Providing inoculum for Didymella bryoniae studies: the effect of light spectrum and storing at low temperature

Abstract The in vitro sporulation of Didymella bryoniae is of great importance for studies that require pure inoculum and in large quantities. Thus, the objectives of this study were to identify the best condition for D. bryoniae sporulation combining different light spectra (UV-A or UV-B light, white light, and continuous dark), with distinct culture media (PDA, V8, ML, and PDAB) and, to evaluate fungus survivability stored at -20°C over time. The fungus samples were only able to sporulate when subjected to the UV-B light treatment, regardless of the culture medium. The highest appearance of spores conidium type was observed in the PDAB medium, and the lowest production occurred in the ML medium. Reproductive structures, such as perithecia and pycnidia, were observed in all culture media. However, there was considerable variation in the amount of each structure between the different culture media. The ML and V8 media showed a greater number of perithecia and the PDA and PDAB media presented a greater proportion of pycnidia compared to perithecia. The storage duration at -20°C did not affect mycelial growth or mycelial growth rate. In conclusion, the UV-B light is essential for D. bryoniae in vitro sporulation. Moreover, the culture medium composition influences the type of fungal structure produced, as well as spores size and quantity. Freezing at -20°C is an efficient technique that can be used to store D. bryoniae for at least five months without loss of viability.

Resumo A esporulação de Didymella bryoniae in vitro é de grande importância para estudos que requerem inóculo puro e em grandes quantidades. Assim, os objetivos deste estudo foram identificar a melhor condição para esporulação de D. bryoniae combinando diferentes espectros de luz (luz UV-A ou UV-B, luz branca e escuro contínuo) com distintos meios de cultura (PDA, V8, ML e PDAB) e, avaliar a sobrevivência do fungo armazenado a -20°C ao longo do tempo. As amostras de fungo só esporularam quando submetidas ao tratamento com luz UV-B, independentemente do meio de cultura. Maior aparecimento de esporos do tipo conídio foi observado no meio PDAB, e a menor produção ocorreu no meio ML. Estruturas reprodutivas, como peritécios e picnídeos, foram observadas em todos os meios de cultura. No entanto, houve uma variação considerável na quantidade de cada estrutura entre os diferentes meios de cultura. Os meios ML e V8 apresentaram maior número de peritécios e os meios PDA e PDAB apresentaram maior proporção de picnídeos em relação aos peritécios. A duração do armazenamento a -20°C não afetou o crescimento micelial ou a taxa de crescimento micelial. Em conclusão, a luz UV-B é essencial para a esporulação de D. bryoniae in vitro. Além disso, a composição do meio de cultura influencia o tipo de estrutura fúngica produzida, bem como o tamanho e a quantidade dos esporos. O congelamento a -20°C é uma técnica eficiente que pode ser usada para armazenar D. bryoniae por pelo menos cinco meses sem perda de viabilidade

Providing inoculum for Didymella bryoniae studies: the effect of light spectrum and storing at low temperature / Fornecendo inóculo para estudos de Didymella bryoniae: o efeito do espectro de luz e armazenamento em baixa temperatura

Abstract The in vitro sporulation of Didymella bryoniae is of great importance for studies that require pure inoculum and in large quantities. Thus, the objectives of this study were to identify the best condition for D. bryoniae sporulation combining different light spectra (UV-A or UV-B light, white light, and continuous dark), with distinct culture media (PDA, V8, ML, and PDAB) and, to evaluate fungus' survivability stored at -20°C over time. The fungus samples were only able to sporulate when subjected to the UV-B light treatment, regardless of the culture medium. The highest appearance of spores conidium type was observed in the PDAB medium, and the lowest production occurred in the ML medium. Reproductive structures, such as perithecia and pycnidia, were observed in all culture media. However, there was considerable variation in the amount of each structure between the different culture media. The ML and V8 media showed a greater number of perithecia and the PDA and PDAB media presented a greater proportion of pycnidia compared to perithecia. The storage duration at -20°C did not affect mycelial growth or mycelial growth rate. In conclusion, the UV-B light is essential for D. bryoniae in vitro sporulation. Moreover, the culture medium composition influences the type of fungal structure produced, as well as spores' size and quantity. Freezing at -20°C is an efficient technique that can be used to store D. bryoniae for at least five months without loss of viability.

Resumo A esporulação de Didymella bryoniae in vitro é de grande importância para estudos que requerem inóculo puro e em grandes quantidades. Assim, os objetivos deste estudo foram identificar a melhor condição para esporulação de D. bryoniae combinando diferentes espectros de luz (luz UV-A ou UV-B, luz branca e escuro contínuo) com distintos meios de cultura (PDA, V8, ML e PDAB) e, avaliar a sobrevivência do fungo armazenado a -20°C ao longo do tempo. As amostras de fungo só esporularam quando submetidas ao tratamento com luz UV-B, independentemente do meio de cultura. Maior aparecimento de esporos do tipo conídio foi observado no meio PDAB, e a menor produção ocorreu no meio ML. Estruturas reprodutivas, como peritécios e picnídeos, foram observadas em todos os meios de cultura. No entanto, houve uma variação considerável na quantidade de cada estrutura entre os diferentes meios de cultura. Os meios ML e V8 apresentaram maior número de peritécios e os meios PDA e PDAB apresentaram maior proporção de picnídeos em relação aos peritécios. A duração do armazenamento a -20°C não afetou o crescimento micelial ou a taxa de crescimento micelial. Em conclusão, a luz UV-B é essencial para a esporulação de D. bryoniae in vitro. Além disso, a composição do meio de cultura influencia o tipo de estrutura fúngica produzida, bem como o tamanho e a quantidade dos esporos. O congelamento a -20°C é uma técnica eficiente que pode ser usada para armazenar D. bryoniae por pelo menos cinco meses sem perda de viabilidade

Characterization of a Clostridioides difficile ST-293 isolate from a recurrent infection in Argentina

Resumen Clostridioides difficile es un patógeno esporulado oportunista responsable de diarrea asociada a antibióticos en humanos. C. difficile produce 2 toxinas principales: TcdAy TcdB, además de la toxina binaria (CDT), también asociada a la virulencia. Este estudio buscó caracterizar el aislamiento ALCD3, involucrado en un episodio de recurrencia de una infección nosocomial. La caracterización molecular mostró que dicho aislamiento pertenece al toxinotipo 0/v y el análisis por MLST demostró un perfil alélico adk:91, atpA:1, dxr:2, glyA: 1, recA:27, sodA: 1 y tpi:1, lo cual corresponde al ST293 (MLST clado 1). Durante el crecimiento, el aislamiento ALCD3 mostró un incremento temprano de la tasa de esporulación y valores máximos de formas termorresistentes luego de 2 días de incubación. Tanto la cinética de esporulación como la producción de formas termorresistentes fueron más rápidas en el aislamiento ALCD3 que en la cepa de referencia VPI 10463. La germinación en presencia del germinante natural taurocolato fue más rápida en el aislamiento ALCD3 que en la cepa VPI 10463, lo que indica que aquel comienza la hidrólisis del córtex antes. También, el co-germinante glicina indujo una rápida liberación de ácido dipicolínico en ALCD3. Estos hallazgos indican que el aislamiento ALCD3 es particularmente eficiente en la esporulación y en la germinación. El presente trabajo representa el primer informe de la circulación de C. difficile ST293 en Argentina. La habilidad del aislamiento ALCD3 para producir toxinas y su alta capacidad de esporulación/germinación son características claves compatibles con un alto potencial de diseminación e inducción de infecciones recurrentes.

Abstract Clostridioides difficile is an opportunistic spore-forming pathogen responsible for antibiotic-associated diarrhea in humans. C. difficile produces two main toxins: TcdA and TcdB as well as a third toxin named binary toxin (CDT) that is also involved in virulence. The present study aimed at characterizing the C. difficile isolate ALCD3 involved in a relapse episode of nosocomial infection. Molecular characterization showed that isolate ALCD3 belongs to tox-inotype 0/v and the MLST analysis demonstrated allelic profile adk:91, atpA:1, dxr:2, glyA: 1, recA:27, sodA: 1 and tpi:1 which corresponds to ST293 (MLST clade: 1). During growth, isolate ALCD3 showed an early increase in the sporulation ratio as well as maximal values of heat resis-tant forms after 2 days of incubation. Both sporulation kinetics and production of heat resistant forms were faster for isolate ALCD3 than for the reference strain VPI 10463. Germination in the presence of the natural germinant taurocholate was faster for isolate ALCD3 than for strain VPI 10463, which indicates that isolate ALCD3 starts cortex hydrolysis earlier than strain VPI 10463. Furthermore, the co-germinant glycine, induces rapid release of dipicolinic acid (DPA) in isolate ALCD3. These findings indicate that isolate ALCD3 is particularly efficient in both sporulation and germination. The present work represents the first report of the circulation of C. difficile ST293 in Argentina. The ability of isolate ALCD3 to produce toxins and its high sporulation/germination capacity are key features compatible with a microorganism with high dissemination potential and the possibility of inducing recurrent infections.

The Potential Effects of Fungicides on Association of Rhizophagus irregularis with Maize and Wheat

Abstract The effect of different fungicides on mycorrhizal fungi should be investigated in different plants and environmental conditions. Thus, the purpose of this study was to appraise the effect of simultaneous fungicides application (including benomyl, rovral TS, mancozeb, and tilt) on the efficiency of Rhizophagus irregularis in cultivations of maize and wheat. This study was conducted in two separate experiments in the laboratory and greenhouse. The results of the laboratory stage showed that the use of all four fungicides significantly reduced the spore number compared to the conditions of non-use of the fungicide, although only rovral TS and mancozeb led to a significant reduction in root colonization percentage of R. irregularis. In the greenhouse, the benomyl significantly increased root dry weight in maize although tilt significantly reduced root colonization of maize with R. irregularis. The tilt and rovral TS had a positive effect and benomyl had a negative effect on wheat growth traits, but the root colonization of wheat with R. irregularis was not affected by fungicides. Generally, benomyl (2 g L-1) in maize and tilt (2 mL L-1) in wheat and rovral TS in both plants could be recommended with the combined application of R. irregularis inoculants. Therefore, depending on the type of fungicide and the host plant, the effect of the fungicide on colonization and association of mycorrhiza varies.

Mulberry extract as an ecofriendly anticoccidial agent: in vitro and in vivo application / Extrato de amoreira como agente anticoccidiano ecologicamente correto: aplicação in vitro e in vivo

Abstract Natural products are ecofriendly agents that can be used against parasitic diseases. Eimeria species cause eimeriosis in many birds and mammals and resistance to available medications used in the treatment of eimeriosis is emerging. We investigated the in vitro and in vivo activity of Morus nigra leaf extracts (MNLE) against sporulation of oocysts and infection of mice with Eimeria papillata. Phytochemical analysis of MNLE showed the presence of seven compounds and the in vitro effects of MNLE, amprolium, DettolTM, formalin, ethanol, and phenol were studied after incubation with oocysts before sporulation. Furthermore, infection of mice with E. papillata induced an oocyst output of approximately 12 × 105 oocysts/g of feces. MNLE significantly decreased oocyst output to approximately 86% and the total number of parasitic stages in the jejunum by approximately 87%. In addition, the reduction in the number of goblet cells in the jejuna of mice was increased after treatment. These findings suggest that mulberry exhibited powerful anticoccidial activity.

Resumo Os produtos naturais são agentes ecologicamente corretos que podem ser usados ​​contra doenças parasitárias. As espécies de Eimeria causam eimeriose em muitas aves e mamíferos e a resistência aos medicamentos disponíveis usados ​​no tratamento da eimeriose está emergindo. Foram investigadas as atividades in vitro e in vivo dos extratos de folhas de Morus nigra (MNLE) contra esporulação de oocistos e infecção de camundongos com Eimeria papillata. A análise fitoquímica do MNLE mostrou a presença de sete compostos e os efeitos in vitro do MNLE, amprolium, DettolTM, formalina, etanol e fenol foram estudados após incubação com oocistos antes da esporulação. Além disso, a infecção de camundongos com E. papillata induziu uma produção de oocistos de aproximadamente 12 × 105 oocistos / g de fezes. O MNLE reduziu significativamente a produção de oocistos para aproximadamente 86%, e o número total de estágios parasitários no jejuno em aproximadamente 87%. Além disso, a redução no número de células caliciformes no jejuno de camundongos aumentou após o tratamento. Esses achados sugerem que a amoreira exibia uma poderosa atividade anticoccidiana.

Isolation and characterization of Pyricularia oryzae isolated from lowland rice in Sarawak, Malaysian Borneo

@#Aims: Rice blast disease caused by Pyricularia oryzae is one of the major biotic diseases of rice in Sarawak, Malaysian Borneo. This study aims to isolate and characterize rice blast fungus obtained from infected leaf collected from four different divisions in Sarawak, viz, Miri, Serian, Sri Aman, and Kuching. Methodology and results: Twelve succeeded isolates were pre-identified as P. oryzae by morphological characteristics of spores, followed by verification through (internal transcribed spacer) ITS sequencing. The isolates were evaluated for morphological characteristics, growth rate and sporulation rate, which were grown on two types of media, (filtered oatmeal agar) FOMA and (potato dextrose agar) PDA. Morphological characterization showed that the colony surface of the different isolates varied from smooth and fluffy to rough and flattened mycelia; some were with the present of concentric rings, and some with aerial mycelia. The growth rate and sporulation rate of each isolate varied based on types of media used. Most of the isolates grew faster on PDA than on FOMA but produced higher number of spores on FOMA as compared to PDA. Conclusion, significance and impact of study: This preliminary study showed that there were variations observed based on morphological and physiological characterization for the different isolates collected in Sarawak, Malaysian Borneo. This study is the first step towards understanding variation in the population of P. oryzae from Sarawak.

Evaluation of Antifungal Activities of Five Plant Extracts against Pseudoperenospora cubensis (Downy Mildew) in Muskmelon (Cucumis melo L.)

Laboratory study was conducted to evaluate the effect of leaf extracts of five indigenous plant on conidia germination, growth and sporulation of Pseudoperenospora cubensis causing downy mildew disease of muskmelon. Extracts of five plant; mexican sunflower (Tithonia diversifolia), bush banana (Uvaria chamae), salt and oil tree (Cleistopholis patens), goat weed (Ageratum conyzoides) and African eggplant (Solanum macrocarpon) at Four concentrations (15, 30, 45 and 60%) were tested against the growth, conidial germination and sporulation of Pseudoperenospora cubensis in vitro. Results show that all the plant extracts significantly inhibited conidia germination and radial growth compared to the control. The extracts had no significant (p≤0.05) effect on sporulation. The rate of inhibition of growth and conidia germination was concentration dependent being highest at 60% for the extracts. The extracts of Solanum macrocarpon was the most effective followed by Ageratum conyzoides, Cleistopholis patens and Uvaria chamea whileTithonia diversifolia caused the least inhibition of growth and conidia germination. At 15, 30, 45 and 60% concentrations growth of Pseudoperenospora cubensis on PDA modified with Solanum macrocqrponwere 3.79, 3.65, 3.33 and 2.87; and 4.25, 4.12, 3.92 and 3.89 for PDA modified with Tithonia diversifolia. Similarly, conidia germination percentages recorded at same concentration of extracts S. macrocarpon were 87, 85, 70 and 62% while that of T. diversifolia were 91, 87, 84 and 72%. The study shows that the plant extracts has the potential for inhibition of the pathogen.

Desenvolvimento tecnológico do Bacillus coagulans BVB5 como potencial cepa probiótica / Technological development of Bacillus coagulans BVB5 as potential probiotic strains

Introdução: O aumento da demanda por alimentos funcionais, os quais incluem os suplementados ou fermentados por microrganismos probióticos, resultou no avanço da pesquisa e desenvolvimento de novas cepas potencialmente probióticas. Os microrganismos probióticos pertencentes ao gênero Bacillus spp. são atraentes devido a sua estabilidade inerente de bactérias formadoras de esporos. Os esporos permitem uma vida de prateleira prolongada e aumentam a capacidade do microrganismo de sobreviver às barreiras gástricas, que se revelam uma vantagem sobre os lactobacilos. Objetivo: Foram realizados experimentos para o desenvolvimento tecnológico de duas cepas formadoras de esporos de Bacillus coagulans BVB1 e BVB5 como potenciais microrganismos probióticos objetivando avaliar o potencial probiótico dos mesmos. Método: Como primeira etapa, as caracterizações fenotípica e genotípica identificaram que a cepa BVB1 não era B. coagulans e sim B. subtilis. Os estudos seguiram com a cinética da fermentação/esporulação apenas da cepa de Bacillus coagulans BVB5. Conclusão: O desafio da esporulação de Bacillus coagulans BVB5 foi vencido, fato que pode ser verificado na cinética da fermentação que apresentou resultados superiores a 99% de grau de esporulação

Introduction: The increased demand for functional foods, which include those supplemented or fermented by probiotic microorganisms, resulted in the advancement of research and development of new potentially probiotic strains. Probiotic microorganisms Bacillus spp. Are attractive because of their inherent stability of spore forming bacteria. Spores allow prolonged shelf life and increase the ability to survive gastric barriers, which prove to be an advantage over lactobacilli. Objective: Experiments were performed for the technological development of Bacillus coagulans BVB1 and BVB5 as potential probiotic microorganisms with two strains of Bacillus coagulans aiming to evaluate the efficacy of probiotic product composed of spore forming microorganism (Bacillus coagulans strains BVB1 and BVB5). Method: As a first step, phenotypic and genotypic characterization identified that the BVB1 strain was not B. coagulans but B. subtilis. Although Bacillus subtilis strain BVB1 presented a technological potential to be used as a probiotic strain in food additives, the studies followed with the kinetics of fermentation/sporulation of Bacillus coagulans BVB5, in agreement with the master's project originally proposed. Conclusion: The challenge of sporulation of Bacillus coagulans BVB5 was overcome, a fact that can be verified with the results of fermentation kinetic which presented sporulation degree more than 99%

Culture medium and inoculation methodology for the study of soft root rot caused by Phytopythium sp / Meio de cultura e metodologia de inoculação para o estudo da podridão mole de raízes causada por Phytopythium sp

ABSTRACT: Cassava (Manihot esculenta Crantz) is an important crop in Brazil and Pará is the major producer of roots. High temperature and humidity of tropical regions favor the development of various diseases, among them the cassava root rot. The objective of this study was to evaluate the effect of luminosity and culture medium on the mycelial growth and sporulation of Phytopythium sp. associated with different methods of inoculation on cassava roots. In vitro tests for pathogen growth were established in a 2 x 6 factorial design (luminosity x culture medium) with five replicates and the means were compared by t test (P≤0.05). The culture medium containing sweet cassava root produced greater mycelial development and higher pathogen sporulation and it was the most suitable medium for pathogen culture. The culture under absence of light generated better mycelial growth than culture under 12 hour of light. Regarding the type of inoculation, the response was better when deeper injuries were induced.

RESUMO: A mandioca (Manihot esculenta Crantz) é uma importante cultura para o Brasil, onde o Pará é o principal produtor de raízes. Regiões tropicais com alta umidade e temperatura favorecem o desenvolvimento de diversas doenças, como as podridões de raiz. O presente estudo tem como objetivo avaliar o efeito da luminosidade e de meios de cultura no crescimento micelial e na esporulação de Phytopythium sp. e analisar métodos de inoculação do patógeno em raízes de mandioca destacadas. Os ensaios in vitro foram instalados em esquema fatorial 2x6 (luminosidade x meio de cultura), com cinco repetições e as médias comparadas pelo teste t (p≤0,05). O meio de cultura contendo raiz de mandioca mansa proporcionou maior desenvolvimento micelial e maior esporulação do patógeno e é o mais adequado para o cultivo do patógeno. O cultivo sob ausência de luz gerou melhor crescimento micelial do que o cultivo sob 12 horas de luz. Quanto ao tipo de inoculação, a resposta foi melhor nas raízes que obtiveram ferimentos mais profundos.

Mutation studies of the gene encoding YuiC, a stationary phase survival protein in Bacillus subtilis

Aims@#YuiC is a stationary phase survival (Sps) protein from the Firmicute Bacillus subtilis that possesses muralytic activity to cleave bacterial cell-wall peptidoglycan. It has a small lytic transglycosylase (MltA) fold analogous to the resuscitation promoting factors (Rpfs) of Actinobacteria which have a hybrid of a mini lysozyme and soluble lytic transglycosylase (Slt35/70) fold. The present study aimed at identifying key residues of YuiC/Sps that are catalytically active and studying the effect of B. subtilis cell growth upon sps/yuiC deletion. @*Methodology and results@#Four forms of mutated yuiC were created through Site-directed, Ligase-Independent Mutagenesis Polymerase Chain Reaction (SLIM PCR) that include the substitutions of D129A, D151A, D162A and K102A. These individual mutated yuiC genes were cloned and expressed in the Escherichia coli BL21 (DE3) expression system and subsequently purified to homogeneity using affinity, cation exchange and size exclusion chromatography. The D129A variant was shown to be insoluble, indicating its role in maintaining the right protein folding of YuiC. The remaining three variants resulted in soluble proteins but were inactive on zymograms indicating that they may be responsible for catalysis. B. subtilis cells harbouring individual sps genes (yuiC, yabE, yocH and yorM) knocked out showed stationary phase defects and altered colony morphologies compared to the wild type. @*Conclusion, significance and impact of study@#This study has identified the key residues involved in catalysis of YuiC, which are the D151, D162 and K102. These are conserved in Sps domains. The catalytic mechanism of YuiC is similar to the mechanism reported for Neisseria gonorrhoeae MltA. sps/yuiC knock outs have implied that each sps/yuiC has a significant role on B. subtilis late growth stage. The B. subtilis YuiC/Sps model has given an insight into Sps functions in the final growth stage of the Firmicutes, which members include etiologic agents of anthrax, botulism and listeriosis. Inhibition of Sps protein may inactivate pathogen replication and facilitate entrance into a non-contagious dormant sporulation stage.

Evaluation of the gelatin technique for the preservation of phytopathogenic fungi / Avaliação da técnica da gelatina para preservação de fungos fitopatogênicos

The preservation methods for fungi have great importance in ex situ collections, representing important biological heritage, useful for mycologists and plant pathologists in several scientific works. However, there is a lack of studies for a suiTable and efficient preservation method for the different groups of fungi. Although, the most appropriate is the one that maintain, even after long periods, the original characteristics of culture: viability, sporulation and pathogenicity, excluding mutations and undesirable contamination. The choice will depend of the laboratory infrastructure, microorganism, objectives, preferences and knowledge of the researcher. We conducted this study inside the Laboratory of Mycology and Plant Protection (LAMIP) in UFU (Universidade Federal de Uberlândia), localized in Uberlândia (MG), Brazil. The objective was to evaluate the gelatin preservation method (17 cultures), never used before for phytopathogenic fungi. Other classical methods were concomitantly evaluated, such as sterile soil (68 cultures), resistant structures (Sclerotinia sclerotiorum) in 4°C (10 strains) and mineral oil (31 cultures). We examined the time for maintaining the viability, sporulation and colonization in host tissues preserved in different dates. The gelatin method remained viability in 10 cultures; this method is suiTable for preservation of the genera and species: Colletotrichum spp., Septoria spp., Fusarium spp., F. moniliforme var. subglutinans, Macrophomina spp., Phomopsis spp. and Verticillium spp. The viability remained in 38 strains of sterile soil, three of mineral oil, and one strain of sclerotia reached a maximum preservation time in 4°C of four years.

Os métodos de preservação de fungos têm grande importância em coleções ex situ, representando importante patrimônio biológico, útil para micologistas e fitopatologista como suporte para vários trabalhos científicos. Não existe um método de preservação adequado e eficiente para os diferentes grupos de fungos, entretanto, o mais apropriado é o que mantém, mesmo após longos períodos, as características originais da cultura: viabilidade, esporulação e patogenicidade, excluindo mutações e contaminação indesejável. A escolha dependerá da infra-estrutura laboratorial, microorganismo, objetivos, preferências e conhecimentos do pesquisador. Este trabalho foi realizado no Laboratório de Micologia e Proteção Vegetal (LAMIP) da UFU (Universidade Federal de Uberlândia), localizado em Uberlândia (MG), Brasil. O objetivo foi avaliar o método de preservação da gelatina (17 culturas), nunca testado antes para fungos fitopatogêncios. Concomitantemente foram avaliados métodos clássicos, como solo estéril (68 culturas), escleródios (Sclerotinia sclerotiorum) em 4 ° C (10 isolados) e óleo mineral (31 culturas). Avaliou-se a manutenção na viabilidade, esporulação e patogenicidade dos isolados. O método da gelatina manteve a viabilidade em 10 culturas, sendo adequado para a preservação dos gêneros e espécies: Colletotrichum spp., Septoria spp., Fusarium spp., F. moniliforme var. subglutinans, Macrophomina spp., Phomopsis spp. e Verticillium spp. A viabilidade foi mantida em 38 isolados em solo estéril, três isolados em óleo mineral e um apenas um isolado de escleródio atingiu um tempo máximo de preservação de quatro anos.

The role of spo0A gene in growth and sporulation of Clostridium difficile / 中国感染与化疗杂志

Objective To investigate the role of spo0A gene in growth and sporulation of Clostridium difficile clinical isolates. Methods ClosTron gene knock-out system was used to knock out the spo0A gene of C. difficile strain C25. Bacterial growth curve was plotted by measuring D600 with spectrophotometer in different phases of bacterial growth. Malachite green staining technique was used to count the number of vegetative cells and spores under optical microscope. The sporulation rate was calculated. Results The spo0A mutant and its C25 parental strain showed similar patterns of growth. However, after knock-out of spo0A gene, an asporogenous phenotype was built, while the parental strain could produce spores as usual.Conclusions The spo0A gene plays a key role in sporulation but not growth of C. difficile strain.

In-vitro anticoccidial potential of Saccharum officinarum extract against Eimeria oocysts / Potencial in vitro anti coccidial de un extracto de Saccharum officinarum contra oocistos de Eimeria

Present study was conducted to evaluate the effect of aqueous methanolic extract from Saccharum officinarum on the sporulation and morphology of oocysts of four Eimeria species (Eimeria tenella, E. necatrix, E. mitis, E. brunetti) of poultry. Sporulation inhibition bioassay was used to evaluate the activity of Saccharum officinarum extract (SOE) on the sporulation of coccidian oocysts. In this assay, unsporulated oocysts were exposed to six concentrations of S. officinarum in 10 percent dimethyl sulfoxide solution (w/v; 10, 5, 2.5, 1.25, 0.625 and 0.31 percent) while DMSO and potassium dichromate solution (K2Cr2O7) served as control groups. The Petri dishes were partially covered to allow the passage of oxygen and incubated at 25-29° C for 48 h, providing 60-80 percent humidity. The sporulation of the oocyst was confirmed by examining sporocysts under inverted microscope at 40x. Results showed anticoccidial activity of SOE against all Eimeria species as proved by its ability to inhibit the sporulation of the oocysts under laboratory conditions. Inhibition of sporulation was observed in dose dependent manner. S. officinarum extract at higher dose also damaged the normal morphology and shape of oocysts of Eimeria species.

El presente estudio se llevó a cabo para evaluar el efecto del extracto metanólico acuoso a partir de Saccharum officinarum en la esporulación de los ooquistes y la morfología de cuatro especies de Eimeria tenella (Eimeria, E. necatrix, E. mitis, E. brunetti) de aves de corral. Bioensayos de la inhibición de la esporulación se utilizaron para evaluar la actividad de extracto de Saccharum officinarum (SOE) en la esporulación de ooquistes de coccidios. En este ensayo, los ooquistes no esporulados se expusieron a seis concentraciones de S. officinarum en solución de dimetil sulfóxido 10 por ciento (w / v; 10, 5, 2,5, 1,25, 0,625 y 0,31 por ciento), mientras DMSO y una solución de dicromato de potasio (K2Cr2O7) sirvió como grupos de control. Las placas de Petri se cubren parcialmente para permitir el paso de oxígeno y se incubaron a 25-29° C durante 48 h, proporcionando el 60-80 por ciento de humedad. La esporulación de los ooquistes fue confirmado mediante el examen de esporoquistes bajo microscopio invertido a 40x. Los resultados mostraron actividad anticoccidial de SOE contra todas las especies de Eimeria como se ha demostrado por su capacidad para inhibir la esporulación de los ooquistes en condiciones de laboratorio. Se observó una inhibición de la esporulación de manera dependiente de la dosis. Extracto de S. officinarum en dosis más alta también dañó la morfología normal y la forma de ooquistes de las especies de Eimeria.

Morphological changes and growth of filamentous fungi in the presence of high concentrations of PAHs

In this study, we evaluated the effect of low and high molecular weight polycyclic aromatic hydrocarbons (PAHs), i.e., Phenanthrene, Pyrene and Benzo[a]pyrene, on the radial growth and morphology of the PAH-degrading fungal strains Aspergillus nomius H7 and Trichoderma asperellum H15. The presence of PAHs in solid medium produced significant detrimental effects on the radial growth of A. nomius H7 at 4,000 and 6,000 mg L⁻¹ and changes in mycelium pigmentation, abundance and sporulation ability at 1,000–6,000 mg L⁻¹. In contrast, the radial growth of T. asperellum H15 was not affected at any of the doses tested, although sporulation was observed only up to 4,000 mg L⁻¹ and as with the H7 strain, some visible changes in sporulation patterns and mycelium pigmentation were observed. Our results suggest that fungal strains exposed to high doses of PAHs significantly vary in their growth rates and sporulation characteristics in response to the physiological and defense mechanisms that affect both pigment production and conidiation processes. This finding is relevant for obtaining a better understanding of fungal adaptation in PAH-polluted environments and for developing and implementing adequate strategies for the remediation of contaminated soils.

The Effect of Media and Light on In Vitro Sporulation of Alternaria Solani.

Early blight of tomato incited by Alternaria solani is an economically significant disease especially in commercial tomato cultivation under greenhouse and field conditions. Since, A. solani is a shy sporulator, the present investigation was taken to assess the optimum in vitro conditons for growth and sporulation of early blight pathogen. Ten A. solani isolates obtained from diseased leaf samples collected different crop growing areas of India were used in the present study. The effect of different incubation periods, fluorescent light, cold-water treatment and media were evaluated. Our results revealed maximum sporulation of A. solani on tomato fruit extract agar medium (TFEA) under continuous light for 7 days at 250C, followed by cold-water treatment and further incubated in darkness at 200C for 48 h. The sporulation of test pathogen was however sparse on V-8 juice agar. Further, the A. solani isolates on V-8 juice agar did not respond to the treatments imposed. Among the isolates, JAS (Jhajjar) isolate, that is more versatile in its ability to produce spores recorded irregular margin with abundant aerial mycelium.

Variación temporal a Microcyclus ulei en los clones de caucho FX 3864 y FX 4098 en condiciones controladas / Temporal variation to Microcyclus ulei in rubber tree clones FX 3864 and FX 4098 under controlled conditions

Este estudio tuvo como objetivo evaluar la variación temporal de los principales componentes fisiológicos asociados a la reacción de dos clones (FX 3864 y FX 4098) de Hevea brasiliensis a Microcyclus ulei en condiciones controladas. Las inoculaciones se realizaron en cámara húmeda a 23 ºC, una humedad relativa superior al 90% y un fotoperíodo de 12 h. Con un modelo de medidas repetidas en el tiempo y mediante correlaciones se analizaron cuatro variables: severidad de ataque (SA), intensidad de esporulación conidial (IE), tamaño de lesión (TL) y frecuencia de infección (FI). M. ulei generó síntomas en los dos clones a los 4 días después de la inoculación. Los signos de la enfermedad (IE= 3) se manifestaron a los 8 días en el clon FX 3864 y a los 12 días en el clon FX 098. FX 3864 presentó la mayor susceptibilidad a M. ulei (mayor severidad y una esporulación alta, IE= 4). En general, se observó una influencia del tiempo en la reacción de cada clon para las variables SA, IE y TL. Altas correlaciones significativas se encontraron entre SA y las variables IE, TL y FI, excepto en el día 12 para IE. Este estudio permite concluir que en condiciones controladas la intensidad y la frecuencia de los síntomas así como el grado de esporulación producidos por M. ulei cambian significativamente a través del tiempo y esta variación está influenciada a su vez por el nivel de susceptibilidad a M. ulei presentado por cada clon de H. brasiliensis.

This study aimed to evaluate the effect of temporal variation on the main physiological components associated with the reaction to Microcyclus ulei of two rubber tree clones of Hevea brasiliensis under controlled conditions. Inoculations were performed in a moist chamber at 23 °C, a relative humidity higher than 90% and a photoperiod of 12 h. The variables severity of attack (SA), conidial sporulation intensity (IE), lesion size (TL) and infection frequency (IF) were analyzed with a repeated over time measurements model and also with correlations. M. ulei produced symptoms (SA, TL, FI) in both rubber tree clones 4 days after inoculation. Signs of the disease (IE = 3) appeared in the FX 3864 at day 8th and in the FX 4098 at day 12th. FX 3864 presented higher susceptibility to M. ulei (greater severity and high sporulation, with IE = 4). In general, the time influenced the reaction of each clone, especially in the variables SA, IE and TL. High significant correlations were found between SA and the variables IE, TL and FI, except on day 12 for IE. It was concluded that intensity and frequency of symptoms and the degree of sporulation produced by M. ulei change significantly over time under controlled conditions and this variation is influenced also by the level of susceptibility to M. ulei showed by each clone of H. brasiliensis.

Suppression of Eimeria tenella Sporulation by Disinfectants

The disinfectant effects (DEs) of 10 types of chemicals, defined by their ability to destroy or inhibit oocysts and consequently prevent sporulation of Eimeria tenella field isolate, were evaluated in vitro. Correct species assignments and sample purities were confirmed by the singular internal transcribed spacer (ITS)-PCR analysis. A total of 18 treatments were performed, and the disinfection suppression levels were 75.9% for 39% benzene + 22% xylene (1:10 dilution), 85.5% for 30% cresol soup (1:1 dilution), and 91.7% for 99.9% acetic acid (1:2 dilution) group. The results indicate that acetic acid, cresol soup, and benzene+xylene are good candidates for suppression of E. tenella oocyst sporulation.

Formación de endosporas en Clostridium y su interacción con el proceso de solventogénesis / Endospore formation in Clostridium and its interaction with solventogenesis

La solventogénesis y la esporulación son mecanismos de las células de Clostridium para resistir ambientes hostiles. Este segundo proceso ha sido estudiado utilizando como modelo lo que sucede con Bacillus, aunque se reconocen diferencias marcadas entre los dos géneros especialmente en el inicio, específicamente en los eventos por medio de los que se da la fosforilación del regulador maestro Spo0A. En la actualidad se ha avalado la teoría que afirma que tres histidin quinasas huérfanas, diferentes a las proteínas Spo0B (histidin quinasa) y Spo0F(fosfotransferasa) en Bacillus, son las encargadas de fosforilar en Clostridium de forma directa a Spo0A, que posteriormente activa la transcripción de diferentes factores sigma relacionados, de forma similar en los dos géneros bacterianos. La proteína Spo0A, perteneciente a la familia de reguladores de respuesta, se comporta como una entidad regulatoria global que tiene injerencia sobre los procesos de formación de esporas y solventes, modulando genes necesarios para que se produzcan acetona y butanol, además de genes de esporulación. El entendimiento de este proceso ha llevado a los investigadores a emplear diferentes técnicas moleculares que permitan incrementar la producción de solventes, así como eliminar la propiedad de las células de producir endosporas. Por tal razón, este escrito presenta un resumen de estas dos redes de expresión de genes, conectadas por el regulador maestro Spo0A.

Solventogenesis and sporulation are mechanisms used by Clostridium cells to resist hostile environments. Sporulation has been studied using as a model what happens with Bacillus, but marked differences were recognized, particularly in the events that led the phosphorylation of the master controller Spo0A. Currently, a theory that claims that three orphan histidine kinases, different from Spo0B (histidin kinase) and spo0F (phosphotransferase) proteins in Bacillus, phosphorilate directly Spo0A in Clostridium activating transcription of different sigma factors which are similar in the two bacterial genera, has been supported. Spo0A protein, which belongs to the family of response regulators, behaves as an entity that has global regulatory interference on the processes of spore formation and solvents, modulating genes necessary to produce acetone and butanol. The understanding of this process has led researchers to employ different molecular techniques that increase the production of solvents, and remove the property of the cells to produce endospores. Reason why, this paper presents an updated summary of these two gene expression networks, connected by the master regulator spo0A.

Concomitant production of two proteases and alpha-amylase by a novel strain of Bacillus subtilis in a microprocessor controlled bioreactor

We describe the simultaneous production of Bacillus subtilis based proteases and alpha amylase using a computer controlled laboratory scale 7.5 L batch bioreactor. The present strain is the first to be reported that concomitantly produces these two industrially important enzymes. The growth and sporulation of Bacillus subtilis was monitored and maximum production of alkaline protease and alpha amylase was found to coincide with maximum sporulation. Two types of proteases were detected in the fermentation broth; a neutral and an alkaline protease most active in a pH range of 7.0-8.0 and 8.0-10, respectively. Maximum production of proteases was observed at an incubation temperature of 37ºC while that of alpha amylase was observed at 40ºC. The optimum aeration and agitation levels for protease production were 0.6 L/L/min and 200rpm, respectively, and for alpha amylase were 0.6 L/L/min and 150 rpm. The kinetic parameters Yp/x and qp were also found to be significant at the given fermentation conditions.

A novel method to optimize culture conditions for biomass and sporulation of the entomopathogenic fungus Beauveria Bassiana IBC1201

Biomass yields and sporulation of Beauveria bassiana was concerned on culture conditions, environmental factors and cultivation method. We optimized the best culture conditions for biomass yields of B. bassiana IBC1201 with the novel "two-stage" cultivation method as well as orthogonal matrix method. Firstly, we cultured spore suspension on the basal medium (sucrose 19.00 g, soy peptone 4.06 g, K2HPO4 1.00 g, KCl 0.50 g, MgSO4 0.50 g, FeSO4 0.10 g and 17.00 g Bactor) for the first stage culture of 4 days under room condition. Then, we transferred them to another defined medium (Cellobiose 9.52 g, urea 1.70 g, ZnSO4•7H2O 0.05 g/L, MnSO4•H2O 0.005 g/L, CaCl2 1.00 g/L, CuSO4•5H2O 0.05 g/L and 17.00 g Bactor) for more 4 days cultivation with the environmental factors combination of water potential -1.2 MPa /pH 3 /12 h light cycle/ 23 ℃ for biomass yields, and with the environmental factors combination of water potential -0.8 MPa /pH 3 /24 h light cycle/ 23 ℃ for spore yields. These results provided important information for mass production (including biomass and spore yields) of this great potential biocontrol fungus.