Martha L. Macías-Rubalcava

Naphthoquinone spiroketal with allelochemical activity from the newly discovered endophytic fungus Edenia gomezpompae.

Chemical investigation of the mycelium of Edenia gomezpompae, a newly discovered endophytic fungus isolated from the leaves of Callicarpa acuminata (Verbenaceae) collected from the ecological reserve El Eden, Quintana Roo, Mexico, resulted in the isolation of four naphthoquinone spiroketals, including three new compounds and palmarumycin CP2 (4). We elucidated the structures of the metabolites by extensive NMR spectroscopy studies, including DEPT, COSY, NOESY, HSQC, HMBC, and chiroptical methods. The trivial names proposed for these compounds are preussomerin EG1 (1), preussomerin EG2 (2) and preussomerin EG3 (3). In addition, the X-ray data for 4 were obtained. The bioactivity of the mycelial organic extracts and the pure compounds was tested against three endophytic fungi (Colletotrichum sp., Phomopsis sp., and Guignardia manguifera) isolated from the same plant species (C. acuminata, Verbenaceae) and against four economically important phytopathogenic microorganisms (two fungoid oomycetes, Phythophtora capsici and Phythophtora parasitica, and the fungi Fusarium oxysporum and Alternaria solani). Spiroketals 1-3 displayed significant growth inhibition against all the phytopathogens. IC50 values for the four phytopathogens were from 20 to 170 microg/ml. Palmarumycin CP2 (4) was not bioactive against any of the fungi tested. Compound 1 showed the strongest bioactivity. The acetylated derivatives of preussomerin EG1 (1), 1a and 1b, were obtained and their biological activity was tested on endophytes and phytopathogens. Preussomerin EG1 1, 1a and 1b exhibited significant bioactivity against all microorganisms tested with the exception of Alternaria solani. This is the first report of allelochemicals with antifungal activity from the newly discovered endophytic fungus E. gomezpompae.

Antifungal, anti-oomycete and phytotoxic effects of volatile organic compounds from the endophytic fungus Xylaria sp. strain PB3f3 isolated from Haematoxylon brasiletto.

To determine the antifungal, anti‐oomycete and phytotoxic activity; and chemical composition of the volatile organic compounds (VOCs) produced by endophytic fungus Xylaria sp. PB3f3 isolated from Haematoxylon brasiletto Karst.

Antifungal Volatile Organic Compounds from the Endophyte Nodulisporium sp. Strain GS4d2II1a: a Qualitative Change in the Intraspecific and Interspecific Interactions with Pythium aphanidermatum.

This study demonstrates volatile organic compounds (VOCs) production as one of the defense mechanisms of the antagonistic endophyte Nodulisporium sp. GS4d2II1a, and the volatile changes in two times of the fungal growth; and, as result of its intra and interspecific interactions with the plant pathogen Pythium aphanidermatum. The antifungal activity of the volatile and diffusible metabolites was evaluated by means of three types of antagonism bioassays and by organic extract agar dilution. VOCs were obtained by gas chromatography coupled to mass spectrometry from 3- and 5-day Nodulisporium sp. cultures, as well as from its interspecific in vitro antagonistic interaction with the oomycete P. aphanidermatum, and its intraspecific Nodulisporium sp.–Nodulisporium sp. interaction. The GS4d2II1a strain completely inhibited the growth of two fungi and seven oomycetes by replacing their mycelia in simple antagonism bioassays and by producing in vitro volatile and diffusible metabolites that acted synergistically in multiple antagonism bioassays. Additionally, VOCs inhibited the growth of three oomycetes and one fungus in antagonism bioassays using divided plates. A total of 70 VOCs were detected, mainly including mono and sesquiterpenes, especially eucalyptol and limonene. Multiple correspondence analysis revealed four different volatile profiles, showing that volatiles changed with the fungus age and its intra and interspecific interactions. The metabolites produced by Nodulisporium sp. GS4d2II1a could be useful for biological control of fungal and oomycetes plant pathogens of economically important crops.

Naphthoquinone Spiroketals and Organic Extracts from the Endophytic Fungus Edenia gomezpompae as Potential Herbicides

From the fermentation mycelium of the endophytic fungus Edenia gomezpompae were obtained several phytotoxic compounds including two new members of the naphthoquinone spiroketal family, namely, palmarumycin EG1 (1) and preussomerin EG4 (4). In addition, preussomerins EG1-EG3 (7-9) and palmarumycins CP19 (2), CP17 (3), and CP2 (6), as well as ergosta-4,6,8(14),22-tetraen-3-one (5), were obtained. Compounds 2, 3, and 5 are new to this species. The structures of palmarumycins CP19 (2) and CP17 (3) were unambiguously determined by X-ray analysis. The isolates and mycelium organic extracts from four morphological variants of E. gomezpompae caused significant inhibition of seed germination, root elongation, and seedling respiration of Amaranthus hypochondriacus, Solanum lycopersicum, and Echinochloa crus-galli. The treatments also affected respiration on intact mitochondria isolated from spinach.

Acremoxanthone E, a novel member of heterodimeric polyketides with a bicyclo[3.2.2]nonene ring, produced by Acremonium camptosporum W. GAMS (Clavicipitaceae) endophytic fungus.

Bioactivity‐directed fractionation of the organic mycelium extract of the endophytic fungus Acremonium camptosporum W. Gams (Clavicipitaceae), isolated from the leaves of Bursera simaruba (Burseraceae), led to the isolation of six major heterodimeric polyketides, including one not previously characterized acremoxanthone derivative. In addition, the already known acremoxanthone C, acremonidins A and B, and acremoxanthones A and B were obtained. The structure of the new compound was established by extensive NMR studies, including DEPT, COSY, NOESY, HSQC, and HMBC methods. The trivial name proposed for this compound is acremoxanthone E. In addition, the structure of acremoxanthone C was unequivocally established for the first time, through X‐ray crystal‐structure analysis. The anti‐oomycete activities of the pure compounds were tested against four economically important phytopathogenic oomycetes. Inhibitory concentration for 50% diameter growth reduction, IC50, values for the four phytopathogens ranged from 6 to 38 μM. Also, in parallel, the cytotoxic activities against six cancer cell lines were evaluated showing IC50 values similar to those of cisplatin. To the best of our knowledge, this is the first report on three different groups of heterodimeric polyketides, linked by a bicyclo[3.2.2]nonene, such as xanthoquinodins, acremonidins, and acremoxanthones, which are isolated from an endophytic fungus. In addition, a common biosynthetic origin could be proposed.

Comparative study of two GH19 chitinase-like proteins from Hevea brasiliensis, one exhibiting a novel carbohydrate-binding domain

Plants express chitinase and chitinase‐like proteins (CLPs) belonging to the glycosyl hydrolases of the GH18 and GH19 families, which exhibit varied functions. CLPs in the GH18 family have been structurally and functionally characterized; however, there are no structures available for any member of the GH19 family. In this study, two CLPs of the GH19 family from the rubber tree Hevea brasiliensis (HbCLP1 and HbCLP2) were cloned, expressed and characterized. HbCLP1 was identical to the allergen Hev b 11.0101 previously described by others, while HbCLP2 was a novel isoform exhibiting an unusual half chitin‐binding domain before the catalytic domain. Sequence alignments showed that in the two proteins the catalytic residues Glu117 and Glu147 in HbCLP1 and HbCLP2, respectively, were mutated to Ala, accounting for the lack of activity. Nonetheless, both CLPs bound chitin and chitotriose (GlcNAc)3 with high affinities, as evaluated with chitin‐affinity chromatography and tryptophan fluorescence experiments. The chitin‐binding domains also bound chitotriose with even higher affinities. The crystal structures of the HbCLP1‐isolated domains were determined at high resolution. The analysis of the crystallographic models and docking experiments using (GlcNAc)6 oligosaccharides provides evidence of the residues involved in sugar binding. Endochitinase activity was restored in both proteins by mutating residues A117E (HbCLP1) and A147E (HbCLP2); the distance between the catalytic proton donor and the catalytic nucleophile in the in silico mutated residues was 9.5 Å, as occurs in inverting enzymes. HbCLP1 and HbCLP2 were highly thermostable and exhibited antifungal activity against Alternaria alternata, suggesting their participation in plant defense mechanisms.

Selected phytotoxins and organic extracts from endophytic fungus Edenia gomezpompae as light reaction of photosynthesis inhibitors

In a search for natural herbicides, we investigated the action mechanism of the naphthoquinone spiroketals, isolated from the endophytic fungus Edenia gomezpompae: preussomerins EG1 (1) and EG4 (2), and palmarumycins CP17 (3), and CP2 (4) on the photosynthesis light reactions. The naphthoquinone spiroketals 1-4 inhibited the ATP synthesis in freshly lysed spinach thylakoids from water to MV, and they also inhibited the non-cyclic electron transport in the basal, phosphorylating and uncoupled conditions from water to MV. Therefore, they act as Hill reaction inhibitors. The results suggested that naphthoquinone spiroketals 1-4 have two interactions and inhibition site on the PSII electron transport chain. The first one involves the water splitting enzyme inhibition; and, the second on the acceptor site of PSII in a similar way that herbicide Diuron, studied by polaroghaphy and corroborated by fluorescence of the chlorophyll a of PSII. The culture medium and mycelium organic extracts from four morphological variants of E. gomezpompae were phytotoxic, and the culture medium extracts were more potent than mycelium extracts. They also act as Hill reaction inhibitors.

Phytotoxic Potential of Secondary Metabolites and Semisynthetic Compounds from Endophytic Fungus Xylaria feejeensis Strain SM3e-1b Isolated from Sapium macrocarpum

Bioactivity-directed fractionation of the combined culture medium and mycelium extract of the endophytic fungus Xylaria feejeensis strain SM3e-1b, isolated from Sapium macrocarpum, led to the isolation of three known natural products: (4S,5S,6S)-4-hydroxy-3-methoxy-5-methyl-5,6-epoxycyclohex-2-enone or coriloxine, 1; 2-hydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, 2; and 2,6-dihydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione or fumiquinone B, 3. This is the first report of compound 3 being isolated from this species. Additionally, four new derivatives of coriloxine were prepared: (4R,5S,6R)-6-chloro-4,5-dihydroxy-3-methoxy-5-methylcyclohex-2-enone, 4; 6-hydroxy-5-methyl-3-(methylamino)cyclohexa-2,5- diene-1,4-dione, 5; (4R,5R,6R)-4,5-dihydroxy-3-methoxy-5-methyl-6-(phenylamino)cyclohex-2-enone, 6; and 2-((4-butylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, 7. X-ray analysis allowed us to unambiguously determine the structures and absolute configuration of semisynthetic derivatives 4, 5, and 6. The phytotoxic activity of the three isolated natural products and the coriloxine derivatives is reported. Germination of the seed, root growth, and oxygen uptake of the seedlings of Trifolium pratense, Medicago sativa, Panicum miliaceum, and Amaranthus hypochondriacus were significantly inhibited by all of the tested compounds. In general, they were more effective inhibiting root elongation than suppressing the germination and seedling oxygen uptake processes as shown by their IC50 values.

Calmodulin Inhibitors from Aspergillus stromatoides

An organic extract was prepared from the culture medium and mycelia of the marine fungus Aspergillus stromatoides Raper & Fennell. The extract was fractionated via column chromatography, and the resulting fractions were tested for their abilities to quench the fluorescence of the calmodulin (CaM) biosensor hCaM M124C‐mBBr. From the active fraction, emodin (1) and ω‐hydroxyemodin (2) were isolated as CaM inhibitors. Anthraquinones 1 and 2 quenched the fluorescence of the hCaM M124C‐mBBr biosensor in a concentration‐dependent manner with Kd values of 0.33 and 0.76 μM, respectively. The results were compared with those of chlorpromazine (CPZ), a classical inhibitor of CaM, with a Kd value of 1.25 μM. Docking analysis revealed that 1 and 2 bind to the same pocket of CPZ. The CaM inhibitor properties of 1 and 2 were correlated with some of their reported biological properties. Citrinin (3), methyl 8‐hydroxy‐6‐methyl‐9‐oxo‐9H‐xanthene‐1‐carboxylate (4), and coniochaetone A (5) were also isolated in the present study. The X‐ray structure of 5 is reported for the first time.

Volatile organic compounds from endophytic fungi as innovative postharvest control of Fusarium oxysporum in cherry tomato fruits

To assess their potential as biopesticides, the effect on the growth of phytopathogen Fusarium oxysporum of six volatile organic compounds from endophytic fungi was studied in vivo and in vitro; compounds were used both as a mixture and individually. In vivo studies were performed inoculating the pathogen into cherry tomatoes, while the in vitro antifungal effect was studied using agar dilution and gas phase methods. Also, the morphology of the hyphae exposed to these compounds was analyzed. Moreover, the possible mechanism of action of these compounds was determined by studying the respiration and cell membrane permeability. Results show that the compounds have a significant concentration-dependent antifungal effect individually and act in a synergic manner. Additionally, changes in cell membrane permeability, damage to the hyphal morphology, and an inhibitory effect on the respiration were observed. The mixture of the six compounds may be used for postharvest control of F. oxysporum in tomatoes.