In vitro nematicidal and acaricidal effect of biosurfactants produced by Bacillus against the root-knot nematode Nacobbus aberrans and the dust mite Tyrophagus putrescentiae.

In the present study, the nematicidal and acaricidal activity of three biosurfactants (BS) produced by strains of the Bacillus genus was evaluated. The BS produced by the Bacillus ROSS2 strain presented a mortality of 39.29% in juveniles (J2) of Nacobbus aberrans at a concentration of 30 mg/mL, this same strain is the one that presented the highest mortality in Tyrophagus putrescentiae, which was 57.97% at a concentration of 39 mg/mL. The BS were qualitatively identified by thin layer chromatography and are lipid in nature based on the retention factor (Rf). While the GC-MS analysis identified two main compounds that are 4,7-Methano-1H-indene-2,6-dicarboxylic acid, 3a,4,7,7a-tetrahydro-1, and Methyl 4-(pyrrol-1-yl)-1,2,5-oxadiazole-3-carboxylate1, which is the polar part indicated by the presence of dicarboxylic acid and carboxylate groups; while the non-polar portion can be interpreted as a hydrocarbon chain of variable length. Based on the present results, BS can be an alternative for the biocontrol of the root-knot nematode N. aberrans and the mite T. putrescentiae.

Exploring plant growth-promoting rhizobacteria as stress alleviators: a methodological insight.

Rhizospheric and root-endophyte bacteria can stimulate plant growth through diverse biochemical mechanisms and pathways, particularly under biotic and abiotic stresses. For this reason, biotechnological trends on plant growth-promoting rhizobacteria (PGPR) application as biofertilizers, bioremediators, and stress alleviators are gaining increasing interest as ecofriendly strategies for sustainable agriculture management and soil restoration. The first steps needed to implement these technologies are isolation, screening, and characterization of PGPR that can be potentially applied as bioinoculants to alleviate biotic and/or abiotic stresses. Therefore, a complete and accurate methodological study and laboratory techniques are required to warrant the correct achievement of these steps. This review compiles and details the fundamentals, methods, and procedures of key protocols used in isolation and characterization of PGPR for plant stress alleviation.

Effect of pH and Carbon Source on Phosphate Solubilization by Bacterial Strains in Pikovskaya Medium.

Phosphate-solubilizing bacteria (PSB) transform precipitated inorganic phosphorus into soluble orthophosphates. This study evaluated the efficiency of tricalcium and iron phosphate solubilization in Pikovskaya medium using five bacterial strains (A1, A2, A3, A5, and A6) cultured in acidic and alkaline pH levels. The bacterial strain that proved to be more efficient for P solubilization and was tolerant to pH variations was selected for assessing bacterial growth and P solubilization with glucose and sucrose in the culture medium. The bacterial strains were identified through 16S rRNA gene sequencing as Pseudomonas libanensis A1, Pseudomonas libanensis (A2), Bacillus pumilus (A3), Pseudomonas libanensis (A5), and Bacillus siamensis (A6). These five bacterial strains grew, tolerated pH changes, and solubilized inorganic phosphorus. The bacterial strain A3 solubilized FePO4 (4 mg L-1) and Ca3(PO4)2 (50 mg L-1). P solubilization was assayed with glucose and sucrose as carbon sources for A3 (Bacillus pumilus MN100586). After four culture days, Ca3(PO4)2 was solubilized, reaching 246 mg L-1 with sucrose in culture media. Using glucose as a carbon source, FePO4 was solubilized and reached 282 mg L-1 in six culture days. Our findings were: Pseudomonas libanensis, and Bacillus siamensis, as new bacteria, can be reported as P solubilizers with tolerance to acidic or alkaline pH levels. The bacterial strain B. pumilus grew using two sources of inorganic phosphorus and carbon, and it tolerated pH changes. For that reason, it is an ideal candidate for inorganic phosphorus solubilization and future production as a biofertilizer.

Nematicidal Activity of the Endophyte Serratia ureilytica against Nacobbus aberrans in Chili Plants (Capsicum annuum L.) and Identification of Genes Related to Biological Control.

The genus Serratia is widely distributed in soil, water, plants, animals, invertebrates, and humans. Some species of this genus have antifungal, antibacterial, and nematicidal activity. In this work, the nematicidal activity of the endophytic strain of Serratia sp. in chili, Capsicum annuum L., is reported, where at a bacterial concentration of 4 × 109 cel/mL, the penetration of nematodes into the roots significantly decreased by 91 and 55% at 7 and 21 days after inoculation. This bacterial concentration also significantly decreased the number of galls, eggs, egg masses and reproduction factor produced by Nacobbus aberrans in Chili plants, with respect to the control where this bacterial strain was not applied. In the analysis of the genome of the strain, based on average nucleotide identity (ANI), the isolate could be affiliated to the species Serratia ureilytica. The size of the genome is 5.4 Mb, with a 59.3% content of GC. Genes related to the synthesis of chitinases, siderophores, proteases C, serralisins, hemolysin, and serrawettin W2 that have been reported for biocontrol of nematodes were identified in the genome. It is the first report of Serratia ureilytica with nematicidal activity. Based on these results of nematicidal activity, this strain can be evaluated in the field as an alternative in the biocontrol of Nacobbus aberrans in chili cultivation.

IDENTIFICATION OF ENDOPHYTIC BACTERIA OF SEEDS FROM Cedrela odorata L. (Meliaceae) WITH BIOTECHNOLOGICAL CHARACTERISTICS / Identificación de bacterias endófitas de semillas de Cedrela odorata L. (Meliaceae) con características biotecnológicas

ABSTRACT In the present study, 62 endophytic bacterial strains of cedar seeds (Cedrela odorata L.), collected in the municipalities of Huehuetán, Motozintla, and Pijijiapan in the state of Chiapas, Mexico were isolated. The goal was to identify characteristics of biotechnological interest such as biocontrol, promotion ofplant growth, and growth in aromatic compounds. The strains were identified by the partial sequence of the 16S ribosomal gene as belonging to the Bacillus genus. The biocontrol capacity of phytopathogenic fungi, production of indoleacetic acid (IAA), solubilization of phosphate, and growth in xenobiotic compounds (phenanthrene, benzene, anthracene, or phenol) were detected in 26 strains of the 62 isolates. 21 % of the strains inhibited the mycelial growth of Alternaria solani and Fusarium sp., and 13 % of the Phytophthora capsici oomycete. IAA production was detected in 24 isolates, phosphate solubilizing activity was identified in 18 isolates, while the ability to grow in the presence of phenanthrene and benzene was found in 26 isolates; 24 isolates grew in the presence of anthracene and only two isolates grew in phenol as the only carbon sources. This is the first report of the isolation and identification of endophytic bacteria from cedar seeds, where biotechnological characteristics were detected for biological control, promotion of plant growth, and growth in the presence of xenobiotic compounds.

RESUMEN En el presente estudio se aislaron 62 cepas bacterianas endófitas de semillas de cedro (Cedrela odorata L.) colectadas en los municipios de Huehuetán, Motozintla y Pijijiapan en el estado de Chiapas, México, con el objetivo de identificar características de interés biotecnológicas como biocontrol, promoción del crecimiento vegetal y crecimiento en compuestos aromáticos. Las cepas se identificaron por la secuencia parcial del gen 16S ribosomal como pertenecientes al género Bacillus. En 26 cepas de las 62 aisladas se detectaron la capacidad de biocontrol de hongos fitopatógenos, la producción de ácido indolacético (AIA), la solubilización de fosfato y el crecimiento en compuestos xenobióticos (fenantreno, benceno, antraceno o fenol). El 21 % de las cepas inhibió el crecimiento miceliar de Alternaria solani y Fusarium sp., y el 13 % del oomiceto Phytophthora capsici. La producción de ácido indolacético se detectó en 24 aislados y la actividad solubilizadora de fosfato se encontró en 18 aislados, mientras que la capacidad de crecer en presencia de fenantreno y benceno se manifestó en 26 aislados (24 aislados crecieron en presencia de antraceno y solo dos aislados crecieron en fenol como únicas fuentes de carbono). Es importante mencionar que este es el primer reporte del aislamiento e identificación de bacterias endófitas de semillas de cedro, en el que se detectaron características biotecnológicas para el control biológico, la promoción del crecimiento vegetal y el crecimiento en presencia de compuestos xenobióticos.

Isolated Phosphate-Solubilizing Soil Bacteria Promotes In vitro Growth of Solanum tuberosum L.

The capacity of four bacterial strains isolated from productive soil potato fields to solubilize tricalcium phosphate on Pikovskaya agar or in a liquid medium was evaluated. A bacterial strain was selected to evaluate in vitro capacity of plant-growth promotion on Solanum tuberosum L. culture. Bacterial strain A3 showed the highest value of phosphate solubilization, reaching a 20 mm-diameter halo and a concentration of 350 mg/l on agar and in a liquid medium, respectively. Bacterial strain A3 was identified by 16S rDNA analysis as Bacillus pumilus with 98% identity; therefore, it is the first report for Bacillus pumilus as phosphate solubilizer. Plant-growth promotion assayed by in vitro culture of potato microplants showed that the addition of bacterial strain A3 increased root and stems length after 28 days. It significantly increased stem length by 79.3%, and duplicated the fresh weight of control microplants. In this paper, results reported regarding phosphorus solubilization and growth promotion under in vitro conditions represent a step forward in the use of innocuous bacterial strain biofertilizer on potato field cultures.

A bacterial strain of Pseudomonas aeruginosa B0406 pathogen opportunistic, produce a biosurfactant with tolerance to changes of pH, salinity and temperature.

A bacterial strain of Pseudomonas aeruginosa B0406 catalogued as pathogen opportunistic was capable to grow with waste cooking oil as only carbon source and produce a biosurfactant. Stability to pH (from 2 to 12), salinity (% NaCl from 0 to 20%) and temperature (from -20 °C up to 120 °C), of biosurfactants was evaluated using a response surface methodology. Biosurfactants reduced surface tension from 50 to 29 ± 1.0 mN/m. Pseudomonas aeruginosa B0406 showed a high biosurfactant yield 4.17 g/L ± 0.38. Biosurfactants stability applying a response surface methodology was observed with combining effect of pH, salinity and temperature. The three factors combined do not affect surface tension of biosurfactants produced by Pseudomonas aeruginosa B0406. Therefore, this biosurfactants are of interest for medical, cosmetic even environmental applications.

Bactericidal effect of silver nanoparticles against propagation of Clavibacter michiganensis infection in Lycopersicon esculentum Mill.

This study explored the use of silver nanoparticle as a bactericidal against the propagation of Clavibacter michiganensis onto tomatoes (Lycopersicon esculentum Mill). In Mexico, tomato production covers about 73% of the total vegetable production but it is affected by outbreak of bacteria canker caused by Clavibacter michiganensis subspecies michiganensis (Cmm). Silver ions possess inhibitor properties, bactericides and high specter antimicrobials. In this study, 6 groups of culture were prepared using 6 different petri dishes where silver nanoparticles of varying concentrations (120, 84, 48, 24, 12 and 0 µg) were added. Furthermore, each group was observed for 20 min, 1, 2, 12 and 24 h. The optimum concentration is 84 µg, which shows an average of 2 Cmm colonies after 20 min. Further increase to 120 µg shows no significant change. However, the average colonies was observed for 48 µg after 1, 2, 12, and 24 h. The obtained results indicate that silver nanoparticles are a promising inhibitor, bactericide and high a specter antimicrobial for treatment or prevention of Cmm.

Partial characterization of a biosurfactant extracted from Pseudomonas sp. B0406 that enhances the solubility of pesticides.

Biodegradation of some organochlorine and organophosphate pesticides is difficult because of their low solubility in water and, therefore, their low bioavailability. To overcome the hydrophobicity problem and the limited pesticide availability, biosurfactants play a major role. In this study, we evaluated the effect of an extract from Pseudomonas sp. B0406 strain with surfactant properties, on the solubility of two pesticides: endosulfan (ED) and methyl parathion (MP). Such a process was performed in order to increase the aqueous solubility of both pesticides, to increase its availability to microorganisms and to promote their biodegradation. The extract from Pseudomonas sp. B0406 showed a critical micellar concentration of 1.4 g/L and the surface tension at that point was 40.4 mN/m. The preliminary chemical and physical partial characterization of the extract with surfactant properties indicated that it is an anionic glycolipid, which increases the solubility of both pesticides of 0.41 at 0.92 mg/L for ED and of 34.58 at 48.10 mg/L for MP. The results of this study suggest the effectiveness of this extract in improving the solubility of both pesticides ED and MP in water and, therefore, of its potential use to enhance the degradation of these pesticides.

Burkholderia species associated with legumes of Chiapas, Mexico, exhibit stress tolerance and growth in aromatic compounds / Especies de Burkholderia asociadas a leguminosas de Chiapas, México exhiben tolerancia a estrés y crecimiento en compuestos aromáticos

Leguminous plants have received special interest for the diversity of β-proteobacteria in their nodules and are promising candidates for biotechnological applications. In this study, 15 bacterial strains were isolated from the nodules of the following legumes: Indigofera thibaudiana, Mimosa diplotricha, Mimosa albida, Mimosa pigra, and Mimosa pudica, collected in 9 areas of Chiapas, Mexico. The strains were grouped into four profiles of genomic fingerprints through BOX-PCR and identified based on their morphology, API 20NE biochemical tests, sequencing of the 16S rRNA, nifH and nodC genes as bacteria of the Burkholderia genus, genetically related to Burkholderia phenoliruptrix, Burkholderia phymatum, Burkholderia sabiae, and Burkholderia tuberum. The Burkholderia strains were grown under stress conditions with 4% NaCl, 45°C, and benzene presence at 0.1% as the sole carbon source. This is the first report on the isolation of these nodulating species of the Burkholderia genus in legumes in Mexico.

Las plantas leguminosas han recibido especial interés por la diversidad de β-proteobacteria que albergan en sus nodulos; algunas de estas bacterias son candidatas prometedoras para aplicaciones biotecnológicas. En el presente trabajo se aislaron 15 cepas bacterianas de los nodulos de las leguminosas Indigofera thibaudiana, Mimosa diplotricha, Mimosa albida, Mimosa pigra y Mimosa púdica, colectadas en 9 áreas de Chiapas, México. Las cepas fueron agrupadas en 4 perfiles de huellas genómicas por BOX-PCR e identificadas sobre la base de su morfología, pruebas bioquímicas API 20NE y secuenciación de los genes 16S ARNr, nifH y nodC como bacterias del género Burkholderia relacionadas genéticamente con Burkholderia phenoliruptrix, Burkholderia phymatum, Burkholderia sabiae y Burkholderia tuberum. Las cepas de Burkholderia crecieron en condiciones de estrés con NaCl al 4%, a una temperatura de 45°C y en presencia de benceno al 0,1% como única fuente de carbono. Este es el primer reporte del aislamiento de especies de Burkholderia nodulantes en leguminosas en México.

Burkholderia species associated with legumes of Chiapas, Mexico, exhibit stress tolerance and growth in aromatic compounds.

Leguminous plants have received special interest for the diversity of ß-proteobacteria in their nodules and are promising candidates for biotechnological applications. In this study, 15 bacterial strains were isolated from the nodules of the following legumes: Indigofera thibaudiana, Mimosa diplotricha, Mimosa albida, Mimosa pigra, and Mimosa pudica, collected in 9 areas of Chiapas, Mexico. The strains were grouped into four profiles of genomic fingerprints through BOX-PCR and identified based on their morphology, API 20NE biochemical tests, sequencing of the 16S rRNA, nifH and nodC genes as bacteria of the Burkholderia genus, genetically related to Burkholderia phenoliruptrix, Burkholderia phymatum, Burkholderia sabiae, and Burkholderia tuberum. The Burkholderia strains were grown under stress conditions with 4% NaCl, 45°C, and benzene presence at 0.1% as the sole carbon source. This is the first report on the isolation of these nodulating species of the Burkholderia genus in legumes in Mexico.

Characterization of Bacteria Isolation of Bacteria from Pinyon Rhizosphere, Producing Biosurfactants from Agro-Industrial Waste.

Two hundred and fifty bacterial strains were isolated from pinyon rhizosphere and screened for biosurfactants production. Among them, six bacterial strains were selected for their potential to produce biosurfactants using two low cost wastes, crude glycerol and lactoserum, as raw material. Both wastes were useful for producing biosurfactants because of their high content in fat and carbohydrates. The six strains were identified by 16S rDNA with an identity percentage higher than 95%, three strains belonged to Enterobacter sp., Pseudomonas aeruginosa, Bacillus pumilus and Rhizobium sp. All strains assayed were able to grow and showed halos around the colonies as evidence of biosurfactants production on Cetyl Trimethyl Ammonium Bromide agar with crude glycerol and lactoserum as substrate. In a mineral salt liquid medium enriched with both wastes, the biosurfactants were produced and collected from free cell medium after 72 h incubation. The biosurfactants produced reduced the surface tension from 69 to 30 mN/m with an emulsification index of diesel at approximately 60%. The results suggest that biosurfactants produced by rhizosphere bacteria from pinyon have promising environmental applications.

[Agroindustrial wastes methanization and bacterial composition in anaerobic digestion]. / Residuos agroindustriales con potencial para la producción de metano mediante la digestión anaerobia.

The tons of organic waste that are annually generated by agro-industry, can be used as raw material for methane production. For this reason, it is important to previously perform biodegradability tests to organic wastes for their full scale methanization. This paper addresses biodegradability, methane production and the behavior of populations of eubacteria and archaeabacteria during anaerobic digestion of banana, mango and papaya agroindustrial wastes. Mango and banana wastes had higher organic matter content than papaya in terms of their volatile solids and total solid rate (94 and 75% respectively). After 63 days of treatment, the highest methane production was observed in banana waste anaerobic digestion: 63.89ml CH4/per gram of chemical oxygen demand of the waste. In the PCR-DGGE molecular analysis, different genomic footprints with oligonucleotides for eubacteria and archeobacteria were found. Biochemical methane potential results proved that banana wastes have the best potential to be used as raw material for methane production. The result of a PCR- DGGE analysis using specific oligonucleotides enabled to identify the behavior of populations of eubacteria and archaeabacteria present during the anaerobic digestion of agroindustrial wastes throughout the process.

Residuos agroindustriales con potencial para la producción de metano mediante la digestión anaerobia / Agroindustrial wastes methanization and bacterial composition in anaerobic digestion

Las toneladas de residuos orgánicos que se generan anualmente en la agroindustria pueden aprovecharse como materia prima para la producción de metano. Para que los residuos orgánicos se puedan convertir a metano a gran escala, es importante que previamente se realicen sobre ellos pruebas de biodegradabilidad; un parámetro importante que conviene establecer es su potencial bioquímico de metano. En el presente trabajo se estudió la biodegradabilidad, la producción de metano y el comportamiento de poblaciones de eubacterias y arqueobacterias durante la digestión anaerobia de residuos de plátano, mango y papaya provenientes de la agroindustria, adicionando un inóculo microbiano. Los residuos de mango y plátano tenían mayor contenido de materia orgánica (94 y 75 %, respectivamente) que el residuo de papaya con base en su relación sólidos volátiles/sólidos totales. Después de 63 días de tratamiento, la mayor producción de metano se observó en la digestión anaerobia del residuo de plátano: 63,89 ml de metano por g de demanda química de oxígeno del residuo. Los resultados del potencial bioquímico de metano demostraron que el residuo de plátano tiene el mejor potencial para ser usado como materia prima en la producción de metano. A través de un análisis por PCR-DGGE con oligonucleótidos específicos se logró evaluar el tamaño y la composición de las poblaciones de eubacterias y arqueobacterias presentes en la digestión anaerobia de residuos agroindustriales a lo largo del proceso.

The tons of organic waste that are annually generated by agro-industry, can be used as raw material for methane production. For this reason, it is important to previously perform biodegradability tests to organic wastes for their full scale methanization. This paper addresses biodegradability, methane production and the behavior of populations of eubacteria and archaeabacteria during anaerobic digestion of banana, mango and papaya agroindustrial wastes. Mango and banana wastes had higher organic matter content than papaya in terms of their volatile solids and total solid rate (94 and 75 % respectively). After 63 days of treatment, the highest methane production was observed in banana waste anaerobic digestion: 63.89 ml CH4/per gram of chemical oxygen demand of the waste. In the PCR-DGGE molecular analysis, different genomic footprints with oligonucleotides for eubacteria and archeobacteria were found. Biochemical methane potential results proved that banana wastes have the best potential to be used as raw material for methane production. The result of a PCR- DGGE analysis using specific oligonucleotides enabled to identify the behavior of populations of eubacteria and archaeabacteria present during the anaerobic digestion of agroindustrial wastes throughout the process.

Removal of methyl parathion and tetrachlorvinphos by a bacterial consortium immobilized on tezontle-packed up-flow reactor.

A tezontle-packed up-flow reactor (TPUFR) with an immobilized bacterial consortium for biological treatment of methyl-parathion and tetrachlorvinphos was evaluated. These organophosphate pesticides are widely used in Mexico for insect and mite control, respectively. With the aim of developing a tool for pesticide biodegradation, four flow rates (0.936, 1.41, 2.19, and 3.51 l/h) and four hydraulic residence times (0.313, 0.206, 0.133, and 0.083 h) were evaluated in a TPUFR. In the bioreactor, with an operating time of 8 h and a flow of 0.936 l/h, we obtained 75% efficiency in the removal of methyl-parathion and tetrachlorvinphos. Their adsorptions in the volcanic rock were 9% and 6%, respectively. It was demonstrated that the removal of pesticides was due to the biological activity of the immobilized bacterial consortium. We confirmed the decrease in toxicity in the treated effluent from the bioreactor through the application of acute toxicity tests on Eisenia foetida. Immobilization of a bacterial consortium using tezontle as a support is innovative and an economical tool for the treatment of mixtures of organophosphorus pesticide residues.

Removal of two organophosphate pesticides by a bacterial consortium immobilized in alginate or tezontle.

In order to remove methyl-parathion (MP) and tetrachlorvinphos (TCF), a bacterial consortium was immobilized with two supports consisting of alginate beads or stones of tezontle colonized by biofilm. Removal kinetics were recorded for suspended and immobilized consortium using a mineral salt medium supplemented with MP and TCF at 25mg/L and with 0.1% (w/v) glucose as a co-substrate. The viability of the consortium cultivated in suspension was maintained for 6 days, whereas the viability of the consortium immobilized in alginate and tezontle supports was maintained for up to 11 and 13 days, respectively. Growth was enhanced when using glucose as a co-substrate. The percentage of MP removed was significantly higher (alpha=0.05) when consortium was immobilized in alginate beads and biofilm on tezontle as compared to suspension culture.