Antifungal Potential Of Secondary Metabolites Involved in The Interaction Between Citrus Pathogens and Cistanche

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Citrus fruits have an important impact on the world’s economy since they have the largest production compared with other fruits; for example, the global orange production for 2018/19 is forecast to reach 54.3 million tons. The major factor affecting the quality of citrus is the postharvest fungal diseases, in particular, the green mold caused by Penicillium digitatum that is responsible for 90% of citrus losses during the postharvest period1,3. To control post-harvest diseases, synthetic fungicides are widely used, causing health and environmental issues. Furthermore, some fungi strains have developed resistance to commonly used fungicides. As antifungal resistance is becoming a significant concern, the search for new bioactive compounds has an important role to bypass the extensive use of fungicides7. Microorganisms are one of the main sources for natural products with useful biological activities, i.e., potential antifungals, antibiotics, anticancer agents, surfactants. However, besides the genetic potential and diversity of microbes, many microbial biosynthetic genes are not activated in the unnatural cultivation conditions used in the laboratory and only a few parts of the metabolites are accessible10. Several strategies are implemented to overcome the limitations in natural products discovery from microbial sources, i.e., OSMAC approach10,11, the use of epigenetic modifications, and co-cultivation14. Co-cultures that mimic natural environments, exhibiting microbial competition for limited space and nutrients, have been revealed to be a major ecological force that could activate silent gene clusters and defense mechanisms that can lead to the production of bioactive secondary metabolites. Co-culture experiments are highly 1 Institute of Chemistry, University of Campinas, CP 6154, 13083-970, Campinas, SP, Brazil. 2 Center for Natural and Human Sciences, Federal University of ABC, 09210-580, Santo André, SP, Brazil. 3Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute, Jena, Germany. 4Chair of Natural Product Chemistry, Friedrich Schiller University Jena, 07743, Jena, Germany. relevant for allowing not only the identification of new compounds but also to investigate chemical events that govern interactions between microorganisms in nature. To date, there is no information about how citrus pathogen fungi interact with other microorganisms in the same environment and what are the mechanisms of attack and defense against endophytic microorganisms or other phytopathogens. This information may lead to the discovery of new secondary metabolites involved in microbial interactions and provide better knowledge about the importance of microbial ecology during the infection process. Here we show an interaction between Penicillium digitatum and Penicillium citrinum, aiming to search for new antifungal compounds that could be used to control postharvest diseases. Considering this purpose, co-culture strategy and MSI were applied to induce the production of secondary metabolites and provide initial insights concerning their biological role based on their spatial distribution in the interaction. The metabolites of interest were isolated and all the structures were elucidated based on Mass spectrometry and NMR experiments. Yet, antifungal assays and confocal microscopy analyses were performed in order to investigate the potential of the fungal metabolites as new antifungal agents.

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Materials and Methods

Fungi culture. Te P. digitatum (PD) strain used in the studies is deposited with the Spanish Type Culture Collection (CECT) under the accession code CECT20796. P. citrinum (PC) strain was provided by Sylvio Moreira Citrus Center (Cordeirópolis, SP, Brazil). P. digitatum and P. citrinum were cultivated on commercial potato dextrose agar (PDA) (Acumedia). PDA was autoclaved at 103 KPa (121 °C) for 15 min. PDA plates were stored at 25 °C for 7 days in darkness. Spores were harvested by washing the agar surface with sterile distilled water and diluting to a final concentration of 106 or 105 spore mL−1.

Co-culture growth conditions fungal spore solution (106 spore mL−1) was inoculated, on opposite sides. Te plates were incubated in darkness at 25 °C for 7 days.

For MSI analyses and confocal microscopy, the in vitro co-culture was prepared by placing a sterile microscope slide in the Petri dish, followed by the pouring of 11ml of PDA22, and the inoculums were made above the microscope slide. The plates were incubated in darkness at 25 °C for 72h. For in vivo co-culture, mature oranges (Citrus sinensis) were surface sterilized and wounded23. A small piece of PDA containing P. citrinum was inoculated in the wound site. Infected and control oranges were stored in sterile 500mL beakers, in darkness at 25 °C. After 10 days, the fruits were wounded on the opposite equatorial region of P. citrinum inoculum and infected with P. digitatum 106 spore mL−1 solution. The fruits were stored for more than 5 days in darkness at 25 °C.

Mass Spectrometry Imaging (MSI) analysis and MS image generation.

After the incubation period, the microscope slides were removed from the Petri dishes and put in a vacuum desiccator, for 1 hour, for complete agar dehydration (Angolini et al., 2015). MSI analyses were performed directly on the microscope containing the co-culture, in positive mode, using a desorption electrospray ionization (DESI) source Prosolia Model Omni Spray 2D®-3201) coupled to a Thermo Scientific QExactive® Hybrid Quadrupole-Orbitrap Mass Spectrometer. MSI data were acquired with a mass resolving power of 70.000 at m/z 200. The DESI configuration used was the same set by the previous work22. Images were generated with a bin width of Δm/z = ± 0.07 using Firefly data conversion software (version 2.1.05) and processed using BioMap software (version 3.8.0.4) developed by Novartis Institutes for BioMedical Research. In BioMap, color scaling was adjusted to a fixed value during the processing of each image. MS spectra were processed with Xcalibur software (version 3.0.63) developed by Thermo Fisher Scientific.

Extraction of metabolites from the co-culture experiments.

The whole contents of the co-culture in vitro were cut into small pieces and transferred to an Erlenmeyer flask. The extraction was performed using methanol. The flasks were sonicated for 1 h in an ultrasonic bath and vacuum filtered. The solvent was removed under reduced pressure and the final extract was stored at −20 °C.

For in vivo co-culture, the orange peels were cut (2 cm×2 cm) in the interface zone between the microorganisms, and extraction was performed with 5mL of methanol for 1h in the ultrasonic bath. Extracts were filtered, dried under N2, and stored at −20 °C.

Mass Spectrometry analysis (MS).

Extracts were diluted in methanol and analyzed on a Thermo Scientific QExactive® Hybrid Quadrupole-Orbitrap Mass Spectrometer. Analyses were performed in the positive mode with an m/z range of 115–1500, the capillary voltage of 3.4 kV, inlet capillary temperature of 280 °C, S-lens 100V. 5 µL of the sample were injected. Stationary phase: Termo Scientific column Accucore C18 2.6 µm (2.1mm×100mm). Mobile phase: 0.1% formic acid (A) and acetonitrile (B). Eluent profile (A/B): 95/5 up to 2/98 within 15min, hold for 5min, up to 95/5 within 1.2min and hold for 7.8min. The total run time was 29min for each run and the flow rate, was 0.2mLmin−1. Injection volume: 5 µL.MS/MS was performed by the collision-induced dissociation (CID) with an m/z range of 100–800 and the collision energy ranged from 10 to 50V. The samples were directly infused by electrospray with a 5.0 µL min−1 flow rate. MS and MS/MS data were processed with Xcalibur software (version 3.0.63) developed by Thermo Fisher Scientific.

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Metabolite separation (HPLC analysis).

Secondary metabolites separations were achieved using a Phenomenex column Luna 5µm Phenyl-Hexyl (250×4.6mm) and a SHIMADZU prominence HPLC LC-20AT, equipped with CBM-20A communication bus module, SPD-M20A photodiode array detector, and SIL-20A autosampler. Mobile phase: water (0.1% formic acid) (A) and acetonitrile (B). Eluent profile (A/B): 65/35 up to 50/50 within 50 min, up to 40/60 within 20 min. The total run time was 70 min and the flow rate of 1.0 ml min−1. The injection volume was 5µL. Preparative HPLC purification was performed on a Phenomenex column Luna 5µm Phenyl-Hexyl (250×10mm) using a Waters 1525 Binary HPLC Pump equipped with Waters 2998 Photodiode Array Detector and Waters Fraction Collector III using the same optimized gradient conditions. The flow rate was set at 4.7mLmin−1 and the injection volume was 200 µL.

NMR Spectroscopy.

1H NMR, 13C NMR, and 2D experiments were performed on a Bruker Avance III 500 (1H 500.13MHz and 13C 125.7MHz) and Bruker Avance III 600 (1H 600.17MHz). Deuterated chloroform (CDCl3; 7.23 ppm), dimethyl sulfoxide (DMSO; 2.50 ppm and 39.51 ppm), and tetramethylsilane (TMS; 0.0 ppm) were used as a solvent and internal reference. Chemical shifts (δ) were expressed in (ppm) and the coupling constants (J) in Hertz (Hz).

Molecular Networking analysis.

A molecular network for P. citrinum metabolites was created using the online workflow at Global Natural Products Social Molecular Networking (GNPS) (http://gnps.ucsd.edu). The data were filtered by removing all MS/MS peaks within+/−17 Da of the precursor m/z. MS/MS spectra were window filtered by choosing only the top 6 peaks in the+/−50Da window throughout the spectrum. The data were then clustered with MS-Cluster with a parent mass tolerance of 0.2 Da and an MS/MS fragment ion tolerance of 0.2Da to create consensus spectra. Further, consensus spectra that contained less than 2 spectra were discarded. A network was then created where edges were filtered to have a cosine score above 0.65 and more than 2 matched peaks. Further edges between two nodes were kept in the network only if each of the nodes appeared in each other’s respective top 10 most similar nodes. The spectra in the network were then searched against GNPS’ spectral libraries. The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.5 and at least 5 matched peaks.

Antifungal assays.

A stock solution of the co-culture extract was prepared in methanol and further diluted in PDA to the concentration of 0.5mgmL−1. 15mL of the resultant solution was poured into a Petri dish followed by the inoculation of 15μl of a 105 spore mL−1 P. digitatum solution on the center of the agar plate. A control assay was also performed. The plates were incubated in darkness at 25 °C for 96h. For antifungal assays, 2.5mL of PDA was supplemented with 6, 9, and 10 (400µgmL−1 ), and each solution was poured into a 6-well microplate. 5μl of a 105 spore mL−1 P. digitatum solution was inoculated on the center of each agar plate. Negative controls were performed in triplicate. The microplate was incubated in darkness at 25 °C for 96h. For the determination of minimum inhibitory concentration (MIC) of compounds 1 and 4, a micro broth dilution assay was performed as recommended by Clinical and Laboratory Standards Institute (2008)24 with few modifications. Stock solutions of 1 and 4, were prepared in water (5% methanol) and further diluted in YES media in a range of concentrations to 600 µg mL−1 to 1 µg mL−1. 195 μl of each solution were transferred to a 96-well microplate followed by the inoculation of 5 μl of a 105 spore mL−1 P. digitatum solution. Assays were made in duplicate and controls in triplicate. Itraconazole (100 µgmL−1 ) was used as a positive control. Negative controls were performed with methanol in YES. The microplates were incubated in darkness at 25 °C for 96h.

Confocal laser scanning microscopy.

After the incubation period, the microscopes slides were removed from the Petri dish. Te in vitro co-culture samples was stained with Congo Red (0.25% w/v in water) for 20minutes and briefly washed in distilled water. Samples were analyzed with a Leica TCS SP5 microscope. Excitation was by the 543nm emission line of the He-Ne laser, and light emitted between 570 and 680nm was collected.

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Results and Discussion

MSI reveals potential antifungals in the interaction between P. digitatum and P. citrinum.

To screen for new antifungal compounds with the potential to protect citrus fruits and control postharvest diseases, we applied a co-culture strategy involving P. digitatum and another citrus pathogen, P. citrinum. Co-culture is a strategy inspired by nature in which the competition between the microorganisms can induce the production of new metabolites. In previous work, the co-cultivation between Trichophyton rubrum and Bionectria ochroleuca induced the production of a new sulfated analog of PS-990, suggesting that this compound is further sulfated during the fungal interaction26. Also, another example of a compound derived from fungi interaction is the tetrapeptide cyclo-(L-leucyl-trans-4-hydroxy-L-prolyl-D-leucyl-trans-4-hydroxy-L-proline) isolated from the co-culture broth of Phomopsis sp. K38 and Alternaria sp. E33; the cyclic tetrapeptide exhibited moderate to high inhibitory activity against phytopathogenic fungi when compared to the commercial fungicide triadimefon27. Thus, co-cultivation experiments are a viable approach to find compounds that can inhibit the main citrus phytopathogens, especially, the green mold caused by P. digitatum.

In co-cultivation performed in both orange (in vivo) and synthetic media (in vitro) we visually observed a long-distance growth inhibition between P. citrinum and P. digitatum. In a fungi interaction, silent genes can be activated and harmful metabolites can be defused from one partner to the other16,28. Tese-induced metabolites are usually localized at the zone of confrontation in solid media of co-cultures16. However, regular approaches used to detect and elucidate metabolites such as mass spectrometry coupled to liquid (LC-MS) or gas (GC-MS) chromatography do not provide information about the spatial distribution of the molecules.

Te information about molecular spatial distribution can be obtained by mass spectrometry imaging (MSI), a powerful tool that generates images for each ion detected in the mass spectrum16. The use of MSI to understand microbial systems and their secondary metabolites is not new and studies, where this technique was successfully applied, can be found in the literature30. For example, MSI was applied to investigate the interaction between Bacillus subtilis 3610 and Streptomyces coelicolor A3. DESI imaging of the bacterial co-culture revealed 57 signals spatially localized to bacterial colonies, leading to the identification of some secondary metabolites such as surfactin and plipastatin31. Furthermore, MSI analysis also showed that S. coelicolor has the production of certain secondary metabolites inhibited in the presence of B. subtilis, revealing an interaction between bacteria31.

Therefore, to detect the secondary metabolites involved in the interaction between the citrus pathogens, we applied DESI-MSI directly on the surface of the co-culture agar, to visualize the diffusion of compounds to the zone of confrontation. MSI signals were obtained for ions [M+H]+ at m/z 519.1857, m/z 503.1908, m/z 475.1590, m/z 460.1960, m/z 459.1645, m/z 625.3942, m/z 609.3988, m/z 527.2862, m/z 511.2894 and m/z 448.2938 (Figs. S1–S10). We observed that all the ions mentioned were detected and concentrated in the zone of confrontation between the fungi (Fig. 1). These compounds may be related to the fungus-fungus interaction and could be potentially new antimicrobial agents. Ions at m/z 625, 609, 527, 511, and 448 were produced by P. citrinum, while ions at m/z 519, 503, 475, 460, and 459, seemed to be a counter-attack of P. digitatum, revealing chemical warfare between these two citrus pathogens. Te ions detected in vitro through MSI analyses were also detected in the extracts of the co-cultures in vivo (Figs. S11–S20) using oranges as substrate (Fig. 2).

To characterize the metabolites involved in the interaction, a co-culture extract was obtained from a scale-up cultivation experiment and the compounds of interest, detected initially through MSI analyses, were isolated by preparative HPLC and characterized through tandem mass spectrometry and NMR analyses.

Through the exact masses obtained by DESI-MSI analyses (Table 1), it was possible to confirm the presence of indole alkaloids produced by P. digitatum. Ions [M+H]+ at m/z 519.1857, m/z 503.1908, m/z 475.1590, m/z 460.1960 and m/z 459.1645 which correspond respectively to tryptoquialanine A (1), tryptoquialanine C (2), tryptoquialanone (3), 15-dimethyl-2-epi-fumiquinazoline A (4) and deoxytryptoquialanone (5) (chemical structures represented in Fig. 3). These compounds are part of the tryptoquialanines biosynthetic pathway32 and were previously detected during DESI-MSI analysis of oranges infected with the green mold disease23. Compound 1 was reported as the major secondary metabolite produced by P. digitatum33. Yet, the deletion of that gene, responsible to regulate 1 biosynthesis, showed that 1 is not involved in the pathogenicity of P. digitatum against citrus since the infection ability of the mutants was not altered34. The exact biological role of the tryptoquialanines is still unknown34 and this study provides a new biological activity of the tryptoquialanines with the involvement of these alkaloids in the fungal interaction. The MS/MS of the ion [M+H]+ at m/z 625.3951 yielded to fragments at m/z 594.3533, 576.3427, 449.2430, and 431.2325 (Fig. S21), the same fragmentation pattern obtained for citrinin A (6) in a study involving co-culture between P. citrinum and Pseudoalteromonas sp. OT5919. Also, the GNPS database suggested that the ion at m/z 625 could be citrinin A. Te tandem mass spectrum obtained for this compound shared five fragments in common with the tandem mass spectrum of 6 deposited in the database (Fig. S22). In addition, it shares a similar MS/MS fragmentation pattern with citrinin A reported by Moree et al. (2014). Citrinadins were reported being higher produced in co-culture situations and concentrated in co-culture interfaces, suggesting a defensive response of P. citrinum against other micro-organisms Molecular networking analysis of the isolated compounds revealed that another citrinin is involved in the co-culture interaction. We observed that ion at m/z 609, detected initially by MSI analysis, is grouped in the same cluster of compound 6 (m/z 625) (Fig. S23), suggesting that this compound is a citrinin-like metabolite. In a molecular networking analysis, related metabolites are grouped in the same clusters since they have a similar MS/MS spectrum.

The exact mass obtained for the ion [M+H]+ at m/z 609.4010 corresponds to a compound with molecular formula C35H53N4O5. Fragmentation pattern yielded to fragments at m/z 578.3583, 464.2905, 451.2586, and 433.2482 (Supplementary Fig. S24). 1 H NMR and 1 H-1 H COSY spectra obtained for the purified metabolite (Figs. S25–S26 and Table S1) exhibited similar signals of a synthesized deoxycitrinadin A that was reported by Bian et al. (2013)37. Te epoxide characteristic signal at δH 4.0 was absent in this derivative and a signal for vynilic hydrogen could be observed at δH 6.9, indicating the lack of the epoxide group and the presence of a double bond in comparison with citrinin A37. It is the first time that deoxycitrinadin A (7) is reported in the literature as a secondary metabolite produced by a microorganism.

Exact masses obtained by DESI-MSI for ions [M+H]+ at m/z 527.2862 and 511.2894 indicated compounds with the elemental composition of C28H38N4O6 and C28H38N4O5, respectively. MS/MS revealed that the ion at m/z 511 has a similar structure compared to m/z 527, except by an absence of an oxygen atom. Fragmentation of the ion at m/z 527 yielded to fragments m/z 281, 247, 219, 182, and 120 (Fig. S27), while ion at m/z 511 yielded to m/z 265, 247, 219, 166, and 120 (Fig. S30). The same fragmentation pattern was reported for the sequence of tetrapeptides Phe-Val-Val-Tyr (8) of Penicillium canescens. Yet, the tetrapeptides 8 and Phe-Val-Val-Phe (9) were recently reported as secondary metabolites produced by Penicillium roqueforti. 1 H and 13C NMR analyses of the isolated compounds (Figs. S28–S32 and Tables S2-S3) confirmed the results obtained through MS/MS, concluding that ions at m/z 527 and m/z 511 correspond to 8 and 9, respectively. The production of these tetrapeptides in fungal chemical warfare is not surprising because small peptides are known for their antimicrobial activity40. This is the first report of 8 and 9 as secondary metabolites of P. citrinum. For ion [M+H]+ at m/z 448.2938, the exact mass suggested a compound with molecular formula C28H38N3O2, the same composition of the secondary metabolite chrysogenamide A (10) (error=−4.6 ppm). Compound 10 was isolated and 1 H and 13C NMR analyses (Figs. S33–S34 and Table S4) confirmed its structure; it is the first report that chrysogenamide A is involved in a fungal interaction. Compound 10 was first reported as a secondary metabolite of Penicillium chrysogenum No. 005, an endophytic fungus associated with the plant Cistanche deserticola. Also, 10 was reported as a secondary metabolite of a P. citrinum strain42. The structures of the metabolites produced by P. citrinum are represented in Fig. 4.

Antifungal assays.

We investigated the antifungal activity of the metabolites produced in the co-culture and P. digitatum was inoculated in agar media containing co-culture extract. Compared to the control, we observed a reduction of 67% in P. digitatum radial growth (Fig. S35), indicating that the metabolites involved in the fungal warfare have potential as antifungal agents. To confirm this hypothesis, we tested the isolated compounds and observed that compounds 6, 9, and 10 (400 µg mL−1 ) reduced 48%, 41%, and 61% of P. digitatum radial growth, respectively, when compared to control (Fig. 5), confirming the antifungal activity. Citrinadins were found to be involved in the response of P. citrinum against other microorganisms in co-cultures, but their biological role in biological environments is still unknown to this date19. Compound 6 was tested for Anti-Buruli ulcer activity on Mycobacterium ulcerans MN209, but no interesting MIC was observed43; also, cytotoxicity activity of 6 against leukemia and carcinoma cells was reported44. Our results are the first report of antimicrobial activity of citrinadins in literature and can provide the first insights into the biological role of these compounds in fungal-fungal interactions. In addition, chrysogenamide A was never reported as an antimicrobial agent until now. Compound 10 exhibited a protective effect on neurocytes against oxidative stress-induced cell death41, however, no other biological activity for 10 was reported in the literature. Antifungal assays applying D-Phe-L-Val-D-Val-L-Tyr revealed that this tetrapeptide has inhibitory activity against B. subtitles and the soybean phytopathogen Fusarium virguliforme38. In contrast, no inhibition of E. coli, B. subtilis, and S. cerevisiae in presence of D-Phe-L-Val-D-Val-L-Tyr or D-Phe-L-Val-D-Val-L-Phe was observed39. The antifungal activity of the tryptoquialanines was also evaluated since these compounds seemed to be a chemical response of P. digitatum against P. citrinum in chemical warfare. Tryptoquialanines 1 and 4 were tested and revealed an antifungal activity against P. citrinum. Compounds 1 and 4 had MIC of 300µgmL−1, inhibiting P. citrinum spore production (Fig. S36). To the best of our knowledge, it is the first report of antimicrobial activity of the tryptoquialanines. Recently, 1 was demonstrated as an insecticidal compound against Ae. aegypti larvae23; the antifungal activity can provide more understanding about the role of the tryptoquialanines in the citrus-pathogen environment once these compounds are not required for P. digitatum virulence.

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Chemical warfare alters P. digitatum cell wall in co-culture.

To investigate the action of the secondary metabolites during the fungal interaction, co-culture samples were stained with Congo Red and observed through confocal laser scanning microscopy. Congo Red is commonly used to stain polysaccharides containing β 1,4 linkages as, for example, the fungal cell wall component chitin45,46. P. digitatum hyphae were observed in the confrontation zone (sample) and compared with hyphae distant to the interface region (control) (Fig. S37). Control hyphae were homogeneously stained with Congo Red while hyphae in the interface region exhibited an altered staining pattern (Fig. 6), with irregular patches. Similar staining patterns were obtained for knockout mutant fungi in which the deleted genes had a role in cell wall organization; as result, mutants exhibited defective cell walls and irregular staining25,46,47. Yet, abnormal staining with Calcofluor White was observed for P. ostreatus P89 treated with 36 °C; high temperature altered chitin distribution and cell wall integrity48. This data shows that P. digitatum hyphae, in contact with the metabolites diffused during the co-culture, have a defective cell wall since Congo Red bounds to fungal cell wall structures. The fungal cell wall is an attractive target of antimicrobials because they are not present in mammalian cells49,50. In conclusion, the microscopy analysis and the antifungal assays reinforce that the metabolites involved in the fungal interaction have potential as antifungal agents and may be the mechanism in nature that these phytopathogens developed to compete against other microorganisms for the host (Fig. 7).

Conclusions

The search for new natural antimicrobials is a promising field in natural products research concerning the economic impact of postharvest diseases on worldwide agriculture. Furthermore, the appearance of fungi strains resistant to fungicides makes the discovery of new antifungal agents to replace synthetic compounds extremely importantly. Using co-cultivation between phytopathogens that compete for the same host, P. digitatum and P. citrinum, we observed a fungal interaction. Through the MSI technique, we detected secondary metabolites defused to the interface zone between the microorganisms. Tryptoquialanines, citrinadins, chyrsogenamide A and tetrapeptides exhibited great antifungal activity, confirming that co-cultures and MSI technique are a good combination in the search of new natural antimicrobials.

Until this date, there has been no information about the interaction between citrus pathogenic fungi. Our data revealed compounds that play a role in the citrus microbial ecology. In addition, we demonstrated that the metabolites studied have great potential as antifungal agents since fungal cell walls are one of the main targets of antifungal compounds. The use of the identified compounds as natural antifungals instead of synthetic fungicides should be further investigated. This paper opens new research possibilities and contributes to environmental and human health, helping in the search for safer strategies for agriculture through the use of compounds obtained from natural sources.

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