ABSTRACT
Macrophomina phaseolina and Rhizoctonia solani are considered two major soil-borne pathogens of Phaseolus vulgaris in Cuba. Their management is difficult, not only due to their intrinsic biology as soil-borne pathogens, but also because the lack of active ingredients available against these pathogens. Actinobacteria, a heterogeneous bacterial group traditionally known as actinomycetes have been reported as promising biological control agents (BCAs) in crop protection. Thus, the main objective of this study was to evaluate the effectiveness of 60 actinobacterial strains as BCAs against M. phaseolina and R. solani in vitro by dual culture assays. The most effective strains were characterized according to their cellulolytic, chitinolytic and proteolytic extracellular enzymatic activity, as well as by their morphological and biochemical characters in vitro. Forty and 25 out of the 60 actinobacteria strains inhibited the mycelial growth of M. phaseolina and R. solani, respectively, and 18 of them showed a common effect against both pathogens. Significant differences were observed on their enzymatic and biochemical activity. The morphological and biochemical characters allow us to identify all our strains as species belonging to the genus Streptomyces. Streptomyces strains CBQ-EA-2 and CBQ-B-8 showed the highest effectiveness in vitro. Finally, the effect of seed treatments by both strains was also evaluated against M. phaseolina and R. solani infections in P. vulgaris cv. Quivicán seedlings. Treatments combining the two Streptomyces strains (CBQ-EA-2 + CBQ-B-8) were able to reduce significantly the disease severity for both pathogen infections in comparison with the non-treated and inoculated control. Moreover, they showed similar effect than that observed for Trichoderma harzianum A-34 and with Celest® Top 312 FS (Syngenta®; Basilea, Switzerland) treatments, which were included for comparative purposes.
ABSTRACT
This study aimed to characterize six Streptomyces strains associated with potato scab in south Brazil through polyphasic taxonomy involving morphology, pathogenicity and genetic features. These strains were compared with other potato-scab Streptomyces species mainly S. europaeiscabiei, S. scabiei and S. stelliscabiei. South-Brazilian Streptomyces strains were morphologically distinct from the type strains of S. scabiei (CFBP 4517T) and their genomospecies S. europaeiscabiei (CFBP 4497 T) and S. stelliscabiei (CFBP 4521T), producing a brown substrate mycelium with red borders and cream-grey brown aerial spores. Red-brown diffusible pigment on YME was also observed. The carbon sources L-Arabinose, D-Fructose, D-Glucose, D-Mannitol, meso-Inositol, Raffinose, Rhamnose, Sucrose, D-Xylose were tested for these strains. All strains were pathogenic causing symptoms of necrosis on radish and several potato cultivars commonly used in potato growing areas in Brazil. In greenhouse conditions, the strains caused scab disease and produced deep-pitted lesions covering large areas of the tuber. These results were correlated with presence of pathogenicity marker genes (txtAB, tomA or nec1) detected by PCR amplifications. In both phylogenetic analyses, 16S rRNA and MLSA, Streptomyces sp. Brazilian strains were closely related to S. europaeiscabiei, S. scabiei and S. stelliscabiei species, but they were allocated in separated branches supported by high bootstrap values and/or with low sequence similarity values. Sequencing of whole genome showed an 10,846,379 bp linear chromosome with high GC content (71.3%) consisting of 9179 putative genes, 3 rRNAs, 89 tRNAs and 1 CRISPRS. The molecular data, including genomic features, associated with morphological, biochemical and pathogenic characteristics warrant that the six Streptomyces Brazilian strains represent a new species associated with potato scab in Brazil, which would be named Streptomyces brasiliscabiei with IBSBF 2867T as the type strain.
Subject(s)
Solanum tuberosum , Streptomyces , Brazil , Phylogeny , Plant Diseases , RNA, Ribosomal, 16S/genetics , Streptomyces/geneticsABSTRACT
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)
Subject(s)
Pseudomonas aeruginosa/growth & development , Staphylococcus aureus/growth & development , Streptomyces/metabolism , Bacillus cereus/growth & development , Escherichia coli/growth & development , Anti-Bacterial Agents/metabolism , Peptide Synthases/genetics , Streptomyces/genetics , Gene Amplification , Polymerase Chain Reaction , Sequence Analysis, DNA , DNA Primers , Polyketide Synthases/genetics , Anti-Bacterial Agents/pharmacologyABSTRACT
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)
Subject(s)
Pseudomonas aeruginosa/growth & development , Staphylococcus aureus/growth & development , Streptomyces/metabolism , Bacillus cereus/growth & development , Escherichia coli/growth & development , Anti-Bacterial Agents/metabolism , Peptide Synthases/genetics , Streptomyces/genetics , Gene Amplification , Polymerase Chain Reaction , Sequence Analysis, DNA , DNA Primers , Polyketide Synthases/genetics , Anti-Bacterial Agents/pharmacologyABSTRACT
In spite of the acknowledged importance of growth-promoting bacteria, only a reduced number of studies were conducted with these microorganisms on Theobroma cacao. The objectives of this work were to study the population densities and genetic diversity of actinomycetes associated with the rhizosphere of cacao as a first step in their application in plant growth promotion and biological control. The populations densities of actinomycetes in soil and cacao roots were similar, with mean values of 1,0 x 10(6) CFU/g and 9,6 x 10(5) CFU/g, respectively. All isolates selected and used in this study were identified through sequencing analyses of a fragment of the rpoB gene that encodes the [beta]-subunit of the RNA polymerase as species of the genus Streptomyces. In vitro cellulolytic, xilanolytic and chitinolytic activity, indolacetic acid production and phosphate solubilization activities were observed in most of the isolates tested. The data obtained in this study demonstrate that actinomycetes account for a higher percentage of the total population of culturable bacteria in soil than on cacao roots. Additionally, actinomycetes from the cacao rhizosphere are genetically diverse and have potential applications as agents of growth promotion.
Apesar da reconhecida importância das bactérias promotoras de crescimento, apenas um reduzido número de estudos foi conduzido com este grupo de microrganismos na cultura do cacaueiro. Os objetivos deste trabalho foram o estudo da densidade populacional e da diversidade genética de actinomicetos associados à rizosfera do cacaueiro como o primeiro passo para sua utilização na promoção de crescimento de mudas desta cultura e no controle biológico de doenças. As densidades populacionais de actinomicetos em amostras de solo e de raízes de cacaueiro foram semelhantes, com valores médios de 1,0 x 10(6) UFC/g e de 9,6 x 10(5) UFC/g, respectivamente. Todos os isolados selecionados para este estudo foram identificados através de análises de seqüências de um fragmento do gene rpoB, que codifica a beta-subunidade da RNA polimerase, como pertencentes ao gênero Streptomyces. Dentre os isolados testados, constatou-se in vitro, a produção de celulase, xilanase, quitinase, ácido indolacético e a capacidade de solubilização de fosfato. Os dados obtidos demonstram que os actinomicetos representam uma maior proporção da população total de bactérias cultiváveis em solo do que em raízes. Adicionalmente, os actinomicetos da rizosfera do cacaueiro são geneticamente diversos e apresentam potencial para atuarem como agentes de promoção de crescimento.
Subject(s)
Actinobacteria/isolation & purification , Cacao/growth & development , Genetic Variation , In Vitro Techniques , RNA-Dependent RNA Polymerase , Plant Roots , Rhizophoraceae/growth & development , Sequence Analysis , Food Samples , Methods , MethodsABSTRACT
In spite of the acknowledged importance of growth-promoting bacteria, only a reduced number of studies were conducted with these microorganisms on Theobroma cacao. The objectives of this work were to study the population densities and genetic diversity of actinomycetes associated with the rhizosphere of cacao as a first step in their application in plant growth promotion and biological control. The populations densities of actinomycetes in soil and cacao roots were similar, with mean values of 1,0 x 10(6) CFU/g and 9,6 x 10(5) CFU/g, respectively. All isolates selected and used in this study were identified through sequencing analyses of a fragment of the rpoB gene that encodes the ß-subunit of the RNA polymerase as species of the genus Streptomyces. In vitro cellulolytic, xilanolytic and chitinolytic activity, indolacetic acid production and phosphate solubilization activities were observed in most of the isolates tested. The data obtained in this study demonstrate that actinomycetes account for a higher percentage of the total population of culturable bacteria in soil than on cacao roots. Additionally, actinomycetes from the cacao rhizosphere are genetically diverse and have potential applications as agents of growth promotion.
ABSTRACT
In spite of the acknowledged importance of growth-promoting bacteria, only a reduced number of studies were conducted with these microorganisms on Theobroma cacao. The objectives of this work were to study the population densities and genetic diversity of actinomycetes associated with the rhizosphere of cacao as a first step in their application in plant growth promotion and biological control. The populations densities of actinomycetes in soil and cacao roots were similar, with mean values of 1,0 x 10(6) CFU/g and 9,6 x 10(5) CFU/g, respectively. All isolates selected and used in this study were identified through sequencing analyses of a fragment of the rpoB gene that encodes the -subunit of the RNA polymerase as species of the genus Streptomyces. In vitro cellulolytic, xilanolytic and chitinolytic activity, indolacetic acid production and phosphate solubilization activities were observed in most of the isolates tested. The data obtained in this study demonstrate that actinomycetes account for a higher percentage of the total population of culturable bacteria in soil than on cacao roots. Additionally, actinomycetes from the cacao rhizosphere are genetically diverse and have potential applications as agents of growth promotion.
Apesar da reconhecida importância das bactérias promotoras de crescimento, apenas um reduzido número de estudos foi conduzido com este grupo de microrganismos na cultura do cacaueiro. Os objetivos deste trabalho foram o estudo da densidade populacional e da diversidade genética de actinomicetos associados à rizosfera do cacaueiro como o primeiro passo para sua utilização na promoção de crescimento de mudas desta cultura e no controle biológico de doenças. As densidades populacionais de actinomicetos em amostras de solo e de raízes de cacaueiro foram semelhantes, com valores médios de 1,0 x 10(6) UFC/g e de 9,6 x 10(5) UFC/g, respectivamente. Todos os isolados selecionados para este estudo foram identificados através de análises de seqüências de um fragmento do gene rpoB, que codifica a -subunidade da RNA polimerase, como pertencentes ao gênero Streptomyces. Dentre os isolados testados, constatou-se in vitro, a produção de celulase, xilanase, quitinase, ácido indolacético e a capacidade de solubilização de fosfato. Os dados obtidos demonstram que os actinomicetos representam uma maior proporção da população total de bactérias cultiváveis em solo do que em raízes. Adicionalmente, os actinomicetos da rizosfera do cacaueiro são geneticamente diversos e apresentam potencial para atuarem como agentes de promoção de crescimento.