Identification of Biomarkers Associated with Phyllosticta citricarpa Tolerance.

Citrus black spot (CBS) is a fungal disease caused by Phyllosticta citricarpa Kiely, (McAlpine Van der Aa), with most cultivars being susceptible to infection. Currently, disease control is based on the application of protective fungicides, which is restricted due to resistance, health and environmental concerns. Although using natural products for disease management is gaining momentum, more advances are required. This study obtained the metabolic profiles of the essential oil and cuticular waxes of two citrus cultivars with a varying susceptibility to CBS infection using gas chromatography-mass spectrometry. A multivariate data analysis identified possible biomarker compounds that contributed to the difference in susceptibility between the two cultivars. Several identified biomarkers were tested in vitro for their antifungal properties against P. citricarpa. Two biomarkers, propanoic acid and linalool, were able to completely inhibit pathogen growth at 750 mg/L and 2000 mg/L, respectively.

Bioassay-Guided Identification of Natural Products for Biocontrol by Thin Layer Chromatography-Direct Bioautography.

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are antagonistic against a target pathogen. It is a rapid, inexpensive, and simple option for the bioassay-guided isolation and identification of NPs that hinges on separation by TLC coupled with the direct application of a target pathogen to examine bioactivity. It is typically used for the analysis of bioactive plant extracts, detecting inhibitory activity against bacteria, fungi, and enzymes. That being said, it has great potential in bacterial NP discovery, particularly for evaluating bacterial NPs against pertinent agricultural pathogens, which is valuable for discovering and developing novel biopesticides for the agriculture industry. Furthermore, it is a tunable protocol that could be applied to other target pathogens or sources of NPs in research programs concerning the discovery and identification of bioactive compounds. Herein, we describe a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum.

Amorosia littoralis Mantle & D. Hawksw. survives as root endophyte of mangroves and produces water soluble melanin pigment.

Melanins are heterogenous biopolymers produced by many macro and microorganisms. They enhance the ecological fitness of the producer organisms by improving their virulence and protecting them from radiation, osmotic and heat stresses. Fungi synthesise either DOPA or DHN melanin and deposit them on their cell walls. Some fungal isolates produce water soluble melanin (pyomelanin) which is excerted out of the hyphae into the surrounding environment. Pyomelanin, a polymer of homogentisate, exhibits antimicrobial, UV screening, antioxidative, and anti-inflammatory activities and recent studies also show that pyomelanin could find use in space travel as radiation shield. However, scant literature is available on fungi with ability to produce pyomelanin. We report for the first time that Amorosia littoralis occurs as a root endophyte in mangrove species including Avicennia marina, Bruguiera cylindrica and Bruguiera gymnorhiza and produces pyomelanin. Considering the various technological uses of pyomelanin, this study underscores the need to explore fungi of different habitats to identify hyperproducers and to obtain chemically diverse pyomelanin.

Discovery of Novel Antioomycete Metabolites from the Marine-Derived Fungus Paraconiothyrium sporulosum.

In our screening program for natural products that are effective in controlling plant diseases, we found that the culture filtrate of Paraconiothyrium sporulosum SFC20160907-M11 effectively suppressed the development of tomato late blight disease caused by Phytophthora infestans. Using a bioassay-guided fractionation of antioomycete activity, 12 active compounds (1-12) were obtained from an ethyl acetate extract of the culture filtrate. Chemical structures of five new compounds 1-5 were determined by the extensive analyses of nuclear magnetic resonance (NMR), high resolution mass spectrometry (HRMS), and circular dichroism (CD) data. Interestingly, mycosporulonol (1) and botrallin (8) completely inhibited the growth of P. infestans at concentrations of 8 and 16 µg/mL, respectively. Furthermore, the spray treatment of 1 and 8 (500 µg/mL) successfully protected tomato seedlings against P. infestans with disease control values of 92%. Taken together, these results suggest that the culture filtrates of P. sporulosum SFC20160907-M11 and their bioactive metabolites can be used as new antioomycete agents for Phytophthora late blight control.

Structure Revision of the Fungal Phytotoxin Cavoxin and of Its Corresponding Chroman-4-one Cavoxone by X-ray Crystallography.

Cavoxin (1) was isolated as the main phytotoxin produced by Phoma cava Schulzer, a toxigenic fungus isolated from Castanea spp. Its structure was determined by 1D NMR and MS in 1985 along with that of the corresponding chroman-4-one cavoxone (2), an artifact formed by acid treatment of 1. Since that time cavoxin was shown to be phytotoxic, antifungal, antifeedant, herbicidal, and antirust with potential application in agriculture and medicine. During a study aimed at improving cavoxin's production by P. cava, single crystals for X-ray diffractometric analysis were obtained. The X-ray crystallography characterization confirmed only in part the structure proposed for cavoxin (1), revealing a different substitution pattern on the aromatic ring, as depicted in the revised structure 3.

Expanding the Structural Diversity of Tubulin-Targeting Agents: Development of Highly Potent Benzimidazoles for Treating Fungal Diseases.

Benzimidazoles, the representative pharmacophore of fungicides, have excellent antifungal potency, but their simple structure and single site of action have hindered their wider application in agriculture. In order to extend the structural diversity of tubulin-targeted benzimidazoles, novel benzimidazole derivatives were prepared by introducing the attractive pyrimidine pharmacophore. 2-((6-(4-(trifluoromethyl)phenoxy)pyrimidin-4-yl)thio)-1H-benzo[d]imidazole (A25) exhibited optimal antifungal activity against Sclerotinia sclerotiorum (S. s.), affording an excellent half-maximal effective concentration (EC50) of 0.158 µg/mL, which was higher than that of the reference agent carbendazim (EC50 = 0.594 µg/mL). Pot experiments revealed that compound A25 (200 µg/mL) had acceptable protective activity (84.7%) and curative activity (78.1%), which were comparable with that of carbendazim (protective activity: 90.8%; curative activity: 69.9%). Molecular docking displayed that multiple hydrogen bonds and π-π interactions could be formed between A25 and ß-tubulin, resulting in a stronger bonding effect than carbendazim. Fluorescence imaging revealed that the structure of intracellular microtubules can be changed significantly after A25 treatment. Overall, these remarkable antifungal profiles of constructed novel benzimidazole derivatives could facilitate the application of novel microtubule-targeting agents.

Determination of the amino linkage of the D-Dab residue of the cyclic lipo-octapeptide occidiofungins A-D and the antifungal activity of their analogues.

Recently, the demand for new antifungal drugs has increased due to the presence of antimicrobial resistant bacteria and their side effects. Occidiofungins (Ocfs) are cyclic lipo-octapeptides that possess unusual amino acids and potent antifungal activities. However, the chemical structure of the 2,4-diamino butyric acid (Dab) residue in the backbone of Ocfs has not been clarified thus far. Therefore, we conducted a structural analysis of the tripeptides around the Dab residue in Ocfs using 1H-NMR spectroscopy. We determined that the D-Dab residue in the peptide backbone of Ocfs has an α-amino linkage. Additionally, we found that Ocf A (5) and Bk-1119 have the same chemical structure. Moreover, the analogue possessing D-αDab (13) showed potent antifungal activity against A. oryzae.

Degradation-Risk-Inspired Optimization of the Antifungal Oxazolinyl Aniline Lead by a Fusion of Triazole with Nicotinamide.

The discovery of readily available and easily modifiable new models is a crucial and practical solution for agrochemical innovation. Antifungal function-oriented fusion of triazole with the prevalidated lead (R)-LE001 affords a novel framework with a broad and enhanced antifungal spectrum. Characterized by the easy accessibility and adjustability of [1,2,4]triazolo[4,3-a]pyridine, modular fine-tuning provided a set of unprecedented leads (e.g., Z23, Z25, Z26, etc.) with superior antifungal potentials than the positive control boscalid. Candidate Z23 exhibited a more promising antifungal activity against Sclerotinia sclerotiorum, Botrytis cinerea, and Phytophthora capsici with EC50 values of 0.7, 0.6, and 0.5 µM, respectively. This candidate could effectively control boscalid-resistant B. cinerea strains and also exhibit good vivo efficacy in controlling gray mold. Noteworthily, both the SDH-inhibition and the efficiency against Oomycete P. capsici are quite distinct from that of the positive control boscalid. A molecular docking simulation also differentiates Z23 from boscalid. These findings highlight the potential of [1,2,4]triazolo[4,3-a]pyridine amide as a novel antifungal model.

Discovery of Novel Acethydrazide-Containing Flavonol Derivatives as Potential Antifungal Agents.

In this study, a series of novel hydrazide-containing flavonol derivatives was designed, synthesized, and evaluated for antifungal activity. In the in vitro antifungal assay, most of the target compounds exhibited potent antifungal activity against seven tested phytopathogenic fungi. In particular, compound C32 showed the best antifungal activity against Rhizoctonia solani (EC50 = 0.170 µg/mL), outperforming carbendazim (EC50 = 0.360 µg/mL) and boscalid (EC50 = 1.36 µg/mL). Compound C24 exhibited excellent antifungal activity against Valsa mali, Botrytis cinerea, and Alternaria alternata with EC50 values of 0.590, 0.870, and 1.71 µg/mL, respectively. The in vivo experiments revealed that compounds C32 and C24 were potential novel agricultural antifungals. 3D quantitative structure-activity relationship (3D-QSAR) models were used to analyze the structure-activity relationships of these compounds. The analysis results indicated that introducing appropriate electronegative groups at position 4 of a benzene ring could effectively improve the anti-R. solani activity. In the antifungal mechanism study, scanning electron microscopy and transmission electron microscopy analyses revealed that C32 disrupted the normal growth of hyphae by affecting the structural integrity of the cell membrane and cellular respiration. Furthermore, compound C32 exhibited potent succinate dehydrogenase (SDH) inhibitory activity (IC50 = 8.42 µM), surpassing that of the SDH fungicide boscalid (IC50 = 15.6 µM). The molecular dynamics simulations and docking experiments suggested that compound C32 can occupy the active site and form strong interactions with the key residues of SDH. Our findings have great potential for aiding future research on plant disease control in agriculture.

Evaluation of Ointments with Daldinia eschscholtzii in Wound Healing in an In Vivo Model.

Fungi are a source of a variety of secondary metabolites of importance in different areas of biotechnology. Several compounds have been characterized with antioxidant, antimicrobial, and anti-inflammatory activity from fungi of the division of the Ascomycota, among which is the species Daldinia eschscholtzii, an endophyte fungus of pantropical distribution. In this study, we evaluated the effect of an ointment made with D. eschscholtzii on the wound healing of BALB/c mice. The species was corroborated using a molecular marker Internal Transcribed Spacer (ITS1 and ITS4). The extracts and dust of the fungus were considered nontoxic as they caused a mortality of <15% in the nematode Panagrellus redivivus, and experimental ointments had no adverse effects on the skin of BALB/c mice. Wounds treated with the D. eschscholtzii ointments had 99.9-100% wound contraction after 17 days, which was similar to commercial healing (positive control). As such, the ointment of D. eschscholtzii is a natural alternative to improve wound healing.

Antifungal effects of volatile organic compounds produced by Trichoderma hamatum against Neocosmospora solani.

Neocosmospora solani causes Fusarium wilt disease and root rot, which are serious problems worldwide. To determine the growth inhibition of Neocosmospora solani by Trichoderma hamatum volatile organic compounds (VOCs), the major chemical components of Trichoderma hamatum VOCs and the differences in their contents at different times were analysed, and the activity of these components was evaluated. The antifungal activity of Trichoderma hamatum was measured by a screening test, as Trichoderma hamatum exhibited strong antagonism against Neocosmospora solani in vitro. The double plate technique was used to verify the activity of Trichoderma hamatum VOCs, and the inhibition rate was 63.77%. Neocosmospora solani mycelia were uneven and expanded, the contents of the cells leaked, and the mycelia shrank and presented a diaphragm in the hyphae upon Trichoderma hamatum VOCs treatment. Trichoderma hamatum VOCs and their contents at different times were analysed by using gas chromatography-mass spectrometry. 6-Pentyl-2H-pyran-2-one clearly presented in greater amounts than the other components on day 3, 4, 5, and 6. VOCs from Trichoderma hamatum exhibited evident effects on the percentage of healthy fruit after day 3. Moreover, Trichoderma hamatum can improve the biological control of diseases caused by soilborne pathogens, and can be applied in biocontrol fields.

Escuzarmycins A-D, Potent Biofungicides to Control Septoria tritici Blotch.

A study targeting novel antifungal metabolites identified potent in vitro antifungal activity against key plant pathogens in acetone extracts of Streptomyces sp. strain CA-296093. Feature-based molecular networking revealed the presence in this extract of antimycin-related compounds, leading to the isolation of four new compounds: escuzarmycins A-D (1-4). Extensive structural elucidation, employing 1D and 2D NMR, high-resolution mass spectrometry, Marfey's analysis, and NOESY correlations, confirmed their structures. The bioactivity of these compounds was tested against six fungal phytopathogens, and compounds 3 and 4 demonstrated strong efficacy, particularly against Zymoseptoria tritici, with compound 3 exhibiting the highest potency (EC50: 11 nM). Both compounds also displayed significant antifungal activity against Botrytis cinerea and Colletotrichum acutatum, with compound 4 proving to be the most potent. Despite moderate cytotoxicity against the human cancer cell line HepG2, compounds 3 and 4 emerge as promising fungicides for combating Septoria tritici blotch, anthracnose, and gray mold.

Onychocolone A produced by the fungus Onychocola sp. targets cancer stem cells and stops pancreatic cancer progression by inhibiting MEK2-dependent cell signaling.

Pancreatic cancer (PC) shows a high fatality rate that can only be faced with a combination of surgery and chemotherapy or palliative treatment in the case of advanced patients. Besides, PC tumors are enriched with subpopulations of cancer stem cells (CSCs) that are resistant to the existing chemotherapeutic agents, which raises an important need for the identification of new drugs. To fill this gap, we have tested the anti-tumoral activity of microbial extracts, which chemical diversity offers a broad spectrum of potential new bioactive compounds. Extracts derived from the fungus Onychocola sp. CF-107644 were assayed via high throughput screening followed by bioassay-guided fractionation and resulted in the identification and isolation of six benzophenone derivatives with antitumoral activity: onychocolones A-F (#1-6). The structures of the compounds were established by spectroscopic methods, including ESI-TOF MS, 1D and 2D NMR analyses and X-ray diffraction. Compounds #1-4 significantly inhibited the growth of the pancreas tumoral cell lines, with low-micromolar Median Effective Doses (ED50s). Compound #1 (onychocolone A) was prioritized for further profiling due to its pro-apoptotic effect, which was further validated on 3D spheroids and pancreatic CSCs. Protein expression assays showed that the effect was mechanistically linked to the inhibition of MEK onco-signaling pathway. The efficacy of onychocolone A was also demonstrated in vivo by the reduction of tumor growth in a pancreatic xenograft mouse model generated by CSCs. Altogether, the data support that onychocolone A is a promising new small molecule for hit-to-lead development of a new treatment for PC.

Formation of Biogenic Manganese Oxide Nodules on Hyphae of a New Fungal Isolate of Periconia That Immobilizes Aqueous Copper.

Mn(II)-oxidizing microorganisms are considered to play significant roles in the natural geochemical cycles of Mn and other heavy metals because the insoluble biogenic Mn oxides (BMOs) that are produced by these microorganisms adsorb other dissolved heavy metals and immobilize them as precipitates. In the present study, a new Mn(II)-oxidizing fungal strain belonging to the ascomycete genus Periconia, a well-studied plant-associating fungal genus with Mn(II)-oxidizing activity that has not yet been exami-ned in detail, was isolated from natural groundwater outflow sediment. This isolate, named strain TS-2, was confirmed to oxidize dissolved Mn(II) and produce insoluble BMOs that formed characteristic, separately-located nodules on their hyphae while leaving major areas of the hyphae free from encrustation. These BMO nodules also adsorbed and immobilized dissolved Cu(II), a model analyte of heavy metals, as evidenced by elemental mapping ana-lyses of fungal hyphae-BMO assemblages using a scanning electron microscope with energy-dispersive X-ray spectroscopy (SEM-EDX). Analyses of functional genes within the whole genome of strain TS-2 further revealed the presence of multiple genes predicted to encode laccases/multicopper oxidases that were potentially responsible for Mn(II) oxidation by this strain. The formation of BMO nodules may have functioned to prevent the complete encrustation of fungal hyphae, thereby enabling the control of heavy metal concentrations in their local microenvironments while maintaining hyphal functionality. The present results will expand our knowledge of the physiological and morphological traits of Mn(II)-oxidizing Periconia, which may affect the natural cycle of heavy metals through their immobilization.

Six undescribed 23-norursane triterpenoids from the biotransformation of ilexgenin a by endophytic fungi and their vascular protective activity.

Biotransformation of ursane-type triterpenoid ilexgenin A by endophytic fungi Lasiodiplodia sp. MQD-4 and Pestalotiopsis sp. ZZ-1, isolated from Ilex pubescences and Callicarpa kwangtungensis respectively, was investigated for the first time. Six previously undescribed metabolites (1-6) with 23-norursane triterpenoids skeleton were isolated and their structures were unambiguously established by the analysis of spectroscopic data and single-crystal X-ray crystallographic experiments. Decarboxylation, oxidation, and hydroxylation reactions were observed on the triterpenoid skeleton. Especially, the decarboxylation of C-23 provided definite evidence to understand the biogenetic process of 23-norursane triterpenoids. Moreover, the qualitative analysis of the extract of I. pubescences showed metabolites 1, 3, 4, and 6 could be detected in the originated plant, indicating biotransformation by endophytic fungi is a practical strategy for the isolation of novel natural products. Finally, all isolates were evaluated for the protective activities against H2O2-induced HUVECs dysfunction in vitro. Compound 5 could improve the viability of endothelial cells and decrease the level of intracellular ROS.

Bioactivity-Based Molecular Networking-Guided Isolation of Epicolidines A-C from the Endophytic Fungus Epicoccum sp. 1-042.

Bioactivity-based molecular networking-guided fractionation enabled the isolation of three new polycyclic tetramic acids bearing cis-decalin, epicolidines A-C (1-3), along with one known compound, PF 1052 (4), from the endophytic fungus Epicoccum sp. 1-042 collected in Tibet, China. Their structures were assigned on the basis of extensive spectroscopic data, partial hydrolysis, advanced Marfey's method, quantum chemistry calculations, and X-ray diffraction analysis. Compounds 2-4 displayed promising activities against Gram-positive bacteria in vitro. Particularly, compound 4 displayed remarkable potential against vancomycin-resistant Enterococcus faecium (VRE) with an MIC value of 0.25 µg/mL, lower than the MIC (0.5 µg/mL) of the antibiotic combination quinupristin/dalfopristin (Q/D). In a further in vivo study, compound 4 increased the survival rate to 100% in the VRE-G. mellonella infection model at a concentration of 10 mg/kg.

Xylarkarynone A and B: Two Bioactive Eremophilane Sesquiterpenes from Xylaria sp. HHY-2.

A new compound xylarkarynone A (1), a first reported natural product compound xylarkarynone B (2) and eight known compounds (3-10) were isolated from Xylaria sp. HHY-2. Their structures were elucidated by spectroscopic methods, DP4+ probability analyses and electronic circular dichroism (ECD) calculations. The bioactivities of isolated compounds were assayed. Compound 1 exhibited obvious activity against A549 cells with an IC50 value of 6.12±0.28 µM. Additionally, compound 1 showed moderate antifungal activities against Plectosphaerella cucumerina and Aspergillus niger with minimum inhibitory concentrations (MICs) of both 16 µg/mL, which was at the same grade with positive control nystatin. Most compounds exhibited varying degrees of inhibitory activity against P. cucumerina, indicating that Xylaria sp. has potential as inhibitors against P. cucumerina.

Bioactive Alkaloids from the Mangrove-Derived Fungus Nigrospora oryzae SYSU-MS0024.

Chemical investigation of marine fungus Nigrospora oryzae SYSU-MS0024 cultured on solid-rice medium led to the isolation of three new alkaloids, including a pair of epimers, nigrosporines A (1) and B (2), and a pair of enantiomers, (+)-nigrosporine C (+)-3, and (-)-nigrosporine C (-)-3, together with eight known compounds (4-11). Their structures were elucidated based on extensive mass spectrometry (MS) and 1D/2D nuclear magnetic resonance (NMR) spectroscopic analyses and compared with data in the literature. The absolute configurations of compounds 1-3 were determined by a combination of electronic circular dichroism (ECD) calculations, Mosher's method, and X-ray single-crystal diffraction technique using Cu Kα radiation. In bioassays, compound 2 exhibited moderate inhibition on NO accumulation induced by lipopolysaccharide (LPS) on BV-2 cells in a dose-dependent manner at 20, 50, and 100 µmol/L and without cytotoxicity in a concentration of 100.0 µmol/L. Moreover, compound 2 also showed moderate acetylcholinesterase (AChE) inhibitory activities with IC50 values of 103.7 µmol/L. Compound 5 exhibited moderate antioxidant activity with EC50 values of 167.0 µmol/L.

Eudistomins Y-Inspired Design and Divergent Optimization of Heteroaryl Ketones for New Antifungal Leads.

The discovery of structurally distinct leads is imperative in modern agrochemical science. Inspired by eudistomins Y and the framework-related pharmaceuticals, aryl heteroaryl ketone was drawn as a common model intriguing the design and divergent synthesis of 14 kinds of heteroaryl ketones aligned with their oxime derivatives. Antifungal function-oriented phenotypical screen protruded benzothiazolyl-phenyl oxime 5a as a promising model, and the concomitant modification led to benzothiazolyl oxime 5am (EC50 = 5.17 µM) as a superior lead than fluoxastrobin (EC50 = 7.54 µM) against Sclerotinia sclerotiorum. Scaffold hopping of the phenyl subunit identified benzothiazolyl-pyridyl oxime as a novel antifungal scaffold accompanied by acquiring oxime 5bm with remarkable activity (EC50 = 3.57 µM) against Pyricularia oryzae. Molecular docking showed that candidate 5am could form more hydrogen bonds with the amino acid residues of actin than metrafenone. This compound also demonstrated better curative efficacy than that of fluoxastrobin and metrafenone in controlling the plant disease caused by S. sclerotiorum. These results rationalize the discovery of antifungal candidates based on aryl heteroaryl ketone.

Chemical Composition and Antimicrobial Properties of Morchella crassipes (Ascomycota) from Kashmir Valley (India).

In the current era, wild macrofungi are being focused for developing and overing novel bioactive compounds for the management of agricultural, horticultural, and other infectious diseases. In that view, current research work was designed to evaluate the biochemical composition and medicinal properties of Morchella crassipes mushroom. The mycochemical screening of aqueous extract exposed the incidence of glycosides, free amino acids and proteins, alkaloids, carbs, flavonoids, terpenoids, phenolic compounds and tannins, except volatile oils, resins, steroids, and anthraquinones. However, hexane extract exhibited the occurrence of glycosides, alkaloids, volatile oils, steroids and terpenoids while as all other phytochemicals were not detected. The gas chromatography mass spectrometry profiling has disclosed the identification of three predominant naturally occurring bioactive volatile monoterpenoids, namely neral, citral, and epoxy-linalool oxide with well-known biological activities. The methanolic extract resulted in strong antifungal efficacy against the tested fungal strains such as Penicillium chrysogenum (20.33 ± 0.57 mm) followed by Pythium ultimum (15.33 ± 0.76 mm) and Aspergillus niger (12.50 ± 0.50 mm) at highest concentrations. Likewise, marked antibacterial effects were reported in case of Staphylococcus aureus (15.16 ± 0.76 mm), followed by Salmonella gallinarum (14.33 ± 0.57 mm) and Escherichia coli (13.66 ± 0.57 mm), respectively. This data may offer baseline information regarding the bioactive metabolites and opening new ways for conducting trails to find natural management strategies to combat multi drug resistant pathogens in horticulture, agriculture, and aquaculture.