This study aimed to explore potential biocontrol mechanisms involved in the interference of antagonistic bacteria with fungal pathogenicity
in planta
. To do this, we conducted a comparative transcriptomic analysis of the “take-all” pathogenic fungus
Gaeumannomyces graminis
var.
tritici
(
Ggt
) by examining
Ggt
-infected wheat roots in the presence or absence of the biocontrol agent
Bacillus velezensis
CC09 (
Bv
) compared with
Ggt
grown on potato dextrose agar (PDA) plates. A total of 4,134 differentially expressed genes (DEGs) were identified in
Ggt
-infected wheat roots, while 2,011 DEGs were detected in
Bv
+
Ggt
-infected roots, relative to the
Ggt
grown on PDA plates. Moreover, 31 DEGs were identified between wheat roots, respectively infected with
Ggt
and
Bv
+
Ggt
, consisting of 29 downregulated genes coding for potential
Ggt
pathogenicity factors – e.g., para-nitrobenzyl esterase, cutinase 1 and catalase-3, and two upregulated genes coding for tyrosinase and a hypothetical protein in the
Bv
+
Ggt
-infected roots when compared with the
Ggt
-infected roots. In particular, the expression of one gene, encoding the ABA3 involved in the production of
Ggt
’s hormone abscisic acid, was 4.11-fold lower in
Ggt
-infected roots with
Bv
than without
Bv
. This is the first experimental study to analyze the activity of
Ggt
transcriptomes in wheat roots exposed or not to a biocontrol bacterium. Our results therefore suggest the presence of
Bv
directly and/or indirectly impairs the pathogenicity of
Ggt
in wheat roots through complex regulatory mechanisms, such as hyphopodia formation, cell wall hydrolase, and expression of a papain inhibitor, among others, all which merit further investigation.