A synergistic interaction between salt-tolerant Pseudomonas and Mesorhizobium strains improves growth and symbiotic performance of liquorice (Glycyrrhiza uralensis Fish.) under salt stress.

Chinese liquorice (Glycyrrhiza uralensis Fish.) is a salt-tolerant medicinal legume that could be utilized for bioremediation of salt-affected soils. We studied whether co-inoculation of the symbiotic Mesorhizobium sp. strain NWXJ19 or NWXJ31 with the plant growth-promoting Pseudomonas extremorientalis TSAU20 could restore growth, nodulation, and shoot/root nitrogen contents of salt-stressed G. uralensis, which was grown in potting soil and irrigated with 0, 50, and 75 mM NaCl solutions under greenhouse conditions. Irrigation with NaCl solutions clearly retarded the growth of uninoculated liquorice, and the higher the NaCl concentration (75 and 100 mM NaCl), the more adverse is the effect. The two Mesorhizobium strains, added either alone or in combination with P. extremorientalis TSAU20, responded differently to the salt levels used. The strain NWXJ19 was a good symbiont for plants irrigated with 50 mM NaCl, whereas the strain NWXJ31 was more efficient for plants irrigated with water or 75 mM NaCl solution. P. extremorientalis TSAU20 combined with single Mesorhizobium strains alleviated the salt stress of liquorice plants and improved yield and nodule numbers significantly in comparison with single-strain-inoculated liquorice. Both salt stress and inoculation raised the nitrogen content of shoots and roots. The nitrogen contents were at their highest, i.e., 30 and 35 % greater compared to non-stressed uninoculated plants, when plants were inoculated with P. extremorientalis TSAU20 and Mesorhizobium sp. NWXJ31 as well as irrigated with 75 mM NaCl solution. From this study, we conclude that dual inoculation with plant growth-promoting rhizobacteria could be a new approach to improve the tolerance of G. uralensis to salt stress, thereby improving its suitability for the remediation of saline lands.

Diversity of sporadic symbionts and nonsymbiotic endophytic bacteria isolated from nodules of woody, shrub, and food legumes in Ethiopia.

Fifty-five bacterial isolates were obtained from surface-sterilized nodules of woody and shrub legumes growing in Ethiopia: Crotalaria spp., Indigofera spp., and Erythrina brucei, and the food legumes soybean and common bean. Based on partial 16S rRNA gene sequence analysis, the majority of the isolates were identified as Gram-negative bacteria belonging to the genera Achromobacter, Agrobacterium, Burkholderia, Cronobacter, Enterobacter, Mesorhizobium, Novosphingobium, Pantoea, Pseudomonas, Rahnella, Rhizobium, Serratia, and Variovorax. Seven isolates were Gram-positive bacteria belonging to the genera Bacillus, Paenibacillus, Planomicrobium, and Rhodococcus. Amplified fragment length polymorphism (AFLP) fingerprinting showed that each strain was genetically distinct. According to phylogenetic analysis of recA, glnII, rpoB, and 16S rRNA gene sequences, Rhizobium, Mesorhizobium, and Agrobacterium were further classified into six different genospecies: Agrobacterium spp., Agrobacterium radiobacter, Rhizobium sp., Rhizobium phaseoli, Mesorhizobium sp., and putative new Rhizobium species. The strains from R. phaseoli, Rhizobium sp. IAR30, and Mesorhizobium sp. ERR6 induced nodules on their host plants. The other strains did not form nodules on their original host. Nine endophytic bacterial strains representing seven genera, Agrobacterium, Burkholderia, Paenibacillus, Pantoea, Pseudomonas, Rhizobium, and Serratia, were found to colonize nodules of Crotalaria incana and common bean on co-inoculation with symbiotic rhizobia. Four endophytic Rhizobium and two Agrobacterium strains had identical nifH gene sequences with symbiotic Rhizobium strains, suggesting horizontal gene transfer. Most symbiotic and nonsymbiotic endophytic bacteria showed plant growth-promoting properties in vitro, which indicate their potential role in the promotion of plant growth when colonizing plant roots and the rhizosphere.

Phylogenetically diverse groups of Bradyrhizobium isolated from nodules of Crotalaria spp., Indigofera spp., Erythrina brucei and Glycine max growing in Ethiopia.

Ethiopian Bradyrhizobium strains isolated from root nodules of Crotalaria spp., Indigofera spp., Erythina brucei and soybean (Glycine max) represented genetically diverse phylogenetic groups of the genus Bradyrhizobium. Strains were characterized using the amplified fragment length polymorphism fingerprinting technique (AFLP) and multilocus sequence analysis (MLSA) of core and symbiotic genes. Based on phylogenetic analyses of concatenated recA-glnII-rpoB-16S rRNA genes sequences, Bradyrhizobium strains were distributed into fifteen phylogenetic groups under B. japonicum and B. elkanii super clades. Some of the isolates belonged to the species B. yuanmingense, B. elkanii and B. japonicum type I. However, the majority of the isolates represented unnamed Bradyrhizobium genospecies and of these, two unique lineages that most likely represent novel Bradyrhizobium species were identified among Ethiopian strains. The nodulation nodA gene sequence analysis revealed that all Ethiopian Bradyrhizobium isolates belonged to nodA sub-clade III.3. Strains were further classified into 14 groups together with strains from Africa, as well as some originating from the other tropical and subtropics regions. Strains were also clustered into 14 groups in nodY/K phylogeny similarly to the nodA tree. The nifH phylogenies of the Ethiopian Bradyrhizobium were generally also congruent with the nodA gene phylogeny, supporting the monophyletic origin of the symbiotic genes in Bradyrhizobium. The phylogenies of nodA and nifH genes were also partially congruent with that inferred from the concatenated core genes sequences, reflecting that the strains obtained their symbiotic genes vertically from their ancestor as well as horizontally from more distantly related Bradyrhizobium species.

Association of toxic indoor air with multi-organ symptoms in pupils attending a moisture-damaged school in Finland.

BACKGROUND: There is an on-going debate on how best to test toxic indoor air. Toxicological methods based on condensed water samples and cell culture technique are newly introduced research tools which were tested in this study. METHODS: Pupils (n=47) from a water-damaged and (n=56) healthy schools were interviewed using a questionnaire. Indoor air was collected with a novel condensed water sampling technique and human THP-1 macrophages were exposed to the condensate. The cytotoxicity of cotton wool swab samples was tested using human BJ fibroblasts. Conventional microbiological culture methods were also performed. RESULTS: Gastrointestinal problems (GI) were reported by 51% from the study cohort but only 4% of the control cohort, relative risk RR=14.30. For any neurological or neuropsychological symptoms, the RR was 63.04, muscular-skeletal pain RR=58.28, headache RR=31.00, respiratory symptoms RR=22.64, fatigue RR=21.45, sub febrility RR=15.49, ear infections RR=7.74, skin rash RR=5.96, all being statistically significant (P<0.001). All indoor air (n=7) and cotton wool samples (n=2) taken from the water-damaged classroom or in proximity of the problematic classrooms were toxic in cell culture assays. Low numbers of moisture-damage indicators were recovered from wall, passive air, and swab samples, namely Aspergillus ochraceus species group, Aspergillus, Eurotium species group, Fusarium, Tritirachium, Scopulariopsis genus group and Aspergillus versicolores species group. CONCLUSIONS: Indoor air toxicity and dampness-related microbiota recovered from the classrooms were associated with multi-organ morbidity of the school occupants. These results corroborated our previous reports from two adult cohorts i.e. evidence of causality. These new toxicological methods based on condensed water and cell culturing techniques seem to be superior to conventional microbiological methods in correlating with clinical symptoms.

Endotoxins associated with cyanobacteria and their removal during drinking water treatment.

The aim of this study was to investigate endotoxin concentrations in cyanobacterial water blooms and strains, and to assess the removal of endotoxins during drinking water treatment. Endotoxin concentrations were measured from 151 hepatotoxic, neurotoxic and non-toxic cyanobacterial water blooms by using Limulus amebocyte lysate (LAL) assay, and the results were compared to bacterial data. Endotoxin activities ranged from 20 to 3.8 x 10(4) endotoxin units (EU) per ml. Endotoxicity of the samples correlated with phycobiliprotein concentration that was used to assess cyanobacterial abundance, heterotrophic plate count, and Aeromonas spp. but it did not correlate with the number of coliforms or streptococci. The high endotoxin concentrations occasionally detected in the water bloom samples were probably due to Gram negative bacteria that existed together with cyanobacteria since the 26 axenic cyanobacterial strains from different genera that were studied showed very low endotoxin activity. No differences in endotoxin activity were detected between hepatotoxic, neurotoxic and non-toxic strains. Removal of endotoxins during drinking water treatment was studied at nine waterworks that previously had been associated with high numbers of cyanobacteria and that used different processes for water purification. Endotoxin concentration in raw waters ranged from 18 to 356 EU ml(-1). The treatment processes reduced 59-97% of the endotoxin activity; in the treated water the concentration ranged from 3 to 15 EU ml(-1). The most significant reduction occurred at the early stages of water treatment, during coagulation, settling and sand filtration. Activated carbon filtration either increased or had no effect on endotoxin concentration. Ozonation and chlorination had little effect on the endotoxin concentrations.

Effects of biotic and abiotic constraints on the symbiosis between rhizobia and the tropical leguminous trees Acacia and Prosopis.

N2-fixing, drought tolerant and multipurpose Acacia and Prosopis species are appropriate trees for reforestation of degraded areas in arid and semiarid regions of the tropics and subtropics. Acacia and Prosopis trees form N2-fixing nodules with a wide range of rhizobia, for example African acacias mainly with Sinorhizobium sp. and Mesorhizobium sp., and Australian acacias with Bradyrhizobium sp. Although dry and hot seasons restrict formation of N2-fixing nodules on Acacia and Prosopis spp., fully grown trees and their symbiotic partners are well adapted to survive in harsh growth conditions. This review on one hand deals with major constraints of arid and semiarid soils, i.e. drought, salinity and high soil temperature, which affect growth of trees and rhizobia, and on the other hand with adaptation mechanisms by which both organisms survive through unfavourable periods. In addition, defects in infection and nodulation processes due to various abiotic and biotic constraints are reviewed. This knowledge is important when Acacia and Prosopis seedlings are used for forestation of degraded areas in arid and semiarid tropics.

Stable isotope labelling reveals that NaCl stress decreases the production of Ensifer (Sinorhizobium) arboris lipochitooligosaccharide signalling molecules.

Ensifer (Sinorhizobium) arboris is a symbiont of salt-tolerant leguminous trees in the genera Acacia and Prosopis that are utilized in the prevention of soil erosion and desertification and in phytoremediation of salinized soil. Signalling between the plant and the rhizobia is essential for the formation of effective symbiosis that increases the success of reclaiming saline sites. We assessed the effect of salt stress on the growth and the production of lipochitooligosaccharide signalling molecules (LCOs) of S. arboris HAMBI 2361, an LCO-overproducing derivative of the S. arboris type strain HAMBI 1552. The strain tolerated NaCl up to 750 mM. To obtain both qualitative and quantitative information on the LCO production under salt stress, we devised a method where LCOs were differentially labelled by stable isotopes of nitrogen, (14)N and (15)N, and analysed by mass spectrometry. Under control conditions, the strain produced altogether 27 structural LCO variants. In 380 mM NaCl, 13 LCO variants were produced in detectable amounts, and six of these were reliably quantified, ranging from one-tenth to one-third of the non-stressed one.

Biogeography of symbiotic and other endophytic bacteria isolated from medicinal Glycyrrhiza species in China.

A total of 159 endophytic bacteria were isolated from surface-sterilized root nodules of wild perennial Glycyrrhiza legumes growing on 40 sites in central and northwestern China. Amplified fragment length polymorphism (AFLP) genomic fingerprinting and sequencing of partial 16S rRNA genes revealed that the collection mainly consisted of Mesorhizobium, Rhizobium, Sinorhizobium, Agrobacterium and Paenibacillus species. Based on symbiotic properties with the legume hosts Glycyrrhiza uralensis and Glycyrrhiza glabra, we divided the nodulating species into true and sporadic symbionts. Five distinct Mesorhizobium groups represented true symbionts of the host plants, the majority of strains inducing N2-fixing nodules. Sporadic symbionts consisted of either species with infrequent occurrence (Rhizobium galegae, Rhizobium leguminosarum) or species with weak (Sinorhizobium meliloti, Rhizobium gallicum) or no N2 fixation ability (Rhizobium giardinii, Rhizobium cellulosilyticum, Phyllobacterium sp.). Multivariate analyses revealed that the host plant species and geographic location explained only a small part (14.4%) of the total variation in bacterial AFLP patterns, with the host plant explaining slightly more (9.9%) than geography (6.9%). However, strains isolated from G. glabra were clearly separated from those from G. uralensis, and strains obtained from central China were well separated from those originating from Xinjiang in the northwest, indicating both host preference and regional endemism.

Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia.

The diversity and phylogeny of 32 rhizobial strains isolated from nodules of common bean plants grown on 30 sites in Ethiopia were examined using AFLP fingerprinting and MLSA. Based on cluster analysis of AFLP fingerprints, test strains were grouped into six genomic clusters and six single positions. In a tree built from concatenated sequences of recA, glnII, rpoB and partial 16S rRNA genes, the strains were distributed into seven monophyletic groups. The strains in the groups B, D, E, G1 and G2 could be classified as Rhizobium phaseoli, R. etli, R. giardinii, Agrobacterium tumefaciens complex and A. radiobacter, respectively, whereas the strains in group C appeared to represent a novel species. R. phaseoli, R. etli, and the novel group were the major bean nodulating rhizobia in Ethiopia. The strains in group A were linked to R. leguminosarum species lineages but not resolved. Based on recA, rpoB and 16S rRNA genes sequences analysis, a single test strain was assigned as R. leucaenae. In the nodC tree the strains belonging to the major nodulating groups were clustered into two closely linked clades. They also had almost identical nifH gene sequences. The phylogenies of nodC and nifH genes of the strains belonging to R. leguminosarum, R. phaseoli, R. etli and the putative new species (collectively called R. leguminosarum species complex) were not consistent with the housekeeping genes, suggesting symbiotic genes have a common origin which is different from the core genome of the species and indicative of horizontal gene transfer among these rhizobia.

Persistence, population dynamics and competitiveness for nodulation of marker gene-tagged Rhizobium galegae strains in field lysimeters in the boreal climatic zone.

Abstract A non-indigenous wild-type strain Rhizobium galegae HAMBI 540, which specifically nodulates perennial goat's rue (Galega orientalis), and its marker gene-tagged derivatives R. galegae HAMBI 2363(luc), R. galegae HAMBI 2368(gusA21) and R. galegae HAMBI 2364(gusA30) were used to evaluate the persistence, population dynamics and competitiveness for nodulation of rhizobia under field conditions in Finland. The lysimeters were filled with clean or diesel oil-polluted (3000 mug g(-1)) agricultural soil. During the first 2 years of the field release luc- and gusA21-tagged strains could be effectively detected by cultivation, reinforced with colony polymerase chain reaction. The population densities remained relatively stable from 10(4) to 10(5) cfu g(-1) dry soil from spring until late autumn. Replicate limiting dilution polymerase chain reaction analysis gave comparable results with cultivation with strain HAMBI 2363 until 49 weeks after inoculation. GUS activity of strain HAMBI 2368 could be stably detected in nodules and soil. On the other hand, luc activity weakened clearly in cold conditions along with decreased metabolic activity of rhizobia. The competitive ability for nodulation of the gusA30-tagged strain decreased slowly with time compared to the wild-type strain. Moderate soil pollution did not have significant effects on target bacteria or plant growth. Limited vertical movement of target bacteria outside the rhizosphere was detected from percolated water.