P. van Berkum

Corresponding 16S rRNA gene segments in Rhizobiaceae and Aeromonas yield discordant phylogenies

Previous evidence has indicated that the 16S rRNA genes in certain species of Aeromonas may have a history of lateral transfer and recombination. A comparative analysis of patterns of 16S nucleotide sequence polymorphism among species of Rhizobium and Agrobacterium was conducted to determine if there is similar evidence for chimeric 16S genes in members of the Rhizobiaceae. Results from phylogenetic analyses and comparison of patterns of nucleotide sequence polymorphism in portions of rhizobial 16S genes revealed the same type of segment-dependent polymorphic site partitioning that was previously reported for Aeromonas. These results support the hypothesis that certain 16S segments in rhizobia may have a history of lateral transfer and recombination.

Ensifer, Phyllobacterium and Rhizobium species occupy nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a Canadian site without a history of cultivation.

Phage-resistant and -susceptible bacteria from nodules of alfalfa and sweet clover, grown at a site without a known history of cultivation, were identified as diverse genotypes of Ensifer, Rhizobium and Phyllobacterium species based on sequence analysis of ribosomal (16S and 23S rRNA) and protein-encoding (atpD and recA) genes, Southern hybridization/RFLP and a range of phenotypic characteristics. Among phage-resistant bacteria, one genotype of Rhizobium sp. predominated on alfalfa (frequency approximately 68 %) but was recovered infrequently ( approximately 1 %) from sweet clover. A second genotype was isolated infrequently only from alfalfa. These genotypes fixed nitrogen poorly in association with sweet clover and Phaseolus vulgaris, but were moderately effective with alfalfa. They produced a near-neutral reaction on mineral salts agar containing mannitol, which is atypical of the genus Rhizobium. A single isolate of Ensifer sp. and two of Phyllobacterium sp. were recovered only from sweet clover. All were highly resistant to multiple antibiotics. Phylogenetic analysis indicated that Ensifer sp. strain T173 is closely related to, but separate from, the non-symbiotic species Sinorhizobium morelense. Strain T173 is unique in that it possesses a 175 kb symbiotic plasmid and elicits ineffective nodules on alfalfa, sweet clover, Medicago lupulina and Macroptilium atropurpureum. The two Phyllobacterium spp. were non-symbiotic and probably represent bacterial opportunists. Three genotypes of E. meliloti that were symbiotically effective with alfalfa and sweet clover were encountered infrequently. Among phage-susceptible isolates, two genotypes of E. medicae were encountered infrequently and were highly effective with alfalfa, sweet clover and Medicago polymorpha. The ecological and practical implications of the findings are discussed.

Enumeration and N2 fixation potential of Rhizobium leguminosarum biovar trifolii grown in soil with varying pH values and heavy metal concentrations

Abstract The potential risk to Rhizobium leguminosarum bv. trifolii and white clover ( Trifolium repens cv. ‘Regal’) from biosolids-induced heavy metal toxicity is of great concern because of their symbiotic association and capacity for N 2 fixation. A greenhouse experiment was conducted to assess the effects of heavy metals from biosolids on the population and N 2 fixing potential of Rhizobium leguminosarum bv. trifolii under two pH regimes. In 1994, soils (Typic Paleudults) were collected from plots that had previously received 224 Mg ha −1 heat-treated and 100 Mg ha −1 Nu-Earth biosolids (applied in 1976 and 1978, respectively). Six soil treatments were used for the study: a control with low and high pH and two biosolids treatments, each with low and high pH. Soil pH and biosolids application significantly affected uptake of metals with phytotoxicity observed in the low pH soil amended with biosolids. The number of Rhizobium was significantly reduced in all low pH treatments. This resulted in no or ineffective nodulation by plants grown in these treatments. High numbers of Rhizobium were found in all high pH treatments, irrespective of metal content. Heat-treated biosolids-amended soil had higher numbers of Rhizobium than the control, but Nu-Earth biosolids-amended soil had lower numbers than the control. Nitrogen fixation, as measured by acetylene reduction activity, was greater in all high pH treatments compared with low pH treatments. When soil pH from the acidic plots was adjusted above 6.0, most of the isolates remained ineffective. Shoot yield and the number of Rhizobium did not show any significant increase with the increase in soil pH. Adjustment of high pH soil to low soil pH significantly reduced the number of Rhizobium irrespective of whether biosolids were applied. In conclusion, few significant effects of biosolids-borne heavy metals on plants, N 2 fixation, and on numbers of Rhizobium leguminosarum bv. trifolii were observed at concentrations of metals studied, as long as soil pH was maintained near 6.0. Where reductions in rhizobial number and plant parameters were observed, the decrease was primarily attributed to low soil pH and to a lesser extent heavy metal toxicity from biosolids.

Chromate-Tolerant Bacteria for Enhanced Metal Uptake by Eichhornia Crassipes (MART.)

A total of 85 chromate-resistant bacteria were isolated from the rhizosphere of water hyacinth grown in Mariout Lake, Egypt, as well as the sediment and water of this habitat. Only 4 (11%), 2 (8%), and 2 (8%) of isolates from each of the environments, respectively, were able to tolerate 200 mg Cr (VI) L−1. When these eight isolates were tested for their ability to tolerate other metals or to reduce chromate, they were shown to also be resistant to Zn, Mn, and Pb, and to display different degrees of chromate reduction (28% to 95%) under aerobic conditions. The isolates with the higher chromate reduction rates from 42% to 95%, (RA1, RA2, RA3, RA5, RA7, and RA8) were genetically diverse according to RAPD analysis using four different primers. Bacterial isolates RA1, RA2, RA3, RA5, and RA8 had 16 S rRNA gene sequences that were most similar to Pseudomonas diminuta, Brevundimonas diminuta, Nitrobacteria irancium, Ochrobactrum anthropi, and Bacillus cereus, respectively. Water hyacinth inoculated with RA5 and RA8 increased Mn accumulation in roots by 2.4- and 1.2-fold, respectively, compared to uninoculated controls. The highest concentrations of Cr (0.4 g kg−1) and Zn (0.18 g kg−1) were accumulated in aerial portions of water hyacinth inoculated with RA3. Plants inoculated with RA1, RA2, RA3, RA5, RA7, and RA8 had 7-, 11-, 24-, 29-, 35-, and 21-fold, respectively, higher Cr concentrations in roots compared to the control. These bacterial isolates are potential candidates in phytoremediation for chromium removal.

Survival of Bradyrhizobium sp. (Arachis) on fungicide-treated peanut seed in relationship to plant growth and yield

Survival and viability of Bradyrhizobium inoculant on fungicide-treated peanut seed and the resulting effects on nitrogen fixation, plant growth and seed yield were determined. Vitavax and Benomyl had the most and least lethal actions against Bradyrhizobium strains grown on YEM medium containing a fungicide, respectively, while Thiram and Captan effects were intermediate. Survival of Bradyrhizobium USDA 3384 and USDA 3456, as single strain peat inoculants, on peanut (Arachis hypogaea L. var. Florunner) seeds treated with Benomyl or Vitavax at the rate of 3g/kg seed was also examined. Both fungicides inhibited the growth and affected the survival of strain USDA 3384 on peanut seed. Vitavax killed the inoculant in 9 h. In contrast, USDA 3456 resisted both fungicides, and survived for up to 72h. Nodule formation on greenhouse-grown plants inoculated with USDA 3384 was inhibited by all fungicides. Shoot dry weight and plant nitrogen content significantly decreased as compared to controls. Fungicides, except Vitavax, had a slight effect on nodulation and plant growth when USDA 3456 was used as inoculant. The agronomic importance of fungicide-inoculant interaction was examined in field experiments conducted in Egypt in soil free of peanut-nodulating Bradyrhizobium, where seeds were treated with a combination of two fungicides and a single strain peat inoculant of either USDA 3384 or USDA 3456. All fungicides decreased nodulation, nitrogen fixation, plant growth and seed yield, especially with USDA 3384 as inoculant. Fungicides inhibited viability and survival of Bradyrhizobium on peanut seeds which decreased nodule formation leading to reduced peanut seed yield.

Report from the Roundtable on Rhizobium Taxonomy

During recent years, there has been an explosion in the field oF Rhizobium taxonomy (Rhizobium is defined here as all root and stem nodulating bacteria). In particular, recent emphasis has been on tropical rhizobia and the vast biodiversity of this group of bacteria has become evident. Molecular methods nowadays offer sophisticated means of constructing phylogenies for bacteria and they also facilitate rapid and accurate genomic fingerprinting at the strain level. The Subcommittee on the Taxonomy of Agrobacterium and Rhizobium is a forum for discussions and recommendations in the field of taxonomy. However, the committee cannot make decisions as to whether new species and genera should be designated and named, since articles proposing those undergo normal peer review and follow standard publication policies. During the conference, a roundtable workshop covering recent advances in Rhizobium and Agrobacterium taxonomy was organized.