Optimization of Paenibacillus sp. NMA1017 Application as a Biocontrol Agent for Phytophthora tropicalis and Moniliophthora roreri in Cacao-Growing Fields in Chiapas, Mexico.

In Mexico, cacao production is endangered by pathogenic fungi, such as Phytophthora spp. and Moniliophthora rorei, that cause black pod rot and moniliasis, respectively. In this study the biocontrol agent Paenibacillus sp. NMA1017 was tested in cacao fields against the previous diseases. The treatments applied were shade management, inoculation of the bacterial strain with or without an adherent, and use of chemical control. The statistical analysis showed that the incidence of black pod rot in tagged cacao trees diminished when the bacterium was applied (reduction of 44.24 to 19.11%). The same result was observed with moniliasis when the pods were tagged (reduction of 66.6 to 27%). The use of Paenibacillus sp. NMA1017 with an integrated management might be a solution to cacao diseases and to having a sustainable production of cacao in Mexico.

The Viable but Non-Culturable (VBNC) State, a Poorly Explored Aspect of Beneficial Bacteria.

Many bacteria have the ability to survive in challenging environments; however, they cannot all grow on standard culture media, a phenomenon known as the viable but non-culturable (VBNC) state. Bacteria commonly enter the VBNC state under nutrient-poor environments or under stressful conditions. This review explores the concept of the VBNC state, providing insights into the beneficial bacteria known to employ this strategy. The investigation covers different chemical and physical factors that can induce the latency state, cell features, and gene expression observed in cells in the VBNC state. The review also covers the significance and applications of beneficial bacteria, methods of evaluating bacterial viability, the ability of bacteria to persist in environments associated with higher organisms, and the factors that facilitate the return to the culturable state. Knowledge about beneficial bacteria capable of entering the VBNC state remains limited; however, beneficial bacteria in this state could face adverse environmental conditions and return to a culturable state when the conditions become suitable and continue to exert their beneficial effects. Likewise, this unique feature positions them as potential candidates for healthcare applications, such as the use of probiotic bacteria to enhance human health, applications in industrial microbiology for the production of prebiotics and functional foods, and in the beer and wine industry. Moreover, their use in formulations to increase crop yields and for bacterial bioremediation offers an alternative pathway to harness their beneficial attributes.

Contributions and difficulties of soil metagenomics and its impact on agricultura / Aportes y dificultades de la metagenómica de suelos y su impacto en la agricultura

RESUMEN Los microorganismos son de gran interés porque colonizan todo tipo de ambiente, sin embargo, uno de los problemas al que nos enfrentamos para conocer su diversidad biológica es que no todos los microorganismos son cultivables. El desarrollo de nuevas tecnologías como la generación de vectores de clonación aunado al desarrollo de técnicas de secuenciación de alto rendimiento ha favorecido el surgimiento de una nueva herramienta llamada metagenómica, la cual nos permite estudiar genomas de comunidades enteras de microorganismos. Debido a que ningún ambiente es idéntico a otro, es importante mencionar que dependiendo del tipo de muestra a analizar será el tipo de reto al cual nos enfrentaremos al trabajar con metagenómica, en el caso específico del suelo existen diversas variantes como la contaminación del suelo con metales pesados o diversos compuestos químicos que podrían limitar los estudios. Sin embargo, pese a las limitaciones que el mismo ambiente presenta, la metagenómica ha permitido tanto el descubrimiento de nuevos genes como la caracterización de las comunidades microbianas que influyen positivamente en el desarrollo de plantas, lo cual en un futuro podría generar un gran impacto en la agricultura. En este artículo se realizó una revisión de diversas investigaciones que han empleado metagenómica, reportadas en las bases de datos de PudMed y Google Schoolar, con el objetivo de examinar los beneficios y limitaciones de las diversas metodologías empleadas en el tratamiento del ADN metagenómico de suelo y el impacto de la metagenómica en la agricultura.

ABSTRACT Microorganisms are of great interest because they colonize all types of environment, however, one of the problems we face in knowing biological diversity is that not all microorganisms are cultivable. The development of new technologies such as the generation of cloning vectors coupled with the development of high performance sequencing techniques, have favored the emergence of a new tool in science called metagenomics, which allows us to study genomes of entire communities. Since all environments are different, the type of challenge that we will face when working with metagenomics is going to change depending of the type of sample, in the specific case of soils, there are several variables, such as soil contamination with heavy metals or chemical compounds that could limit metagenomic studies. However, despite the limitations that the environment presents, with the help of metagenomics, both gene discovery and the characterization of microbial communities that positively influence plant development have been achieved, which could generate a greater impact on agriculture in the future. In this article a review of several investigations that have used metagenomics, reported in the PudMed and Google Schoolar databases was carried out, with the aim of examining the benefits and limitations of the various methodologies used in the treatment of metagenomic DNA from soil and the impact of metagenomics in agriculture.

Growth inhibition of pathogenic microorganisms by Pseudomonas protegens EMM-1 and partial characterization of inhibitory substances.

The bacterial strain, EMM-1, was isolated from the rhizosphere of red maize ("Rojo Criollo") and identified as Pseudomonas protegens EMM-1 based on phylogenetic analysis of 16S rDNA, rpoB, rpoD, and gyrB gene sequences. We uncovered genes involved in the production of antimicrobial compounds like 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, and lectin-like bacteriocins. These antimicrobial compounds are also produced by other fluorescent pseudomonads alike P. protegens. Double-layer agar assay showed that P. protegens EMM-1 inhibited the growth of several multidrug-resistant (MDR) bacteria, especially clinical isolates of the genera Klebsiella and ß-hemolytic Streptococcus. This strain also displayed inhibitory effects against diverse fungi, such as Aspergillus, Botrytis, and Fusarium. Besides, a crude extract of inhibitory substances secreted into agar was obtained after the cold-leaching process, and physicochemical characterization was performed. The partially purified inhibitory substances produced by P. protegens EMM-1 inhibited the growth of Streptococcus sp. and Microbacterium sp., but no inhibitory effect was noted for other bacterial or fungal strains. The molecular weight determined after ultrafiltration was between 3 and 10 kDa. The inhibitory activity was thermally stable up to 60°C (but completely lost at 100°C), and the inhibitory activity remained active in a wide pH range (from 3 to 9). After treatment with a protease from Bacillus licheniformis, the inhibitory activity was decreased by 90%, suggesting the presence of proteic natural compounds. All these findings suggested that P. protegens EMM-1 is a potential source of antimicrobials to be used against pathogens for humans and plants.

Long-Chain Hydrocarbons (C21, C24, and C31) Released by Bacillus sp. MH778713 Break Dormancy of Mesquite Seeds Subjected to Chromium Stress.

Volatile organic compounds (VOCs) produced by rhizobacteria have been proven to stimulate plant growth during germination and seedling stages. However, the modulating effect of bacterial volatiles on the germination of seeds subjected to heavy metal stress is scarcely studied. In this work, the ability of volatiles released by Bacillus sp. MH778713 to induce seed dormancy breakage in Prosopis laevigata and Arabidopsis thaliana seeds were examined. The minimal inhibitory concentration of chromium (Cr) VI that prevents seed germination of P. laevigata and A. thaliana on water-Cr-agar plates was 2500 and 100 mg L-1, respectively. Remarkably, partitioned Petri-dish co-cultivation of Bacillus sp. MH778713 and plant seeds under Cr-stress showed the beneficial effect of volatiles emitted by Bacillus sp. MH778713, helping plant seeds to overcome Cr-stress. Among the metabolites emitted by Bacillus sp. MH778713, octadecane, heneicosane, 2,4-di-tert-butylphenol, hexadecane, eicosane, octacosane, and tetratriacontane were the most abundant. To confirm that these long-chain compounds produced by Bacillus sp. MH778713 could be responsible for the seed dormancy breakage, high pure organic compounds (2,4-di-tert-butylphenol, heneicosane, hentriacontane, and tetracosane) were used directly in germination assays of P. laevigata and A. thaliana seeds instead of volatiles emitted by Bacillus sp. MH778713. All organic compounds allowed Prosopis and Arabidopsis seeds to overcome Cr-toxicity and germinate. The results of this study provide new insight into the role of long-chain bacterial compounds produced by Bacillus sp. MH778713 as triggers of seed abiotic stress tolerance, surmounting chromium stress and stimulating seedling development.

Importancia de compuestos antimicrobianos producidos por bacterias benéficas en el biocontrol de fitopatógenos / The importance of antimicrobial compounds produced by beneficial bacteria on the biocontrol of phytopathogens

ABSTRACT Bacteria produce antimicrobial compounds to compete for nutrients and space in a particular habitat. Antagonistic interactions can be evaluated by several methodologies including the double-layer agar and simultaneous inhibition assays. Among the well-known inhibitory substances produced by bacteria are the broad-spectrum antibiotics, organic acids, siderophores, antifungal, and bacteriocins. The most studied bacterial genera able to produce these inhibitory substances are Enterococcus, Lactococcus, Streptomyces, Bacillus, Pseudomonas, Klebsiella, Escherichia, and Burkholderia. Some beneficial bacteria can promote plant growth and degrade toxic compounds in the environment representing an attractive solution to diverse issues in agriculture and soil pollution, particularly in fields with damaged soils where pesticides and fertilizers have been indiscriminately used. Beneficial bacteria may increase plant health by inhibiting pathogenic microorganisms; some examples include Gluconacetobacter diazotrophicus, Azospirullum brasilense, Pseudomonas fluorescens, Pseudomonas protegens, and Burkholderia tropica. However, most studies showing the antagonistic potential of these bacteria have been performed in vitro, and just a few of them have been evaluated in association with plants. Several inhibitory substances involved in pathogen antagonism have not been elucidated yet; in fact, we know only 1 % of the bacterial diversity in a natural environment leading us to assume that many other inhibitory substances remain unexplored. In this review, we will describe the characteristics of some antimicrobial compounds produced by beneficial bacteria, the principal methodologies performed to evaluate their production, modes of action, and their importance for biotechnological purposes.

RESUMEN Las bacterias producen compuestos antimicrobianos para competir por nutrientes y espacio en un hábitat particular. Las interacciones antagónicas pueden evaluarse mediante varias metodologías, incluido el agar de doble capa y los ensayos de inhibición simultánea. Las sustancias inhibidoras mejor conocidas producidas por bacterias incluyen antibióticos, ácidos orgánicos, sideróforos, antifúngicos y bacteriocinas. Entre los géneros bacterianos más estudiados que producen sustancias inhibidoras se incluyen Enterococcus, Lactococcus, Streptomyces, Bacillus, Pseudomonas, Klebsiella, Escherichia y Burkholderia. Algunas bacterias beneficiosas tienen la capacidad de promover el crecimiento de las plantas y degradar compuestos tóxicos en el ambiente, por lo que podrían incrementar el rendimiento de los cultivos y disminuir problemas de contaminación del suelo, especialmente donde los pesticidas y fertilizantes han sido utilizados indiscriminadamente. Algunas bacterias beneficiosas pueden aumentar la salud de las plantas al inhibir microorganismos patógenos, por ejemplo, Gluconacetobacter diazotrophicus, Azospirullum brasilense, Pseudomonas fluorescens, Pseudomonas protegens y Burkholderia tropica. Sin embargo, la mayoría de los estudios que muestran el potencial antagónico de estas bacterias se han realizado in vitro, y pocos de ellos se han evaluado en asociación con plantas. Varias sustancias inhibitorias implicadas en el antagonismo de los patógenos aún son desconocidas; de hecho, sabemos que solo se ha aislado el 1 % de la diversidad bacteriana en un ambiente natural, lo que sugiere que hay muchas otras sustancias inhibitorias que no han sido exploradas. En esta revisión describimos las características de algunos compuestos antimicrobianos producidos por bacterias beneficiosas, las principales metodologías usadas para evaluar su producción, modos de acción y su importancia para fines biotecnológicos.

Diversity and phenotypic analyses of salt- and heat-tolerant wild bean Phaseolus filiformis rhizobia native of a sand beach in Baja California and description of Ensifer aridi sp. nov.

In northern Mexico, aridity, salinity and high temperatures limit areas that can be cultivated. To investigate the nature of nitrogen-fixing symbionts of Phaseolus filiformis, an adapted wild bean species native to this region, their phylogenies were inferred by MLSA. Most rhizobia recovered belong to the proposed new species Ensifer aridi. Phylogenetic analyses of nodC and nifH show that Mexican isolates carry symbiotic genes acquired through horizontal gene transfer that are divergent from those previously characterized among bean symbionts. These strains are salt tolerant, able to grow in alkaline conditions, high temperatures, and capable of utilizing a wide range of carbohydrates and organic acids as carbon sources for growth. This study improves the knowledge on diversity, geographic distribution and evolution of bean-nodulating rhizobia in Mexico and further enlarges the spectrum of microsymbiont with which Phaseolus species can interact with, including cultivated bean varieties, notably under stressed environments. Here, the species Ensifer aridi sp. nov. is proposed as strain type of the Moroccan isolate LMR001T (= LMG 31426T; = HAMBI 3707T) recovered from desert sand dune.

Structural characterization of scorpion peptides and their bactericidal activity against clinical isolates of multidrug-resistant bacteria.

Scorpion venom peptides represent a novel source of antimicrobial peptides (AMPs) with broad-spectrum activity. In this study, we determined the minimum bactericidal concentration (MBC) of three scorpion AMPs, Uy234, Uy17, and Uy192, which are found in the venomous glands of the Urodacus yaschenkoi scorpion, against the clinical isolates of multidrug-resistant (MDR) bacteria. In addition, we tested the activity of a consensus AMP designed in our laboratory based on some previously reported IsCT-type (cytotoxic linear peptide) AMPs with the aim of obtaining higher antimicrobial activity. All peptides tested showed high antimicrobial activity against MDR clinical isolates, with the highest activity against ß-hemolytic Streptococcus strains. The hemolytic activity was determined against human red blood cells and was significantly lower than that of previously reported AMPs. The α-helical structure of the four AMPs was confirmed by circular dichroism (CD). These results suggest that the four peptides can be valuable tools for the design and development of AMPs for use in the inhibition of MDR pathogenic bacteria. A clear index of synergism and additivity was found for the combination of QnCs-BUAP + Uy234, which makes these peptides the most promising candidates against pathogenic bacteria.

Chromium Hyper-Tolerant Bacillus sp. MH778713 Assists Phytoremediation of Heavy Metals by Mesquite Trees (Prosopis laevigata).

Heavy metal accumulation in mesquite trees (Prosopis laevigata) growing in aluminum, titanium, chromium and zirconium-polluted soils of a semi-arid region in Mexico was investigated using wavelength dispersive X-ray fluorescence analysis. The results showed that P. laevigata trees can hyper accumulate up to 4100 mg/kg of Al, 14000 mg/kg of Fe, 1600 mg/kg of Ti, 2500 mg/kg of Zn, but not chromium, regarding high chromium concentrations found in soils (435 mg/kg). Since plant-associated microorganism can modulate phytoremediation efficiency, the biodiversity of P. laevigata associated bacteria was studied. Eighty-eight isolates from P. laevigata nodules were obtained; all isolates tolerated high concentrations of Al, Fe, Zn and Cr in vitro. The top-six chromium tolerant strains were identified by 16S rRNA sequence analysis as belonging to genus Bacillus. Bacillus sp. MH778713, close to Bacillus cereus group, showed to be the most resistant strain, tolerating up to 15000 mg/L Cr (VI) and 10000 mg/L of Al. Regarding the bioaccumulation traits, Bacillus sp. MH778713 accumulated up to 100 mg Cr(VI)/g of cells when it was exposed to 1474 mg/L of Cr VI. To assess Bacillus sp. MH778713 ability to assist Prosopis laevigata phytoremediation; twenty plants were inoculated or non-inoculated with Bacillus sp. MH778713 and grown in nitrogen-free Jensen's medium added with 0, 10 and 25 mg/L of Cr(VI). Only plants inoculated with Bacillus sp. grew in the presence of chromium showing the ability of this strain to assist chromium phytoremediation. P. laevigata and Bacillus spp. may be considered as good candidates for soil restoration of arid and semiarid sites contaminated with heavy metals.

Desiccation-induced viable but nonculturable state in Pseudomonas putida KT2440, a survival strategy.

The potential of Pseudomonas putida KT2440 to act as a plant-growth promoter or as a bioremediator of toxic compounds can be affected by desiccation. In the present work, the bacterial survival ratio (BSR) in response to air desiccation was evaluated for P. putida KT2440 in the presence of different protectors. The BSR in the presence of nonreducing disaccharides, such as trehalose, was high after 15 days of desiccation stress (occurring at 30°C and 50% relative humidity), whereas in the absence of a protector the bacterial counts diminished to nondetectable numbers (ca 2.8 log CFU/mL). The LIVE/DEAD staining method showed that bacteria protected with trehalose maintained increased numbers of green cells after desiccation while cells without protection were all observed to be red. This indicated that nonprotected bacteria had compromised membrane integrity. However, when nonprotected bacteria subjected to 18 days of desiccation stress were rehydrated for a short time with maize root exudates or for 48 h with water (prolonged rehydration), the bacterial counts were as high as that observed for those not subjected to desiccation stress, suggesting that the cells entered the viable but nonculturable (VBNC) state under desiccation and that they returned to a culturable state after those means of rehydration. Interestingly an increase in the green color intensity of cells that returned to a culturable state was observed using LIVE/DEAD staining method, indicating an improvement in their membrane integrity. Cellular activity in the VBNC state was determined. A GFP-tagged P. putida strain expressing GFP constitutively was subjected to desiccation. After 12 days of desiccation, the GFP-tagged strain lost culturability, but it exhibited active GFP expression, which in turn made the cells green. Furthermore, the expression of 16S rRNA, rpoN (housekeeping), mutL, mutS (encoding proteins from the mismatch repair complex), and oprH (encoding an outer membrane protein) were examined by RT-PCR. All evaluated genes were expressed by both types of cells, culturable and nonculturable, indicating active molecular processes during the VBNC state.