André Luiz Martinez de Oliveira

Composition and activity of endophytic bacterial communities in field-grown maize plants inoculated with Azospirillum brasilense

The application of agricultural practices in which non-leguminous plants are inoculated with growth-promoting diazotrophic bacteria is gaining importance worldwide. Nevertheless, an efficient strategy for using this inoculation technology is still lacking, and a better comprehension of the environmental factors that influence a plant’s ability to support its associative bacterial community is indispensable to achieving standardized inoculation responses. To address the effects of nitrogen (N)-fertilization on the diversity of both the total and metabolically active endophytic bacterial communities of field-grown maize plants, we extracted total DNA and RNA from maize plants inoculated with Azospirillum brasilense strain Ab-V5 that were growing in Oxisol and treated with regular and low levels of N-fertilizers (RN and LN, respectively). Four clonal libraries were constructed and sequenced and the dominant populations analyzed. Partial description of the bacterial diversity indicated that plants receiving RN- and LN-treatments can maintain bacterial communities with similar diversity indexes for the total endophytic bacterial community, although the communities of Novosphingobium and Methylobacterium were unevenly distributed. Fertilization management had a stronger effect on the dominant populations of the metabolically active bacterial community, and 16S rRNA gene libraries from RN plants suggested a lower diversity of such populations in comparison with libraries from LN plants. The agronomic parameters obtained at the end of the crop season indicated that the inoculation treatment was efficient in promoting plant growth. However, the combination of regular treatments with N-fertilizers and plant inoculation did not have an additive effect and actually tended to decrease crop productivity.

Maize Inoculation with Azospirillum brasilense Ab-V5 Cells Enriched with Exopolysaccharides and Polyhydroxybutyrate Results in High Productivity under Low N Fertilizer Input

Although Azospirillum strains used in commercial inoculant formulations presents diazotrophic activity, it has been reported that their ability to produce phytohormones plays a pivotal role in plant growth-promotion, leading to a general recommendation of its use in association with regular N-fertilizer doses. In addition, a high variability in the effectiveness of Azospirillum inoculants is still reported under field conditions, contributing to the adoption of the inoculation technology as an additional management practice rather than its use as an alternative practice to the use of chemical inputs in agriculture. To investigate whether the content of stress-resistance biopolymers would improve the viability and performance of Azospirillum inoculants when used as substitute of N-fertilizers, biomass of A. brasilense strain Ab-V5 enriched in exopolysaccharides (EPS) and polyhydroxybutirate (PHB) was produced using a new culture medium developed by factorial mixture design, and the effectiveness of resulting inoculants was evaluated under field conditions. The culture medium formulation extended the log phase of A. brasilense cultures, which presented higher cell counts and increased EPS and PHB contents than observed in the cultures grown in the OAB medium used as control. An inoculation trial with maize conducted under greenhouse conditions and using the biopolymers-enriched Ab-V5 cells demonstrated the importance of EPS and PHB to the long term bacterial viability in soil and to the effectiveness of inoculation. The effectiveness of liquid and peat inoculants prepared with Ab-V5 cells enriched with EPS and PHB was also evaluated under field conditions, using maize as target crop along different seasons, with the inoculants applied directly over seeds or at topdressing under limiting levels of N-fertilization. No additive effect on yield resulted from inoculation under high N fertilizer input, while inoculated plants grown under 80% reduction in N fertilizer showed yields at levels compared to fully fertilized plants, regardless the inoculation method. The presented data highlights the feasibility to partially substitute the N-fertilizer demand in non-legume crops using high-quality inoculant formulations, prepared with diazotrophic bacteria enriched with stress-resistance biopolymers that confer increased viability an effectiveness to the bacterial cells.

Plant growth-promoting bacteria associated with nitrogen fertilization at topdressing in popcorn agronomic performance

The use of plant growth-promoting bacteria is a promising alternative with low environmental impact to increase the efficiency of use of chemical fertilizers, ensuring high yield with better cost-effective ratio. In maize crops, several studies have demonstrated an increased yield when Azospirillum-based inoculants are used. In the case of popcorn, there are no available studies related to use of inoculation and its response on yield parameters. Thus, the aim of this study was to evaluate the field performance of popcorn when inoculated with the commercial product Masterfix L (A. brasilense Ab-V5 and A. brasilense Ab-V6) and the non-commercial inoculant UEL (A. brasilense Ab-V5 + Rhizobium sp. 53GRM1) associated with nitrogen fertilization. The trials were conducted in Londrina and Maringa, Parana State, Brazil, in a randomized block design with four replications, in a split plot design with the inoculation treatments located in the plots (uninoculated, Masterfix L, and UEL) and the different N rates located in the subplots where ammonium sulphate was applied in the topdressing at the V6 stage (0, 50, 100, and 150 kg∙ha–1). The variance analysis showed significant effects (p < 0.05) of inoculation (Londrina environment) and N rates (both environments) only for grain yield. There was no inoculation effect in the grain yield when inoculants were applied together with N-fertilization at topdressing. In the absence of N-fertilization at topdressing, the inoculants Masterfix L. and UEL promoted higher grain yield as compared to the uninoculated plants, with resulting increases of 13.21 and 26.61% in yield, respectively.

Culturable bacterial pool from aged petroleum-contaminated soil: identification of oil-eating Bacillus strains

Information gleaned from soil microbiota may provide access to new economically important species. Here, we describe the isolation, identification, and genetic diversity of high-density bacterial populations isolated from aged oil-contaminated soil. Twenty different morphotypes were identified in populations present at densities of up to 107 cells g−1 soil, encompassing seven bacterial genera based on 16S rRNA sequencing. Six isolates of the genus Bacillus were identified, three of which appear to consume oil. The genetic clusters defined by the DNA fingerprinting analysis suggest that there is a close relationship between these oil-eating Bacillus species. Isolates able to grow using crude oil as a carbon source were biochemically characterized and found to exhibit high lipolytic activity in liquid medium and to produce alkaline-stable biosurfactants. Fluorescence spectroscopy analysis of the cell-free extract from the oil-eating Bacillus sp. strain MO.04b showed an increase in the relative fluorescence intensity of low-molecular-mass aromatics concomitantly with an increase in the protein content, suggesting the transformation of aromatic hydrocarbons to the liquid phase in response to biodegradation. The approach adopted in this study suggests a low diversity of the high-density bacterial population colonizing an aged oil-contaminated soil and may prove useful in selecting bacterial isolates for bioremediation studies and biotechnological applications such as biosurfactant production.

Identification of Genes Involved in Indole-3-Acetic Acid Biosynthesis by Gluconacetobacter diazotrophicus PAL5 Strain Using Transposon Mutagenesis

Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds than the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao. cccA, and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with tryptophan and provides evidence for the involvement of an L-amino acid oxidase gene cluster in the biosynthesis of IAA. Furthermore, we showed that the mutant strains with reduction in IAA biosynthesis ability, in consequence of the lower transcription levels of genes of the lao cluster, had remarkable effects on development of rice roots.

Invasion ecology applied to inoculation of plant growth promoting bacteria through a novel SIMPER-PCA approach

AimsPlant growth promoting bacteria (PGPB) have been used on crops for years, but inoculants that are efficient in some locations may not be efficient in others. Here, we applied classical invasion ecology theory to PGPB inoculation in order to identify patterns that can be used to predict plant growth promoting (PGP) efficiency. The hypotheses that the inoculant that causes most impact will be the most efficient PGPB, and that the most invasible locations would have higher PGP efficiency, were tested. We also aim to present our statistical approach to analyze SIMPER results.MethodsUsing next generation sequencing targeting the 16S rDNA gene in metagenomics samples, we analyzed samples of pre-planting bulk soil and rhizosphere of inoculated maize plants. Bacterial communities of inoculated plants were compared to the non-inoculated controls, in order to estimate the inoculant invasion impact. Crop yield was compared to different indexes, and a novel data exploration approach was employed.ResultsThe most efficient inoculant was not the most invasive, and a nutrient per diversity ratio was unable to predict inoculant efficiency or invasion impact. However, the efficient inoculation treatment presented an enrichment of specific pre-planting taxa.ConclusionsInvasion ecology frameworks could not anticipate field results of inoculated plants. Nonetheless, our data exploration approach, which is explained in detail, can be useful to raise new hypothesis and improve the visualization of dissimilarity data.

Selection of Leuconostoc strains isolated from artisanal Serrano Catarinense cheese for use as adjuncts in cheese manufacture: Selection of Leuconostoc strains for use as adjuncts

BACKGROUND Serrano Catarinense cheese is a raw bovine milk cheese produced in the region of Santa Catarina, Brazil. Twelve representative strains of Leuconostoc isolated from 20 samples of this artisanal cheese were selected and submitted for evaluation of the acidifying, proteolytic, autolytic, aminopeptidase and lipolytic activities, NaCl and acid resistance, production of dextran and biogenic amines and antimicrobial activity. The aim was to genetically and technologically characterize the Leuconostoc strains in order to use them in mixed starter cultures for cheese manufacture. RESULTS Leuconostoc mesenteroides subsp. mesenteroides was the species that accounted for the largest proportion of isolates of Leuconostoc genus. Two leuconostoc isolates stood out in the acidifying activity, with reduction in pH of 1.12 and 1.04 units. The isolates showed low proteolytic and autolytic activity. Most of the isolates were dextran producers, presented good resistance to the salt and pH conditions of the cheese and showed antimicrobial activity against cheese pathogen bacteria, and none of them produced biogenic amines. CONCLUSION These results allowed the selection of five strains (UEL 04, UEL 12, UEL 18, UEL 21 and UEL 28) as good candidates for use as adjunct cultures for cheese manufacture.

The influence of topdressing nitrogen on Azospirillum spp. inoculation in maize crops through meta-analysis

Bragantia, Campinas, v. 77, n. 3, p.493-500, 2018 ABSTRACT: The Azospirillum is considered one of the most studied plant growth promoter genus. These bacteria are capable of promoting plant growth through several factors. However, the effect of Azospirillum spp. associated to nitrogen fertilization on maize grain yield has brought about controversial results. Thus, the objective of this study was to verify the influence of topdressing nitrogen on the effect of Azospirillum spp. inoculation on maize crops through meta-analysis. Data were collected from articles published in scientific journals, obtained from the Web of Science®, Scopus® and Google Scholar® databases. The bibliographic review included only articles with direct comparisons between maize yield in the presence and absence of Azospirillum spp. under field conditions in Brazil. The meta-analysis SOIL AND PLANT NUTRITION Article

Bactérias Promotoras do Crescimento Vegetal no Controle in vitro de Colletotrichum gloeosporioides, Agente Causal da Antracnose em Frutos de Pimenta

Universidade Estadual de Londrina – UEL, Departamento de Agronomia Caixa Postal 10.011 – CEP 86.057-970, Londrina – PR E-mail: vybaba15@gmail.com Universidade Estadual de Londrina – UEL, Departamento de Biologia Geral Caixa Postal 10.011 – CEP 86.057-970, Londrina – PR Universidade Estadual de Londrina – UEL, Departamento de Bioquímica e Biotecnologia Caixa Postal 10.011 – CEP 86.057-970, Londrina – PR

Atividade Alelopática de Exsudatos Radiculares de Milho sobre a Germinação de um Milho

Introducao: Os exsudatos radiculares produzidos por diferentes especies vegetais sao capazes de induzir respostas quimiotaticas sobre a microbiota do solo, mediando interacoes planta bacteria. Neste trabalho, exsudatos radiculares de hibridos de milho com alta (DKB 390) e baixa (DKB 240) capacidade associativa a para estudar o potencial alelopatico sobre o desenvolvimento inicial do hibrido de baixa capacidade DKB240. Metodo H2O); controle 2 (DKB240 + Ab mL); e DKB 390 (DKB240 + exsudato DKB 390, 1 mg mL inicial foi realizada apos sete dias de germinacao sob temperatura controlada (28 determinados: diâmetro de raizes, profundidade de comprimento especifico, area, comprimento total e volume, em 10 repeticoes por tratamento. Os resultados foram submetidos ao teste de medias de Tukey a 5% de significância. As avaliacoes indicaram que houve efeito alel de desenvolvimento do hibrido houve efeito alelopatico dos exsudatos testados sobre os parâmetros de desenvolvimento do hibrido DKB240, com destaque em relacao ao tratamento controle 1 (H planta (40,2%); perimetro de raizes 37,3%); area superficial de raizes (41%); comprimento total de raizes (40,3%); e volume de raizes (34,7%). Estes parâmetros nao diferiram entre os tratamentos controle 1 e controle 2 (A. brasilense Ab molecular dos exsudatos testados esta sendo realizada, e a identificacao das moleculas responsaveis pelos efeitos observados podera levar ao desenvolvimento de aditivos comerciais para uso como tratamento de sementes.