Jeffry J. Fuhrmann

Evolutionary relationships among the soybean bradyrhizobia reconstructed from 16S rRNA gene and internally transcribed spacer region sequence divergence.

From sequence divergence of 16S rRNA genes and the internally transcribed spacer (ITS) region it is reported that variation in phylogenetic placement exists among the 17 different serotype strains of Bradyrhizobium that have been isolated from nodules of soybean. Evolutionary relationships among the bradyrhizobia were more resolved using reconstructions derived from ITS than from 16S rRNA gene sequence divergence. Strain USDA 129 was placed together with USDA 62, 110, 122 and 126, but did not cluster with USDA 123 and 127, with which it shares antigenic determinants. The results from the phylogenetic analysis were supported with data from determinations of genetic diversity among additional strains within each of these serogroups using amplified fragment length polymorphism analysis. From these results it was concluded that strains of serogroup 129 were more similar to strains of serogroups 62, 110 and 122 than they were to strains of serogroups 123 and 127. The serotype strain of Bradyrhizobiumjaponicum USDA 135 and the type strain for Bradyrhizobium liaoningense possessed identical 16S rRNA gene and ITS region sequences. Also, the type strain for B. liaoningense cross-reacted with antisera prepared against somatic antigens of USDA 135. Therefore, it was not possible to distinguish B. liaoningense from serogroup 135 in our analysis of B. japonicum and Bradyrhizobium elkanii.

Microbial community responses to atrazine exposure and nutrient availability: linking degradation capacity to community structure.

Repeated pesticide exposure may enhance biodegradation through selective enrichment of pesticide-metabolizing microorganisms, particularly when the compound is used as a C and energy source. The relationship between pesticide application history and degradation rate is unclear when the chemical is utilized as a nutrient source other than C. Atrazine, a poor source of C and energy, was chosen as a model compound because it can serve as an N source for some microorganisms. Soils with (H-soil) and without (NH-soil) prior s-triazine treatment history were repeatedly exposed to atrazine and a variety of C and N source amendments. Exposure to atrazine and inorganic-N availability were the dominant factors leading to the development of microbial communities with an enhanced capacity to degrade atrazine. The density of the atrazine-degrading microorganisms increased immediately, up to 1000-fold, with atrazine exposure in the H-soil, but comparable increases were not observed in the NH-soil until 12 weeks following laboratory acclimation, despite high rates of atrazine mineralization in these soils immediately following the acclimation period. Whole-soil fatty acid methyl ester (FAME) analysis showed that the application of alternative C and N sources in addition to atrazine resulted in a microbial community composition that was distinctly different from that in either the atrazine-alone treatment or water controls for both the H- and NH-soils. These data suggest that the microbial communities in both soils were altered differently in response to the treatments but developed a similar enhanced capacity to mineralize atrazine.

Atrazine and phenanthrene degradation in grass rhizosphere soil

Abstract Organic contaminants often disappear more quickly from planted than from non-planted soils. Five grass species (Sudan grass, ryegrass, tall fescue, crested wheatgrass and switch grass) were grown in soils without (Phase I) or with (Phase II) prior atrazine (ATR) and phenanthrene (PHE) amendment to study the degradation of these compounds by rhizosphere microorganisms. In suspensions of soil without prior chemical exposure, no significant loss of ATR was observed after 16 days incubation. The most probable number (MPN) of ATR-degrading bacteria in the soils was below detection. Phenanthrene degradation was observed in suspensions inoculated with all soils, but the rates of degradation were not significantly different among them. The number of PHE-degrading bacteria was similar in planted and non-planted soils (105 cells g−1 soil) except the number in tall fescue soil was significantly higher than in non-planted soil. In the Phase II study, both compounds were mineralized whether or not soils had been conditioned with ATR or PHE. Prior amendment with either ATR or PHE significantly reduced the acclimation period preceding the onset of mineralization. However, enumeration procedures detected ATR-degrading bacteria only in ATR-amended soils. Prior exposure to PHE did not alter the number of PHE-degrading bacteria significantly.

Characterization of rhizosphere microbial community structure in five similar grass species using FAME and BIOLOG analyses

Abstract Accelerated biodegradation of organic contaminants in planted soil is frequently reported yet our current understanding of plant–microbe interactions does not allow us to predict which plant species can encourage the development of rhizosphere communities with enhanced degradation capacity. In a companion study, five grass species (Sudan grass, ryegrass, tall fescue, crested wheatgrass, and switch grass) were grown in a Matapeake silt loam soil to study the degradation of atrazine and phenanthrene by rhizosphere microorganisms (see Fang et al., 2000 , this vol., Fang, C., Radosevich, M., Fuhrmann, J. J., 2000. Atrizine and phenanthrene degradation in grass rhizosphere soil. Soil Biology & Biochemistry, in press). In the present paper substrate utilization patterns (BIOLOG ® ), and fatty acid methyl ester (FAME) profiles of the same rhizosphere microbial communities were determined. Both FAME and BIOLOG ® analyses detected changes in soil microbial community structure among treatments. However, community structure did not directly correlate to either ATR or PHE degradation rates.

Response of microbial community structure and function to short-term biochar amendment in an intensively managed bamboo (Phyllostachys praecox) plantation soil: Effect of particle size and addition rate

Biochar incorporated into soil has been known to affect soil nutrient availability and act as a habitat for microorganisms, both of which could be related to its particle size. However, little is known about the effect of particle size on soil microbial community structure and function. To investigate short-term soil microbial responses to biochar addition having varying particle sizes and addition rates, we established a laboratory incubation study. Biochar produced via pyrolysis of bamboo was ground into three particle sizes (diameter size<0.05mm (fine), 0.05-1.0mm (medium) and 1.0-2.0mm (coarse)) and amended at rates of 0% (control), 3% and 9% (w/w) in an intensively managed bamboo (Phyllostachys praecox) plantation soil. The results showed that the fine particle biochar resulted in significantly higher soil pH, electrical conductivity (EC), available potassium (K) concentrations than the medium and coarse particle sizes. The fine-sized biochar also induced significantly higher total microbial phospholipid fatty acids (PLFAs) concentrations by 60.28% and 88.94% than the medium and coarse particles regardless of addition rate, respectively. Redundancy analysis suggested that the microbial community structures were largely dependent of particle size, and that improved soil properties were key factors shaping them. The cumulative CO2 emissions from biochar-amended soils were 2-56% lower than the control and sharply decreased with increasing addition rates and particle sizes. Activities of α-glucosidase, β-glucosidase, β-xylosidase, N-acetyl-β-glucosaminidase, peroxidase and dehydrogenase decreased by ranging from 7% to 47% in biochar-amended soils over the control, indicating that biochar addition reduced enzyme activities involved carbon cycling capacity. Our results suggest that biochar addition can affect microbial population abundances, community structure and enzyme activities, that these effects are particle size and rate dependent. The fine particle biochar may additionally produce a better habitat for microorganisms compared to the other particle sizes.

Bamboo invasion of native broadleaf forest modified soil microbial communities and diversity

The effect of plant invasion on soil microbial communities in various ecosystems has increasingly become the focus of research over the last decade. Moso bamboo (Phyllostachys edulis) invasion of native forests in Tianmushan National Nature Reserve located in southeastern China has resulted in greatly decreased biodiversity of plants and birds. We combined three different microbial community techniques (Biolog, cellular fatty acids, and 16S-PCR–DGGE) to examine whether changes in the overstory of plant taxa, or any associated environmental changes, modified soil microbial communities. Three types of forests were examined: mono-bamboo forest, mixed forest of bamboo and broadleaf, and native broadleaf forest. The fatty acid and DGGE results showed that bamboo invasion of the native forest influenced soil community structure and increased microbial biomass and taxonomic diversity despite decreased plant diversity. The Biolog results indicated no change in microbial functional diversity as a result of bamboo invasion. Evidence from bacterial PCR–DGGE suggested that bamboo stimulated the growth of otherwise undetected soil bacterial species. Overall, the results indicate that bamboo invasion may significantly affect associated soil microbial communities.

Fatty acid methyl ester (FAME) analysis for monitoring Nocardia levels in activated sludge

The overall goal of this study was to evaluate the ability of fatty acid methyl ester (FAME) technology to quantify Nocardia levels in activated sludge samples. In order to distinguish Nocardia cells in activated sludge, fatty acids associated with N. amarae pure culture were determined and compared to fatty acid profiles of non-foaming activated sludge. Seven fatty acid peaks were observed for N. amarae cells grown in pure culture. Two of these seven peaks appeared to be unique to Nocardia when compared to the FAME profiles for the activated sludge sample and, therefore, can potentially serve as signature fatty acids for detecting and quantifying Nocardia in similar systems. In addition, we performed FAME analysis on mixed liquor samples containing various levels of N. amarae cells. Greater amounts of the signature fatty acids were extracted from the mixed liquors containing higher levels of Nocardia, and these amounts additionally correlated well with the conventional filamentous counting technique.

Direct assessment of viral diversity in soils by random PCR amplification of polymorphic DNA.

ABSTRACT Viruses are the most abundant and diverse biological entities within soils, yet their ecological impact is largely unknown. Defining how soil viral communities change with perturbation or across environments will contribute to understanding the larger ecological significance of soil viruses. A new approach to examining the composition of soil viral communities based on random PCR amplification of polymorphic DNA (RAPD-PCR) was developed. A key methodological improvement was the use of viral metagenomic sequence data for the design of RAPD-PCR primers. This metagenomically informed approach to primer design enabled the optimization of RAPD-PCR sensitivity for examining changes in soil viral communities. Initial application of RAPD-PCR viral fingerprinting to soil viral communities demonstrated that the composition of autochthonous soil viral assemblages noticeably changed over a distance of meters along a transect of Antarctic soils and across soils subjected to different land uses. For Antarctic soils, viral assemblages segregated upslope from the edge of dry valley lakes. In the case of temperate soils at the Kellogg Biological Station, viral communities clustered according to land use treatment. In both environments, soil viral communities changed along with environmental factors known to shape the composition of bacterial host communities. Overall, this work demonstrates that RAPD-PCR fingerprinting is an inexpensive, high-throughput means for addressing first-order questions of viral community dynamics within environmental samples and thus fills a methodological gap between narrow single-gene approaches and comprehensive shotgun metagenomic sequencing for the analysis of viral community diversity.

Evidence from Internally Transcribed Spacer Sequence Analysis of Soybean Strains that Extant Bradyrhizobium spp. Are Likely the Products of Reticulate Evolutionary Events

ABSTRACT The internally transcribed spacer (ITS) sequences of several members within each of 17 soybean bradyrhizobial serogroups were determined to establish whether the regions within all members of each serogroup were identical. The rationale was to provide a sequence-based alternative to serology. The objective also was to link the extensive older literature on soybean symbiosis based on serology with ITS sequence data for more recent isolates from both soybean and other legumes nodulated by rhizobia within the genus Bradyrhizobium. With the exception of serogroup 31 and 110 strains, sequence identity was established within each serogroup. Variation ranged from 0 to 23 nucleotides among serogroup 31 strains, and the regions in the type strains USDA 31 (serogroup 31) and USDA 130 (serogroup 130) were identical. Sequence identity was established among most strains within serogroup 110. The exceptions were USDA 452 and USDA 456, which had ITS sequences that were identical with those of the serotype 124 strain, USDA 124. Perhaps this would imply that USDA 452, USDA 456, and serogroup 31 strains are members of rhizobial lineages resulting from genetic exchange and homologous recombination events. This conclusion would be supported by the construction of a phylogenetic network from the ITS sequence alignment implying that the genomes of extant members of the genus Bradyrhizobium are likely the products of reticulate evolutionary events. A pairwise homoplasy index (phi or Φw) test was used to obtain further evidence for recombination. The ITS sequences of USDA 110 and USDA 124 were more divergent (53 nucleotides) than this region between the type strain Bradyrhizobium japonicum USDA 6T and the proposed species Bradyrhizobium yuanmingense (28 nucleotides) and Bradyrhizobium liaoningense (48 nucleotides). Therefore, support for assigning discrete species boundaries among these three proposed species appears limited, considering the evidence for recombination, the narrow divergence of the ITS sequence, and their relative placement on the phylogenetic network.

Comparison of rhizobitoxine-induced inhibition of β-cystathionase from different bradyrhizobia and soybean genotypes

The enzyme β-cystathionase catalyzes the conversion of cystathionine to homocysteine in both plants and bacteria. Preparations of this enzyme taken from both Salmonella and spinach (Spinacia oleracea L.) have been shown to be irreversibly inhibited by low concentrations of rhizobitoxine (RT), a chlorosis-inducing phytotoxin produced by some strains of soybean bradyrhizobia. The sensitivities of β-cystathionase from bradyrhizobia and soybean are not well characterized. Therefore, we purified β-cystathionase from selected bradyrhizobia and soybean genotypes that have been shown to exhibit differences in RT production and apparent RT sensitivities, respectively. Enzyme purified from E. coli strain DH52 was used for comparison. The enzymes differed in their physiological properties and RT sensitivities. Overall, the β-cystathionase enzymes purified from bradyrhizobia were more sensitive to RT than were those from the soybean cultivars. Kinetic studies showed that the nature of the RT-induced inhibition also differed between the two sources. The enzymes from bradyrhizobia exhibited inhibition that was [RT]-dependent, whereas the enzymes from soybean showed a time-dependent inhibition. These contrasting characteristics may in part reflect differences in active site accessibility, amino acid components, and associated RT diffusion rates. However, in all cases the inhibition caused by RT showed a typical substrate-competitive inhibition pattern.