Abstract: Solid and liquid formulations based on the plant growth-promoting bacteria Bacillus subtilis Bs006 were designed as biological inoculant for vegetable nursery production. Considering the importance of microbial survival from the production process to the soil application, spore viability (CFU) in each formulation was assessed during twelve months of storage at 20, 30, and 40°C. At the three temperature levels evaluated, survival was higher than 85 and 90% for solid and liquid formulations, respectively. The bacterial biological activity was evaluated as plant growth promotion on lettuce, broccoli, and tomato in nursery. The formulations were applied at three concentrations (1x107, 5x107, 1x108 CFU/mL) at sowing and 21 days after seeding. Root and aerial part length and dry weight were the response variables evaluated. A positive effect was observed, particularly with the liquid formulation at 1x108 spores/mL, showing the longest length of root and aerial part and the highest dry weight values in the root and foliar parts. Regarding the endophytism, B. subtilis colonized roots, stems, and leaves, achieving concentrations between 8x102 and 1x105 CFU/g.

Resumen: La loque americana (LA) (Paenibacillus larvae) es una enfermedad bacteriana que afecta a las abejas (Apis mellifera), en particular a la etapa larvaria, y puede causar graves efectos sobre la salud de las colmenas. Las esporas pueden mantenerse en forma latente en la miel sin desarrollar síntomas, por lo que es posible realizar su aislamiento en colmenas aparentemente sanas. La presencia de esporas en miel constituye además una importante limitación para el comercio internacional. En el Uruguay esta enfermedad se encuentra, en la actualidad, con una baja prevalencia. El objetivo del presente trabajo fue realizar el monitoreo de mieles en la zona este-noreste del país, regiones donde nunca se han reportado casos clínicos de esta enfermedad. Durante la zafra 2021-2022 (setiembre-febrero) se colectaron muestras de miel (n=80) en los departamentos de Rivera, Cerro Largo, Treinta y Tres, Rocha, Lavalleja y Maldonado. En ninguna de las muestras se aislaron colonias de P. larvae. Los resultados indican que es posible establecer zonas libres de LA. Las actividades de investigación y extensión con los apicultores son fundamentales para mantener y desarrollar buenas prácticas apícolas vinculadas a la sanidad de las abejas, así como para la obtención de mieles diferenciadas que puedan tener mejores condiciones para la exportación a mercados específicos.

Abstract: American foulbrood (AF) (Paenibacillus larvae) is a bacterial disease that affects bees (Apis mellifera), particularly their larvae stage, and can cause serious effects on the health of hives. The spores can remain latent in honey without developing symptoms, so it is possible to isolate the bacteria from apparently healthy colonies. The presence of spores in honey is also an important limitation for international trade. In Uruguay this disease is currently with a low prevalence. The objective of this work was to made an AF surveillance of honey in the east-northeast area of the country, where clinical cases of this disease have never been reported. During the 2021-2022 harvest season (September-February), honey samples (n=80) were collected from the departments of Rivera, Cerro Largo, Treinta y Tres, Rocha, Lavalleja and Maldonado. In none of the honey samples were isolated colonies of P. larvae. The results indicate that it is possible to establish AF-free zones. Research and extension activities with beekeepers are essential to maintain and develop good beekeeping practices linked to the health of bees, as well as to obtain differentiated honeys that may have better conditions for export to specific markets.

Resumo: A loque americana (AL) (Paenibacillus larvae) é uma doença bacteriana que afeta as abelhas (Apis mellifera), principalmente na fase larval, podendo causar sérios efeitos na saúde das colmeias. Os esporos podem permanecer latentes no mel sem desenvolver sintomas, por isso é possível isolá-los de colmeias aparentemente saudáveis. A presença de esporos no mel também é uma limitação importante para o comércio internacional. No Uruguai esta doença está atualmente com baixa prevalência. O objetivo deste trabalho foi monitorar o mel na região leste-nordeste do país, regiões onde nunca foram relatados casos clínicos desta doença. Durante a safra 2021-2022 (setembro-fevereiro), foram coletadas amostras de mel (n=80) dos departamentos de Rivera, Cerro Largo, Treinta y Tres, Rocha, Lavalleja e Maldonado. Em nenhuma das amostras foram isoladas colônias de P. larvae. Os resultados indicam que é possível estabelecer zonas livres de LA. Atividades de pesquisa e extensão com apicultores são essenciais para manter e desenvolver boas práticas apícolas ligadas à sanidade das abelhas, bem como obter méis diferenciados que possam ter melhores condições de exportação para mercados específicos.

Abstract Pressure-assisted thermal sterilization (PATS) is a new technology to inactivate bacterial spores and ensure food safety. Little has been known about the effects of PATS combining with ε-PL on the spore’s nucleic acid, enzymes and other key substances. This study aimed to investigate the inactivation effect of PATS combining with ε-PL on the spores of B. subtilis. The spores were treated with pressure 600 MPa at 25 °C, 65 °C and 75 °C, and ε-PL at 0.1% and 0.3%. After treatment, the survival rate of B. subtilis spores, leakage of nucleic acid and protein, the change in the cell membrane ATPase activity, the leakage of dipicolinic acid, and the damage on protein and nucleic acid of the spores were determined. The results showed that PATS combining with ε-PL inactivated more spores, and significantly increased the release of protein and nucleic acid compared to the control. ATPase activity reached the lowest value after the treatment of 600 MPa/75 °C combining with 0.3% ε-PL. The release of dipicolinic acid from the spores was increased by 600 MPa/75 °C combining with 0.3% ε-PL as compared with 600 MPa/75 °C treatment alone. FTIR analysis showed that a combination of PATS with ε-PL changed the spectral features of B. subtilis functional groups of proteins and nucleic acids. The PATS treatments when combined with ε-PL were found to shift the symmetric and antisymmetric stretching vibrational absorption peaks of phosphodiester group in nucleic acid molecules (P=O). This change suggested that the combined treatment denatured nucleic acid. The combined treatment also changed the protein from an ordered state to a disordered state, and decreased protein stability. The results improved our understanding on the principle of spore inactivation by PATS combining with ε-PL. Pressureassisted Pressure assisted (PATS safety εPL ε PL s substances B 60 2 C 6 7 01 0 1 0.1 03 3 determined control MPa75 MPa/7 0.3 εPL. PL. alone acids P=O. PO P=O . P O (P=O) stability 0. MPa7 MPa/ (P=O

Abstract The antimicrobial activities of certain spices and herbs are well-documented. Herbs and spices are often exposed to bacterial contamination, spore-forming bacteria in particular. The spores introduced during the production process result in a contamination that causes food alteration during storage. The aim of this study was to isolate spore-forming bacteria from commercially available spices and herbs, such as ginger, cinnamon, black pepper, curcumin, saffron, and clove. The API 50 CHB/E medium was used to identify different genera of spore-forming bacteria isolated from samples. We found that these belonged to the genus Bacillus, such as Bacillus cereus, which was isolated from cinnamon, and Bacillus subtilis, and isolated from curcumin and black pepper. The third species belonged to the genus Brevibacillus, Brevibacillus laterosporus, which was isolated from ginger. The D-values (decimal reduction) of each bacterium were determined at 80, 85, 90, 95, and 100 °C. The Z-value was determined for each bacterium. The whole-cell protein profiles of the identified bacteria were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Since spore-forming bacterial contamination of herbs and spices can occur during their harvest, exsiccation, or through cross-contamination during packaging, we propose different methods for spore elimination.

Abstract Rice is prone to contamination with Bacillus cereus which causes food poisoning. The aim of this study was to investigate the presence of Bacillus cereus in cooked and uncooked rice. A total of forty samples of rice were collected from local supermarkets and tested before and after cooking for the presence of B. cereus. Half of the uncooked rice samples were B. cereus positive. In contrast B. cereus was not detected in the cooked samples when they were immediately examined for the presence of B. cereus. Storing the cooked sample at room temperature (25 °C) for twelve hours, however, gave the spores the chance to grow, and two samples showed bacterial growth higher than 1×103 Cfu/g. The D-value of B. cereus ATCC 14579 spores was found to be 2.7, 1.55, 1.35, 1.2 and 0.6 min at 80, 85, 90, 95, and 100 °C respectively. The Z-value was 9 °C. The level of bacterial growth was below the limits but it was sufficient to suggest that cooked rice should be routinely checked for B. cereus to control bacterial contamination and reheating should reach 80 °C to insure the safety.

Resumen Introducción: Clostridioides difficile es una causa importante de diarrea a nivel hospitalario y comunitario. Esta bacteria se transmite por medio de la ingestión de endosporas, las cuales son difíciles de erradicar por su resistencia intrínseca a diferentes agentes químicos de desinfección. La cepa de referencia CD630 está bien caracterizada, es poco virulenta, no ha causado brotes, y es altamente susceptible a los desinfectantes. Además, pertenece al mismo clado MLST y es filogenéticamente muy cercana a la cepa NAPCR1. Sin embargo, solo la última ha causado brotes en Costa Rica y se ha convertido en una cepa endémica en varios hospitales locales. La cepa NAPCR1 causa enfermedad por mecanismos poco usuales y es genotípicamente diferente a la cepa CD630. Por lo tanto, su potencial epidémico podría estar influenciado por cambios fenotípicos en sus esporas, como una resistencia incrementada a los desinfectantes. Objetivo: Determinar si la cepa NAPCR1 presenta mayor resistencia que CD630 a un desinfectante de alta eficacia utilizado a nivel hospitalario y dilucidar posibles mecanismos a nivel genómico. Métodos: Se utilizó el método de dilución-neutralización para evaluar la actividad esporicida in vitro del dicloroisocianurato de sodio (DCC) contra esporas de 3 subtipos de la cepa NAPCR1 (LIBA-2945, LIBA-5761, y LIBA-6276), CD630 y un aislamiento representativo de la cepa epidémica e hipervirulenta NAP1 (LIBA-5758). Esta caracterización fenotípica fue complementada con una búsqueda genómica de polimorfismos en 15 genes relacionados con la estructura de la endospora o el proceso de esporulación. Resultados: El DCC a una concentración final de 0.1 % (p/v) erradicó las endosporas de la cepa CD630 con gran eficacia (factor de reducción logarítmica; FRL ≥ 5) y eliminó parcialmente las de las cepas NAPCR1 (FRL promedio = 1.77-3.64) y NAP1 (FRL promedio = 3.58). El perfil de susceptibilidad del aislamiento NAPCR1 LIBA-5761 fue único, ya que mostró un mayor nivel de resistencia hacia el DCC que los otros aislamientos NAPCR1 y la cepa NAP1 examinada (ANOVA, P < 0.05). Los tres aislamientos NAPCR1 mostraron deleciones en bclA1 y los aislamientos LIBA-5761 y LIBA-6276 tenían deleciones adicionales en bclA2. Conclusiones: Nuestros experimentos in vitro confirman la resistencia incrementada a los desinfectantes de la cepa NAPCR1 y una susceptibilidad diferencial en sus tres subtipos. Adicionalmente, señalan la importancia de evaluar continuamente la eficacia de los desinfectantes contra cepas circulantes y asignan un posible papel en la resistencia a los desinfectantes gracias a las proteínas del exosporio de C. difficile.

Abstract Introduction: Clostridioides difficile is a significant cause of diarrhea in hospitals and the community. This bacterial pathogen is transmitted through the ingestion of endospores, which are challenging to eliminate due to intrinsic resistance to a variety of chemical disinfection agents. The well-characterized laboratory strain CD630 displays low virulence, has not caused outbreaks, and is highly susceptible to disinfectants. Nonetheless, a closely related strain termed NAPCR1 caused outbreaks in Costa Rica and later became endemic in many hospitals from this country. This strain causes disease through unusual mechanisms and is genotypically distinct from CD630. Consequently, its epidemic potential could be influenced by as yet unknown spore phenotypes, such as increased resistance to disinfectants. Objective: To determine whether the NAPCR1 strain is more resistant to a conventional and highly effective C. difficile sporicidal agent than strain CD630 and to identify potential explanatory mechanisms at the genomic level. Methods: We used an in vitro dilution-neutralization method to calculate the sporicidal activity of sodium dichloroisocyanurate (DCC) against purified spores from three subtypes of NAPCR1 isolates (LIBA-2945, LIBA-5761, and LIBA-6276), CD630, and a representative of the highly virulent and epidemic NAP1 strain (LIBA-5758). This phenotypic characterization was complemented with a genomics-steered search of polymorphisms in 15 spore- or sporulation-related genes. Results: Whereas DCC at a final concentration of 0.1 % (w/v) eradicated CD630 endospores with high efficacy (log10 reduction factor (LFR) ≥ 5), it only partially inactivated NAPCR1 (average LFR range: = 1.77-3.37) and NAP1 endospores (average LRF = 3.58). As hypothesized, the three NAPCR1 subtypes tested were more resistant to DCC than strain CD630 (ANOVA, P < 0.05), with LIBA-5761 showing the highest level of DCC resistance overall (ANOVA, P < 0.05). All three NAPCR1 isolates showed large deletions in bclA1. Besides, isolates LIBA-5761 and LIBA-6276 had deletions in bclA2. Conclusions: Our in vitro tests revealed a differential resistance of spores from the C. difficile NAPCR1 strain to DCC. They highlight the importance of continuously evaluating the efficacy of deployed disinfection agents against circulating strains and hint to a potential role of structural proteins from the exosporium in resistance to disinfectants in C. difficile.

Resumen Introducción. La broca del café (Hypothenemus hampei, CBB) es una de las plagas más devastadoras en plantaciones de café alrededor del mundo. Aunque CBB es susceptible al efecto de las δ-endotoxinas del Bacillus thuringiensis subs. israelensis (Bti) a nivel de laboratorio, la eficacia de este método de control es deficiente en campo, posiblemente debido a la inactivación ocasionada por proteasas digestivas diferentes a las requeridas para la activación de las protoxinas. Objetivo. Determinar si la incorporación de un extracto de harina de soya con inhibidores de proteasas (PI) mezclado con cristales y esporas de Bti (Bti-sc) en una dieta artifical, podría mejorar la toxicidad de Bti contra CBB. Materiales y métodos. Este estudio se realizó en San José, Costa Rica entre 2012 y 2013. Se expuso un conjunto de insectos hembras adultas de CBB a una mezcla que incluyó diferentes concentraciones del extracto de soya parcialmente purificado con PI activo y un liofilizado de Bti-sc, y fueron evaluadas mediante un bioensayo en dieta artificial para estimar la concentración subletal (CL50). Los resultados de mortalidad se validaron mediante observación del intestino medio diseccionado, cuya ultraestructura se analizó mediante microscopía electrónica de transmisión. Resultados. El extracto de soya degradó parcialmente el complejo Bti-sc, redujo la CL50 en casi cuatro veces (de 1,135 a 0,315 µg µl-1) y potenció la mortalidad de CBB de manera concentración-dependiente. Los análisis histológicos del intestino medio confirmaron este efecto sinérgico, dado que se visualizaron daños severos en el epitelio intestinal de CBB expuestos a Bti-sc + PI comparado con Bti-sc solo. Conclusiones. La combinación del extracto de soya enriquecido con PI y Bti-sc potenció la letalidad sobre CBB, que se confirmó por el colapso intestinal. La harina de soya es un suplemento económico que podría aumentar la efectividad de Bti para controlar CBB y retrasar el desarrollo de resistencia biológica.

Abstract Introduction. The coffee berry borer (Hypothenemus hampei Ferrari, CBB) is one of the most devastating pests on coffee plantations around the world. Although CBB is susceptible to the effect of δ–endotoxins of Bacillus thuringiensis subs. israelensis (Bti) at laboratory level, the efficacy of this control method is poor in the field, presumably due to the inactivation by digestive proteases different to those required for protoxin activation. Objective. To study whether the addition of a soybean flour extract enriched with protease inhibitors (PI), mixed with Bti crystals and spores (Bti-sc) in an artificial diet, could improve the toxicity of Bti against CBB. Materials and methods. This study was performed in San José, Costa Rica, between 2012 and 2013. A set of adult female CBB insects was exposed to a mixture containing different concentrations of a partially purified soybean meal extract with active PI and lyophilized Bti-sc, and were tested through a bioassay in artificial diet to estimate the sub-lethal concentration (LC50). The mortality results were validated by observing the dissected midgut, whose ultrastructure was analyzed by transmission electron microscopy. Results. The soybean extracts partially degraded the Bti-sc complex, it reduced its LC50 by almost four times (from 1.135 to 0.315 µg µl-1) and enhanced CBB mortality in a concentration-dependent manner. Histological analyses of the midgut confirmed this synergistic effect, since severe epithelial damage to the intestinal epithelium of CBB exposed to Bti-sc + PI was visualized compared to Bti-sc alone. Conclusions. The combination of a soybean extract enriched in PI and Bti-sc enhanced the mortality effect over CBB, which was confirmed by the midgut collapse. Soybean flour is a cost-effective supplement that could increase Bti effectiveness against CBB and delay the appearance of biological resistance.

Resumo Este estudo tem como objetivo avaliar a influência da adição de esporos de B. subtilis AP91 nas propriedades mecânicas de argamassas. Para tanto, a bactéria B. subtilis AP91 isolada de folhas de arroz da variedade agulha precoce foi utilizada na concentração de 105 esporos/mL em argamassas com traço de 1:3 (em massa) e relação água/cimento (a/c) de 0,63. Os esporos bacterianos foram adicionados de duas diferentes formas: na água de amassamento e por imersão em solução contendo os esporos. A análise por Microscopia Eletrônica de Varredura (MEV) com Sistema de Energia Dispersiva (EDS) mostrou cristais com picos de cálcio, o que possivelmente indica a presença de carbonato de cálcio bioprecipitado. Os resultados obtidos a partir da análise mecânica mostraram que a bioprecipitação de CaCO3 pela bactéria B. subtilis AP91 foi satisfatória, particularmente quando os esporos foram adicionados à água de amassamento, aumentando a resistência à compressão em até 31%. Portanto, conclui-se que a adição de esporos de B. subtilis AP91 na água de amassamento das argamassas induziu a biocimentação, que aumentou a resistência à compressão. A bioprecipitação de carbonato de cálcio pode ter outras consequências benéficas, como o fechamento de poros e fissuras em materiais cimentícios, que poderiam melhorar a durabilidade, embora mais pesquisas neste aspecto ainda sejam necessárias.

Abstract This study aims to evaluate the influence of B. subtilis AP91 spores addition on the mechanical properties of mortars. B. subtilis strain AP91, isolated from rice leaves of the needle variety, which has an early cycle of production, was used at the concentration of 105 spores/mL in mortars with cement-to-sand ratio of 1:3 (by weight) and water-to-cement ratio (w/c) of 0.63. These spores were added in two different ways: in the mixing water and by immersion in a solution containing bacterial spores. Scanning Electron Microscope (SEM) analysis showed crystals with calcium peaks on the EDS, which possibly indicates the presence of bioprecipitated calcium carbonate. The results obtained in the mechanical analysis showed that the bioprecipitation of CaCO3 by B. subtilis strain AP91 was satisfactory, particularly when the spores were added in the mixing water, increasing the compressive strength up to 31%. Thus, it was concluded that the addition of B. subtilis AP91 spores in the mixing water of cement mortars induced biocementation, which increased the compressive strength. This bioprecipitation of calcium carbonate may very well have other advantageous consequences, such as the closure of pores and cracks in cementitious materials that could improve durability properties, although more research is still needed on this matter.

The aim of this study was to determine the presence of bacterial agents and to characterize the histopathological lesions in ornamental tetra bleeding heart (Hyphessobrycon erytrhostigma) fish from a commercial aquarium in the city of Iquitos, Peru. For the microbiological study 60 fish were used, samples were taken from spleen and kidney. Isolation was done on MacConkey agar, Trypticase Soy agar, Pseudomonas Aeromonas selective agar (GSP) and Cytophaga agar and the genus identification was done by Gram staining and biochemical tests. Another 60 fish were used for the histopathological study where the skin, eye, gills, stomach, intestine, liver, spleen, muscle, kidney and peritoneum tissues were analyzed. Seven bacterial genera were isolated: Pseudomonas, Aeromonas, Lactobacillus, Bacillus, Flavobacterium, Staphylocccus and Escherichia. Spores of Mixosporidium sp, hyperplasia and lamella fusion were observed in gills. In the liver and peritoneum, parasitic and bacterial granulomas were found. In muscle, kidney and spleen, bacterial granulomas were found, and in stomach and intestine epithelial cell hyperplasia were found, among other lesions. Results showed that 52.3% (22/42) of the bacterial granulomas were positive for Ziehl Neelsen staining, which showed acid-fast bacilli bacteria. No lesions were found in the eyes.

El objetivo del presente estudio fue determinar la presencia de agentes bacterianos y caracterizar las lesiones histopatológicas en tejidos del pez ornamental ‘tetra bleeding heart’ (Hyphessobrycon erytrhostigma) procedentes de un acuario comercial de la ciudad de Iquitos, Perú. Para el estudio microbiológico se utilizaron 60 peces, tomándose muestras de bazo y riñón. El aislamiento se hizo en agar MacConkey, agar Tripticasa de Soya, agar selectivo para Pseudomonas-Aeromonas (GSP) y agar Cytophaga. La identificación de los géneros bacterianos se hizo mediante la coloración Gram y pruebas bioquímicas. Para el estudio histopatológico se utilizaron otros 60 peces, evaluándose los tejidos de piel, ojo, branquias, estómago, intestino, hígado, bazo, músculo, riñón y peritoneo. Se aislaron siete géneros bacterianos: Pseudomonas, Aeromonas, Lactobacillus, Bacillus, Flavobacterium, Staphylocccus y Escherichia. En los estudios histopatológicos, se observó en las branquias la presencia de esporas de Mixosporidium sp, hiperplasia y fusión de lamelas; en hígado y peritoneo se hallaron granulomas parasitarios y bacterianos; en músculo, riñón y bazo se encontraron granulomas bacterianos; y en el estómago e intestino se observó hiperplasia de las células epiteliales, entre otras lesiones. El 52.3% (22/42) de los granulomas bacterianos fueron positivos a la tinción de Ziehl Neelsen, donde se evidenció bacterias bacilares largas acidorresistentes. No se observaron lesiones en los ojos.

RESUMO. Elasmopalpus lignosellus (Zeller, 1848) (Lepidoptera, Pyralidae) é considerada praga para mais de 60 espécies de plantas cultivadas como o milho, cana-de-açúcar, trigo, soja, arroz, feijão, sorgo, amendoim e algodão. O objetivo deste trabalho foi selecionar e caracterizar isolados de Bacillus thuringiensis virulentos a E. lignosellus atuando como método alternativo de controle. Assim, 47 isolados foram avaliados em bioensaios seletivos contra lagartas de 1º ínstar de E. lignosellus. Para os isolados mais tóxicos, a concentração letal (CL50) foi estimada e a sua caracterização foi realizada por métodos morfológicos, bioquímicos e moleculares. Dos 47 isolados testados, 12 causaram mortalidade acima 85% e apresentaram CL50 entre 0.038E+8 a 0.855E+8 esporos mL-1. Os menores valores de CL50 foram obtidos pelos isolados BR83, BR145, BR09, BR78, S1534 e S1302, os quais não diferiram da linhagem padrão HD-1, com exceção do BR83. O perfil proteico revelou proteínas Cry entre 60 e 130 kDa, e a caracterização molecular mostrou a presença dos genes cry1, cry2, cry3 e cry11. A análise morfológica identificou três diferentes inclusões cristalinas: bipiramidais, esféricas e cuboides. Entre os isolados avaliados, 12 apresentam potencial biotecnológico para controle de E. lignosellus via formulação de novos bioinseticida ou produção de plantas transgênicas.

ABSTRACT. Elasmopalpus lignosellus (Zeller, 1848) (Lepidoptera, Pyralidae) is an insect pest of 60 economically important crops, including sugarcane, wheat, soybean, rice, beans, sorghum, peanuts, and cotton. The aim of this work was to select and characterize Bacillus thuringiensis isolates with insecticidal activity against E. Lignosellus that could be used as an alternative method of control. Selective bioassays were done to evaluate the toxicity of 47 isolates against first instar larvae of E. lignosellus. For the most toxic bacterial strains, the lethal concentration (LC50) was estimated and morphological, biochemical and molecular methods were used to characterize the isolates. Among the 47 isolates tested, 12 caused mortality above 85% and showed LC50 values from 0.038E+8 to 0.855E+8 spores mL-1. Isolates BR83, BR145, BR09, BR78, S1534, and S1302 had the lowest LC50 values and did not differ from the standard HD-1 strain; the exception was BR83.The protein profiles produced bands with molecular masses of 60-130 kDa. The genes cry1, cry2, cry3, and cry11 were identified in the molecular characterization. The morphological analysis identified three different crystal inclusions: bipyramidal, spherical and cuboidal. Among the tested isolates, 12 isolates have potential for biotechnological control of E. Lignosellus by development of new biopesticides or genetically modified plants.

Among the various rhizospheric bacterial isolates screened from Westren Ghat regions of Kerala, a Bacillus sp. with both antagonistic activity to Pythium myriotylum and plant growth promoting properties was identified. Scanning electron microscopic analysis revealed significant ultrastructural changes of hyphae and spores of P. myriotylum upon treatment with Bacillus sp.WG4. For the structural characterization of antagonistic compound in the crude exract, GC MS analysis was carried out and this has resulted in the identification of presence of pyrrolo [1,2- a] pyrazine-1,4-dione, hexahydro-3-(phenylmethyl) as basis of antifungal activity. The in vivo antifungal activity of Bacillus strain was analysed by pre-inoculation of this to ginger rhizome. Here the presence of Bacillus strain was found to provide effective protection to ginger rhizome upon inoculation with Pythium myriotylum. Very interestingly, when the Bacillus strain was checked for its synergistic antifungal activity with commercially available biocontrol agent Trichoderma, the combination was found to have increased antifungal activity. Due to its antifungal and growth enhancement effects, the isolated strain can be of promising application as antifungal plant probiotic agent alone or in combination with other agents like Trichoderma.

RESUMO: Os esporos de bactérias estão amplamente difundidos no ambiente e podem contaminar o leite. Os esporos conferem resistência aos processos térmicos e sua germinação pode reduzir a vida útil do leite pasteurizado, uma vez que bactérias esporuladas aeróbias são muito frequentemente produtoras de proteases e lipases. O objetivo do presente trabalho foi identificar Paenibacillus sp. na microbiota deteriorante, oriunda da germinação de esporos no leite cru, e avaliar o seu potencial deteriorante. Vinte amostras de leite de propriedades distintas foram tratadas a 80ºC/12min e semeadas para isolamento de bactérias formadoras de esporos. Essas cepas foram repicadas em ágar leite e tributirina para verificação do seu potencial proteolítico e lipolítico, respectivamente. Foi realizada a amplificação e sequenciamento do gene 16S do rRNA para identificação das cepas por similaridade com as sequências de DNA depositadas no GenBank. Foram obtidos 137 isolados, dos quais 40 (29,2%) apresentaram atividade deteriorante do leite. Dessas, 3 (7,5%) cepas foram identificadas como Paenibacillus sp., todas lipolíticas. Paenibacillus sp. são considerados os principais responsáveis pela deterioração do leite pasteurizado de longa vida útil em outros países. Para o aumento da vida útil do leite pasteurizado brasileiro, é importante conhecer a microbiota esporulada, de forma a determinar sua origem e poder controlar a contaminação do leite tanto pelas formas vegetativas como por seus esporos.

ABSTRACT: Bacterial spores are widespread in the environment and can contaminate milk. Spores are resistant to thermal conditions and your germination reduces milk shelf-life because the aerobic bacteria that are sporulated produce proteases and lipases. The aim of this study was identify Paenibacillus sp., the spoilage microbiota, arising from the germination of spores in raw milk and your spoilage potential. Twenty different milk samples were treated at 80°C/12min and plated to isolate spore-forming bacteria. These strains were picked in milk agar and tributyrin agar for verification of their potential proteolytic and lipolytic activities, respectively. Amplification and sequencing of the 16S rRNA gene of the strains for identification by similarity to the DNA sequences deposited in GenBank was performed. One hundred and thirty-seven isolates were obtained, of which 40 (29.2%) showed spoilage activity for milk. Of these, three (7.5%) were identified as strains of Paenibacillus sp., and all were lipolytic. Paenibacillus sp. have been identified as primarily responsible for the spoilage of pasteurized milk with a long shelf-life in other countries. To increase the shelf-life of Brazilian pasteurized milk, it is important to identify the sporulated microbes to determine their origin and to control the contamination of milk by vegetative forms such as spores.

Introdução: Bacillus thuringiensis, tem sido usado mundialmente para o controle de culicídeos, especialmente no combate ao Aedes aegypti. Várias formulações vêm sendo fabricadas, no entanto, a efetividade desses inseticidas variam bastante de acordo com as condições ambientais. Objetivo: avaliar a influência dos fatores abióticos na efetividade de B. thuringiensis var. israelensis para larvas de Ae. Aegypti. Métodos: realizou-se bioensaio experimental no campus do CESC/UEMA, com utilização de 20 tanques de cimento, destes, 10 ficaram expostos aos fatores ambientais, e apenas 5 foram tratados e 5 serviram como controle. Os outros 10 tanques foram colocados à sombra, também 5 tratados e 5 não tratados (controle). Após o tratamento foram colocadas 25 larvas em estádio L 3 em cada tanque e realizadas leituras de mortalidade após 24 horas e a cada 7 dias. Semanalmente foram coletadas amostras de água de todos os tanques para medir o número de esporos viáveis e avaliar a reciclagem bacteriana. Resultados e Discussão: o larvicida apresentou eficácia inicial, matando 100 % das larvas nos tanques que estavam ao sol e a sombra. Os fatores ambientais, insolação, temperatura e pluviometria afetaram negativamente a efetividade do produto, pois se observou diminuição na eficácia do produto ao longo da realização do experimento. Observou-se variação do número de esporos ao longo do desenvolvimento do experimento, demonstrando que a bactéria apresenta potencial de reciclagem. Conclusão: esses dados servem como parâmetros para melhoramento de inseticidas à base de Bti, no sentido de se fabricar formulações mais adequadas às condições ambientais da nossa região.

Introduction: Bacillus thuringiensis , has been used worldwide for the control of mosquitoes, particularly in combating Aedes aegypti. Various formulations have been made however, the effectiveness of these insecticides vary varies considerably according to the environmental conditions. Objective: to evaluate the influence of abiotic factors on the effectiveness of B. thuringiensis var. israelensis to against larvae of Ae. aegypti. Methods: we conducted bioassay experiments in the campus of the CESC / UEMA. With use of 20 Twenty cement tanks, of these, were used, 10 of which were exposed to environmental factors, and only. Of these, 5 were treated and 5 served as controls. The other 10 tanks were placed in the shade, also again 5 were treated and 5 untreated were not (control). After treatment, the 25 stage L 3 larvae were placed in 25 L 3 each stage tank and mortality. Mortality readings were taken after 24 hours and every 7 days. Weekly water samples were collected from all tanks to measure once a week to check the number of viable spores and evaluate bacterial recycling. Results and discussion: the larvicidal efficacy: The larvicide showed initial efficacy, killing 100 % of the larvae in tanks that were located both in the sun and in the shade. Environmental factors, sunlight, temperature and rainfall negatively affected the effectiveness of the product, because for there was a reduction in the effectiveness of the product throughout the experiment. It was observed variation in the number of spores during the development of the experiment demonstrating, showing that the bacterium has potential recyclability. Conclusion: these data serve as parameters for improving to in Bti-based insecticides, in order to make the with a view to making formulations more suited toin keeping with the environmental conditions of our region.

Introducción: la Bacillus thuringiensis se ha utilizado en muchas regiones del mundo para el control de los mosquitos, sobre todo en el combate contra el Aedes aegypti. Se han elaborado varias formulaciones; sin embargo, la efectividad de esos insecticidas varía considerablemente según las condiciones ambientales. Objetivo: evaluar la influencia de los factores abióticos en la efectividad de la B. thuringiensis var. israelensis contra larvas de Ae. aegypti. Métodos: se realizaron bioensayos experimentales en el campus de la CESC / UEMA. Se utilizaron 20 tanques de cemento, 10 de los cuales quedaron expuestos a factores ambientales. De ellos, 5 fueron tratados y 5 sirvieron como controles. Los otros 10 tanques fueron ubicados en la sombra, y también en este caso 5 fueron tratados y 5 no (controles). Después del tratamiento, se colocaron 25 larvas en estadio larval L3 en cada tanque. Se realizaron lecturas de mortalidad luego de 24 horas y cada 7 días. Se tomaron muestras de agua de todos los tanques una vez a la semana para comprobar el número de esporas viables y evaluar el reciclaje bacteriano. Resultados y discusión: el larvicida mostró eficacia inicial, matando el 100 % de las larvas de tanques ubicados tanto al sol como en la sombra. Los factores ambientales, la luz solar, la temperatura y la lluvia afectaron negativamente la efectividad del producto, constatándose una reducción de la misma a lo largo del experimento. Se observaron variaciones en el número de esporas durante el experimento, lo que demuestra que la bacteria presenta potencial de reciclaje. Conclusión: los datos obtenidos sirven de parámetros para el mejoramiento de los insecticidas a base de Bti, con vistas a elaborar formulaciones más acordes con las condiciones ambientales de nuestra región.

No outono de 2011, em fazendas de ciprinídeos localizadas em Iasi, no rio Jijia, diversas infecções bacterianas e cistos externos macroscópicos na pele se desenvolveram como resultado do estresse induzido por fatores bióticos e abióticos. No exame do conteúdo dos cistos, a presença de diversos esporos foi observada, a maioria do gênero Dermocystidium sp. As amostras foram colhidas das seguintes espécies: carpa comum (Cyprinus carpio) e carpa cruciana (Carassius auratus gibelio) de fazenda piscícola, além do rio Jijia. Assim sendo, 35 peixes foram examinados, todos demonstrando cistos, fragmentação da barbatana dorsal e congestão das guelras. O exame histológico da pele mostrou um campo de múltiplos cistos dérmicos com formações circulares claras eosinofílicas (14-16µm) contendo corpo central refratado similar ao relatado para Dermocystidium sp. Amostras de guelras foram retiradas das áreas afetadas para exame MEV, com o propósito de se avaliar não apenas os aspectos da morfologia normal, mas também os aspectos de algumas modificações das áreas afetadas, além da presença da bactéria etiologicamente incriminada: Pseudomonas fluorescens. Os isolados foram identificados por meio de métodos fenotípicos. Todas as amostras que mostraram mobilidade e positividade-oxidase foram testadas usando-se fita API 20 NE. Consequentemente, estas foram taxonomicamente agrupadas na espécie Pseudomonas fluorescens. O microscópio eletrônico de varredura (MEV) foi usado pela primeira vez na caracterização de lesões bacterianas produzidas por Pseudomonas nas guelras de Cyprinus carpio e Carassius auratus gibelio. O diagnóstico de septicemia com espécies condicionais de patogênico de Pseudomonas fluorescens foi correlacionado com os resultados das investigações físico-químicas da água e de dados sobre as condições de reprodução dos animais investigados.

In autumn 2011 in cyprinid farms located in Iasi on the Jijia river, several infections with bacterial strains and macroscopical external cysts on the skin were diagnosedwhich developed as a result of the stress induced by biotic and abiotic factors. On the examination of the cyst contents the presence of numerous spores was observed, mostly of the Dermocystidium spgenusThe samples were taken from the common carp (Cyprinus carpio) and crucian carp (Carassius auratus gibelio)species from the fish farm as well as from the Jijia River. 35 fish were examined, all of them showing cysts, fragmentation of their dorsal fin and congestion of the gills. Histological examination of the skin showed a field of multiple dermal cysts with round light eosinophilic formations (14-16µm) containing a central refractable body similar to that reported for Dermocystidium sp.Gills samples were taken from the affected areas for the SEM examination with the purpose of evaluating not only aspects of normal morphology, but also aspects of some modifications of the affected areas as well as the presence of the etiologically incriminated bacteria Pseudomonas fluorescens. The isolates were identified through phenotypic methods. All the strains that showed mobility and oxidase-positivity were tested using API 20 NE strip. Consequently, they were taxonomically grouped into the species Pseudomonas fluorescens.The scanning electron microscope (SEM) was used for the first time in the characterization of the bacterial lesions produced by Pseudomonasstrains on Cyprinus carpioand Carassius auratus gibeliogills. The diagnosis of septicemia with conditional pathogen species of Pseudomonas fluorescens was correlated with the results of the physico-chemical investigations of water and the data concerning the breeding conditions of the investigated livestock.

A produtividade e sustentabilidade das culturas têm sido frequentemente relacionadas com a matéria orgânica e a biomassa microbiana do solo, especialmente devido ao seu papel na ciclagem de nutrientes do solo. Este trabalho teve como objetivo avaliar a biomassa de fungos e bactérias do solo por microscopia de epifluorescência e a densidade de esporos de fungos micorrízicos arbusculares (FMAs) em lavouras de cana de açúcar (Saccharum officinarum L.) sob diferentes manejos. Foram coletadas amostras de solo de lavouras manejadas com ou sem queima, com ou sem colheita mecanizada, com ou sem aplicação de vinhaça e de matas ciliares adjacentes. As amostras de solo foram coletadas a 10 cm de profundidade e mantidas a 4°C até o momento das análises. A biomassa fúngica variou de 25 a 37µg C g-1 de solo seco e a bacteriana, de 178 to 263µg C g-1 de solo seco. A razão média entre a biomassa fúngica e bacteriana do solo nos diferentes manejos de canaviais foi de 0,14. A densidade de esporos de FMAs variou de 9 a 13 esporos g-1 de solo seco. Os diferentes manejos da cana de açúcar não afetaram significativamente a densidade de esporos de FMAs. Em geral, não foram encontradas mudanças significativas na biomassa microbiana nos diferentes manejos de solo com cana de açúcar em comparação com a mata ciliar. Todavia, a soma das biomassas fúngicas e bacterianas (208 a 301µg C g-1 de solo seco) determinadas por microscopia de epifluorescência foram similares a resultados da biomassa total do solo encontrados em outros estudos com métodos tradicionais (e.g. fumigação-extração). Os resultados evidenciam que a determinação da razão fungo/bactéria por microscopia de epifluorescência, associada a outros parâmetros, é uma metodologia promissora para compreender a funcionalidade microbiana e ciclagem de nutrientes sob diferentes manejos do solo.

Crop productivity and sustainability have often been related to soil organic matter and soil microbial biomass, especially because of their role in soil nutrient cycling. This study aimed at measuring fungal and bacterial biomass by epifluorescence microscopy and arbuscular mycorrhizal fungal (AMF) spore density in sugarcane (Saccharum officinarum L.) fields under different managements. We collected soil samples of sugarcane fields managed with or without burning, with or without mechanized harvest, with or without application of vinasse and from nearby riparian native forest. The soil samples were collected at 10cm depth and storage at 4°C until analysis. Fungal biomass varied from 25 to 37µg C g-1 dry soil and bacterial from 178 to 263µg C g-1 dry soil. The average fungal/bacterial ratio of fields was 0.14. The AMF spore density varied from 9 to 13 spores g-1 dry soil. The different sugarcane managements did not affect AMF spore density. In general, there were no significant changes of microbial biomass with crop management and riparian forest. However, the sum of fungal and bacterial biomass measured by epifluorescence microscopy (i.e. 208-301µg C g-1 dry soil) was very close to values of total soil microbial biomass observed in other studies with traditional techniques (e.g. fumigation-extraction). Therefore, determination of fungal/bacterial ratios by epifluorescence microscopy, associated with other parameters, appears to be a promising methodology to understand microbial functionality and nutrient cycling under different soil and crop managements.