| Summary: | <i>Trichoderma harzianum </i>and <i>Aneurinobacillus migulanus </i>were tested separately and in combination as potential biological control agents against <i>F. oxysporum </i>f. sp. <i>gladioli </i>on gladiolus in soil-less culture. The major secondary metabolites produced by <i>Trichoderma harzianum </i>against <i>F. oxysporum </i>f. sp. <i>gladioli </i>in the treated gladiolus corms as well as in the potato dextrose broth were studied. The impact of re-application times on the effectiveness of <i>Trichoderma harzianum </i>and <i>Aneurinobacillus migulanus </i>against gladiolus corm rot and Gladiolus production in soil-less culture was examined. Results showed that <i>T. harzianum </i>proved to use different mechanisms against <i>F. oxysporum </i>f. sp. <i>gladioli </i>depending on the inoculation method. <i>T. harzianum </i>and <i>A. migulanus </i>reduced host defence enzymes. Also, they increased GA <sub>3</sub> and IAA content in the corms. SEM showed that the suppressive mechanisms of <i>T. harzianum </i>and <i>A. migulanus </i>differed <i>T. harzianum </i>appeared to operate through a combination of antibiosis and substrate competition, 6-n-pentyl-6H-pyran-2-one (6PP) and harzianic acid (HA) were found as the main metabolites secreted by <i>T. harzianum. </i>A significant positive correlation was revealed between qPCR and CFU for both <i>T. harzianum </i>and <i>F. oxysporum </i>f. sp. <i>gladioli. </i>Multiple applications of <i>T. harzianum </i>and <i>A. migulanus </i>re- application was a successful strategy for suppression of <i>F. oxysporum </i>f. sp. <i>gladioli. </i>Briefly, <i>T. harzianum </i>was more effective than <i>A. migulanus </i>in <i>F. oxysporum </i>f. sp. <i>gladioli </i>suppression and Gladiolus growth enhancement in soil-less culture. The mixing strategy between <i>T. harzianum </i>and <i>A. migulanus </i>failed.
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