, β-1,3-glucanases, aquaporins, late blight resistance protein (CA11g02470), and PR protein (CA04g13070), to either complete or partial resistance of selected fruits and vegetable crops (particularly in pepper cultivars against \u003cem\u003eP. capsici)\u003c/em\u003e have attracted considerable research attention (Cheng et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, the contributions of plant-specific metabolites (phytochemicals) derived from \u003cem\u003eP. capsici\u003c/em\u003e-induced metabolome reprogramming events to the resistance of pepper cultivars against blight pathogens are not fully understood.\u003c/p\u003e \u003cp\u003eClasses of plant metabolites reported to accumulate in host tissues or infection sites include terpenes (monoterpenes, sesquiterpenes, diterpenes, and polyterpenes), phenolics (flavonoids, isoflavonoids, tannins, coumarin, etc.), and sulfur-containing compounds (alkaloids), inter alia (Jan et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Flavonoids constitute a predominant proportion of plant secondary metabolites (G\u0026oacute;rniak et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Flavanoids, including apigenin, anthocyanins, cyanidin, caffeic acid, chlorogenic acid, and daidzin, represent essential antioxidant and anti-microbial compounds with significant pharmacological value (Chagas et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recent research has revealed that differences in the accumulation of caffeic acid and chlorogenic acid in ripe and unripe pepper fruits during anthrax infection determine pepper\u0026rsquo;s resistance to anthrax (Baba et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough studies have identified apigenin and apigenin-7-O-β-d-glucoside, natural plant flavonoids found in celery (\u003cem\u003eApium graveolens\u003c/em\u003e), parsley (\u003cem\u003ePetroselinum crispum\u003c/em\u003e), and other fruits and vegetables (Williams and Grayer \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Petrussa et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), with significant anticancer, antioxidant, anti-inflammatory, antibacterial, and antiviral properties (Clavin et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Benavente-Garcia and Castillo \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Rossato et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), the inherent contributions of glycosylated and non-glycosylated apigenin to plant defense against phytopathogenic microbes, including \u003cem\u003eP. capsici\u003c/em\u003e, are just beginning to attract research attention.\u003c/p\u003e \u003cp\u003eResults from comparative metabolome profiling of \u003cem\u003eP. capsici-susceptible\u003c/em\u003e, moderately resistant, and resistant pepper cultivars challenged with \u003cem\u003eP. capsici\u003c/em\u003e revealed an increasing accumulation of apigenin and apigenin-7-O-β-d-glucoside that correlated with the resistance capabilities of the individual pepper cultivars. Further, we demonstrated that both apigenin and apigenin-7-O-β-d-glucoside significantly inhibited the vegetative growth of economically important phytopathogenic oomycete and fungal species, including \u003cem\u003eP. capsici, Phytophthora sojae, Pyricularia oryzae, Fusarium graminearum\u003c/em\u003e, and \u003cem\u003eColletotrichum siamense.\u003c/em\u003e This study provides crucial insights into the antimicrobial efficacy of apigenin and apigenin-7-O-β-d-glucoside, underscoring the potential of glycosylated and non-glycosylated apigenin in developing biopesticides to support the environment-friendly prevention and control of essential plant pathogenic oomycete and fungal pathogens.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAccumulation of glycosylated and non-glycosylated apigenins in resistant pepper seedlings in response to\u003c/b\u003e \u003cb\u003eP. capsici\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the possible connection between the accumulation of apigenin and apigenin-7-O-β-d-glucoside and resistance variations in different pepper cultivars (HNUCB0081, HNUCB0226, and XIAO ZHOU PI) against \u003cem\u003eP. capsici\u003c/em\u003e, we analyzed non-targeted metabolomics data from samples collected at different stages of \u003cem\u003eP. capsici\u003c/em\u003e-pepper interactions. The results revealed a significant increase in apigenin and apigenin-7-O-β-d-glucoside levels in the completely resistant (XIAO ZHOU PI) and moderately resistant (HNUCB0226) cultivars, compared to the highly susceptible HNUCB0081 cultivar in response to \u003cem\u003eP. capsici\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B, and Supporting Information, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe quantitatively analyzed apigenin and apigenin-7-O-β-d-glucoside levels in inoculated leaf tissues of HNUCB0081, HNUCB0226, and XIAO ZHOU PI seedlings under \u003cem\u003eP. capsici\u003c/em\u003e-induced biotic stress using targeted metabolomics. Our analyses revealed a significant increase in intracellular apigenin levels, particularly in the HNUCB0081 and HNUCB0226 cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E). Unexpectedly, the intracellular level of apigenin-7-O-β-d-glucoside in XIAO ZHOU PI before inoculation was significantly higher than in both inoculated and control groups of HNUCB0081 and HNUCB0226. During \u003cem\u003eP. capsici\u003c/em\u003e infection, apigenin-7-O-β-d-glucoside levels in XIAO ZHOU PI were significantly higher than in HNUCB0081 and HNUCB0226 under the same conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eApigenin and Apigenin-7-O-β-d-glucoside inhibit vegetative growth of phytopathogens, including\u003c/b\u003e \u003cb\u003eP. capsici\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe investigated the inhibitory effects of apigenin and apigenin-7-O-β-d-glucoside on \u003cem\u003eP. capsici\u003c/em\u003e by assessing the vegetative growth of LT1534 strains on TA agar medium, independently supplemented with different concentrations of apigenin or apigenin-7-O-β-d-glucoside. Compared to the control groups, we observed a significant reduction in the vegetative growth of \u003cem\u003eP. capsici\u003c/em\u003e on media containing either compound, with stable IC\u003csub\u003e50\u003c/sub\u003e values of 126.9 \u0026micro;M and 110.6 \u0026micro;M, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B, D-E, and G-H). These results confirm that both apigenin and apigenin-7-O-β-d-glucoside significantly inhibit the vegetative growth of \u003cem\u003eP. capsici\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAdditionally, we examined the inhibitory effects of a combination of apigenin and apigenin-7-O-β-d-glucoside at a 1:1 molar ratio on TA agar medium. At a concentration of 5 \u0026micro;M or higher inhibited vegetative growth and at 150 \u0026micro;M, almost complete inhibition was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, F). The IC\u003csub\u003e50\u003c/sub\u003e values calculated for apigenin, apigenin-7-O-β-d-glucoside, and their combination demonstrated that the combined treatment exerted a higher inhibitory effect on \u003cem\u003eP. capsici\u003c/em\u003e growth, with an IC\u003csub\u003e50\u003c/sub\u003e of 35.63 \u0026micro;M compared to 126.9 \u0026micro;M and 110.6 \u0026micro;M, for apigenin and apigenin-7-O-β-d-glucoside alone. These findings suggest that the moderate accumulation of these compounds plays a crucial role in enhancing the resistance of pepper cultivars against \u003cem\u003eP. capsici\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFurther, we evaluated the effects of apigenin, apigenin-7-O-β-d-glucoside, and their combination on the vegetative growth of economically important phytopathogenic fungi and oomycetes, including \u003cem\u003eP. sojae\u003c/em\u003e, \u003cem\u003eP. oryzae\u003c/em\u003e, \u003cem\u003eC. siamense\u003c/em\u003e, and \u003cem\u003eF. graminearum\u003c/em\u003e. Exogenous application of each compound showed significant inhibitory efficacy across all species (Supporting Information, Figs. S2-S5). Interestingly, unlike \u003cem\u003eP. capsici\u003c/em\u003e, there was no substantial difference in the concentrations required to cause 50% inhibition in the vegetative growth of \u003cem\u003eP. sojae\u003c/em\u003e, \u003cem\u003eP. oryzae\u003c/em\u003e, \u003cem\u003eC. siamense\u003c/em\u003e, and \u003cem\u003eF. graminearum\u003c/em\u003e. The IC\u003csub\u003e50\u003c/sub\u003e values for \u003cem\u003eP. capsici\u003c/em\u003e were 2\u0026ndash;3 times lower than for these other species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-I) and Supporting Information, Fig. S2, and S5 G-I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExogenous apigenins trigger apoptotic-like collapse in vegetative hyphae of\u003c/b\u003e \u003cb\u003eP. capsici\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo gain further insight into the effects of apigenin, apigenin-7-O-β-d-glucoside, and their combination on the morphology and cellular dynamics of vegetative hyphae in \u003cem\u003eP. capsici\u003c/em\u003e, LT1534 strains were cultured on TA agar media supplemented with exogenous apigenin (126.9 \u0026micro;M), apigenin-7-O-β-d-glucoside (110.6 \u0026micro;M), a cocktail of apigenin and apigenin-7-O-β-d-glucoside (35.63 \u0026micro;M), DMSO (2% \u003csup\u003ev\u003c/sup\u003e/\u003csub\u003ev\u003c/sub\u003e), and the mock control group, then incubated under optimal growth conditions for 24 h.\u003c/p\u003e \u003cp\u003eSEM and TEM analyses revealed that treatment with apigenin and apigenin-7-O-β-d-glucoside induced significant collapse of hyphal cells, compared to DMSO and the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). TEM analysis further demonstrated dynamic changes, including increased liposome formation, nuclear alterations, mitochondrial disintegration, vacuolar enlargement, and abnormal plasma membrane and cell wall morphology in hyphal cells treated with apigenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), suggesting that the accumulation of glycosylated and non-glycosylated apigenin disrupts vital cellular processes, thereby interfering with the pathophysiological development of \u003cem\u003eP. capsici.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eApigenins significantly suppress asexual reproduction in\u003c/b\u003e \u003cb\u003eP. capsici\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSporangia play a crucial role in the overwintering, reproduction, and dissemination of \u003cem\u003eP. capsici\u003c/em\u003e. To investigate the influence of apigenin, apigenin-7-O-β-d-glucoside, or a cocktail of both on sporangium production in \u003cem\u003eP. capsici\u003c/em\u003e, we assessed the sporangium production of LT1534 strains grown on TA agar medium supplemented with various concentrations of these compounds. Treatments included apigenin, apigenin-7-O-β-d-glucoside, a cocktail of both compounds, DMSO (2% v/v), and a mock control group.\u003c/p\u003e \u003cp\u003eThe results showed that apigenin and apigenin-7-O-β-d-glucoside, at concentrations of 100 \u0026micro;M and the cocktail at 25 \u0026micro;M, significantly reduced the sporangium production capabilities of \u003cem\u003eP. capsici\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-F) and Supporting Information, Fig. S6). We concluded that the accumulation of apigenin and apigenin-7-O-β-d-glucoside in host tissues during pepper-P. \u003cem\u003ecapsici\u003c/em\u003e interactions also acts to directly compromise the ability of P. \u003cem\u003ecapsici\u003c/em\u003e to reproduce, thereby limiting its pathogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eApigenins inhibit zoosporogenesis and suppress zoospore germination in\u003c/b\u003e \u003cb\u003eP. capsici\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the effects of apigenin, apigenin-7-O-β-d-glucoside, and their combination on zoospore release in \u003cem\u003eP. capsici\u003c/em\u003e, we used the IC50 value from the growth experiment along with two concentrations (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;10 \u0026micro;M) of each compound to prepare sporangium suspensions. A 30 \u0026micro;L sporangium suspension was inoculated on hydrophobic coverslips, placed in a moist sample box, and incubated at 4\u0026deg;C for 30 and 60 min. Microscopy examinations revealed that compared to 0.2% v/v DMSO and the mock control groups, treatment with apigenin, apigenin-7-O-β-d-glucoside, or their combination significantly suppressed the rate of zoospore release in \u003cem\u003eP. capsici\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003eAdditionally, we assessed the effects of apigenin and apigenin-7-O-β-d-glucoside on zoospore germination by treating zoospores harvested from LT1534 wild-type strains with various concentrations of each compound and their combination. Microscope examinations of the treated zoospores revealed a substantial reduction in germination efficiency, with the cocktail of apigenin and apigenin-7-O-β-d-glucoside exerting the most significant inhibitory effect on zoospore germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExogenous apigenin and apigenin-7-O-β-d-glucoside enhance resistance in susceptible pepper cultivars against\u003c/b\u003e \u003cb\u003eP. capsici\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the effects of apigenin, apigenin-7-O-β-d-glucoside, and their combination on the pathogenicity of \u003cem\u003eP. capsici\u003c/em\u003e, we pre-treated \u003cem\u003eP. capsici\u003c/em\u003e-susceptible pepper seedlings with apigenin (126.9 \u0026micro;M), apigenin-7-O-β-d-glucoside (110.6 \u0026micro;M), and a combination of both compounds (126.9 \u0026micro;M) and apigenin-7-O-β-d-glucoside (35.63 \u0026micro;M), and incubated the treated seedlings in the dark for 30 min before inoculating the pre-treated leaves with sporangia suspensions (concentration of 3\u0026times;10⁵ sporangia/mL) prepared from LT1534 strains. Infection assessment results at 72 h post-inoculation (hpi) revealed a significant reduction in lesion expansion in seedlings that were pre-treated. The cocktail of apigenin and apigenin-7-O-β-d-glucoside recorded the highest inhibitory effect on the infection capabilities of \u003cem\u003eP. capsici.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAdditionally, we confirmed that apigenin, apigenin-7-O-β-d-glucoside, the cocktail, and the 0.2% v/v DMSO treatment had no observable adverse effects on the pepper seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Based on these results, we inferred that the accumulation of apigenin and apigenin-7-O-β-d-glucoside possibly plays a direct role in enhancing pepper immunity against \u003cem\u003eP. capsici.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eApigenin is a naturally occurring flavonoid found in plants. Its two forms are the non-glycosylated (aglycone) form and the glycosylated (glycone) form (Lefort and Blay \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The biosynthesis of both forms of apigenin has been reported in numerous plants, including parsley, celery, and chamomile, inter alia (Tang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, there are limited reports on the generation and accumulation of apigenin in pepper (Castillejo et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Islam et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Here, we demonstrated the generation and accumulation of apigenin and apigenin-7-O-β-d-glucoside in the leaf metabolome of three pepper cultivars. These observations suggest that the regulatory pathways and enzymes involved in apigenin biosynthesis and glycosylation, including flavone synthases (FNS) (Righini et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tian et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Luo et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), Chalcone Isomerase (CHI), chalcone synthase (CHS), 4-coumarate ligase (4-CL), cinnamate-4-hydroxylase (C4H) (Cheng et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), phenylalanine ammonia-lyase (PAL), UDP-glucose-7-O-glucosyltransferase (UGT) (Mar\u0026iacute;n et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hou et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and other cytochrome P450 enzymes (Peng et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), are relatively conserved in plants.\u003c/p\u003e \u003cp\u003eMoreover, the accumulation of apigenin in the leaf metabolome of the three pepper cultivars increased in response to \u003cem\u003eP. capsici\u003c/em\u003e infection, further underscoring the direct and indirect contributions of apigenin in boosting plant immunity against invading pathogens. This finding is supported by previous research indicating that the biosynthesis and accumulation of apigenin enhance plant immunity against biotic stress by activating multiple plant defense mechanisms, including the generation of defense hormones like salicylic acid and jasmonic acid, and the expression of genes coding for pathogenesis-related (PR) (Falcone Ferreyra et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)\u003c/p\u003e \u003cp\u003ePlant flavonoids, including apigenin, generally possess antimicrobial properties against pathogenic bacterial and fungal groups, either by directly inhibiting the growth of pathogenic microbes inside or around the host tissues or indirectly by providing cellular cues that trigger the generation of defense-related compounds and hormones (Nayaka et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Previous studies confirmed the inhibitory effects of apigenin against diverse human pathogenic bacteria, including \u003cem\u003ePseudomonas aeruginosa, Salmonella typhimurium, Proteus mirabilis\u003c/em\u003e, and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (Nayaka et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). We demonstrated that the exogenous application of apigenin, apigenin-7-O-β-d-glucoside, or their combination significantly inhibited the vegetative morphogenesis of economically significant oomycete and fungal species, including \u003cem\u003eP. capsici\u003c/em\u003e, \u003cem\u003eP. sojae\u003c/em\u003e, \u003cem\u003eP. oryzae\u003c/em\u003e, \u003cem\u003eC. siamense\u003c/em\u003e, and \u003cem\u003eF\u003c/em\u003e. \u003cem\u003egraminearum\u003c/em\u003e. These observations are consistent with previous findings regarding the significant inhibitory activities of non-glycosylated apigenin against sporangium formation of \u003cem\u003eP. capsici\u003c/em\u003e (Kato et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and a wide range of soybean pathogens, including \u003cem\u003eColletotrichum truncatum\u003c/em\u003e, \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e, \u003cem\u003ePhoma exigua\u003c/em\u003e, \u003cem\u003ePhytophthora sojae\u003c/em\u003e, \u003cem\u003ePythium ultimum\u003c/em\u003e, \u003cem\u003eRhizoctonia solani\u003c/em\u003e, and \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e (Jiang et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These observations indicated that both non-glycosylated and glycosylated apigenin exhibit broad antimicrobial effects against pathogenic bacteria, oomycetes, and fungi. This partly supports the reasoning that differences in the levels of apigenin and apigenin-7-O-β-d-glucoside between the different pepper cultivars during \u003cem\u003eP. capsici\u003c/em\u003e infection possibly account for the variations in resistance among the three pepper cultivars against \u003cem\u003eP. capsici\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn Candida albicans, apigenin treatment triggered cell shrinkage and significantly compromised membrane integrity (Lee et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Apigenin suppresses human carcinogenesis by accelerating apoptosis in cancer cells (Woo et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Here, we showed that apigenin and apigenin-7-O-β-d-glucoside treatment caused hyphal cells in \u003cem\u003eP. capsici\u003c/em\u003e to collapse, triggered the formation of lytic vacuoles, aggregation of lipid bodies, the disintegration of cellular organelles, including nuclear and mitochondrial, and compromised cell wall and membrane integrity. Additionally, we demonstrated that the application of apigenin or apigenin-7-O-β-d-glucoside, either alone or in combination, significantly compromised the survival of zoospores in \u003cem\u003eP. capsici\u003c/em\u003e. Accordingly, we speculate that the accumulation of glycosylated and non-glycosylated apigenin in plants disrupts disease development by inducing cell death in the invading pathogen. Future studies should investigate the direct influence of apigenin on the initiation of programmed cell death processes, including apoptosis or autophagy, in phytopathogenic oomycetes and fungi.\u003c/p\u003e \u003cp\u003eApigenin plays a buffering role by regulating the generation and accumulation of cytotoxic compounds, including reactive nitrogen species (RNS) and reactive oxygen species (ROS). For instance, SH-SY5Y neuroblastoma cell lines treated with apigenin were protected from the cytotoxic effects of reactive radicals (Kim et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Unlike neuroblastoma cells, treating malignant mesothelioma cell lines with apigenin modulates ROS generation and compromises mitochondrial integrity (Lee et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Further, we showed that pre-treatment of \u003cem\u003eP. capsici\u003c/em\u003e-susceptible pepper cultivars with apigenin, apigenin-7-O-β-d-glucoside, and a combination of both pre-enhances the resistance of pepper seedlings against \u003cem\u003eP. capsici\u003c/em\u003e without causing physical or cellular injuries in the pre-treated plants. These observations, coupled with previous research findings that implicated apigenin in promoting growth and stress tolerance in rice seedlings (Mekawy et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), suggest that apigenin may modulate different molecular targets in plants than in humans, bacteria, or fungi.\u003c/p\u003e \u003cp\u003eIn conclusion, these findings provide a solid framework for screening protein targets of glycosylated and non-glycosylated apigenin in phytopathogenic oomycetes and fungi, advancing the development of sustainable apigenin-based strategies for controlling \u003cem\u003eP. capsici\u003c/em\u003e and other important fungal pathogens.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrated that the intracellular levels of apigenin and apigenin-7-O-β-d-glucoside correlate with the varying resistance levels of three pepper cultivars to \u003cem\u003eP. capsici\u003c/em\u003e. The results suggest that the production of these compounds in response to \u003cem\u003eP. capsic\u003c/em\u003ei infection significantly inhibits disease progression by suppressing the reproduction, pathogenic differentiation, and morphological development of \u003cem\u003eP. capsici\u003c/em\u003e. Additionally, apigenin and apigenin-7-O-β-d-glucoside also inhibit a wide range of phytopathogenic oomycetes and fungi, including \u003cem\u003eP. sojae\u003c/em\u003e, \u003cem\u003eP. oryzae\u003c/em\u003e, \u003cem\u003eC. siamense\u003c/em\u003e, and \u003cem\u003eF. graminearum\u003c/em\u003e. These findings indicate that these compounds could serve as valuable natural agents for sustainable control of oomycete and fungal diseases, opening new opportunities for integrated disease management and the development of pepper cultivars with enhanced resistance.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTest materials and reagents\u003c/h2\u003e \u003cp\u003eApigenin (HPLC\u0026thinsp;\u0026ge;\u0026thinsp;98% purity) was purchased from Hebei Co-create Biotechnology Co., Ltd., Apigenin-7-O-β-d-glucoside (HPLC\u0026thinsp;\u0026ge;\u0026thinsp;98% purity) from Nanjing Chunqiu Biological Engineering Co., Ltd. and DMSO from Biofroxx, Germany.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTest strains and pepper cultivar\u003c/h3\u003e\n\u003cp\u003eThe wild-type strains of \u003cem\u003ePhytophthora capsici\u003c/em\u003e LT1534, \u003cem\u003ePyricularia oryza\u003c/em\u003ee Guy11, \u003cem\u003ePhytophthora sojae\u003c/em\u003e P6497, \u003cem\u003eColletotrichum siamense\u003c/em\u003e, and \u003cem\u003eFusarium graminearum\u003c/em\u003e PH-1 were obtained from the strains library of Tropical Plant Disease Control, Microbial Genomics, and Microbial Resource Utilization Laboratory at the Tropical Agriculture and Forestry College of Hainan University. Pepper cultivars HNUCB0081 (highly susceptible) and HNUCB0226 (moderately resistant) were a gift from Professor Zhiwei Wang from the Tropical Agriculture and Forestry College of Hainan University. The completely resistant cultivar, XIAO ZHOU PI, was purchased from the Chunhua Seed Industry Center in Yichun City, Jiangxi Province, P.R. China.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCultivation and preparation of samples from pepper seedlings for targeted metabolomic analysis\u003c/h2\u003e \u003cp\u003eLeaves from six-week-old susceptible pepper cultivar (HNUCB0081), moderately resistant pepper cultivar (HNUCB0226), and highly resistant pepper cultivar (XIAO ZHOU PI) were inoculated with sporangium suspensions at a concentration of approximately 5\u0026times;10⁵ sporangia/mL. Forty microliters of the sporangium suspension were pre-incubated in the dark at 25\u0026deg;C for 24 h and then exposed to natural light for 48 h, along with a mock control group treated with autoclaved double-deionized water. The leaves were collected, ground into a powder with liquid nitrogen, and lyophilized for 48 h. Each sample was weighed (30 mg) and mixed with 1 mL of 70% methanol, vortexed at room temperature at 100% power for 20 min, and centrifuged at 12,000 rpm at 4\u0026deg;C for 10 min. The resulting supernatants were pipetted into sterilized centrifuge tubes. One hundred microliters of supernatant were diluted with 900 \u0026micro;L of 70% methanol, vortexed, centrifuged, and 100 \u0026micro;L was used for quantitative metabolic analysis, the data for which data were obtained using Waters ACQUITY UPLC-XEVO TQ-S MS triple quadrupole liquid mass spectrometry instruments at the Metabolomics Research Center, Straits Joint Research Institute, Fujian Agriculture and Forestry University.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssays for assessing the inhibitory effects of apigenin and its glucoside derivatives on the vegetative growth of oomycetes and fungi\u003c/b\u003e \u003c/p\u003e \u003cp\u003eVegetative growth assays for \u003cem\u003eP. capsici\u003c/em\u003e and \u003cem\u003eP. sojae\u003c/em\u003e involved inoculating 4 mm media plugs containing vegetative mycelia from actively growing colonies of \u003cem\u003eP. capsici\u003c/em\u003e (LT1534) and \u003cem\u003eP. sojae\u003c/em\u003e (P6497) onto tomato juice agar (TA) medium (10% tomato juice, 0.14% CaCO\u003csub\u003e3\u003c/sub\u003e, 1.5% Agar, for \u003cem\u003eP. capsici\u003c/em\u003e) and V8 agar medium (10% V8 juice, 0.14% CaCO\u003csub\u003e3\u003c/sub\u003e, 1.5% Agar, for \u003cem\u003eP. sojae\u003c/em\u003e). These media were supplemented with varying concentrations of apigenin, apigenin-7-O-β-d-glucoside, or a 1:1 combination of both compounds. Cultures were incubated under dark conditions at 25\u0026deg;C for 4 days (for \u003cem\u003eP. capsici\u003c/em\u003e) and 10 days (for \u003cem\u003eP. sojae\u003c/em\u003e), and colony diameters were measured to assess growth.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eP. oryzae\u003c/em\u003e (Guy11), \u003cem\u003eC. siamense\u003c/em\u003e, and \u003cem\u003eF. graminearum\u003c/em\u003e (PH-1), 4 mm mycelial plugs were similarly taken from actively growing colonies and inoculated onto a complete medium (CM) for \u003cem\u003eP. oryzae\u003c/em\u003e or potato dextrose agar (PDA) for \u003cem\u003eC. siamense\u003c/em\u003e and \u003cem\u003eF. graminearum\u003c/em\u003e. These media were also supplemented with apigenin, apigenin-7-O-β-d-glucoside, or their 1:1 combination, and incubated at 28\u0026deg;C for 10 days (\u003cem\u003eP. oryzae\u003c/em\u003e), 7 days (\u003cem\u003eC. siamense\u003c/em\u003e), and 4 days (\u003cem\u003eF. graminearum\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eThe inhibitory effects of apigenin, apigenin-7-O-β-d-glucoside, and their combination on the vegetative growth of \u003cem\u003eP. capsici\u003c/em\u003e, \u003cem\u003eP. sojae\u003c/em\u003e, \u003cem\u003eP. oryzae\u003c/em\u003e, C. \u003cem\u003esiamense\u003c/em\u003e, and \u003cem\u003eF. graminearum\u003c/em\u003e were evaluated by measuring colony diameters on their respective culture media.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethod for evaluating the efficacy of apigenin and its derivatives against\u003c/b\u003e \u003cb\u003eP. capsici\u003c/b\u003e \u003cb\u003esporangium formation, encystment, and zoospore release\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe LT1534 strain was cultured on a TA medium supplemented with different concentrations of apigenin, apigenin-7-O-β-d-glucoside, and a cocktail preparation of both compounds. The treated strains were incubated at 28\u0026deg;C in darkness for 4 days. The plates were further incubated under continuous light for 1\u0026ndash;2 days to maximize sporulation. The sporangia were washed from the plates with sterile water, and the final volume of the sporangium suspension was adjusted to 1 mL. The suspension was mixed thoroughly, and 10 \u0026micro;L was placed on a hemocytometer and counted under a light microscope.\u003c/p\u003e \u003cp\u003eFor spore encystment and zoospore release, the sporangia were washed from the plates with apigenin, apigenin-7-O-β-d-glucoside, or their combination (1:1 mole ratio) and filtered with sterilized Miracloth to generate sporangium suspensions with a concentration of 1\u0026times;10⁵ sporangia/mL. A 20 \u0026micro;L suspension was inoculated onto hydrophobic coverslips, kept in a moist chamber, and incubated at 4\u0026deg;C for 30 or 60 min. Microscopic assessment of encystment, zoospore release, and survival in response to apigenin, apigenin-7-O-β-d-glucoside, or their combination was performed at 30- and 60 min post-inoculation (mpi) using a light microscope.\u003c/p\u003e \u003cp\u003eFor zoospore germination, sporangia were washed from culture plates with sterilized water and incubated at 4\u0026deg;C for 30 min to induce encystment and zoospore release. The zoospores were filtered through two layers of Miracloth to obtain zoospore suspensions. The concentration of zoospores in suspensions was adjusted to 1\u0026times;10⁵zoospores/mL. Zoospores in suspensions were treated with different concentrations of apigenin, apigenin-7-O-β-d-glucoside, and a combination of both, and 20 \u0026micro;L of the suspension was inoculated on hydrophobic coverslips, placed in moist sample boxes, and incubated at room temperature. The influence of different concentrations of apigenin, apigenin-7-O-β-d-glucoside, and their combination on the germination efficiency of zoospores was monitored at 30 and 60 mpi under a light microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssays to evaluate the effects of apigenin on the pathogenicity and virulence of\u003c/b\u003e \u003cb\u003eP. capsici\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSix-week-old pepper seedlings, at the 5\u0026ndash;8 leaf stage and susceptible to \u003cem\u003eP. capsici\u003c/em\u003e, were pre-sprayed with different concentrations of apigenin, apigenin-7-O-β-d-glucoside, and their combination, and incubated in the dark for 30 min. The pre-treated leaves were inoculated with sporangium suspensions at a final concentration of 3x10⁵ sporangia/mL. For each treatment, 20 \u0026micro;L of sporangia suspension was inoculated on the pre-treated leaves and non-treated control groups. The inoculated seedlings were incubated in a dark growth chamber at 27\u0026deg;C with 80\u0026ndash;95% humidity for 24 h and later transferred to a growth chamber with 12h light-dark cycles. Disease severity was assessed by measuring lesion diameters on infected leaves.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eScanning electron microscopy and transmission electron microscopy investigation\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eP. capsici\u003c/em\u003e LT1534 was inoculated on a TA agar medium containing varying concentrations of apigenin, apigenin-7-O-β-d-glucoside, or a mixture of both. After incubating in the dark at 25\u0026deg;C for 1 day, a mycelium sample was collected, frozen in liquid nitrogen for 10 min at -90\u0026deg;C, and coated with a gold-palladium layer. Images were captured using a Regulus 8100 (HITACHI) scanning electron microscope. For transmission electron microscopy (TEM), \u003cem\u003eP. capsici\u003c/em\u003e LT1534 was inoculated into a TA agar medium containing different concentrations of apigenin, apigenin-7-O-β-d-glucoside, or a combination of both. After a day of dark incubation at 25\u0026deg;C, a mycelium sample was fixed in 2.5% glutaraldehyde for 1 h, rinsed thrice with 0.1 M PBS (phosphate buffer saline) for 15 min each, and subsequently fixed in 0.1 M osmium tetroxide for 1\u0026ndash;2 h. The sample was rinsed with 0.1 M PBS four times for 15 min each and dehydrated in a graded ethanol series (50%, 70%, 80%, 90%, 95%, 100%) for 15 min at each step. The samples were dehydrated with acetone (2\u0026ndash;3 times, 15 min each) before embedding and staining with uranyl acetate and lead citrate. Images were acquired using an H-7650 (HITACHI) transmission electron microscope at the Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eAll statistical analyses were performed using data from at least three biological experiments, each with 3 to 5 technical replicates. Data were computed using GraphPad Prism 9.5.1 software. Paired t-tests were performed for pairwise comparisons, and non-parametric tests (e.g., Kruskal-Wallis) were used for group comparisons when assumptions for parametric ANOVA were not met. Statistical significance was denoted as follows: *P\u0026thinsp;\u0026le;\u0026thinsp;0.05, **P\u0026thinsp;\u0026le;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026le;\u0026thinsp;0.001.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAp Apigenin\u003c/p\u003e\u003cp\u003eApg Apigenin-7-O-β-d-glucoside\u003c/p\u003e\u003cp\u003eCM Complete media\u003c/p\u003e\u003cp\u003eCHI Chalcone Isomerase\u003c/p\u003e\u003cp\u003eCHS Chalcone Synthase\u003c/p\u003e\u003cp\u003eC4H Cinnamate-4-Hydroxylase\u003c/p\u003e\u003cp\u003eCW Cellular wall\u003c/p\u003e\u003cp\u003eCM Cellular membrane\u003c/p\u003e\u003cp\u003eDMSO Dimethyl sulfoxide\u003c/p\u003e\u003cp\u003eFNS Flavone synthases\u003c/p\u003e\u003cp\u003ehpi H post-inoculation\u003c/p\u003e\u003cp\u003eHPLC High High-performance liquid chromatography\u003c/p\u003e\u003cp\u003eLV Lytic vacuoles\u003c/p\u003e\u003cp\u003eLB Lipid bodies\u003c/p\u003e\u003cp\u003empi Min post-inoculation\u003c/p\u003e\u003cp\u003eMI Mitochondrion\u003c/p\u003e\u003cp\u003eN Cellular nuclei\u003c/p\u003e\u003cp\u003ePBS Phosphate buffer saline\u003c/p\u003e\u003cp\u003ePR Pathogenesis-related\u003c/p\u003e\u003cp\u003ePDA Potato dextrose agar\u003c/p\u003e\u003cp\u003ePAL Phenylalanine Ammonia-Lyase\u003c/p\u003e\u003cp\u003eRNS Reactive nitrogen species\u003c/p\u003e\u003cp\u003eROS Reactive oxygen species\u003c/p\u003e\u003cp\u003eSEM Scanning electron microscopy\u003c/p\u003e\u003cp\u003eTEM Transmission electron microscopy\u003c/p\u003e\u003cp\u003eTA Tomato-juice agar\u003c/p\u003e\u003cp\u003eUGT UDP-Glucose-7-O-Glucosyltransferase\u003c/p\u003e\u003cp\u003eUPLC Ultra performance liquid chromatography\u003c/p\u003e\u003cp\u003e4-CL 4-Coumarate Ligase\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003c/p\u003e\u003cp\u003ein this study comply with the ethical standards of China, where this research was conducted.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eAll authors have read and consented to the publication of the study.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003eSupporting information\u003c/h2\u003e \u003cp\u003eSupporting information may be found in the online version of this article.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Open Fund of the Key Laboratory of Agricultural Microbiome to JB and JN, Hainan University Research Start-up Fund to JN (KYQD(ZR)-21043) and the National Undergraduate Training Program for Innovation and Entrepreneurship research fund to BL (202410589029)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJN, JB, LL＋, and JR conceived and designed the experiment. JN, JB, and BL sourced funding for the research. JN, LL＋, JR, BL, XQ, RS and KZ performed the experiments. JN, JB, LL, MS, and WL conducted data and method validation. LL＋, JR, BL, and XQ carried out visualization. LL＋, JR, BL, XQ, WL, and LL drafted the manuscript, while JN, JB, MS, and LL revised the same. All authors contributed to the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are grateful to the members of JN laboratory for their insightful discussions.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eThe data supporting the findings of this study are available and can be obtained from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaba VY, Constantino LV, Ivamoto ST, Moreira AFP, Madeira TB, Nixdorf SL, et al. 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Host-induced gene silencing of PcCesA3 and PcOSBP1 confers resistance to \u003cem\u003ePhytophthora capsici\u003c/em\u003e in Nicotiana benthamiana through NbDCL3 and NbDCL4 processed small interfering RNAs. Int J Biol Macromol. 2022;222:1665\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams CA, Grayer RJ. Anthocyanins and other flavonoids. Nat Prod Rep. 2004;21:539\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoo J-S, Choo G-S, Yoo E-S, Kim S-H, Lee J-H, Han S-H, et al. Apigenin induces apoptosis by regulating Akt and MAPK pathways in human melanoma cell A375SM. Mol Med Rep. 2020;22:4877\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Plant immunity, Apigenin-7-O-β-d-glucoside, Apoptosis, Pathogen-host interaction, Green prevention","lastPublishedDoi":"10.21203/rs.3.rs-6565949/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6565949/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eApigenin is a natural plant flavonoid known for its remarkable biological activity. While numerous studies have documented the accumulation of glycosylated and non-glycosylated apigenin in fruits and vegetable crops, their direct and indirect effects on plant resistance remain poorly understood. This study employed non-targeted metabolomics strategies to monitor metabolome-reprogramming events in susceptible, moderately resistant, and highly resistant pepper (\u003cem\u003eCapsicum annuum)\u003c/em\u003e cultivars challenged with \u003cem\u003ePhytophthora capsici\u003c/em\u003e. Results revealed a positive correlation between the accumulation of apigenin and apigenin-7-O-β-d-glucoside in pepper seedlings inoculated with \u003cem\u003eP. capsici\u003c/em\u003e and the resistance characteristics of the individual pepper cultivars. Further, we demonstrated that both apigenin and apigenin-7-O-β-d-glucoside significantly inhibited the vegetative growth of economically important phytopathogenic oomycetes and fungi, including \u003cem\u003eP. capsici\u003c/em\u003e, \u003cem\u003ePhytophthora sojae, Pyricularia oryzae, Fusarium graminearum\u003c/em\u003e, and \u003cem\u003eColletotrichum siamense.\u003c/em\u003e These findings highlight the potential applications of glycosylated and non-glycosylated apigenin in developing biopesticides to support the environment-friendly prevention and control of critical crop fungal and oomycete diseases.\u003c/p\u003e","manuscriptTitle":"Apigenin and Apigenin-7-O-β-d-Glucoside Enhance Pepper Defense Against Phytophthora capsici by Inhibiting Pathogen Growth and Reproduction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 12:00:46","doi":"10.21203/rs.3.rs-6565949/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6ddcea51-d903-4372-ac5b-7c707ef07406","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:23:13+00:00","versionOfRecord":{"articleIdentity":"rs-6565949","link":"https://doi.org/10.1186/s42483-026-00414-z","journal":{"identity":"phytopathology-research","isVorOnly":false,"title":"Phytopathology Research"},"publishedOn":"2026-03-26 16:09:47","publishedOnDateReadable":"March 26th, 2026"},"versionCreatedAt":"2025-05-16 12:00:46","video":"","vorDoi":"10.1186/s42483-026-00414-z","vorDoiUrl":"https://doi.org/10.1186/s42483-026-00414-z","workflowStages":[]},"version":"v1","identity":"rs-6565949","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6565949","identity":"rs-6565949","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}