Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt / Bacillus subtilis P10 endofítico de Prunus cerasifera como agente de biocontrole contra murcha de Verticillium de tomate

Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.(AU)

As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.(AU)

Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt / Bacillus subtilis P10 endofítico de Prunus cerasifera como agente de biocontrole contra murcha de Verticillium de tomate

Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.

As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.

Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt / Bacillus subtilis P10 endofítico de Prunus cerasifera como agente de biocontrole contra murcha de Verticillium de tomate

Abstract Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.

Resumo As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.

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.

Endophytic bacteria Bacillus safensis and Pseudomonas hibiscicola and their ability to increase rice seedling growth

Endophytic bacteria Bacillus safensis RS95 and Pseudomonas hibiscicola RS121 were evaluated for their ability to promote the growth of rice seedlings and produce indole-acetic acid (IAA) and siderophores and to solubilize phosphates. 'Guri' rice seeds were immersed in bacterial endophyte cell suspensions (separated and two-strain mixed), as well as in Escherichia coli DH5α, phosphate-buffered saline (PBS) and water treatments (negative controls). Seeds were sown on agar-water in Petri plates placed vertically at an angle of 65°. The ability of plant growth-promoting endophytic bacteria (PGPEB) to produce IAA and siderophores was determined by Salkowski colorimetric and chrome azurol S (CAS) assays, respectively. Mineral phosphate solubilization activity was calculated by inoculating the endophytes onto medium containing insoluble phosphate. PGPEB showed a positive effect on the growth of rice seedlings, causing a mean growth of shoots and primary-roots of 60 and 67%, respectively. Bacterial strains also showed positive traits for IAA and siderophore production, as well as phosphate-solubilization activity

Evaluation of the incidence of endophytic fungi in leaves, stems and roots of bean plants in the State of Paraná / Avaliação da incidência de fungos endofíticos em folhas, caules e raízes de plantas de feijão no Estado do Paraná

The present study aimed to isolate endophilic fungi from plant tissues (leaves, stems and roots) of beans, in addition to identifying them at the genus level. The experiment was conducted at IDR-Londrina. Bean samples were collected at the Londrina Research Station and at UENP-Bandeirantes. The leaves, stems and roots were separated; fragments of 5 mm diameter were cut and disinfested. They were later transferred to BDA culture medium. The plates were incubated at room temperature for seven days for growth of the microorganisms present. The presence and absence, colonization rate (TC) for each tissue and gender identification of fungi were evaluated. The experiment was conducted in a completely randomized design, with five replications. Forty-eight endophilic isolates were found, 13 in root, 17 in stem and 18 in leaf. CT ranged from 17-100% (leaf), 17-83% (stem) and 16-100% (root). The genus Alternaria sp. and Cladosporium sp. have greater specificity with the leaf and stem, Penicillium sp. root and stem. The genera Fusarium sp. and Aspergillus sp. are found in all organs, Curvularia sp., Nigrospora sp. and Thichoderma sp. demonstrated leaf specificity, Macrophomina sp. the root, and Colletotrichum sp. to the stem. Thus, the identified fungi present potential for future studies in their application in the biocontrol of various plant diseases.

O presente estudo teve como objetivo realizar o isolamento de fungos endofíticos dos tecidos vegetais (folhas, caules e raízes) de feijão, além de identifica-los a nível de gênero. O experimento foi conduzido no IDR-Londrina. Amostras de feijão foram coletadas na Estação de Pesquisa de Londrina e na UENP-Bandeirantes. Foram separadas as folhas, caules e raízes, cortados fragmentos de 5 mm diâmetro e desinfestados. Posteriormente foram transferidos para meio de cultura BDA. As placas foram incubadas à temperatura ambiente por sete dias, para crescimento dos microrganismos presentes. Foram avaliados a presença e ausência, taxa de colonização (TC) para cada tecido e identificação a nível de gênero dos fungos. O experimento foi conduzido em delineamento inteiramente casualizado, com cinco repetições. Encontrou-se 48 isolados endofíticos, sendo 13 em raiz, 17 em caule e 18 em folha. A TC variou de 17-100% (folha), de 17-83% (caule) e 16-100% (raiz). O gênero Alternaria sp. e Cladosporium sp. apresentam especificidade maior com a folha e caule, o Penicillium sp. com raiz e caule. Os gêneros Fusarium sp. e Aspergillus sp. são encontrados em todos os órgãos, Curvularia sp., Nigrospora sp. e Thichoderma sp. demonstraram especificidade em folha, Macrophomina sp. a raiz, e Colletotrichum sp. ao caule. Dessa forma, os fungos identificados apresentam-se com potencial para futuros estudos em sua aplicação no biocontrole de diversas doenças de plantas.

Ornamental plant Pachystachys lutea as a source of promising endophytes for plant growth and phytoprotective activity

Endophytes are growth-promoting agents capable of synthesizing phytohormones, uptaking nutrients, and controlling pathogens. There is a strong potential to exploit them in the agriculture field like biofertilizers and biocontrol agents. In this work, we aimed to evaluate endophytic fungi isolated from Pachystachys lutea for their potential to solubilize phosphate, synthesise indole acetic acid (IAA), antagonize phytopathogens, and promote plant growth under greenhouse conditions. The phosphate solubilization efficiency was assessed on Pikovskayas agar medium. For analysis of IAA production, mycelia plugs of endophytes were cultured in Potato Dextrose Broth medium supplemented with L-tryptophan, with Salkowski Reagent, and the absorbance of the culture was measured. The antagonism evaluation of strain Alternaria sp. PL75 against phytopathogens was performed using the paired-culture technique. The promotion of plant growth provided by Alternaria sp. PL75 was evaluated in tomato plants. All strains evaluated were able to solubilize phosphate; however, the strain Alternaria sp. PL75 was the most effective (4.29). Two strains, Nemania sp. PL27 and Alternaria sp. PL75, produced 1.86 and 1.73 & 956;g mL-1 of IAA, respectively. In the antagonism assay, the endophyte Alternaria sp. PL75 and its fungal extract showed the best results against the pathogen Moniliophthora perniciosa. The greenhouse experiment result showed the endophyte Alternaria sp. PL75 increased the plantlets emergency speed index and the percentage of germination from 60 to 81.63%. It was also observed a statistical significance in the shoot length of the treated plants with the endophyte suspension (55.38 cm) compared to the control (41.67 cm).(AU)

Ornamental plant Pachystachys lutea as a source of promising endophytes for plant growth and phytoprotective activity

Endophytes are growth-promoting agents capable of synthesizing phytohormones, uptaking nutrients, and controlling pathogens. There is a strong potential to exploit them in the agriculture field like biofertilizers and biocontrol agents. In this work, we aimed to evaluate endophytic fungi isolated from Pachystachys lutea for their potential to solubilize phosphate, synthesise indole acetic acid (IAA), antagonize phytopathogens, and promote plant growth under greenhouse conditions. The phosphate solubilization efficiency was assessed on Pikovskaya’s agar medium. For analysis of IAA production, mycelia plugs of endophytes were cultured in Potato Dextrose Broth medium supplemented with L-tryptophan, with Salkowski Reagent, and the absorbance of the culture was measured. The antagonism evaluation of strain Alternaria sp. PL75 against phytopathogens was performed using the paired-culture technique. The promotion of plant growth provided by Alternaria sp. PL75 was evaluated in tomato plants. All strains evaluated were able to solubilize phosphate; however, the strain Alternaria sp. PL75 was the most effective (4.29). Two strains, Nemania sp. PL27 and Alternaria sp. PL75, produced 1.86 and 1.73 & 956;g mL-1 of IAA, respectively. In the antagonism assay, the endophyte Alternaria sp. PL75 and its fungal extract showed the best results against the pathogen Moniliophthora perniciosa. The greenhouse experiment result showed the endophyte Alternaria sp. PL75 increased the plantlets emergency speed index and the percentage of germination from 60 to 81.63%. It was also observed a statistical significance in the shoot length of the treated plants with the endophyte suspension (55.38 cm) compared to the control (41.67 cm).

Biosurfactants synthesized by endophytic Bacillus strains as control of Moniliophthora perniciosa and Moniliophthora roreri

Ecuador is responsible for up to 70 % of the world’s production of fine flavor cocoa. Although fungal diseases have reduced cocoa production, eco–friendly alternatives have been implemented to control the spread of phytopathogens. This study aimed to explore the potential of endophytic strains isolated from fine flavor Nacional cocoa trees to synthetize compounds with antimicrobial and surface active properties. It was determined that under in vitro conditions, seven strains (DS03, DS07, DS18, DS23, DS31, DS34 and DS50) exhibited antifungal activity that inhibited the mycelial growth of Moniliophthora roreri and Moniliophthora perniciosa. The strains were identified based on their morphological (pattern forming Bacilli) and genetic characteristics using the 16S SSU rRNA gene sequences. Furthermore, we determined that the endophytic strains contained genes that encoded the lipopeptides Fengycin (fenA, fenB, fenC, fenE), Iturin (ituB, ituC, ituD), and Bacyllomicin D (bamC, bamD). The production of biosurfactant compounds was determined by a reduction in surface tension in the mineral medium. Additionally, the biosurfactant crude extract (BCE) was tested for its inhibitory activity against M. perniciosa and M. roreri. The minimal inhibitory concentration (MIC) was determined to be 0.07 and 0.035 mg mL−1, respectively, in both cases in a fungistatic action mode. The swelling granulation and fragmentation of both phytopathogens’ hyphae were observed using the BCE treatment. These results suggested the biocontrol capacity of the endophytic strains, which under in vitro conditions, have the ability to synthesize antifungal and surface active compounds that might be used as an ecological alternative in cocoa plants to prevent infection by phytopathogens.

Biosurfactants synthesized by endophytic Bacillus strains as control of Moniliophthora perniciosa and Moniliophthora roreri

Ecuador is responsible for up to 70 % of the worlds production of fine flavor cocoa. Although fungal diseases have reduced cocoa production, eco–friendly alternatives have been implemented to control the spread of phytopathogens. This study aimed to explore the potential of endophytic strains isolated from fine flavor Nacional cocoa trees to synthetize compounds with antimicrobial and surface active properties. It was determined that under in vitro conditions, seven strains (DS03, DS07, DS18, DS23, DS31, DS34 and DS50) exhibited antifungal activity that inhibited the mycelial growth of Moniliophthora roreri and Moniliophthora perniciosa. The strains were identified based on their morphological (pattern forming Bacilli) and genetic characteristics using the 16S SSU rRNA gene sequences. Furthermore, we determined that the endophytic strains contained genes that encoded the lipopeptides Fengycin (fenA, fenB, fenC, fenE), Iturin (ituB, ituC, ituD), and Bacyllomicin D (bamC, bamD). The production of biosurfactant compounds was determined by a reduction in surface tension in the mineral medium. Additionally, the biosurfactant crude extract (BCE) was tested for its inhibitory activity against M. perniciosa and M. roreri. The minimal inhibitory concentration (MIC) was determined to be 0.07 and 0.035 mg mL−1, respectively, in both cases in a fungistatic action mode. The swelling granulation and fragmentation of both phytopathogens hyphae were observed using the BCE treatment. These results suggested the biocontrol capacity of the endophytic strains, which under in vitro conditions, have the ability to synthesize antifungal and surface active compounds that might be used as an ecological alternative in cocoa plants to prevent infection by phytopathogens.(AU)

Characterization of Pseudomonas bacteria of Piper tuberculatum regarding the production of potentially bio-stimulating compounds for plant growth / Caracterização de bactérias Pseudomonas de Piper tuberculatum quanto à produção de compostos potencialmente bioestimulantes para o crescimento de plantas

Despite advances in the identification and characterization of endophytic bacteria in various plant species worldwide, little is known about such microorganisms in plants from the Amazon region. Previous studies reported that Piper tuberculatum endophytic Pseudomonas (isolates Pt12 and Pt13, identified as Pseudomonas putida and Pseudomonas sp., respectively) were able to inhibit the in vitro growth of Fusarium solani f. sp. piperis, which causes root rot in black pepper (Piper nigrum), and that Pt13 promoted the growth of P. nigrum. Therefore, the aim here was to characterize these bacteria regarding their ability to produce plant growth-promoting substances [siderophores, indol acetic acid (IAA) and soluble phosphate]. Chrome azurol S assays were performed for the detection of siderophores. For qualitative and quantitative assays of IAA production and phosphate solubilization, Salkowski´s reagent and NBRIP medium with molybdenum blue reagent, respectively, were used. Results revealed that Pt12 and Pt13 were able to synthesize IAA, mainly under a high concentration of L-tryptophan, indicating that they are IAA-producing bacteria, probably through a tryptophan-dependent biosynthesis pathway. The presence of P. nigrum extract positively influenced the IAA production by Pt12 and Pt13, with highest values of 125 and 90 µg mL-1, respectively. In addition, Pt12 was positive for the production of siderophores and produced 56.56 µg mL-1 of soluble phosphate. In contrast, Pt13 showed no ability to produce siderophores or to solubilize phosphate. Besides their potential in controlling plant diseases, Pt12 and Pt13 have potential as biofertilizers, favoring sustainable agriculture.(AU)

Apesar dos avanços na identificação e caracterização de bactérias endofíticas em espécies vegetais em todo o mundo, pouco se sabe sobre esses microrganismos em plantas da região amazônica. Estudos anteriores mostraram que Pseudomonas de Piper tuberculatum (isolados Pt12 e Pt13, identificados como Pseudomonas putida e Pseudomonas sp., respectivamente) são capazes de inibir Fusarium solani f. sp. piperis, que causa a podridão das raízes da pimenteira-do-reino (Piper nigrum), e que Pt13 promoveu o crescimento de P. nigrum. Portanto, o objetivo do presente trabalho foi caracterizar essas bactérias quanto à capacidade de produzir substâncias potencialmente bioestimulantes para o crescimento vegetal [sideróforos, ácido indol acético (AIA) e fosfato solúvel]. Ensaios de Cromo Azurol S foram realizados para detecção de sideróforos. Para os ensaios qualitativos e quantitativos de produção de AIA e solubilização de fosfato, foram utilizados o reagente de Salkowski e o meio NBRIP com azul de molibdênio, respectivamente. Os resultados revelaram que Pt12 e Pt13 sintetizaram AIA, principalmente sob alta concentração de L-triptofano, indicando que provavelmente utilizam uma via de biossíntese dependente deste aminoácido. A presença do extrato de P. nigrum influenciou positivamente a produção de AIA por Pt12 e Pt13, com valores máximos de 125 e 90 µg mL-1, respectivamente. Além disso, Pt12 foi positiva para produção de sideróforos e produziu 56.56 µg.mL-1 de fosfato solúvel. Em contraste, Pt13 não produziu sideróforos, nem solubilizou fosfato. Além do potencial de controle de doenças de plantas, Pt12 e Pt13 têm potencial como biofertilizantes, favorecendo a agricultura sustentável.(AU)

Adaptation of plants to high-calcium content kart regions: possible involvement of symbiotic microorganisms and underlying mechanisms / Adaptação de plantas a regiões de kart com alto teor de cálcio: possível envolvimento de microrganismos simbióticos e mecanismos subjacentes

Rhizosphere microorganisms and endophytes can help their hosts absorb nutrients and regulate the levels of plant hormones. Moreover, they can modulate the expressions of host genes, assist hosts in eliminating reactive oxygen species (ROS) and secreting volatile organic compounds. Therefore, rhizosphere microorganisms and endophytes are considered as determinant factors driving processes involved in the growth of host plants. However, the physiological and ecological functions, as well as the molecular mechanism underlying the behavior of rhizosphere microorganisms and endophytes in their role in the adaptive capacity of host plants in the karstic high-calcium environment have not been systematically studied. This review summarizes the physiological and molecular mechanisms of rhizosphere microorganisms and endophytes which help host plants to adapt to various kinds of adverse environments. The adaptive capacities of plants growing in adverse environments, partly, or totally, depends on microorganisms co-existing with the host plants.(AU)

Os microorganismos e endófitos da rizosfera podem ajudar seus hospedeiros a absorver nutrientes e regular os níveis de hormônios vegetais. Além disso, eles podem modular as expressões dos genes hospedeiros, auxiliar os hospedeiros na eliminação de espécies reativas de oxigênio (ROS) e secretar compostos orgânicos voláteis. Portanto, microorganismos e endófitos da rizosfera são considerados determinantes dos processos envolvidos no crescimento de plantas hospedeiras. No entanto, as funções fisiológicas e ecológicas, bem como o mecanismo molecular subjacente ao comportamento dos microrganismos e endofíticos da rizosfera no seu papel na capacidade adaptativa das plantas hospedeiras no ambiente cárstico de alto teor de cálcio, não foram sistematicamente estudados. Esta revisão resume os mecanismos fisiológicos e moleculares de microrganismos e endófitos da rizosfera que ajudam as plantas hospedeiras a se adaptarem a vários tipos de ambientes adversos. As capacidades adaptativas das plantas que crescem em ambientes adversos, em parte ou totalmente, dependem de microrganismos coexistentes com as plantas hospedeiras.(AU)

Produção de enzimas por fungos filamentosos / Production of enzymes by filamental fungi

Estudos utilizando fungos endofíticos como produtores de metabólitos secundários de interesse biotecnológico vem sendo explorados. Assim, o presente trabalho objetivou avaliar a produção de enzimas por fungos filamentosos endofíticos, sendo escolhida a cepa 50 (C50), proveniente da coleção de cultura do Laboratório de Produtos Naturais e Biotecnologia (LPNBio) da UESB. A produção das enzimas amilase, celulase, invertase, lipase e poligalacturonase foi avaliada pelo o índice enzimático, atividade enzimática e verificado o tempo de fermentação de maior produtividade. Com exceção da invertase, a C50 apresentou atividade para as demais enzimas, destaque para lipase e poligalacturonase no tempo de 96 horas de fermentação. Estes resultados mostraram que a C50 tem potencial para ser explorada como produtora de enzimas.(AU)

Produção de enzimas por fungos filamentosos / Production of enzymes by filamental fungi

Estudos utilizando fungos endofíticos como produtores de metabólitos secundários de interesse biotecnológico vem sendo explorados. Assim, o presente trabalho objetivou avaliar a produção de enzimas por fungos filamentosos endofíticos, sendo escolhida a cepa 50 (C50), proveniente da coleção de cultura do Laboratório de Produtos Naturais e Biotecnologia (LPNBio) da UESB. A produção das enzimas amilase, celulase, invertase, lipase e poligalacturonase foi avaliada pelo o índice enzimático, atividade enzimática e verificado o tempo de fermentação de maior produtividade. Com exceção da invertase, a C50 apresentou atividade para as demais enzimas, destaque para lipase e poligalacturonase no tempo de 96 horas de fermentação. Estes resultados mostraram que a C50 tem potencial para ser explorada como produtora de enzimas.

Mangrove endophyte promotes reforestation tree (Acacia polyphylla) growth

Mangroves are ecosystems located in the transition zone between land and sea that serve as a potential source of biotechnological resources. Brazil's extensive coast contains one of the largest mangrove forests in the world (encompassing an area of 25,000 km2 along all the coast). Endophytic bacteria were isolated from the following three plant species: Rhizophora mangle, Laguncularia racemosa and Avicennia nitida. A large number of these isolates, 115 in total, were evaluated for their ability to fix nitrogen and solubilize phosphorous. Bacteria that tested positive for both of these tests were examined further to determine their level of indole acetic acid production. Two strains with high indole acetic acid production were selected for use as inoculants for reforestation trees, and then the growth of the plants was evaluated under field conditions. The bacterium Pseudomonas fluorescens (strain MCR1.10) had a low phosphorus solubilization index, while this index was higher in the other strain used, Enterobacter sp. (strain MCR1.48). We used the reforestation tree Acacia polyphylla. The results indicate that inoculation with the MCR1.48 endophyte increases Acacia polyphylla shoot dry mass, demonstrating that this strain effectively promotes the plant's growth and fitness, which can be used in the seedling production of this tree. Therefore, we successfully screened the biotechnological potential of endophyte isolates from mangrove, with a focus on plant growth promotion, and selected a strain able to provide limited nutrients and hormones for in plant growth.(AU)

Metabolites from endophytic Aspergillus fumigatus and their in vitro effect against the causal agent of tuberculosis / Metabolitos de Aspergillus fumigatus endofítico e seu efeito in vitro contra o agente causal da tuberculose

Tuberculosis (TB) remains one of the most deadly communicable infectious diseases, causing 1.4 million deaths in 2015 worldwide due to many conditions, including the inadequate treatment and the emergence of multidrug-resistant strains of the causal agent, Mycobacterium tuberculosis. Therefore, drugs developed from natural sources, as microorganisms and plant extracts, are a frequent target for the research and discovery of antimicrobial compounds. The current study started the characterization of compounds produced by an Aspergillus fumigatus isolated from copaíba (Copaifera multijuga) that efficiently inhibits M. tuberculosis by releasing the compounds into the fermentation broth under specific culture conditions. A preliminary assay was carried out with a correlate species, M. smegmatis, aiming to detect an antimicrobial effect related to A. fumigatus fermentation broth. The direct use of this substrate in antibiosis assays againstM. tuberculosis H37Rv strain (ATCC 27294) allowed the detection of antimicrobial activity with a minimal inhibitory concentration of 256 g mL-1, demonstrating that purification processes developed by the Biotage Flash Chromatography System are robust and reliable techniques for purification of compounds from natural sources. Also, this chromatographic system can be used in combination with specific biochemical tests, improving the search for reliable results. We conclude that this fraction can express a broad action range, inhibiting both Mycobacterium species used as target organisms.(AU)

A tuberculose continua a ser uma das doenças infecciosas transmissíveis mais mortais, causando 1,4 milhão de mortes em 2015 em todo o mundo devido a vários fatores, incluindo o tratamento inadequado e o surgimento de cepas multirresistentes do agente causal, Mycobacterium tuberculosis. Portanto, as drogas desenvolvidas a partir de fontes naturais, como micro-organismos e extratos de plantas, são um alvo freqüente para a pesquisa e descoberta de compostos antimicrobianos. O presente estudo foi um ponto de partida para caracterizar compostos produzidos por um Aspergillus fumigatus isolado de copaíba (Copaifera multijuga) que inibe eficientemente M. tuberculosis, liberando os compostos no caldo de fermentação em condições de cultura específicas. Realizou-se um ensaio preliminar com uma espécie correlata, M. smegmatis, com o objetivo de detectar um efeito antimicrobiano relacionado ao caldo de fermentação de A. fumigatus. O uso direto deste substrato em ensaios de antibiose contra a estirpe H37Rv de M. tuberculosis (ATCC 27294) permitiu a detecção de atividade antimicrobiana com uma concentração inibitória mínima de 256 g mL-1, demonstrando que os processos de purificação desenvolvidos pelo Biotage Flash Chromatography System são técnicas robustas e confiáveis para purificar compostos de fontes naturais. Além disso, este sistema cromatográfico pode ser usado em combinação com testes bioquímicos específicos, melhorando a busca de resultados confiáveis. Concluímos que esta fração pode expressar uma ampla gama de ação, inibindo ambas as espécies de Mycobacterium utilizadas como organismos-alvo.(AU)

Dark septate endophyte decreases stress on rice plants

Abiotic stress is one of the major limiting factors for plant development and productivity, which makes it important to identify microorganisms capable of increasing plant tolerance to stress. Dark septate endophytes can be symbionts of plants. In the present study, we evaluated the ability of dark septate endophytes isolates to reduce the effects of water stress in the rice varieties Nipponbare and Piauí. The experiments were performed under gnotobiotic conditions, and the water stress was induced with PEG. Four dark septate endophytes were isolated from the roots of wild rice (Oryza glumaepatula) collected from the Brazilian Amazon. Plant height as well as shoot and root fresh and dry matter were measured. Leaf protein concentrations and antioxidant enzyme activity were also estimated. The dark septate endophytes were grown in vitro in Petri dishes containing culture medium; they exhibited different levels of tolerance to salinity and water stress. The two rice varieties tested responded differently to inoculation with dark septate endophytes. Endophytes promoted rice plant growth both in the presence and in the absence of a water deficit. Decreased oxidative stress in plants in response to inoculation was observed in nearly all inoculated treatments, as indicated by the decrease in antioxidant enzyme activity. Dark septate endophytes fungi were shown to increase the tolerance of rice plants to stress caused by water deficiency.(AU)

Two methods for isolation of endophytic and edaphic Bacillus spp. from sugarcane fields / Dois métodos para isolamento de Bacillus spp. endofíticos e edáficos de canaviais

Bacillus has been widely studied and used for the control of pests and diseases. The adapted protocol proposed by POLANCZYK (2004) proved to be more efficient than the one by the World Health Organization (WHO, 1985) to isolate edaphic strains of Bacillus. However, it has not been assessed for isolation of endophytic strains, which are much less abundant in the nature and more difficult to be isolated. This study aimed to compare two methodological procedures for isolation of Bacillus, established by the WHO (1985) and by POLANCZYK (2004), regarding their efficiency for isolation of endophytics and edaphics Bacillus strains from inside the root tissue of sugarcane, as well as from the associated soil sample, collected from 11 locations; and to compare the density of bacteria in both environments. Endophytic and edaphic strains of Bacillus were isolated by both procedures. However, the isolation protocol performed by POLANCZYK (2004) made more efficient by having a greater number of colony forming units (CFU) per gram of soil and root indicating that this procedure is more useful, especially for isolation of endophytic strains of Bacillus, which are much less abundant in the nature than edaphic strains, being therefore more difficult to be isolated. Using the Polanczyk protocol (2004), Bacillus strains were recovered from all roots (endophytic) and soil (edaphic) samples of all the 11 fields, suggesting that the plant root may be another important source for isolation of Bacillus besides the soil. Higher densities of Bacillus were isolated from the edaphic environment compared with the endophytic environment, with significant differences when isolated by Polanczyk method (2004).

Bacillus tem sido amplamente estudado e usado para o controle de pragas e doenças. O protocolo adaptado proposto por POLANCZYK (2004) mostrou-se mais eficiente do que o da Organização Mundial de Saúde (WHO, 1985) para isolar cepas edáficas de Bacillus. No entanto, não foi avaliado quanto ao isolamento de estirpes endofíticas, que são muito menos abundantes na natureza e mais difíceis de isolar. Este estudo teve como objetivos comparar dois procedimentos metodológicos para o isolamento de Bacillus, o estabelecido pela OMS (WHO, 1985) e o de POLANCZYK (2004), quanto a sua eficiência para o isolamento de estirpes endofitas e edáficas de Bacillus originárias do interior do tecido radicular de cana-de-açúcar, bem como de amostras de solos associados, coletada de 11 locais; e comparar a densidade de bactérias em ambos os ambientes. As cepas endofíticas e edáficas de Bacillus foram isoladas por ambos os procedimentos. No entanto, o protocolo de isolamento realizado por POLANCZYK (2004) demonstrou-se mais eficiente por gerar maior número de unidades de formação de colônias (CFU) por grama de solo e raiz, indicando que esse procedimento é mais útil, especialmente para isolamento de estirpes endofíticas de Bacillus, que são muito menos abundantes na natureza do que as cepas edáficas, sendo, portanto, mais difíceis de serem isoladas. Usando o protocolo de POLANCZYK (2004), as cepas de Bacillus foram isoladas de todas as amostras de raízes (endofíticas) e de solo (edáficas) de todos os 11 campos, sugerindo que a raiz da planta pode ser outra fonte importante de isolamento de Bacillus além do solo. As densidades mais altas de Bacillus foram isoladas do ambiente edáfico em comparação com o ambiente endofítico, com diferenças significativas quando isoladas pelo método de POLANCZYK (2004).

Endophytic fungus Purpureocillium sp. A5 protect mangrove plant Kandelia candel under copper stress

Mangrove is an important ecosystem in the world. Mangrove ecosystems have a large capacity in retaining heavy metals, and now they are usually considered as sinks for heavy metals. However, the mechanism of why the soil of mangrove ecosystems can retain heavy metal is not certain. In this research, endophytic fungus Purpureocillium sp. A5 was isolated and identified from the roots of Kandelia candel. When this fungus was added, it protected the growth of K. candel under Cu stress. This can be illustrated by analyzing chlorophyll A and B, RWC and WSD to leaves of K. candel. Purpureocillium sp. A5 reduces uptake of Cu in K. candel and changes the pH characterization of soil. Furthermore, A5 increase the concentration of Cu complexes in soil, and it enhanced the concentration of carbonate-bound Cu, Mn-Fe complexes Cu and organic-bound Cu in soil. Nevertheless, a significant reduction of the Cu ion was noted among A5-treated plants. This study is significant and illustrates a promising potential use for environmental remediation of endophytes, and also may partially explain the large capacity of mangrove ecosystems in retaining heavy metals.(AU)

Two methods for isolation of endophytic and edaphic Bacillus spp. from sugarcane fields / Dois métodos para isolamento de Bacillus spp. endofíticos e edáficos de canaviais

Bacillus has been widely studied and used for the control of pests and diseases. The adapted protocol proposed by POLANCZYK (2004) proved to be more efficient than the one by the World Health Organization (WHO, 1985) to isolate edaphic strains of Bacillus. However, it has not been assessed for isolation of endophytic strains, which are much less abundant in the nature and more difficult to be isolated. This study aimed to compare two methodological procedures for isolation of Bacillus, established by the WHO (1985) and by POLANCZYK (2004), regarding their efficiency for isolation of endophytics and edaphics Bacillus strains from inside the root tissue of sugarcane, as well as from the associated soil sample, collected from 11 locations; and to compare the density of bacteria in both environments. Endophytic and edaphic strains of Bacillus were isolated by both procedures. However, the isolation protocol performed by POLANCZYK (2004) made more efficient by having a greater number of colony forming units (CFU) per gram of soil and root indicating that this procedure is more useful, especially for isolation of endophytic strains of Bacillus, which are much less abundant in the nature than edaphic strains, being therefore more difficult to be isolated. Using the Polanczyk protocol (2004), Bacillus strains were recovered from all roots (endophytic) and soil (edaphic) samples of all the 11 fields, suggesting that the plant root may be another important source for isolation of Bacillus besides the soil. Higher densities of Bacillus were isolated from the edaphic environment compared with the endophytic environment, with significant differences when isolated by Polanczyk method (2004).(AU)

Bacillus tem sido amplamente estudado e usado para o controle de pragas e doenças. O protocolo adaptado proposto por POLANCZYK (2004) mostrou-se mais eficiente do que o da Organização Mundial de Saúde (WHO, 1985) para isolar cepas edáficas de Bacillus. No entanto, não foi avaliado quanto ao isolamento de estirpes endofíticas, que são muito menos abundantes na natureza e mais difíceis de isolar. Este estudo teve como objetivos comparar dois procedimentos metodológicos para o isolamento de Bacillus, o estabelecido pela OMS (WHO, 1985) e o de POLANCZYK (2004), quanto a sua eficiência para o isolamento de estirpes endofitas e edáficas de Bacillus originárias do interior do tecido radicular de cana-de-açúcar, bem como de amostras de solos associados, coletada de 11 locais; e comparar a densidade de bactérias em ambos os ambientes. As cepas endofíticas e edáficas de Bacillus foram isoladas por ambos os procedimentos. No entanto, o protocolo de isolamento realizado por POLANCZYK (2004) demonstrou-se mais eficiente por gerar maior número de unidades de formação de colônias (CFU) por grama de solo e raiz, indicando que esse procedimento é mais útil, especialmente para isolamento de estirpes endofíticas de Bacillus, que são muito menos abundantes na natureza do que as cepas edáficas, sendo, portanto, mais difíceis de serem isoladas. Usando o protocolo de POLANCZYK (2004), as cepas de Bacillus foram isoladas de todas as amostras de raízes (endofíticas) e de solo (edáficas) de todos os 11 campos, sugerindo que a raiz da planta pode ser outra fonte importante de isolamento de Bacillus além do solo. As densidades mais altas de Bacillus foram isoladas do ambiente edáfico em comparação com o ambiente endofítico, com diferenças significativas quando isoladas pelo método de POLANCZYK (2004).(AU)