Real-time PCR quantification and live-cell imaging of endophytic colonization of barley (Hordeum vulgare) roots by Fusarium equiseti and Pochonia chlamydosporia
Article first published online: 19 JAN 2009
© The Authors (2009). Journal compilation © New Phytologist (2009)
Volume 182, Issue 1, pages 213–228, April 2009
How to Cite
Maciá-Vicente, J. G., Jansson, H.-B., Talbot, N. J. and Lopez-Llorca, L. V. (2009), Real-time PCR quantification and live-cell imaging of endophytic colonization of barley (Hordeum vulgare) roots by Fusarium equiseti and Pochonia chlamydosporia. New Phytologist, 182: 213–228. doi: 10.1111/j.1469-8137.2008.02743.x
- Issue published online: 6 MAR 2009
- Article first published online: 19 JAN 2009
- Received: 17 September 2008, Accepted: 22 November 2008
- Agrobacterium tumefaciens-mediated transformation;
- Fusarium equiseti;
- green fluorescent protein;
- Hordeum vulgare (barley);
- live cell imaging;
- Pochonia chlamydosporia;
- real-time PCR;
- root endophytes
- •New tools were developed for the study of the endophytic development of the fungal species Fusarium equiseti and Pochonia chlamydosporia in barley (Hordeum vulgare) roots. These were applied to monitor the host colonization patterns of these potential candidates for biocontrol of root pathogens.
- • Molecular beacons specific for either F. equiseti or P. chlamydosporia were designed and used in real-time polymerase chain reaction (PCR) quantification of fungal populations in roots. Genetic transformation of isolates with the green fluorescent protein (GFP) gene was carried out using an Agrobacterium tumefaciens-mediated transformation protocol, and spatial patterns of root colonization were investigated by laser confocal microscopy.
- • Quantification of endophytes by real-time PCR in roots of barley gave similar results for all fungi, and was more accurate than culturing methods. Conversely, monitoring of root colonization by GFP-expressing transformants showed differences in the endophytic behaviours of the two species, and provided evidence of a plant response against endophyte colonization.
- • Both F. equiseti and P. chlamydosporia colonized barley roots endophytically, escaping attempts by the host to prevent fungal growth within root tissues. This strongly supports a balanced antagonism between the virulence of the colonizing endophyte and the plant defence response. Development of real-time PCR techniques and GFP transformants of these fungal species will facilitate future work to determine their biocontrol capacity.