Selection of phosphate-solubilizing diazotrophic Herbaspirillum and Burkholderia strains and their effect on rice crop yield and nutrient uptake

Background and Aims

Plant growth-promoting bacteria, mainly diazotrophs and phosphate solubilizers, can reduce the use of chemical fertilizers for rice crops. Here, diazotrophic bacteria isolated from rice were screened for their ability to solubilize inorganic P (P_i) in vitro and in association with rice plants cultivated in pots.

Methods

Forty-nine isolates were tested for the ability to solubilize P_i on NBRIP and GL agar plate media and seven selected strains were further evaluated in NBRIP liquid medium. Three of these strains were inoculated in rice plants grown in soil pots containing ¹⁵N-labeled fertilizer and two sources of P: tricalcium phosphate (TCP) or simple superphosphate (SSP). The dry matter, yield, N, P, and the ¹⁵N content accumulated in plant tissues were measured at 135 days after planting.

Results

Seven strains belonging to the genera Herbaspirillum and Burkholderia formed a halo of solubilized P_i on agar plates. The Burkholderia strains showed peak soluble P (around 200 mg P L^?1) on the fifth day when grown in NBRIP liquid medium for 14 days. Inoculation of Herbaspirillum strains (H18, ZA15) and a Burkholderia vietaminensis strain (AR114) increased rice grain yield from 33 to 47 % with TCP and 18 to 44 % with TSS, respectively. The bacterial inoculation led to enhanced N-use efficiency of the ¹⁵N-labeled fertilizer.

Conclusion

These results suggest that the selection and use of P-solubilizing diazotrophic bacteria are a good strategy to promote P solubilization and/or N use efficiency in rice plants.     

Screening of plant growth promoting Rhizobacteria isolated from sunflower (Helianthus annuus L.)

Background and Aims

This study was aimed at assessing the diversity of putatively diazotrophic rhizobacteria associated with sunflower (Helianthus annuus L.) cropped in the south of Brazil, and to examine key plant growth promotion (PGP) characteristics of the isolates for the purposes of increasing plant productivity.

Methods

299 strains were isolated from the roots and rhizosphere of sunflower cultivated in five different areas using N-free media. 16S rDNA PCR-RFLP and 16S rRNA partial sequencing were used for identification and the Shannon index was used to evaluate bacterial diversity. Production of siderophores and indolic compounds (ICs), as well phosphate solubilization activities of each isolate were also evaluated in vitro. On the basis of multiple PGP activities, eight isolates were selected and tested for their N-fixation ability, and their capacity as potential PGPR on sunflower plants was also assessed.

Results

All except three Gram-positive strains (phylum Actinobacteria) belonged to the Gram-negative Proteobacteria subgroups [Gamma (167), Beta (78), and Alpha (50)] and the family Flavobacteriaceae (1)]. Shannon indexes ranged from 0.96 to 2.13 between the five sampling sites. Enterobacter and Burkholderia were the predominant genera isolated from roots and rhizosphere, respectively. Producers of siderophores and ICs were widely found amongst the isolates, but only 19.8% of them solubilized phosphate. About 8% of the isolates exhibited all three PGP traits, and these mostly belonged to the genus Burkholderia. Four isolates were able to stimulate the growth of sunflower plants under gnotobiotic conditions.

Conclusions

Enterobacter and Burkholderia were the dominant rhizospheric bacterial genera associated with sunflower plants. Inoculation with isolates belonging to the genera Achromobacter, Chryseobacterium, Azospirillum, and Burkholderia had a stimulatory effect on plant growth.     

The effect of plant growth-promoting rhizobacteria on the growth of rice (Oryza sativa L.) cropped in southern Brazilian fields

Background and Aims

Several strains of rhizobacteria may be found in the rhizospheric soil, on the root surface or in association with rice plants. These bacteria are able to colonize plant root systems and promote plant growth and crop yield through a variety of mechanisms. The objectives of this study were to isolate, identify, and characterize putative plant growth-promoting rhizobacteria (PGPR) associated with rice cropped in different areas of southern Brazil.

Methods

Bacterial strains were selectively isolated based on their growth on three selective semi-solid nitrogen-free media. Bacteria were identified at the genus level by PCR-RFLP 16S rRNA gene analysis and partial sequencing methodologies. Bacterial isolates were evaluated for their ability to produce indolic compounds and siderophores and to solubilize phosphate. In vitro biological nitrogen fixation and the ability to produce 1-aminocyclopropane-1-carboxylate deaminase were evaluated for each bacterial isolate used in the inoculation experiments.

Results

In total, 336 bacterial strains were isolated representing 31 different bacterial genera. Strains belonging to the genera Agrobacterium, Burkholderia, Enterobacter, and Pseudomonas were the most prominent isolates. Siderophore and indolic compounds producers were widely found among isolates, but 101 isolates were able to solubilize phosphate. Under gnotobiotic conditions, eight isolates were able to stimulate the growth of rice plants. Five of these eight isolates were also field tested in rice plants subjected to different nitrogen fertilization rates.

Conclusions

The results showed that the condition of half-fertilization plus separate inoculation with the isolates AC32 (Herbaspirillum sp.), AG15 (Burkholderia sp.), CA21 (Pseudacidovorax sp.), and UR51 (Azospirillum sp.) achieved rice growth similar to those achieved by full-fertilization without inoculation, thus highlighting the potential of these strains for formulating new bioinoculants for rice crops.     

Delineation of Paraburkholderia tuberum sensu stricto and description of Paraburkholderia podalyriae sp. nov. nodulating the South African legume Podalyria calyptrata

Since the discovery of Paraburkholderia tuberum, an indigenous South African species and one of the first beta-rhizobia described, several other South African rhizobial Paraburkholderia species have been recognized. Here, we investigate the taxonomic status of 31 rhizobial isolates from the root nodules of diverse South African legume hosts in the Core Cape Subregion, which were initially identified as P. tuberum. These isolates originate from the root nodules of genera in the Papilionoideae as well as Vachellia karroo, from the subfamily Caesalpinioideae. Genealogical concordance analysis of five loci allowed delineation of the isolates into two putative species clusters (A and B). Cluster A included P. tuberum STM678^T, suggesting that this monophyletic group represents P. tuberum sensu stricto. Cluster B grouped sister to P. tuberum and included isolates from the Paarl Rock Nature Reserve in the Western Cape Province. Average Nucleotide Identity (ANI) analysis further confirmed that isolates of Cluster A shared high genome similarity with P. tuberum STM678^T compared to Cluster B and other Paraburkholderia species. The members of Cluster B associated with a single species of Podalyria, P. calyptrata. For this new taxon we accordingly propose the name Paraburkholderia podalyriae sp. nov., with the type strain WC7.3b^T (= LMG 31413^T; SARCC 750^T). Based on our nodA and nifH phylogenies, P. podalyriae sp. nov. and strains of P. tuberum sensu stricto (including one from V. karroo) belong to symbiovar africana, the symbiotic loci of which have a separate evolutionary origin to those of Central and South American Paraburkholderia strains.     

Burkholderia sp. Induces Functional Nodules on the South African Invasive Legume Dipogon lignosus (Phaseoleae) in New Zealand Soils

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678^T which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.     

Root associated iron oxidizing bacteria increase phosphate nutrition and influence root to shoot partitioning of iron in tolerant plant Typha angustifolia

Aims

Typha angustifolia is a heavy metal tolerant plant that grows in a uranium mine tailings highly contaminated with iron. In this study three iron oxidizing microbes (FeOBs) isolated from Typha rhizoplane were investigated for their role in plant growth promotion (PGP). Their effect on iron nutrition in Typha under iron replete and excess condition was also evaluated.

Methods

The PGP activities of the FeOBs were studied by measuring their influence on plant growth. To investigate the mechanism of growth promotion their ability to solubilize phosphate, and to produce Indole acetic acid and siderophores were studied. The influence of the FeOBs on root to shoot partitioning of iron was tested by measuring total iron content in roots and shoots treated with microbes.

Results

The FeOBs were named as Paenibacillus cookii JGR8, (MTCC12002), Pseudomonas jaduguda JGR2 (LMG25820) and Bacillus megaterium JGR9 (MTCC12001). The siderophore producers, influenced iron accumulation in the plant root. Additionally P. pseudoalcaligenes JGR2 increased shoot iron content overcoming the root- shoot barrier that allows Typha to exclude metals from its shoot. Among the PGP mechanisms tested, ability to solubilize phosphate appeared to be most significant for increasing the plant biomass.

Conclusion

FeOBs that produce siderophore increased iron content in plant and therefore can be of immense biotechnological importance. However Biomass increase was directly correlated with increased phosphate acquisition and not with enhanced iron accumulation in Typha.     

Biological nitrogen fixation and phosphate solubilization by bacteria isolated from tropical soils

Introduction

In addition to fixing atmospheric nitrogen, some bacterial isolates can also solubilize insoluble phosphates, further contributing to plant growth.

Aims

The objectives of this study were the following: isolate, select, and identify nodulating bacteria in the cowpea that are efficient not only in biological nitrogen fixation (BNF) but also in the solubilization of insoluble inorganic phosphates; identify and quantify the organic acids produced; and establish the relationship between those acids and the solubilizing capacity.

Methods

The bacteria were captured from two soils containing high concentrations of insoluble phosphorus from the cities of Lavras and Patos de Minas, using the cowpea [Vigna unguiculata (L.) Walp.] as bait. We obtained 78 strains, which were characterized according to their cultural attributes in culture medium 79 with the strains UFLA 03-84, INPA 03-11B, and BR3267 (approved by the Ministry of Livestock and Supply Agriculture—MAPA, as inoculants for the cowpea) and Burkholderia cepacia (LMG1222^T), which was used as a positive control for phosphate solubilization. Strains that were selected for their efficiency in both processes were identified by 16S rDNA sequence analysis. We evaluated the symbiotic efficiency (BNF) in a greenhouse and the solubilization efficiency of CaHPO₄, Al(H₂PO₄)₃, and FePO₄.2H₂O in solid and liquid GELP media. Strains that excelled at the solubilization of these phosphate sources were also evaluated for the production of the following organic acids: oxalic, citric, gluconic, lactic, succinic, and propionic.

Results

The presence of Acinetobacter, Bacillus, Firmicutes, Microbacterium, Paenibacillus, and Rhizobium was detected by 16S rDNA sequencing and analysis. Bacterial strains obtained from cowpea nodules varied greatly in the efficiency of their BNF and phosphate solubilization processes, especially in the strains UFLA 03-09, UFLA 03-10, UFLA 03-12, and UFLA 03-13, which were more efficient in both processes. More strains were able to solubilize insoluble inorganic calcium and iron phosphates in liquid medium than in solid medium. The production of organic acids was related to the solubilization of CaHPO₄ and FePO₄.2H₂O for some strains, and the type and concentration of the acid influenced this process.

Conclusions

These are the first results obtained with bacterial isolates from tropical soils in which the production of organic acids was detected and quantified to examine the solubilization of insoluble inorganic phosphates.     

Acacia,climate, and geochemistry in Australia

Background

Nitrogen-fixing legumes are key species in grassland ecosystems, as their ability to fix atmospheric nitrogen can facilitate neighboring plants. However, little is known about the fate of this legume effect in the face of extreme weather events, which are increasingly expected to occur.

Methods

Here, we examined experimentally how the presence of a legume modifies above-ground net primary production (ANPP) and nitrogen supply of neighboring non-legumes under annually recurrent pulsed drought and heavy rainfall events by comparing responses of three key species in European grassland versus without legume presence over 4 years.

Results

Legume presence facilitated community productivity of neighboring non-legumes under ambient weather conditions and also under experimental heavy rainfall. However, no facilitation of community productivity by the legume was found under experimental drought. Productivity of the three target species responded species-specifically to legume presence under different weather conditions: Holcus lanatus was facilitated only under control conditions, Plantago lanceolata was facilitated only under heavy rainfall, and Arrhenatherum elatius was facilitated irrespective of climate manipulations. The legume effects on δ ¹⁵N, leaf N concentration, and N uptake were also species-specific, yet irrespective of the climate manipulations. The data suggest that the missing legume effect on community productivity under the pulsed drought was rather caused by reduced N-uptake of the target species than by reduced N-fixation by the legume.

Conclusions

In contrast to heavy rain, the presence of a legume could not effectively buffer community ANPP against the negative effects of extreme drought events in an experimental temperate grassland. Facilitation also depends on the key species that are dominating a grassland community.     

Burkholderia phymatum Strains Capable of Nodulating Phaseolus vulgaris Are Present in Moroccan Soils

Phylogenetic analysis of 16S rRNA, nodC, and nifH genes of four bacterial strains isolated from root nodules of Phaseolus vulgaris grown in Morocco soils were identified as Burkholderia phymatum. All four strains formed N₂-fixing nodules on P. vulgaris and Mimosa, Acacia, and Prosopis species and reduced acetylene to ethylene when cultured ex planta.Until 2001 all known bacteria involved in root nodule symbioses with leguminous plants were classified as members of the order Rhizobiales of the Alphaproteobacteria, including Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium (28, 36, 38). Moulin et al. (21), however, described two Burkholderia nodule-forming strains isolated from Machaerium lunatum in French Guiana and Aspalathus carnosa in South Africa, respectively, this being the first report on the presence of a betaproteobacterium within root nodules of legumes. Later, the strains were formally classified as Burkholderia phymatum STM815^T and Burkholderia tuberum STM678^T, respectively (33). Burkholderia species are the predominant isolates from nodules of mimosoid legumes from Panama (2), Costa Rica (3), Taiwan (4, 6), Brazil (5, 7), Venezuela (5), and Madagascar (24), which indicates a high affinity of Burkholderia for forming effective symbioses with Mimosa. Diazotrophy is well represented in Burkholderia; among the more than 55 species presently classified as Burkholderia, 9 have been shown to fix N₂ ex planta by using either the acetylene reduction activity (ARA) assay or the presence of nifH genes encoding nitrogenase reductase (3, 5, 11, 24) and more recently by ¹⁵N₂ isotopic dilution experiments (17).Common bean (Phaseolus vulgaris) is an herbaceous leguminous plant which establishes N₂-fixing symbiosis with at least 5 species of the genus Rhizobium. Rhizobium etli is the predominant species in America (29) and is also detected in Europe and Africa (13, 20). Rhizobium leguminosarum bv. phaseoli is commonly found in Europe (13) and has also been reported to be present in Tunisia (20) and Colombia (10). Rhizobium tropici is found in acid soils of South America and is also present in Europe and several African countries (18). Rhizobium giardinii has been detected only in European and Tunisian soils (1, 20), and Rhizobium gallicum has been found nodulating beans in Europe, North Africa, and Mexico (1, 13). In this study we report on the isolation and characterization of B. phymatum from root nodules of P. vulgaris grown in alkaline soils from Morocco. Our results show that strains formed effective nodules on species of Mimosa, Acacia, and Prosopis and fixed atmospheric N₂ under free-living conditions.Soil was taken from a field near Oulade Mansour (34°47′N, 2°15′W, Oujda province, Morocco) where maize and common bean have traditionally been grown as rotational crops without N fertilization. Soil had a sandy-clay texture and the following characteristics: pH (in water), 8.1; 55.18% sand; 17.17% silt; 27.65% clay; 6.1% carbonates; 7.69% organic carbon; 0.069 total nitrogen. Seeds of P. vulgaris cv. Flamingo were surface sterilized (96% ethanol for 30 s followed by immersion in 15% [vol/vol] H₂O₂ for 8 min), washed several times with sterile water, germinated in the dark, and planted in 1-kg pots containing soil and sterile sand (1:1, vol/vol). Plants were grown for 30 days in controlled environmental chambers under conditions previously described (9). Nodules were collected, pooled together, surface sterilized with 2.5% HgCl₂ for 5 min, and rinsed thoroughly with sterile distilled water. Then, 12 nodules were placed independently on petri dishes and crushed in a drop of sterile water with a sterile glass rod. The resulting suspension was streaked onto petri dishes containing either yeast extract-mannitol (YEM) medium (35) or peptone-mineral salts-yeast extract (PSY) medium (25). After incubation of the plates at 30°C for 7 days, CFU which represented all of the colony types that could be distinguished by microscopic observation of living cells were chosen. After identification, Burkholderia strains were routinely grown in BAc medium (12).For DNA extraction and PCR amplifications, genomic DNA was isolated from bacterial cells using the RealPure genomic DNA extraction kit (Durviz, Spain) according to the manufacturer''s instructions. Repetitive extragenic palindromic (REP) fingerprinting was performed using primers REPIR-I and REP2-I according to the method of de Bruijn (8). PCR amplifications of the 16S rRNA gene fragments were done with the Bphym-F and Bphym-R species-specific primer pair (37).After isolation from root nodules, 52 strains forming morphologically different colonies were obtained and grouped in two main clusters after REP-PCR fingerprinting (data not shown), a technique that is extensively used to group bacteria at subspecies or strain level (8, 34) and has proven to be a powerful tool for studies of microbial ecology and evolution (14). The nearly complete sequence of the 16S rRNA gene from a representative strain of each REP-PCR group was obtained and compared with those held in GenBank. Forty-six strains in cluster I were members of the family Rhizobiaceae from the Alphaproteobacteria. Another 4 strains, here referred to as GR strains (GR01 to GR04), grouped in cluster II and were classified into the family Burkholderiaceae within the Betaproteobacteria. The remaining two strains have not been clearly classified as yet. The four GR strains have almost identical 16 rRNA gene sequences, and BLAST searches showed that they were phylogenetically close (99% identity) to B. phymatum STM815^T, a strain originally isolated from the papilionoid legume Machaerium lunatum (21, 33). A phylogenetic analysis including 30 Burkholderia reference strains showed that strains from root nodules of P. vulgaris form a tight cluster with B. phymatum STM815^T (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Phylogenetic tree showing the positions of four P. vulgaris-isolated strains, GR01, GR03, GR05, and GR06, within the genus Burkholderia based on 16S rRNA gene sequence comparisons. One thousand bootstrap samplings were performed. The NCBI GenBank accession number for each strain is shown in parentheses. The bar represents one nucleotide substitution per 1,000 nucleotides. The multiple alignments of the sequences were performed with CLUSTAL W software (30). The tree topology was inferred by the neighbor-joining method (27), based on 1,310 DNA sites, and the distance matrix method was performed according to the method of Jukes and Cantor (15) using the program MEGA version 2.1 (16).The nodC gene was amplified with the primer pairs and conditions previously described (11). Amplification of the nodC gene from each GR strain yielded a DNA fragment of about 0.4 kb (data not shown) whose nucleotide sequences were identical for all four strains and showed 99% identity to those of B. phymatum strains STM815^T and NGR195A (11). A phylogenetic tree inferred from NodC sequences from B. phymatum strains and members of the order Rhizobiales is shown in Fig. ​Fig.2.2. Primers IGK (23) and NDR-1 (31) were used for amplification of the nifH genes as indicated earlier (22). PCR amplifications of the nifH gene and further sequencing from each GR strain revealed that they all had almost identical DNA sequences, which were 99% identical to those of B. phymatum STM815^T and NGR195A (11). A phylogenetic tree based on NifH sequences showing the relationships between B. phymatum and other Burkholderia and rhizobial species is shown in Fig. ​Fig.33.Open in a separate windowFIG. 2.Phylogenetic tree inferred from NodC sequences shows the positions of four P. vulgaris-isolated strains, GR01, GR03, GR05, and GR06, within the genus Burkholderia. The tree topology was inferred by the neighbor-joining method (27) based on 195 sites. The bar represents the number of amino acid substitutions per site. One thousand bootstrap samplings were performed. The NCBI GenBank accession number for each strain is shown in parentheses.Open in a separate windowFIG. 3.Phylogenetic tree inferred from NifH sequences shows the positions of four P. vulgaris-isolated strains, GR01, GR03, GR05, and GR06, within the genus Burkholderia. The tree topology was inferred by the neighbor-joining method (27) based on 195 sites. The bar represents the number of amino acid substitutions per site. One thousand bootstrap samplings were performed. The NCBI GenBank accession number for each strain is shown in parentheses.For nodulation tests, seeds of Glycine max, Cicer arietinum, Pisum sativum, Lens culinaris, Lotus corniculatus, and Medicago sativa were surface sterilized as described above for common beans. Seeds of Mimosa, Leucaena, Prosopis, and Acacia were surface sterilized with concentrated sulfuric acid for 10 min followed by 3% sodium hypochlorite for 10 min and then washed thoroughly with sterile water. Plant cultivation was carried out as indicated above. Acetylene reduction activity (ARA) by nodulated plants was assayed on detached root systems excised at the cotyledonary node as previously described (19). The GR strains are true symbionts of P. vulgaris as, after nodule isolation, they were able to establish new effective symbiosis with common beans, with values of ARA ranging from 492 to 525 μmol ethylene/plant/h. B. phymatum STM815^T also infected P. vulgaris, but the efficiency of the symbiosis, determined as plant dry weight (1.06 ± 0.18 g/plant/h), was half of that found in plants nodulated by the GR strains. These strains also nodulated Mimosa pigra, Acacia cochliacantha, Acacia bilimeki, Leucaena glauca, and Prosopis laevigata but were unable to form nodules on P. sativum, L. culinaris, L. corniculatus, M. sativa, G. max, and C. arietinum.Diazotrophy is common among Burkholderia species, as shown recently by N₂ isotopic dilution studies (17). Under free-living conditions, ARA by the GR strains was tested in semisolid JMV medium as indicated earlier (26). At the end of the experiments, the culture purity was routinely checked by plating to verify uniform colony morphology. Like strain STM815^T, strains isolated from P. vulgaris also had nitrogenase activity when grown ex planta. Values of activity, however, were about half of that detected in strain STM815^T (83 ± 15 nmol C₂H₄/h).Preparation of whole-cell proteins and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) assays were performed as described previously (12). Protein profiles were compared with type and reference strains of legume-nodulating Burkholderia species. P. vulgaris-isolated strains showed SDS-PAGE protein profiles (evaluated by visual comparison) almost identical to those from the STM815^T type strain of B. phymatum but clearly different from those of other legume-nodulating Burkholderia species (Fig. ​(Fig.4).4). No differences were found when API 20 NE and API 50 CH strips were used to check for differences in nitrogen and carbon sources between B. phymatum STM815^T and the GR strains (data not shown).Open in a separate windowFIG. 4.Protein electrophoregrams (SDS-PAGE) of P. vulgaris-isolated strains and type strains of known legume-nodulating Burkholderia species. Lane 1, B. tuberum STM678^T; lane 2, B. phymatum STM815^T; lanes 3 to 6, B. phymatum GR01, GR03, GR04, and GR06, respectively; lane 7, B. mimosarum PAS44^T; lane 8, B. nodosa Br3437^T.Based on 16S rRNA gene sequences and protein profiles, which provide strong evidence for the delineation of bacterial species (32), the Phaseolus-isolated strains could be assigned to the species B. phymatum. Moreover, analysis of the phylogenetic relationships of such sequences and other Burkholderia species showed that they formed a robust clade with B. phymatum STM815^T. In addition, sequencing of the nodC and nifH genes revealed that the phylogenetically closest bacterial species was B. phymatum STM815^T. All these results support the affiliation of Phaseolus-isolated strains as B. phymatum. Since genomic DNAs from the GR strains had the same DNA band pattern after REP-PCR fingerprinting and extremely similar 16S rRNA and nifH gene sequences, as well as identical nodC sequences, the four strains could be derived from a single clone. Our results also suggest that strains of B. phymatum isolated from Mimosa and Phaseolus have acquired their symbiosis genes either from a common ancestor or by lateral transfer between them, the direction of transfer being unknown. Although limited to three isolates, strains NGR114 and NGR195A from Mimosa invisa and Mimosa pudica in Papua New Guinea, respectively (11), and STM815^T from M. lunatum in French Guiana (21, 33), and four strains from P. vulgaris in Morocco, our results raise questions concerning the biogeographical, environmental, and host taxon distribution of B. phymatum nodule symbionts. B. phymatum was originally discovered in nodules from M. lunatum in French Guiana, and most other strains in this lineage have been found associated with host legumes in the genus Mimosa, primarily in the Neotropics. Thus, the current results extend both the known host distribution and geographic range of this group of nodule symbionts. Whether B. phymatum is prevalent on rhizobial species within nodules of P. vulgaris in the geographic site where soil samples were taken cannot be elicited from the present results. Accordingly, it will be important in future work to survey additional sites both within the native geographic range and elsewhere to understand the consistency of the association between Phaseolus and Burkholderia.     

Comparative genome analysis of rice-pathogenic Burkholderia provides insight into capacity to adapt to different environments and hosts

Background

In addition to human and animal diseases, bacteria of the genus Burkholderia can cause plant diseases. The representative species of rice-pathogenic Burkholderia are Burkholderia glumae, B. gladioli, and B. plantarii, which primarily cause grain rot, sheath rot, and seedling blight, respectively, resulting in severe reductions in rice production. Though Burkholderia rice pathogens cause problems in rice-growing countries, comprehensive studies of these rice-pathogenic species aiming to control Burkholderia-mediated diseases are only in the early stages.

Results

We first sequenced the complete genome of B. plantarii ATCC 43733^T. Second, we conducted comparative analysis of the newly sequenced B. plantarii ATCC 43733^T genome with eleven complete or draft genomes of B. glumae and B. gladioli strains. Furthermore, we compared the genome of three rice Burkholderia pathogens with those of other Burkholderia species such as those found in environmental habitats and those known as animal/human pathogens. These B. glumae, B. gladioli, and B. plantarii strains have unique genes involved in toxoflavin or tropolone toxin production and the clustered regularly interspaced short palindromic repeats (CRISPR)-mediated bacterial immune system. Although the genome of B. plantarii ATCC 43733^T has many common features with those of B. glumae and B. gladioli, this B. plantarii strain has several unique features, including quorum sensing and CRISPR/CRISPR-associated protein (Cas) systems.

Conclusions

The complete genome sequence of B. plantarii ATCC 43733^T and publicly available genomes of B. glumae BGR1 and B. gladioli BSR3 enabled comprehensive comparative genome analyses among three rice-pathogenic Burkholderia species responsible for tissue rotting and seedling blight. Our results suggest that B. glumae has evolved rapidly, or has undergone rapid genome rearrangements or deletions, in response to the hosts. It also, clarifies the unique features of rice pathogenic Burkholderia species relative to other animal and human Burkholderia species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1558-5) contains supplementary material, which is available to authorized users.     

Mixotrophic metabolism in Burkholderia kururiensis subsp. thiooxydans subsp. nov., a facultative chemolithoautotrophic thiosulfate oxidizing bacterium isolated from rhizosphere soil and proposal for classification of the type strain of Burkholderia kururiensis as Burkholderia kururiensis subsp. kururiensis subsp. nov.

A thiosulfate-oxidizing facultative chemolithoautotrophic Burkholderia sp. strain ATSB13^T was previously isolated from rhizosphere soil of tobacco plant. Strain ATSB13^T was aerobic, Gram-staining-negative, rod shaped and motile by means of sub-terminal flagellum. Strain ATSB13^T exhibited mixotrophic growth in a medium containing thiosulfate plus acetate. A phylogenetic study based on 16S rRNA gene sequence analysis indicated that strain ATSB13^T was most closely related to Burkholderia kururiensis KP23^T (98.7%), Burkholderia tuberum STM678^T (96.5%) and Burkholderia phymatum STM815^T (96.4%). Chemotaxonomic data [G+C 64.0 mol%, major fatty acids, C_18:1 ω7c (28.22%), C_16:1 ω7c/15 iso 2OH (15.15%), and C_16:0 (14.91%) and Q-8 as predominant respiratory ubiquinone] supported the affiliation of the strain ATSB13^T within the genus Burkholderia. Though the strain ATSB13^T shared high 16S rRNA gene sequence similarity with the type strain of B. kururiensis but considerably distant from the latter in terms of several phenotypic and chemotaxonomic characteristics. DNA–DNA hybridization between strain ATSB13^T and B. kururiensis KP23^T was 100%, and hence, it is inferred that strain ATSB13^T is a member of B. kururiensis. On the basis of data obtained from this study, we propose that B. kururiensis be subdivided into B. kururiensis subsp. kururiensis subsp. nov. (type strain KP23^T = JCM 10599^T = DSM 13646^T) and B. kururiensis subsp. thiooxydans subsp. nov. (type strain ATSB13^T = KACC 12758^T).     

N2 fixation, N transfer, and yield in grassland communities including a deep-rooted legume or non-legume species

Background and aims

Legumes are important components of grassland mixtures due to their ability to sustain high yields with moderate nitrogen inputs. This study investigates nitrogen relationships in mixtures of Trifolium pratense and grasses into which a deep-rooted forb was included, and particularly whether these realtionships differ when the forb is a legume or a non-legume species.

Methods

A field experiment in which mixtures of T. pratense, Phleum pratense, Lolium perenne, and Medicago sativa or Cichorium intybus, and monocropped stands of all species was established in 2007 and harvested in 2008 and 2009. The experiment received a total input of 100 kg?ha^?1?N yearly. Yield and botanical composition were determined in seven harvests. Species were analysed for ¹⁵N abundance, and N₂ fixation and N transfer were calculated. Soil samples were analysed twice for inorganic N.

Results

Non-legumes benefitted from the presence of legumes in terms of N concentration, and the yield of mixtures exceeded that of monocropped non-legumes but not monocropped legumes. The mixture containing M. sativa did not yield more DM or N than did the mixture containing C. intybus. A total of 17.08 kg?N ha^?1 was transferred from T. pratense to the non-legumes in the mixture in which it was the sole legume species.

Conclusions

It is concluded that there was a synergy effect in species mixtures, but the effect did not differ between the two deep-rooted species.     

Boron delays dehydration and stimulates root growth in Eucalyptus urophylla (Blake,S.T.) under osmotic stress

Background

Water and nutritional restrictions are limiting factors for the growth of Eucalyptus trees in tropical climates. In the dry season, boron (B) uptake is severely affected.

Aims

The objectives of this study were to evaluate the phloem mobility of B and whether its deficiency can increase plant sensitivity to osmotic stress. It was also tested to what extent foliar application of B could mitigate the negative effects of drought under low B supply.

Methods

Seedlings of a drought tolerant Eucalyptus urophylla (Blake, S. T.) clone were grown in nutrient solution, subjected to low availability of B for 25 days, and then submitted to a progressive osmotic stress. After imposition of osmotic stress, B was applied to young or mature leaves.

Results

B applications, mainly to mature leaf, stimulated root growth and delayed dehydration under osmotic stress and led to an increased B translocation and carbon isotopic composition. The expression of B transporters and pectin metabolism genes were also increased in water-stressed plants supplied with B by foliar application.

Conclusions

B deficiency led to increased plant dehydration and decreased root growth under osmotic stress. The application of B to mature leaf of water-stressed plants proved effective in mitigating the negative effects of water deficit in root growth.     

Paenibacillus yonginensis sp. nov., a potential plant growth promoting bacterium isolated from humus soil of Yongin forest

Strain DCY84^T, a Gram-stain positive, rod-shaped, aerobic, spore-forming bacterium, motile by means of peritrichous flagella, was isolated from humus soil from Yongin forest in Gyeonggi province, South Korea. Strain DCY84^T shared the highest sequence similarity with Paenibacillus barengoltzii KACC 15270^T (96.86 %), followed by Paenibacillus timonensis KACC 11491^T (96.49 %) and Paenibacillus phoenicis NBRC 106274^T (95.77 %). Strain DCY84^T was found to able to grow best in TSA at temperature 30 °C, at pH 8 and at 0.5 % NaCl. MK-7 menaquinone was identified as the isoprenoid quinone. The major polar lipids were identified as phosphatidylethanolamine, an unidentified aminophospholipid, two unidentified aminolipids and an unidentified polar lipid. The peptidoglycan was found to contain the amino acids meso-diaminopimelic acid, alanine and d-glutamic acid. The major fatty acids of strain DCY84^T were identified as branched chain anteiso-C_15:0, saturated C_16:0 and branched chain anteiso-C_17:0. The cell wall sugars of strain DCY84^T were found to comprise of ribose, galactose and xylose. The major polyamine was identified as spermidine. The DNA G+C content was determined to be 62.6 mol%. After 6 days of incubation, strain DCY84^T produced 52.96 ± 1.85 and 72.83 ± 2.86 µg/ml l-indole-3-acetic acid, using media without l-tryptophan and supplemented with l-tryptophan, respectively. Strain DCY84^T was also found to be able to solubilize phosphate and produce siderophores. On the basis of the phenotypic characteristics, genotypic analysis and chemotaxonomic characteristics, strain DCY84^T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus yonginensis sp. nov. is proposed. The type strain is DCY84^T (=KCTC 33428^T = JCM 19885^T).     

Soil organic C and nutrient contents under trees with different functional characteristics in seasonally dry tropical silvopastures

Aims

The selection of tree characteristics is critical for the outcome of the tree effects on soil fertility in silvopastoral pastures. This study aims to quantify the effects of trees on soil nutrient and C stocks, as well as assessing differences on the effects between legume (Albizia saman; Enterolobium cyclocarpum) and non-legume tree species (Tabebuia rosea; Guazuma ulmifolia).

Methods

In Central Nicaragua, soil was sampled (0–10 cm deep) in paired plots, under both a canopy and in open grassland, in 12 sites per tree species and analysed for organic C, total N stocks, available P and extractable K⁺, Ca²⁺ and Mg²⁺. To assess the effects of herbaceous composition and cattle to soil proprieties, we recorded the cover of plant groups and assessed the mass of dung in each plot.

Results

Soil organic C and N, available P and extractable K⁺ and Ca²⁺ were higher under the tree canopy than under paired open grassland. The basal area of trees was positively related with the canopy effect on soil variables, thus suggesting that the age or sizes of the trees are relevant factors associated with the content of soil C and nutrients. No specific effects related to the legume species group were detected.

Conclusions

Our results indicate that in fertile seasonally dry subtropical pastures, scattered trees have an overall effect on soil fertility, and that the magnitude of the effect depends more on the tree characteristics (i.e. basal area, crown area) than on whether the species is a legume or not.     

Phenotypic and genotypic characterisation of root nodule bacteria nodulating Millettia pinnata (L.) Panigrahi, a biodiesel tree

Aims

Milletia pinnata is a leguminous tropical tree that produces seed oil suitable for biodiesel and is targeted to be planted on marginal land associated with nitrogen poor soil. This study aimed to identify effective rhizobia species for M. pinnata.

Methods

Soil samples were collected from M. pinnata grown in Kununurra, Australia. Rhizobia were trapped, characterised and sequenced for 16S rRNA, atpD, dnaK and recA genes.

Results

Forty isolates tolerated pH 7 – 9, temperatures 29 – 37 °C, salinity below 1 % NaCl, and had optimal growth on mannitol, arabinose or glutamate as a single carbon source, a few grew on sucrose and none grew on lactose. Inoculation of isolates increased shoot dry weight of M. pinnata’s seedlings in nitrogen minus media. Slow-growing isolates were closely related to Bradyrhizobium yuanmingense, Bradyrhizobium sp. DOA10, Bradyrhizobium sp. ORS305 and B. liaoningense LMG 18230^T. The fast-growing isolates related to Rhizobium sp. 8211, R. miluonense CCBAU 41251^T, R miluonense CC-B-L1, Rhizobium sp. CCBAU 51330 and Rhizobium sp. 43015

Conclusions

Millettia pinnata was effectively nodulated by slow-growing isolates related to Bradyrhizobium yuanmingense, Bradyrhizobium sp. DOA10 Bradyrhizobium sp. ORS305, B. liaoningense LMG 18230^T and fast-growing isolates related Rhizobium sp. 8211, R. miluonense, Rhizobium sp. CCBAU 51330 and Rhizobium sp. 43015     

Nitrogen fixation in annual and perennial legume-grass mixtures across a fertility gradient

Background and aims

The selection of legume species and species mixtures influences agroecosystem nitrogen (N) and carbon cycling. We utilized a fertility gradient to investigate the effects of plant species interactions on biological N fixation of an annual and perennial legume in response to shifting soil resource availability.

Methods

Legume N fixation of annual field pea (Pisum sativum) and perennial red clover (Trifolium pratense) grown in monoculture and mixtures with oats (Avena sativa) or orchardgrass (Dactylis glomerata) was estimated using the ¹⁵N natural abundance method across 15 farm fields and we measured six soil N pools ranging from labile to more recalcitrant.

Results

Evidence of complementary and facilitative species interactions was stronger for the perennial red clover-orchardgrass mixture than for the annual field pea-oat mixture (N Land Equivalency Ratios were 1.6 and 1.2, respectively). We estimated that the transfer of fixed N from red clover to orchardgrass increased aboveground N fixation estimates by 15% from 33 to 38?kg?N ha^?1. Despite a more than 2-fold range in soil organic matter levels and more than 3-fold range in labile soil N pools across field sites, the N fertility gradient was not a strong predictor of N fixation. While grass N assimilation was positively correlated with soil N pools, we found only weak, inverse correlations between legume N fixation and soil N availability. In grass-legume mixtures, soil N availability indirectly influenced N fixation through plant competition.

Conclusions

These results suggest that increasing diversity of cropping systems, particularly through the incorporation of perennial mixtures into rotations, could improve overall agroecosystem N cycling efficiency.     

Contribution of Cd-EDTA complexes to cadmium uptake by maize: a modelling approach

Background and aims

Chelant-enhanced phytoextraction has given variable and often unexplained experimental results. This work was carried out to better understand the mechanisms of Cd plant uptake in the presence of EDTA and to evaluate the contributions of Cd-EDTA complexes to the uptake.

Method

A 1-D mechanistic model was implemented, which described the free Cd²⁺ root absorption, the dissociation and the direct absorption of the Cd-EDTA complexes. It was used to explain Cd uptake by maize in hydroponics and in soil.

Results

In hydroponics, the addition of EDTA caused a decrease in Cd uptake by maize, particularly when the ratio of total EDTA ([EDTA] _T ) to total Cd ([Cd] _T ) was greater than 1. At [Cd] _T = 1 μM, when [EDTA] _T /[Cd] _T < 1, the model indicated that Cd uptake was predominantly due to the absorption of free Cd²⁺, whose pool was replenished by the dissociation of Cd-EDTA. When [EDTA] _T /[Cd] _T > 1, the low Cd uptake was mostly due to Cd-EDTA absorption. In soil spiked with 5 mg Cd kg^?1, Cd uptake was not affected by the various EDTA additions, because of the buffering capacity of the soil solid phase.

Conclusions

Addition of EDTA to soil increases Cd solubility but dissociation of Cd-EDTA limits the availability of the free Cd²⁺ at the root surface, which finally reduces the plant uptake of the metal.     

Soil nitrogen balance resulting from N fixation and rhizodeposition by the symbiotic association Anthyllis vulneraria/Mesorhizobium metallidurans grown in highly polluted Zn,Pb and Cd mine tailings

Background and aims

The association of the legume Anthyllis vulneraria and the grass Festuca arvernensis, was found to be very efficient for the phytostabilisation of highly multi-metal contaminated mine tailings. Our objective was to quantify the contribution of Anthyllis inoculated with its symbiotic bacteria Mesorhizobium metallidurans to the soil N pool and to test whether a starter nitrogen fertilization may improve symbiotic nitrogen fixation and the growth of Festuca.

Methods

Plants of Festuca and of Anthyllis inoculated with M. metallidurans were grown separately during eight months in pots filled with mine contaminated soil. Estimation of the N fluxes was realized using ¹⁵?N isotopic methods.

Results

Starter N fertilization (28 kg N ha^?1) improved symbiotic N₂ fixation and the growth of both species. Belowground N balance (N rhizodeposition – soil N uptake) of the non-fertilized Anthyllis at maturity was negative (?30.6 kg N ha^?1). However, the amount of N derived from fixation, including above- and belowground parts, was 78.6 kg N ha^?1, demonstrating the ability of this symbiotic association to improve soil N content after senescence.

Conclusions

i) soil N enrichment by the N₂-fixing symbiotic association occurs after plant senescence, when decaying leaves and shoots are incorporated into the soil; ii) application of a starter fertilization is an efficient solution to improve phytostabilisation of highly contaminated sites.