Non-Target Effects of Trichoderma- and Bacillus- Based Products on the Citrus Microbiome

Microbial communities associated with the rhizosphere and phyllosphere are recognized as fundamental components influencing essential plant processes, including nutrient acquisition, growth promotion, and tolerance to stress. Biological control agents (BCAs), such as Trichoderma spp. and Bacillus spp., are widely applied in citrus crops. However, while BCAs effectiveness against plant pathogens is widely established, their resulting impact on indigenous, non-target bacterial and fungal communities remains poorly understood. The aim of this study was to evaluate the non-target effects of two commercial microbial formulations—one containing Trichoderma asperellum ICC 012 and T. gamsii ICC 080, and the other Bacillus amyloliquefaciens QST 713—on the resident microbiomes of Citrus volkameriana seedlings by using the amplicon-based metagenomic analysis, targeting the 16S rRNA and ITS1 regions. The application of the Trichoderma formulation as a soil drench in the rhizosphere resulted in minimal changes to the overall composition and diversity (α- and β-diversity) of the bacterial communities. This stability is considered a desirable trait for overall soil health. However, specific taxonomic changes were observed, such as a notable decrease in the genus Rhodococcus (0.4% vs. 1.5% in controls) among bacteria. In the fungal communities, the treatment led to a significant shift in phylum relative abundance, characterized by an increase in Basidiomycota (38% vs. 28% in controls) and a corresponding decrease in Ascomycota (51% vs. 56% in controls). Successful colonization was confirmed by a substantially higher relative abundance of the inoculated Trichoderma genus compared to control plants (1.4% vs. 0.1% in controls). Conversely, the foliar application of the Bacillus product induced a substantial restructuring of the phyllosphere bacterial community. This treatment caused a statistically significant reduction in bacterial α-diversity and a clear differentiation in community composition (β-diversity) relative to untreated controls. The successful colonization by the BCA resulted in the dominance of the Bacillus genus in the treated samples (27% vs. 2% in controls). Importantly, this ecological shift was accompanied by the enrichment of other beneficial bacterial taxa, including Sphingomonas (15% vs. 4% in controls) and the Burkholderia-Caballeronia-Paraburkholderia group (4% vs. 2% in controls). While fungal phyla abundances remained generally stable in the phyllosphere, specific genera such as Cladosporium (15% vs. 23% in controls) and Symmetrospora (21% vs. 13% in controls) prevailed post-treatment. In conclusion, these findings highlight the importance of considering non-target microbiome shift when implementing microbial biocontrol strategies in citrus production systems, since in this study was demonstrated that commercial BCAs exert a markedly differential influence based on the compartment of application: Trichoderma promoted ecological stability in the rhizosphere, whereas Bacillus induced a directional community shift in the phyllosphere.