Diversity of Pectobacterium strains by biochemical, physiological, and molecular characterization

Pectobacterium is a complex taxon of strains with diverse characteristics. It comprises several genera, including Erwinia, Brenneria, Pectobacterium, Dickeya, and Pantoea. Pectobacterium and Dickeya cause diseases in a wide range of plants, including potatoes, where they are causative agents of soft rot in tubers and blackleg in field-grown plants. Characterizing Pectobacterium species allows for the analysis of the diversity of pectinolytic bacteria, which may support control strategies for plant bacterial diseases. The aim of this study was to perform biochemical, physiological, and molecular characterizations of Pectobacterium spp. from different sites and host plants. The isolated strains were characterized by the glucose fermentation test, Gram staining, catalase activity, oxidase activity, growth at 37 ºC, reducing substances from sucrose, phosphatase activity, indole production, acid production from different sources (sorbitol, melibiose, citrate, and lactose), pathogenicity in potato, and hypersensitivity reactions. Molecular characterization was performed with species-specific primers ECA1f/ECA2r and EXPCCF/EXPCCR, which identify P. atrosepticum and P. carotovorum subsp. carotovorum (Pcc), respectively, and with primers 1491f/L1RA/L1RG and Br1f/L1RA/L1RG that differentiate Pcc from Dickeya chrysanthemi and from P. carotovorum subsp. brasiliensis. The strains were identified as belonging to the genus Pectobacterium, though they did not fit the biochemical nor the molecular classification standards for subspecies differentiation, indicating significant diversity among the strains

Inoculation of maize with Azospirillum brasilense in the seed furrow 1 Inoculação do milho com Azospirillum brasilense no sulco de semeadura

ABSTRACT -Several studies addressing the inoculation of cereals with diazotrophic microorganisms can be found in the literature. However, in many experiments, investigators have overlooked the feasibility of applying these microorganisms to the furrow together with the seed, and the effect of bacterial concentration on phytostimulation. The aim of this work was to evaluate the effect of doses of an inoculant based on Azospirillum brasilense, applied to the seed furrow when planting maize, combined with different doses of nitrogen fertiliser. The experiment was carried out in the field, in soil of the cerrado region of Brazil. An experimental design of randomised blocks in bands was adopted, comprising nitrogen (40, 100, 200 and 300 kg ha -1 ) and doses of an A. brasilense-based liquid inoculant applied to the seed furrow (0, 100, 200, 300 and 400 mL ha -1 ). The dose of 200 mL ha -1 Azospirillum was noteworthy for grain production. This is the first report of the effective application of Azospirillum in the seed furrow when planting maize in the cerrado region of Brazil

Nitrogen application and Azospirillum brasilense inoculation on maize hybrids

Maize is one of the world s most important cultivated cereals; however, it presents great challenges for correct fertilization management. Its high nitrogen demand has been the major cause of excessive applications of synthetic nitrogen fertilizers in the crop. As a result, both environmental and economical problems have increased. Therefore, alternative methods that promote plant growth and ensure productivity need to be better studied to associate efficiency, economic viability and environment safety. Diazotrophic bacteria are able to fix atmospheric nitrogen and synthesize plant growth promoting substances, which may reduce the need for nitrogen fertilizers. To test this hypothesis, this work evaluated inoculation with Azospirillum brasilense combined with nitrogen fertilization on maize plants under Brazilian Cerrado conditions. The research consisted of three trials, in a randomized block design. The first experiment was done in the field at Iraí de Minas, during the crop season of 2009/2010. Four maize hybrids inoculated or not with the Azospirillum bacteria and subjected to different nitrogen rates (50, 100, 150, 200, 250 and 300 kg of N ha-1) were evaluated. The second trial, under greenhouse conditions at Uberlândia, was set up as a 3 x 3 factorial. The first factor corresponded to nitrogen rates (0, 100 and 200 kg ha-1) and the second factor to Azospirillum inoculant (0, 100 and 200 mL ha-1). At the end of this experiment, another greenhouse trial was done to determine the possible residual effect of inoculation and nitrogen fertilization. According to the field study, it was concluded that maize inoculation with Azospirillum brasilense is a promising technology capable of significantly enhancing crop yield, in both high and low investment systems. In general, greenhouse evaluations showed that nitrogen application promoted growth, increased chlorophyll and nutrient contents, and intensified the activity of rhizosphere enzymes related to ammonium and inorganic phosphorus availability in maize plants. Also, nitrogen fertilization had residual effect on plant development in the test evaluating the residual effect of the fertilizer.Conselho Nacional de Desenvolvimento Científico e TecnológicoMestre em AgronomiaO milho é um dos principais cereais cultivados no mundo, porém apresenta desafios quanto ao correto manejo da adubação. Sua alta exigência por nitrogênio tem sido a principal responsável pelas excessivas doses de fertilizantes nitrogenados sintéticos aplicados na cultura. Como consequência, há aumento no custo de produção da lavoura e impactos negativos ao meio ambiente. Dessa forma, é necessário estudar métodos alternativos que promovam o crescimento das plantas e garantam a produtividade, buscando-se aliar eficiência, viabilidade econômica e segurança ambiental. Bactérias diazotróficas podem fixar nitrogênio atmosférico e produzir substâncias promotoras do crescimento, podendo reduzir a utilização de fertilizantes nitrogenados na cultura do milho. Para testar essa hipótese, este trabalho teve como objetivo avaliar a inoculação com Azospirillum brasilense em combinação com adubação nitrogenada em híbridos de milho, nas condições do cerrado brasileiro. A pesquisa foi composta por três ensaios, todos em delineamento experimental de blocos ao acaso. O primeiro foi realizado a campo, no município de Iraí de Minas, durante a safra 2009/2010. Foram avaliados quatro híbridos de milho inoculados ou não com a bactéria do gênero Azospirillum e submetidos a diferentes doses de adubação nitrogenada (50, 100, 150, 200, 250 e 300 kg de N ha-1). O segundo ensaio, realizado em casa de vegetação, no município de Uberlândia, foi conduzido em esquema fatorial 3 x 3, sendo o primeiro fator correspondente a doses de nitrogênio (0, 100 e 200 kg ha-1) e o segundo, à dose do inoculante líquido à base de Azospirillum (0, 100 e 200 mL ha-1). Ao final desse experimento, outro ensaio em casa de vegetação foi realizado para verificar o possível efeito residual da inoculação e da adubação nitrogenada. Em campo, concluiu-se que a inoculação de milho com Azospirillum brasilense é uma tecnologia promissora capaz de incrementar significativamente a produtividade da cultura, em sistemas de alto e baixo investimento. As avaliações em casa de vegetação permitiram concluir que a adição de nitrogênio, de maneira geral, promoveu maior desenvolvimento das plantas de milho, incrementou os teores de clorofila e de nutrientes e aumentou a atividade das enzimas relacionadas à disponibilização de amônia e fósforo inorgânico na rizosfera. Além disso, apresentou efeito residual no desenvolvimento das plantas na semeadura subsequente

In vitro and in planta characterization of a chimeric antimicrobial protein against the phytopathogen Ralstonia solanacearum

The phytobacterium Ralstonia solanacearum [(SMITH, 1896) YABUUCHI et al. 1996], causative agent of bacterial wilt and Moko disease, is considered one of the world s most destructive plant pathogen. In Brazil this xylem-restricted bacterium reduces yields of agriculturally important crops and calls for effective disease management strategies, so far limited to preventive actions. Antimicrobial peptides have been considered powerful compounds for plant protection due to their antiviral, antifungal, and antibacterial activities. Hence, they are promising candidates to the development of novel rationally-designed therapies for the control of R. solanacearum. Mirroring the function and properties of cecropin B, a well-studied α-helical antimicrobial peptide (AH-AMP), several candidates were selected by bioinformatic tools and tested in vitro against the bacterium. The identified peptides included a linear AH-AMP within the existing structure of phosphoenolpyruvate carboxylase, named PPC20. This peptide stood out as the most efficient in killing the pathogen without jeopardizing human cells. In order to investigate whether the combination of two innate immune functions provides a robust class of antimicrobial therapeutics, this lytic domain was combined to a putative plant-derived elastase (the pathogenesis-related protein SlP14a), leading to the development of a chimeric protein. To characterize and validate this novel antimicrobial chimera as a biocontrol agent, bioassays were conducted in vitro and in planta. SlP14a and SlP14a-PPC20 were expressed in both bacterial and plant (transient expression) systems. Purified proteins showed in vitro antibacterial activity by inhibiting R. solanacearum growth. In order to explore the in vivo biological function of SlP14a-PPC20, transgenic lines of tomato cultivar MoneyMaker were obtained and characterized. To assess whether these lines acquired enhanced tolerance to the pathogen, they were challenged with R. solanacearum by stem inoculation. The survival rates and the reduction of disease symptoms were significantly higher in transgenic plants compared with the non-transgenic ones. This study proposes an alternative strategy for bacterial wilt control based on expression of a newly designed therapeutic antimicrobial protein in tomato plants.Conselho Nacional de Desenvolvimento Científico e TecnológicoDoutor em AgronomiaA fitobactéria Ralstonia solanacearum [(SMITH, 1896) YABUUCHI et al. 1996], agente causal da murcha-bacteriana e da doença do Moko, é considerada um dos mais destrutivos patógenos de plantas em todo o mundo. No Brasil, sua ocorrência compromete o rendimento de culturas agronomicamente importantes, destacando a necessidade de estratégias eficazes para o manejo da doença, até então limitadas a ações preventivas. Peptídeos antimicrobianos (AMPs) participam da defesa inata de inúmeros organismos e são considerados potenciais agentes terapêuticos no combate a ampla variedade de patógenos, em virtude de suas propriedades antivirais, antifúngicas e antibacterianas. Visto isso, são candidatos promissores para o desenvolvimento de novas terapias no controle de R. solanacearum. Mediante o uso de ferramentas de bioinformática, vários AMPs foram selecionados baseando-se na estrutura e função da cecropina B, um conhecido peptídeo antimicrobiano α-helicoidal (AH-AMP), e testados in vitro contra a bactéria. Dentre os peptídeos identificados, um AH-AMP derivado da enzima fosfoenolpiruvato carboxilase, denominado PPC20, destacou-se como o mais eficiente para controlar o patógeno, simultaneamente configurando baixa toxicidade a células humanas. No intuito de verificar se a combinação de duas funções imunes inatas presentes na mesma molécula potencializa seu efeito antimicrobiano, esse domínio lítico foi fusionado a uma elastase putativa derivada de plantas (a proteína relacionada à patogênese, SlP14a), resultando no desenvolvimento de uma quimera. A caracterização e validação dessa nova proteína quimérica foi realizada por bioensaios conduzidos in vitro e in planta. Os genes SlP14a e SlP14a-PPC20 foram clonados e expressos em células bacterianas e em plantas de tabaco (expressão transiente). As proteínas extraídas e purificadas de ambos os sistemas de expressão apresentaram atividade antibacteriana in vitro através da inibição do crescimento de R. solanacearum. A fim de verificar a função biológica in vivo da quimera (SlP14a-PPC20), linhagens transgênicas de tomate (cultivar MoneyMaker) foram obtidas e inoculadas com R. solanacearum. Os índices de sobrevivência e a redução dos sintomas da murcha-bacteriana foram significativamente mais elevados em plantas transgênicas quando comparados com aqueles relativos às plantas não transformadas. Este estudo propõe uma estratégia alternativa para o controle da murcha-bacteriana mediante a expressão de uma nova proteína terapêutica antimicrobiana em plantas de tomate

Biotecnologia aplicada ao melhoramento genético do cafeeiro Biotechnology applied to the genetic improvement of coffee plant

O melhoramento genético do cafeeiro mediante técnicas convencionais é trabalhoso e demorado. A biotecnologia oferece estratégias alternativas para auxiliar na multiplicação e no desenvolvimento de novas variedades com resistência a estresses bióticos e abióticos, melhor qualidade de bebida e maturação mais uniforme dos frutos. As técnicas de cultura de tecidos têm possibilitado a obtenção de grande número de plantas e a garantia da uniformidade genética do material. O emprego de marcadores moleculares, principalmente através da seleção assistida, facilitou o rápido progresso do melhoramento genético da cultura, assim como a transformação genética, via cultura e fusão de protoplastos, biobalística ou mediada por Agrobacterium sp. Esta revisão objetiva sumarizar o histórico, situação atual e perspectivas da biotecnologia no melhoramento genético do cafeeiro.<br>Genetic improvement of coffee through classical breeding is laborious and time consuming. Biotechnology offers alternative strategies to assist multiplication and development of new and improved coffee varieties, including those resistant to biotic and abiotic stresses, with better cup quality, and with uniform fruit maturation. Tissue culture techniques have enabled the production of a large number of plants with genetic uniformity. The use of molecular markers, especially through assisted selection, led to rapid progress of coffee plant breeding, as well as the use of genetic transformation by protoplasts culture and fusion, biobalistics, or Agrobacterium-mediated. This review provides a summary of biotechnology history, current situation and directions applied to the genetic improvement of coffee plant

Inoculation of maize with Azospirillum brasilense in the seed furrow

ABSTRACT Several studies addressing the inoculation of cereals with diazotrophic microorganisms can be found in the literature. However, in many experiments, investigators have overlooked the feasibility of applying these microorganisms to the furrow together with the seed, and the effect of bacterial concentration on phytostimulation. The aim of this work was to evaluate the effect of doses of an inoculant based on Azospirillum brasilense, applied to the seed furrow when planting maize, combined with different doses of nitrogen fertiliser. The experiment was carried out in the field, in soil of the cerrado region of Brazil. An experimental design of randomised blocks in bands was adopted, comprising nitrogen (40, 100, 200 and 300 kg ha-1) and doses of an A. brasilense-based liquid inoculant applied to the seed furrow (0, 100, 200, 300 and 400 mL ha-1). The dose of 200 mL ha-1Azospirillum was noteworthy for grain production. This is the first report of the effective application of Azospirillum in the seed furrow when planting maize in the cerrado region of Brazil