Assessment of differences in ascomycete communities in the rhizosphere of field-grown wheat and potato.

To assess effects of plant crop species on rhizosphere ascomycete communities in the field, we compared a wheat monoculture and an alternating crop rotation of wheat and potato. Rhizosphere soil samples were taken at different time points during the growing season in four consecutive years (1999-2002). An ascomycete-specific primer pair (ITS5-ITS4A) was used to amplify internal transcribed spacer (ITS) sequences from total DNA extracts from rhizosphere soil. Amplified DNA was analyzed by denaturing gradient gel electrophoresis (DGGE). Individual bands from DGGE gels were sequenced and compared with known sequences from public databases. DGGE gels representing the ascomycete communities of the continuous wheat and the rotation site were compared and related to ascomycetes identified from the field. The effect of crop rotation exceeded that of the spatial heterogeneity in the field, which was evident after the first year. Significant differences between the ascomycete communities from the rhizospheres of wheat in monoculture and one year after a potato crop were found, indicating a long-term effect of potato. Sequencing of bands excised from the DGGE gels revealed the presence of ascomycetes that are common in agricultural soils.

Ascomycete communities in the rhizosphere of field-grown wheat are not affected by introductions of genetically modified Pseudomonas putida WCS358r.

A long-term field experiment (1999-2002) was conducted to monitor effects on the indigenous microflora of Pseudomonas putida WCS358r and two transgenic derivatives constitutively producing phenazine-1-carboxylic acid (PCA) or 2,4-diacetylphloroglucinol (DAPG). The strains were introduced as seed coating on wheat into the same field plots each year. Rhizosphere populations of ascomycetes were analysed using denaturing gradient gel electrophoresis (DGGE). To evaluate the significance of changes caused by the genetically modified microorganisms (GMMs), they were compared with effects caused by a crop rotation from wheat to potato. In the first year, only the combination of both GMMs caused a significant shift in the ascomycete community. After the repeated introductions this effect was no longer evident. However, cropping potato significantly affected the ascomycete community. This effect persisted into the next year when wheat was grown. Clone libraries were constructed from samples taken in 1999 and 2000, and sequence analysis indicated ascomycetes of common genera to be present. Most species occurred in low frequencies, distributed almost evenly in all treatments. However, in 1999 Microdochium occurred in relatively high frequencies, whereas in the following year no Microdochium species were detected. On the other hand, Fusarium-like organisms were low in 1999, and increased in 2000. Both the DGGE and the sequence analysis revealed that repeated introduction of P. putida WCS358r had no major effects on the ascomycete community in the wheat rhizosphere, but demonstrated a persistent difference between the rhizospheres of potato and wheat.

Occurrence of Escherichia coli O157 and Other Verocytotoxin-Producing E. coli in Retail Raw Meats in the Netherlands.

Raw meats obtained from retail outlets in the Netherlands were examined for the presence of Escherichia coli of serogroup O157 and other verocytotoxin (VT)-producing E. coli (VTEC), in three different surveys. In the first survey O157 VTEC were detected and isolated by selective plating onto sorbitol MacConkey agar following selective enrichment in modified tryptone soy broth with acriflavin. The organisms were isolated from 2 (0.3%) of 770 samples of minced mixed beef and pork, but not detected in samples of raw minced beef (n = 1,000), minced pork (n = 260), or poultry products (n = 300). In the second survey an additional 360 raw meats were examined with the 3M Petrifilm™ Test Kit-HEC, after selective enrichment in modified E. coli broth containing novobiocin. VT-negative E. coli O157 strains were isolated from 22 (6.1%) samples. In the third survey 180 enrichment cultures of the first survey were screened for the presence of VT1 and VT2 genes with a polymerase chain reaction (PCR). Twenty-nine (16.1%) of the 180 enrichment cultures showed a positive PCR: one for the VT1 gene only, 17 for the VT2 gene only, and 11 for both the VT1 and VT2 gene. A total of 46 VTEC strains were isolated from 10 randomly selected PCR-positive samples. Serotyping revealed that 41 of the 46 VTEC isolates belonged to nine different O serogroups; the remaining five were unidentifiable. A number of the serogroups recovered have been associated with human disease.

Quantification of ectomycorrhizal mycelium in soil by real-time PCR compared to conventional quantification techniques.

Mycelial biomass estimates in soils are usually obtained by measuring total hyphal length or by measuring the amount of fungal-specific biomarkers such as ergosterol and phospholipid fatty acids (PLFAs). These methods determine the biomass of the fungal community as a whole and do not allow species-specific identification. Molecular methods based on the extraction of total soil DNA and the use of genes as biomarkers enable identification of mycelia directly from the environment. Three molecular techniques were compared to determine the most reliable method for determining the biomass of individual fungal species in soil. The growth of extramatrical mycelium of two ectomycorrhizal (EM) fungal species (Suillus bovinus and Paxillus involutus) in soil was monitored by denaturing gradient gel electrophoresis, a cloning technique and real-time quantitative polymerase chain reaction, and the results were compared with those obtained with hyphal length determination and PLFA analysis. The molecular methods enabled identification and relative quantification of both species separately in an environment with several fungal species present and showed consistent results. Amounts of target DNA per gram soil were used to quantitatively compare soil samples. Increasing amounts of S. bovinus DNA and decreasing amounts of P. involutus DNA were detected over time in an environment containing a more complex community. This work demonstrates that molecular methods provide tools to determine the biomass of individual fungal species in soil.

Molecular identification of ectomycorrhizal mycelium in soil horizons.

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (> or = 99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had > or = 98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.

Effects of Pseudomonas putida modified to produce phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol on the microflora of field grown wheat.

Pseudomonas putida WCS358r, genetically modified to have improved activity against soil-borne pathogens, was released into the rhizosphere of wheat. Two genetically modified derivatives carried the phz or the phl biosynthetic gene loci and constitutively produced either the antifungal compound phenazine-1-carboxylic acid (PCA) or the antifungal and antibacterial compound 2,4-diacetylphloroglucinol (DAPG). In 1997 and 1998, effects of single introductions of PCA producing derivatives on the indigenous microflora were studied. A transient shift in the composition of the total fungal microflora, determined by amplified ribosomal DNA restiction analysis (ARDRA), was detected. Starting in 1999, effects of repeated introduction of genetically modified microorganisms (GMMs) were studied. Wheat seeds coated with the PCA producer, the DAPG producer, a mixture of the PCA and DAPG producers, or WCS358r, were sown and the densities, composition and activities of the rhizosphere microbial populations were measured. All introduced strains decreased from 10(7) CFU per gram of rhizosphere sample to below the detection limit after harvest of the wheat plants. The phz genes were stably maintained in the PCA producers, and PCA was detected in rhizosphere extracts of plants treated with this strain or with the mixture of the PCA and DAPG producers. The phl genes were also stably maintained in the DAPG producing derivative of WCS358r. Effects of the genetically modified bacteria on the rhizosphere fungi and bacteria were analyzed by using amplified ribosomal DNA restriction analysis. Introduction of the genetically modified bacterial strains caused a transient change in the composition of the rhizosphere microflora. However, introduction of the GMMs did not affect the several soil microbial activities that were investigated in this study.