Performance of lactating cows fed sugarcane silage treated with microbial additives

Background: No studies have been conducted evaluating sugarcane silage associated with both Lactobacillus plantarum and Pediococcus pentosaceus for lactating dairy cows. Objective: To evaluate diets containing different roughages with and without microbial inoculants on intake, digestibility and milk yield of medium-producing cows. Methods: A total of 15 Holstein cows distributed into a randomized block design were used. Dietary treatments were: 1) a corn silage-based diet (CS), 2) a fresh sugarcane-based diet (SC), 3) a sugarcane silage ensiled without inoculant (SS), 4) sugarcane silage ensiled with Lactobacillus buchneri (SSLB), and 5) sugarcane silage ensiled with Lactobacillus plantarum and Pediococcus pentosaceus (SSLP). Results: Digestible organic matter intake (DOMI) was lower (p<0.05) in cows fed SSLB (9.77 kg day-1) when compared with cows fed CS (13.29 kg day-1) and SSLP (12.42 kg day-1). Ensiling of sugarcane increased intake of neutral detergent fiber (NDF) compared to SC (mean of 6.00 kg day-1 versus 4.97 kg day-1; p<0.05). Dry matter digestibility was greater (p<0.05) in CS (77.80%) compared with diets based on sugarcane silage, whereas NDF digestibility was lower for treatments with sugarcane (p<0.05). Milk yield was similar among CS (27.99 kg), SC (25.59 kg), and silages with additives (25.47 and 27.07 for SSLB and SSLP, respectively). Cows fed CS produced more fat-corrected milk (25.89 kg) than those fed sugarcane-based diets (p<0.05). Conclusions: Fresh sugarcane or sugarcane silage with additives can be used as a roughage source for dairy cows producing up to 23.43 kg d-1 fat-corrected milk, considering the total diet is properly balanced.

Antecedentes: No existen estudios que evalúen la adición de Lactobacillus plantarum y Pediococcus pentosaceus en el ensilaje de caña de azúcar para vacas lactantes. Objetivo: Evaluar dietas con diferentes forrajes y ensilajes con y sin inoculantes microbianos sobre el consumo, digestibilidad y producción lactea de vacas de mediana producción. Metodología: Quince vacas Holstein se distribuyeron en un diseño de bloques al azar. Los tratamientos fueron: 1) dieta con ensilaje de maíz (CS), 2) dieta con caña de azúcar fresca (SC), 3) dieta con ensilaje de caña sin inoculantes (SS), 4) dieta con ensilaje de caña de azúcar ensilada con Lactobacillus buchneri (SSLB), y 5) dieta con ensilaje de caña de azúcar ensilada con Lactobacillus plantarum y Pediococcus pentosaceus (SSLP). Resultados: El consumo de materia orgánica digestible fue menor (p<0,05) en la dieta SSLB (9,77 kg day-1) en comparación con CS (13,29 kg dia-1) y SSLP (12,42 kg dia-1). El ensilaje de caña de azúcar promovió un mayor consumo de fibra detergente neutra (NDF) en comparación con SC (promedio de 6,00 kg dia-1 versus 4,97 kg dia-1; p<0,05). La digestibilidad de la materia seca fue mayor (p<0,05) para la dieta CS (77,80%) en comparación con las dietas con ensilaje de caña de azúcar, mientras que la digestibilidad de la NDF fue menor para las dietas a base de caña de azúcar (p<0,05). La producción de leche fue similar entre CS (27,99 kg), SC (25,59 kg) y ensilajes con aditivos (25,47 y 27,07 para SSLB y SSLP, respectivamente). Las vacas alimentadas con ensilaje de maíz produjeron más leche corregida por grasa (25,89 kg) que las alimentadas con dietas a base de caña de azúcar (p<0,05). Conclusión: La caña de azúcar fresca o ensilada con aditivos se puede utilizar como fuente de forraje para vacas que producen hasta 23,43 kg d-1 leche corregida por grasa, siempre que la dieta total esté equilibrada adecuadamente.

Antecedentes: Há carência de estudos avaliando a adição de Lactobacillus plantarum e Pediococcus pentosaceus na silagem de cana-de-açúcar para vacas em lactação. Objetivo: Avaliar dietas contendo diferentes forragens e silagens com e sem inoculantes microbianos sobre o consumo, digestibilidade e produção de leite de vacas de média produção de leite. Métodos: Quinze vacas Holandesas foram distribuídas em um delineamento em blocos casualizados. Os tratamentos foram: 1) dieta com silagem de milho (CS), 2) dieta com cana-de-açúcar fresca (SC), 3) dieta com silagem de cana ensilada sem inoculantes (SS), 4) dieta com silagem de cana-de-açúcar ensilada com Lactobacillus buchneri (SSLB), ou 5) dieta com silagem de cana- de-açúcar ensilada com Lactobacillus plantarum, and Pediococcus pentosaceus (SSLP). Resultados: O consumo de matéria orgânica digestível foi menor (p<0,05) na dieta SSLB (9,77 kg day-1) comparada com CS (13,29 kg dia-1) e SSLP (12,42 kg dia-1). A ensilagem da cana-de-açúcar promoveu maior consumo de fibra em detergente neutro (FDN) em comparação com SC (média de 6,00 kg dia-1 versus 4.97 kg dia-1; p<0.05). A digestibilidade da matéria seca foi maior (p<0,05) para a dieta CS (77,80%) comparada com as dietas com silagem de cana-de-açúcar, enquanto que a digestibilidade da FDN foi menor para as dietas baseadas em cana-de-açúcar (p<0,05). A produção de leite foi similar entre CS (27,99 kg), SC (25,59 kg) e silagens com aditivos (25,47 e 27,07 para SSLB e SSLP, respectivamente). Vacas alimentadas com silagem de milho produziram mais leite corrigido para gordura (25,89 kg) que aquelas alimentadas com dietas baseadas em cana-de-açúcar (p<0,05). Conclusão: A cana-de-açúcar fresca ou ensilada com aditivos pode ser utilizada como fonte volumosa para vacas produzindo até 23,43 kg d-1 leite corrigido para gordura, desde que a dieta total esteja apropriadamente balanceada.

Microbial inoculants and garbage fermentation liquid reduced root-knot nematode disease and As uptake in Panax quinquefolium cultivation by modulating rhizosphere microbiota community / 中草药·英文版

Objective: To find a suitable ecological cultivation measure to solve the problem of root-knot nematode disease of Panax quinquefolium (Panacis Quinquefolii Radix) and the heavy metals accumulating in its roots. Methods: Three-year-old P. quinquefolium was treated with four different combinations of microbial inoculant (MI) and garbage fermentation liquid (GFL) [the joint application of ‘TuXiu’ MI and Fifty potassium MI (TF), the combination use of ‘No. 1′ MI and Fifty potassium MI (NF), ‘Gulefeng’ poly-γ-glutamic acid MI (PGA), GFL], and the untreated control (CK). Here, high-throughput sequencing, ICP-MS and UPLC were employed to systematically characterize changes of microbial diversity and structure composition, heavy metals (As, Cd and Pb) content and ginsenoside content among different treatments. Results: The results revealed that different MIs and GFL could increase the root dry weight of P. quinquefolium, PGA enhanced it by 83.24%, followed by GFL (49.93%), meanwhile, PGA and GFL were able to lessen root-knot nematode disease incidence by 57.25% and 64.35%. The treatment of PGA and GFL can also effectively reduce heavy metals in roots. The As content in GFL and PGA was decreased by 52.17% and 43.48% respectively, while the Cd and Pb contents of GFL and PGA was decreased somewhat. Additionally, the content of total ginsenosides was increased by 42.14% and 42.07%, in response to TF and NF, respectively. Our metagenomic analysis showed that the relative abundance of particular soil microbial community members related to the biocontrol of root-knot nematode disease and plant pathogen (i.e., Chaetomium in NF, Xylari in GFL, and Microascus in PGA), heavy metal bioremediation (Hyphomacrobium in PGA and Xylaria in GFL), and nitrogen fixation (Nordella and Nitrospira in TF) was significantly increased; notably, potential harmful microflora, such as Plectosaphaerella and Rhizobacter, were more abundant in the control group. Conclusion: MI and GFL could improve the quality of P. quinquefolium by modifying its rhizosphere microbial community structure and composition, both of them are beneficial to the development of ecological cultivation of P. quinquefolium.

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.

Desarrollo de marcadores moleculares del tipo SCAR para la identificación de Azospirillum brasilense Az39 / Development of sequence characterized amplified región markers for identification of Azospirillum brasilense Az39

Resumen Azospirillum brasilense Az39 es utilizada por empresas productoras de inoculantespara la formulación de bioinsumos en América del Sur desde hace más de 30 a˜nos. Esta cepapuede promover el crecimiento, desarrollo, así como la capacidad de tolerar diferentes tiposde estrés en las plantas inoculadas, lo que determina un aumento de la productividad de culti-vos de interés agronómico. En la actualidad, no existen protocolos en Argentina que permitanconfirmar la identidad de Az39 en productos comerciales a nivel de laboratorios de control decalidad de inoculantes. Por ello, el objetivo de este trabajo fue desarrollar una metodología enbase molecular que permita la identificación certera de A. brasilense Az39. Con la secuenciacompleta del genoma y mediante herramientas bioinformáticas, se pudieron reconocer frag-mentos de ADN presentes únicamente en el genoma de Az39. Se dise˜naron cebadores dirigidosa amplificar por PCR dichas secuencias. Como resultado se observaron los productos específicosúnicamente en la presencia de la cepa de interés. La reacción pudo detectar un título mínimode 105UFC/ml (4,5 ng/l ADN) o de 102UFC/ml (0,88 ng/l ADN) o una concentración mínimade 0,098 ng/l ADN, dependiendo del método de extracción utilizado. Los cebadores fueronevaluados en el análisis de productos comerciales obtenidos del mercado nacional, arrojandoresultados positivos, tanto en muestras directas como así también en pruebas confirmatoriasa partir de colonias aisladas de tales productos. La metodología desarrollada en este trabajo,permite la detección certera de A. brasilense Az39 en cultivos puros o mezclas complejas demicroorganismos.

Abstract Azospirillum brasilense Az39 has been used since more than 30 years by several companies in South America for biofertilizers production. This strain may promote plants growth and development, as well as the ability of inoculated plants to tolerate environmental stresses, which determines an increase in the productivity under field conditions. At present, there are no protocols in Argentina to confirm the identity of Az39 in commercial products; however, such biofertilizers are formulated almost exclusively with this strain. Therefore, the objective of this paper was to develop a molecular methodology that allows the accurate identification of A. brasilense Az39. Using the complete genome sequence and several bioinformatics tools, fragments of DNA present only in the Az39 genome were recognized. A set of PCR primers to amplify these sequences were designed, and the specific products were observed only in the strain of our interest. The sensitivity of the methodology was evaluated, where the strain could be detected up to a titer of 105 CFU/ml (4.5 ng/pl ADN) or 102 CFU/ml (0.88 ng/pl DNA) or in a minimal concentration of 0.098 ng/pl DNA, depending on the DNA extraction methodology used. Primers were tested against direct samples of commercial inoculants and cultures, in both cases there were specifics products, both in direct samples and in confirmatory tests from isolated colonies from those products. The procedure presented in this paper allows the accurate identification of A. brasilense Az39 in pure cultures, mixtures of microorganisms, and commercial biofertilizers.

Use of bacterial endospore with longer shelf-life in improved retting of jute

Aim: The aim of the present study was to evaluate the retting of jute (Corchorus olitorius L. and C. capsularis L.) using the endospores of microbial consortium of three strains of Bacillus pumilus with extended shelf-life. Methodology: Endospore and vegetative cells of Bacillus pumilus were tested for viability by introducing them into different temperature, pH, UV radiation and antibiotics. Laboratory, as well as field-trials of jute retting was performed with 6 and 18-months-old endospores and vegetative cells of Bacillus pumilus with estimation of enzymatic activities for comparison of their retting efficiency. Results: Endospores of Bacillus pumilus recorded very high colony forming unit (109 to 108ml-1) compared to their vegetative cells (106 to 104ml-1) after 6 to 18 months of their preservation. Endospores also showed higher resistance to temperature, pH, UV irradiation and antibiotic than their vegetative forms. High colony forming unit and higher release of pectinolytic and xylanolytic enzymes during retting of jute by endospores resulted in complete of jute retting in 10 days with good quality jute fibre compared to talc based formulation. Interpretation: It can be concluded from the study that endospores remained highly efficient in rejuvenating higher CFU and quantitatively larger pool of enzymes to accelerate retting of jute after prolonged preservation. Therefore, the endospores of Bacillus pumilus can be used cost effectively in place of their talc based formulation for higher shelf life of the product, faster retting and better fibre quality of jute.

Changes in the microbial community during bioremediation of gasoline-contaminated soil

Abstract We aimed to verify the changes in the microbial community during bioremediation of gasoline-contaminated soil. Microbial inoculants were produced from successive additions of gasoline to municipal solid waste compost (MSWC) previously fertilized with nitrogen-phosphorous. To obtain Inoculant A, fertilized MSWC was amended with gasoline every 3 days during 18 days. Inoculant B received the same application, but at every 6 days. Inoculant C included MSWC fertilized with N–P, but no gasoline. The inoculants were applied to gasoline-contaminated soil at 10, 30, or 50 g/kg. Mineralization of gasoline hydrocarbons in soil was evaluated by respirometric analysis. The viability of the inoculants was evaluated after 103 days of storage under refrigeration or room temperature. The relative proportions of microbial groups in the inoculants and soil were evaluated by FAME. The dose of 50 g/kg of inoculants A and B led to the largest CO2 emission from soil. CO2 emissions in treatments with inoculant C were inversely proportional to the dose of inoculant. Heterotrophic bacterial counts were greater in soil treated with inoculants A and B. The application of inoculants decreased the proportion of actinobacteria and increased of Gram-negative bacteria. Decline in the density of heterotrophic bacteria in inoculants occurred after storage. This reduction was bigger in inoculants stored at room temperature. The application of stored inoculants in gasoline-contaminated soil resulted in a CO2 emission twice bigger than that observed in uninoculated soil. We concluded that MSWC is an effective material for the production of microbial inoculants for the bioremediation of gasoline-contaminated soil.

Aislados de Bacillus provenientes de la rizósfera de cactus incrementan la germinación y la floración en Mammillaria spp. (Cactaceae) / Bacillus isolates from rhizosphere of cacti improve germination and bloom in Mammillaria spp. (Cactaceae)

Las cactáceas son la vegetación característica de las zonas áridas en México, donde las lluvias son escasas, la evapotranspiración es elevada y la fertilidad de los suelos es baja. Las plantas han desarrollado estrategias fisiológicas como la asociación con microorganismos en la zona de la rizósfera para incrementar la captación de nutrientes. En el presente trabajo se obtuvieron 4 aislados bacterianos de la rizósfera de Mammillaria magnimamma y Coryphantha radians, los que fueron nombrados como QAP3, QAP19, QAP22 y QAP24 e identificados genéticamente como pertenecientes al género Bacillus. Estos aislados exhibieron in vitro propiedades bioquímicas como solubilización de fosfatos, producción de ácido indolacético y actividad ACC deaminasa, que se relacionan con la promoción del crecimiento de las plantas. Dicha promoción fue ensayada inoculando semillas de M. magnimamma y evaluando luego algunos parámetros. Se encontró que todos los aislados incrementaron la germinación desde un 17% hasta un 34,3% (con respecto a las semillas testigo sin inocular); el aislado QAP24 fue el que presentó el mayor efecto en este sentido y permitió la germinación de todas las semillas viables (84,7%) 3 días antes que en el testigo. La inoculación de este aislado en plantas de Mammillaria zeilmanniana mostró un efecto positivo sobre la floración: en 2 meses dentro del período de un año se detectó un incremento en el número de plantas en floración con respecto a las plantas testigo, de hasta el 31,0% en uno de ellos. Se concluye que los aislados de Bacillus spp. caracterizados poseen potencial para ser empleados en programas de conservación de especies vegetales de zonas áridas.

Cacti are the most representative vegetation of arid zones in Mexico where rainfall is scarce, evapotranspiration is high and soil fertility is low. Plants have developed physiological strategies such as the association with microorganisms in the rhizosphere zone to increase nutrient uptake. In the present work, four bacterial isolates from the rhizosphere of Mammillaria magnimamma and Coryphantha radians were obtained and named as QAP3, QAP19, QAP22 and QAP24, and were genetically identified as belonging to the genus Bacillus, exhibiting in vitro biochemical properties such as phosphate solubilization, indoleacetic acid production and ACC deaminase activity related to plant growth promotion, which was tested by inoculating M. magnimamma seeds. It was found that all isolates increased germination from 17 to 34.3% with respect to the uninoculated control seeds, being QAP24 the one having the greatest effect, accomplishing the germination of viable seeds (84.7%) three days before the control seeds. Subsequently, the inoculation of Mammillari zeilmanniana plants with this isolate showed a positive effect on bloom, registering during two months from a one year period, an increase of up to 31.0% in the number of flowering plants compared to control plants. The characterized Bacillus spp. isolates have potential to be used in conservation programs of plant species from arid zones.

Scientometric analysis of Colombian research on bio-inoculants for agricultural production / Análisis cienciométrico de investigación colombiana en bio-inoculantes para producción agrícola / Análise cientométrica das pesquisas colombianas em bioinoculantes para a produção agrícola

The excessive use of synthetic chemical inputs in agricultural production has led to the disruption of biogeochemical cycles. One of the alternatives that arose within the systems of sustainable agriculture was the partial or total replacement of chemicals by biological substances. The analysis of relevant scientific literature has become a tool for assessing the quality of knowledge generation and its impact on the environment. A scientometric analysis was conducted of Colombian research on bio-inoculants from 2009 through 2014 in journals added to the Web of SciencesTM in order to identify the characteristics of the main target crops, the microorganisms used, and the beneficial effects on agriculture. In this work, 34 articles were identified: 24 (71 %) were research on bio-fertilizer development and 10 (29 %) on bio-pesticides. Articles mainly focused on the study of Gram-negative bacilli affecting the area (77 %), while others focused on issues and topics surrounding vegetables (30 %).The analysis of co-occurrence of keywords identified: i. several genera of microorganisms (e.g. A%otobacter sp., Bradyrhi%obium sp.) and sustainable agriculture as issues that have a leading role in this scientific field, ii. plant growth promoting rhizobacteria (PGPR) as an emerging issue, iii. biological nitrogen fixation (BNF) as a subject which has risen in a complementary manner and iv. endophytic bacteria and biodiversity as issues in growth. This study showed that research in Colombia could be targeted on issues such as endophytic bacteria, diversity and productivity.

El uso excesivo de insumos sintéticos en la producción agrícola ha llevado a la disrupción de los ciclos biogeoquímicos. Como alternativa a este problema, los sistemas de agricultura sostenible han reemplazado parcial o totalmente los químicos por sustancias biológicas. El análisis de literatura científica relevante se ha convertido en una herramienta para determinar la calidad de la generación de conocimiento en este campo y su impacto en el ambiente.Se llevó a cabo un análisis cienciométrico de la investigación en Colombia sobre bioinoculantes entre 2009 y 2014. Se tuvieron en cuenta revistas indizadas en la Red de Ciencias®. Se buscó identificar las características de los principales cultivos, los microorganismos utilizados y sus efectos benéficos en la agricultura. Se seleccionaron 34 artículos: 24 (71%) sobre desarrollo de biofertilizantes y 10 (29%) sobre biopesticidas. Los artículos se enfocaron principalmente en el estudio de bacilos Gram-negativos (77%), mientras que otros se enfocaron en los temas relativos a hortalizas (30%). El análisis de co-ocurrencia de palabras claves identificó: i. Algunos géneros de microorganismos (Azptobacter sp., Bradyrhizobium sp.) y la agricultura sostenible como temas relevantes en este campo, ii. Rizobacterias promotoras del crecimiento en plantas (PGPR) como un tópico emergente, iii. Fijación biológica de nitrógeno (FBN) como un tema emergente y complementario y iv. Bacterias endofíticas y biodiversidad como tópicos en crecimiento. Este estudio mostró también que la investigación en Colombia podría enfocarse en temas como bacterias endofíticas, diversidad y productividad.

O uso excessivo de produtos químicos sintéticos na produção agrícola levou a uma perturbação dos ciclos biogeoquímicos. Uma das alternativas que surgiram dentro dos sistemas de agricultura sustentável foi a substituição total ou parcial de compostos químicos por substancias biológicas. O estudo de literatura científica relevante se tornou uma ferramenta para avaliar a qualidade da geração de conhecimento e seu impacto no meio ambiente. A análise cientométrica foi realizada em investigacoes colombianas sobre bioinoculantes de 2009 a 2014, em revistas disponíveis em Web of ScienceTM, com o intuito de identificar as características das principais culturas alvos, os microorganismos utilizados, e o efeito benéfico na agricultura. Neste trabalho, 34 artigos foram identificados: 24 (71 %) eram pesquisas sobre o desenvolvimiento de biofertilizantes e 10 (29 %) sobre biopesticidas. Os artigos eram focados principalmente no estudo de bacilos Gram-negativos afetando a área (77 %), enquanto que outros eram voltados a questoes e temas de vegetais (30 %). A análise de coocorréncia de palavras-chave identificou: i. diversos géneros de microorganismos (Ex. Azptobacter sp., Bradyrhizpbium sp.) e agricultura sustentável como questóes fundamentais nesta área científica, ii. rizobactéricas promotoras de crescimento de plantas (PGPR) como um tema emergente, iii. fixação biológica de nitrogénio (BNF) como um assunto que tem surgido de maneira complementar e iv. bactérias endofíticas e biodiversidade como temas em crescimento. Este estudo mostrou que a pesquisa na Colombia poderia ser dirigida a questoes como bactérias endofíticas, diversidade e produtividade.

Symbiotic potential and survival of native rhizobia kept on different carriers

Native rhizobia are ideal for use as commercial legume inoculants. The characteristics of the carrier used to store the inoculants are important for the survival and symbiotic potential of the rhizobia. The objective of this study was to investigate the effects of peat (PEAT), perlite sugarcane bagasse (PSB), carboxymethyl cellulose plus starch (CMCS), and yeast extract mannitol supplemented with mannitol (YEMM) on the survival, nodulation potential and N₂ fixation capacity of the native strains Sinorhizobium mexicanum ITTG R7^T and Rhizobium calliandrae LBP2-1^T and of the reference strain Rhizobium etli CFN42^T. A factorial design (4 × 3) with four repetitions was used to determine the symbiotic potential of the rhizobial strains. The survival of the strains was higher for PEAT (46% for strain LBP2-1^T, 167% for strain CFN42^T and 219% for strain ITTG R7^T) than for the other carriers after 240 days, except for CFN42^T kept on CMCS (225%). All the strains kept on the different carriers effectively nodulated common bean, with the lowest number of nodules found (5 nodules) when CFN42^T was kept on CMCS and with the highest number of nodules found (28 nodules) when ITTG R7^T was kept on PSB. The nitrogenase activity was the highest for ITTG R7^T kept on PEAT (4911 μmol C₂H₄ per fresh weight nodule h⁻¹); however, no activity was found when the strains were kept on YEMM. Thus, the survival and symbiotic potential of the rhizobia depended on the carrier used to store them.

Efecto de la temperatura de almacenamiento sobre la viabilidad de bacterias simbióticas fijadoras de nitrógeno utilizadas en la elaboración de inoculantes biológicos para arveja (Pisum sativum) y soya (Glycine max) / Effect of storage temperature on the viability of symbiotic nitrogen fixing bacteria used in the preparation of biological inoculants for crop pea (Pisum sativum) and soybean (Glycine max)

El nitrógeno es un elemento requerido en grandes cantidades por los cultivos, sin embargo, en el suelo es uno de los elementos más limitantes ya que se encuentra en formas poco disponibles para las plantas y debe ser incorporado a través de la fertilización química o por medio de la fijación biológica llevada a cabo por microorganismos. Las bacterias simbióticas fijadoras de nitrógeno han sido ampliamente utilizadas para la elaboración de inoculantes, constituyéndose en una alternativa viable para mejorar el rendimiento de los cultivos a través de un mejor suministro de este elemento. Estos inoculantes para que puedan ser comercializados deben cumplir con ciertos requisitos de calidad que garanticen el buen funcionamiento del producto. En este estudio se evalúo la viabilidad de las cepas ICA L9 e ICA J96 en inoculantes almacenados a temperaturas de 4±2, 18±3 y 28±2 °C durante 180 días y la actividad biológica en plantas de arveja y soya. Las cepas utilizadas pertenecen al Banco de Germoplasma de Microorganismos de Corpoica; para los ensayos biológicos se emplearon semillas de arveja variedad "Santa Isabel" y soya variedad "Corpoica Superior 6". Al evaluar la viabilidad de los inoculantes almacenados a 4±2 °C, 18±3 °C y 28±2 °C, se evidenció que la temperatura no afectó la supervivencia y la concentración de rizobios después de 180 días de almacenamiento permitiendo observar un número de unidades formadoras de colonias por gramo superior a 108, valor que garantiza la calidad del inoculante. En cuanto a la actividad biológica, se observó que las cepas inoculadas fueron infectivas y efectivas para la fijación biológica del nitrógeno, compa­rado con los testigos absolutos.

Nitrogen is an element required in large amounts by most crops, however, in soil is one of the most limiting and located in ways not available to the plant and must be incorporated through chemical fertilization or by biological fixation conducted by microorganisms. Fixing symbiotic bacteria nitrogen has been widely used for the production of inoculants, becoming a viable alternative to improve crop yields through a better supply of this element. These inoculants to be marketed must meet certain requirements quality to ensure the smooth operation of the product. This study assessed the viability of the strains ICA L9 and ICA J96 inoculants stored at temperatures 4±2, 18±3 y 28±2°C for 180 days and the biological activity in pea and soybean plants. The strains used belong to the collection of work Germplasm Bank CORPOICA Microorganisms; for biological assays were used pea seeds variety "Santa Isabel" and soybean variety "Superior Corpoica 6". In assessing the viability of inoculants stored at 4±2°C, 18±3°C y 28±2°C, evidenced that the temperature did not affect the survival and the concentration of rhizobia after 180 days of storage allowing to observe a number of colony forming units per gram than 10s, value that guarantees the quality of the inoculant. Concerning the biological activity, it was observed that the strains were inoculated infective and effective biological nitrogen fixation, absolute compared to controls.

Comportamento ingestivo de vacas leiteiras alimentadas com silagem de milheto ou milho / Ingestive behavior of dairy cattle fed millet and corn silage

Objetivou-se com este estudo avaliar o comportamento ingestivo de vacas leiteiras alimentadas com dietas contendo como volumoso silagem de milho ou silagem de milheto com 5 ou 20 mm de tamanho de partículas, com ou sem inoculante bacteriano. Foram utilizadas cinco vacas mestiças Holandês × Gir, com aproximadamente 100 dias de lactação ao início do experimento e peso corporal aproximado de 550 kg, produzindo, em média, 15,2 ± 2,3 kg de leite por dia. Os animais foram arranjados num delineamento em quadrado latino 5×5. O tempo total de alimentação, ruminação e ócio, não foi alterado pelo tipo de silagem na dieta. As variáveis que compõem o comportamento ingestivo não foram afetadas quando as vacas receberam as diferentes silagens de milheto comparadas com silagem de milho. O tamanho de partículas entre 5 e 20 mm, a presença ou não de inoculante e o tipo de silagem com teores de MS em 28%, não afeta o tempo total de alimentação, ruminação e ócio em vacas mestiças Holandês Gir com média de produção de 15,2 kg por dia. Silagem de milho ou milheto, tamanho de partículas 5 ou 20 mm, e o uso ou não de inoculante em silagem de milheto, não altera o tempo total despendido com alimentação, ruminação e ócio em dietas para vacas leiteiras mestiças Holandês × Gir com média de produção de 15,2 kg por dia.

The objective of this study was to evaluate the ingestive behavior of dairy cows fed corn and millet silage with a 5 mm particle size without inoculant, millet silage with a 5 mm particle size with inoculant, millet silage with a 20 mm particle size without inoculant, millet silage with a 20 mm particle size with inoculant. Five cows Holstein x Gir, with approximately 100 days of lactation at the beginning of the experiment and a mean body weight of 550 kg, producing an average of 15 kg of milk per day were used. The animals were arranged in a randomized 5 × 5 Latin square. The total feeding time, rumination time, chewing time, number of alimentary bolus, rumination time for bolus and number of chews was determined. The variables that make up the feeding behavior were not affected when the cows were fed different millet silages compared with corn silage. Cows fed corn silage and millet silage of 5 mm particle size had a higher intake of dry matter and neutral detergent fiber. A particle size between 5 and 20 mm, the presence or absence of inoculum and the type of silage with DM at 28%, does not affect the total feeding time , ruminating and resting of cows with an average production of 15.2 kg per day. The presence of the inoculant does not affect the intake of dry matter and neutral detergent fiber. Cows fed diets containing corn silage or pearl millet silage with a 5 mm particle size fed more DM.

The use of lacZ marker in enumeration of Azotobacter chroococcum in carrier based inoculants

A transconjugant of Azotobacter chroococcum Mac 27 tagged with lac Z(A. chroococcum Mac27 L) was found to possess high levels of β-galactosidase activity constitutively.Further, the lac Z marker was found to be stably integrated into the chromosome of the A. chroococcum Mac 27 and did not have any adverse effect on growth, nitrogen fixation and excretion of ammonia. A quick method to determine the viable cell number in broth culture and carrier based inoculants has been developed on the basis of β-galactosidase assay. It was found that there was a direct relationship between the number of cell as determined by standard plate count and intensity of colour that developed upon degradation of ONPG due to β-galactosidase activity .The method was found to be sensitive enough to determine 1.7 x 10(6) CFU mL-1 in broth culture as well as carrier based Azotobacter inoculants. Further, it was observed that when A. chroococcum Mac27 L was inoculated on Brassica campestris, it could be detected in the presence of other bacteria capable of growing on Burks agar medium containing X-gal on the basis of lac Z genetic marker.

Morphological characteristics of maize plants in estimate the silage chemical composition / Características morfológicas de plantas de milho estimam a composição bromatológica da silagem

The aim of this study was to estimate the chemical composition of maize silage based on the morphological characteristics of maize plants and to evaluate the effect of nitrogen fertilization and the inclusion of a microbial inoculant during the ensiling process on the production of maize silage and its morphological, qualitative and fermentative characteristics. The experimental treatments consisted of four levels of nitrogen fertilization with urea (0, 100, 200 and 300 kg ha-1) and the inclusion or exclusion of the microbial inoculants during the ensiling process. A completely randomized design was used in a 4 × 2 factorial arrangement of treatments. The maize silage chemical composition was estimated by evaluating the plant height (PH) and ear characteristics (NRE= number of rows per ear; NKE= number of kernels per ear; ELS= ear length with straw; EL= ear length without straw) using the following equations:CP= -12.44 + 5.871 × PH + 0.01814 × NRE² (R²= 0.89; P < 0.0001); NDF= 587.93-0.78×NKE-11.67×ELS-0.47×EL+0.0000007×NKE³+0.006× EL³ (R²=0.92; P = 0.003); ADF= 41.48 -0.046 × NRE2 (R2 = 0.42; P = 0.02);TDN= 57.81 - 0.0319 × NRE2 (R2 = 0.42; P = 0.02);EDDM= 56.58 + 0.035 × NRE2 (R2 = 0.42; P = 0.02) andNEL= 1.31 + 0.000757 × NRE2 (R2 = 0.41; P = 0.02). In conclusion, nitrogen fertilization increases the silage energy and protein content; while the inclusion of microbial inoculants during the ensiling process does not alter the chemical and fermentative characteristics of the maize silage.

O objetivo do presente estudo foi estimar a composição bromatológica da silagem por meio de características morfoló- gicas das plantas de milho, e avaliar o efeito de níveis de adubação nitrogenada e da inclusão de inoculante microbiano na ensilagem sobre as características produtivas, morfológicas, bromatológicas e fermentativas de silagem de milho. Os tratamentos consistiram em quatro níveis de adubações com ureia: 0; 100; 200 e 300 Kg ha-1, e da inclusão ou não de inoculante microbiano. Foi utilizado um delineamento inteiramente casualizado, com arranjo fatorial de tratamentos 4 × 2. A composição bromatológica da silagem foi estimada pela altura das plantas (AP) de milho e pelas características da espiga (NLE = número de linhas de grãos por espiga; NGE = Número de grãos por espiga; CCP = comprimento da espiga com palha; CSP = comprimento da espiga sem palha) por meio das seguintes equações: PB = -12,44 + 5,871 × AP + 0,01814 × NLE² (R² = 0,89; P < 0,0001); FDN = 587,93 – 0,78 × NGE – 11,67 × CCP – 0,47 × CSP + 0,0000007 × NGE³ + 0,006 × CCP³ (R² = 0,92; P = 0,003); FDA = 41,48 - 0,046 × NLE2 (R2 = 0,42; P = 0,02); NDT = 57,81 – 0,0319 × NLE2 (R2 = 0,42; P = 0,02); DMSE = 56,58 + 0,035 × NLE2 (R2 = 0,42; P = 0,02) e ELL = 1,31 + 0,000757 × NLE2 (R2 = 0,41; P = 0,02). Pode-se concluir que a adubação nitrogenada aumenta o teor de energia e de proteína da silagem; enquanto a inclusão de inoculante microbiano não altera as características bromatológicas e fermentativas da silagem de milho.

Caracterização da região espaçadora 16-23S rDNA para diferenciação de estirpes de rizóbios utilizadas na produção de inoculantes comerciais no Brasil / Characterization of the spacer region 16-23S rDNA for differentiation of strains of rhizobia used in the production of commercial inoculants in Brazil

A identificação de estirpes de rizóbio tem sido feita pela especificidade por hospedeiros e ensaios microbiológicos tradicionais. Por constituírem um grupo filogeneticamente heterogêneo, diferentes técnicas moleculares têm sido empregadas para auxiliar na caracterização genética e na identificação de estirpes eficientes e competitivas para a produção de inoculantes. Este trabalho teve por objetivos caracterizar a região espaçadora 16S-23S rDNA das estirpes de rizóbios utilizadas nos inoculantes comercializados no Brasil para espécies leguminosas, utilizando a técnica da PCR em combinação com a de RFLP, e avaliar a possibilidade do uso desse marcador molecular como método auxiliar para identificação das estipes. A amplificação da região espaçadora 16-23 S rDNA das estirpes de rizóbios gerou fragmentos com tamanhos que variaram entre 700pb e 1350pb. Os produtos resultantes da amplificação foram submetidos à digestão com as endonucleases. Mps I, Dde I e Hae III. Os resultados obtidos neste estudo indicam a possibilidade do uso da técnica de PCR-RFLP da região espaçadora 16S-23S rDNA como marcador molecular para a diferenciar as estirpes de rizóbios, em complemento às técnicas microbiológicas tradicionais. Contudo, este marcador não é suficientemente discriminatório para ser usado na identificação das estirpes recomendadas para a produção de inoculantes comerciais.

The identification of strains of rhizobia has been made by host specificity and regular microbiological tests. By forming a phylogenetically heterogeneous group, different molecular techniques have been employed to assist in the genetic characterization and identification of efficient and competitive strains for production of inoculants. This study aimed to characterize the spacer region 16S-23S rDNA of the strains of rhizobia used in commercial inoculants in Brazil for legume species, using PCR combined with RFLP, and assess the possibility of using this molecular marker as an auxiliary method for identification of strains. The amplification of the 16-23 S rDNA spacer region of rhizobium strains generated fragments with sizes ranging between 700 and 1350bp. Products from the amplification were subjected to digestion with Mps I, Dde I and Hae III endonucleases. The results indicated the possibility of using the technique of PCR-RFLP of 16S-23S spacer region rDNA as molecular marker to differentiate most strains tested and recommended for production of inoculants, in addition to the traditional microbiological techniques. However, this marker is not sufficiently discriminatory to be used in the identification of the strains recommended for the production of commercial inoculants.

Diseño de un medio para la producción de un co־cultivo de bacterias fosfato solubilizadoras con actividad fosfatasa / Design of a culture media for the production of a phosphate-solubilizing bacteria co-culture / Desenho de um meio para a produção de uma co-cultura de bactérias solubilizadoras de fosfato com actividade da fosfatase

Objetivo. Diseñar un medio de cultivo complejo para la producción de biomasa y fosfatasas ácidas a partir de bacterias solubilizadoras de fosfatos aisladas de suelo. Materiales y métodos. A partir de muestras de suelo de cultivos de palma de aceite se realizaron los aislamientos y la selección de bacterias fosfato solubilizadoras (BFS) en agar SMRS1, las cuales fueron sometidas a pruebas de antagonismo con el fin de verificar su aptitud para la formación de co-cultivos. Posteriormente, se realizó un diseño experimental Box-Behnken para evaluar el efecto de cada uno de los componentes del medio de cultivo sobre la producción de biomasa y enzimas fosfatasas a escala de laboratorio. Finalmente se realizaron curvas de crecimiento y de producción de enzima para determinar los tiempos de producción. Resultados. Se obtuvieron 5 bacterias fosfato solubilizadoras, de las cuales 3 fueron seleccionadas con base en el índice de solubilización; dichas cepas, de morfología bacilar Gram negativa, fueron identificadas como A, B y C, cuyos índices de solubilización correspondieron a 2,03, 2,12 y 2,83, respectivamente. De acuerdo con los análisis de ANOVA para el diseño experimental de Box Behnken, el factor que tuvo efecto significativo sobre la actividad fosfatasa (p<0,01), fue el hidrolizado de levadura, y el formulado que generó la mayor concentración de biomasa y actividad fosfatasa (p<0,01) fue el que contenía 10, 15 y 2,5 gL-1 de roca fosfórica sacarosa e hidrolizado de levadura, respectivamente, obteniendo valores máximos de biomasa y actividad fosfatasa de 11,8 unidades logarítmicas de UFC y 12,9 unidades fosfatasa con incubación por 24 horas a 100 rpm. Conclusión. Se determinó que el medio con formulación 10gL-1 de roca fosfórica, 2,5gL-1 de hidrolizado de levadura y 15gL-1 de sacarosa comercial, fue ideal para la producción de biomasa y enzimas fosfatasas a partir de las cepas evaluadas. Así mismo, se comprobó que el hidrolizado de levadura fue el único factor significativamente influyente en la producción de enzimas fosfatasas.

Objective. To design a complex culture media for the production of biomass and acid phosphatases from phosphate-solubilizing bacteria isolated from soil. Materials and methods. Phosphate-solubilizing bacteria were isolated from oil palm crop soil samples and selected on SMRS1 agar, which were then assessed with antagonism tests to verify their aptitude to form a co-culture. A Box-Behnken experimental design was applied to evaluate the effect of each one of the culture media components on the production of biomass and phosphatase enzymes at a laboratory scale. Finally, microbial growth and enzyme production curves were carried out in order to determine their production times. Results. Five phosphate-solubilizing bacterial strains were isolated and three of them were selected based on their solubilization indices. These Gram negative strains with bacillus morphology were identified as A, B and C; their solubilization indices were 2.03, 2.12, and 2.83, respectively. According to the ANOVA analyses for the Box-Behnken design, the only factor which had a significant effect on the phosphatase activity (p<0.01) was hydrolyzed yeast, and the formulation that generated the highest biomass concentration and phosphatase activity (p<0.01) contained 10, 15 and 2.5 gL-1 of phosphoric rock, sucrose and hydrolyzed yeast, respectively. After 24 hours of incubation at 100 rpm, the highest values of biomass and phosphatase activity were obtained: 11.8 logarithmic units of CFU and 12.9 phosphatase units. Conclusion. We determined that the culture media based on phosphoric rock 10 gL-1, hydrolyzed yeast 2.5 gL-1 and commercial sucrose 15 gL-1 was ideal for the production of biomass and phosphatases by the strains evaluated; likewise, we proved that the hydrolyzed yeast was the only factor significantly influential for the production of phosphatases.

Objetivo. Desenhar um meio de cultura complexo para a produção de biomassa e fosfatase ácida a partir de bactérias solubilizadoras de fosfato isoladas do solo. Materiais e métodos. De amostras de solo de plantações de dendezeiros foram isoladas e selecionadas bactérias solubilizadoras de fosfato (BFS) em ágar SMRS1, que foram testadas em provas de antagonismo para verificar sua capacidade de formar co-culturas. Subsequentemente, foi realizado um desenho experimental do tipo Box-Behnken para avaliar o efeito de cada um dos componentes do meio de cultura na produção de biomassa e de enzimas fosfatase a escala de laboratório. Finalmente foram realizadas curvas de crescimento e de produção da enzima para determinar os tempos de produção. Resultados. Foram obtidas 5 bactérias solubilizadoras de fosfato, das quais 3 foram selecionadas com base no índice de solubilização, tais cepas, de morfologia bacilar Gram negativas, foram identificadas como A, B e C, cujos índices de solubilização corresponderam a 2,03, 2 12 e 2,83, respectivamente. De acordo com a análise ANOVA para o desenho experimental do tipo Box Behnken, o fator que teve efeito significativo na atividade da fosfatase (p <0,01), foi o hidrolisado de levedura, e o formulado que gerou a maior concentração de biomassa e atividade da fosfatase (p <0,01) foi aquel que contive 10, 15 e 2,5 gL-1 de rocha fosfato sacarose e hidrolisado de levedura, respectivamente, obtendo-se valores máximos de biomassa e atividade de fosfatase de 11,8 unidades log de UFC e 12,9 unidades de fosfatase com incubação durante 24 horas a 100 rpm. Conclusão. Foi determinado que o meio com formulação 10gL-1 de rocha fosfórica, 2,5 gL-1 de hidrolisado de levedura e 15gL-1 de sacarose comercial, foi ideal para a produção de biomassa e enzimas fosfatase a partir das cepas avaliadas. Da mesma forma, verificou-se que o hidrolisado de levedura foi o único fator significativo influente na produção de enzimas fosfatase.

Role of bioinoculants and organic fertilizers in fodder production and quality of leguminous tree species.

The comparative effect of dual inoculation of native N fixer (Rhizobium) and AM fungi consortia with different organic fertilizers (vermicompost and farm yard manure) on fodder production and quality of two leguminous tree species (Leucaena leucocephala (Lam) de. Wit. and Sesbania sesban (L.) Merr.) in silvopastoral system and their impact on the fodder production of un-inoculated Panicum maximum Jacq. under cut and carry system. After three years of plantation maximum tree survival was in L. leucocephala in all the treatments in comparison to S. sesban while fodder production was more in S. sesban for initial two years and in third year it accelerated in L. leucocephala. Dual inoculation with vermicompost significantly improved fodder production, fodder quality and rhizosphere microflora in L. leucocephala but in S. sesban dual inoculation was at par with single inoculation of N fixer, AM fungi and control (without inoculation). The grass production was higher with L. leucocephala for two years while in third year it was more with S. sesban. The association of Rhizobium with AM fungi in L. leucocephala was better than in S. sesban.

Environmental Factors and Bioremediation of Xenobiotics Using White Rot Fungi

This review provides background information on the importance of bioremediation approaches. It describes the roles of fungi, specifically white rot fungi, and their extracellular enzymes, laccases, ligninases, and peroxidises, in the degradation of xenobiotic compounds such as single and mixtures of pesticides. We discuss the importance of abiotic factors such as water potential, temperature, and pH stress when considering an environmental screening approach, and examples are provided of the differential effect of white rot fungi on the degradation of single and mixtures of pesticides using fungi such as Trametes versicolor and Phanerochaete chrysosporium. We also explore the formulation and delivery of fungal bioremedial inoculants to terrestrial ecosystems as well as the use of spent mushroom compost as an approach. Future areas for research and potential exploitation of new techniques are also considered.