title: Quantification in soil and the rhizosphere of the nematophagous fungus Verticillium chlamydosporium by competitive PCR and comparison with selective plating.
abstract: 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.
authors.affiliation: IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, United Kingdom.
authors.forename: T H
authors.initials: TH
authors.lastname: Mauchline
authors.forename: B R
authors.initials: BR
authors.lastname: Kerry
authors.forename: P R
authors.initials: PR
authors.lastname: Hirsch
mesh_terms: D000818:Animals; D015169:Colony Count, Microbial; D004271:DNA, Fungal / Q000032:analysis / Q000302:isolation & purification; D018551:Solanum lycopersicum / Q000469:parasitology; D009348:Nematoda / Q000382:microbiology*; D010572:Pest Control, Biological; D010935:Plant Diseases / Q000469:parasitology; D018517:Plant Roots / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012988:Soil Microbiology; D011198:Solanum tuberosum / Q000469:parasitology; D020172:Verticillium / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification
chemical_list: D004271:DNA, Fungal