Marcos Antônio Soares

Functional role of an endophytic Bacillus amyloliquefaciens in enhancing growth and disease protection of invasive English ivy (Hedera helix L.)

BackgroundWe hypothesize that invasive English ivy (Hedera helix) harbors endophytic microbes that promote plant growth and survival. To evaluate this hypothesis, we examined endophytic bacteria in English ivy and evaluated effects on the host plant.MethodsEndophytic bacteria were isolated from multiple populations of English ivy in New Brunswick, NJ. Bacteria were identified as a single species Bacillus amyloliquefaciens. One strain of B. amyloliquefaciens, strain C6c, was characterized for indoleacetic acid (IAA) production, secretion of hydrolytic enzymes, phosphate solubilization, and antibiosis against pathogens. PCR was used to amplify lipopeptide genes and their secretion into culture media was detected by MALDI-TOF mass spectrometry. Capability to promote growth of English ivy was evaluated in greenhouse experiments. The capacity of C6c to protect plants from disease was evaluated by exposing B+ (bacterium inoculated) and B− (non-inoculated) plants to the necrotrophic pathogen Alternaria tenuissima.ResultsB. amyloliquefaciens C6c systemically colonized leaves, petioles, and seeds of English ivy. C6c synthesized IAA and inhibited plant pathogens. MALDI-TOF mass spectrometry analysis revealed secretion of antifungal lipopeptides surfactin, iturin, bacillomycin, and fengycin. C6c promoted the growth of English ivy in low and high soil nitrogen conditions. This endophytic bacterium efficiently controlled disease caused by Alternaria tenuissima.ConclusionsThis study suggests that B. amyloliquefaciens plays an important role in enhancing growth and disease protection of English ivy.

Electrochemical biosensor for carbofuran pesticide based on esterases from Eupenicillium shearii FREI-39 endophytic fungus

In this work, a biosensor was constructed by physical adsorption of the isolated endophytic fungus Eupenicillium shearii FREI-39 esterase on halloysite, using graphite powder, multi-walled carbon nanotubes and mineral oil for the determination of carbofuran pesticide by inhibition of the esterase using square-wave voltammetry (SWV). Specific esterase activities were determined each 2 days over a period of 15 days of growth in four different inoculation media. The highest specific activity was found on 6th day, with 33.08 U on PDA broth. The best performance of the proposed biosensor was obtained using 0.5 U esterase activity. The carbofuran concentration response was linear in the range from 5.0 to 100.0 µg L(-1) (r=0.9986) with detection and quantification limits of 1.69 µg L(-1) and 5.13 µg L(-1), respectively. A recovery study of carbofuran in spiked water samples showed values ranging from 103.8±6.7% to 106.7±9.7%. The biosensor showed good repeatability and reproducibility and remained stable for a period of 20 weeks. The determination of carbofuran in spiked water samples using the proposed biosensor was satisfactory when compared to the chromatographic reference method. The results showed no significant difference at the 95% confidence level with t-test statistics. The application of enzymes from endophytic fungi in constructing biosensors broadens the biotechnological importance of these microorganisms.

Disease protection and allelopathic interactions of seed-transmitted endophytic pseudomonads of invasive reed grass ( Phragmites australis )

Background and aimsNon-native Phragmites australis (haplotype M) is an invasive grass that decreases biodiversity and produces dense stands. We hypothesized that seeds of Phragmites carry microbes that improve seedling growth, defend against pathogens and maximize capacity of seedlings to compete with other plants.MethodsWe isolated bacteria from seeds of Phragmites, then evaluated representatives for their capacities to become intracellular in root cells, and their effects on: 1.) germination rates and seedling growth, 2.) susceptibility to damping-off disease, and 3.) mortality and growth of competitor plant seedlings (dandelion (Taraxacum officionale F. H. Wigg) and curly dock (Rumex crispus L.)).ResultsTen strains (of 23 total) were identified and characterized; seven were identified as Pseudomonas spp. Strains Sandy LB4 (Pseudomonas fluorescens) and West 9 (Pseudomonas sp.) entered root meristems and became intracellular. These bacteria improved seed germination in Phragmites and increased seedling root branching in Poa annua. They increased plant growth and protected plants from damping off disease. Sandy LB4 increased mortality and reduced growth rates in seedlings of dandelion and curly dock.ConclusionsPhragmites plants associate with endophytes to increase growth and disease resistance, and release bacteria into the soil to create an environment that is favorable to their seedlings and less favorable to competitor plants.

Functional Role of Bacteria from Invasive Phragmites australis in Promotion of Host Growth

We hypothesize that bacterial endophytes may enhance the competitiveness and invasiveness of Phragmites australis. To evaluate this hypothesis, endophytic bacteria were isolated from P. australis. The majority of the shoot meristem isolates represent species from phyla Firmicutes, Proteobacteria, and Actinobacteria. We chose one species from each phylum to characterize further and to conduct growth promotion experiments in Phragmites. Bacteria tested include Bacillus amyloliquefaciens A9a, Achromobacter spanius B1, and Microbacterium oxydans B2. Isolates were characterized for known growth promotional traits, including indole acetic acid (IAA) production, secretion of hydrolytic enzymes, phosphate solubilization, and antibiosis activity. Potentially defensive antimicrobial lipopeptides were assayed for through application of co-culturing experiments and mass spectrometer analysis. B. amyloliquefaciens A9a and M. oxydans B2 produced IAA. B. amyloliquefaciens A9a secreted antifungal lipopeptides. Capability to promote growth of P. australis under low nitrogen conditions was evaluated in greenhouse experiments. All three isolates were found to increase the growth of P. australis under low soil nitrogen conditions and showed increased absorption of isotopic nitrogen into plants. This suggests that the Phragmites microbes we evaluated most likely promote growth of Phragmites by enhanced scavenging of nitrogenous compounds from the rhizosphere and transfer to host roots. Collectively, our results support the hypothesis that endophytic bacteria play a role in enhancing growth of P. australis in natural populations. Gaining a better understanding of the precise contributions and mechanisms of endophytes in enabling P. australis to develop high densities rapidly could lead to new symbiosis-based strategies for management and control of the host.

Phosphate solubilization and phytohormone production by endophytic and rhizosphere Trichoderma isolates of guanandi (Calophyllum brasiliense Cambess)

This work aimed to isolate and evaluate the phosphate solubilization and phytohormone production abilities of endophytic and rhizosphere fungi belonging to the genus Trichoderma isolated from guanandi (Calophyllum brasiliense Cambess). From the guanandi collected from the field, 12 isolates obtained were grown in potato dextrose broth and supplemented with the phosphate sources, to test their capacity to solubilize phosphates. A strain of Trichoderma asperellum from a commercial inoculant was also used. One isolate was able to solubilize calcium phosphate, 12 solubilized iron phosphate and two solubilized aluminum phosphate. Only the rhizosphere isolates were able to synthesize indole acetic acid (IAA) and none of the rhizosphere or endophytic isolates produced cytokinin or gibberellin. There are Trichoderma isolates that can benefit plant development, both for their known antagonistic ability against phytopathogenic fungi and for their ability to provide phosphates and or to produce phytohormones. Key words: Trichoderma, inoculants, calcium, iron, aluminum, solubilization, plant growth promotion.

Endophytic bacterium, Bacillus amyloliquefaciens, enhances ornamental hosta resistance to diseases and insect pests

A total of 84 bacterial endophytes were isolated from seeds of 6 cultivars of ornamental hostas, and they were identified to 5 species based on morphological characteristics and 16S rDNA sequence analysis. Among them, the strain ‘Blu-v2’, which was isolated from the seeds of cultivar ‘Blue Umbrella’ and identified to be Bacillus amyloliquefaciens, showed highest antifungal activity and capacity to deter feeding by Fall armyworms (Spodoptera fruigiperda). Lipopeptides in cultures of Blu-v2 were determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and its antifungal activities were verified. However, the lipopeptide preparation did not show toxicity to larvae of Fall armyworms. In a greenhouse experiment, Blu-v2 was inoculated into small plantlets of hosta (cultivar ‘Rainforest Sunrise’). The leaves of plants with bacteria (endophyte-infected = E+) and without bacteria (endophyte-free = E−) were used in seven-day feeding experiments employing fourth-instar larvae of Fall armyworms. We found that there was a significant decrease in the weights of larvae fed with E+ compared to E− plants; and the mortality rate of larvae fed with E− leaves was lower (3.33%) compared to that of larvae fed with E+ leaves (30%). Based on our studies, we suggest that endophytic B. amyloliquefaciens strain Blu-v2 has potential value as a biocontrol agent to reduce damage from fungal diseases and insect pests of hosta cultivars.

Diversity of fungi associated with plants growing in geothermal ecosystems and evaluation of their capacities to enhance thermotolerance of host plants

Diversity and heat-adaptation of endophytic fungi (EF) and rhizospheric fungi associated with plants growing in geothermal ecosystems, Southwest China, as well as their benefit in improving host plant thermotolerances were investigated. A total of 1589 culturable fungi belonging to 38 taxa were isolated, in which Curvularia, Acrophialophora, Penicillium, and Aspergillus were the dominant genera. The Shannon indices of EF and rhizospheric fungi ranged from 1.80 to 2.56 and 0.73 to 2.11, respectively. Phylogenetic analysis indicated that the EF have a close relationship with rhizospheric fungi. However, some fungi exhibited apparently species-specific habitat distribution patterns. Growth temperature tests indicated that 60.22% of the tested isolates were thermotolerant fungi and only 39.78% were mesophiles, and the number of heat-adapted fungi increased with increasing environmental temperatures. The strain G1-29, which was isolated from the roots of Hedyotis diffusa and identified as Curvularia crepinii, significantly improved host plant thermotolerance under laboratory conditions: the death rate of endophyte-infected plants was significantly lower than that of endophyte-free plants (t-test, p = .0158, df = 4). Our results suggested that the EF and rhizospheric fungi associated with plants growing in geothermal ecosystems are diverse, and many of them have adapted to the high environmental temperatures. Some fungi have come to be the dominant endemic inhabitants of specific niches, and some played an important role in improving host plant thermotolerances.

Diversity of cultivable bacterial endophytes in Paullinia cupana and their potential for plant growth promotion and phytopathogen control

Endophytic bacteria occupy the same niche of phytopathogens and may produce metabolites that induce the host plant systemic resistance and growth. Host and environmental variables often determine the endophytic communitys structure and composition. In this study, we addressed whether the plant genotype, organ, and geographic location influence the structure, composition, and functionality of endophytic bacterial communities in Paullinia cupana. To characterize the communities and identify strains with potential application in agriculture, we analyzed two P. cupana genotypes cultivated in two cities of the State of Amazonas, Brazil. Endophytic bacteria were isolated from surface-disinfested root, leaf, and seed tissues through the fragmentation and maceration techniques. The colonization rate, number of bacteria, richness, diversity, and functional traits were determined. The plant growth-promoting ability of selected bacterial strains was assessed in Sorghum bicolor. We identified 95 bacterial species distributed in 29 genera and 3 phyla (Proteobacteria, Actinobacteria, and Firmicutes). The colonization rate, richness, diversity, and species composition varied across the plant organs; the last parameter also varied across the plant genotype and location. Some strains exhibited relevant plant growth-promoting traits and antagonistic traits against the main phytopathogens of P. cupana, but they were not separated by functional traits. The main bacterial strains with plant growth-promoting traits induced S. bicolor growth. Altogether, our findings open opportunities to study the application of isolated endophytic bacterial strains in the bioprospection of processes and products.

PacCl, a pH-responsive transcriptional regulator, is essential in the pathogenicity of Colletotrichum lindemuthianum, a causal agent of anthracnose in bean plants

In fungi, the expression of genes encoding proteins related to parasitism is regulated by several factors, including pH. This study reports the structural and functional characterization of the pacCl gene, which encodes the transcription factor PacC of C. lindemuthianum. The pacCl gene showed reduced expression in acidic pH, and its transcription was activated by elevated extracellular pH. The importance of this gene was demonstrated by the development of a pacC1 disruption mutant line of C. lindemuthianum. The mutant line was able to penetrate the host tissue through differentiation of primary hyphae. However, it was not able to cause maceration on the infected plant tissue. The results suggest that PacCl is a regulator of gene activation, and its expression is required for fungal growth in alkaline conditions, as well as for the transcription of genes necessary for the passage from the biotrophic to the necrotrophic phase.

Potencialidade de produção de biodiesel por óleos e gorduras residuais na cidade de Itabira-MG

Grande parte da energia produzida no Brasil provem de fontes renovaveis. Porem, as energias nao-renovaveis sao mais utilizadas, em especial o petroleo, cujas reservas sao finitas. Uma alternativa para substituicao do diesel e o biodiesel, biocombustivel produzido por fontes renovaveis ou recicladas, como oleos e gorduras residuais, OGRs, que geram menos poluentes que os combustiveis derivados do petroleo. O objetivo deste trabalho foi verificar, por meio de questionario, a geracao de OGRs pelos estabelecimentos comerciais da cidade de Itabira-MG, de forma a avaliar e discutir a potencialidade de producao de biodiesel na localidade em funcao da oferta de OGRs, retorno financeiro e fatores logisticos. Para isso, na Prefeitura de Itabira foram levantados dados de todos os estabelecimentos que possuem alvara de funcionamento, escolhendo-se os que estao localizados em areas centrais da cidade. Verificou-se a geracao de quantidades insuficientes de oleo de soja e de gordura hidrogenada residuais, inviabilizando no momento a producao de biodiesel em grande escala. Contudo, em relacao a simulacao feita com os custos de producao do biocombustivel, os valores encontrados sao compativeis aos do mercado consumidor. Alem disso, um dado preocupante levantado foi a doacao dos oleos de soja residuais para a reutilizacao por pessoas carentes da cidade, um destino perigoso a saude dessas pessoas.