Fungal interactions in the rhizosphere are difficult to monitor in situ: filamentous hyphae are morphologically similar and their growth is difficult to estimate in soil and the rhizosphere, especially when there are several forms (hyphae, spores derived from single or multiple cells).

Growth of P. chlamydosporia on roots

Plate counts on V. chlamydosporium-selective agar

P. chlamydosporia plate counts

 Primers for the Pochonia chlamydosporia beta-tubulin gene have been used to confirm the presence of the fungus; in quantitative PCR they can monitor population dynamics in soil and the rhizosphere (Hirsch et al., 2000; 2001; Mauchline et al., 2002).  A semi-selective medium for Pochonia chlamydosporia enables quantification of fungal propagules extracted from roots and soil and PCR fingerprinting can discriminate between colonies on agar plates arising from different isolates.  However, the use of quantitative PCR to estimate the relative numbers of different isolates offers the potential of comparing population dynamics of different fungal species or isolates of the same species.

Competitive PCR estimates

In addition to P. chlamydosporia, we have developed primers specific for the nematophagous fungi Plectosphaerella cucumerina and Paecilomyces lilacinus (Atkins et al., 2003; 2005).  We have developed specific primers to investigate competition and interactions between different P. chlamydosporia isolates with different host nematode affinities.  (Simon Atkins, Belkis Peteira, Brian Kerry and Penny Hirsch)

References:

Atkins, S.D., Hildago-Diaz, L., Kalisz, H., Mauchline, T.H., Hirsch, P.R,. & Kerry, B.R. (2003) Development of a new management strategy for the control of root-knot nematodes (Meloiogyne spp.) in organic vegetable production. Pest Management Science 59: 183-189.

Atkins, S.D., Clark, I.M., Pande, S., Hirsch, P.R,. & Kerry, B.R. (2005)  The use of real-time PCR and species-specific primers for the identification and monitoring of Paecilomyces lilacinus  FEMS Microbiology Ecology 51, 257-264.

Hirsch, P.R., Mauchline, T.M., Mendum, T.A & Kerry, B.R. (2000) Detection of the nematophagous Verticillium chlamydosporium in nematode-infested plant roots using PCR. Mycological Research 104: 435-439.

Hirsch, P.R,., Atkins, S.D., Mauchline, T.H., Morton, C.O., Davies, K.G. & Kerry, B.R. (2001)  Methods for studying the nematophagous fungus Verticillium chlamydosporium in the root environment.  Plant & Soil 232: 21-30.

Mauchline, T. H., Kerry, B. R. & Hirsch, P. R. (2002). Quantification in soil and the rhizosphere of the nematophagous fungus Verticillium chlamydosporium by competitive PCR and comparison with selective plating. Applied and Environmental Microbiology 68, 1846-1853.

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