Strain FP2 Research Articles

Impact of seed-applied fungicide and insecticide on Azospirillum brasilense survival and wheat growth-promoting ability.

The use of Azospirillum brasilense as a crop inoculant has increased in recent years. Thus, the compatibility of the inoculation technology with seed treatments using pesticides needs to be evaluated. In this study, we evaluated the effect of an insecticide and fungicide formulation on A. brasilense strain FP2 population by culturing and culture-independent approaches. In addition, we evaluated the impact of these pesticides on the ability of A. brasilense to promote plant growth by monitoring biometric traits (root and shoot dry mass and length) of wheat grown in Greenhouse conditions. Seed pesticide dressings, mainly fungicide, led to a significant mortality of A. brasilense over time. The ability of A. brasilense to promote wheat growth also decreased due to pesticide treatments combined with sowing delay. Considering that pesticides confer fitness advantages to the wheat in field condition, our results suggest that sowing within the first 4 h after inoculation maintain the beneficial effects of A. brasilense on wheat growth promotion. Furthermore, we conclude that inoculation and treatment of seeds with pesticides may be compatible techniques when carried out immediately before sowing.

Whole-Genome Sequencing of Clostridium sp. Strain FP2, Isolated from Spoiled Venison

Clostridium sp. strain FP2 was isolated from vacuum-packaged refrigerated spoiled venison in New Zealand. This report describes the generation and annotation of the 5.6-Mb draft genome sequence of Clostridium sp. FP2, which will facilitate future functional genomic studies to improve our understanding of premature spoilage of red meats.

Process optimization of butachlor bioremediation by Enterobacter cloacae using Plackett Burman design and response surface methodology

Abstract The present study was undertaken to assess the biodegradation efficiency of native butachlor catabolizing bacterial strains isolated from the soil contaminated with the effluents from pesticide formulation units of Odisha, India. Butachlor, a chloroacetanilide class of herbicide, is widely used for the control of unwanted annual grasses and broadleaf weeds. The microbial strains designated as FP1, FP2 and FP4 showed better butachlor tolerance capability than the rest of the isolates. Out of these, the strain FP2 demonstrated the highest degradation efficiency and hence been subjected to morphological, biochemical and genetic characterisation. The bacterial isolate was identified as Enterobacter cloacae as per the 16 s rRNA gene sequencing. Two-step statistical approach was employed to optimised various parameters that affect the butachlor degradation efficiency of the bacteria. Parameters such as growth temperature, pH and media components were screened applying Plackett–Burman design and were optimised further by employing the response surface methodology. Fitting the experimental results via a second order quadratic polynomial equation having a correlation coefficient of 0.99 has been achieved. Under optimised conditions, the microbial strain was able to display a biodegradation efficiency of 1.67 mg/L/hr of butachlor. The obtained results proved the ability of the isolated strain to be used for the remediation of butachlor-contaminated effluents efficiently.

Azospirillum brasilense FP2 modulates respiratory burst oxidase gene expression in maize seedlings

Azospirillum brasilense is free-living gram-negative bacteria isolated from the rhizosphere of grasses. The plant growth promoting rhizobacteria, such as A. brasilense, can promote plant growth through biological nitrogen fixation and phytohormones production. Our aim was to evaluate the expression of several maize genes involved in stress response of maize (Pioneer 30F53 variety) after inoculation with A. brasilense strain FP2. Samples were collected 1, 4, 7 and 10 days after inoculation (DAI). Significant increase of the transcript level was observed for Auxin transporter-like protein 1 (Zmaux1), Respiratory burst oxidase protein (ZmrbohA and ZmrbohB), MAP kinase 5 (Zmmpk5), Gibberellin 20 oxidase 4 (Zmga20ox4) and Ent-kaurene oxidase (Zmko1) when comparing inoculated to non-inoculated samples. For root samples, increasing transcripts levels for Zmmpk5 gene were observed from 1 to 7 DAI, and a decrease at 10 DAI in inoculated samples compared to non-inoculated. The modulation of gene expression observed for genes involved in the oxidative burst (ZmrbohA and ZmrbohB) and for the signal transduction chain (Zmmpk5), indicates that the plant respond to the presence of the bacterium.

Microscopic and proteomic analysis of Zea mays roots (P30F53 variety) inoculated with Azospirillum brasilense strain FP2

Plant growth-promoting rhizobacteria (PGPR) are considered to have a beneficial effect on host plants. Azospirillum brasilense is a diazotroph associated with important agricultural crops such as maize and wheat, and has been used as model organism to investigate associative plant growth promotion. In this study, we have employed scanning electronic microscopic to observe the presence of bacterium on the root surface. Two-dimensional gel electrophoresis and mass spectrometry were used to analyze the changes in protein profile of maize roots in response to A. brasilense strain FP2 7 days after inoculation and we observed 46 differently expressed protein spots. We report the identification of three of these proteins.

Dual RNA-seq transcriptional analysis of wheat roots colonized by Azospirillum brasilense reveals up-regulation of nutrient acquisition and cell cycle genes.

BackgroundThe rapid growth of the world’s population demands an increase in food production that no longer can be reached by increasing amounts of nitrogenous fertilizers. Plant growth promoting bacteria (PGPB) might be an alternative to increase nitrogenous use efficiency (NUE) in important crops such wheat. Azospirillum brasilense is one of the most promising PGPB and wheat roots colonized by A. brasilense is a good model to investigate the molecular basis of plant-PGPB interaction including improvement in plant-NUE promoted by PGPB.ResultsWe performed a dual RNA-Seq transcriptional profiling of wheat roots colonized by A. brasilense strain FP2. cDNA libraries from biological replicates of colonized and non-inoculated wheat roots were sequenced and mapped to wheat and A. brasilense reference sequences. The unmapped reads were assembled de novo. Overall, we identified 23,215 wheat expressed ESTs and 702 A. brasilense expressed transcripts. Bacterial colonization caused changes in the expression of 776 wheat ESTs belonging to various functional categories, ranging from transport activity to biological regulation as well as defense mechanism, production of phytohormones and phytochemicals. In addition, genes encoding proteins related to bacterial chemotaxi, biofilm formation and nitrogen fixation were highly expressed in the sub-set of A. brasilense expressed genes.ConclusionsPGPB colonization enhanced the expression of plant genes related to nutrient up-take, nitrogen assimilation, DNA replication and regulation of cell division, which is consistent with a higher proportion of colonized root cells in the S-phase. Our data support the use of PGPB as an alternative to improve nutrient acquisition in important crops such as wheat, enhancing plant productivity and sustainability.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-378) contains supplementary material, which is available to authorized users.

Real time PCR detection targeting nifA gene of plant growth promoting bacteria Azospirillum brasilense strain FP2 in maize roots

The plant growth promoting bacteria (PGPB) Azospirillum brasilense has been recommended for use in commercial inoculants in Brazil. Effective methods are necessary to monitor PGPB strains in the rhizosphere. Our purpose was to develop a real time PCR method for detection of A. brasilense strain FP2 in maize seedlings targeting nifA. Primer pairs were designed and their specificity was verified using DNA from 12 different bacterial species. Standard curves were prepared for DNA quantification using serial dilution of A. brasilense DNA extracts. PCR efficiencies and correlation coefficient presented values within the acceptable range for qPCR, mean PCR efficiency was 95 % and correlation coefficient was 0.98, indicating that nifA gene was suitable for the quantitative analysis of the target bacterial genome. Inoculated maize seedlings were grown in vitro or in pots, bacterial DNA copy number per gram of fresh root was quantified 1, 4, 7 and 10 days after inoculation. The developed primers targeting nifA will be useful for monitoring Azospirillum brasilense strain FP2 in crops.

Identification of six differentially accumulated proteins of Zea mays seedlings (DKB240 variety) inoculated with Azospirillum brasilense strain FP2

The plant growth promoting bacteria (PGPB) Azospirillum brasilense has been known to exert beneficial influence on inoculated plants, such as maize and wheat. The successful PGPB-plant association depends on plant and PGPB genotypes and it was not fully understood. In this study we have employed two-dimensional gel electrophoresis and mass spectrometry to analyze the changes in protein profile of maize roots in response to A. brasilense strain FP2 ten days after inoculation and 87 differently expressed protein spots were revealed. We report the identification of six of these proteins in detail: Two down-regulated proteins in inoculated roots, Zea mays annexin2 (2-fold) and Z. mays hypothetical protein LOC100275372 (4-fold), that is 60% similar to an Arabidopsis thaliana hydroxyproline-rich glycoprotein-like protein; two up-regulated proteins in inoculated roots, A. brasilense F-type H+-transporting ATPase beta chain (3-fold) and A. brasilense major outer membrane protein OmaA precursor (14-fold). Two spots exclusively expressed in inoculated roots were identified as A. brasilense malate dehydrogenase.

Marinobacter lacisalsi sp. nov., a moderately halophilic bacterium isolated from the saline-wetland wildfowl reserve Fuente de Piedra in southern Spain

A Gram-negative, non-spore-forming, motile, moderately halophilic, aerobic, rod-shaped bacterium, designated strain FP2.5(T), was isolated from the inland hypersaline lake Fuente de Piedra, a saline-wetland wildfowl reserve located in the province of Málaga in southern Spain. Strain FP2.5(T) was subjected to a polyphasic taxonomic study. It produced colonies with a light-yellow pigment. Strain FP2.5(T) grew at salinities of 3-15 % (w/v) and at temperatures of 20-40 degrees C. The pH range for growth was 5-9. Strain FP2.5(T) was able to utilize various organic acids as sole carbon and energy source. Its major fatty acids were C(16 : 0), C(18 : 1)omega9c and C(16 : 1)omega9c. The DNA G+C content was 58.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain FP2.5(T) appeared to be a member of the genus Marinobacter and clustered closely with the type strains of Marinobacter segnicrescens, Marinobacter bryozoorum and Marinobacter gudaonensis (levels of 16S rRNA gene sequence similarity of 98.1, 97.4 and 97.2 %, respectively). However, DNA-DNA relatedness between the new isolate and the type strains of its closest related Marinobacter species was low; levels of DNA-DNA relatedness between strain FP2.5(T) and M. segnicrescens LMG 23928(T), M. bryozoorum DSM 15401(T) and M. gudaonensis DSM 18066(T) were 36.3, 32.1 and 24.9 %, respectively. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data, strain FP2.5(T) is considered to represent a novel species of the genus Marinobacter, for which the name Marinobacter lacisalsi sp. nov. is proposed. The type strain is FP2.5(T) (=CECT 7297(T)=LMG 24237(T)).

In vitro uridylylation of the Azospirillum brasilense N-signal transducing GlnZ protein

Azospirillum brasilense is a diazotroph which associates with important agricultural crops. The nitrogen fixation process in this organism is highly regulated by ammonium and oxygen, and involves several proteins including the two PII-like proteins, GlnB and GlnZ. Although these proteins are structurally very similar, they play different roles in the control of nitrogen fixation. In this work, we describe the expression, purification, and uridylylation of the GlnZ protein of A. brasilense strain FP2. The amplified glnZ gene was sub-cloned and expressed as a His-tagged fusion protein. After purification, we obtained 30–40 mg of purified GlnZ per liter of culture. This protein was purified to 99% purity and assayed for in vitro uridylylation using a partially purified Escherichia coli GlnD as a source of uridylylyl-transferase activity. Analyses of the uridylylation reactions in non-denaturing and denaturing polyacrylamide gel electrophoresis showed that up to 74% of GlnZ monomers were modified after 30 min reaction. This covalent modification is strictly dependent on ATP and 2-ketoglutarate, while glutamine acts as an inhibitor and promotes deuridylylation.

Monitoring A zospirillum-wheat interactions using the gfp and gusA genes constitutively expressed from a new broad-host range vector

To monitor the colonization of wheat roots by Azospirillum brasilense, we constructed several plasmids based on the pBBR1 replicon expressing the gfp and gusA genes constitutively. Both genes were placed under control of the gentamycin resistance gene promoter resulting in high levels of expression in Escherichia coli and A. brasilense. The constructed plasmids were stably maintained in A. brasilense strains even in the absence of selective pressure. The colonization of wheat plants grown under controlled conditions in sterilized vermiculite by A. brasilense strain FP2 (a Sp7-derivative) transconjugants containing these plasmids was monitored. Bacteria expressing GFP were easily observed in fresh plant material by fluorescence microscopy. Cell aggregates and single bacteria were visualized on the surfaces of young root zones, such as roots hairs and lateral roots. Large cellular clumps were observed at the points of lateral root emergence or at intercellular spaces of root epidermal cells 30 days after inoculation. Although we failed to detected bacteria in internal cortical and xylem tissues of wheat roots, the initial stage of endophytic colonization by A. brasilense may involve the sites detected in this work.

ThentrBCgenes ofAzospirillum brasilenseare part of anifR3-like–ntrB–ntrCoperon and are negatively regulated

A cosmid able to complement the Nif- and nitrate-dependent growth phenotypes of the Azospirillum brasilense mutant FP9 was isolated from a genomic library of the wild-type strain FP2. A 6-kb DNA region was sequenced and showed two open reading frames (ORFs) identified as the ntrB and ntrC genes. An ORF1 located upstream from the ntrB gene and coding for a 36-kDa polypeptide showed similarity to the nifR3 gene of Rhodobacter capsulatus and the ORF1 of Rhizobium leguminosarum, both located upstream from the ntrB gene in a complex operon. Two other unidentified ORFs (ORF5 and partial ORF4) coding for hydrophobic polypeptides were also observed. delta ORF1-ntrBC, ORF1, ntrB, and ntrC mutants obtained by recombination of suicide plasmids containing an insertion of a promoterless lacZ kanamycin cassette showed decreased nitrogenase activities and were unable to grow on nitrate as the sole N source. These phenotypes were restored by complementation with plasmids containing the ntrC gene. Analysis of lacZ transcriptional fusions suggested that the ORF1-ntrBC operon in Azospirillum brasilense is expressed from a promoter located upstream from the ORF1 and that it is negatively regulated by the ntrC gene product.

Excretion of ammonium by Azospirillum brasilense mutants resistant to ethylenediamine

Several spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense strain FP2 (Sp7, NalR SmR) were isolated. Four mutants, HM053, HM14, HM26, and HM210, were found to fix nitrogen constitutively in the presence of high concentration of NH4+ and to excrete NH4+ derived from nitrogen fixation. They also showed lower rates of NH4+ uptake than the wild-type strain, FP2. All of the mutants were prototrophic for glutamine or glutamate. Their glutamate synthase and glutamate dehydrogenase activities were similar to those of the wild-type strain. However, they presented different patterns of glutamine synthetase activity. Mutant HM14 showed low levels of normally regulated glutamine synthetase activity, while the other mutants showed low levels (HM053) or wild-type levels (HM26 and HM210) of constitutively adenylylated glutamine synthetase activity. The mutants are probably defective in the adenylylation system. Key words: Azospirillum brasilense, ammonium excretion, ethylenediamine resistance, glutamine synthetase.

Production and Comparison of Indigenous and Commercial Baker’s Yeasts

A comparative investigation on the characteristics, production and leavening action of two indigenous (isolate FR-1 and DP-4) and three commercial baker’s yeasts (Saccharomyces cerevisiae strain FT, FP-1 and FP-2) was carried out in laboratory scale. The indigenous baker’s yeasts, FR-1 and DP-4, were isolated from fermented rice and decomposed pineapple and they were presumptively identified as Saccharomyces cerevisiae and Saccharomyces species respectively. Among these five yeast isolates, FR-1, FT, FP-1 and FP-2 were found similar in morphological and cultural characteristics. The effect of physicochemical properties on the growth and production of cell mass was studied. The maximum cell mass was found after 36 h of incubation during growth in MYGP and MYMP media containing 1.5% glucose at 30°C and pH 5.0. The maximum cell mass was produced by DP-4, followed by FP-2, FR-1, FT and FP-1. The effect of different bread ingredients on leavening was also investigated and it was found that oil, butter, egg and milk influenced the leavening action by these yeasts. The pathogenicity test of FR-1 and DP-4 on animal models (rabbits) exhibited no abnormalities in the experimental animals. The indigenous isolates, FR-1 and DP-4, seemed potential as candidates for bread making.Keywords: Baker’s yeast, Saccharomyces cerevisiae, Physicochemical parameters, Cell mass, Leavening actionDOI: http://dx.doi.org/10.3329/bjm.v23i2.868 Bangladesh J Microbiol, Volume 23, Number 2, December 2006, pp 89-92