Broad-Spectrum Antifungal, Biosurfactants and Bioemulsifier Activity of Bacillus subtilis subsp. spizizenii-A Potential Biocontrol and Bioremediation Agent in Agriculture.

In this study, the antifungal, biosurfactant and bioemulsifying activity of the lipopeptides produced by the marine bacterium Bacillus subtilis subsp. spizizenii MC6B-22 is presented. The kinetics showed that at 84 h, the highest yield of lipopeptides (556 mg/mL) with antifungal, biosurfactant, bioemulsifying and hemolytic activity was detected, finding a relationship with the sporulation of the bacteria. Based on the hemolytic activity, bio-guided purification methods were used to obtain the lipopeptide. By TLC, HPLC and MALDI-TOF, the mycosubtilin was identified as the main lipopeptide, and it was further confirmed by NRPS gene clusters prediction based on the strain's genome sequence, in addition to other genes related to antimicrobial activity. The lipopeptide showed a broad-spectrum activity against ten phytopathogens of tropical crops at a minimum inhibitory concentration of 400 to 25 µg/mL and with a fungicidal mode of action. In addition, it exhibited that biosurfactant and bioemulsifying activities remain stable over a wide range of salinity and pH and it can emulsify different hydrophobic substrates. These results demonstrate the potential of the MC6B-22 strain as a biocontrol agent for agriculture and its application in bioremediation and other biotechnological fields.

Transcriptome analysis of the spider Phonotimpus pennimani reveals novel toxin transcripts.

Background: Phonotimpus pennimani (Araneae, Phrurolithidae) is a small-sized (3-5 mm) spider endemic to the Tacaná volcano in Chiapas, Mexico, where it is found in soil litter of cloud forests and coffee plantations. Its venom composition has so far not been investigated, partly because it is not a species of medical significance. However, it does have an important impact on the arthropod populations of its natural habitat. Methods: Specimens were collected in Southeastern Mexico (Chiapas) and identified taxonomically by morphological characteristics. A partial sequence from the mitochondrial gene coxI was amplified. Sequencing on the Illumina platform of a transcriptome library constructed from 12 adult specimens revealed 25 toxin or toxin-like genes. Transcripts were validated (RT-qPCR) by assessing the differential expression of the toxin-like PpenTox1 transcript and normalising with housekeeping genes. Results: Analysis of the coxI-gene revealed a similarity to other species of the family Phrurolithidae. Transcriptome analysis also revealed similarity with venom components of species from the families Ctenidae, Lycosidae, and Sicariidae. Expression of the toxin-like PpenTox1 gene was different for each developmental stage (juvenile or adult) and also for both sexes (female or male). Additionally, a partial sequence was obtained for the toxin-like PpenTox1 from DNA. Conclusion: Data from the amplification of the mitochondrial coxI gene confirmed that P. pennimani belongs to the family Phrurolithidae. New genes and transcripts coding for venom components were identified.

Bacterial Diversity Associated with Anastrepha obliqua Males Change Under Mass-Rearing Conditions and with Irradiation.

Recent advances in understanding the symbiotic interactions between bacteria and fruit flies have shown that they are relevant for mass rearing and the sterile insect technique (SIT). SIT involves mass production and release of sterile insects that would copulate with their wild conspecifics and thus decrease the population growth rate. The irradiation process used to sterilize mass-reared flies can modify the diversity and structure of the midgut bacterial communities, which could affect sterile male survival, flight capacity, and sexual competitiveness. Our aim was to compare bacterial communities in the midgut of wild and mass-reared Anastrepha obliqua (Macquart) males irradiated at 0, 60, and 80 Gy. After adult's emergence, their midguts were dissected, DNA was extracted, and high-throughput sequencing of the V3-V4 region of the 16S rDNA gene was performed. A total of 11 phyla, 17 classes, 47 families, and 52 genera of bacteria were identified. The most representative phylum was Proteobacteria and the predominant family was Enterobacteriaceae. We found that wild males had a different intestinal bacterial community from mass-reared males. In addition, irradiation at 60 and 80 Gy caused changes in the diversity and structure of the midgut microbiota of these sterile males, suggesting that mass rearing and irradiation cause artificial selection of the bacterial communities in the gut of A. obliqua males.

Fungal diversity in shade-coffee plantations in Soconusco, Mexico.

Background: As forested natural habitats disappear in the world, traditional, shade-coffee plantations offer an opportunity to conserve biodiversity and ecosystem services. Traditional coffee plantations maintain a diversity of tree species that provide shade for coffee bushes and, at the same time, are important repositories for plants and animals that inhabited the original cloud forest. However, there is still little information about shade-coffee plantation's fungal diversity despite their relevance for ecosystem functioning as decomposers, symbionts and pathogens. Specifically, it is unknown if and what mycorrhizae-forming fungi can be found on the branches and trunks of coffee bushes and trees, which hold a diversity of epiphytes. Here, we evaluate fungal communities on specific plant microsites on both coffee bushes and shade trees. We investigate the ecological roles played by this diversity, with a special focus on mycorrhizae-forming fungi that may enable the establishment and development of epiphytic plants. Methods: We collected 48 bark samples from coffee bushes and shade trees (coffee; tree), from four plant microsites (upper and lower trunks, branches and twigs), in two shade-coffee plantations in the Soconusco region in southern Mexico, at different altitudes. We obtained ITS amplicon sequences that served to estimate alpha and beta diversity, to assign taxonomy and to infer the potential ecological role played by the detected taxa. Results: The bark of shade trees and coffee bushes supported high fungal diversity (3,783 amplicon sequence variants). There were no strong associations between community species richness and collection site, plant type or microsite. However, we detected differences in beta diversity between collection sites. All trophic modes defined by FUNGuild database were represented in both plant types. However, when looking into guilds that involve mycorrhizae formation, the CLAM test suggests that coffee bushes are more likely to host taxa that may function as mycorrhizae. Discussion: We detected high fungal diversity in shade-coffee plantations in Soconusco, Chiapas, possibly remnants of the original cloud forest ecosystem. Several mycorrhiza forming fungi guilds occur on the bark of coffee bushes and shade trees in this agroecosystem, with the potential of supporting epiphyte establishment and development. Thus, traditional coffee cultivation could be part of an integrated strategy for restoration and conservation of epiphytic populations. This is particularly relevant for conservation of threatened species of Orchidaceae that are highly dependent on mycorrhizae formation.

Effect of a Probiotic-Enriched Diet on Sexual Competitiveness, Pheromone Emission, and Cuticular Hydrocarbons of Sterile and Fertile Anastrepha ludens (Diptera: Tephritidae).

The sterile insect technique has been used for the eradication or control of numerous tephritid fruit flies. However, mass-rearing and sterilization can affect the microbiota and sexual performance of male tephritid fruit flies. Despite the addition of postteneral protein food which contributes to the enhancement of the sexual performance of mass-reared males, in some cases, they are less competitive than their wild counterparts. Alternatively, the addition of probiotics may improve the sexual performance of mass-reared sterile males. In this study, we evaluated the effect of a postteneral Lactobacillus casei-enriched diet on the sexual competitivity, pheromone emission, and cuticular hydrocarbons of mass-reared sterile and fertile Anastrepha ludens (Loew) (Diptera: Tephritidae) males. Flies were fed either with sugar, standard diet (sugar and protein, 3:1), sugar + probiotic, or standard diet + probiotic. The addition of the probiotic improved the sexual competitivity of fertile and sterile males that were devoid of protein but led to a negative effect on males fed with a standard diet. As compared to males that were fed with the standard diet + probiotic/only sugar, the males fed with the standard diet or those fed on sugar + probiotic displayed a higher number of mating instances. Sterile males that fed on sugar + probiotic had a higher relative amount of anastrephine, epianastrephine, n-methyl octacosane, and 2-methyl triacontane than those fed on sugar only. Overall, these compounds were common in the treatments where males had the best sexual performance. Our results suggest that the probiotics offer nutritional advantages to males whose food lacks protein.

Tween 80 Improves the Acid-Fast Bacilli Quantification in the Magnetic Nanoparticle-Based Colorimetric Biosensing Assay (NCBA).

Despite its reduced sensitivity, sputum smear microscopy (SSM) remains the main diagnostic test for detecting tuberculosis in many parts of the world. A new diagnostic technique, the magnetic nanoparticle-based colorimetric biosensing assay (NCBA) was optimized by evaluating different concentrations of glycan-functionalized magnetic nanoparticles (GMNP) and Tween 80 to improve the acid-fast bacilli (AFB) count. Comparative analysis was performed on 225 sputum smears: 30 with SSM, 107 with NCBA at different GMNP concentrations, and 88 with NCBA-Tween 80 at various concentrations and incubation times. AFB quantification was performed by adding the total number of AFB in all fields per smear and classified according to standard guidelines (scanty, 1+, 2+ and 3+). Smears by NCBA with low GMNP concentrations (≤1.5 mg/mL) showed higher AFB quantification compared to SSM. Cell enrichment of sputum samples by combining NCBA-GMNP, incubated with Tween 80 (5%) for three minutes, improved capture efficiency and increased AFB detection up to 445% over SSM. NCBA with Tween 80 offers the opportunity to improve TB diagnostics, mainly in paucibacillary cases. As this method provides biosafety with a simple and inexpensive methodology that obtains results in a short time, it might be considered as a point-of-care TB diagnostic method in regions where resources are limited.

Hemicellulolytic bacteria in the anterior intestine of the earthworm Eisenia fetida (Sav.).

Tropical agriculture produces large amounts of lignocellulosic residues that can potentially be used as a natural source of value-added products. The complexity of lignocellulose makes industrial-scale processing difficult. New processing techniques must be developed to improve the yield and avoid this valuable resource going to waste. Hemicelluloses comprise a variety of polysaccharides with different backbone compositions and decorations (such as methylations and acetylations), and form part of an intricate framework that confers structural stability to the plant cell wall. Organisms that are able to degrade these biopolymers include earthworms (Eisenia fetida), which can rapidly decompose a wide variety of lignocellulosic substrates. This ability probably derives from enzymes and symbiotic microorganisms in the earthworm gut. In this work, two substrates with similar C/N ratios but different hemicellulose content were selected. Palm fibre and coffee husk have relatively high (28%) and low (5%) hemicellulose contents, respectively. A vermicomposting mixture was prepared for the earthworms to feed on by mixing a hemicellulose substrate with organic market waste. Xylanase activity was determined in earthworm gut and used as a selection criterion for the isolation of hemicellulose-degrading bacteria. Xylanase activity was similar for both substrates, even though their physicochemical properties principally pH and electrical conductivity, as shown by the MANOVA analysis) were different for the total duration of the experiment (120 days). Xylanolytic strains isolated from earthworm gut were identified by sequence analysis of the 16S rRNA gene. Our results indicate that the four Actinobacteria, two Proteobacteria, and one Firmicutes isolated are active participants of the xylanolytic degradation by microbiota in the intestine of E. fetida. Most bacteria were more active at pH 7 and 28 °C, and those with higher activities are reported as being facultatively anaerobic, coinciding with the microenvironment reported for the earthworm gut. Each strain had a different degradative capacity.

Brevipalpus mites associated with coffee plants (Coffea arabica and C. canephora) in Chiapas, Mexico.

Tenuipalpid mites of the genus Brevipalpus are of significant economic and quarantine importance in agriculture. They can damage and vector phytopathogenic viruses in coffee plantations and other crops. In this study, we focused on: identification of the Brevipalpus species, assessment of the spread of Brevipalpus-associated viruses (CoRSV, CiLV-N, CiLVC and CiLVC2), and mite population fluctuations over the course of 1 year. The study was conducted in coffee plantations in Soconusco, a coffee-producing region in Chiapas, Mexico. The collected mites of the Brevipalpus phoenicis sensu lato species complex (635) were identified as Brevipalpus papayensis (80.2%) and B. yothersi (19.8%) based on morphological and molecular characteristics. Their population abundance was low and there were no indications for virosis. The highest mite abundance was recorded in August-September and the lowest in February-March. An interaction was observed between mite abundance and coffee species in open-growth and shaded cultivation at various altitudes. Brevipalpus papayensis was most abundant in Coffea arabica var. Bourbon, in shaded (80%) growing conditions at an altitude of 1300 m above sea level. In C. canephora (in open-growth cultivation conditions at low altitude), B. yothersi was more abundant than in C. arabica, and as abundant as B. papayensis. We are of the opinion that, at this moment, B. papayensis and B. yothersi do not present risks to the production of coffee for the studied plantations. However, as the coffee-producing regions of Mexico are ecologically diverse, it will be important to continue examining the status of Brevipalpus mite populations in other regions in Mexico.

Biodegradation of NSAIDs and their effect on the activity of ligninolytic enzymes from Pleurotus djamor.

In this work, the white-rot fungus Pleurotus djamor was used for the first time to determine the degradation kinetics of the nonsteroidal anti-inflammatory drugs diclofenac, naproxen and, ketoprofen, either individually or in mixtures, in submerged cultures. Removal of 93% individual diclofenac and 99% diclofenac in mixtures with naproxen and ketoprofen at 6 h of incubation with the fungus was achieved. The elimination levels of naproxen and ketoprofen individually were 90% and 87%, respectively, after 48 h of incubation. However, the removal levels of these compounds in mixtures were 85% and 83%, respectively. On the other hand, during the degradation kinetics analysis, the enzymatic activities of laccases, manganese peroxidases, and lignin peroxidases were evaluated, yielding values of 3700, 270 and 31 U/L, respectively. Additionally, it was demonstrated that during degradation of diclofenac or the three drugs mixed in the submerged cultures, the enzymatic activity of extracellular laccases expressed by P. djamor increased by 200% and 300%, respectively. The activity of manganese peroxides increased by 126% with diclofenac and 138% when the mixture of drugs was added to the cultures. On the other hand, lignin peroxidase only increased activity by 123% with the drug mixture.

Variation in the cuticular hydrocarbons of the Mexican fruit fly Anastrepha ludens males between strains and age classes.

In this study cuticular hydrocarbons (CHCs) were characterized from wings of individual unmated males of different Anastrepha ludens (Loew) mass-reared strains of different ages (3 and 19-day-old): (a) a standard mass-reared colony (control), (b) a genetic sexing strain, (c) a selected strain, (d) a hybrid strain, and (e) wild males. We found that the hydrocarbon profiles in all males included two n-alkanes, five monomethyl alkanes, and two alkenes. CHCs ranged from C26 to C31 . The most prominent peaks were 2-methyloctacosane (2-Me-C28), n-nonacosene (C29:1), 2-methyltriacontane (2-Me-C30), and n-hentriacontene (C31:1). Significant variations in the CHC amounts of the mass-reared strains were observed from Day 9 and thereafter. Comparison of CHCs using multivariate and canonical analyses across ages and among mass-reared strains and wild males revealed qualitative and quantitative differences. The relative amounts of C29:1 and 2-Me-C30 were significantly higher across age groups in the mass-reared strains than those in the wild males. In contrast, amounts of n-nonacosane (C29) significantly increased in wild males as they aged. Through statistical analyses, we inferred that CHC amounts vary with age. Wild males differed significantly from the mass-reared strains in the amount of C29, and the genetic sexing strain Tap-7 had significantly higher values for 2-methylhexacosane (2-Me-C26). In contrast the selected and control strain differed from the other strains in amounts of C29:1 and 2-Me-C30. We suggest that differential profiles in hydrocarbon composition among the strains may be mainly due to environmental pressures.

Bacillus subtilis with endocellulase and exocellulase activities isolated in the thermophilic phase from composting with coffee residues / Bacillus subtilis con actividades de endocelulasa y exocelulasa aislado en la fase termofílica del compostaje con residuos de café

The goal of this study was to isolate, select and characterize bacteria with cellulolytic activity from two different coffee residue composting piles, one of which had an internal temperature of 57 -#9702;C and pH 5.5 and the other, a temperature of 61 -#9702;C, and pH 9.3. Culture media were manipulated with carboxymethylcellulose and crystalline cellulose as sole carbon sources. The enzyme activity was assessed by hydrolysis halo formation, reducing sugar production and zymograms. Three out of twenty isolated strains showed higher enzymatic activity and were identified as Bacillus subtilis according to their morphological, physiological, biochemical characteristics and based on the sequence analysis of 16S rDNA regions. The enzymatic extracts of the three selected strains showed exocellulase and endocellulase maximum activity of 0.254 and 0.519 U/ml, respectively; the activity of these enzymes was maintained even in acid pH (4.8) and basic (9.3) and at temperatures of up to 60°C. The enzymatic activities observed in this study are within the highest reported for cellulose produced by bacteria of the genus Bacillus. Endocellulase activity was shown in the zymograms from 24 h until 144 h of incubation. Furthermore, the pH effect on the endocellulase activity is reported for the first time by zymograms. The findings in this study entail the possibility to use these enzymes in the procurement of fermentable substrates for the production of energy from the large amount of residues generated by the coffee agroindustry.

El objetivo de este estudio fue aislar, seleccionary caracterizar bacterias con actividad celulolítica a partir de 2 diferentes pilas de compostaje de residuos de café, una con temperatura interna de 57°C y pH 5,5; la otra con temperatura interna de 61 °C y pH 9,3. Se utilizaron medios de cultivo con carboximetilcelulosa y celulosa cristalina como únicas fuentes de carbono. La actividad enzimàtica fue evaluada por formación de halos de hidrólisis, producción de azúcares reductores y zimogramas. De 20 cepas aisladas, 3 presentaron mayor actividad enzimàtica y fueron identificadas como Bacillus subtilis sobre la base de sus características morfológicas, fisiológicas y bioquímicas y del análisis de las secuencias de la región 16S del ADNr. Los extractos enzimáticos de las 3 cepas seleccionadas presentaron actividad de exocelulasa y de endocelulasa, con máximos de 0,254 y 0,519 U/ml, respectivamente; la actividad de estas enzimas se mantuvo incluso a pH ácido (4,8) o básico (9,3) y a temperaturas de hasta 60 °C. Las actividades enzimáticas halladas en este estudio se ubican dentro de las más altas reportadas para celulasas producidas por bacterias del género Bacillus. En los zimogramas se demostró actividad de endocelulasa desde las 24h hasta las 144h de incubación. Asimismo, se reporta por primera vez el efecto del pH sobre la actividad de endocelulasa observado por zimogramas. Los resultados de este estudio abren la posibilidad de hacer uso de estas enzimas en la obtención de sustratos fermentables para la producción de energía a partir de los residuos generados en grandes cantidades por la agroindustria del café.

Bacillus subtilis with endocellulase and exocellulase activities isolated in the thermophilic phase from composting with coffee residues.

The goal of this study was to isolate, select and characterize bacteria with cellulolytic activity from two different coffee residue composting piles, one of which had an internal temperature of 57°C and pH 5.5 and the other, a temperature of 61°C, and pH 9.3. Culture media were manipulated with carboxymethylcellulose and crystalline cellulose as sole carbon sources. The enzyme activity was assessed by hydrolysis halo formation, reducing sugar production and zymograms. Three out of twenty isolated strains showed higher enzymatic activity and were identified as Bacillus subtilis according to their morphological, physiological, biochemical characteristics and based on the sequence analysis of 16S rDNA regions. The enzymatic extracts of the three selected strains showed exocellulase and endocellulase maximum activity of 0.254 and 0.519 U/ml, respectively; the activity of these enzymes was maintained even in acid pH (4.8) and basic (9.3) and at temperatures of up to 60°C. The enzymatic activities observed in this study are within the highest reported for cellulose produced by bacteria of the genus Bacillus. Endocellulase activity was shown in the zymograms from 24h until 144h of incubation. Furthermore, the pH effect on the endocellulase activity is reported for the first time by zymograms. The findings in this study entail the possibility to use these enzymes in the procurement of fermentable substrates for the production of energy from the large amount of residues generated by the coffee agroindustry.

Enzymatic dynamics into the Eisenia fetida (Savigny, 1826) gut during vermicomposting of coffee husk and market waste in a tropical environment.

Epigeic worms modify microbial communities through their digestive processes, thereby influencing the decomposition of organic matter in vermicomposting systems. Nevertheless, the enzyme dynamics within the gut of tropically adapted earthworms is unknown, and the enzymes involved have not been simultaneously studied. The activities of 19 hydrolytic enzymes within three different sections of the intestine of Eisenia fetida were determined over a fasting period and at 24 h and 30, 60, and 90 days of vermicomposting, and data were evaluated by multivariate analyses. There were found positive correlations between the maximal activity of glycosyl hydrolases and one esterase with the anterior intestine (coincident with the reduction of hemicellulose in the substrate) and the activity of the protease α-chymotrypsin with posterior intestine. The results suggest that activities of enzymes change in a coordinated manner within each gut section, probably influenced by selective microbial enzyme enrichment and by the availability of nutrients throughout vermicomposting.

Degradation of the herbicide paraquat by macromycetes isolated from southeastern Mexico.

Fifty-four macromycetes, isolated from southeastern Mexico, were used in order to evaluate their capacity for degradation and tolerance to the herbicide paraquat. Ten of these strains were capable of growing in a solid culture medium in the presence of 200 ppm paraquat. Subsequently, assays to evaluate the degradation of the xenobiotic in a liquid medium were carried out. Of the ten strains evaluated, three presented the highest levels of degradation of the compound, which were Trametes pavonia (54.2%), Trametes versicolor (54.1%) and Hypholoma dispersum. They presented the highest overall degradation percentage (70.7%) after 12 days culture. The presence of ligninolytic enzymes in these strains was evaluated. H. dispersum only presented aryl alcohol oxidase activity; however, with the data obtained, it was not possible to conclude whether this specific enzyme is responsible for paraquat degradation. The level of degradation obtained is above the one reported for Pseudomonas putida, one of the few reports on paraquat degradation. This is the first report on the contaminant degradation capacity of H. dispersum.

Producción de enzimas ligninolíticas durante la degradación del herbicida paraquat por hongos de la pudrición blanca / Ligninolytic enzyme production by white rot fungi during paraquat (herbicide) degradation

El paraquat es un herbicida utilizado ampliamente en la agricultura. Debido a su gran distribución y uso inadecuado, representa un problema grave de contaminación del suelo y el agua. Se ha encontrado que los hongos de la podredumbre blanca son capaces de degradar compuestos contaminantes que poseen estructuras similares a la lignina, como es el caso del paraquat. En el presente trabajo se evaluó la degradación de este herbicida y su efecto en la producción de enzimas ligninolíticas por parte de algunos hongos de la podredumbre blanca aislados del sur de México. Seis cepas fúngicas mostraron tolerancia al herbicida durante el cultivo en medio sólido. Tres de las 6 cepas evaluadas, correspondientes a las especies Polyporus tricholoma, Cilindrobasidium laeve y Deconica citrispora, mostraron niveles de degradación del 32, el 26 y el 47%, en ese orden, a los 12 días de cultivo en presencia del xenobiótico. Se detectó un incremento en las actividades de las enzimas lacasa y Mn-peroxidasa en las cepas que presentaron el mayor porcentaje de degradación, probablemente asociado a la disminución del herbicida. Adicionalmente, se realizaron ensayos con extractos enzimáticos procedentes del medio de cultivo extracelular de las 2 cepas que presentaron mayor degradación. Después de 24 h de incubación, se obtuvo una degradación del 49% del paraquat inicial con los extractos de D. citrispora. Los resultados obtenidos indican que la degradación del herbicida estaría asociada a la presencia de enzimas extracelulares en los hongos de la podredumbre blanca. En este trabajo se muestran las primeras evidencias del potencial de biodegradación de diferentes especies de hongos de la pudrición blanca.

Paraquat is a widely used herbicide in agriculture. Its inappropriate use and wide distribution represents a serious pollution problem for soil and water. White rot fungi are capable of degrading pollutants having a similar structure to that of lignin, such as paraquat. This study evaluated the degradation effect of paraquat on the production of ligninolytic enzymes by white rot fungi isolated from the South of Mexico. Six fungal strains showed tolerance to the herbicide in solid culture. Three of the six evaluated strains showed levels of degradation of 32, 26 and 47% (Polyporus tricholoma, Cilindrobasidium laeve and Deconica citrispora, respectively) after twelve days of cultivation in the presence of the xenobiotic. An increase in laccase and manganese peroxidase (MnP) activities was detected in the strains showing the highest percentage of degradation. Experiments were done with enzyme extracts from the extracellular medium with the two strains showing more degradation potential and enzyme production. After 24 hours of incubation, a degradation of 49% of the initial paraquat concentration was observed for D. citrispora. These results suggest that paraquat degradation can be attributed to the presence of extracellular enzymes from white rot fungi. In this work the first evidence of the biodegradation potential of D. citrispora and Cilindrobasidium leave is shown.

[Ligninolytic enzyme production by white rot fungi during paraquat (herbicide) degradation]. / Producción de enzimas ligninolíticas durante la degradación del herbicida paraquat por hongos de la pudrición blanca.

Paraquat is a widely used herbicide in agriculture. Its inappropriate use and wide distribution represents a serious pollution problem for soil and water. White rot fungi are capable of degrading pollutants having a similar structure to that of lignin, such as paraquat. This study evaluated the degradation effect of paraquat on the production of ligninolytic enzymes by white rot fungi isolated from the South of Mexico. Six fungal strains showed tolerance to the herbicide in solid culture. Three of the six evaluated strains showed levels of degradation of 32, 26 and 47% (Polyporus tricholoma, Cilindrobasidium laeve and Deconica citrispora, respectively) after twelve days of cultivation in the presence of the xenobiotic. An increase in laccase and manganese peroxidase (MnP) activities was detected in the strains showing the highest percentage of degradation. Experiments were done with enzyme extracts from the extracellular medium with the two strains showing more degradation potential and enzyme production. After 24hours of incubation, a degradation of 49% of the initial paraquat concentration was observed for D. citrispora. These results suggest that paraquat degradation can be attributed to the presence of extracellular enzymes from white rot fungi. In this work the first evidence of the biodegradation potential of D. citrispora and Cilindrobasidium leave is shown.

Proteomic and morphometric study of the in vitro interaction between Oncidium sphacelatum Lindl. (Orchidaceae) and Thanatephorus sp. RG26 (Ceratobasidiaceae).

Orchidaceae establish symbiotic relationships with fungi in the Rhizoctonia group, resulting in interactions beneficial to both organisms or in cell destruction in one of them (pathogenicity). Previous studies have focused mostly on terrestrial species with a few, preliminary studies, on epiphytes. To further our understanding of the molecular mechanisms involved in these symbioses, we evaluated the interaction between Oncidium sphacelatum Lindl. and the mycorrhizal fungus Thanatephorus sp. strain RG26 (isolated from a different orchid species) in vitro using morphometric and proteomic analyses. Evidence from the morphometric and microscopic analysis showed that the fungus promoted linear growth and differentiation of orchid protocorms during 98 days interaction. On day 63, protocorm development was evident, so we analyzed the physiological response of both organisms at that moment. Proteome results suggest that orchid development stimulated by the fungus apparently involves cell cycle proteins, purine recycling, ribosome biogenesis, energy metabolism, and secretion that were up-regulated in the orchid; whereas in the fungus, a high expression of proteins implicated in stress response, protein-protein interaction, and saccharides and protein biosynthesis were found in the symbiotic interaction. This is the first work reporting proteins differentially expressed in the epiphytic orchid-fungus interaction and will contribute to the search for molecular markers that will facilitate the study of this symbiosis in both wild orchids and those in danger of extinction.

Sexual behavior and male volatile compounds in wild and mass-reared strains of the Mexican fruit fly Anastrepha ludens (Diptera: Tephritidae) held under different colony management regimes.

We compared the calling and mating behavior and volatile release of wild males Anastrepha ludens (Loew) with males from 4 mass-reared strains: (i) a standard mass-reared colony (control), (ii) a genetic sexing strain (Tap-7), (iii) a colony started from males selected on their survival and mating competitiveness abilities (selected), and (iv) a hybrid colony started by crossing wild males with control females. Selected and wild males were more competitive, achieving more matings under field cage conditions. Mass-reared strains showed higher percentages of pheromone calling males under field conditions except for Tap-7 males, which showed the highest percentages of pheromone calling males under laboratory cage conditions. For mature males of all strains, field-cage calling behavior increased during the last hour before sunset, with almost a 2 fold increase exhibited by wild males during the last half hour. The highest peak mating activity of the 4 mass-reared strains occurred 30 min earlier than for wild males. By means of solid phase microextraction (SPME) plus gas chromatography-mass spectrometry (GC-MS), the composition of volatiles released by males was analyzed and quantified. Wild males emitted significantly less amounts of (E,E)-α-farnesene but emitted significantly more amounts of (E,E)-suspensolide as they aged than mass-reared males. Within the 4 mass-reared strains, Tap-7 released significantly more amounts of (E,E)-α-farnesene and hybrid more of (E,E)-suspensolide. Differences in chemical composition could be explained by the intrinsic characteristics of the strains and the colony management regimes. Characterization of calling behavior and age changes of volatile composition between wild and mass-reared strains could explain the differences in mating competitiveness and may be useful for optimizing the sterile insect technique in A. ludens.

Complex regulation of Hsf1-Skn7 activities by the catalytic subunits of PKA in Saccharomyces cerevisiae: experimental and computational evidences.

BACKGROUND: The cAMP-dependent protein kinase regulatory network (PKA-RN) regulates metabolism, memory, learning, development, and response to stress. Previous models of this network considered the catalytic subunits (CS) as a single entity, overlooking their functional individualities. Furthermore, PKA-RN dynamics are often measured through cAMP levels in nutrient-depleted cells shortly after being fed with glucose, dismissing downstream physiological processes. RESULTS: Here we show that temperature stress, along with deletion of PKA-RN genes, significantly affected HSE-dependent gene expression and the dynamics of the PKA-RN in cells growing in exponential phase. Our genetic analysis revealed complex regulatory interactions between the CS that influenced the inhibition of Hsf1/Skn7 transcription factors. Accordingly, we found new roles in growth control and stress response for Hsf1/Skn7 when PKA activity was low (cdc25Δ cells). Experimental results were used to propose an interaction scheme for the PKA-RN and to build an extension of a classic synchronous discrete modeling framework. Our computational model reproduced the experimental data and predicted complex interactions between the CS and the existence of a repressor of Hsf1/Skn7 that is activated by the CS. Additional genetic analysis identified Ssa1 and Ssa2 chaperones as such repressors. Further modeling of the new data foresaw a third repressor of Hsf1/Skn7, active only in the absence of Tpk2. By averaging the network state over all its attractors, a good quantitative agreement between computational and experimental results was obtained, as the averages reflected more accurately the population measurements. CONCLUSIONS: The assumption of PKA being one molecular entity has hindered the study of a wide range of behaviors. Additionally, the dynamics of HSE-dependent gene expression cannot be simulated accurately by considering the activity of single PKA-RN components (i.e., cAMP, individual CS, Bcy1, etc.). We show that the differential roles of the CS are essential to understand the dynamics of the PKA-RN and its targets. Our systems level approach, which combined experimental results with theoretical modeling, unveils the relevance of the interaction scheme for the CS and offers quantitative predictions for several scenarios (WT vs. mutants in PKA-RN genes and growth at optimal temperature vs. heat shock).

Assessment of methods to recover DNA from bacteria, fungi and archaea in complex environmental samples.

DNA extraction from environmental samples is a critical step for metagenomic analysis to study microbial communities, including those considered uncultivable. Nevertheless, obtaining good quality DNA in sufficient quantities for downstream methodologies is not always possible, and it depends on the complexity and stability of each ecosystem, which could be more problematic for samples from tropical regions because those ecosystems are less stable and more complex. Three laboratory methods for the extraction of nucleic acids from samples representing unstable (decaying coffee pulp and mangrove sediments) and relatively stable (compost and soil) environments were tested. The results were compared with those obtained using two commercial DNA extraction kits. The quality of the extracted DNA was evaluated by PCR amplification to verify the recovery of bacterial, archaeal, and fungal genetic material. The laboratory method that gave the best results used a lysis procedure combining physical, chemical, and enzymatic steps.