Development of a defined compost system for the study of plant-microbe interactions.

Plant growth promoting rhizobacteria can improve plant health by providing enhanced nutrition, disease suppression and abiotic stress resistance, and have potential to contribute to sustainable agriculture. We have developed a sphagnum peat-based compost platform for investigating plant-microbe interactions. The chemical, physical and biological status of the system can be manipulated to understand the relative importance of these factors for plant health, demonstrated using three case studies: 1. Nutrient depleted compost retained its structure, but plants grown in this medium were severely stunted in growth due to removal of essential soluble nutrients - particularly, nitrogen, phosphorus and potassium. Compost nutrient status was replenished with the addition of selected soluble nutrients, validated by plant biomass; 2. When comparing milled and unmilled compost, we found nutrient status to be more important than matrix structure for plant growth; 3. In compost deficient in soluble P, supplemented with an insoluble inorganic form of P (Ca3(PO4)2), application of a phosphate solubilising Pseudomonas strain to plant roots provides a significant growth boost when compared with a Pseudomonas strain incapable of solubilising Ca3(PO4)2. Our findings show that the compost system can be manipulated to impose biotic and abiotic stresses for testing how microbial inoculants influence plant growth.

Old meets new: most probable number validation of metagenomic and metatranscriptomic datasets in soil.

Metagenomics and metatranscriptomics provide insights into biological processes in complex substrates such as soil, but linking the presence and expression of genes with functions can be difficult. Here, we obtain traditional most probable number estimates (MPN) of Rhizobium abundance in soil as a form of sample validation. Our work shows that in the Highfield experiment at Rothamsted, which has three contrasting conditions (>50 years continual bare fallow, wheat and grassland), MPN based on host plant nodulation assays corroborate metagenomic and metatranscriptomic estimates for Rhizobium leguminosarum sv. trifolii abundance. This validation is important to legitimize soil metagenomics and metatranscriptomics for the study of complex relationships between gene function and phylogeny. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has demonstrated for the first time a functional assay validation of metagenomic and metatranscriptomic datasets by utilizing the clover and Rhizobium leguminosarum sv. trifolii mutualism. The results show that the Most Probable Number results corroborate the results of the 'omics approaches and gives confidence to the study of other biological systems where such a cross-check is not available.

An analysis of Pseudomonas genomic diversity in take-all infected wheat fields reveals the lasting impact of wheat cultivars on the soil microbiota.

Manipulation of the soil microbiota associated with crop plants has huge promise for the control of crop pathogens. However, to fully realize this potential we need a better understanding of the relationship between the soil environment and the genes and phenotypes that enable microbes to colonize plants and contribute to biocontrol. A recent 2 years of investigation into the effect of wheat variety on second year crop yield in the context of take-all fungal infection presented the opportunity to examine soil microbiomes under closely defined field conditions. Amplicon sequencing of second year soil samples showed that Pseudomonas spp. were particularly affected by the wheat cultivar grown in year one. Consequently, 318 rhizosphere-associated Pseudomonas fluorescens strains were isolated and characterized across a variety of genetic and phenotypic traits. Again, the wheat variety grown in the first year of the study was shown to exert considerable selective pressure on both the extent and nature of Pseudomonas genomic diversity. Furthermore, multiple significant correlations were identified within the phenotypic/genetic structure of the Pseudomonas population, and between individual genotypes and the external wheat field environment. The approach outlined here has considerable future potential for our understanding of plant-microbe interactions, and for the broader analysis of complex microbial communities.

Soil organic matter and the extracellular microbial matrix show contrasting responses to C and N availability.

An emerging paradigm in soil science suggests microbes can perform 'N mining' from recalcitrant soil organic matter (SOM) in conditions of low N availability. However, this requires the production of extracellular structures rich in N (including enzymes and structural components) and thus defies stoichiometric expectation. We set out to extract newly synthesised peptides from the extracellular matrix in soil and compare the amino acid (AA) profiles, N incorporation and AA dynamics in response to labile inputs of contrasting C/N ratio. Glycerol was added both with and without an inorganic source of N (10% 15N labelled NH4NO3) to a soil already containing a large pool of refractory SOM and incubated for 10 days. The resulting total soil peptide (TSP) and extracellular pools were compared using colorimetric methods, gas chromatography, and isotope ratio mass spectrometry. N isotope compositions showed that the extracellular polymeric substance (EPS) contained a greater proportion of products formed de novo than did TSP, with hydrophobic EPS-AAs (leucine, isoleucine, phenylalanine, hydroxyproline and tyrosine) deriving substantially more N from the inorganic source provided. Quantitative comparison between extracts showed that the EPS contained greater relative proportions of alanine, glycine, proline, phenylalanine and tyrosine. The greatest increases in EPS-peptide and EPS-polysaccharide concentrations occurred at the highest C/N ratios. All EPS-AAs responded similarly to treatment whereas the responses of TSP were more complex. The results suggest that extracellular investment of N (as EPS peptides) is a microbial survival mechanism in conditions of low N/high C which, from an evolutionary perspective, must ultimately lead to the tendency for increased N returns to the microbial biomass. A conceptual model is proposed that describes the dynamics of the extracellular matrix in response to the C/N ratio of labile inputs.

Assessment of core and accessory genetic variation in Rhizobium leguminosarum symbiovar trifolii strains from diverse locations and host plants using PCR-based methods.

UNLABELLED: The nitrogen-fixing symbiosis between Rhizobium leguminosarum and host legumes is recognized as a key part of sustainable agriculture. A culture collection containing rhizobia isolated from legumes of economic importance in the UK and worldwide, maintained at Rothamsted Research for many years, provided material for this study. We aimed to develop and validate efficient molecular diagnostics to investigate whether the host plant or geographical location had a greater influence on the genetic diversity of rhizobial isolates, and the extent to which the core bacterial genome and the accessory symbiosis genes located on plasmids were affected. To achieve this, core housekeeping genes and those involved in symbiosis interactions were sequenced and compared with genome-sequenced strains in the public domain. Results showed that some Rh. leguminosarum symbiovar trifolii strains nodulating clovers and Rh. leguminosarum sv. viciae strains nodulating peas and vicias shared identical housekeeping genes, clover nodule isolates from the same location could have divergent symbiosis genes, and others isolated on different continents could be very similar. This illustrates the likely co-migration of rhizobia and their legume hosts when crops are planted in new areas and indicates that selective pressure may arise from both local conditions and crop host genotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: The nitrogen-fixing symbiosis between Rhizobium leguminosarum and host legumes has been recognized as a key part of sustainable agriculture for many years; this study provides new tools to study rhizobial biogeography which will be invaluable for extending the cultivation of legumes and indicating whether or not inoculation is necessary.

Biodiesel Co-Product (BCP) Decreases Soil Nitrogen (N) Losses to Groundwater.

This study compares a traditional agricultural approach to minimise N pollution of groundwater (incorporation of crop residues) with applications of small amounts of biodiesel co-product (BCP) to arable soils. Loss of N from soil to the aqueous phase was shown to be greatly reduced in the laboratory, mainly by decreasing concentrations of dissolved nitrate-N. Increases in soil microbial biomass occurred within 4 days of BCP application-indicating rapid adaptation of the soil microbial community. Increases in biomass-N suggest that microbes were partly mechanistic in the immobilisation of N in soil. Straw, meadow-grass and BCP were subsequently incorporated into experimental soil mesocosms of depth equal to plough layer (23 cm), and placed in an exposed netted tunnel to simulate field conditions. Leachate was collected after rainfall between the autumn of 2009 and spring of 2010. Treatment with BCP resulted in less total-N transferred from soil to water over the entire period, with 32.1, 18.9, 13.2 and 4.2 mg N kg-1 soil leached cumulatively from the control, grass, straw and BCP treatments, respectively. More than 99 % of nitrate leaching was prevented using BCP. Accordingly, soils provided with crop residues or BCP showed statistically significant increases in soil N and C compared to the control (no incorporation). Microbial biomass, indicated by soil ATP concentration, was also highest for soils given BCP (p < 0.05). These results indicate that field-scale incorporation of BCP may be an effective method to reduce nitrogen loss from agricultural soils, prevent nitrate pollution of groundwater and augment the soil microbial biomass.

A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts.

Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts.

A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans.

AIMS: To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans. METHODS AND RESULTS: An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. CONCLUSIONS: Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. SIGNIFICANCE AND IMPACT OF THE STUDY: These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.

Rapid and reliable DNA extraction and PCR fingerprinting methods to discriminate multiple biotypes of the nematophagous fungus Pochonia chlamydosporia isolated from plant rhizospheres.

AIMS: To develop a simple, rapid, reliable protocol producing consistent polymerase chain reaction (PCR) fingerprints of Pochonia chlamydosporia var. chlamydosporia biotypes for analysing different fungal isolates during co-infection of plants and nematodes. METHODS AND RESULTS: DNA extracted from different P. chlamydosporia biotypes was fingerprinted using enterobacterial repetitive intragenic consensus (ERIC)-PCR. Four extraction methods (rapid alkaline lysis; microLYSIS-PLUS; DNeasy; FTA cards) gave consistent results within each protocol but these varied between protocols. Reproducible fingerprints were obtained only if DNA was extracted from fresh fungal cultures that were free of agar. Some DNA degradation occurred during storage, except with the FTA cards, used with this fungus for the first time, which provide a method for long-term archiving. Rapid alkaline lysis and ERIC-PCR identified fungal isolates from root and nematode egg surfaces when plants were treated with different combinations of fungal biotypes; the dominant biotype isolated from the rhizosphere was not always the most abundant in eggs. CONCLUSIONS: ERIC-PCR fingerprinting can reliably detect and identify different P. chlamydosporia biotypes. It is important to use fresh mycelium and the same DNA isolation method throughout each study. SIGNIFICANCE AND IMPACT OF THE STUDY: This evaluation of methods to assess genetic diversity and identify specific P. chlamydosporia biotypes is relevant to other mycelial fungi.

Impact of the nematophagous fungus Pochonia chlamydosporia on nematode and microbial populations.

The microbial and nematode populations associated with two plants (tomato and cabbage) inoculated with the nematophagous fungus, Pochonia chlamydosporia var. chlamydosporia or root knot nematode (Meloidogyne incognita), or both, were compared with those in unplanted controls. The dominant factor affecting culturable microbial populations was found to be the presence or absence of tomato plants. Generally microbial colony counts were lowest in unplanted soil, small increases were associated with cabbage and significantly greater numbers with tomato plants. Differences in microbial diversity (estimated from community profiles of carbon substrate utlisation, using Biolog) were observed between planted and unplanted soils, however, there were few differences between soils with either of the two plants. The presence of P. chlamydosporia was associated with a reduction in the numbers of plant parasitic nematodes (51%-78%) including the migratory ectoparasites, whereas free-living nematodes, culturable bacteria and bacterial populations assessed by Biolog were unaffected by the application of fungus.

Detection and quantification of Plectosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selective media, and baiting.

Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitor P. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P. cucumerina in soil, root, and nematode samples. PCR was combined with a bait method to identify P. cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A second P. cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance of P. cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity of P. cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.

Quantification in soil and the rhizosphere of the nematophagous fungus Verticillium chlamydosporium by competitive PCR and comparison with selective plating.

A competitive PCR (cPCR) assay was developed to quantify the nematophagous fungus Verticillium chlamydosporium in soil. A gamma-irradiated soil was seeded with different numbers of chlamydospores from V. chlamydosporium isolate 10, and samples were obtained at time intervals of up to 8 weeks. Samples were analyzed by cPCR and by plating onto a semiselective medium. The results suggested that saprophytic V. chlamydosporium growth did occur in soil and that the two methods detected different phases of growth. The first stage of growth, DNA replication, was demonstrated by the rapid increase in cPCR estimates, and the presumed carrying capacity (PCC) of the soil was reached after only 1 week of incubation. The second stage, an increase in fungal propagules presumably due to cell division, sporulation, and hyphal fragmentation, was indicated by a less rapid increase in CFU, and 3 weeks was required to reach the PCC. Experiments with field soil revealed that saprophytic fungal growth was limited, presumably due to competition from the indigenous soil microflora, and that the PCR results were less variable than the equivalent plate count results. In addition, the limit of detection of V. chlamydosporium in field soil was lower than that in gamma-irradiated soil, suggesting that there was a background population of the fungus in the field, although the level was below the limit of detection. Tomatoes were infected with the root knot nematode (RKN) or the potato cyst nematode (PCN) along with a PCN-derived isolate of the fungus (V. chlamydosporium isolate Jersey). Increases in fungal growth were observed in the rhizosphere of PCN-infested plants but not in the rhizosphere of RKN-infested plants after 14 weeks using cPCR. In this paper we describe for the first time PCR-based quantification of a fungal biological control agent for nematodes in soil and the rhizosphere, and we provide evidence for nematode host specificity that is highly relevant to the biological control efficacy of this fungus.

Characterization of two novel Rhizobium leguminosarum bacteriophages from a field release site of genetically-modified rhizobia.

Two Rhizobium leguminosarum biovar viceae bacteriophages with contrasting properties were isolated from a field site in which the survival of genetically modified R. leguminosarum inoculants had been monitored for several years. Inoculant strain RSM2004 was used as the indicator for phage isolation and propagation. One phage, RL1RES, was temperate and could not replicate in any of the 42 indigenous R. leguminosarum field isolates tested although nested PCR indicated that phage sequences were present in six of the isolates. The second phage, RL2RES, was virulent, capable of generalised transduction, contained DNA with modified cytosine residues, and was capable of infecting all field isolates tested although the GM inoculant strain CT0370 was resistant. Sequence with homology to RL2RES was detected by nested PCR in six of the 42 field-isolates. These were not the same isolates that showed homology to RL1RES. The implication of these findings for the survival of rhizobial inoculants, and the ecology of phages and their host bacteria, are discussed.

The PCR amplification of non-tuberculous mycobacterial 16S rRNA sequences from soil.

Non-tuberculous mycobacteria are free living saprophytic organisms commonly found in soil and water. Some are major causes of opportunistic infection, particularly in immuno-compromised patients, and may influence the efficacy of bacille Calmette-Guérin vaccinations. Many of these organisms are not amenable to culture, so information about their distribution is limited. PCR primers designed to amplify part of the mycobacterial 16S rRNA gene were applied to DNA extracted from cultured organisms and soil. The PCR products from soil contained sequences with similarity to slow growing mycobacteria similar to Mycobacterium lentiflavum, and to fast growing mycobacteria such as the xenobiotic degraders PYR-I and RJGII.

Use of molecular and isotopic techniques to monitor the response of autotrophic ammonia-oxidizing populations of the beta subdivision of the class proteobacteria in arable soils to nitrogen fertilizer.

This study examined the effects of NH(4)NO(3) fertilizer on the size and activity of nitrifying, autotrophic, ammonia-oxidizing populations of the beta subdivision of the class Proteobacteria in arable soils. Plots under different long-term fertilizer regimes were sampled before and after NH(4)NO(3) additions, and the rates of nitrification were determined by (15)N isotopic pool dilution assays. Ammonia-oxidizing populations in the plots were quantified by competitive PCR assays based on the amoA and ribosomal 16S genes. Prior to fertilizer addition, ammonium concentrations and nitrification rates in the plots were comparatively low; ammonia-oxidizing populations were present at 10(4) to 10(5) gene copies g of soil(-1). Three days after the application of fertilizer, nitrification rates had risen considerably but the size of the ammonia-oxidizing population was unchanged. Six weeks after fertilizer treatment, ammonium concentrations and nitrification rates had fallen while the ammonia-oxidizing populations in plots receiving fertilizer had increased. The rapidity of the rise in nitrification rates observed after 3 days suggests that it results from phenotypic changes in the ammonia-oxidizing bacterial population. Associated increases in population sizes were only observed after 6 weeks and did not correlate directly with nitrifying activity. Phylogenetic analyses of PCR products from one of the plots revealed a population dominated by Nitrosospira-type organisms, similar to those prevalent in other soils.

Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction.

Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn5; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strain provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g-1 soil in soil-extracted DNA.

Occurrence of flavonoids and nucleosides in agricultural soils.

AN ECOLOGICALLY RELEVANT SOIL EXTRACTION PROCEDURE SEPARATED TWO TYPES OF MOLECULES IMPORTANT FOR BACTERIA: flavonoids and small hydrophilic organic compounds. Two flavonoids, identified previously as inducers of nodulation genes in Rhizobium meliloti, were detected in rhizosphere soil from alfalfa (Medicago sativa L.). In addition, biologically significant quantities (micromoles per kilogram) of ribonucleosides and deoxyribonucleosides were found in all soils tested. Long-term wheat (Triticum aestivum L.) plots that had received manure contained elevated amounts of nucleosides, and in a separate experiment, the presence of legumes in a wheat-cropping sequence increased soil nucleosides. Intact bacterial cells accounted for less than 1% of the free nucleosides detected. These results suggest new testable hypotheses for molecular ecologists and differ from those obtained with older, harsher techniques.

Rhizobium meliloti fixGHI sequence predicts involvement of a specific cation pump in symbiotic nitrogen fixation.

We present genetic and structural analyses of a fix operon conserved among rhizobia, fixGHI from Rhizobium meliloti. The nucleotide sequence of the operon suggests it may contain a fourth gene, fixS. Adjacent open reading frames of this operon showed an overlap between TGA stop codons and ATG start codons in the form of an ATGA motif suggestive of translational coupling. All four predicted gene products contained probable transmembrane sequences. FixG contained two cysteine clusters typical of iron-sulfur centers and is predicted to be involved in a redox process. FixI was found to be homologous with P-type ATPases, particularly with K+ pumps from Escherichia coli and Streptococcus faecalis but also with eucaryotic Ca2+, Na+/K+, H+/K+, and H+ pumps, which implies that FixI is a pump of a specific cation involved in symbiotic nitrogen fixation. Since prototrophic growth of fixI mutants appeared to be unimpaired, the predicted FixI cation pump probably has a specifically symbiotic function. We suggest that the four proteins FixG, FixH, FixI, and FixS may participate in a membrane-bound complex coupling the FixI cation pump with a redox process catalyzed by FixG.

Construction of a Tn5 derivative determining resistance to gentamicin and spectinomycin using a fragment cloned from R1033.

A physical and genetic map of the IncP plasmid R1033 was constructed: restriction fragments were subcloned and antibiotic resistance genes were located. The map is consistent with previous reports that R1033 is a derivative of RP4 carrying a 16-kb transposon Tn1696 which contains the antibiotic-resistance determinants present on R1033 but not on RP4. A BamHI fragment from R1033, determining resistance to gentamicin, spectinomycin and streptomycin, was cloned into Tn5, replacing the central Bg/II fragment that determined kanamycin resistance, producing a recombinant transposon Tn5-GmSpSm. This was shown to transpose in Rhizobium leguminosarum at a frequency similar to that of the parental Tn5.