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.

Nematicidal, Acaricidal and Plant Growth-Promoting Activity of Enterobacter Endophytic Strains and Identification of Genes Associated with These Biological Activities in the Genomes.

In the present study, the nematicidal and acaricidal activity of three Enterobacter endophytic strains isolated from Mimosa pudica nodules was evaluated. The percentages of mortality of Enterobacter NOD4 against Panagrellus redivivus was 81.2%, and against Nacobbus aberrans 70.1%, Enterobacter NOD8 72.4% and 62.5%, and Enterobacter NOD10 64.8% and 58.7%, respectively. While against the Tyrophagus putrescentiae mite, the mortality percentages were 68.2% due to Enterobacter NOD4, 64.3% due to Enterobacter NOD8 and 77.8% due to Enterobacter NOD10. On the other hand, the ability of the three Enterobacter strains to produce indole acetic acid and phosphate solubilization, characteristics related to plant growth-promoting bacteria, was detected. Bioinformatic analysis of the genomes showed the presence of genes related to IAA production, phosphate solubilization, and nitrogen fixation. Phylogenetic analyzes of the recA gene, phylogenomics, and average nucleotide identity (ANI) allowed us to identify the strain Enterobacter NOD8 related to E. mori and Enterobacter NOD10 as E. asburiae, while Enterobacter NOD4 was identified as a possible new species of this species. The plant growth-promoting, acaricidal and nematicidal activity of the three Enterobacter strains makes them a potential agent to include in biocontrol alternatives and as growth-promoting bacteria in crops of agricultural interest.

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.

Caracterización de bacterias ácido lácticas con actividad antimicrobiana aisladas del queso crema de Chiapas, México / Characterization of lactic acid bacteria with antimicrobial activity isolated from cream cheese from Chiapas, Mexico

Resumen El queso crema de Chiapas es un producto artesanal fabricado en diferentes regiones de México. La capacidad de las bacterias lácticas, de producir grandes cantidades de ácido láctico y acético, tiene como efecto la disminución del pH, considerado uno de los factores primarios en la inhibición de microorganismos indeseables, como patógenos y bacterias coliformes, en alimentos lácteos. El objetivo de este trabajo fue aislar bacterias ácido lácticas y evaluar su efecto antagónico contra bacterias patógenas in vitro y contra patógenos presentes en la leche cuando se adicionan durante la elaboración del queso crema. Se aislaron cepas de bacterias lácticas del queso crema de Chiapas, México. Los aislados fueron identificados mediante pruebas bioquímicas API 50CH y secuencias del gen ribosomal 16S. Las bacterias de interés se usaron en pruebas de inhibición del crecimiento con cepas patógenas. Se cuantificó la producción de ácido acético y láctico por cromatografía de gases acoplado a masas en los quesos, al inicio y a los 10 d posteriores a su elabo ración. Se aislaron 203 cepas bacterianas, de las cuales 82 tuvieron la capacidad de inhibir el crecimiento de 7 cepas bacterianas patógenas. Las cepas fueron identificadas por la secuencia parcial del gen ribosomal 16S como pertenecientes al género Lactobacillus; la adición de estas cepas en la elaboración del queso crema redujo o eliminó coliformes, Staphylococcus aureus, mohos y levaduras, mientras que en el queso elaborado con leche pasteurizada se detectaron 290 UFC/mL de mohos y levaduras. Estos resultados sugieren que las cepas de Lactobacillus aisladas del queso crema artesanal de Chiapas tienen potencial para su uso como cultivo iniciador con actividad de bioconservación en este y productos similares.

Abstract Chiapas cream cheese is an artisanal product manufactured in different regions of Mexico. The ability of lactic bacteria to produce large amounts of lactic and acetic acid has the effect of decreasing the pH, considered one of the primary factors in the inhibition of undesirable microorganisms such as pathogens and coliform bacteria, in dairy foods. The objective of this work was to isolate lactic acid bacteria and evaluate their antagonistic effect against pathogenic bacteria in vitro, as well as their effect against pathogens that are present in milk when they are added in the preparation of cream cheese. Strains of lactic bacteria were isolated from cream cheese from Chiapas, Mexico. Isolates were identified by API 50CH biochemical tests and 16S ribosomal gene sequences. The bacteria under study were used in pathogen growth inhibition tests. The production of acetic and lactic acid was quantified by gas chromatography coupled to masses in the cheeses at the beginning and 10 d after its elaboration. 203 bacterial strains were isolated, 82 of which had the ability to inhibit the growth of 7 pathogenic bacterial strains. The strains were identified by the partial sequence of the 16S ribosomal gene as belonging to the genus Lactobacillus; The addition of these strains in the elaboration of cream cheese reduces or eliminates coliforms, Staphylococcus aureus, molds and yeasts, while in the cheese made with pasteurized milk 290 CFU/mL of molds and yeasts were detected. These results suggest that Lactobacillus strains isolated from Chiapas artisanal cream cheese has the potential to be used as a starter culture with bioconservation activity in this and similar products.

Serratia sp., an endophyte of Mimosa pudica nodules with nematicidal, antifungal activity and growth-promoting characteristics.

In the present study, the nematicidal activity of an isolated strain of Mimosa pudica nodules was evaluated against the Nacobbus aberrans (J2) phytonymatodes with a mortality of 88.8%, while against the gastrointestinal nematode Haemonchus contortus (L3) and free-living Panagrellus redivivus was 100%. The ability to inhibit the growth of phytopathogenic fungi Fusarium sp., and Alternaria solani, as well as the oomycete Phytophthora capsici, this antifungal activity may be related to the ability to produce cellulases, siderophores and chitinases by this bacterial strain. Another important finding was the detection of plant growth promoter characteristics, such as auxin production and phosphate solubilization. The strain identified by sequences of the 16S and rpoB genes as Serratia sp. is genetically related to Serratia marcescens and Serratia nematodiphila. The promoter activity of plant growth, antifungal and nematicide of the Serratia sp. strain makes it an alternative for the biocontrol of fungi and nematodes that affect both the livestock and agricultural sectors, likewise, candidate as a growth-promoting bacterium.

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.

Isolation and characterization of endophytes from nodules of Mimosa pudica with biotechnological potential.

Legumes establish symbiotic relationships with different microorganisms, which could function as plant growth promotion microorganisms (PGPM). The finding of new PGPM strains is important to increase plant production avoiding or diminishing the use of industrial fertilizers. Thus, in this work we evaluated the plant growth promotion traits of ten strains isolated from Mimosa pudica root nodules. According to the 16S rDNA sequence, the microorganisms were identified as Enterobacter sp. and Serratia sp. To the best of our knowledge this is the first report describing and endophytic interaction between Mimosa pudica and Enterobacter sp. These strains have some plant growth promoting traits such as phosphate solubilization, auxin production and cellulase and chitinase activity. Strains identified as Serratia sp. inhibited the growth of the phytopathogenic fungi Fusarium sp., and Alternaria solani and the oomycete Phytophthora capsici. According to their biochemical characteristics, three strains were selected to test their plant growth promoting activity in a medium with an insoluble phosphate source. These bacteria show low specificity for their hosts as endophytes, since they were able to colonize two very different legumes: Phaseolus vulgaris and M. pudica. Seedlings of P. vulgaris were inoculated and grown for fifteen days. Enterobacter sp. NOD1 and NOD10, promoted growth as reflected by an increase in shoot height as well as an increase in the size and emergence of the first two trifolia. We could localize NOD5 as an endophyte in roots in P. vulgaris by transforming the strain with a Green Fluorescent Protein carrying plasmid. Experiments of co-inoculation with different Rhizobium etli strains allowed us to discard that NOD5 can fix nitrogen in the nodules formed by a R. etli Fix- strain. The isolates described in this work show biotechnological potential for plant growth promoting activity and production of indoleacetic acid and siderophores.

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.

Rapid identification of nitrogen-fixing and legume-nodulating Burkholderia species based on PCR 16S rRNA species-specific oligonucleotides.

Several novel N(2)-fixing Burkholderia species associated with plants, including legume-nodulating species, have recently been discovered. Presently, considerable interest exists in studying the diazotrophic Burkholderia species, both for their ecology and their great potential for agro-biotechnological applications. However, the available methods used in the identification of these Burkholderia species are time-consuming and expensive. In this study, PCR species-specific primers based on the 16S rRNA gene were designed, which allowed rapid, easy, and correct identification of most known N(2)-fixing Burkholderia. With this approach, type and reference strains of Burkholderia kururiensis, B. unamae, B. xenovorans, B. tropica, and B. silvatlantica, as well as the legume-nodulating B. phymatum, B. tuberum, B. mimosarum, and B. nodosa, were unambiguously identified. In addition, the PCR species-specific primers allowed the diversity of the diazotrophic Burkholderia associated with field-grown tomato and sorghum plants to be determined. B. tropica and B. xenovorans were the predominant species found in association with tomato, but the occurrence of B. tropica with sorghum plants was practically exclusive. The efficiency of the species-specific primers was validated with the detection of B. tropica and B. xenovorans from DNA directly recovered from tomato rhizosphere soil samples. Additionally, using PCR species-specific primers, all of the legume-nodulating Burkholderia were correctly identified, even from single nodules collected from inoculated common bean plants. These primers could contribute to rapid identification of the diazotrophic and nodulating Burkholderia species associated with important crop plants and legumes, as well as revealing their environmental distribution.