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The purpose of this work was to isolate and identify native bacteria from plants collected in the State of Yucatán, México with the ability to promote growth of chili pepper (Capsicum annuum L. cv Jalapeño). We identified nine bacterial isolates that belong to five species of Bacillus (i.e. Bacillus subtilis, B. flexus, B. cereus, B. megaterium and B. endophyticus) that produced indoleacetic acid (4.0-24.3 µg/mL) with solubilization index of 1.3-1.6. All the bacterial isolates were evaluated based on their ability to promote growth of chili pepper. Plants inoculated with B. subtilis ITC-N67 showed an increase in stem diameter and root volume, whereas inoculation with B. cereus ITC-BL18 increased the number of flower buds, fresh biomass of roots and total fresh biomass. Conversely, B. flexus ITC-P4 and B. flexus ITC-P22 showed deleterious effect on root volume and total biomass. In summary, our data showed that native B. cereus TC-BL18 and B. subtilis ITC-N67 have potential to be used as growth promoting microorganism for chili pepper, particularly in the state of Yucatán, México.
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Induced systemic resistance (ISR) is a mechanism involved in the plant defense response against pathogens. Certain members of the Bacillus genus are able to promote the ISR by maintaining a healthy photosynthetic apparatus, which prepares the plant for future stress situations. The goal of the present study was to analyze the effect of the inoculation of Bacillus on the expression of genes involved in plant responses to pathogens, as a part of the ISR, during the interaction of Capsicum chinense infected with PepGMV. The effects of the inoculation of the Bacillus strains in pepper plants infected with PepGMV were evaluated by observing the accumulation of viral DNA and the visible symptoms of pepper plants during a time-course experiment in greenhouse and in in vitro experiments. The relative expression of the defense genes CcNPR1, CcPR10, and CcCOI1 were also evaluated. The results showed that the plants inoculated with Bacillus subtilis K47, Bacillus cereus K46, and Bacillus sp. M9 had a reduction in the PepGMV viral titer, and the symptoms in these plants were less severe compared to the plants infected with PepGMV and non-inoculated with Bacillus. Additionally, an increase in the transcript levels of CcNPR1, CcPR10, and CcCOI1 was observed in plants inoculated with Bacillus strains. Our results suggest that the inoculation of Bacillus strains interferes with the viral replication, through the increase in the transcription of pathogenesis-related genes, which is reflected in a lowered plant symptomatology and an improved yield in the greenhouse, regardless of PepGMV infection status.
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The aim of this study was to evaluate the effect of inoculation with Bacillus spp. isolates on the photosynthetic apparatus of Capsicum chinense plants infected with PepGMV. In vitro and greenhouse experiments were performed to evaluate whether the inoculation improved plants' performance through the increase in photosynthetic efficiency to control PepGMV. The results showed that despite PepGMV infection, the plants inoculated with some isolates of Bacillus spp. had a healthy photosynthetic mechanism, as the photochemical parameters and gas exchange increased. The maximum photochemical quantum yield of PSII (Fv/Fm) of plants with PepGMV and inoculated with Bacillus isolates (M9, K46, and K47) increased (7.85, 7.09, and 7.77%, respectively) with respect to uninoculated controls. In inoculated plants, the CO2 assimilation rate increased and the transpiration rate decreased, therefore indicating an increased water use efficiency. This effect was reflected by the less severe symptoms caused by PepGMV in the plants obtained from seeds inoculated with different Bacillus spp. Plants inoculated with K47 isolates showed an increase in fruit yield and quality. This study suggests that it is possible to protect, at the greenhouse level, C. chinense plants from PepGMV through selected rhizobacteria inoculation.
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Practically all Bacillus thuringiensis strains contain a set of self-replicating, extrachromosomal DNA molecules or plasmids, which vary in number and size in the different strains. The plasmid patterns obtained from gel electrophoresis have previously been used as a tool to characterize strains, but comparison of the plasmid patterns has been limited in the number and diversity of strains analyzed. In this report, we were able to compare the plasmid patterns of 83 type strains (out of 84) and 47 additional strains from six serotypes. The information obtained from this comparison showed the importance of this tool as a strain characterization procedure and indicates the complexity and uniqueness of this feature. For example, with one exception, all type strains showed a unique plasmid pattern. All were unique in such a way that none showed even a single comigrating plasmid in the agarose gels, and therefore, cluster analysis was impossible, indicating that plasmid patterns are qualitative rather than quantitative features. Furthermore, comparison between strains belonging to the same serotype showed a great difference in variability. Some serotypes (e.g., israelensis) showed the same basic pattern among all its strains, while other serotypes (e.g., morrisoni) showed a great diversity of patterns. These results indicate that plasmid patterns are valuable tools to discriminate strains below the serotype level.
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Bacillus thuringiensis/genética , DNA Bacteriano/genética , Plasmídeos/isolamento & purificação , Bacillus thuringiensis/classificação , Técnicas de Tipagem Bacteriana/métodos , Impressões Digitais de DNA , Eletroforese em Gel de Ágar , Genótipo , Polimorfismo Genético , SorotipagemRESUMO
Abstract The Central American Locust Schistocerca piceifrons piceifrons is one of the most damaging plant pest in Mexico and Central America. The present work was carried out to evaluate the seasonal population fluctuation of S. p. piceifrons and vegetation diversity and their association with weather factors and edaphic conditions in the gregarious zone of the Yucatán Península. The study was performed in seven sites during three seasons: North-wind (December 2013), rainy (June 2014) and dry (April 2014). The locust density was sampled in transect of 100 m2, as well as the vegetation in 16 m2: plant species richness (PSR) and relative species density (RSD), and analyzed by generalized linear models. Additionally, soil samples were obtained at 10 cm depth into a 4 × 6 m quadrat, land use in the sites was classified and temperature, precipitation and evaporation of each site were obtained from the database and they were analyzed with multiple factor analysis. The population density of S. p. piceifrons was higher in the sites Panaba, Tizimin, Tunkas and Cenotillo (F= 74.3, P < 0.0001). Characterization of vegetation showed that PSR and RSD were higher during the rainy season relative to those in the dry season (F= 50.4, P < 0.0001). RSD was identified as the most important group associated with locust density (0.86), followed by isotherm/isohyets (0.63), maximum precipitation and temperature (0.60), as well as the land use (0.65); no correlation between locust density and soil characteristics was found. Locust density was positively correlated with the abundance of the grass Panicum maximum (Sr2= 0.85, PC5= 0.87). This work shows that the population of S. p. piceifrons is high in the rainy season and influenced primarily by the abundance of the grass P. maximum and the precipitation. The results indicate that surveys for early detection and control of the locust on the Yucatán Península can focus on areas with the grass P. maximum to predict risk areas and target survey efforts. Rev. Biol. Trop. 66(1): 403-414. Epub 2018 March 01.
Resumen La langosta Centroamericana Schistocerca piceifrons piceifrons es una de las plagas más dañinas en México y Centroamérica. El presente trabajo se realizó para evaluar la fluctuación estacional de poblaciones de S. p. piceifrons y su asociación con la diversidad de vegetación, factores climáticos y edáficos en la zona gregarígena de la Península de Yucatán. Se seleccionaron siete sitios y se muestrearon a lo largo de tres estaciones: nortes (diciembre 2013), lluvias (junio 2014) y sequías (abril 2014). La densidad poblacional de la langosta fue muestreada en transectos de 100 m2, así como la vegetación en 16 m2 obteniendo la riqueza de especies vegetales (REV) y la densidad relativa de especies (DRE), y fueron analizadas por modelos lineales generalizados. Adicionalmente se obtuvieron muestras de suelo de 10 cm de profundidad en un área de 4 × 6 m, se clasificó el uso del suelo de cada sitio y se obtuvieron en una base de datos las condiciones de temperatura, precipitación y evaporación para cada sitio, estos datos se analizaron con análisis de factores múltiples. La densidad poblacional de S. p. piceifrons fue mayor en los sitios de Panaba, Tizimin, Tunkas y Cenotillo (F= 74.3, P < 0.0001). La caracterización de la vegetación mostró que la REV y la DRE fueron mayores en la estación de lluvias que en la de sequías (F= 50.4, P < 0.0001). La DRE fue el grupo más importante asociado a la densidad de la langosta (0.86), seguido por las isotermas/isoyetas (0.63) y la precipitación-temperatura máxima (0.60), así como el uso del suelo (0.65); no hubo correlación con las características del suelo. La densidad de la langosta fue correlacionada positivamente con la abundancia del pasto Panicum maximum (Sr2= 0.85, PC5= 0.87). El estudio mostró que las poblaciones de S. p. piceifrons fueron mayores en la estación lluviosa e influenciadas principalmente por la abundancia del pasto P. maximum y la precipitación. Los resultados indican que las exploraciones, la detección temprana y el control de la langosta en la Península de Yucatán puede enfocarse sobre áreas cultivadas con P. maximum para predecir áreas con riesgo y eficientar los recursos.
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A total of 119 Bacillus thuringiensis strains (83 type strains and 26 native isolates), as well as five B. cereus group species, were analyzed by repetitive extragenic palindromic sequence-based PCR analysis (Rep-PCR) fingerprinting. Primers Bc-REP-1 and Bc-REP-2 were specifically designed according to an extragenic 26-bp repeated sequence found in the six B. cereus group genomes reported. A total of 47 polymorphic bands were detected, and the patterns varied from 5 to 13 bands in number and from 0.2 to 3.8 kb in size. Virtually each type strain showed a distinctive B. cereus (Bc)-Rep-PCR pattern, except for B. thuringiensis serovars dakota (H serotype 15 [H15]) and sotto (H4a,4b), as well as serovars amagiensis (H29) and seoulensis (H35), which shared the same patterns. As expected, serovar entomocidus (H6) and its biovar subtoxicus showed an identical pattern; similarly, serovars sumiyoshiensis (H3a,3d) and fukuokaensis (H3a,3d,3e), which share two antigenic determinants, also showed identical Bc-Rep-PCR patterns. Interestingly, serovars israelensis (H14) and malaysiensis (H36), which share several phenotypic attributes, also showed identical Bc-Rep-PCR patterns. Native, coleopteran-active strains, including the self-agglutinated LBIT-74 strain, showed Bc-Rep-PCR patterns identical or very similar to that of the tenebrionis strain. Likewise, native mosquitocidal strains (including some self-agglutinated strains) also showed patterns identical or very similar to that of the serovar israelensis IPS-82 strain. Additionally, native beta-exotoxin-producing strains from serovar thuringiensis showed patterns identical to that of the B. thuringiensis type strain. The B. cereus group-specific Bc-Rep-PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify B. thuringiensis serotypes, including nonflagellar and self-agglutinated strains.
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Bacillus cereus/genética , Bacillus thuringiensis/genética , Impressões Digitais de DNA/métodos , Bacillus cereus/classificação , Bacillus thuringiensis/classificação , Bacillus thuringiensis/isolamento & purificação , Técnicas de Tipagem Bacteriana , Sequência de Bases , Primers do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Filogenia , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido NucleicoRESUMO
El objetivo de este trabajo fue la selección de cepas nativas del género Bacillus con actividad quitinolíticay proteolítica, en suelo tropical en la costa de Oaxaca, México. Se aislaron 150 cepas, de las cuales 22fueron seleccionadas por presentar actividad quitinolítica y proteolítica. Dicha actividad se evaluó porla formación de halo de hidrólisis alrededor de la colonia en medios de cultivo suplementados con quitinacoloidal al 5% y leche descremada al 1% respectivamente. Las cepas LUM B001, B003, B013, B015y B065 presentaron mayor actividad quitinolítica y proteolítica, por lo que tienen el potencial para serevaluadas en control biológico de hongos fitopatógenos. Se encontró al género Bacillus distribuido ensuelos cultivados y no cultivados, no se encontraron diferencias estadísticas según el cultivo establecido(P<0,05), sin embargo se encontraron diferencias significativas (P<0,05) entre las zonas estudiadas, correspondiendolas menores recuperaciones de cepas a los terrenos del municipio de Tututepec, Oaxaca.
This work was aimed at selecting native strains from the Bacillus genus having chitinolytic and proteolytic activityfrom soil from the tropical coast of Oaxaca, Mexico. 150 strains were isolated, 22 of which were selectedas they presented chitinolytic and proteolytic activity. Such activity was assessed by the formation of a hydrolysishalo around the colony in culture media supplemented with 5% colloidal chitin and 1% skimmed milk.The LUM B001, B003, B013, B015 and B065-chitin strains presented higher quitinolytic and proteolytic activity,thereby having the potential for being evaluated in the biological control of phytopathogenic fungi. TheBacillus genus was found in cultivated and uncultivated soils; no statistical differences were found accordingto established crop (p <0.05); however, significant differences (p <0.05) were found between the areas beingstudied regarding the smaller amount of strains collected from land in the municipality of Tututepec, Oaxaca.