A Nematode Crude Extract Acts as an Elicitor of the Nematocidal Activity of Nematophagous Fungi Liquid Culture Filtrates Against Haemonchus contortus (Nematoda: Trichostrongylidae).

AIM: This study was designed to investigate if culturing nematophagous fungi (NF) in the presence of a Haemonchus contortus larva crude extract (HcCE) enhances the nematocidal activity of nematophagous fungi liquid culture filtrates (NFCF). MATERIALS AND METHODS: Four NF Arthrobotrys oligospora, A. musiformis, Duddingtonia flagrans and Clonostachys rosea were cultured in flasks (n = 5) containing Czapek-Dox broth medium (CDB) in the presence or absence of HcCE. NFCF recovered by filtration of each fungus (200 mg/mL) were assessed on H. contortus infective larvae (L3) using 96-well micro-titer plates (n = 4). Additionally, CDB and water were considered negative controls, while Ivermectin acted as a positive control. After 48 h confrontation, ten 10-µL aliquots of each well were deposited on slides and observed under the microscope (40 ×). Dead and alive larvae in the aliquots were quantified, and a mortality rate (MR) was estimated. RESULTS: The MR of the different NFCF was greatly enhanced by the presence of HcCE. The four NF incubated in the absence of HcCE showed low mortality percentages from 8.2 to 25.8%; in contrast, when the assessed NF growth in the presence of HcCE showed a lethal activity ranging from 66.8 to 80.5%. Only C. rosea showed a moderate increase in the presence of the elicitor (42.7%). CONCLUSION: This study shows evidence about the HcCE enhances the production of nematocidal activity in NFCF. Future studies should be performed to elucidate the compounds responsible of the nematocidal activity that could have important implications in the control of sheep haemonchosis.

Enzymatic Bioprospecting of Fungi Isolated from a Tropical Rainforest in Mexico.

The humid tropical environment provides an ideal place for developing a high diversity of plants; this is why it is an interesting site for the enzymatic bioprospecting of fungi that are responsible for the recycling of organic matter in an efficient and accelerated way and whose enzymes could have multiple biotechnological applications. For this study, 1250 isolates of macroscopic and microscopic fungal morphotypes were collected from soil, leaf litter, and wood. One hundred and fifty strains (50 from each source) were selected for the enzymatic screening. From the first phase, 51 strains with positive activity for laccase, protease, amylase, xylanase, and lipase enzymes were evaluated, of which 20 were isolated from leaf litter, 18 from the soil, and 13 from wood. The 10 best strains were selected for the enzymatic quantification, considering the potency index and the production of at least two enzymes. High laccase activity was detected for Trametes villosa FE35 and Marasmius sp. CE25 (1179 and 710.66 U/mg, respectively), while Daedalea flavida PE47 showed laccase (521.85 U/mg) and protease activities (80.66 U/mg). Fusarium spp. PH79 and FS400 strains had amylase (14.0 U/mg, 49.23 U/mg) and xylanase activities (40.05 U/mg, 36.03 U/mg) respectively. These results confirm the enzymatic potential of fungi that inhabit little-explored tropical rainforests with applications in industry.

Antitubercular Activity of the Fungus Gliocladium sp. MR41 Strain.

Tuberculosis (TB) is a leading cause of death worldwide from infectious diseases and its inadequate treatment has led to emergence of resistant strains. The emergence of these strains renders the search for new drugs for the treatment of TB. The aim of this study was the evaluation of the anti-TB activity of the extract from fungus Gliocladium sp. MR41, and bioassay-guided fractionation and identification of majority compounds was carried out. Fungal strain culture was lyophilized and extracted by maceration in Ethyl Acetate (EtOAc). This extract was fractionated by liquid-liquid partitioning and chromatographic techniques, and the compounds were identified by their spectroscopic data. Furthermore, the EtOAc extract, fractions, and pure compounds were tested on Mycobacterium tuberculosis using the Microplate Alamar Blue Assay. From the bioactive AcetoNitrile Fraction (AcNF; MIC = 3.13 µg/mL) of the EtOAc extract, four compounds were isolated: ergosterol (1), ergosterol-5, 8-peroxide (2), 1, 6-di-O-acetyl-2,3,4,5-tetrahydroxy-hexane (3), and allitol (4). Only 2 exhibited potent activities against M. tuberculosis (MIC = 0.78 µg/mL). Additionally, this is the first report, to our knowledge, of polyols 3 and 4 from this fungus.

Bactericidal Activity of Chrysomya rufifacies and Cochliomyia macellaria (Diptera: Calliphoridae) Larval Excretions-Secretions Against Staphylococcus aureus (Bacillales: Staphylococcaceae).

The inhibitory effect of Chrysomya rufifacies (Macquart) and Cochliomyia macellaria (Fabricius) larval excretions-secretions (ES) on Staphylococcus aureus was determined using a portable colorimetric method without the need for any dedicated spectral instrument. Polystyrene 96 well microplates were used and 100 µl of the bacterial inoculum (5 × 105 CFU/ml) plus 100 µl of the dipteran exosecretions at different concentrations were added to each well. Subsequently, 50 µl of a 1% solution of the triphenyl tetrazolium chloride stain was added to each well to determine the bacterial viability. The color development in each well was measured with the ImageJ software S. aureus was exposed to different concentrations of the ES of both species individually. At a concentration of 800 ppm ES of C. rufifacies or Co. macellaria, bacterial growth was inhibited 97.45 ± 1.70% and 82.21 ± 1.88%, respectively. As expected, exposure to a lower concentration (i.e., 50 ppm) was less inhibitory (C. rufifacies ES, 77.65 ± 4.25% and Co. macellaria ES, 43.54 ± 4.63%). This study demonstrates for the first time the bactericidal activity of C. rufifacies and Co. macellaria ES against S. aureus. This finding is promising as it could result in the identification and synthesis of proteins capable of suppressing pathogen development in wounds. Additionally, the proposed method can simplify the use of expensive laboratory instruments for antimicrobial activity determination.

In Vitro Lethal Activity of the Nematophagous Fungus Clonostachys rosea (Ascomycota: Hypocreales) against Nematodes of Five Different Taxa.

This study was aimed to evaluate the in vitro lethal activity of the nematophagous fungi Clonostachys rosea against 5 nematodes species belonging to different taxa. Two groups of 35 Petri dishes (PD) each were divided into 5 series of 7 (PD). Group 1 (series 1, 2, 3, 4, and 5) contained only water agar; meanwhile group 2 plates (series 6, 7, 8, 9, and 10) contained C. rosea cultures growth on water agar. Every plate from the two groups was added with 500 nematodes corresponding to the following genera/specie: Haemonchus contortus, Caenorhabditis elegans, Rhabditis sp., Panagrellus redivivus, and Butlerius sp. After 5-day incubation at room temperature, free (nontrapped) larvae were recovered from plates using the Baermann funnel technique. Recovered nematodes were counted and compared with their proper controls. Results shown an important reduction percentage of the nematode population attributed to the fungal lethal activity as follows: H. contortus (L3) 87.7%; C. elegans 94.7%; Rhabditis sp. 71.9%; P. redivivus 92.7%; and Butlerius sp. 100% (p ≤ 0.05). The activity showed by C. rosea against the H. contortus can be crucial for further studies focused to the biological control of sheep haemonchosis, although the environmental impact against beneficial nematodes should be evaluated.

Insect deterrent and nematicidal screening of microfungi from Mexico and anti-aphid compounds from Gliomastix masseei.

Fungal metabolites are promising alternatives for the development of biorational pesticides. In this sense, microfungi from tropical regions are valuable sources of natural compounds for pest management. With the aim of broadening the search for new eco-friendly products to manage plant pests, this study was carried out to evaluate the biological activity of 23 tropical fungal extracts on three species of phytophagous insects and a plant parasitic nematode. In addition, the active principles of the most effective extract were identified. The insect deterrent activity of fungal extracts was evaluated on the settling of aphids Myzus persicae and Rhopalosiphum padi, and on the feeding of lepidoptera larva Spodoptera littoralis; the nematostatic activity was evaluated on the mobility of Meloidogyne javanica. Active metabolites from Gliomastix masseei were identified by GC-MS techniques and by comparison with commercial standards. Results showed seven extracts with strong effect on the settling of M. persicae and R. padi (settling inhibition >80%). The calculated median of effective concentration (EC50) values ranged from 8 to 38µg/cm2 for the extracts of Clonostachys rosea and G. masseei, respectively. Bioassay-guided separation of the ethyl acetate extract of G. masseei revealed the presence of fatty acids and their derivatives, where methyl 9-octadecenoate was the most active compound with EC50 values of 16µg and 35µg/cm2 for M. persicae and R. padi, respectively. Extracts of C. rosea and G. masseei could be a promising option in the control of pest aphids in agriculture.

Insect deterrent and nematicidal screening of microfungi from Mexico and anti-aphid compounds from Gliomastix masseei / Evaluación de la actividad disuasiva de insectos y nematotóxica de microhongos de México y compuestos anti-áfidos de Gliomastix masseei

Fungal metabolites are promising alternatives for the development of biorational pesticides. In this sense, microfungi from tropical regions are valuable sources of natural compounds for pest management. With the aim of broadening the search for new eco-friendly products to manage plant pests, this study was carried out to evaluate the biological activity of 23 tropical fungal extracts on three species of phytophagous insects and a plant parasitic nematode. In addition, the active principles of the most effective extract were identified. The insect deterrent activity of fungal extracts was evaluated on the settling of aphids Myzus persicae and Rhopalosiphum padi, and on the feeding of lepidoptera larva Spodoptera littoralis; the nematostatic activity was evaluated on the mobility of Meloidogyne javanica. Active metabolites from Gliomastix masseei were identified by GC-MS techniques and by comparison with commercial standards. Results showed seven extracts with strong effect on the settling of M. persicae and R. padi (settling inhibition >80%). The calculated median of effective concentration (EC50) values ranged from 8 to 38 µg/cm² for the extracts of Clonostachys rosea and G. masseei, respectively. Bioassay-guided separation of the ethyl acetate extract of G. masseei revealed the presence of fatty acids and their derivatives, where methyl 9-octadecenoate was the most active compound with EC50 values of 16 µg and 35 µg/cm² for M. persicae and R. padi, respectively. Extracts of C. rosea and G. masseei could be a promising option in the control of pest aphids in agriculture.

Los metabolitos fúngicos son agentes prometedores para el desarrollo de plaguicidas biorracionales. En este sentido, los hongos microscópicos de zonas tropicales representan una valiosa fuente de compuestos naturales para el manejo de plagas. Con la finalidad de ampliar la investigación en productos amigables con el medio ambiente, en este estudio se evaluó la actividad biológica de 23 extractos de hongos sobre 3 especies de insectos fitófagos y un nematodo fitoparásito; además se identificaron los componentes del extracto más activo. El efecto disuasivo de los extractos fúngicos se evaluó en el asentamiento de Myzus persicae y Rhopalosiphum padi, así como en la alimentación de Spodoptera littoralis; la actividad nematostática se evaluó sobre la movilidad de Meloidogyne javanica. Los metabolitos activos de Gliomastix masseei se identificaron por cromatografía de gases-espectrometría de masas y por comparación con muestras comerciales. Los resultados mostraron 7 extractos con fuerte efecto en la inhibición del asentamiento (> 80%) de M. persicae y R. padi. Los valores de la concentración efectiva media (CE50) estuvieron en el rango de 8 a 38 µg/cm² para los extractos de Clonostachys rosea y G. masseei, respectivamente. El extracto de acetato de etilo de G. masseei se fraccionó por un proceso biodirigido y reveló la presencia de ácidos grasos y sus derivados, donde el 9-octadecenoato de metilo fue el más activo, con una CE50 de 16 µg/cm² para M. persicae y 35 µg/cm² para R. padi. Los extractos fúngicos de C. rosea y G. masseei pueden ser una alternativa promisoria en el control de áfidos que son plaga en la agricultura.

Bioweathering Potential of Cultivable Fungi Associated with Semi-Arid Surface Microhabitats of Mayan Buildings.

Soil and rock surfaces support microbial communities involved in mineral weathering processes. Using selective isolation, fungi were obtained from limestone surfaces of Mayan monuments in the semi-arid climate at Yucatan, Mexico. A total of 101 isolates representing 53 different taxa were studied. Common fungi such as Fusarium, Pestalotiopsis, Trichoderma, and Penicillium were associated with surfaces and were, probably derived from airborne spores. In contrast, unusual fungi such as Rosellinia, Annulohypoxylon, and Xylaria were predominantly identified from mycelium particles of biofilm biomass. Simulating oligotrophic conditions, agar amended with CaCO3 was inoculated with fungi to test for carbonate activity. A substantial proportion of fungi, in particular those isolated from mycelium (59%), were capable of solubilizing calcium by means of organic acid release, notably oxalic acid as evidenced by ion chromatography. Contrary to our hypothesis, nutrient level was not a variable influencing the CaCO3 solubilization ability among isolates. Particularly active fungi (Annulohypoxylon stygium, Penicillium oxalicum, and Rosellinia sp.) were selected as models for bioweathering experiments with limestone-containing mesocosms to identify if other mineral phases, in addition to oxalates, were linked to bioweathering processes. Fungal biofilms were seen heavily covering the stone surface, while a biomineralized front was also observed at the stone-biofilm interface, where network of hyphae and mycogenic crystals was observed. X-ray diffraction analysis (XRD) identified calcite as the main phase, along with whewellite and wedellite. In addition, lower levels of citrate were detected by Attenuated Total Reflectance-Fourier-Transform Infrared Spectroscopy (ATR-FTIR). Overall, our results suggest that a diverse fungal community is associated with limestone surfaces insemi-arid climates. A subset of this community is geochemically active, excreting organic acids under quasi-oligotrophic conditions, suggesting that the high metabolic cost of exuding organic acids beneficial under nutrient limitation. Oxalic acid release may deteriorate or stabilize limestone surfaces, depending on microclimatic dynamics.

Emulsifying activity and stability of a non-toxic bioemulsifier synthesized by Microbacterium sp. MC3B-10.

A previously reported bacterial bioemulsifier, here termed microbactan, was further analyzed to characterize its lipid component, molecular weight, ionic character and toxicity, along with its bioemulsifying potential for hydrophobic substrates at a range of temperatures, salinities and pH values. Analyses showed that microbactan is a high molecular weight (700 kDa), non-ionic molecule. Gas chromatography of the lipid fraction revealed the presence of palmitic, stearic, and oleic acids; thus microbactan may be considered a glycolipoprotein. Microbactan emulsified aromatic hydrocarbons and oils to various extents; the highest emulsification index was recorded against motor oil (96%). The stability of the microbactan-motor oil emulsion model reached its highest level (94%) at 50 °C, pH 10 and 3.5% NaCl content. It was not toxic to Artemia salina nauplii. Microbactan is, therefore, a non-toxic and non-ionic bioemulsifier of high molecular weight with affinity for a range of oily substrates. Comparative phylogenetic assessment of the 16S rDNA gene of Microbacterium sp. MC3B-10 with genes derived from other marine Microbacterium species suggested that this genus is well represented in coastal zones. The chemical nature and stability of the bioemulsifier suggest its potential application in bioremediation of marine environments and in cosmetics.

Antimycobacterial and antileishmanial effects of microfungi isolated from tropical regions in México.

A total of 82 fungal extracts were selected and screened against Mycobacterium tuberculosis and promastigotes of Leishmania mexicana strains. Results showed inhibitory activity in 29 % of the fungal strains against at least one of the targets tested. The most significant antituberculosis (antiTB) effects were presented by Cylindrocarpon sp. XH9B, Fusarium sp. TA54, Fusarium XH1Ga, Gliocladium penicillioides TH04 and TH21, Gliocladium sp. TH16, Kutilakesa sp. MR46, and Verticillium sp. TH28 strains (minimal inhibition concentration (MIC) = 1.56-25 µg/ml). Mortality of L. mexicana promastigotes was displayed by only four strains, Fusarium sp. TA50, Fusarium sp. TA54, Verticillium sp. TH28, and the unidentified 2TA2 strain (IC(50) = 14.23-100 µg/ml and IC(100) = 50-100 µg/ml). Seven of these most active strains were defatted and their corresponding fractions evaluated again. The results showed the best antiTB activity in Gliocladium sp. TH16 (MIC = 1.56 µg/ml) and the highest leishmanicidal potential in Fusarium sp. TA54 (IC(50) = 6.36 µg/ml). These results show that fungi living in the tropical regions of México have the ability to produce bioactive metabolites that could be used in the near future as natural products to control neglected tropical diseases.

Succession of fungi colonizing porous and compact limestone exposed to subtropical environments.

Little is known about the dynamics of succession of fungi on limestone exposed in subtropical environments. In this study, the colonization of experimental blocks of compact and porous limestone by a fungal community derived from natural biofilms occurring on Structure X from the archaeological site of Becán (México), was studied using a cultivation-dependent approach after short-term (9 m) exposure in order to provide a preliminary insight of the colonization process under seminatural conditions. Microbial growth seen as the change of colour of stone surfaces to black/dark green was more abundant on the porous limestone. There was a fairly clear difference in microbial colonization between the onset of the experiment and the 6th month for both limestone types, but no significant increase in the colonization of coupons occurred between months 6 and 9. This could be related to the low rainfall expected for this period, corresponding to the dry season. A total of 977 isolates were obtained. From these, 138 sterile fungi were unidentified, 380 could only be assigned to the order Sphaeropsidales; the remaining isolates (459) were grouped into 27 genera and 99 different species. Nearly all detected fungal species belonged to the Ascomycota (90 %). Rare taxa (species represented by one to three isolates) included the recently described genus Elasticomyces, several species of genera Hyalodendron, Monodyctis, Papulospora, Curvularia, and Septoria. Other taxa were Minimedusa and Gliomastix luzulae, which have not been previously described for stone environments. Abundant fungi included several species of the common genera Cladosporium, Alternaria, and Taeniolella typical for a range of habitats. Succession of populations was observed for certain taxa, this shift in the composition of fungal communities was more evident in porous limestone. After 6 m of exposure, species of the genera Scolecobasidium, Hyalodendron, and Taeniolella were predominant, while after 9 m, the predominant species belonged to the genera Curvularia and Alternaria, particularly on porous stone. These results suggest that Curvularia and Alternaria replaced other fungi, due to a higher tolerance towards low levels of available water during the dry season. Higher levels of water within the porous stone, keep longer periods of microbial activity, minimizing the impact of desiccation. This study contributes to understand the diversity of fungal communities in stone surfaces in subtropical settings and the dynamics of colonization on limestone.