Genome mining of Burkholderia ambifaria strain T16, a rhizobacterium able to produce antimicrobial compounds and degrade the mycotoxin fusaric acid.

Burkholderia ambifaria T16 is a bacterium isolated from the rhizosphere of barley plants that showed a remarkable antifungal activity. This strain was also able to degrade fusaric acid (5-Butylpyridine-2-carboxylic acid) and detoxify this mycotoxin in inoculated barley seedlings. Genes and enzymes responsible for fusaric acid degradation have an important biotechnological potential in the control of fungal diseases caused by fusaric acid producers, or in the biodegradation/bio catalysis processes of pyridine derivatives. In this study, the complete genome of B. ambifaria T16 was sequenced and analyzed to identify genes involved in survival and competition in the rhizosphere, plant growth promotion, fungal growth inhibition, and degradation of aromatic compounds. The genomic analysis revealed the presence of several operons for the biosynthesis of antimicrobial compounds, such as pyrrolnitrin, ornibactin, occidiofungin and the membrane-associated AFC-BC11. These compounds were also detected in bacterial culture supernatants by mass spectrometry analysis. In addition, this strain has multiple genes contributing to its plant growth-promoting profile, including those for acetoin, 2,3-butanediol and indole-3-acetic acid production, siderophores biosynthesis, and solubilisation of organic and inorganic phosphate. A pan-genomic analysis demonstrated that the genome of strain T16 possesses large gene clusters that are absent in the genomes of B. ambifaria reference strains. According to predictions, most of these clusters would be involved in aromatic compounds degradation. One genomic region, encoding flavin-dependent monooxygenases of unknown function, is proposed as a candidate responsible for fusaric acid degradation.

Contribution of the Siderophores Pyoverdine and Enantio-Pyochelin to Fitness in Soil of Pseudomonas protegens Pf-5.

Pseudomonas protegens synthesizes two major iron-chelating metabolites (siderophores): pyoverdine (Pvd) and enantio-pyochelin (E-Pch). Although iron sequestration and uptake seem to be the main biological role of these siderophores, other functions including metal homeostasis and antibiotic activity have been proposed. The aim of this study was to evaluate the contribution of Pvd and E-Pch to the survival of P. protegens in soil using wild type and isogenic mutant strains unable to produce Pvd, E-Pch or both siderophores. Survival of these strains in sterile soil microcosms, in soil microcosms containing the native microflora and in sterile soil microcosms containing fusaric acid (a mycotoxin able to chelate iron and other metals), was compared by determination of colony forming units (CFU) per gram dry soil over time. In sterile soil, cell densities of Pvd-producing strains were significantly higher than that of non-producers after 21 days of permanence in the microcosms. In non-sterile soil, viability of all strains declined faster than in sterile soil and Pvd producers showed higher CFU × (g dry weight soil)-1 values than non-producers. The presence of fusaric acid negatively affected viability of strains unable to produce Pvd, while had no effect on the viability of strains able to produce Pvd. Altogether, these results show that the ability to produce Pvd increases survival of P. protegens in soil, while the ability to synthesize E-Pch does not, indicating that under the conditions which prevail in soil, iron scavenging via Pvd is more beneficial than via E-Pch.

A novel Burkholderia ambifaria strain able to degrade the mycotoxin fusaric acid and to inhibit Fusarium spp. growth.

Fusaric acid (FA) is a fungal metabolite produced by several Fusarium species responsible for wilts and root rot diseases of a great variety of plants. Bacillus spp. and Pseudomonas spp. have been considered as promising biocontrol agents against phytopathogenic Fusarium spp., however it has been demonstrated that FA negatively affects growth and production of some antibiotics in these bacteria. Thus, the capability to degrade FA would be a desirable characteristic in bacterial biocontrol agents of Fusarium wilt. Taking this into account, bacteria isolated from the rhizosphere of barley were screened for their ability to use FA as sole carbon and energy source. One strain that fulfilled this requirement was identified according to sequence analysis of 16S rRNA, gyrB and recA genes as Burkholderia ambifaria. This strain, designated T16, was able to grow with FA as sole carbon, nitrogen and energy source and also showed the ability to detoxify FA in barley seedlings. This bacterium also exhibited higher growth rate, higher cell densities, longer survival, higher levels of indole-3-acetic acid (IAA) production, enhanced biofilm formation and increased resistance to different antibiotics when cultivated in Luria Bertani medium at pH 5.3 compared to pH 7.3. Furthermore, B. ambifaria T16 showed distinctive plant growth-promoting features, such as siderophore production, phosphate-solubilization, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, in vitro antagonism against Fusarium spp. and improvement of grain yield when inoculated to barley plants grown under greenhouse conditions. This strain might serve as a new source of metabolites or genes for the development of novel FA-detoxification systems.

Identification and expression analysis of 11 subtilase genes during natural and induced senescence of barley plants.

Subtilases are one of the largest groups of the serine protease family and are involved in many aspects of plant development including senescence. In wheat, previous reports demonstrate an active participation of two senescence-induced subtilases, denominated P1 and P2, in nitrogen remobilization during whole plant senescence. The aim of the present study was to examine the participation of subtilases in senescence-associated proteolysis of barley leaves while comparing different senescence types. With this purpose, subtilase enzymatic activity, immunodetection with a heterologous antiserum and gene expression of 11 subtilase sequences identified in barley databases by homology to P1 were analyzed in barley leaves undergoing dark-induced or natural senescence at the vegetative or reproductive growth phase. Results showed that subtilase specific activity as well as two inmunoreactive bands representing putative subtilases increased in barley leaves submitted to natural and dark-induced senescence. Gene expression analysis showed that two of the eleven subtilase genes analyzed, HvSBT3 and HvSBT6, were up-regulated in all the senescence conditions tested while HvSBT2 was expressed and up-regulated only during dark-induced senescence. On the other hand, HvSBT1, HvSBT4 and HvSBT7 were down-regulated during senescence and two other subtilase genes (HvSBT10 and HvSBT11) showed no significant changes. The remaining subtilase genes were not detected. Results demonstrate an active participation of subtilases in protein degradation during dark-induced and natural leaf senescence of barley plants both at the vegetative and reproductive stage, and, based on their expression profile, postulate HvSBT3 and HvSBT6 as key components of senescence-associated proteolysis.

Evaluation of native bacteria and manganese phosphite for alternative control of charcoal root rot of soybean.

Plant growth promoting rhizobacteria (PGPR) are potential agents to control plant pathogens and their combined use with biopesticides such as phosphites may constitute a novel strategy to incorporate in disease management programs. In the present study, 11 bacterial isolates were selected on the basis of their antagonistic activity against Macrophomina phaseolina in dual-culture tests, and their plant growth promoting traits. Selected isolates were characterised on the basis of auxin and siderophore production, phosphate solubilisation and rep-PCR genomic fingerprinting. Two of these isolates, identified as Pseudomonas fluorescens 9 and Bacillus subtilis 54, were further evaluated for their inhibitory capacity against M. phaseolina using in vitro (on soybean seeds) and in vivo (greenhouse assay) tests. Both bacteria were applied individually as well as in combined treatment with manganese phosphite as seed treatments. Damage severity on soybean seeds was significantly reduced, compared with the untreated control, by both bacterial strains; however, the individual application of phosphite showed to be least effective in controlling M. phaseolina. Interestingly, the phosphite treatment improved its performance under greenhouse conditions compared to the results from the in vitro assays. In the greenhouse trials, the greatest reductions in disease severity were achieved when strain P. fluorescens 9 was applied singly or when strain B. subtilis 54 was combined with manganese phosphite, achieving 82% of control in both cases. This work is the first to report the control of M. phaseolina using combined treatment with PGPR and phosphite under greenhouse conditions.

Analysis of ascorbate peroxidase genes expressed in resistant and susceptible wheat lines infected by the cereal cyst nematode, Heterodera avenae.

Changes in ascorbate peroxidase (APX) enzyme activity in response to nematode (Heterodera avenae) attack were studied in roots of three hexaploid wheat lines carrying Cre2, Cre5, or Cre7 nematode resistance genes and the susceptible Triticum aestivum cv. Anza. A spectrophotometric analysis was carried out with root extracts of infected plants 4, 7, 11, and 14 days after nematode inoculation using uninfected plant as control. APX induction in infected resistant genotypes was similar and higher than in the susceptible control. The introgression wheat/Aegilops ventricosa H-93-8 line, carrying the Cre2 gene, and its parental line H-10-15 as susceptible control were used to analyze whether this increase of activity was correlated with the induction of APX gene expression. Genes encoding cytosolic forms of APX were induced in roots of both lines in response to nematode infection. This induction took place both earlier and with greater intensity in the resistant line than in the susceptible one, and it was also higher in the root area at the site of nematode attachment.

Analysis of class III peroxidase genes expressed in roots of resistant and susceptible wheat lines infected by Heterodera avenae.

The response of resistant wheat-Aegilops ventricosa introgression line H-93-8 and its susceptible parent, Triticum aestivum H-10-15, to Ha71 Spanish population of Heterodera avenae was studied to determine the changes in peroxidase gene expression during incompatible and compatible wheat-nematode interactions. Twenty peroxidase genes were characterized from both 211 expressed sequence tags and 259 genomic DNA clones. Alignment of deduced amino acid sequences and phylogenetic clustering with peroxidases from other plant species showed that these enzymes fall into seven different groups (designated TaPrx108 to TaPrx114) which represent peroxidases secreted to the apoplast by a putative N-terminal peptide signal. TaPrx111, TaPrx112, and TaPrx113 were induced by nematode infection in both genotypes but with differing magnitude and timing. TaPrx112 and TaPrx113 groups increased more in resistant than in susceptible infected lines. In addition, in situ hybridization analyses of genes belonging to TaPrx111, TaPrx112, and TaPrx113 groups revealed a more intense signal in cells close to the vascular cylinder and parenchyma vascular cells of resistant than susceptible wheat when challenged by nematodes. These data seem to suggest that wheat apoplastic peroxidases, because of their different expression in quantity and timing, play different roles in the plant response to nematode infection.