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The effect of barley seed treated with chlorothalonil fungicide on mycorrhizal root colonization was evaluated. The treatments were: 1) Seed with Mycorrhizal INIFAP?, one, two and three doses, and uninoculated seed; 2) Seed with and without fungicide. Monthly seed plantings were performed with different storage time, looking forward to assessing the colonization degree of the roots. As revealed by the results, the seed treated with chlorothalonil did not lower the root colonization by Mycorrhizal INIFAP?; the chlorothalonil treatment had a 32.63% average root colonization, whereas without chlorothalonil, it was 36.46%. When the seed was treated with root colonization by Mycorrhizal INIFAP?, this was lower in the treatment with one dose; no significant difference was revealed by the treatments with either double or triple doses. The colonization percentage was progressively decreased by seed storage. The root colonization by Mycorrhizal INIFAP?, throughout the inoculated seed storage time, remained constant and unchanged for the first six months, but then decreased in 50% within a 10-month period for both treated and not treated fungicide seed.