Summary: The use of rhizobacteria has the ability to colonize the root system, promote plant growth and biocontrol or suppress phytopathogenic fungi. The objective of this research was to select and analyze fluorescent rhizobacteria solubilizing nitrogen, phosphorus and potassium, producing lytic enzymes with antagonistic activity towards P. palmivora and L. theobromae. The bacteria were isolated from root and leaf tissue of endemic CCN-51 cacao cultivars from the provinces of Los Ríos, Santa Elena and Esmeralda. Characterization of antagonistic activity was based on the selection of proteolytic activity, siderophore production and fluorescent pigmentation under ultra violet light. In addition, antagonistic activity was performed using cell extracts. Nine bacterial strains (BF 567, FZ 9-7, LH 5-10, MH 4-20, MN 5-20, MN 5-19, AC3, PV-25) with protease production, siderophore production and fluorescence emission were selected. The ability of the bacteria to solubilize complex minerals (nitrogen, phosphorus and potassium) was analyzed and their solubilization efficiency varied, ranging from 50 to 100 %. The antagonistic factor of cell supernatants from FZ 9-7, BF 567 and PV-25 inhibited mycelial growth toward P. palmivora and L. theobromae by 40, 60 and 85%, respectively. The use of rhizobacteria could be used as an alternative biofertilizer to chemical fertilizers and biological control of phytopathogenic soil fungi through their volatile emissions.

Resumen: El empleo de rizobacterias tiene la capacidad de colonizar el sistema radicular, promover el crecimiento en plantas y la capacidad biocontroladora o suprimir hongos fitopatógenos. El objetivo de esta investigación fue seleccionar y analizar las rizobacterias fluorescentes solubilizadoras de nitrógeno, fósforo y potasio, productoras de enzimas líticas de actividad antagonistas hacia P. palmivora y L. theobromae. Las bacterias se aislaron del tejido radicular y foliar de cultivares endémicos de cacao CCN-51 de las provincias de Los Ríos, Santa Elena y Esmeralda. La caracterización de la actividad antagonista se apoyó en la selección de actividad proteolítica, producción de sideróforos y pigmentación fluorescente bajo luz ultra violeta. Además, se realizó la actividad antagonista mediante los extractos celulares. Se seleccionaron nueve cepas bacterianas (BF 567, FZ 9-7, LH 5-10, MH 4-20, MN 5-20, MN 5-19, AC3, PV-25) con producción de proteasa, sideróforos y emisión de fluorescencia. Se analizó la capacidad de las bacterias para solubilizar minerales complejos (nitrógeno, fosforo y potasio) su eficiencia de solubilización vario, oscilando entre 50 y 100%. El factor antagónico de los sobrenadantes celulares de FZ 9-7, BF 567 y PV-25 inhiben el crecimiento del micelio hacia P. palmivora y L. theobromae en un 40, 60 y 85%, respectivamente. La utilización de las rizobacterias podría usarse como biofertilizante alternativo a los fertilizantes químicos y control biológico de hongos fitopatógenos del suelo a través de sus emisiones volátiles.

Resumen Los esclerocios de algunos hongos albergan bacterias nativas para el control biológico de hongos fitopatógenos. Los objetivos de esta investigación fueron: i) explorar poblaciones bacterianas asociadas a esclerocios de Claviceps gigantea en maíces de seis localidades del Estado de México, ii) evaluar el antagonismo in vitro de estas poblaciones bacterianas contra tres especies de Fusarium fitopatógenos de maíz, iii) identificar molecularmente los antagonistas más eficientes y caracterizar la producción de metabolitos in vitro. Poblaciones bacterianas en los esclerocios se calcularon por conteo directo en placa; el antagonismo in vitro contra Fusarium graminearum, F. sublgutinans y F. verticillioides se evaluó por confrontación en medio Waksman agar. Las bacterias antagonistas se identificaron por secuenciación parcial del gen 16S rRNA y se evaluaron in vitro para la producción de ácido indol-3-acético, sideróforos, actividad lipolítica, proteolítica y solubilización de fosfato mineral. La densidad bacteriana estuvo entre 2.023 a 2.397 Log10UFC g-1 de esclerocio. Veintidos cepas mostraron antagonismo in vitro contra al menos una especie de Fusarium. Se identificaron como miembros de los géneros Bacillus, Delftia, Micromonospora, Pseudomonas, Sphingobacterium, Staphylococcus y Stenotrophomonas. Los 22 antagonistas mostraron in vitro actividad lipolítica, proteolítica, produjeron sideróforos y solubilizaron fosfato; únicamente 12 (55%) produjeron ácido indol-3-acético. Las cepas Bacillus subtilis (BA1), Pseudomonas syringae (BA2) y Bacillus amyloliquefaciens (BA18) fueron antagonistas contra las tres especies de Fusarium y produjeron todos los metabolitos evaluados.

Abstract Fungal sclerotia house native bacterial populations of interest for biological control of plant pathogenic fungi. The objectives of this research were: i) to explore the bacterial populations associated with Claviceps gigantea sclerotia in maize from six locations in the State of Mexico, ii) to evaluate the in vitro antagonism of these bacterial populations against three species of Fusarium phytopathogens of maize, iii) to molecularly identify the more efficient antagonists and characterize the in vitro production of metabolites. Bacterial populations in the sclerotia were calculated by direct plate count; in vitro antagonism against Fusarium graminearum, F. sublgutinans, and F. verticillioides was evaluated by confrontation in Waksman agar medium. Antagonistic bacteria were identified by partial sequencing of 16S rRNA gene and evaluated in vitro for the production of indole-3 acetic acid, siderophores, lipolytic and proteolytic activity, and mineral phosphate solubilization. The bacterial density ranged from 2.023 to 2.397 Log10UFC g-1 of sclerotia. Twenty-two strains showed in vitro antagonism against at least one Fusarium species and were identified as members of the genera Bacillus, Delftia, Micromonospora, Pseudomonas, Sphingobacterium, Staphylococcus, and Stenotrophomonas. The 22 antagonists showed in vitro lipolytic, proteolytic, siderophore-producing and phosphate-solubilizing activity; only 12 (55%) produced indole-3-acetic acid. Bacillus subtilis (BA1), Pseudomonas syringae (BA2), and Bacillus amyloliquefaciens (BA18) strains were antagonists against the three Fusarium species and produced all the metabolites evaluated.

Abstract Fluorescent Pseudomonas and Bacillus spp. are among the bacteria with the greatest potential for the biological control of plant diseases. The objectives of this research were: i) to identify fluorescent Pseudomonas and Bacillus species from the rhizosphere of wild agaves antagonists of pectinolytic bacteria, and ii) to detect the presence of genes for the biosynthesis of antimicrobial lipopeptides (AMPs) in the antagonist strains. The in vitro antagonist efficiency index (IEA) of 109 strains of fluorescent Pseudomonas and 119 of Bacillus spp. isolated from the rhizosphere of wild agaves against five pectinolytic bacterial strains of the genera Pectobacterium and Dickeya were evaluated. By PCR, antagonistic bacteria were identified by sequencing the 16S rRNA gene and AMPs genes: fenD (fengicin), ituC (iturin), srfAA (surfactin), and bmy (bacillomycin) for Bacillus spp., prnD (pyrrolnitrin), and phlD (2,4-diacetiylphloroglucinol) for fluorescent Pseudomonas were detected with specific primers. The in vitro IEA resulted in the selection of 25 antagonist strains against one or more pectinolytic bacteria. Comparison of the 16S rRNA gene sequences identified 11 (44%) strains in the Bacillus genus and 14 (56%) in Pseudomonas with similarities between 97 and 100%. Among the Bacillus and Pseudomonas species, most harbored between two to four AMPs genes. The ItuC, srfAA, and phlD genes were more frequent in all antagonist strains; all the genes evaluated were detected in the Bacillus subtilis (9B14) and Pseudomonas fluorescens (G3, D4, and H6) strains.

Resumen Pseudomonas fluorescentes y Bacillus spp. se encuentran entre las bacterias con mayor potencial para el control biológico de enfermedades en plantas. Los objetivos de esta investigación fueron: i) identificar especies de Pseudomonas fluorescentes y Bacillus de la rizósfera de agaves silvestres antagonistas de bacterias pectinolíticas, y ii) detectar la presencia de genes para la biosíntesis de lipopéptidos antimicrobianos (AMPs) en las cepas antagonistas. Se evaluó el índice de eficiencia antagonista (IEA) in vitro de 109 cepas de Pseudomonas fluorescentes y 119 de Bacillus spp. aisladas de la rizósfera de agaves silvestre contra cinco cepas bacterianas pectinolíticas de los géneros Pectobacterium y Dickeya. Por PCR, las bacterias antagonistas se identificaron por secuenciación del gen 16S rRNA y se detectaron los genes AMPs: fenD (fengicina), ituC (iturina), srfAA (surfactina), y bmy (bacilomicina) para Bacillus spp.; prnD (pirrolnitrina) y phlD (2,4 diacetilfloroglucinol) para Pseudomonas fluorescentes con iniciadores específicos. Los resultados del IEA in vitro resultó en la selección de 25 cepas antagonistas contra una o más bacterias pectinolítcas. La comparación de las secuencias del gen 16S rRNA identificaron 11 (44%) cepas en el género Bacillus y 14 (56%) en Pseudomonas con similitud entre el 97 y 100%. Entre las especies de Bacillus y Pseudomonas, la mayoría albergó entre dos a cuatro genes AMPs. Los genes ItuC, srfAA y phlD fueron de mayor frecuencia en todas las cepas antagonistas; en las cepas Bacillus subtilis (9B14) y Pseudomonas fluorescens (G3, D4 y H6) se detectaron todos los genes evaluados.

Resumo O fungo micotoxigênico Fusarium verticillioides é o principal patógeno do milho e causa doenças do colmo e da podridão da espiga com perdas econômicas significativas. Além disso, o uso excessivo de fungicidas no controle de F. verticillioides constitui uma ameaça ao meio ambiente e à saúde humana. Assim, alternativas sustentáveis, como o controle biológico, são necessárias para minimizar os riscos associados ao método atual. Este estudo isolou e caracterizou bactérias e fungos que colonizam o estigma do milho para avaliar novos agentes de biocontrole em potencial. As amostras foram coletadas em campos de milho nas localidades brasileiras de Sete Lagoas-MG, Sidrolândia-MS, Sertaneja-PR e Goiânia-GO. Cento e sessenta e sete microrganismos foram isolados, 46% endofíticos e 54% epifíticos. O teste de antagonismo empregando a técnica de disco de difusão em meio sólido, mostrou que 83% dos isolados apresentaram atividade antagonista contra F. verticillioides. Em seguida, 42 isolados mais eficientes foram identificados a partir do sequenciamento parcial do gene 16S rRNA bacteriano e da região ITS de fungos. Os isolados bacterianos pertencem ao gênero Bacillus (57,1%), Burkholderia (23,8%), Achromobacter (7,1%), Pseudomonas (2,4%) e Serratia (2,4%), enquanto os fungos são Penicillium (2,4%), Candida (2.4), e Aspergillus (2,4%). Os resultados mostraram que microrganismos do estigma do milho podem representar novos agentes promissores para o controle de F. verticillioides.

Abstract The mycotoxigenic fungus Fusarium verticillioides is the primary maize pathogen and causes the maize stalk and ear rot diseases with significant economic losses. Furthermore, the excessive use of fungicides to control F. verticillioides constitutes threats to the environment and human health. Thus, sustainable alternatives such as biological control are needed to minimize the hazards associated with the current method. Although much is known about the vulnerability of the maize silks as a gateway for several fungal pathogens invading the developing grains, studies on the chemical properties of silk extracts and their resident microbiota are scarce. This study isolated and characterized bacteria and fungi that colonize the maize stigma to assess new potential biocontrol agents. The samples were collected from maize fields in the Brazilian localities of Sete Lagoas-MG, Sidrolândia-MS, Sertaneja-PR, and Goiânia-GO. One hundred sixty-seven microorganisms were isolated, 46% endophytic and 54% epiphytic. First, the antagonist activity was evaluated by the agar disc diffusion method performed in triplicate, and 83% of the isolates showed antagonist activity against F. verticillioides. Then, the 42 most efficient isolates were identified based on the partial sequencing of the bacterial 16S rRNA gene and fungi ITS region. The bacteria belong to the genera Bacillus (57.1%), Burkholderia (23.8%), Achromobacter (7.1%), Pseudomonas (2.4%), and Serratia (2.4%), while the fungi are Penicillium (2.4%), Candida (2.4), and Aspergillus (2.4%). The results showed that microorganisms from maize stigma might represent new promising agents for F. verticillioides control.

ABSTRACT The presence of ants in apiaries has long been considered a problem for beekeeping. The health of the hives is a key issue in beekeeping since honey bees are affected by viruses, bacteria, fungi and parasites. Penicillium, Aspergillus and Fusarium are mycotoxigenic fungi, which are widely distributed throughout the world. The presence of these fungi has been observed in both the digestive tract of bees and honey. One of the most studied bacteria as antagonists is Bacillus. The objective of the study was to determine the antifungal activity of Bacillus subtilis against Aspergillus section nigri and Fusarium solani. The isolation was carried out in nutritive agar of B. subtilis from cuticles of ants from apiaries of the temperate valleys of the province of Jujuy, Argentina. The fungal strains were obtained from the strain collection of the Laboratory of Agricultural Microbiology and Beekeeping/ Stingless beekeeping Health of the Faculty of Agrarian Sciences, UNJu. The antagonism tests were performed on plates with potato dextrose agar medium with explants of the fungi against Bacillus subtilis incubated at 28 ° C for one week. Colony diameters were recorded, and the percentage of growth inhibition was determined at 5, 10 and 15 days. The inhibition percentages were higher after 5 days of incubation. The inhibition percentage was greater than 50% in Aspergillus section nigri and greater than 65% in Fusarium solani. Bacillus subtilis showed antifungal activity against the pathogenic fungi of bees under study.

RESUMEN La presencia de hormigas en los apiarios ha sido considerada durante mucho tiempo un problema para la apicultura. En la producción apícola es muy importante el aspecto sanitario de las colmenas, ya que las abejas melíferas son afectadas por virus, bacterias, hongos y parásitos. Penicillium, Aspergillus y Fusarium son micotoxigénicos con una amplia distribución en el mundo. Se ha demostrado la presencia de estos hongos en el tubo digestivo de las abejas y en la miel. Una de las bacterias más estudiadas como antagonistas es Bacillus. El objetivo del estudio fue determinar la actividad antifúngica de Bacillus subtilis frente Aspergillus sección nigri y Fusarium solani. Se realizó el aislamiento en Agar nutritivo de B. subtilis de cutículas de hormigas de colmenares de los valles templados de la provincia de Jujuy, Argentina. Los hongos fueron provistos por el cepario del Laboratorio de Microbiología Agrícola y Sanidad Apícola y Meliponícola de la Facultad de Ciencias Agrarias, UNJu. Las pruebas de antagonismo se realizaron en placas con medio agar papa dextrosa con explantes de los hongos frente a Bacillus subtilis incubadas a 28°C durante una semana. Se registraron los diámetros de las colonias y se determinó el porcentaje de inhibición a los 5, 10 y 15 días. Los porcentajes de inhibición fueron mayores a los 5 días de incubación. El porcentaje de inhibición fue mayor al 50% en Aspergillus sección nigri y mayor al 65% en Fusarium solani. Bacillus subtilis mostró actividad antifúngica contra los hongos patógenos de abejas en estudio.

ABSTRACT Introduction: The use of Plant Growth Promoting Bacteria (PGPB) allows for diminishing the use of chemical products in agriculture. These products are dangerous for environments and human health. The objective of the present work was to characterize isolates from Bacilli class to select potentially efficient strains for their use in sustainable agriculture. Methods: To isolate bacteria; the samples from rhizosphere and rice, maize and coffee plants were heated to 80 ºC. The isolates were characterized according to PGPB and antagonistic traits. Several strains were selected to study the colonization, diversity and their effect on plant growth. Results: One hundred forty-five aerobic Gram-positive endospore forming bacteria were isolated from rice, maize and coffee. The studied strains showed several traits for plant growth. In thirty-seven selected strains from rice, maize and coffee, antagonistic activity was demonstrated against phytopathogenic fungi. After that, two strains were selected and they show their ability to colonize rice plants, move and form biofilms. The promised strains were identified as belonging to Bacillus genus by DNAr 16S sequencing. The molecular study of Bacillus from rice demonstrated the high genetic diversity and the occurrence of virulence determinants in few strains. The inoculation of maize and tomato in semicontrolled conditions showed positive effects in plant growth. Conclusions: An integrally characterized bacterial native collection, efficient for plant growth promotion and antagonistic activity is available, which constitutes the base for new bioproducts obtainment. This work proposes an efficient methodology for prospecting and characterizing Bacillus for their use in agricultural sustainability.

RESUMEN Introducción: El uso de las bacterias promotoras del crecimiento vegetal (PGPB) permiten disminuir el empleo de químicos en la agricultura, perjudiciales para el ambiente y los seres humanos. El objetivo del presente trabajo fue caracterizar integralmente aislados de la clase Bacilli para la selección de cepas potencialmente eficientes para su uso en la agricultura sostenible. Métodos: Se aislaron las bacterias calentando a 80 ºC las muestras de rizosfera y plantas desinfectadas de arroz, maíz y cafeto. Se caracterizaron según sus atributos como PGPB y antagonistas de patógenos. Se seleccionaron cepas para estudiar la colonización, la diversidad poblacional y el efecto en la promoción del crecimiento. Resultados: Se aislaron y caracterizaron 145 bacterias aerobias Gram positivas formadoras de endosporas endófitas y rizosféricas. Las cepas estudiadas muestran varios atributos para la promoción del crecimiento. Se demostró el antagonismo de 37 cepas de arroz, maíz cafeto frente a 11 hongos fitopatógenos. Se seleccionaron dos cepas que demostraron su capacidad para colonizar plantas de arroz y de formar biopelículas y de movimiento. Se identificaron las cepas promisorias por secuenciación del DNAr 16S como pertenecientes al género Bacillus. El estudio molecular de la población de Bacillus aislados del arroz demostró su elevada diversidad genética y la presencia de determinantes de virulencia en algunas cepas. La inoculación de maíz y tomate en condiciones semicontroladas mostró efectos positivos en el crecimiento de la planta. Conclusiones: Se dispone de una colección microbiana nativa, caracterizada integralmente, eficiente en la promoción del crecimiento vegetal y con actividad antagonista de patógenos, que constituyen la base para la obtención de nuevos bioproductos. Se propone una metodología eficiente para la prospección y caracterización de Bacillus para la sostenibilidad agrícola.

Resumen Introducción: La actividad antimicrobiana realizada por las bacterias asociadas con los corales, además de promover la salud de su huésped, representa una fuente para obtener nuevos compuestos bioactivos. Objetivo: Analizar la actividad antimicrobiana de las bacterias asociadas con el zoantario Palythoa caribaeorum de los arrecifes de Carapibus, Paraíba, Brasil. Metodología: El análisis filogenético de la bacterias se realizó con base en secuencias parciales del gen RNAr 16S utilizando herramientas moleculares y de bioinformática. La actividad antimicrobiana de las cepas se probó contra cuatro cepas bacterianas y una cepa de levadura: Bacillus cereus (CCT0198), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa y Candida albicans (ATCC 10231), utilizando ensayos antibiosis y antibiograma, y la concentración inhibitoria mínima (CIM) que se determinó por el método de microdilución. Resultados: Las cepas bacterianas pertenecían a Firmicutes (84 %) y Gammaproteobacteria (16 %). Entre 49 cepas se encontraron cinco géneros de bacterias: Bacillus, Vibrio, Pseudomonas, Staphylococcus y Alteromonas. Un total de 19 cepas exhibieron actividad antimicrobiana, siendo el género Bacillus el responsable del mayor número de bacterias antagonistas, con 12 cepas positivas en el ensayo de antibiosis y cuatro en la prueba de antibiograma. El mayor número de bacterias antagonistas se encontró en Bacillus (12 aislamientos), seguido por Vibrio (tres aislamientos) y Pseudomonas (un aisladmiento). El NC8, clasificado como Bacillus subtilis, inhibió todas las cepas estándar en el ensayo de antibiosis y las cepas de B. cereus, S. aureus y C. albicans en la prueba de antibiograma. El material liofilizado del B. subtilis NC8 mostró acción bacteriostática contra B. cereus, con un valor de CIM de 125 μg/μl. En la prueba de citotoxicidad, el grado de hemólisis fue del 4.8 % para el material liofilizado a las concentraciones probadas, lo que indica su baja citotoxicidad. Conclusión: Los resultados muestran el potencial antimicrobiano de algunos aislamientos bacterianos asociados al P. caribaeourum, especialmente los pertenecientes al género Bacillus.

Abstract Introduction: The coral-associated bacteria with antimicrobial activity may be important to promote the health of their host through various interactions, and may be explored as a source of new bioactive compounds. Objective: To analyze the antimicrobial activity of bacteria associated with the zoanthid Palythoa caribaeorum from the coral reefs of Carapibus, Paraiba state, Brazil. Methods: The phylogenetic analysis of the bacteria was conducted based on partial sequences of the 16S rRNA gene using molecular and bioinformatics tools. The antimicrobial activity of the 49 isolates was tested against four bacterial strains and one yeast strain: Bacillus cereus (CCT0198), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa and Candida albicans (ATCC 10231). The antibiosis and antibiogram assays were conducted and the Minimal Inhibitory Concentration (MIC) was determined by the microdilution method. Results: The bacterial isolates belonged to Firmicutes phylum (84 % of the isolates) and the Proteobacteria phylum (16 % of the isolates). Among the 49 isolates five genera were found, with the Bacillus genus being the most abundant (82 % of the isolates), followed by Vibrio (10 %), Pseudomonas (4 %), Staphylococcus (2 %) and Alteromonas (2 %). Antibiosis test revealed that 16 isolates (33 %) showed antimicrobial activity against one or more of five tested reference strains. The highest number of antagonistic bacteria were found in the Bacillus genus (12 isolates), followed by Vibrio (three isolates) and Pseudomonas (one isolate) genera. The B. subtilis NC8 was the only isolate that inhibited all tested strains in the antibiosis assay. However, antibiogram test with post-culture cell-free supernatant of NC8 isolate showed the inhibition of only B. cereus, S. aureus and C. albicans, and the lyophilized and dialyzed material of this isolate inhibited only B. cereus. The lyophilized material showed bacteriostatic activity against B. cereus, with a MIC value of 125 μg/μl, and in the cytotoxicity assay, the hemolysis value was of 4.8 %, indicating its low cytotoxicity. Conclusions: The results show the antimicrobial potential of some bacterial isolates associated with the P. caribaeourum tissue, especially those belonged to Bacillus genus.

RESUMO A fim de se obter agentes de biocontrole para controle da antracnose em frutos de mamão em pós-colheita, buscou-se: i) selecionar bactérias epifíticas de mamoeiro antagonistas a Colletotrichum brevisporum pela produção de compostos antimicrobianos difusíveis e voláteis, quitinase e inibição da germinação de conídios; ii) quantificar a produção de biofilme pelos isolados selecionados; iii) obter uma concentração de bactérias que proporcionasse o controle da antracnose em discos de frutos; iv) avaliar a redução da severidade da doença em frutos de mamão. De 224 bactérias, 74 exibiram um mecanismo de ação contra o patógeno, 13 isolados dois mecanismos, quatro apresentaram três, e uma bactéria exibiu todos. Mediante a verificação da compatibilidade entre antagonistas, e a quantificação da formação de biofiolme, estabeleceram-se cinco “Mix” compostos por quatro isolados bacterianos. Em discos de frutos as bactérias foram avaliadas nas concentrações 106, 107 e 108 UFC mL-1. À exceção do Mix 2, os demais reduziram significativamente a antracnose nos discos quando comparados ao controle. Os “Mix” 1, 3, 5, na concentração 108 UFC mL-1 exibiram melhor desempenho reduzindo a doença em até 75 %, 89,78 % e 90,8 %, respectivamente. Os Mix 3 e 5, na concentração de 108 UFC mL-1, foram avaliados em frutos juntamente com o fungicida sintético Piraclostrobina. Não se verificou diferença significativa entre os Mix e o fungicida, que atingiu níveis de redução da doença superiores a 97 %, e as combinações com 98 %, frente ao controle não tratado.

ABSTRACTUMO Aiming to obtain biocontrol agents for post-harvest anthracnose control in papaya fruits, the present study sought to: i) select, from papaya fruits, epiphytic bacteria that are antagonistic to Colletotrichum brevisporum, based on the production of diffusible and volatile antimicrobial compounds, chitinase, and conidial germination inhibition; ii) quantify biofilm production by the selected strains; iii) obtain a concentration of bacteria that would provide anthracnose control in fruit discs; iv) evaluate the disease severity reduction in papaya fruits. Of 224 bacteria, 74 exhibited one mechanism of action against the pathogen, 13 isolates showed two mechanisms, four isolates had three mechanisms, and one bacterium showed all mechanisms. Verification of compatibility between antagonists, as well as biofilm quantification, led to the formation of five “Mix” composed of four bacterial isolates. In fruit discs, bacteria were evaluated at the concentrations 106, 107 and 108 CFU mL-1. Except Mix 2, the remaining combinations significantly reduced anthracnose in the discs when compared to control. Mix 1, 3 and 5, at the concentration 108 CFU mL-1, showed the best performance, reducing the disease severity by up to 75.0%, 89.78% and 90.8%, respectively. Mix 3 and 5, at the concentration 108 CFU mL-1, were evaluated together with the synthetic fungicide Pyraclostrobin in fruits. There was no significant difference between the Mix and the fungicide, which reached disease reduction levels superior to 97%, while combinations reached 98%, compared to the untreated control.

Abstract The objective of the present study was to select bacterial strains from the rhizosphere of common bean crop and to determine their potential to control Sclerotium rolfsii under in vitro and in planta conditions. Soil samples were collected in the Municipalities of Ahome, Guasave and Angostura, Sinaloa, México. The strains were evaluated under in vitro conditions, afterwards the best isolates were tested in planta, and were identified based on 16S region of ribosomal DNA (three strains). Sixty-five bacterial isolates were tested against Sclerotium rolfsii with an inhibition from 2.5 to 65%. Acinetobacter pittii (COHUI06), Pseudomonas putida (SANMI02) and Burkholderia sp. (GLS06) inhibited 55, 60 and 65% under in vitro conditions; and resulted not hemolytic and their molecular identification was based on 16S rDNA. Only Burkholderia sp. exherted the highest percentage inhibition in planta to control S. rolfsii with a reduction of incidence and disease severity of 40 and 50% respectively, and promoted the dry weight of the plant. A. pittii and P. putida were not efficient in controlling the fungus in planta. Greenhouse and field studies with these bacteria are suggested.

Resumen El objetivo del presente estudio fue seleccionar cepas bacterianas de la rizósfera del cultivo de frijol y determinar su potencial para el control de Sclerotium rolfsii bajo condiciones in vitro e in planta. Se recolectaron muestras de suelo en los municipios de Ahome, Guasave y Angostura, Sinaloa, México, durante el ciclo agrícola 2018-2019. Los aislados se evaluaron bajo condiciones in vitro y se seleccionaron los mejores para la evaluación in planta e identificación molecular (tres aislados) con base a la región 16S del ADN ribosomal. Se evaluaron 65 aislados bacterianos in vitro contra S. rolfsii con un porcentaje de inhibición de 2.5 a 65%. Acinetobacter pittii (COHUI06), Pseudomonas putida (SANMI02) y Burkholderia sp. (GLS06) inhibieron 55, 60 y 65% bajo condiciones in vitro; además resultaron no hemolíticas. Solo Burkholderia sp. ejerció mayor porcentaje de inhibición in planta para el control de S. rolfsii, con reducción de incidencia y severidad de la enfermedad en un 40 y 50% respectivamente, y promovió el peso seco de la planta. A. pittii o P. putida no fueron eficientes para el control del hongo in planta. Se sugiere hacer estudios con las bacterias en invernadero y campo.

Abstract. The objective was to evaluate the antagonistic effect in vitro of native strains of Trichoderma asperellum, Trichoderma harzianum and two isolates of Bacillus spp. against Fusarium oxysporum and Dickeya chrysanthemi. Bacillus, isolated from soil samples of Aloe vera plantations, was morphologically identified and a modified dual culture confrontation was carried out, placing the F. oxysporum in the center of the Petri dish and Bacillus in the four cardinal points; it was arranged under a completely randomized experimental design with five repetitions, the variables registered were percentage of antagonism, inhibition halo and concentration of conidia. For Trichoderma they were confronted against F. oxysporum and D. chrysanthemi with a dual confrontation, a percentage of inhibition and antagonism classification were determined. In the confrontation of T. asperellum it obtained a percentage of inhibition of 70.5% against F. oxysporum and 41.9% against D. chrysanthemi. Regarding the inhibition halos of Bacillus (B5 and B4), they obtained 4 mm. In the conidia concentration B5 showed 1.3, B4 2.6 and the control 12.6 spores per dilution. Trichoderma and Bacillus represent a viable alternative for the control of F. oxysporum and D. chrysanthemi.

Resumen. El objetivo fue evaluar el efecto antagónico in vitro de cepas nativas de Trichoderma asperellum, Trichoderma harzianum y dos aislados de Bacillus spp. frente a Fusarium oxysporum y Dickeya chrysanthemi. Se identificó morfológicamente a Bacillus, aislado de muestras de suelo de plantaciones de Aloe vera y se realizó una confrontación de cultivos duales modificada, colocando en el centro de la caja Petri F. oxysporum y en los cuatro puntos cardinales las bacterias de Bacillus; para esto se dispuso bajo un diseño experimental completamente al azar con cinco repeticiones, las variables registradas fueron porcentaje de antagonismo, halo de inhibición y concentración de conidios. Para Trichoderma se confrontaron frente a F. oxysporum y D. chrysanthemi con una confrontación dual y se determinó porcentaje de inhibición y clasificación de antagonismo. En la confrontación de T. asperellum obtuvo un porcentaje de inhibición de 70.5 % frente a F. oxysporum y 41.9 % frente a D. chrysanthemi. En cuanto a los halos de inhibición de Bacillus (B5 y B4) obtuvieron 4 mm. En la concentración de conidios B5 mostró 1.3, B4 2.6 y el testigo 12.6 esporas por dilución. Trichoderma y Bacillus representan una alternativa viable para el control de F. oxysporum y D. chrysanthemi.

RESUMO A brusone (Magnaporthe oryzae) limita a produção do arroz (Oryza sativa), no Brasil e no mundo. Os objetivos foram caracterizar seis isolados bacterianos morfológica, bioquímica e molecularmente, e determinar seu potencial como antagonistas a M. oryzae. Utilizaram se os isolados bacterianos BRM 32109, BRM 32110, BRM 32111, BRM 32112, BRM 32113 e BRM 32114. Avaliou-se o formato e a cor das colônias, coloração de Gram e produção de pigmentos fluorescentes. Identificou-se a produção de celulase, quitinase, fosfatase, ácido indolacético, sideróforo e formação de biofilme. A identificação molecular foi realizada utilizando-se oligonucleotídeos iniciadores específicos para amplificação por PCR da região 16S rRNA, seguidos de sequenciamento. Os dois ensaios envolvendo antagonismo foram realizados em laboratório, em delineamento inteiramente casualizados, um com cultivo pareado entre isolado de bactérias e M. oryzae, e outro com filtrados bacterianos. Um terceiro ensaio em casa de vegetação para avaliar a supressão da brusone. Identificou-se Pseudomonas sp. (BRM 32111 e BRM 32112), Burkholderia sp. (BRM 32113), Serratia sp. (BRM 32114) e Bacillus spp. (BRM 32110 e BRM 32109 ). Os isolados BRM 32112, BRM 32111 e BRM 32113 inibiram o crescimento da colônia de M. oryzae em 68, 65 e 48%, respectivamente. As suspensões bacterianas BRM 32111, BRM 32112 e seu filtrado suprimiram a brusone foliar em 81,0, 79,2 e 66,3%, respectivamente. BRM 32111 e BRM 32112 são antagonistas ao fungo M. oryzae, solubilizam fosfato, produzem sideróforos, celulase e biofilme e suprimiram a brusone foliar, mostrando-se potenciais agentes biológicos para o controle da brusone foliar.

ABSTRACT Rice blast (Magnaporthe oryzae) limits rice (Oryza sativa) grain yields worldwide. The objective of this investigation was to morphologically, biochemically, and molecularly characterize six bacterial isolates, BRM 32109, BRM 32110, BRM 32111, BRM 32112, BRM 32113, and BRM 32114, and to determine their potential as antagonists to M. oryzae. Morphological characterization was based on colony formation and color, Gram staining, and fluorescent pigment production. Biochemical studies were based on cellulase, chitinase, phosphatase, indoleacetic acid, and siderophore production, as well as biofilm formation. The molecular identification used specific primers for PCR amplification of the 16S rRNA region, followed by sequencing. The antagonism studies involved three experiments, which had randomized designs. Two of them were conducted in laboratory conditions, pairing bacterial colonies and M. oryzae, using bacterial filtrates, and the third was conducted in greenhouse conditions. BRM 32111 and BRM 32112 were identified as Pseudomonas sp., BRM 32113 as Burkholderia sp., BRM 32114 as Serratia sp., and BRM 32110 and BRM 32109 as Bacillus spp. BRM 32112, BRM 32111, and BRM 32113 inhibited the colony of M. oryzae by 68%, 65%, and 48%, respectively. The bacterial suspensions of the BRM 32111, BRM 32112, and BRM 3212 filtrates suppressed leaf blast by 81.0, 79.2, and 66.3%, respectively. BRM 32111 and BRM 32112 were determined to be antagonists of M. oryzae and were found to solubilize phosphate, produce siderophores and cellulose, form biofilms, and suppress leaf blast. These isolates should be further investigated as potential biological control agents for leaf blast control.

Abstract Introduction: The plants harbor a microbiome that includes plant growth-promoting bacteria, which can act as biological agents that antagonize the growth of phytopathogens, diminishing or eliminating their harmful effects in plants. In this work, the presence and association of the phlD and hcnC antifungal genes with the production of siderophores (Fe chelating agents) in bacteria isolated from the endosphere and rhizosphere of blackberry plants (Rubus fruticosus L.) was evaluated. Method: In total, 410 bacterial isolates associated with blackberry plants (Rubus fruticosus L.), from the rhizosphere (205) and endosphere (205) were analyzed for the detection of the phlD and hcnC antimicrobial genes by polymerase chain reaction and subsequent sequencing. The search of the genes was carried out only in siderophore producing strains, using the CAS medium (Chromium Azurol) for its production. Of the strains selected, antagonism bioassays were carried out against the plant pathogens Botrytis cinerea, Fusarium oxysporum, F. solani and Rhizoctonia solani. Results: The results showed that from the 410 bacterial isolates analyzed here (50% endophytes and 50% rhizospheric), it was confirmed that 70 strains were siderophores-producing (24 from rhizosphere and 46 endophytes). It was observed that 70% (49/70) of the siderophore producing strains present either one or two of the phlD or hcnC genes. The nucleotide sequences of the phlD and hcnC genes showed high identity to homologous genes to species Pseudomonas sp., P. fluorescens, P. chlororaphis, P. protegens, P. putida and P. brassicacearum. The abundance of the hcnC gene was also higher in the endophytic and rhizospheric isolates (36/70), with respect to the phlD gene (13/70). Greater detection of phlD or hcnC genes was found in strains isolated from the endosphere (43/70), compared with those of rhizospheric origin (6/70). Finally, a high ratio was observed between the production of siderophores, the presence of genes phlD and hcnC with the antagonistic activity towards important phytopathogenic fungi, like Botrytis cinerea, Fusarium oxysporum, Fusarium solani and Rhizoctonia solani. Conclusion: These results showed the potential to identify quickly, efficiently and at low cost, mechanisms of antagonism towards phytopatogens in bacteria associated with blackberry plants (Rubus fruticosus L.), such as siderophore production.

Resumen Introducción: Las plantas albergan un microbioma que incluye bacterias promotoras del crecimiento vegetal, las cuales pueden fungir como agentes biológicos que antagonizan el crecimiento de fitopatógenos, disminuyendo o eliminando su efectos nocivos en plantas. En este trabajo se evaluó la presencia de los genes antifúngicos phlD y hcnC con la producción de sideróforos (agentes quelantes de Fe) en bacterias aisladas de la endósfera y rizósfera de plantas de zarzamora (Rubus fruticosus L.). Método: Se analizaron 410 aislados bacterianos asociados a plantas de zarzamora (Rubus fruticosus L.), provenientes de la rizósfera (205) y endosfera (205) para la detección de los genes antimicrobianos phlD y hcnC por reacción en cadena de la polimerasa y posterior secuenciación. La búsqueda de los genes se realizó únicamente en cepas productoras de sideróforos, utilizando el medio CAS (Cromo Azurol) para su producción. De las cepas seleccionadas, se realizaron los bioensayos de antagonismo contra los patógenos de plantas Botrytis cinerea, Fusarium oxysporum, F. solani y Rhizoctonia solani. Resultados: Los resultados de este trabajo muestran que, de los 410 aislados bacterianos analizados (50% rizosféricos y 50% endófitos), 70 aislados fueron productores de sideróforos (24 rizosféricas y 46 endófitas). Se observó que el 70% (49/70) de las cepas productoras de sideróforos, presentaron uno o dos de los genes phlD y/o hcnC. Las secuencias nucleotídicas de los genes phlD y/o hcnC mostraron una alta identidad con genes homólogos a las especies Pseudomonas sp., P. fluorescens, P. chlororaphis, P. protegens, P. putida y P. brassicacearum. La abundancia del gen hcnC fue mayor en los aislados endófitos y rizosféricos (36/70), con respecto al gen phlD (13/70). Se detectaron mayoritariamente los genes phlD y/o hcnC en cepas aisladas de la endósfera (43/70), comparado con aquellas cepas rizosféricas (6/70). Finalmente, se observó una alta relación entre la producción de sideróforos, la presencia de genes phlD y/o hcnC y la actividad antagónica hacia los fitopatógenos Botrytis cinerea, Fusarium oxysporum, Fusarium solani y Rhizoctonia solani. Conclusión: Estos resultados muestran el potencial de identificar de forma rápida, eficiente y a bajo costo, mecanismos de antagonismo hacia fitópatógenos en bacterias asociadas a plantas de zarzamora (Rubus fruticosus L.), tales como la producción de sideróforos.

Abstract An alternative to promote the quality of tomato fruits (Solanum lycopersicon L.) and reduce the indirect effects of phytopathogenic fungi that cause root and crown diseases, might be the use of antagonistic bacteria of the Bacillus genus. The aim of the study was to determine the effect, of three Bacillus strains alone and together in interaction with phytopathogenic fungi on the plant growth promotion of tomato cv. Merlice and fruit quality. For this purpose, seedlings were inoculated with three Bacillus species (B. amyloliquefaciens, B. methylotrophicus and B. subtilis subsp. inaquosorum) and then with three phytopathogenic fungi of tomato (Alternaria solani, Fusarium oxysporum and Phytophthora infestans). At the end of the production cycle, the height and weight of plants, the length and weight of roots, chlorophyll in leaves, and the fruit yield, was determined. The fruits harvested were characterized for color, weight, size, mesocarp thickness, total soluble solids, titratable acidity, bromatological analysis, firmness, and number and weight of seeds. Bacillus inoculation with the three strains resulted in higher values among the variables evaluated. Bacillus amyloliquefaciens significantly increased plant height and root length and yield by 30 %, whereas the phytopathogen F. oxysporum significantly decreased yield in 30 %. Bacillus subtilis strain showed positive effects on fruit diameter and firmness, protein content and root length. The three Bacillus strains promoted the growth of tomato plants, yield and improved some of their fruit quality parameters.

Resumen Una alternativa para promover la calidad del fruto de jitomate (Solanum lycopersicon L.) y aminorar los efectos indirectos de hongos fitopatógenos causantes de enfermedades de raíz y cuello puede ser el uso de bacterias antagonistas del género Bacillus. El objetivo del estudio fue determinar el efecto de tres cepas de Bacillus solas y en interacción con tres hongos fitopatógenos en la promoción del crecimiento de plantas de jitomate cv. Merlice y calidad del fruto. Para este propósito, se inocularon plántulas con tres especies de Bacillus (B. amyloliquefaciens, B. methylotrophicus y B. subtilis subsp. inaquosorum), y después con tres hongos fitopatógenos del jitomate (Alternaria solani, Fusarium oxysporum y Phytophthora infestans). Al final del ciclo de producción, se determinaron, la altura y peso de las plantas; longitud y peso de raíces; y clorofila en hojas y el rendimiento. Los frutos cosechados se caracterizaron por color, peso, tamaño, espesor del mesocarpio, sólidos solubles totales, acidez titulable, composición bromatológica, firmeza, y número y peso de semillas. La inoculación con las tres cepas de Bacillus resultó en los valores más altos en la mayoría de las variables evaluadas. B. amyloliquefaciens incrementó significativamente la altura de la planta, longitud de raíz y el rendimiento en un 30 %, mientras que el fitopatógeno F. oxysporum redujo el rendimiento 30 %. Bacillus subtilis mostró efectos positivos en el diámetro y firmeza del fruto, contenido de proteínas y longitud de raíz. Las tres cepas de Bacillus promovieron el crecimiento de las plantas y el rendimiento y mejoraron algunos parámetros de calidad del fruto.

ABSTRACT Phymatotrichopsis omnivore affect a great number of fruit species causing great economic losses and the use of agrochemicals to combat them, resulting in adverse environmental effects, so it would be very useful to find alternatives that contribute to reduce the use of these chemicals. There are soil-borne microorganisms with mechanisms of inhibition towards various pathogens, which contribute to the use of biological control methods. The objective of this work was to select and identify bacteria strains capable of inhibiting the growth of P. omnivora. To do this, bacteria were isolated from roots of fruit trees with the presence of the disease to evaluate their antagonistic effect in vitro. A total of 88 bacterial isolates were obtained, of which the antifungal capacity was evaluated by simultaneous confrontation of the fungus and the bacteria in triplicate assays. Cottonseeds were inoculated with strains that showed significant inhibition, using in vitro cultures. The bacterial strains M1, N8 and N15 presented greater biocontrol capacity and, considering the results obtained, they were identified by 16S ribosomal DNA sequencing.

RESUMEN Phymatotrichopsis omnívora afecta a un gran número de especies de frutales ocasionando grandes pérdidas económicas, y el uso de agroquímicos para combatirlos ocasionan efectos negativos para el medioambiente, por lo que sería muy útil encontrar alternativas que contribuyan a disminuir el uso de estos químicos. Existen microorganismos nativos del suelo con mecanismos de inhibición hacia diversos patógenos, lo que contribuye al empleo de métodos de control biológico. El objetivo de este trabajo fue seleccionar e identificar cepas de bacterias con capacidad de inhibir el crecimiento de P. omnivora, para lo cual se aislaron bacterias de raíces de árboles frutales con presencia de la enfermedad, con el fin de evaluar su efecto antagónico in vitro. Se obtuvieron 88 aislamientos bacterianos, de los cuales se evaluó la capacidad antifúngica mediante enfrentamiento simultáneo del hongo y la bacteria en ensayos por triplicado. Se inocularon semillas de algodón con las cepas que presentaron inhibición importante utilizando cultivos in vitro. Las cepas bacterianas M1, N8 y N15 presentaron mayor capacidad biocontroladora y teniendo en cuenta los resultados obtenidos, se identificaron mediante secuenciación del ADN ribosómico 16S.

ABSTRACT The present work was carried out with the objective of updating the state of existing information on the endophytic microorganisms associated with Theobroma cacao L., as agents of biological control of moniliasis, caused by Moniliophthora roreri Evans et al. It is summarized on the origin and evolution of the definitions of endophytic microorganisms, emphasizing the biological control of cocoa diseases with emphasis on moniliasis, one of the most important phytosanitary problems worldwide; as well as an assessment of their employment prospects. Fungal and bacterial endophytes have several mechanisms of action, which include direct competition for space and nutrients, antibiosis, mycoparasitism, induced systemic resistance and promotion of growth; functions that are part of the strategies for an effective and sustainable management of the frosty pod rot.

RESUMEN El presente trabajo se realizó con el objetivo de actualizar el estado de información existente sobre los microorganismos endófitos asociados a Theobroma cacao L. como agentes de control biológico de la moniliasis, causada por Moniliophthora roreri Evans et al. Se resume sobre el origen y evolución de las definiciones de microorganismos endófitos, enfatizando en el control biológico de las enfermedades del cacao con énfasis en la moniliasis, uno de los problemas fitosanitarios de mayor importancia a nivel mundial; así como se hace una valoración sobre sus perspectivas de empleo. Los hongos y bacterias endófitos presentan varios mecanismos de acción entre los que se encuentran: competencia directa por el espacio y nutrientes, antibiosis, micoparasitismo, resistencia sistémica inducida y promoción del crecimiento; funciones que son parte de las estrategias para el manejo eficaz y sostenible de la moniliasis.