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The fungus (Verticillium hemileiae Bouriquet) associated with pustules of coffee rust (Hemileia vastatrix) was isolated of samples collected in the municipality of Cacahoatán, Chiapas, México. In the isolation grew only (V. hemileiae), since in the case of (H. vastatrix) as obligate parasite does not develop in culture medium. Work was carried out in laboratory and nursery, using a completely randomized experimental design, and in field with a randomized block distribution. The best culture medium to propagate (V. hemileiae) was Sabouruad Dextrose Agar plus malt extract, spore germination of 90% was observed at a concentration of 2.57105 conidia per milliliter. The medium pH was 5.5 with which presented 91.8% germination, pathogenicity 92.2% and a concentration of 2.56105 conidia per milliliter. The best substrate to propagate massively (V. hemileiae) was wheat bran, which recorded concentration of 2.31105 conidia per milliliter, viability of 90.96%, growth of 66.2%, pathogenicity 91.1%; and applying plants in nursery and field conditions the largest number of pustules and infection occurred in the control without application.

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