Cloning, expression and antifungal effect of the recombinant chitinase from Streptomyces sampsonii KJ4 / Clonagem, expressão e efeito antifúngico da quitinase recombinante de Streptomyces sampsonii KJ40

Streptomyces sampsonii is a kind of biocontrol bacterium with antifungal effects, and chitinase is one of the main antifungal substances. To improve and further study the structure and function of the chitinase gene of S. sampsonii, we amplified the target fragment by PCR, ligated the fragment to the expression vector pET-32a, introduced the resulting plasmid into Escherichia coli BL21 (DE3) and induced expression of the chitinase. Then, the recombinant chitinase was purified by is-labelled protein micro purification kit. A chitinase gene, Sschi61, was cloned from the genome and expressed in a prokaryote. The antifungal effect of the recombinant protein was also studied. Finally, the chitinase gene Sschi61 with a length of 1755 bp was obtained, and the expression of the 82 kDa recombinant chitinase was induced in E. coli by IPTG. The recombinant chitinase could inhibit the black spot pathogen of Eucommia ulmoides (Pestalotiopsis trachicarpicola). After the hyphae of the pathogen of black spot of Eucommia ulmoides (Pestalotiopsis trachicarpicola) were soaked with recombinant chitinase, the hyphae cells expanded, broke, and dissolved.

Streptomyces sampsonii é uma espécie de bactéria de biocontrole com efeitos antifúngicos, sendo a quitinase uma das principais substâncias desse tipo. Para melhorar e estudar mais a estrutura e função do gene da quitinase de S. sampsonii, amplificamos o fragmento alvo por PCR, ligamos o fragmento ao vetor de expressão pET-32a, introduzimos o plasmídeo resultante em Escherichia coli BL21 (DE3) e induzimos expressão da quitinase. Em seguida, a quitinase recombinante foi purificada pelo kit de micropurificação de proteína marcada. Um gene da quitinase, Sschi61, foi clonado do genoma e expresso em um procarioto. O efeito antifúngico da proteína recombinante também foi estudado. Finalmente, o gene da quitinase Sschi61 foi obtido, contando comprimento de 1755 pb, e a expressão da quitinase recombinante de 82 kDa foi induzida em E. coli por IPTG. A quitinase recombinante pode inibir o patógeno da mancha preta de Eucommia ulmoides (Pestalotiopsis trachicarpicola). Após as hifas do patógeno da mancha preta de Eucommia ulmoides (Pestalotiopsis trachicarpicola) serem embebidas com quitinase recombinante, as células das hifas se expandiram, quebraram e se dissolveram.

Cloning, expression and antifungal effect of the recombinant chitinase from Streptomyces sampsonii KJ40 / Clonagem, expressão e efeito antifúngico da quitinase recombinante de Streptomyces sampsonii KJ40

ABSTRACT: Streptomyces sampsonii is a kind of biocontrol bacterium with antifungal effects, and chitinase is one of the main antifungal substances. To improve and further study the structure and function of the chitinase gene of S. sampsonii, we amplified the target fragment by PCR, ligated the fragment to the expression vector pET-32a, introduced the resulting plasmid into Escherichia coli BL21 (DE3) and induced expression of the chitinase. Then, the recombinant chitinase was purified by is-labelled protein micro purification kit. A chitinase gene, Sschi61, was cloned from the genome and expressed in a prokaryote. The antifungal effect of the recombinant protein was also studied. Finally, the chitinase gene Sschi61 with a length of 1755 bp was obtained, and the expression of the 82 kDa recombinant chitinase was induced in E. coli by IPTG. The recombinant chitinase could inhibit the black spot pathogen of Eucommia ulmoides (Pestalotiopsis trachicarpicola). After the hyphae of the pathogen of black spot of Eucommia ulmoides (Pestalotiopsis trachicarpicola) were soaked with recombinant chitinase, the hyphae cells expanded, broke, and dissolved.

RESUMO: Streptomyces sampsonii é uma espécie de bactéria de biocontrole com efeitos antifúngicos, sendo a quitinase uma das principais substâncias desse tipo. Para melhorar e estudar mais a estrutura e função do gene da quitinase de S. sampsonii, amplificamos o fragmento alvo por PCR, ligamos o fragmento ao vetor de expressão pET-32a, introduzimos o plasmídeo resultante em Escherichia coli BL21 (DE3) e induzimos expressão da quitinase. Em seguida, a quitinase recombinante foi purificada pelo kit de micropurificação de proteína marcada. Um gene da quitinase, Sschi61, foi clonado do genoma e expresso em um procarioto. O efeito antifúngico da proteína recombinante também foi estudado. Finalmente, o gene da quitinase Sschi61 foi obtido, contando comprimento de 1755 pb, e a expressão da quitinase recombinante de 82 kDa foi induzida em E. coli por IPTG. A quitinase recombinante pode inibir o patógeno da mancha preta de Eucommia ulmoides (Pestalotiopsis trachicarpicola). Após as hifas do patógeno da mancha preta de Eucommia ulmoides (Pestalotiopsis trachicarpicola) serem embebidas com quitinase recombinante, as células das hifas se expandiram, quebraram e se dissolveram.

Thermo-resistant enzyme-producing microorganisms isolated from composting / Microrganismos produtores de enzimas termo-resistentes isolados de compostagem

Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Os fertilizantes organo-minerais suplementados com aditivos biológicos são uma alternativa aos adubos químicos. Neste estudo, microrganismos termoresistentes foram isolados de compostagem por procedimentos de duas etapas. Inicialmente, as amostras tomadas em diferentes períodos e temperaturas (33 dias a 52 ºC, 60 dias a 63 ºC e mais de 365 dias a 26 ºC) foram pré-incubadas a 80 oC por 30 minutos. Posteriormente, a seleção microbiana foi conduzida por métodos baseados em cultura in vitro e choque térmico a 60 oC e 100 oC por 2h e 4h. Quarenta e um isolados foram capazes de crescer a 60 °C por 4h; vinte e sete a 100 °C por 2h e dois a 100 °C por 4h. A identificação molecular por sequenciamento parcial do gene ribossomal 16S usando primers universais revelou que trinta e cinco isolados eram de oito espécies de Bacillus, um Brevibacillus borstelensis, três Streptomyces thermogriseus e dois fungos (Thermomyces lanuginosus e T. dupontii). Os dados dos ensaios de atividade de amilase, fitase e celulase e o índice enzimático (IE) mostraram que 38 dos 41 isolados termorresistentes produziram pelo menos uma enzima. Para a atividade da amilase, o maior valor de IE foi observado em Bacillus licheniformis (isolado 21C2, IE = 4,11), seguido por Brevibacillus borstelensis (isolado 6C2, IE = 3,66), Bacillus cereus (isolado 18C2, IE = 3,52) e Bacillus paralicheniformis (isolado 20C2, IE = 3,34). Para a fitase, os maiores valores de IE foram observados para B. cereus (isolado 18C2, IE = 2,30) e B. licheniformis (isolado 3C1, IE = 2,15). Em relação à produção de celulose, B. altitudinis (isolado 6C1) foi o mais eficiente (IE = 6,40), seguido por três Bacillus subtilis (isolados 9C1, 16C2 e 19C2) com valores de IE de 5,66, 5,84 e 5,88, respectivamente, e um B. pumilus (isolado 27C2, IE = 5,78). Pode-se inferir que os microrganismos selecionados são potencialmente úteis como aditivos biológicos em fertilizantes organo-minerais e outros processos biotecnológicos.

Thermo-resistant enzyme-producing microorganisms isolated from composting / Microrganismos produtores de enzimas termo-resistentes isolados de compostagem

Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.(AU)

Os fertilizantes organo-minerais suplementados com aditivos biológicos são uma alternativa aos adubos químicos. Neste estudo, microrganismos termoresistentes foram isolados de compostagem por procedimentos de duas etapas. Inicialmente, as amostras tomadas em diferentes períodos e temperaturas (33 dias a 52 ºC, 60 dias a 63 ºC e mais de 365 dias a 26 ºC) foram pré-incubadas a 80 oC por 30 minutos. Posteriormente, a seleção microbiana foi conduzida por métodos baseados em cultura in vitro e choque térmico a 60 oC e 100 oC por 2h e 4h. Quarenta e um isolados foram capazes de crescer a 60 °C por 4h; vinte e sete a 100 °C por 2h e dois a 100 °C por 4h. A identificação molecular por sequenciamento parcial do gene ribossomal 16S usando primers universais revelou que trinta e cinco isolados eram de oito espécies de Bacillus, um Brevibacillus borstelensis, três Streptomyces thermogriseus e dois fungos (Thermomyces lanuginosus e T. dupontii). Os dados dos ensaios de atividade de amilase, fitase e celulase e o índice enzimático (IE) mostraram que 38 dos 41 isolados termorresistentes produziram pelo menos uma enzima. Para a atividade da amilase, o maior valor de IE foi observado em Bacillus licheniformis (isolado 21C2, IE = 4,11), seguido por Brevibacillus borstelensis (isolado 6C2, IE = 3,66), Bacillus cereus (isolado 18C2, IE = 3,52) e Bacillus paralicheniformis (isolado 20C2, IE = 3,34). Para a fitase, os maiores valores de IE foram observados para B. cereus (isolado 18C2, IE = 2,30) e B. licheniformis (isolado 3C1, IE = 2,15). Em relação à produção de celulose, B. altitudinis (isolado 6C1) foi o mais eficiente (IE = 6,40), seguido por três Bacillus subtilis (isolados 9C1, 16C2 e 19C2) com valores de IE de 5,66, 5,84 e 5,88, respectivamente, e um B. pumilus (isolado 27C2, IE = 5,78). Pode-se inferir que os microrganismos selecionados são potencialmente úteis como aditivos biológicos em fertilizantes organo-minerais e outros processos biotecnológicos.(AU)

Biocontrol potential of actinobacteria against Pantoea ananatis, the causal agent of maize white spot disease / Potencial de biocontrole de actinobactérias contra Pantoea ananatis, agente causal da doença mancha branca do milho

Pantoea ananatis is the causal agent of maize white spot, a foliar disease responsible for significant maize yield reduction worldwide, especially in Brazil. In general, the maize foliar diseases control involves the adoption of resistant genotypes and pesticides application. However, the use of agrochemicals can significantly cause increase production costs, damage to human health and negative environmental impacts. In this sense, the use of biological control agents has been considered among the most promising eco-friendly technologies for sustainable agriculture. Actinobacteria, particularly of Streptomyces genus, has been widely recognized as agroindustrially important microorganism due to its potential in producing diverse range of secondary metabolites, including antibiotics and enzymes. Thus, the aim of this work is to characterize and to evaluate the potential of soil actinobacteria for P. ananatis control. We observed that 59 actinobacteria strains (85%) exhibited proteolytic or chitinolytic activity. Only the strains Streptomyces pseudovenezuelae ACSL 470, that also exhibited high proteolytic activity, S. novaecaesareae ACSL 432 and S. laculatispora ACP 35 demonstrated high or moderate antagonist activity in vitro against P. ananatis. Temporal analysis of metabolites produced by these strains growth in different liquid media indicated greater antibacterial activity at 72 h. In this condition, chromatographic and mass spectrometry analysis revealed that S. pseudovenezuelae ACSL 470 strain produced neomycin, an aminoglycoside antibiotic that displayed high bactericidal activity in vitro against P. ananatis. This is the first report of actinobacteria acting as potential microbial antagonists for P. ananatis control. Further studies are needed to determine the control efficacy of maize white spot disease by Streptomyces strains or their metabolites in greenhouse and field conditions.

Pantoea ananatis é o agente causal da mancha branca do milho, doença foliar responsável pela redução significativa da produtividade do milho em todo o mundo, especialmente no Brasil. Em geral, o controle de doenças foliares do milho envolve a adoção de genótipos resistentes e a aplicação de agrotóxicos. No entanto, o uso de agroquímicos pode causar aumento significativo dos custos de produção, danos à saúde humana e impactos ambientais negativos. Nesse sentido, o uso de agentes de controle biológico tem sido considerado uma das tecnologias ecologicamente corretas mais promissoras para a agricultura sustentável. Actinobactérias, particularmente do gênero Streptomyces, têm sido amplamente reconhecidas como microrganismos de importância agroindustrial devido ao seu potencial de produção de diversos metabólitos secundários, incluindo antibióticos e enzimas. Assim, o objetivo deste trabalho é caracterizar e avaliar o potencial de actinobactérias do solo para o controle de P. ananatis. Observamos que 59 cepas de actinobactérias (85%) apresentaram atividade proteolítica ou quitinolítica. Apenas as cepas Streptomyces pseudovenezuelae ACSL 470, que também exibiu alta atividade proteolítica, S. novaecaesareae ACSL 432 e S. laculatispora ACP 35 demonstraram alta ou moderada atividade antagonista in vitro contra P. ananatis. A análise temporal do crescimento dos metabólitos produzidos por essas cepas em diferentes meios líquidos indicou maior atividade antibacteriana em 72 h. Nesta condição, análises cromatográficas e de espectrometria de massa revelaram que a cepa S. pseudovenezuelae ACSL 470 produziu neomicina, um antibiótico aminoglicosídeo que apresentou alta atividade bactericida in vitro contra P. ananatis. Este é o primeiro relato de actinobactérias atuando como potenciais antagonistas microbianos para o controle de P. ananatis. Mais estudos são necessários para determinar a eficácia do controle da doença da mancha branca do milho por cepas de Streptomyces ou seus metabólitos em condições de casa de vegetação e campo.

Thermo-resistant enzyme-producing microorganisms isolated from composting

Abstract Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Resumo Os fertilizantes organo-minerais suplementados com aditivos biológicos são uma alternativa aos adubos químicos. Neste estudo, microrganismos termoresistentes foram isolados de compostagem por procedimentos de duas etapas. Inicialmente, as amostras tomadas em diferentes períodos e temperaturas (33 dias a 52 ºC, 60 dias a 63 ºC e mais de 365 dias a 26 ºC) foram pré-incubadas a 80 oC por 30 minutos. Posteriormente, a seleção microbiana foi conduzida por métodos baseados em cultura in vitro e choque térmico a 60 oC e 100 oC por 2h e 4h. Quarenta e um isolados foram capazes de crescer a 60 °C por 4h; vinte e sete a 100 °C por 2h e dois a 100 °C por 4h. A identificação molecular por sequenciamento parcial do gene ribossomal 16S usando primers universais revelou que trinta e cinco isolados eram de oito espécies de Bacillus, um Brevibacillus borstelensis, três Streptomyces thermogriseus e dois fungos (Thermomyces lanuginosus e T. dupontii). Os dados dos ensaios de atividade de amilase, fitase e celulase e o índice enzimático (IE) mostraram que 38 dos 41 isolados termorresistentes produziram pelo menos uma enzima. Para a atividade da amilase, o maior valor de IE foi observado em Bacillus licheniformis (isolado 21C2, IE = 4,11), seguido por Brevibacillus borstelensis (isolado 6C2, IE = 3,66), Bacillus cereus (isolado 18C2, IE = 3,52) e Bacillus paralicheniformis (isolado 20C2, IE = 3,34). Para a fitase, os maiores valores de IE foram observados para B. cereus (isolado 18C2, IE = 2,30) e B. licheniformis (isolado 3C1, IE = 2,15). Em relação à produção de celulose, B. altitudinis (isolado 6C1) foi o mais eficiente (IE = 6,40), seguido por três Bacillus subtilis (isolados 9C1, 16C2 e 19C2) com valores de IE de 5,66, 5,84 e 5,88, respectivamente, e um B. pumilus (isolado 27C2, IE = 5,78). Pode-se inferir que os microrganismos selecionados são potencialmente úteis como aditivos biológicos em fertilizantes organo-minerais e outros processos biotecnológicos.

Metagenomic study of the communities of bacterial endophytes in the desert plant Senna Italica and their role in abiotic stress resistance in the plant / Estudo metagenômico das comunidades de endófitos bacterianos na planta do deserto Senna Italica e seu papel na resistência ao estresse abiótico na planta

Plant leaves and roots are home to diverse communities of bacteria, which play a significant role in plant health and growth. Although one of the most unfriendly environments for plant growth is deserts, desert plants can influence their surrounding microbial population and choose favorable bacteria that encourage their growth under these severe circumstances. Senna italica is known for its excellent medicinal values as a traditional medical plant, but little is known about its associated endophytic bacterial community under extreme conditions. In the present study, metagenomic sequencing of 16S rRNA was used to report the diversity of endophytic bacterial communities associated with the leaves and roots of the desert medicinal plant Senna italica that was collected from the Asfan region in northeast Jeddah, Saudi Arabia. Analyses of the 16S rRNA sequences at the taxonomic phylum level revealed that bacterial communities in the roots and leaves samples belonged to five phyla, including Cyanobacteria, Proteobacteria, Actinobacteria, Firmicutes, and unclassified phyla. Results indicated that the most common phyla were Cyanobacteria/Chloroplast and Actinobacteria. Analysis of the 16S rRNA sequences at the taxonomic phylum level revealed that bacterial communities in the roots and leaves samples belonged to twelve genera at the taxonomic genus level. The most abundant ones were highlighted for further analysis, including Okibacterium and Streptomyces found in Actinobacteria, which were the dominant genus in roots samples. However, Streptophyta found in Cyanobacteria/Chloroplast was the dominant genus in leaf samples. Metagenomic analysis of medicinal plants leads to identifying novel organisms or genes that may have a role in abiotic stress resistance in the plant. The study of endophytic microbiome taxonomic, phylogenetic, and functional diversity will better know innovative candidates that may be selected as biological agents to enhance agricultural and industrial processes, especially for crop desert agricultural improvement.

As folhas e raízes das plantas abrigam diversas comunidades de bactérias, que desempenham um papel significativo na saúde e no crescimento das plantas. Embora um dos ambientes mais hostis para o crescimento de plantas sejam os desertos, as plantas do deserto podem influenciar a população microbiana circundante e escolher bactérias favoráveis ​​que encorajem seu crescimento sob essas circunstâncias severas. Senna italica é conhecida por seus excelentes valores medicinais como planta medicinal tradicional, mas pouco se sabe sobre sua comunidade bacteriana endofítica associada em condições extremas. No presente estudo, o sequenciamento metagenômico de 16S rRNA foi usado para relatar a diversidade de comunidades bacterianas endofíticas associadas às folhas e raízes da planta medicinal do deserto Senna italica que foi coletada na região de Asfan no nordeste de Jeddah, Arábia Saudita. Análises das sequências de rRNA 16S no nível taxonômico do filo revelaram que as comunidades bacterianas nas amostras de raízes e folhas pertenciam a cinco filos, incluindo Cyanobacteria, Proteobacteria, Actinobacteria, Firmicutes e filos não classificados. Os resultados indicaram que os filos mais comuns foram Cyanobacteria/Cloroplast e Actinobacteria. A análise das sequências de rRNA 16S no nível taxonômico do filo revelou que as comunidades bacterianas nas amostras de raízes e folhas pertenciam a doze gêneros no nível taxonômico de gênero. Os mais abundantes foram destacados para análise posterior, incluindo Okibacterium e Streptomyces encontrados em Actinobacteria, que foram os gêneros dominantes nas amostras de raízes. No entanto, Streptophyta encontrado em Cyanobacteria/Chloroplast foi o gênero dominante nas amostras de folhas. A análise metagenômica de plantas medicinais leva à identificação de novos organismos ou genes que podem ter um papel na resistência ao estresse abiótico na planta. O estudo da diversidade taxonômica, filogenética e funcional do microbioma endofítico conhecerá melhor os candidatos inovadores que podem ser selecionados como agentes biológicos para melhorar os processos agrícolas e industriais, especialmente para o melhoramento agrícola do deserto.

Effect of pH soil and irrigation regimes on management of potato scab

Potato scab caused by different species of phytopathogenic Streptomyces is considered one of the main bacterial diseases of economic crop importance worldwide. Several studies are being carried out in order to control the disease, but until now, there is no efficient way to do this. Some management strategies have been investigated including application of chemical and biological products and utilization of resistant cultivars of potato but there are few reports about the impact of pH and irrigation regimes on the disease. The present study aimed to evaluate the effects of these last two factors on the incidence and severity of potato scab caused by S. scabiei, S. acidiscabies, Streptomyces sp., S. caviscabies and S. europaeiscabiei in assays at pH 4.0, 4.5, 5.0, 5.5, 6.5 and 7.5; and irrigation regimes of once a week, alternate days and daily in greenhouse conditions. The experimental design for the pH tests was randomized blocks arranged in a 5x2 factorial scheme, with 5 replications and 3x2 for the irrigation regimes with 5 replications. The pH tests showed significant differences between the treatments and pH 4,0 - 4,5 presented lower incidence and severity of the disease for the most species tested but no significant differences were observed between the irrigation regimes. The soil acidification is considered a classic strategy for management of the disease and the results obtained herein corroborated this hypothesis.

Effect of pH soil and irrigation regimes on management of potato scab

ABSTRACT Potato scab caused by different species of phytopathogenic Streptomyces is considered one of the main bacterial diseases of economic crop importance worldwide. Several studies are being carried out in order to control the disease, but until now, there is no efficient way to do this. Some management strategies have been investigated including application of chemical and biological products and utilization of resistant cultivars of potato but there are few reports about the impact of pH and irrigation regimes on the disease. The present study aimed to evaluate the effects of these last two factors on the incidence and severity of potato scab caused by S. scabiei, S. acidiscabies, Streptomyces sp., S. caviscabies and S. europaeiscabiei in assays at pH 4.0, 4.5, 5.0, 5.5, 6.5 and 7.5; and irrigation regimes of once a week, alternate days and daily in greenhouse conditions. The experimental design for the pH tests was randomized blocks arranged in a 5x2 factorial scheme, with 5 replications and 3x2 for the irrigation regimes with 5 replications. The pH tests showed significant differences between the treatments and pH 4,0 - 4,5 presented lower incidence and severity of the disease for the most species tested but no significant differences were observed between the irrigation regimes. The soil acidification is considered a classic strategy for management of the disease and the results obtained herein corroborated this hypothesis.

Evaluation of the production of antifungal metabolites against Colletotrichum gloeosporioides in Streptomyces 5.1 by random mutagenesis

Streptomyces 5.1 is a bacterium isolated from rice soils in the south of the Tolima department (Colombia). This microorganism is characterized by its antagonistic activity against rubber tree phytopathogens like Colletotrichum gloeosporioides, the causal agent of leaf anthracnose. The antifungal activity of this Streptomyces isolate has been associated with secondary metabolites production. However, the identity of those metabolites is unknown because its purification and identification have not been possible through classic chemical studies. Therefore, aiming to contribute in the study of the secondary metabolites produced by 5.1 from a molecular approach, this research seeks to identify -preliminarily- the genomic fingerprint changes associated with the production of antifungal secondary metabolites produced by Streptomyces 5.1 through the evaluation of a mutant library of 5.1 obtained by random mutagenesis using controlled ultraviolet light exposure. The antifungal activity of obtained mutants was evaluated using Colletotrichum gloeosporioides (C1) fungus as a biosensor, isolated by the Biotechnology Institute of Universidad Nacional de Colombia. In this way, the library of mutants of 5.1, initially formed by 300 isolations, was classified into two phenotypic groups of interest: enhanced mutants (1 isolate) and null mutants (11 isolates) of secondary metabolites. The genomic changes in both groups were analyzed by obtaining the genomic profile of the isolates using Repetitive Extragenic Palindromic (Rep-PCR). The obtained profiles evidenced the presence of one additional band in the enhanced mutant, and the absence of a specific band in the non-producing mutants, both in comparison with the original strain. These bands are proposed for a future sequencing study which will define their role in the production process of metabolites with antifungal activity in Streptomyces 5.1.

Evaluation of the production of antifungal metabolites against Colletotrichum gloeosporioides in Streptomyces 5.1 by random mutagenesis

Streptomyces 5.1 is a bacterium isolated from rice soils in the south of the Tolima department (Colombia). This microorganism is characterized by its antagonistic activity against rubber tree phytopathogens like Colletotrichum gloeosporioides, the causal agent of leaf anthracnose. The antifungal activity of this Streptomyces isolate has been associated with secondary metabolites production. However, the identity of those metabolites is unknown because its purification and identification have not been possible through classic chemical studies. Therefore, aiming to contribute in the study of the secondary metabolites produced by 5.1 from a molecular approach, this research seeks to identify -preliminarily- the genomic fingerprint changes associated with the production of antifungal secondary metabolites produced by Streptomyces 5.1 through the evaluation of a mutant library of 5.1 obtained by random mutagenesis using controlled ultraviolet light exposure. The antifungal activity of obtained mutants was evaluated using Colletotrichum gloeosporioides (C1) fungus as a biosensor, isolated by the Biotechnology Institute of Universidad Nacional de Colombia. In this way, the library of mutants of 5.1, initially formed by 300 isolations, was classified into two phenotypic groups of interest: enhanced mutants (1 isolate) and null mutants (11 isolates) of secondary metabolites. The genomic changes in both groups were analyzed by obtaining the genomic profile of the isolates using Repetitive Extragenic Palindromic (Rep-PCR). The obtained profiles evidenced the presence of one additional band in the enhanced mutant, and the absence of a specific band in the non-producing mutants, both in comparison with the original strain. These bands are proposed for a future sequencing study which will define their role in the production process of metabolites with antifungal activity in Streptomyces 5.1.(AU)

Antibacterial activity of phytopathogenic Streptomyces strains against bacteria associated to clinical diseases / Atividade antimicrobiana de Streptomyces fitopatogênicas contra bactérias associadas a doenças de importância clínica

The genus Streptomyces is associated with the ability to produce and excrete a variety of bioactive compounds, such as antibiotic, antifungal and antiviral. Biological active polyketide and peptide compounds with applications in medicine, agriculture and biochemical research are synthesized by PKS-I and NRPS genes. The evaluation of the presence of these genes associated with the biosynthesis of secondary metabolites in different phytopathogenic Streptomyces strains were performed using degenerated primers. The positive signal was observed in 58/63 Streptomyces strains for NRPS gene, 43/63 for PKS-I, and for PKS-II all the 63 strains showed positive signal of amplification. These strains also were tested with double layer agar-well technique against bacterial with clinical importance, and it was possible to observe the Streptomyces spp. strains were able to inhibit the growth of 14, 20, 13 and 3 isolates Gram-positive and Gram-negative bacteria, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 11775) respectively. The Streptomyces sp. strains IBSBF 2019 and IBSBF 2397 showed antibacterial activity against all four bacteria-target tested.(AU)

O gênero Streptomyces apresenta alta capacidade de produzir e excretar uma grande variedade de compostos biologicamente ativos, como antibióticos, antifúngicos e antivirais. Compostos biologicamente ativos de policetídeos e peptídeos com aplicações na medicina, agricultura e pesquisas bioquímicas são sintetizados pelos genes PKS-I e NRPS. A avaliação da presença desses genes associados à biossíntese de metabólitos secundários em diferentes linhagens de Streptomyces fitopatogênicas foi realizada através do uso de primers degenerados. O sinal positivo foi observado em 58/63 linhagens de Streptomyces para o gene NRPS, 43/63 para o gene PKS-I e, para o gene PKS-II, todas as 63 linhagens apesentaram o sinal positivo de amplificação. Essas linhagens também foram testadas através da técnica de dupla camada contra bactérias de importância clínica e foi possível observar que as linhagens de Streptomyces spp. foram capazes de inibir o crescimento de 14, 20, 13 e 3 isolados de bactérias Gram-positivas e Gram-negativas, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) e Escherichia coli (ATCC 11775), respectivamente. As linhagens de Streptomyces sp. ISBSF 2019 e 2397 apresentaram atividade antibacteriana contra todas as bactérias-alvo testadas.(AU)

Antibacterial activity of phytopathogenic Streptomyces strains against bacteria associated to clinical diseases / Atividade antimicrobiana de Streptomyces fitopatogênicas contra bactérias associadas a doenças de importância clínica

The genus Streptomyces is associated with the ability to produce and excrete a variety of bioactive compounds, such as antibiotic, antifungal and antiviral. Biological active polyketide and peptide compounds with applications in medicine, agriculture and biochemical research are synthesized by PKS-I and NRPS genes. The evaluation of the presence of these genes associated with the biosynthesis of secondary metabolites in different phytopathogenic Streptomyces strains were performed using degenerated primers. The positive signal was observed in 58/63 Streptomyces strains for NRPS gene, 43/63 for PKS-I, and for PKS-II all the 63 strains showed positive signal of amplification. These strains also were tested with double layer agar-well technique against bacterial with clinical importance, and it was possible to observe the Streptomyces spp. strains were able to inhibit the growth of 14, 20, 13 and 3 isolates Gram-positive and Gram-negative bacteria, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 11775) respectively. The Streptomyces sp. strains IBSBF 2019 and IBSBF 2397 showed antibacterial activity against all four bacteria-target tested.(AU)

O gênero Streptomyces apresenta alta capacidade de produzir e excretar uma grande variedade de compostos biologicamente ativos, como antibióticos, antifúngicos e antivirais. Compostos biologicamente ativos de policetídeos e peptídeos com aplicações na medicina, agricultura e pesquisas bioquímicas são sintetizados pelos genes PKS-I e NRPS. A avaliação da presença desses genes associados à biossíntese de metabólitos secundários em diferentes linhagens de Streptomyces fitopatogênicas foi realizada através do uso de primers degenerados. O sinal positivo foi observado em 58/63 linhagens de Streptomyces para o gene NRPS, 43/63 para o gene PKS-I e, para o gene PKS-II, todas as 63 linhagens apesentaram o sinal positivo de amplificação. Essas linhagens também foram testadas através da técnica de dupla camada contra bactérias de importância clínica e foi possível observar que as linhagens de Streptomyces spp. foram capazes de inibir o crescimento de 14, 20, 13 e 3 isolados de bactérias Gram-positivas e Gram-negativas, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) e Escherichia coli (ATCC 11775), respectivamente. As linhagens de Streptomyces sp. ISBSF 2019 e 2397 apresentaram atividade antibacteriana contra todas as bactérias-alvo testadas.(AU)

Growth and antioxidant response of juvenile oysters Crassostrea sikamea and Crassostrea corteziensis treated with Streptomyces strains / Crescimento e resposta oxidative de ostras jovens Crassostrea sikamea e Crassostrea corteziensis tratadas com cultura Streptomyces

The effect of three Streptomyces strains (N7, RL8 and V4) and a mixture of Bacillus (BMix) on the growth (Weight, Size) and superoxide dismutase activity (SOD) in hatchery-reared juvenile oysters Crassostrea corteziensis and Crassostrea sikamea was investigated to determine their probiotic potential. Microorganisms were added to culture water at 1×106 CFU/ml once a day during 30 days and all oysters fed daily a microalgae mix. Juveniles of C. sikamea treated with strains N7, RL8 and V4 had a significant weight gain compared to the control group. C. corteziensis juveniles treated with strains RL8 and BMix showed a significantly higher weight gain than the control group. No significant size increase was observed in any treated group for both oyster species. SOD activity significantly increased in C. sikamea treated with RL8 and with RL8, N7 and BMix in C. corteziensis. Streptomyces strains RL8 and N7 emerge as promising probiotic agents to cultivate C. sikamea and C. corteziensis and may also be useful to other molluscs and marine invertebrates .(AU)

O efeito de três culturas Streptomyces (N7, RL8 e V4) e uma mistura de Bacillus (BMix) sobre o cresimento (Peso, Tamanho) e atividade superóxido dismutase (SOD) em ostras jovens Crassostrea corteziensis e Crassostrea sikamea cultivadas artificalmente foi investigado para determinar seu potencial probiótico. Microorganismos foram adicionados à água de cultura a 1×10 6 CFU/ml uma vez por dia durante 30 dias e todas as ostras foram alimentadas diariamente com uma mistura de microalgas. Jovens C. sikamea tratados com culturas N7, RL8 e V4 tiveram ganho de peso significativo quando comparado ao grupo de controle. Jovens C. corteziensis tratados com culturas RL8 e BMix demonstraram peso significativamente mais algo que o grupo de controle. Nenhum aumento em tamanho foi observado em grupos tratados em ambas espécies. A atividade SOD foi significamente aumentada em C. sikamea treatado com RL8 e com RL8, N7 e BMix em C. corteziensis. Culturas Streptomyces RL8 e N7 surgem como agentes probióticos promissores para o cultivo de C. sikamea e C. corteziensis e podem ser úteis para outros moluscos animais marinhos invertebrados.(AU)

Growth and antioxidant response of juvenile oysters Crassostrea sikamea and Crassostrea corteziensis treated with Streptomyces strains / Crescimento e resposta oxidative de ostras jovens Crassostrea sikamea e Crassostrea corteziensis tratadas com cultura Streptomyces

The effect of three Streptomyces strains (N7, RL8 and V4) and a mixture of Bacillus (BMix) on the growth (Weight, Size) and superoxide dismutase activity (SOD) in hatchery-reared juvenile oysters Crassostrea corteziensis and Crassostrea sikamea was investigated to determine their probiotic potential. Microorganisms were added to culture water at 1×106 CFU/ml once a day during 30 days and all oysters fed daily a microalgae mix. Juveniles of C. sikamea treated with strains N7, RL8 and V4 had a significant weight gain compared to the control group. C. corteziensis juveniles treated with strains RL8 and BMix showed a significantly higher weight gain than the control group. No significant size increase was observed in any treated group for both oyster species. SOD activity significantly increased in C. sikamea treated with RL8 and with RL8, N7 and BMix in C. corteziensis. Streptomyces strains RL8 and N7 emerge as promising probiotic agents to cultivate C. sikamea and C. corteziensis and may also be useful to other molluscs and marine invertebrates .(AU)

O efeito de três culturas Streptomyces (N7, RL8 e V4) e uma mistura de Bacillus (BMix) sobre o cresimento (Peso, Tamanho) e atividade superóxido dismutase (SOD) em ostras jovens Crassostrea corteziensis e Crassostrea sikamea cultivadas artificalmente foi investigado para determinar seu potencial probiótico. Microorganismos foram adicionados à água de cultura a 1×10 6 CFU/ml uma vez por dia durante 30 dias e todas as ostras foram alimentadas diariamente com uma mistura de microalgas. Jovens C. sikamea tratados com culturas N7, RL8 e V4 tiveram ganho de peso significativo quando comparado ao grupo de controle. Jovens C. corteziensis tratados com culturas RL8 e BMix demonstraram peso significativamente mais algo que o grupo de controle. Nenhum aumento em tamanho foi observado em grupos tratados em ambas espécies. A atividade SOD foi significamente aumentada em C. sikamea treatado com RL8 e com RL8, N7 e BMix em C. corteziensis. Culturas Streptomyces RL8 e N7 surgem como agentes probióticos promissores para o cultivo de C. sikamea e C. corteziensis e podem ser úteis para outros moluscos animais marinhos invertebrados.(AU)

Surto de intoxicação por monensina sódica em equinos associada a consumo de suplemento mineral à base de melaço de cana-de-açúcar / Outbreak of monensin sodium poisoning in horses associated with consumption of mineral supplement based on sugarcane molasses

Background: Sodium monensin is a molecule of the group of ionophores antibiotics (IAs) of the polyether carboxylic typeproduced from the fermentation of Streptomyces cinnamonensis. Cases of IA poisoning in animals usually occur accidentally. Fatal poisoning in horses, a non-target species and with particular sensitivity usually occurs by the consumption ofthese products from the toxic dose of 2-3 mg/kg. This report aims to describe the epidemiological and clinical-pathologicalaspects of an outbreak of ionophore antibiotic poisoning in horses due to the consumption of mineral supplementationindicated for ruminants based on sugarcane molasses.Case: Two horses were necropsied. During necropsy, fragments of various organs were collected and fixed in 10% bufferedformalin, routinely processed for histology and stained with hematoxylin and eosin. The information obtained from the animaltrainer was that 19 horses received approximately 25 kg of low moisture mixture based on sugarcane molasses, enriched withmacro and micro minerals, vitamins and additives containing 1000 mg of monensin/kg in the trough. One day after receivingthe product, five horses began to develop ataxia, reluctance to move, difficulty of accompanying the herd and arising. Out ofthe five, three died after three days of evolution, one after six days (equine 1) and another after 15 days (equine 2) [morbidity 26.31%]. At necropsy, diffuse pallor was observed in the gluteus medius, quadriceps femoris, semimembranosus, bicepsbrachii and deep pectoral muscles. Microscopically the muscle fibers were tumefied with hypereosinophilic sarcoplasmof homogeneous appearance and with loss of striations, pynotic or absent nuclei (necrosis). Multifocal areas of fibers withsarcoplasmic fragmentation were observed, with clusters of irregular eosinophilic debris, flake (floct necrosis) or granule(granular necrosis)...

Surto de intoxicação por monensina sódica em equinos associada a consumo de suplemento mineral à base de melaço de cana-de-açúcar / Outbreak of monensin sodium poisoning in horses associated with consumption of mineral supplement based on sugarcane molasses

Background: Sodium monensin is a molecule of the group of ionophores antibiotics (IAs) of the polyether carboxylic typeproduced from the fermentation of Streptomyces cinnamonensis. Cases of IA poisoning in animals usually occur accidentally. Fatal poisoning in horses, a non-target species and with particular sensitivity usually occurs by the consumption ofthese products from the toxic dose of 2-3 mg/kg. This report aims to describe the epidemiological and clinical-pathologicalaspects of an outbreak of ionophore antibiotic poisoning in horses due to the consumption of mineral supplementationindicated for ruminants based on sugarcane molasses.Case: Two horses were necropsied. During necropsy, fragments of various organs were collected and fixed in 10% bufferedformalin, routinely processed for histology and stained with hematoxylin and eosin. The information obtained from the animaltrainer was that 19 horses received approximately 25 kg of low moisture mixture based on sugarcane molasses, enriched withmacro and micro minerals, vitamins and additives containing 1000 mg of monensin/kg in the trough. One day after receivingthe product, five horses began to develop ataxia, reluctance to move, difficulty of accompanying the herd and arising. Out ofthe five, three died after three days of evolution, one after six days (equine 1) and another after 15 days (equine 2) [morbidity 26.31%]. At necropsy, diffuse pallor was observed in the gluteus medius, quadriceps femoris, semimembranosus, bicepsbrachii and deep pectoral muscles. Microscopically the muscle fibers were tumefied with hypereosinophilic sarcoplasmof homogeneous appearance and with loss of striations, pynotic or absent nuclei (necrosis). Multifocal areas of fibers withsarcoplasmic fragmentation were observed, with clusters of irregular eosinophilic debris, flake (floct necrosis) or granule(granular necrosis)... (AU)

Genome sequence of Streptomyces mangrovisoli MUSC 149T isolated from intertidal sediments

As the largest genus in Actinobacteria family, Streptomyces species have the ability to synthesize numerous compounds of diverse structures with bioactivities. Streptomyces mangrovisoli MUSC 149T was previously isolated as a novel streptomycete from mangrove forest in east coast of Peninsular Malaysia. The high quality draft genome of MUSC 149T comprises 9,165,825 bp with G + C content of 72.5%. Through bioinformatics analysis, 21 gene clusters identified in the genome were associated with the production of bioactive secondary metabolites. The presence of these biosynthetic gene clusters in MUSC 149T suggests the potential exploitation of the strain for production of medically important compounds.(AU)

Streptomyces globosus DK15 and Streptomyces ederensis ST13 as new producers of factumycin and tetrangomycin antibiotics

Fifty seven soil-borne actinomycete strains were assessed for the antibiotic production. Two of the most active isolates, designed as Streptomyces ST-13 and DK-15 exhibited a broad range of antimicrobial activity and therefore they were selected for HPLC fractionation against the most suppressed bacteria Staphylococcus aureus (ST-13) and Chromobacterium violaceum (DK-15). LC/MS analysis of extracts showed the presence of polyketides factumycin (DK15) and tetrangomycin (ST13). The taxonomic position of the antibiotic-producing actinomycetes was determined using a polyphasic approach. Phenotypic characterization and 16S rRNA gene sequence analysis of the isolates matched those described for members of the genus Streptomyces. DK-15 strain exhibited the highest 16S rRNA gene sequence similarity to Streptomyces globosus DSM-40815 (T) and Streptomyces toxytricini DSM-40178 (T) and ST-13 strain to Streptomyces ederensis DSM-40741 (T) and Streptomyces phaeochromogenes DSM-40073 (T). For the proper identification, MALDI-TOF/MS profile of whole-cell proteins led to the identification of S. globosus DK-15 (accession number: KX527570) and S. ederensis ST13 (accession number: KX527568). To our knowledge, there is no report about the production of these antibiotics by S.globosus and S. ederensis, thus isolates DK15 and ST13 identified as S. globosus DK-15 and S.ederensis ST-13 can be considered as new sources of these unique antibacterial metabolites.(AU)

Genome sequence of Streptomyces gilvigriseus MUSC 26T isolated from mangrove forest

Abstract Streptomycetes remain as one of the important sources for bioactive products. Isolated from the mangrove forest, Streptomyces gilvigriseus MUSC 26T was previously characterised as a novel streptomycete. The high quality draft genome of MUSC 26T contained 5,213,277 bp with G + C content of 73.0%. Through genome mining, several gene clusters associated with secondary metabolites production were revealed in the genome of MUSC 26T. These findings call for further investigations into the potential exploitation of the strain for production of pharmaceutically important compounds.