Analysing scots pine defence-related transcripts and fungal DNA levels in seedlings single- or dual-inoculated with endophytic and pathogenic Rhizoctonia species

P>Fungal DNA and induction of host defence-related transcripts were monitored by real-time PCR in young Scots pine seedlings inoculated with pathogenic uninucleate (UNR) and endophytic binucleate (BNR) Rhizoctonia species. The UNR (teleomorph Ceratobasidium bicorne) causes root dieback in conifer seedlings following invasion of the vascular cylinder via root apex and destroying apical meristems whilst the BNR, representing anastomosis group AG-I of genus Ceratobasidium, is primarily restricted to the cortex in basal root regions. In the experiment 1 the fungi were simultaneously inoculated on roots, while in experiment 2, BNR was pre-inoculated 168 h before inoculation with UNR. Nucleic acids were extracted from infected roots at intervals up to 192 h post-infection (hpi), and the genomic DNA levels of the host and fungi and the transcript levels of a house-keeping gene (glyceraldehyde-3-phosphate dehydrogenase) and nine putative defence genes were quantified. In simultaneous inoculation UNR was more competitive than BNR whereas pre-inoculation of BNR suppressed but did not completely prevent root colonization by UNR. Stilbene synthase (STS) transcription was significantly up-regulated in single-inoculations with both fungi and in dual inoculation in both experiments. Maximum STS transcript levels were observed in roots single-inoculated with UNR; the peak level at 48 hpi in experiment 2 was significantly higher than in seedlings single-inoculated with BNR or co-inoculated with both fungi, the latter two treatments showing relatively similar STS transcript levels. Similarly, transcript levels of phenylalanine ammonia lyase at 48 hpi in experiment 2 were significantly higher in roots single-inoculated with UNR compared with BNR or in UNR+BNR co-inoculations. The other seven putative defence genes monitored did not show any clear-cut up-regulation following fungal inoculation. We conclude that BNR suppresses UNR in Scots pine roots via direct competition for infection sites, since the studied transcripts showed no evidence of BNR induced resistance against UNR.