Penny R. Hirsch

Simple and rapid method fordirect extraction of microbial DNA fromsoil for PCR

Abstract A simple and rapid procedure for direct extraction of DNA from soils was developed to yield DNA of a high purity and quality suitable for amplification using the polymerase chain reaction (PCR). Co-extracted humic material from soil was a major contaminant of DNA and methods were devised to overcome this problem. Oligonucleotide PCR primers were designed to detect and monitor a genetically-modified (GM) Rhizobium leguminosarum bv. viciae strain RSM2004 (marked with Tn5) which had become established in Rothamsted field soils. The key steps of the procedure were alkaline-SDS buffer assisted lysis of indigenous soil bacteria in a bead-beater and the purification of extracted DNA by separate PVPP and Sephadex G-75 spin-column chromatography. The mean yield from Rothamsted soil was 25±1.7xa0 μ g crude DNAxa0g −1 wet soil (i.e. 20xa0 μ gxa0g −1 dry soil), sheared to fragment sizes of about 22–25xa0kb. The recovered DNA was easier to purify and of a higher quality, as verified by PCR amplification of a 442xa0bp target sequence of Tn5, than DNA extracted by a hot-SDS lysis method. The detection limit was demonstrated to be one culturable cell of RSM2004 (i.e. a single copy of Tn5) 10xa0mg −1 soil against a background of 10 7 diverse non-target bacteria.

Absence of nitrogen fixation in clover grown on soil subject to long-term contamination with heavy metals is due to survival of only ineffective Rhizobium

All strains of Rhizobium isolated from ineffective nodules on white clover (> 50 separate isolations) formed in metal-contaminated soils from a field experiment were demonstrated to be wholly ineffective in nitrogen fixation in plant infection tests on N-free nutrient agar. The plasmid profiles of these isolates were all very similar indicating a lack of genetic diversity in the population surviving in high concentrations of heavy metals. Isolates from comparable field plots of uncontaminated soil had a wide diversity of plasmid profiles. n nInoculation of white clover at sowing with a large inoculum of effective Rhizobium leguminosarum bv. trifolii on metal contaminated soil resulted in effective N2-fixation. However, if the inoculated soils were held for 2 months in a moist condition before sowing with white clover, N2-fixation was not detected with inoculum doses of 107 cells pot−1 or less but was obvious where a very large inoculum of 1010 cells pot−1 was added. This indicates that white clover rhizobia are unable to survive (or at least unable to remain effective) in the presence of concentrations of heavy metals close to the current Commission of the European Communities guidelines for environmental protection. A rapid method of assessment of the toxic effects of pollutants on populations of Rhizobium spp is described.

Heavy metals from past applications of sewage sludge decrease the genetic diversity of rhizobium leguminosarum biovar trifolii populations

White clover plants grown at a site contaminated with heavy metals following applications of sewage sludge were found to have small white root nodules containing ineffective rhizobia (S isolates) which had identical plasmid profiles, unlike the diverse profiles of effective rhizobia from root nodules on adjacent control plots. Our paper supports an earlier suggestion that the ineffective S isolates of Rhizobium from nodules of white clover grown on heavy-metal contaminated soil represent a single strain. These new data include restriction fragment length polymorphism (RFLP) studies using probes specific for a chromosomally-located gene (lac), a plasmid-located symbiotic gene (nifH,D) and a repeated sequence specific for Rhizobium leguminosarum bv. trifolii (RtRS). RFLP patterns of isolates from control plots indicated that although these strains showed variation, they were related to one another but not to the S isolates. We also demonstrated that although the S isolates were ineffective on white clover, they formed normal nodules on subterranean clover, which were effective in nitrogen fixation. However, they ineffectively nodulated red clover and were unable to nodulate Vicia hirsuta. Thus the population of R. leguminosarum bv. trifolii had been radically altered by long-term exposure to heavy metal contamination, apparently losing those agronomically-important strains capable of forming effective symbiotic associations with white and red clover.

PCR-based molecular discrimination of Verticillium chlamydosporium isolates

PCR-based assays were performed to resolve the genetic variation between 28 different isolates of Verticillium chlamydosporium using primers designed to amplify ribosomal internal transcribed spacers (ITS) and intergenic spacers (IGS). Different isolates of V. chlamydosporium were also differentiated using primers matching enterobacterial repetitive intergenic consensus (ERIC) sequences and repetitive extragenic palindromic (REP) elements. Restriction fingerprinting of PCR-amplified ITS products failed to yield intraspecific polymorphism, and different levels of discrimination between V. chlamydosporium isolates were not achieved. However restriction patterns of ITS products digested with Hae III and Hinf I were useful in differentiating between some of the closely related isolates of V. chlamydosporium, plant pathogenic Verticillium species and some common soil fungi. PCR amplification of IGS was found to be the most sensitive method which enabled the detection of 22 variants within the sample of 28 isolates of V. chlamydosporium and six different plant pathogenic Verticillium species. By using ERIC and REP-PCR fingerprinting, isolates were categorized in 20 and 13 genotypes, respectively. In general, PCR-based procedures can differentiate between closely related isolates of V. chlamydosporium within IGS genotypes. This also could be achieved by ERIC and REP-PCR, and may be considered a rapid tool for the genetic characterization and detection of different isolates of V. chlamydosporium.

Detection of the nematophagous fungus Verticillium chlamydosporium in nematode-infested plant roots using PCR

PCR-based methods to detect Verticillium chlamydosporium on infected plant roots were developed. Arbitrary ERIC primers and those based on rRNA genes, to identify fungi grown in pure culture, were unsuitable for DNA extracted from nematode-infested roots, because of interference by plant and nematode DNA. A novel method utilizing specific primers designed from an amplified and cloned fragment of the V. chlamydosporium β-tubulin gene was developed. Although it could not discriminate between different isolates of V. chlamydosporium, one primer set could identify the fungus on tomato roots infested with root-knot nematodes. The V. chlamydosporium β-tubulin sequence data showed close homology to sequences from plant endophytic Acremonium and Epichloe species and the saprotrophic Trichoderma viride.

Changes in the population structure of β-group autotrophic ammonia oxidising bacteria in arable soils in response to agricultural practice

Abstract Autotrophic ammonia oxidising bacteria of the β-group proteobacteria are important in the global nitrogen cycle, as they are responsible for the majority of ammonia oxidation in arable soil. This study examined the effects of agricultural practices on the community structure of autotrophic ammonia oxidising populations in arable plots that receive either NH4NO3, farmyard manure plus NH4NO3, or no nitrogen. The 16S rRNA genes of autotrophic ammonia oxidisers were PCR amplified from DNA extracted from these soils, and the products hybridised to oligonucleotide probes specific for each of the previously recognised phylogenetically distinct groups of autotrophic ammonia oxidising bacteria. Only 16S rDNA sequences from group 3 and group 4 organisms were detected in the PCR products. Products from the plot that received NH4NO3 fertiliser were dominated by group 3 sequences, while those that received no nitrogen fertiliser were dominated by group 4 sequences. Ploughing of the plots was associated with an increased abundance of group 3 organisms particularly in plots that had previously received NH4NO3 fertiliser. These data were related to both the size and activity of the ammonia oxidising populations, as determined by a competitive PCR assay based upon the amoA gene, and by 15N isotopic pool dilution, respectively.

Plant genotype and micronutrient status influence colonization of wheat roots by soil bacteria

Abstract Wheat genotypes show differential growth under manganese (Mn) or zinc (Zn) deficiency. A contribution of rhizoplane microorganisms to such a differential response is unclear. This study was conducted to assess changes in bacterial colonization of roots of wheat genotypes differing in growth under Mn or Zn deficiency. Soil microorganisms were extracted from three soils by two‐phase aqueous partitioning and used to inoculate seedlings of four wheat (Triticum aestivum L.) genotypes mounted in growth pouches and supplied with various nutrient solutions. The total count of colony‐forming units (cfu) decreased after extraction compared to soils before extraction, while pseudomonad numbers remained largely unaffected by extraction. Axenic plants grown in pouches and supplied with solutions containing no Zn or Mn showed a decline in shoot growth due to Zn or Mn deficiency. The most Mn‐efficient wheat genotype C8MM (which grows better and yields more than Mn‐inefficient genotypes under Mn‐deficiency condi...

Plant genotype, micronutrient fertilization and take-all infection influence bacterial populations in the rhizosphere of wheat

The relationship between micronutrient efficiency of four wheat (Triticum aestivum L.) genotypes, tolerance to take-all disease (caused by Gaeumannomyces graminis (Sacc.) Arx and Olivier var. tritici Walker), and bacterial populations in the rhizosphere was tested in soil fertilized differentially with Zn and Mn. Plant growth was reduced by Mn or Zn deficiency and also by take-all. There was an inverse relationship between micronutrient efficiency of wheat genotypes when grown in deficient soils and the length of take-all lesions on roots (efficient genotypes had shorter lesions than inefficient ones). In comparison to the rhizosphere of control plants of genotypes Aroona and C8MM receiving sufficient Mn and Zn, the total numbers of bacterial cfu (colony forming units) were greater in the rhizosphere of Zn-efficient genotype Aroona under Zn deficiency and in Mn-efficient genotype C8MM under Mn deficiency. These effects were not observed in other genotypes. Take-all decreased the number of bacterial cfu in the rhizosphere of fully-fertilized plants but not of those subjected to either Mn or Zn deficiency. In contrast, the Zn deficiency treatment acted synergistically with take-all to increase the number of fluorescent pseudomonads in the rhizosphere. Although numbers of Mn-oxidising and Mn-reducing bacteria were generally low, take-all disease increased the number of Mn reducers in the rhizosphere of Mn-efficient genotypes Aroona and C8MM. Under Mn-deficiency conditions, the number of Mn reducers in the rhizosphere increased in Aroona but not in C8MM wheat. The results suggest that bacterial microflora may play a role in the expression of Mn and Zn efficiency and tolerance to take-all in some wheat genotypes.

Methods for studying the nematophagous fungus Verticillium chlamydosporium in the root environment

In order to exploit fully the biocontrol potential of the nematophagous fungus Verticillium chlamydosporium, it is important to understand the ecology of the fungus in soil, and interactions with both plant and nematode hosts. Several approaches for studying the fungus in soil and the root environment are compared. These include a semi-selective medium for V. chlamydosporium, PCR primers specific for the fungal β-tubulin gene, and monoclonal antibodies. In addition to providing a target for species-specific primers, the β-tubulin gene is implicated in resistance to the fungicides used in the semi-selective medium, and the genetic basis for this is investigated. Culture and PCR-based methods were used to screen for the presence of the fungus in field soils known to have been suppressive to cereal cyst nematode and that contained V. chlamydosporium populations. Monoclonal antibodies specific for either hyphae or conidia of the fungus were obtained, and their application as a tool for visualising the infection process on the root was explored.

The effects of pesticides on the diversity of culturable soil bacteria

The numbers of culturable soil bacteria in plots that had received either no pesticides or the full combination (aldicarb, chlorfenvinphos, benomyl, glyphosate, plus chlorotoluron or triadimefon) over a 20 year period were compared. Differences were very small although there were consistently higher numbers on the treated plot, possibly reflecting the greater crop yields which had been reported previously. There was no significant difference in numbers of bacterial colonies with homology to a nif gene probe in soils from the two plots. Genetic fingerprinting of Pseudomonas fluorescens isolates from the plots, using ERIC‐PCR, showed that the dominant strains in the two populations were not the same although there was no obvious difference in the degree of diversity. Substrate utilization by microbial populations from the two plots was compared using Biolog plates. The population from the pesticide‐treated plot showed a higher rate of substrate utilization which could reflect a slightly higher inoculum of heterotrophic bacteria, but could also indicate greater metabolic diversity in the population.