Endophytic species of Induratia from coffee and carqueja plants from Brazil and its potential for the biological control of toxicogenic fungi on coffee beans by means of antimicrobial volatiles.

Several endophytic fungi have been reported to have produced bioactive metabolites. Some of them, including the Induratia species, have the capacity to emit volatile compounds with antimicrobial properties with broad spectrum against human and plant pathogens. The present study aimed to prospect the Induratia species producing volatile organic compounds (VOCs), in carqueja plants used in alternative medicine and coffee plants in Brazil. A total of 11 fungal isolates producing volatile metabolites were obtained by a parallel growth technique, using I. alba 620 as a reference strain. Phylogenetic relationships revealed the presence of at least three distinct species, I. coffeana, I. yucatanensis, and Induratia sp. SPME/GC/MS analyses of the VOCs in the headspace above the mycelium from Induratia species cultured for 10 days on PDA revealed the volatile profile emitted by I. coffeana CCF 572, I. coffeana COAD 2055, I. yucatanensis COAD 2062, and Induratia sp. COAD 2059. Volatile organic compounds produced by I. coffeana isolates presented antimicrobial activity against Aspergillus ochraceus, A. sclerotiorum, A. elegans, A. foetidus, A. flavus, A. tamari, A. tubingensis, A. sydowii, A. niger, A. caespitosus, A. versicolor, and A. expansum, sometimes by decreasing the growth rate or, mainly, by fully inhibiting colony growth. Fifty-eight percent of the target species died after 6 days of exposure to VOCs emitted by I. coffeana CCF 572. In addition, VOCs emitted by the same fungus inhibited the growth in A. ochraceus inoculated into coffee beans, which indicates that plants which have I. coffeana as an endophyte may be protected from attacks by this plant pathogen.

Muscodor brasiliensis sp. nov. produces volatile organic compounds with activity against Penicillium digitatum.

Endophytic fungi belonging to Muscodor genus are considered as promising alternatives to be used in biological control due to the production of volatile organic compounds (VOCs). The strains LGMF1255 and LGMF1256 were isolated from the medicinal plant Schinus terebinthifolius and, by morphological data and phylogenetic analysis, identified as belonging to Muscodor genus. Phylogenetic analysis suggests that strain LGMF1256 is a new species, which is herein introduced as Muscodor brasiliensis sp. nov. The analysis of VOCs production revealed that compounds phenylethyl alcohol, α-curcumene, and E (ß) farnesene until now has been reported only from M. brasiliensis, data that supports the classification of strain LGMF1256 as a new species. M. brasiliensis completely inhibited the phytopathogen P. digitatum in vitro. We also evaluated the ability of VOCs from LGMF1256 to inhibit the development of green mold symptoms by inoculation of P. digitatum in detached oranges. M. brasiliensis reduced the severity of diseases in 77%, and showed potential to be used for fruits storage and transportation to prevent the green mold symptoms development, eventually reducing the use of fungicides.

The potential of compounds isolated from Xylaria spp. as antifungal agents against anthracnose

ABSTRACT Anthracnose is a crop disease usually caused by fungi in the genus Colletotrichum or Gloeosporium. These are considered one of the main pathogens, causing significant economic losses, such as in peppers and guarana. The current forms of control include the use of resistant cultivars, sanitary pruning and fungicides. However, even with the use of some methods of controlling these cultures, the crops are not free of anthracnose. Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to human health and the environment. In order to find natural antifungal agents against guarana anthracnose, endophytic fungi were isolated from Amazon guarana. The compounds piliformic acid and cytochalasin D were isolated by chromatographic techniques from two Xylaria spp., guided by assays with Colletotrichum gloeosporioides. The isolated compounds were identified by spectrometric techniques, as NMR and mass spectrometry. This is the first report that piliformic acid and cytochalasin D have antifungal activity against C. gloeosporioides with MIC 2.92 and 2.46 µmol mL-1 respectively. Captan and difenoconazole were included as positive controls (MIC 16.63 and 0.02 µmol mL-1, respectively). Thus, Xylaria species presented a biotechnological potential and production of different active compounds which might be promising against anthracnose disease.

The potential of compounds isolated from Xylaria spp. as antifungal agents against anthracnose.

Anthracnose is a crop disease usually caused by fungi in the genus Colletotrichum or Gloeosporium. These are considered one of the main pathogens, causing significant economic losses, such as in peppers and guarana. The current forms of control include the use of resistant cultivars, sanitary pruning and fungicides. However, even with the use of some methods of controlling these cultures, the crops are not free of anthracnose. Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to human health and the environment. In order to find natural antifungal agents against guarana anthracnose, endophytic fungi were isolated from Amazon guarana. The compounds piliformic acid and cytochalasin D were isolated by chromatographic techniques from two Xylaria spp., guided by assays with Colletotrichum gloeosporioides. The isolated compounds were identified by spectrometric techniques, as NMR and mass spectrometry. This is the first report that piliformic acid and cytochalasin D have antifungal activity against C. gloeosporioides with MIC 2.92 and 2.46µmolmL-1 respectively. Captan and difenoconazole were included as positive controls (MIC 16.63 and 0.02µmolmL-1, respectively). Thus, Xylaria species presented a biotechnological potential and production of different active compounds which might be promising against anthracnose disease.

Biosynthesis and mass spectral fragmentation pathways of (13)C and (15)N labeled cytochalasin D produced by Xylaria arbuscula.

The fungus Xylaria arbuscula was isolated as an endophyte from Cupressus lusitanica and has shown to be a prominent producer of cytochalasins, mainly cytochalasins C, D and Q. Cytochalasins comprise an important class of fungal secondary metabolites that have aroused attention due to their uncommon molecular structures and pronounced biological activities. Due to the few published studies on the ESI-MS/MS fragmentation of this important class of secondary metabolites, in the first part of our work, we studied the cytochalasin D fragmentation pathways by using an ESI-Q-ToF mass spectrometer coupled with liquid chromatography. We verified that the main fragmentation routes were generated by hydrogen and McLafferty rearrangements which provided more ions than just the ones related to the losses of H2 O and CO as reported in previous studies. We also confirmed the diagnostic ions at m/z 146 and 120 as direct precursor derived from phenylalanine. The present work also aimed the production of structurally diverse cytochalasins by varying the culture conditions used to grow the fungus X. arbuscula and further insights into the biosynthesis of cytochalasins. HPLC-MS analysis revealed no significant changes in the metabolic profile of the microorganism with the supplementation of different nitrogen sources but indicated the ability of X. arbuscula to have access to inorganic and organic nitrogen, such as nitrate, ammonium and amino acids as a primary source of nitrogen. The administration of 2-13 C-glycine showed the direct correlation of this amino acid catabolism and the biosynthesis of cytochalasin D by X. arbuscula, due to the incorporation of three labeled carbons in cytochalasin chemical structure. Copyright © 2017 John Wiley & Sons, Ltd.

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

AIMS: 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. METHODS AND RESULTS: Bioactivity and chemical composition of the VOCs from Xylaria sp. PB3f3 were established by using simple and multiple antagonism bioassays, and gas chromatography/mass spectrometry, respectively. The results showed that Xylaria sp. PB3f3 inhibited the growth of the oomycetes Pythium aphanidermatum (78·3%), Phytophthora capsici (48·3%), and the fungi Alternaria solani (24·5%) and Fusarium oxysporum (24·2%), in multiple antagonism bioassays. Volatile organic compounds, produced at 20 and 30 days of fungal growth, inhibited root elongation on Amaranthus hypochondriacus (27·6%) and on Solanum lycopersicum (53·2%). Forty VOCs were identified at 10, 20 and 30 days in Xylaria sp. PB3f3 cultures. The compounds with the highest fibre affinity were: 3-methyl-1-butanol and thujopsene, at 10 days of fungal growth; an unidentified amine and 2-methyl-1-butanol at 20 days; and 2-methyl-1-propanol at 30 days. In the gas phase assay method 2-methyl-1-propanol and 2-methyl-1-butanol showed significant inhibitory effects on root elongation and germination of Am. hypochondriacus and S. lycopersicum. CONCLUSIONS: Xylaria sp. PB3f3 and its VOCs showed significant phytotoxic effects on root elongation and germination of Am. hypochondriacus and S. lycopersicum. SIGNIFICANCE AND IMPACT OF THE STUDY: The genus Xylaria produces a great variety of secondary metabolites, but, up date, there are no reports of the identification of bioactive volatile compounds. Thus, Xylaria sp. PB3f3 and its VOCs are a possible candidate for the biological control of weeds.

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.

Optimization of zofimarin production by an endophytic fungus, Xylaria sp. Acra L38.

To optimize the medium for high zofimarin production, sucrose maltose, glucose, tryptone and peptone were used in an orthogonal array design experiment, where the highest value of zofimarin produced was 25.6 µg/mL. This value was about 3 times higher than that obtained with Czapek yeast extract (CzYE) culture medium. A study with Plackett-Burman design showed that sucrose, maltose, glucose and NaNO3 were significant factors in zofimarin production. Further studies using central composite design (CCD) showed the significance of glucose and the interactions of these critical components affecting zofimarin production. Multiple regression analysis of the data yielded a poor fit as shown by the mismatch of the model with these variable factors. When a polynomial equation was applied, the maximum zofimarin production was predicted to be 201.9 µg/mL. Experimental verification yielded a much lower amount of zofimarin, at around 70 µg/mL. Reconsideration of the CCD data and repetition of some runs with high zofimarin production resulted in reproducible zofimarin yield at 79.7 µg/mL. Even though the amount was lower than the predicted value, the medium optimization study was considered to be quite successful as the yield increased to around 8 times that obtained with the original CzYE culture medium.

Optimization of zofimarin production by an endophytic fungus, Xylaria sp. Acra L38

To optimize the medium for high zofimarin production, sucrose maltose, glucose, tryptone and peptone were used in an orthogonal array design experiment, where the highest value of zofimarin produced was 25.6 µg/mL. This value was about 3 times higher than that obtained with Czapek yeast extract (CzYE) culture medium. A study with Plackett-Burman design showed that sucrose, maltose, glucose and NaNO3 were significant factors in zofimarin production. Further studies using central composite design (CCD) showed the significance of glucose and the interactions of these critical components affecting zofimarin production. Multiple regression analysis of the data yielded a poor fit as shown by the mismatch of the model with these variable factors. When a polynomial equation was applied, the maximum zofimarin production was predicted to be 201.9 µg/mL. Experimental verification yielded a much lower amount of zofimarin, at around 70 µg/mL. Reconsideration of the CCD data and repetition of some runs with high zofimarin production resulted in reproducible zofimarin yield at 79.7 µ/mL. Even though the amount was lower than the predicted value, the medium optimization study was considered to be quite successful as the yield increased to around 8 times that obtained with the original CzYE culture medium.(AU)

Optimization of zofimarin production by an endophytic fungus, Xylaria sp. Acra L38

To optimize the medium for high zofimarin production, sucrose maltose, glucose, tryptone and peptone were used in an orthogonal array design experiment, where the highest value of zofimarin produced was 25.6 µg/mL. This value was about 3 times higher than that obtained with Czapek yeast extract (CzYE) culture medium. A study with Plackett-Burman design showed that sucrose, maltose, glucose and NaNO3 were significant factors in zofimarin production. Further studies using central composite design (CCD) showed the significance of glucose and the interactions of these critical components affecting zofimarin production. Multiple regression analysis of the data yielded a poor fit as shown by the mismatch of the model with these variable factors. When a polynomial equation was applied, the maximum zofimarin production was predicted to be 201.9 µg/mL. Experimental verification yielded a much lower amount of zofimarin, at around 70 µg/mL. Reconsideration of the CCD data and repetition of some runs with high zofimarin production resulted in reproducible zofimarin yield at 79.7 µ/mL. Even though the amount was lower than the predicted value, the medium optimization study was considered to be quite successful as the yield increased to around 8 times that obtained with the original CzYE culture medium.

Potential role of oxidative exoenzymes of the extremophilic fungus Pestalotiopsis palmarum BM-04 in biotransformation of extra-heavy crude oil.

Large amount of drilling waste associated with the expansion of the Orinoco Oil Belt (OOB), the biggest proven reserve of extra-heavy crude oil (EHCO) worldwide, is usually impregnated with EHCO and highly salinized water-based drilling fluids. Oxidative exoenzymes (OE) of the lignin-degrading enzyme system (LDS) of fungi catalyse the oxidation of a wide range of toxic pollutants. However, very little evidences on fungal degradation or biotransformation of EHCO have been reported, which contain high amounts of asphaltenes and its biodegradation rate is very limited. The aims of this work were to study the ability of Pestalotiopsis palmarum BM-04 to synthesize OE, its potential to biotransform EHCO and to survive in extreme environmental conditions. Enzymatic studies of the LDS showed the ability of this fungus to overproduce high amounts of laccase (LACp) in presence of wheat bran or lignin peroxidase (LIPp) with EHCO as sole carbon and energy source (1300 U mgP(-1) in both cases). FT-IR spectroscopy with Attenuated Total Reflectance (ATR) analysis showed the enzymatic oxidation of carbon and sulfur atoms in both maltenes and asphaltenes fractions of biotreated EHCO catalysed by cell-free laccase-enriched OE using wheat bran as inducer. UV-visible spectrophotometry analysis revealed the oxidation of the petroporphyrins in the asphaltenes fraction of biotreated EHCO. Tolerance assays showed the ability of this fungus to grow up to 50,000 p.p.m. of EHCO and 2000 mM of NaCl. These results suggest that P. palmarum BM-04 is a hopeful alternative to be used in remediation processes in extreme environmental conditions of salinity and EHCO contamination, such as the drilling waste from the OOB.

An endophytic Nodulisporium sp. from Central America producing volatile organic compounds with both biological and fuel potential.

A Nodulisporium sp. (Hypoxylon sp.) has been isolated as an endophyte of Thelypteris angustifolia (Broadleaf Leaf Maiden Fern) in a rainforest region of Central America. It has been identified both on the basis of its morphological characteristics and by scanning electron microscopy as well as ITS sequence analysis. The endophyte produces volatile organic compounds (VOCs) that have both fuel (mycodiesel) and use for biological control of plant disease. When grown on potato dextrose agar, the organism uniquely produces a series of ketones, including acetone; 2-pentanone; 3-hexanone, 4-methyl; 3-hexanone, 2,4- dimethyl; 2-hexanone, 4-methyl, and 5-hepten, 2-one and these account for about 25% of the total VOCs. The most abundant identified VOC was 1,8 cineole, which is commonly detected in this group of organisms. Other prominent VOCs produced by this endophyte include 1-butanol, 2- methyl, and phenylethanol alcohol. Moreover, of interest was the presence of cyclohexane, propyl, which is a common ingredient of diesel fuel. Furthermore, the VOCs of this isolate of Nodulisporium sp. were selectively active against a number of plant pathogens, and upon a 24 h exposure caused death to Phytophthora palmivora, Rhizoctonia solani, and Sclerotinia sclerotiorum and 100% inhibition to Phytophthora cinnamomi with only slight to no inhibition of the other pathogens that were tested. From this work, it is becoming increasingly apparent that each isolate of this endophytic Nodulisporium spp., including the Daldina sp. and Hypoxylon spp. teleomorphs, seems to produce its own unique set of VOCs.

Muscodor albus E-6, an endophyte of Guazuma ulmifolia making volatile antibiotics: isolation, characterization and experimental establishment in the host plant.

Muscodor albus is an endophytic fungus, represented by a number of isolates from tropical tree and vine species in several of the world's rainforests, that produces volatile organic compounds (VOCs) with antibiotic activity. A new isolate, E-6, of this organism, with unusual biochemical and biological properties, has been obtained from the branches of a mature Guazuma ulmifolia (Sterculiaceae) tree growing in a dry tropical forest in SW Ecuador. This unique organism produces many VOCs not previously observed in other M. albus isolates, including butanoic acid, 2-methyl-; butanoic acid, 3-methyl-; 2-butenal, 2-methyl-; butanoic acid, 3-methylbutyl ester; 3-buten-1-ol, 3-methyl; guaiol; 1-octene, 3-ethyl-; formamide, N-(1-methylpropyl); and certain azulene and naphthalene derivatives. Some compounds usually seen in other M. albus isolates also appeared in the VOCs of isolate E-6, including caryophyllene; phenylethyl alcohol; acetic acid, 2-phenylethyl ester; bulnesene; and various propanoic acid, 2-methyl- derivatives. The biological activity of the VOCs of E-6 appears different from the original isolate of this fungus, CZ-620, since a Gram-positive bacterium was killed, and Sclerotinia sclerotiorum and Rhizoctonia solani were not. Scanning electron micrographs of the mycelium of isolate E-6 showed substantial intertwining of the hyphal strands. These strands seemed to be held together by an extracellular matrix accounting for the strong mat-like nature of the mycelium, which easily lifts off the agar surface upon transfer, unlike any other isolate of this fungus. The ITS-5.8S rDNA partial sequence data showed 99 % similarity to the original M. albus strain CZ-620. For the first time, successful establishment of M. albus into its natural host, followed by recovery of the fungus, was accomplished in seedlings of G. ulmifolia. Overall, isolates of M. albus, including E-6, have chemical, biological and structural characteristics that make them potentially useful in medicine, agricultural and industrial applications.