Effect of the ectomycorrhizal fungus Hebeloma hiemale on adventitious root formation in derooted Pinus halepensis shoot hypocotyls

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Abstract
The effect of the ectomycorrhizal fungus Hebeloma hiemale and of its culture filtrate on in vitro rooting of Pinus halepensis derooted shoot hypocotyls was studied in an attempt to determine if ectomycorrhizal fungi could enhance adventitious root formation in gymnosperms. Pinus halepensis hypocotyls did not root in the absence of hormonal treatment, whereas the rooting percentage was 87.3% in the presence of 5 μM indole-3-acetic acid (IAA). In the presence of tryptophan, which is a precursor of IAA, H. hiemale strongly enhanced rooting of hypocotyls cultivated in the absence of any hormonal treatment. In the presence of 0.1 mM tryptophan, the rooting percentage of the inoculated hypocotyls was 96.6%, whereas it was only 7.6% in the absence of the fungus. Hebeloma hiemale culture filtrate obtained in the absence of tryptophan did not contain IAA and did not stimulate rooting of the hypocotyls. In contrast, a culture filtrate obtained in the presence of tryptophan contained IAA; an ethyl acetate extract from this filtrate allowed 100% rooting. Different fractions were isolated by preparative thin-layer chromatography from the IAA-containing filtrate and studied for their effect on rooting. It was demonstrated that IAA was responsible for the rhizogenic activity of H. hiemale. These results suggest that ectomycorrhizal fungi which rapidly metabolize exogenously supplied tryptophan to IAA could be a suitable tool to enhance in vitro rooting of micropropagated gymnosperms. Key words: ectomycorrhizal fungus, indole-3-acetic acid, rooting, shoot hypocotyl, Hebeloma hiemale, Pinus halepensis.