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.

Bioprospection and molecular phylogeny of culturable endophytic fungi associated with yellow passion fruit

Endophytic microorganisms live inside the plants without causing any damage to their hosts. In the agricultural field, these endophytes might be a strategy of biological control for phytopathogens. We aimed to isolate endophytic fungifrom yellowpassion fruit (Passiflora edulis) leaves, evaluating its biocontrol capacity by in vitroantagonism against phytopathogen Colletotrichum sp. CNPU378. We also carried out greenhouse experiments in bean seedlings. A high colonization frequency was obtained (89%), and the molecular identification based on DNA sequencing attested Colletotrichumas the most frequent genus and minor occurrence of Curvulariaendophytes. The endophytes tested showed different types of competitive interactions in in vitro antagonism inhibition rate ranging from 28.8 to 48.8%. There were 10 promising antagonists tested for their antagonist activity of crude extracts of secondary metabolites, in which strain PE-36 (20.8%) stood out among the other strains evaluated. In the greenhouse assay, plants inoculated only with endophyte Colletotrichumsp. PE-36 was symptomless and suggest that the endophyte strengthened the growth promotion in common bean plants, especially in the root length and number of leaves when compared to control plantsand other treatments. Despite many fungiof Colletotrichumgenus being described as causative agents of anthracnose, in this study, the plant sampled was colonized predominantly by Colletotrichumendophytes living in asymptomatic relationship. By the way,we come across a Colletotrichumsp. endophyte able to antagonize a Colletotrichumsp. pathogen

Bioassay on banana rhizome discs for selecting bacteria antagonistic to Fusarium oxysporum f. sp. cubense / Bioensaio em discos de rizomas de bananeiras para a seleção de bactérias antagonistas a Fusarium oxysporum f. sp. cubense

Detached plant organs are alternative materials to in vitro tests for selecting biocontrol agents. On the other hand, the use of scales to quantify injured areas can generate inconsistent results. Rhizospheric and endophytic bacteria were selected as growth inhibitors of Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Panama disease of banana. For this, rhizome discs were treated with 200 µL of antagonist suspension (109 CFU mL−¹) and inoculated with the pathogen. The material was placed in plastic gerbox boxes and incubated in BOD at 25 °C for 12 days. Afterward, the area of rhizome discs (mm²) colonized by Foc was quantified by digital images. The assay was set up in a completely randomized design, with four replications and three discs per replication. The control consisted of untreated and inoculated discs. The results showed the efficiency of this method in selecting the biological control agent, as the 26 isolates were group into five different clusters, with isolates belonging to four of these groups (from '1' to '4') being able to reduce Foc colonization. Isolates 520EB, 993EB, and 531EB had the highest potential for inhibition, with areas of 343.3, 344.1, 364.8 mm², respectively, promoting inhibition ranging from 53 to 56 % of the colonized area compared to the control (782.6 mm²).

O emprego de órgãos vegetais destacados é uma alternativa aos testes in vitro para seleção de agentes de biocontrole. Por outro lado, o uso de escalas para quantificação de área lesionada pode gerar resultados inconsistentes. Bactérias rizosféricas e endofíticas foram selecionadas como inibidoras do crescimento de Fusarium oxysporum f. sp. cubense (Foc), agente causal do mal-do-Panamá da bananeira. Para tanto, discos de rizoma foram tratados com 200 µL de suspensão dos antagonistas (109 UFC mL-¹) e inoculados com patógeno. O material foi disposto em caixas plásticas tipo gerbox e incubado em BOD a 25 ºC, por doze dias. Após, a área dos discos de rizoma (mm²) colonizada por Foc dos discos foi quantificada por meio de imagens digitais. O ensaio foi montado em delineamento inteiramente casualizado, com quatro repetições e três discos por repetição. Discos não tratados e inoculados compuseram o controle. Com base nos resultados obtidos, foi possível verificar a eficiência do método na seleção de agente de controle biológico, uma vez que foi possível o agrupamento dos 26 isolados utilizados em cinco clústeres distintos, sendo que isolados pertencentes a quatro destes grupos (do '1' a '4') foram capazes de proporcionar a redução da colonização por Foc. Os isolados com maior potencial de inibição foram520EB, 993EB e 531EB com 343,3, 344,1, 364,8 mm2, respectivamente, promovendo inibição variando de 53 a 56 % da área colonizada quando comparados com ao controle (782,6 mm²).

Bioassay on banana rhizome discs for selecting bacteria antagonistic to Fusarium oxysporum f. sp. cubense / Bioensaio em discos de rizomas de bananeiras para a seleção de bactérias antagonistas a Fusarium oxysporum f. sp. cubense

Detached plant organs are alternative materials to in vitro tests for selecting biocontrol agents. On the other hand, the use of scales to quantify injured areas can generate inconsistent results. Rhizospheric and endophytic bacteria were selected as growth inhibitors of Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Panama disease of banana. For this, rhizome discs were treated with 200 µL of antagonist suspension (109 CFU mL−¹) and inoculated with the pathogen. The material was placed in plastic gerbox boxes and incubated in BOD at 25 °C for 12 days. Afterward, the area of rhizome discs (mm²) colonized by Foc was quantified by digital images. The assay was set up in a completely randomized design, with four replications and three discs per replication. The control consisted of untreated and inoculated discs. The results showed the efficiency of this method in selecting the biological control agent, as the 26 isolates were group into five different clusters, with isolates belonging to four of these groups (from '1' to '4') being able to reduce Foc colonization. Isolates 520EB, 993EB, and 531EB had the highest potential for inhibition, with areas of 343.3, 344.1, 364.8 mm², respectively, promoting inhibition ranging from 53 to 56 % of the colonized area compared to the control (782.6 mm²).(AU)

O emprego de órgãos vegetais destacados é uma alternativa aos testes in vitro para seleção de agentes de biocontrole. Por outro lado, o uso de escalas para quantificação de área lesionada pode gerar resultados inconsistentes. Bactérias rizosféricas e endofíticas foram selecionadas como inibidoras do crescimento de Fusarium oxysporum f. sp. cubense (Foc), agente causal do mal-do-Panamá da bananeira. Para tanto, discos de rizoma foram tratados com 200 µL de suspensão dos antagonistas (109 UFC mL-¹) e inoculados com patógeno. O material foi disposto em caixas plásticas tipo gerbox e incubado em BOD a 25 ºC, por doze dias. Após, a área dos discos de rizoma (mm²) colonizada por Foc dos discos foi quantificada por meio de imagens digitais. O ensaio foi montado em delineamento inteiramente casualizado, com quatro repetições e três discos por repetição. Discos não tratados e inoculados compuseram o controle. Com base nos resultados obtidos, foi possível verificar a eficiência do método na seleção de agente de controle biológico, uma vez que foi possível o agrupamento dos 26 isolados utilizados em cinco clústeres distintos, sendo que isolados pertencentes a quatro destes grupos (do '1' a '4') foram capazes de proporcionar a redução da colonização por Foc. Os isolados com maior potencial de inibição foram520EB, 993EB e 531EB com 343,3, 344,1, 364,8 mm2, respectivamente, promovendo inibição variando de 53 a 56 % da área colonizada quando comparados com ao controle (782,6 mm²).(AU)

Tripartite symbiosis of Sophora tomentosa, rhizobia and arbuscular mycorhizal fungi

ABSTRACT Sophora tomentosa is a pantropical legume species with potential for recovery of areas degraded by salinization, and for stabilization of sand dunes. However, few studies on this species have been carried out, and none regarding its symbiotic relationship with beneficial soil microorganisms. Therefore, this study aimed to evaluate the diversity of nitrogen-fixing bacteria isolated from nodules of Sophora tomentosa, and to analyze the occurrence of colonization of arbuscular mycorrhizal fungi on the roots of this legume in seafront soil. Thus, seeds, root nodules, and soil from the rhizosphere of Sophora tomentosa were collected. From the soil samples, trap cultures with this species were established to extract spores and to evaluate arbuscular mycorhizal fungi colonization in legume roots, as well as to capture rhizobia. Rhizobia strains were isolated from nodules collected in the field or from the trap cultures. Representative isolates of the groups obtained in the similarity dendrogram, based on phenotypic characteristics, had their 16S rRNA genes sequenced. The legume species showed nodules with indeterminate growth, and reddish color, distributed throughout the root. Fifty-one strains of these nodules were isolated, of which 21 were classified in the genus Bacillus, Brevibacillus, Paenibacillus, Rhizobium and especially Sinorhizobium. Strains closely related to Sinorhizobium adhaerens were the predominant bacteria in nodules. The other genera found, with the exception of Rhizobium, are probably endophytic bacteria in the nodules. Arbuscular mycorrhizal fungi was observed colonizing the roots, but arbuscular mycorhizal fungi spores were not found in the trap cultures. Therefore Sophora tomentosa is associated with both arbuscular mycorhizal fungi and nodulating nitrogen-fixing bacteria.(AU)

Metarhizium spp.: Seleção de isolados, ação antimicrobiana e eficácia de formulações granulares no controle de Rhipicephalus microplus

Metarhizium sp. é encontrado no solo e em raízes de plantas e a pastagem é um ambiente propício para o desenvolvimento desse entomopatogeno, tornando a rizosfera de forrageiras e a presença de hospedeiros como o carrapato Rhipicephalus microplus um nicho ecológico ideal. Porém, fatores bióticos e abióticos interferem na persistência fúngica, sendo fundamental selecionar bons isolados, considerar as interações ecológicas com outros microrganismos e formulá-los. Baseado nisso, o presente trabalho apresenta estudos aplicados ao gênero Metarhizium no controle de pragas. Foram avaliados: a tolerância térmica e a capacidade de diferentes isolados do fungo em colonizar tecidos da planta Vigna radiata; a expressão do gene de adesão na planta em diferentes substratos; o efeito antimicrobiano e acaricida do exsudato fúngico; e a eficácia de formulações granulares de microescleródios (MS) e blastosporos (BLS) para o controle R. microplus. No ensaio de tolerância térmica, suspensões conidiais foram expostas a 25ºC, 40°C e 45°C, por 2h, 4h, 8h e 12h, e posteriormente a germinação foi avaliada após incubação por 48h a 25 ± 1°C, para a atividade dos isolados no frio esses foram submetidos à temperatura de 5ºC durante 14 dias, selecionando assim os termotolerantes. Para o estudo de colonização endofítica, sementes, raiz, caule e folha de V. radiata foram tratadas com suspensões de diferentes isolados, e a colonização dos tecidos do vegetal foi avaliada. No ensaio de expressão gênica, após o cultivo do fungo sobre meio mínimo (MM), meio batata dextrose ágar (BDA) e sobre a planta, a expressão do Gene mad2 foi observada. Para avaliar o efeito do exsudato de Metarhizium sp. no carrapato e em bactérias, o líquido resultante da exsudação de colônias foi coletado e a partir do teste de difusão em disco sobre cepas de duas bactérias isoladas do solo e sobre cepas resistentes e sensíveis de Staphylococcus sp. e Escherichia coli foi avaliado. Para o efeito acaricida, o exsudato fúngico foi inoculado nas fêmeas do carrapato e parâmetros biológicos, mortalidade e resposta celular do artrópode foram avaliadas. Por último, no teste de eficácia de formulações granulares de MS e BLS, vasos contendo Urochloa decumbens foram tratados com os grânulos, e posteriormente fêmeas de R. microplus foram introduzidas. Foram avaliados, a mortalidade, o número de larvas infestantes, assim como a persistência do fungo no solo. Quanto aos resultados, no teste de tolerância térmica, todos os isolados de Metarhizium spp testados germinaram mais que 90% após 12h a 40 °C e após a incubação no frio. Foram considerados bons isolados, aqueles que não sofreram variação drástica na germinação ao longo do tempo de exposição. Ao avaliar a capacidade de colonizar V. radiata, todos os isolados testados foram capazes de colonizar raízes, caules e folhas, havendo variação na colonização dos diferentes tecidos. Quando o fungo se desenvolveu sobre a planta o gene mad2 foi mais expresso que em BDA, porem foi no MM que o gene foi mais expresso. O exsudato fúngico foi capaz de inibir o crescimento das cepas resistente e sensível de Staphylococcus spp., e afetou mais as cepas de bactérias isoladas do solo do que as demais quando comparadas entre si. O exsudato também afetou diretamente o carrapato, reduzindo a resposta celular, causando morte e diminuição da postura de ovos. As formulações granulares de M. robertsii mostraram-se eficazes em controlar o carrapato nas condições testadas e o isolado fúngico colonizou a rizosfera da de U. decumbens. Este estudo fornece dados sobre a aplicação de isolados de Metarhizium spp. no solo e contribui para futuras pesquisas e bioprospecção desse microrganismo.

Metarhizium sp. species could be found in soil and plant roots, and the pasture is ideal environment for this fungus development, where the plant's rhizosphere and the presence of hosts such as the tick Rhipicephalus microplus make the best ecological niche. However, biotic and abiotic factors interfere on its persistence been essential to select good isolates, consider ecological interactions with other microorganisms, and formulate them. Based on this, the present study considers the applicability of the genus Metarhizium spp. in pest control. Were evaluated: thermal tolerance and the ability of different isolates of the fungus to colonize tissues of the Vigna radiata plant; the antimicrobial and acaricidal effect of fungal exudate; and the effectiveness of granular formulations of microsclerotia (MS) and blastospores(BLS) to control cattle ticks. In the thermal tolerance test, conidial suspensions of the fungus were exposed to 25 ° C, 40 ° C and 45 ° C, for 2h, 4h, 8h and 12h, the germination was evaluated after incubation for 48h at 28 ± 1 ° C, and also the cold activity of the isolates (at 5 ºC for 14 days) were evaluated, based on that, thermal tolerant isolates were selected. For the study of endophytic colonization, seeds, root, stem, and leaf of V. radiata were treated with fungus suspensions, and the colonization of the plant tissues by different isolates was evaluated. In the gene expression assay, after cultivating the fungus on minimal medium (MM), potato dextrose agar (BDA) and on the plant, the expression of the mad2 gene was observed. To assess the effect of Metarhizium sp. exudate on ticks and bacteria, the liquid resulting from colonies exudation was collected. Then, using disk diffusion test, the effect on strains of two bacteria isolated from the soil and on resistant and sensitive strains of Staphylococcus and Escherichia coli was evaluated. For the action on ticks, the fungal exudate was inoculated in tick females and the biological parameters, mortality, and immune response of the arthropod were evaluated. Finally, for the test with MS and BLS granular formulations, pots containing Urochloa decumbens were used and their soil was treated with the granular formulations, where later R. microplus females were introduced and the mortality, the number of larvae obtained in each group, as well as the persistence of the fungus in the soil were evaluated. As regards to the results, in the thermal tolerance test, all Metarhizium spp. isolates tested germinated more than 90% after 12h at 40 °C and after the be cooled, been considered good isolates those that did not suffer drastic variation in germination over the time of exposure. When evaluated the ability to colonize V. radiata, all tested isolates were able to colonize roots, stems, and leaves, with variation in the colonization of different tissues. When the fungus developed on the plant, the mad2 gene was more expressed than in PDA, but it was in the MM that the gene obtained greater expression. The fungal exudate was also able to inhibit the growth of the resistant and sensitive strains of Staphylococcus spp., and it affected more the strains of bacteria isolated from the soil than the others. The exudate also directly affected engorged R. microplus females, reducing the cellular response, causing death, and decreased egg production. The treatment with different concentrations of granular formulations of M. robertsii proved to be effective in controlling the cattle tick under the conditions tested and the fungus isolate colonized U. decumbens rhizosphere. This study provides data about the application of Metarhizium spp. in the soil and contributes to future research, showing the bioprospecting of this microorganism.

Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw

Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.(AU)

PRODUÇÃO DE L-ASPARAGINASE E DIVERSIDADE DE FUNGOS ENDOFÍTICOS DE Mandevilla catimbauensis (APOCYNACEAE) DO PARQUE NACIONAL DO VALE DO CATIMBAU

A Caatinga, região exclusivamente brasileira, possui 47 unidades de conservação, entre estas, o Parque Nacional do Vale do Catimbau. O Parque é composto por inúmeras espécies endêmicas, entre elas, Mandevilla catimbauensis, uma espécie rara, pertencente à família Apocynaceae, e que está classificada como vulnerável à extinção. Os fungos endofíticos vivem no interior dos vegetais, sem lhes causar danos. Alguns endófitos possuem potencial para produzir substâncias bioativas como a enzima L-asparaginase. A L-asparaginase é utilizada no tratamento de diversos cânceres em humanos e outros animais. É também utilizada na indústria alimentícia para redução dos níveis de acrilamida dos alimentos. Pequisadores buscam fontes eucarióticas capazes de produzir L-asparaginase com menores efeitos colaterais. O objetivo desse estudo foi estimar a diversidade de fungos endofíticos presentes em M. catimbauensis e avaliar o potencial biotecnológico desses fungos na produção de L-asparaginase. Um total de 66 isolados foram obtidos e, a taxa de colonização dos fragmentos foi de 11,78%. As análises filogenéticas utilizando sequências de ITS e/ou LSU do DNAr revelaram a presença de sete ordens do filo Ascomycota. Um total de 18 táxons foi identificados. Os endófitos mais fequentemente isolados foram membros do gênero Phyllosticta (45,10%). A curva de acumulação de espécies não alcançou o ponto de estabilização. Uma nova espécie do gênero Phyllosticta foi descoberta, descrita e publicada como Phyllosticta catimbauensis. A produção de L-asparaginase foi estudada por 20 isolados. Um total de 14 fungos apresentou capacidade de produzir a enzima (0,48 U g-1 2,22 U g-1). A espécie P. catimbauensis exibiu capacidade significativa, e foi selecionada para realizar um planejamento fatorial 23. A melhor produção enzimática foi 2,25 U g-1, utilizando 1,5 g de L-asparagina, pH 5 e 1,5 g inóculo. Posteriormente, uma sequência experimental foi realizada e foi possível obter um aumento significativo na produção de L-asparaginase de 3,50 U g-1, utilizando 3,5 g de L-asparagina, pH 4,2 e 1 g de inóculo. Esse é o primeiro estudo sobre os fungos endofíticos de M. catimbauensis e também da produção de L-asparaginase por esses fungos. Essa pesquisa, poderá contribuir com o conhecimento da comunidade fúngica presente em M. catimbaunensis, além de auxiliar futuros estudos sobre a produção enzimática desses endófitos.

The Caatinga, an exclusively Brazilian region, has 47 conservation units, among them, the Catimbau Valley National Park. The park is compound of numerous endemic species, including Mandevilla catimbauensis, a rare species belonging to the family Apocynaceae, which is classified as vulnerable to extinction. Endophytic fungi live inside the plants without causing them harm. Some endophytes have the potential to produce bioactive substances like the enzyme L-asparaginase. L-asparaginase is used to treatment various cancers in humans and other animals. It is also used in the food industry to reduce the levels of acrylamide in food. Researchers pursue for eukaryotic sources capable of producing L-asparaginase with lower side effects. The aim of this study was to estimate the diversity of endophytic fungi present in M. catimbauensis and to evaluate the biotechnological potential of these fungi in the production of L-asparaginase. A total of 66 isolates were obtained and the rate of colonization of the fragments was 11.78%. Phylogenetic analyzes using ITS and / or LSU sequences of rDNA revealed the presence of seven orders of the Ascomycota phylum. A total of 18 taxa were identified. The most commonly isolated endophytes were members of the genus Phyllosticta (45.10%). The curve of species accumulation did not reach the point of stabilization. A new species of the genus Phyllosticta was discovered, described and published as Phyllosticta catimbauensis. The production of L-asparaginase was studied by 20 isolates. A total of 14 fungi were efficient to produce the enzyme (0,48 U g-1 2,22 U g-1). The species P. catimbauensis exhibited significant capacity and was selected to perform a factorial design 23. The best enzymatic production was 2,25 U g-1, using 1,5 g of L-asparagine, pH 5 and 1,5 g inoculum . Subsequently, an experimental sequence was performed and a significant increase in L-asparaginase production of 3,50 U g-1 was obtained using 3,5 g of L-asparagine, pH 4,2 and 1 g of inoculum. This is the first study on the endophytic fungi of M. catimbauensis and also on the production of L-asparaginase by these fungi. This research may contribute to the knowledge of the fungal community present in M. catimbaunensis, besides helping future studies on the enzymatic production of these endophytes.

Methylobacterium-plant interaction genes regulated by plant exudate and quorum sensing molecules

Bacteria from the genus Methylobacterium interact symbiotically (endophytically and epiphytically) with different plant species. These interactions can promote plant growth or induce systemic resistance, increasing plant fitness. The plant colonization is guided by molecular communication between bacteria-bacteria and bacteria-plants, where the bacteria recognize specific exuded compounds by other bacteria (e.g. homoserine molecules) and/or by the plant roots (e.g. flavonoids, ethanol and methanol), respectively. In this context, the aim of this study was to evaluate the effect of quorum sensing molecules (N-acyl-homoserine lactones) and plant exudates (including ethanol) in the expression of a series of bacterial genes involved in Methylobacterium-plant interaction. The selected genes are related to bacterial metabolism (mxaF), adaptation to stressful environment (crtI, phoU and sss), to interactions with plant metabolism compounds (acdS) and pathogenicity (patatin and phoU). Under in vitro conditions, our results showed the differential expression of some important genes related to metabolism, stress and pathogenesis, thereby AHL molecules up-regulate all tested genes, except phoU, while plant exudates induce only mxaF gene expression. In the presence of plant exudates there is a lower bacterial density (due the endophytic and epiphytic colonization), which produce less AHL, leading to down regulation of genes when compared to the control. Therefore, bacterial density, more than plant exudate, influences the expression of genes related to plant-bacteria interaction.

Colonization and plant growth promoting characterization of endophytic Pseudomonas chlororaphis strain Zong1 isolated from Sophora alopecuroides root nodules

The endophytic strain Zong1 isolated from root nodules of the legume Sophora alopecuroides was characterized by conducting physiological and biochemical tests employing gfp-marking, observing their plant growth promoting characteristics (PGPC) and detecting plant growth parameters of inoculation assays under greenhouse conditions. Results showed that strain Zong1 had an effective growth at 28 ºC after placed at 4-60 ºC for 15 min, had a wide range pH tolerance of 6.0-11.0 and salt tolerance up to 5% of NaCl. Zong1 was resistant to the following antibiotics (µg/mL): Phosphonomycin (100), Penicillin (100) and Ampicillin (100). It could grow in the medium supplemented with 1.2 mmol/L Cu, 0.1% (w/v) methylene blue and 0.1-0.2% (w/v) methyl red, respectively. Zong1 is closely related to Pseudomonas chlororaphis based on analysis the sequence of 16S rRNA gene. Its expression of the gfp gene indicated that strain Zong1 may colonize in root or root nodules and verified by microscopic observation. Furthermore, co-inoculation with Zong1 and SQ1 (Mesorhizobium sp.) showed significant effects compared to single inoculation for the following PGPC parameters: siderophore production, phosphate solubilization, organic acid production, IAA production and antifungal activity in vitro. These results suggest strains P. chlororaphi Zong1 and Mesorhizobium sp. SQ1 have better synergistic or addictive effect. It was noteworthy that each growth index of co-inoculated Zong1+SQ1 in growth assays under greenhouse conditions is higher than those of single inoculation, and showed a significant difference (p < 0.05) when compared to a negative control. Therefore, as an endophyte P. chlororaphis Zong1 may play important roles as a potential plantgrowth promoting agent.(AU)

Specific plant induced biofilm formation in Methylobacterium species

Two endophytic strains of Methylobacterium spp. were used to evaluate biofilm formation on sugarcane roots and on inert wooden sticks. Results show that biofilm formation is variable and that plant surface and possibly root exudates have a role in Methylobacterium spp. host recognition, biofilm formation and successful colonization as endophytes.

Structural interaction between GFP-labeled diazotrophic endophytic bacterium Herbaspirillum seropedicae RAM10 and pineapple plantlets 'Vitória'

The events involved in the structural interaction between the diazotrophic endophytic bacterium Herbaspirillum seropedicae, strain RAM10, labeled with green fluorescent protein, and pineapple plantlets 'Vitória' were evaluated by means of bright-field and fluorescence microscopy, combined with scanning electron microscopy for 28 days after inoculation. After 6 hours of inoculation, H. seropedicae was already adhered to the roots, colonizing mainly root hair surface and bases, followed by epidermal cell wall junctions. Bacteria adherence in the initial periods occurred mainly in the form of solitary cells and small aggregates with pleomorphic cells. Bacteria infection of root tissue occurred through the cavities caused by the disruption of epidermal cells during the emergence of lateral roots and the endophytic establishment by the colonization of intercellular spaces of the cortical parenchyma. Moreover, within 1 day after inoculation the bacteria were colonizing the shoots. In this region, the preferred sites of epiphytic colonization were epidermal cell wall junctions, peltate scutiform trichomes and non-glandular trichomes. Subsequently, the bacteria occupied the outer periclinal walls of epidermal cells and stomata. The penetration into the shoot occurred passively through stoma aperture followed by the endophytic establishment on the substomatal chambers and spread to the intercellular spaces of spongy chlorenchyma. After 21 days of inoculation, bacterial biofilm were seen at the root hair base and on epidermal cell wall surface of root and leaf, also confirming the epiphytic nature of H. seropedicae.

Occurrence of endophytic bacteria of the genus Azospirillum in lowland rice plants collected in Santa Catarina state / Ocorrência de bactérias endofíticas do gênero Azospirillum em arroz irrigado no estado de Santa Catarina

Bacteria of the genus Azospirillum can endophytically colonize lowland rice plants and contribute to the development of this culture. The objective of this work was to evaluate the occurrence of endophytic Azospirillum spp. isolated from roots and stems of lowland rice plants grown in the State of Santa Catarina, Brazil. Samples of lowland rice plants were collected in eight farms in the cities of Guaramirim (two areas), Massaranduba, Rodeio (two areas), Rio do Sul, Agronômica and Pouso Redondo. Azospirillum spp. occurrence in disinfested roots and stems of the collected plants was evaluated using the More Probable Number method (MPN), in two semi-solid selective broth: NFb (A. lipoferum and A. brasilense) and LGI (A. amazonense), and the similarity among the eight areas was estimated by the principal component analysis (PCA). The bacteria grown in semi-solid broths were purified in their respective broths, isolated in PDA broth to obtain pure and morphologically characterized cultures. The mean population density of Azospirillum spp. varied from 1.6x108 to 1.42x108 cells g-1 of fresh matter (LGI), and from 1.76x108 to 1.56x108 cells g-1 of fresh matter (NFb), in roots and stem, respectively. Sixty two (62) isolates were obtained, most of them from disinfested roots. The results showed that Azospirillum spp. occurs in satisfactory population densities in lowland rice fields in

Bactérias do gênero Azospirillum podem colonizar endofiticamente plantas de arroz irrigado e contribuem para o desenvolvimento da cultura. O objetivo desse estudo foi avaliar a ocorrência de bactérias endofíticas do gênero Azospirillum isoladas de raízes e colmos de arroz irrigado cultivado em Santa Catarina. Amostras de plantas de arroz irrigado foram coletadas em oito propriedades rurais nas cidades de Guaramirim (duas áreas), Massaranduba, Rodeio (duas áreas), Rio do Sul, Agronômica e Pouso Redondo. A ocorrência de bactérias endofíticas do gênero Azospirillum em raízes desinfestadas e colmos foi avaliada através do método do Número Mais Provável de Propágulos (NMP), em meio seletivo semi-sólido NFb (A. lipoferum e A. brasilense) e LGI (A. amazonense), e a similaridade entre as áreas foi estimada através da análise de componentes principais (ACP). As bactérias crescidas nos meios semi-seletivos foram purificadas em seus respectivos meios, isoladas em meio BDA até a obtenção de culturas puras e caracterizadas morfologicamente. A densidade populacional média de Azospirillum spp. foi de 1,6x108 e 1,42x108 células g-1 de matéria fresca (meio LGI), e 1,76x108 e 1,56x108 células g-1 de matéria fresca (meio NFb), para raízes e colmos respectivamente. Ao final do isolamento, foram obtidos 62 isolados, dos quais a maior parte oriunda de raízes desinfestadas. Os resultados obtidos most

Efeito dos ácidos húmicos na inoculação de bactérias diazotróficas endofíticas em sementes de milho

The objective of this research was to evaluate the effect of the humic acids in the inoculation of endophytic diazotrophic bacteria in corn seeds. It was used the bacteria Herbaspirillum seropedicae Z67 BR 11175. The inoculation of the seeds with the bacteria and the addition of humic acids were accomplished by the coating of the corn seeds UENF 506-8. The coating was accomplished with a mixture of lime, semi-solid middle culture, water and cement. The seeds covered were sown in Leonard vases. Forty days after sowing the plants were collected and the bacteria couting was accomplished in the roots by the Most probable Number technique. The results showed that the humic acids doesn't interfere negatively in the bacteria growth of and they stimulate the colonization of the native microbiota. However, in the appraised conditions, the united application of bacteria + humic acids didn't stimulate the growth of the bacteria population inoculated in the corn seedling.

Os ácidos húmicos podem atuar no aumento da população de bactérias diazotróficas introduzidas no interior da planta e, consequentemente, no incremento dos efeitos benéficos sobre a planta hospedeira. Com este trabalho, objetivou-se avaliar o efeito dos ácidos húmicos na inoculação de bactérias diazotróficas endofíticas, em sementes de milho. Foi utilizada a estirpe Herbaspirillum seropedicae Z67 BR 11175. A inoculação das sementes com as bactérias e a adição de ácidos húmicos foram realizadas pelo recobrimento das sementes de milho UENF 506-8, com a mistura de calcário, meio de cultura semisólido, água e cimentante. As sementes recobertas foram semeadas em vasos Leonard. Aos 40 dias após a semeadura, as plântulas foram coletadas, e foi realizada a contagem de bactérias nas raízes pela técnica do Número Mais Provável. Os resultados deste trabalho mostram que os ácidos húmicos não interferem negativamente no crescimento das bactérias e estimulam a colonização da microbiota nativa. No entanto, nas condições avaliadas, a aplicação conjunta de bactérias + ácidos húmicos não estimulou o crescimento da população de bactérias inoculadas nas plântulas de milho.

Efeito dos ácidos húmicos na inoculação de bactérias diazotróficas endofíticas em sementes de milho

The objective of this research was to evaluate the effect of the humic acids in the inoculation of endophytic diazotrophic bacteria in corn seeds. It was used the bacteria Herbaspirillum seropedicae Z67 BR 11175. The inoculation of the seeds with the bacteria and the addition of humic acids were accomplished by the coating of the corn seeds UENF 506-8. The coating was accomplished with a mixture of lime, semi-solid middle culture, water and cement. The seeds covered were sown in Leonard vases. Forty days after sowing the plants were collected and the bacteria couting was accomplished in the roots by the Most probable Number technique. The results showed that the humic acids doesn't interfere negatively in the bacteria growth of and they stimulate the colonization of the native microbiota. However, in the appraised conditions, the united application of bacteria + humic acids didn't stimulate the growth of the bacteria population inoculated in the corn seedling.

Os ácidos húmicos podem atuar no aumento da população de bactérias diazotróficas introduzidas no interior da planta e, consequentemente, no incremento dos efeitos benéficos sobre a planta hospedeira. Com este trabalho, objetivou-se avaliar o efeito dos ácidos húmicos na inoculação de bactérias diazotróficas endofíticas, em sementes de milho. Foi utilizada a estirpe Herbaspirillum seropedicae Z67 BR 11175. A inoculação das sementes com as bactérias e a adição de ácidos húmicos foram realizadas pelo recobrimento das sementes de milho UENF 506-8, com a mistura de calcário, meio de cultura semisólido, água e cimentante. As sementes recobertas foram semeadas em vasos Leonard. Aos 40 dias após a semeadura, as plântulas foram coletadas, e foi realizada a contagem de bactérias nas raízes pela técnica do Número Mais Provável. Os resultados deste trabalho mostram que os ácidos húmicos não interferem negativamente no crescimento das bactérias e estimulam a colonização da microbiota nativa. No entanto, nas condições avaliadas, a aplicação conjunta de bactérias + ácidos húmicos não estimulou o crescimento da população de bactérias inoculadas nas plântulas de milho.

Efeito dos ácidos húmicos na inoculação de bactérias diazotróficas endofíticas em sementes de milho

The objective of this research was to evaluate the effect of the humic acids in the inoculation of endophytic diazotrophic bacteria in corn seeds. It was used the bacteria Herbaspirillum seropedicae Z67 BR 11175. The inoculation of the seeds with the bacteria and the addition of humic acids were accomplished by the coating of the corn seeds UENF 506-8. The coating was accomplished with a mixture of lime, semi-solid middle culture, water and cement. The seeds covered were sown in Leonard vases. Forty days after sowing the plants were collected and the bacteria couting was accomplished in the roots by the Most probable Number technique. The results showed that the humic acids doesn't interfere negatively in the bacteria growth of and they stimulate the colonization of the native microbiota. However, in the appraised conditions, the united application of bacteria + humic acids didn't stimulate the growth of the bacteria population inoculated in the corn seedling.

Os ácidos húmicos podem atuar no aumento da população de bactérias diazotróficas introduzidas no interior da planta e, consequentemente, no incremento dos efeitos benéficos sobre a planta hospedeira. Com este trabalho, objetivou-se avaliar o efeito dos ácidos húmicos na inoculação de bactérias diazotróficas endofíticas, em sementes de milho. Foi utilizada a estirpe Herbaspirillum seropedicae Z67 BR 11175. A inoculação das sementes com as bactérias e a adição de ácidos húmicos foram realizadas pelo recobrimento das sementes de milho UENF 506-8, com a mistura de calcário, meio de cultura semisólido, água e cimentante. As sementes recobertas foram semeadas em vasos Leonard. Aos 40 dias após a semeadura, as plântulas foram coletadas, e foi realizada a contagem de bactérias nas raízes pela técnica do Número Mais Provável. Os resultados deste trabalho mostram que os ácidos húmicos não interferem negativamente no crescimento das bactérias e estimulam a colonização da microbiota nativa. No entanto, nas condições avaliadas, a aplicação conjunta de bactérias + ácidos húmicos não estimulou o crescimento da população de bactérias inoculadas nas plântulas de milho.

Occurrence of endophytic bacteria of the genus Azospirillum in lowland rice plants collected in Santa Catarina state / Ocorrência de bactérias endofíticas do gênero Azospirillum em arroz irrigado no estado de Santa Catarina

Bacteria of the genus Azospirillum can endophytically colonize lowland rice plants and contribute to the development of this culture. The objective of this work was to evaluate the occurrence of endophytic Azospirillum spp. isolated from roots and stems of lowland rice plants grown in the State of Santa Catarina, Brazil. Samples of lowland rice plants were collected in eight farms in the cities of Guaramirim (two areas), Massaranduba, Rodeio (two areas), Rio do Sul, Agronômica and Pouso Redondo. Azospirillum spp. occurrence in disinfested roots and stems of the collected plants was evaluated using the More Probable Number method (MPN), in two semi-solid selective broth: NFb (A. lipoferum and A. brasilense) and LGI (A. amazonense), and the similarity among the eight areas was estimated by the principal component analysis (PCA). The bacteria grown in semi-solid broths were purified in their respective broths, isolated in PDA broth to obtain pure and morphologically characterized cultures. The mean population density of Azospirillum spp. varied from 1.6x108 to 1.42x108 cells g-1 of fresh matter (LGI), and from 1.76x108 to 1.56x108 cells g-1 of fresh matter (NFb), in roots and stem, respectively. Sixty two (62) isolates were obtained, most of them from disinfested roots. The results showed that Azospirillum spp. occurs in satisfactory population densities in lowland rice fields in

Bactérias do gênero Azospirillum podem colonizar endofiticamente plantas de arroz irrigado e contribuem para o desenvolvimento da cultura. O objetivo desse estudo foi avaliar a ocorrência de bactérias endofíticas do gênero Azospirillum isoladas de raízes e colmos de arroz irrigado cultivado em Santa Catarina. Amostras de plantas de arroz irrigado foram coletadas em oito propriedades rurais nas cidades de Guaramirim (duas áreas), Massaranduba, Rodeio (duas áreas), Rio do Sul, Agronômica e Pouso Redondo. A ocorrência de bactérias endofíticas do gênero Azospirillum em raízes desinfestadas e colmos foi avaliada através do método do Número Mais Provável de Propágulos (NMP), em meio seletivo semi-sólido NFb (A. lipoferum e A. brasilense) e LGI (A. amazonense), e a similaridade entre as áreas foi estimada através da análise de componentes principais (ACP). As bactérias crescidas nos meios semi-seletivos foram purificadas em seus respectivos meios, isoladas em meio BDA até a obtenção de culturas puras e caracterizadas morfologicamente. A densidade populacional média de Azospirillum spp. foi de 1,6x108 e 1,42x108 células g-1 de matéria fresca (meio LGI), e 1,76x108 e 1,56x108 células g-1 de matéria fresca (meio NFb), para raízes e colmos respectivamente. Ao final do isolamento, foram obtidos 62 isolados, dos quais a maior parte oriunda de raízes desinfestadas. Os resultados obtidos most