Burkholderia Tropica Research Articles

Draft Genome Sequence of Phosphate-Solubilizing Bacterium Paraburkholderia tropica Strain P-31 Isolated from Pomegranate (Punica granatum) Rhizosphere.

We report the 8.9 Mb draft genome sequence of phosphate-solubilizing bacterium Paraburkholderia tropica strain P-31, isolated from pomegranate (Punica granatum) rhizosphere. The draft genome sequence of Paraburkholderia tropica strain P-31 consists of 8,881,246 bp with a G+C content of 64.7%, 8,039 protein-coding genes, and 49 RNAs.

Selection and evaluation of reference genes for RT-qPCR expression studies on Burkholderia tropica strain Ppe8, a sugarcane-associated diazotrophic bacterium grown with different carbon sources or sugarcane juice.

Among the members of the genus Burkholderia, Burkholderia tropica has the ability to fix nitrogen and promote sugarcane plant growth as well as act as a biological control agent. There is little information about how this bacterium metabolizes carbohydrates as well as those carbon sources found in the sugarcane juice that accumulates in stems during plant growth. Reverse transcription quantitative PCR (RT-qPCR) can be used to evaluate changes in gene expression during bacterial growth on different carbon sources. Here we tested the expression of six reference genes, lpxC, gyrB, recA, rpoA, rpoB, and rpoD, when cells were grown with glucose, fructose, sucrose, mannitol, aconitic acid, and sugarcane juice as carbon sources. The lpxC, gyrB, and recA were selected as the most stable reference genes based on geNorm and NormFinder software analyses. Validation of these three reference genes during strain Ppe8 growth on the same carbon sources showed that genes involved in glycogen biosynthesis (glgA, glgB, glgC) and trehalose biosynthesis (treY and treZ) were highly expressed when Ppe8 was grown in aconitic acid relative to other carbon sources, while otsA expression (trehalose biosynthesis) was reduced with all carbon sources. In addition, the expression level of the ORF_6066 (gluconolactonase) gene was reduced on sugarcane juice. The results confirmed the stability of the three selected reference genes (lpxC, gyrB, and recA) during the RT-qPCR and also their robustness by evaluating the relative expression of genes involved in glycogen and trehalose biosynthesis when strain Ppe8 was grown on different carbon sources and sugarcane juice.

English

The use of inoculants containing growth-promoting diazotrophic bacteria can stimulate mass and nutrient accumulation in sugarcane. The purpose of this study was to evaluate plant growth and accumulation of macroelements in sugarcane, variety RB92579, under bacterial inoculation with or without N fertilization. The field experiment was carried out in a Red-Yellow Podzolic soil in Seropédica, RJ, in a randomized block design with four replications. The treatments consisted of 50 kg N ha-1; 50 kg N ha-1 + inoculation; inoculation; and an absolute control. The following bacteria were inoculated: Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Azospirillum amazonense, and Burkholderia tropica. The plants were sampled at 100, 130, 168, 212, 261, and 295 days after planting (DAP), and growth and nutrient accumulation rates were estimated by functional analysis of plant growth. Nutrient accumulation rates were highest around 180 DAP for N and P, and around 160 DAP for K, in the different treatments, preceding the maximal crop growth rate (between 210 and 220 DAP). The accumulation of biomass, N, P and K was greater and crop growth rates were higher in the treatments with bacterial inoculation fertilized or not with 50 kg N ha-1, compared with the control.   Key words: Saccharum species, inoculum, growth promotion.

Burkholderia tropica UNA BACTERIA CON GRAN POTENCIAL PARA SU USO EN LA AGRICULTURA

The genus Burkholderia whit over 90 species reported to date, is divided into two major groups phylogenetically distant. The first group is made up of pathogenic species which highlights referred opportunistic pathogens such as Burkholderia cepacia (Bcc) complex; The other group consists of nonpathogenic species with skills to promote plant growth and rhizoremediation. Burkholderia tropica is a bacterium capable of fixing nitrogen; isolated from the rhizosphere, rhizoplane, stem and roots of corn plants and sugarcane. Besides its ability diazotrofa, B. tropica presents characteristics that classify it as a plant promoter bacteria, such growth as is the ability to produce siderophores, solubilizing phosphates, producing exo-heteropolysaccharides, and can be used as biocontrol for some plant parasites, which It makes it a promising bacteria to be used in the agricultural sector.

Identificación molecular de bacterias asociadas a plantas ornamentales producidas in vitro

En plantas ornamentales reproducidas in vitro en viveros del Centro de Desarrollo Tecnológico Tezoyuca perteneciente al FIRA, se detectaron síntomas de necrosamiento en hojas y tallo durante la fase de adaptación. El objetivo de este trabajo fue identificar el agente etiológico de la enfermedad en hojas y tallos necrosados de los explantes en la etapa de aclimatación, los cuales se sembraron en condiciones asépticas para detectar el crecimiento de microorganismos. Se obtuvieron doce colonias de bacterias de dichas muestras, a las que se les extrajo ADN con un kit comercial. Los iniciadores de la reacción de PCR fueron rP2 y fD1. Todos los productos de PCR se secuenciaron y se analizaron con el programa Chromas Lite®. Utilizando la búsqueda de homología por BLAST se identificaron las siguientes bacterias: Kosakonia oryzae, Pectobacterium cypripedii, Burkholderia tropica, Serratia marcescens, Pantoea dispersa, Erwinia rhapontici, Pantoea dispersa y Pantoea agglomerans. De éstas, Pectobacterium cypripedii, Pantoea agglomerans y Erwinia rhapontici son de importancia fitosanitaria. No se hicieron pruebas de patogenicidad con estos microorganismos.

Role of phosphate solubilizing Burkholderia spp. for successful colonization and growth promotion of Lycopodium cernuum L. (Lycopodiaceae) in lateritic belt of Birbhum district of West Bengal, India

Profuse growth of Lycpodium cernuum L. was found in phosphate deficient red lateritic soil of West Bengal, India. Interaction of vesicular–arbuscular mycorrhiza (VAM) with Lycopodium rhizoids were described earlier but association of PGPR with their rhizoids were not studied. Three potent phosphate solubilizing bacterial strains (P4, P9 and P10) associated with L. cernuum rhizoids were isolated and identified by 16S rDNA homologies on Ez-Taxon database as Burkholderia tropica, Burkholderia unamae and Burkholderia cepacia respectively. Day wise kinetics of phosphate solubilization against Ca3(PO4)2 suggested P4 (580.56±13.38μgml−1) as maximum mineral phosphate solubilizer followed by P9 (517.12±17.15μgml−1) and P10 (485.18±14.23μgml−1) at 28°C. Release of bound phosphates by isolated strains from ferric phosphate (FePO4), aluminum phosphate (AlPO4) and four different complex rock phosphates indicated their very good phosphate solubilizng efficacy. Nitrogen independent solubilizition also supports their nitrogen fixing capabilities. Inhibition of P solubilization by calcium salts and induction by EDTA suggested pH dependent chelation of metal cations by all of the isolates. Rhizoidal colonization potentials of Burkholderia spp. were confirmed by in planta experiment and also using scanning electron microscope (SEM). Increases of total phosphate content in Lycopodium plants upon soil treatment with these isolates were also recorded. In addition siderophore production on CAS agar medium, tryptophan dependent IAA production and antifungal activities against pathogenic fungi by rhizospheric isolates deep-rooted that they have definite role in nutrient mobilization for successful colonization of L. cernuum in nutrient deficient lateritic soil.

Desenvolvimento Inicial de Duas Variedades de Cana-de-açúcar Inoculadas com Bactérias Diazotróficas

RESUMO A cana-de-açúcar é uma cultura de grande destaque na economia, em razão da produção de açúcar, etanol e energia. Tecnologias que possam contribuir para o aumento da produtividade e qualidade da cultura com mínimos danos ao meio ambiente são necessárias. Objetivou-se avaliar a produção de ácido indol acético de cinco estirpes de bactérias diazotróficas e o efeito da inoculação delas na brotação de duas variedades de cana-de-açúcar, RB867515 e IACSP95-5000. A produção de auxina foi determinada pelo teste colorimétrico, usando o reagente de Salkowski. Para avaliar a germinação, foi conduzido um experimento em casa de vegetação, utilizando-se delineamento experimental em blocos ao acaso com quatro repetições e sete tratamentos: controle não inoculado; inoculação mista com as cinco estirpes e inoculação individual com Gluconacetobacter diazotrophicus (Gd) estirpe BR11281T(PAL-5T), Herbaspirillum seropedicae (Hs - BR11335 = HRC54), Herbaspirillum rubrisubalbicans (Hr - BR11504 = HCC103), Burkholderia tropica (Bt - BR11366T = PPe 8 T) e Azospirillum amazonense (Aa - BR11145 = CBAMc). As bactérias mais eficientes na produção de auxina foram Hs e Hr, declinando 48 h após o crescimento. Hr, Aa e Bt aumentaram o índice de velocidade de germinação e o número de brotações nas duas variedades. Na var. RB867515, a velocidade de germinação ainda foi positivamente influenciada pela inoculação mista, sendo o mesmo observado pela inoculação de Gd na var. IACSP95-5000.

Colonization and plant growth-promotion of tomato by Burkholderia tropica

Several diazotrophic Burkholderia species have been described to exhibit some activities involved in plant growth promotion and biological control. In this work seedlings of tomato plants were inoculated with this bacterium in order to study colonization of different vegetal tissues and plant growth promoting ability under greenhouse conditions. Tomato seedlings inoculated with Burkholderia tropica strain MTo-293 and two derivative strains containing the marker genes gusA and gfp, respectively (constructions described in this work), were grown under gnotobiotic conditions. Colonization was monitored both by colony counting of bacterial suspensions from homogenized tissues with or without previous surface disinfection and by microscopic observation of entire plant tissues. In another set of experiments tomato seedlings were inoculated with B. tropica MTo-293 for evaluation of tomato production under greenhouse conditions. Tomato yields were determined by quantifying total tomato production throughout the crop in two different seasons. B. tropica could be isolated from root surfaces (>7.0 logCFUg−1 fresh weight) and from surface-disinfected and disrupted roots (>5.0 logCFUg−1 fresh weight) and stems (>4.0 logCFUg−1 fresh weight) of inoculated plants. Microscopic studies showed colonizing bacteria on root hairs, root tips, lateral root emergence sites, and stomata. In greenhouse experiments inoculated plants showed a consistent increase of both number and weight of fruits as compared to uninoculated controls. Although this enhancement in fruit production was only statistically significant for fruit weight in the first crop season, our results show a consistent tendency to a higher yield (5–15%) for the inoculated treatments also in the second year. These results show that seedling inoculation with B. tropica led to effective root colonization of tomato plants followed by bacterial spreading to aerial tissues. This significant colonization was accompanied by an enhancement of tomato production in two different crop seasons.

Identification of volatile compounds produced by the bacterium Burkholderia tropica that inhibit the growth of fungal pathogens

It has been documented that bacteria from the Burkholderia genera produce different kinds of compounds that inhibit plant pathogens, however in Burkholderia tropica, an endophytic diazotrophic and phosphate-solubilizing bacterium isolated from a wide diversity of plants, the capacity to produce antifungal compounds has not been evaluated. In order to expand our knowledge about Burkholderia tropica as a potential biological control agent, we analyzed 15 different strains of this bacterium to evaluate their capacities to inhibit the growth of four phytopathogenic fungi, Colletotrichum gloeosporioides, Fusarium culmorum, Fusarium oxysporum and Sclerotium rolffsi. Diverse analytical techniques, including plant root protection and dish plate growth assays and gas chromatography-mass spectroscopy showed that the fungal growth inhibition was intimately associated with the volatile compounds produced by B. tropica and, in particular, two bacterial strains (MTo293 and TTe203) exhibited the highest radial mycelial growth inhibition. Morphological changes associated with these compounds, such as disruption of fungal hyphae, were identified by using photomicrographic analysis. By using gas chromatography-mass spectroscopy technique, 18 volatile compounds involved in the growth inhibition mechanism were identified, including α-pinene and limonene. In addition, we found a high proportion of bacterial strains that produced siderophores during growth with different carbon sources, such as alanine and glutamic acid; however, their roles in the antagonism mechanism remain unclear.

Survival of endophytic bacteria in polymer-based inoculants and efficiency of their application to sugarcane

Background & aims Studies have been conducted to evaluate maintenance of cell viability and stability, as well as to select cheap carriers to extend the shelf life of plant beneficial bacterial inoculants for agricultural crops. The purpose of this study was to evaluate the shelf life and the colonization efficiency of novel liquid and gel-based inoculant formulations for sugarcane. The different inoculant formulations were all composed of a mixture of five strains of diazotrophic bacteria (Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicans, Azospirillum amazonense and Burkholderia tropica), which are recognized as sugarcane growth promoters.

Five novel acid-tolerant oligotrophic thiosulfate-metabolizing chemolithotrophic acid mine drainage strains affiliated with the genus Burkholderia of Betaproteobacteria and identification of two novel soxB gene homologues

Five acid-tolerant thiosulfate-metabolizing bacteria were isolated from acid mine drainage samples from Garubathan, India. 16S rRNA gene analysis revealed that the strains were affiliated with the genus Burkholderia of the class of Betaproteobacteria. Comparative 16S rRNA gene sequence analyses indicated that the strains designated as GAH1 and GAH2 produced a separate phylogenetic branch having Burkholderia pyrrocinia ATCC 51958 T (96–98%) as the closest relative. Strains GAH4 and Burkholderia tropica Ppe8 T (93%) branched out separately in the phylogenetic tree. Strain GMX2 was most closely related to Burkholderia cepacia ATCC 25417 T (99.6%) and Burkholderia vietnamiensis LMG 10929 T (99%). Strain GAH5 was most closely related to B. pyrrocinia ATCC 51958 T (98%). Oligotrophy has been demonstrated in all AMD strains of Burkholderia spp. All strains showed chemolithoautotrophic and mixotrophic growth in thiosulfate. Furthermore, cell-free extracts of all test strains possessed thiosulfate and sulfite dehydrogenase activities. Phylogenetic analysis of the soxB gene revealed that GAH4 and GAH2 strains formed a novel cluster, Betaproteobacteria II, having highest similarity with Allochromatium vinosum, a member of Gammaproteobacteria II.

Burkholderia heleia sp. nov., a nitrogen-fixing bacterium isolated from an aquatic plant, Eleocharis dulcis, that grows in highly acidic swamps in actual acid sulfate soil areas of Vietnam

Nitrogen-fixing bacteria, strains SA41(T), SA42 and SA53, were isolated from an aquatic plant, Eleocharis dulcis, that grows in highly acidic swamps (pH 2-4) in actual acid sulfate soil areas of Vietnam. The isolates were Gram-negative, aerobic, non-spore-forming, rod-shaped bacteria, having a cell width of 0.6-0.7 microm and a length of 1.5-1.7 microm. They showed good growth between pH 3.0 and 7.0, and between 17 and 37 degrees C. The organisms contained ubiquinone Q-8 as the predominant isoprenoid quinone, and C(16 : 0), C(17 : 0) cyclo, C(18 : 1) omega7c and summed feature 3 (C(16 : 1) omega7c and/or iso-C(15 : 0) 2-OH) as major fatty acids. Their fatty acid profiles are similar to those reported for other Burkholderia species. The DNA G+C content of these strains was 64 mol%. On the basis of 16S rRNA gene sequence similarity, these strains were shown to belong to the genus Burkholderia. Although their calculated 16S rRNA gene sequence similarity values to Burkholderia silvatlantica, Burkholderia mimosarum, Burkholderia ferrariae and Burkholderia tropica were 98.5, 98.2, 98.0 and 97.0 %, respectively, the isolates formed a distinct group in phylogenetic trees, and the DNA-DNA relatedness values of strain SA41(T) to these species were 39, 41, 39 and 33 %, respectively. The results of physiological and biochemical tests, including whole-cell protein pattern analysis, allowed phenotypic differentiation of these strains from the published Burkholderia species. Therefore, strains SA41(T), SA42 and SA53 represent a novel species for which the name Burkholderia heleia sp. nov. is proposed. The type strain is SA41(T) (=NBRC 101817(T)=VTCC-D6-7(T)).

First isolation of Burkholderia tropica from a neonatal patient successfully treated with imipenem

We report the first case of a human Burkholderia tropica infection. The patient was a premature neonate who had necrotizing enterocolitis with bowel perforation requiring surgical intervention. The stoma care and difficulties in feeding were a chronic problem. At the age of almost 4 months he developed septicemia due to B. tropica. Three consecutive blood cultures grew this organism. The organism was cleared from the blood after a course of imipenem and resolution of post-operative ileus. Our case suggests that environmental and plant pathogens can cause human infection especially in those in an immunocompromised condition.

Colonization of sugarcane plantlets by mixed inoculations with diazotrophic bacteria

Micropropagated sugarcane plants have been used in Brazil for almost three decades. Besides the improvement in plant health, micropropagated sugarcane carries no endophytic plant growth-promoting bacteria. The Brazilian inoculation technology to reintroduce diazotrophic bacteria in micropropagated sugarcane plantlets revealed a synergistic-like effect in PGP-bacteria mixed inoculations. The infection model of single diazotrophic bacteria species in sugarcane was studied in detail, but still many questions remain open. In this study we used a combined fluorescence in situ hybridization (FISH) and a cultivation based approach (MPN) to evaluate the colonization of sugarcane plantlets by mixed inocula. The highest colonization for three out of the five species studied was obtained with a mixed inoculum, when the Azospirillum amazonense showed an increase by almost 100 times in colonization and Herbaspirillum spp. and Burkholderia tropica was determined at 10 7 cells per gram root fresh weight. All of the inoculated bacterial species could be detected using the FISH probes 12 h after bacterial inoculation. The FISH results confirmed the MPN counts and showed differences in the population numbers and colonization behavior of particular bacterial inoculum strains in the different mixed inocula. A putative antagonistic effect among the inoculated H. seropedicae and H. rubrisubalbicans strains was observed using FISH, as well as the better competitiveness of B. tropica as compared to the A. amazonense strain. The observed data probably reflect also specific interactions with the sugarcane variety used in this particular inoculation system, and may not be generalized as a rule. This is the first study about the competition for sugarcane colonization in a mixed bacterial inoculum.

Burkholderia ferrariae sp. nov., isolated from an iron ore in Brazil

A Gram-negative, non-spore-forming bacterial strain with the ability to solubilize highly insoluble phosphatic minerals was isolated from a high-phosphorous iron ore from Minas Gerais State, Brazil. This strain, designated FeGl01(T), was subjected to a polyphasic taxonomic investigation. Comparative 16S rRNA gene sequence analysis indicated that it formed a distinct phylogenetic lineage within the genus Burkholderia together with several other species of the genus, e.g. Burkholderia sacchari, Burkholderia tropica and Burkholderia unamae. Partial nucleotide sequencing and analysis of the recA gene roughly corroborated the phylogenetic position of strain FeGl01(T) within the genus Burkholderia. The chemotaxonomic properties of strain FeGl01(T), such as ubiquinone Q-8 as the predominant quinone system and C(16 : 0), C(17 : 0) cyclo, C(18 : 1)omega7c and C(19 : 0)omega8c cyclo as the major fatty acids, were also consistent with its classification within the genus Burkholderia. DNA-DNA hybridization experiments between strain FeGl01(T) and the type strains of B. unamae, B. sacchari and B. tropica yielded reassociation values of 40 % or lower, which, together with qualitative and quantitative differences in fatty acid composition and with differences in several phenotypic traits, support the separation of the new isolate from the phylogenetically most closely related species. Therefore, it is suggested that strain FeGl01(T) represents a novel species of the genus Burkholderia, for which the name Burkholderia ferrariae sp. nov. is proposed. The type strain is FeGl01(T) (=LMG 23612(T)=CECT 7171(T)=DSM 18251(T)).

Culture conditions for the production of an acidic exopolysaccharide by the nitrogen-fixing bacterium Burkholderia tropica

The endophytic diazotrophic bacterium Burkholderia tropica, strain Ppe8, produced copious amounts of exopolysaccharide (EPS) on batch growth in liquid synthetic media containing mannitol and glutamate as carbon and nitrogen sources. The effect of various aeration regimes and carbon source concentrations on EPS production was determined, as well as the effects of temperature and time of growth. The degree of aeration had a great influence on the yield of EPS, in contrast with the C:N ratio of the medium. Growth temperature also affected the EPS yield after the first 24 h of culture but seemed to be irrelevant after that. After isolation and purification, the EPS was analyzed by high-performance size exclusion chromatography and multiangle laser light scattering (HPSEC-MALLS), revealing a molecular mass of 300 kDa. The acid hydrolyzate of EPS was examined by HPLC and found to contain Glc, Rha, GlcA, and an aldobiouronic acid. The latter was found to have a GlcA and a Rha unit. Carboxy-reduced EPS contained Glc and Rha (3:2). The monosaccharide composition of the native acidic EPS was calculated as GlcA, Glc, and Rha in a molar ratio of 1:2:2.

Burkholderia tropica sp. nov., a novel nitrogen-fixing, plant-associated bacterium

In an ecological survey of nitrogen-fixing bacteria isolated from the rhizosphere and as endophytes of sugarcane, maize and teosinte plants in Brazil, Mexico and South Africa, a new phylogenetically homogeneous group of N(2)-fixing bacteria was identified within the genus Burkholderia. This polyphasic taxonomic study included microscopic and colony morphology, API 20NE tests and growth on different culture media at different pH and temperatures, as well as carbon source assimilation tests and whole-cell protein pattern analysis. Analysis of 16S rRNA gene sequences showed 99.2-99.9 % similarity within the novel species and 97.2 % similarity to the closest related species, Burkholderia sacchari. The novel species was composed of four distinct amplified 16S rDNA restriction analysis groups. The DNA-DNA reassociation values within the novel species were greater than 70 % and less than 42 % for the closest related species, B. sacchari. Based on these results and on many phenotypic characteristics, a novel N(2)-fixing species is proposed for the genus Burkholderia, Burkholderia tropica sp. nov., with the type strain Ppe8(T) (=ATCC BAA-831(T)=LMG 22274(T)=DSM 15359(T)). B. tropica was isolated from plants grown in geographical regions with climates ranging from temperate subhumid to hot humid.