Microbial biostimulants are formulations of beneficial microorganisms that enhance plant growth, nutrient uptake and stress tolerance through biological mechanisms rather than synthetic chemistry. Among the most widely used players are fungi of the genus Trichoderma and spore-forming bacteria of the genus Bacillus. When these organisms are co-inoculated, they can occupy complementary niches in the rhizosphere, the bustling zone of soil surrounding plant roots, potentially improving nutrient solubilisation, microbial persistence and suppression of pathogenic microbes compared with single-species inoculations. But combining microbes is not trivial. Each species carries its own ecological preferences and metabolic activities, and incompatible pairings can destabilise a product during storage or undermine its performance in the field.

The Johannesburg team, led by Thuso Mudau and Fidele Tugizimana, set out to address a stubborn knowledge gap: the metabolic mechanisms that drive synergistic, neutral or antagonistic interactions among beneficial microorganisms remain largely unexplored, which makes it difficult to predict which combinations will work. Their approach combined comparative untargeted metabolomics of two fungal species, Trichoderma asperellum and Trichoderma harzianum, with laboratory compatibility assays against a defined four-strain Bacillus consortium marketed as Bacstim 100, containing B. licheniformis M017, B. licheniformis 1001, B. amyloliquefaciens and B. laterosporus.

The analytical pipeline was rigorous. Metabolites were extracted from powdered fungal biomass using liquid chromatography-mass spectrometry grade methanol, chosen for its ability to rapidly quench enzymatic activity while pulling out a broad range of polar and semi-polar compounds. The extracts were separated on an ultra-performance liquid chromatography system coupled to a high-resolution SYNAPT XS mass spectrometer, with data-independent acquisition generating fragmentation spectra for each detected molecule. Nine independent biological replicates were prepared per species, and pooled quality-control samples injected throughout the analytical sequence confirmed that instrument performance remained stable from start to finish.

The statistical analysis told a striking story. Principal component analysis separated the two fungal species cleanly, with the model explaining 84.3 percent of the variation and achieving a predictive capacity of 67.5 percent, both comfortably above accepted thresholds for biological data. A supervised orthogonal partial least squares discriminant analysis reinforced the separation, and its statistical significance was confirmed by cross-validated analysis of variance with a p-value below 0.0001. Permutation testing found no evidence of model overfitting, and receiver operating characteristic analysis within the cross-validation framework yielded a perfect area under the curve of 1.00, an internal estimate of how reliably the chemical profiles distinguish the two species. In short, despite belonging to the same genus, T. asperellum and T. harzianum carry unmistakably different chemical signatures.

Of the 3,215 molecular features detected across the samples, 42 were putatively annotated at level 2 of the Metabolomics Standards Initiative, meaning they were assigned to compound classes based on spectral matching rather than confirmed with authentic standards. Seventeen of these emerged as key discriminants between the species, falling largely into three functional classes: aromatic amino acids, indole derivatives and lipids. T. asperellum was characterised by elevated levels of tryptophan, phenylalanine, indole-3-lactic acid, indole-3-propanoic acid, sphingosine and phytosphingosine, while T. harzianum accumulated more tyrosine, guanosine, tryptamine and linoleoyl ethanolamide. The pattern suggests two distinct metabolic strategies, with T. asperellum leaning towards lipid-associated chemistry and T. harzianum towards indole and nitrogen-related compounds.

These chemical differences carry potential functional weight. Tryptophan is the biological precursor of indole compounds, including indole-3-acetaldehyde, which can be oxidised to indole-3-acetic acid, the classic plant hormone auxin, by microbial alcohol dehydrogenase enzymes. Auxin production in beneficial microbes is known to promote fungal growth, stimulate spore germination and elongate fungal hyphae, and the enrichment of tryptamine and indole-3-acetaldehyde in T. harzianum hints at enhanced downstream metabolism along this pathway. Meanwhile, the elevated glutamate in T. asperellum and higher arginine in T. harzianum point to divergent nitrogen management, since glutamate can be converted to glutamine as a nitrogen source while arginine serves as a storage and transport molecule. Lipid-derived metabolites such as linoleoyl ethanolamide and 13-oxo-ODE, both polyunsaturated fatty acid derivatives, may influence membrane integrity and stress responses.

Pathway analysis added another layer of insight. Using a hypergeometric enrichment test and relative betweenness centrality as a topology measure, the researchers mapped the annotated metabolites onto fungal metabolic pathways and found significant enrichment in sphingolipid metabolism, purine metabolism, phenylalanine metabolism, the biosynthesis of phenylalanine, tyrosine and tryptophan, ubiquinone and terpenoid-quinone biosynthesis, and tyrosine and tryptophan metabolism. Sphingolipid metabolism showed the most pronounced impact, driven by the elevated sphingosine and phytosphingosine in T. asperellum. These molecules are far from incidental: sphingolipids govern cell division, hyphal formation and spore germination in fungi, and phytosphingosine contributes to membrane stability. The authors suggest that the lipid-rich profile of T. asperellum could confer enhanced membrane stability and adaptive capacity under fluctuating environmental conditions, potentially aiding persistence in the rhizosphere.

The compatibility experiments delivered the study’s most commercially relevant verdict. In potato dextrose broth, co-cultivation of either fungal species with the Bacillus consortium increased total dry biomass substantially, by 168.4 percent for T. asperellum and 98.6 percent for T. harzianum compared with monocultures, although the researchers are careful to note that this reflects additive growth of two organisms rather than demonstrated synergy, since the individual contributions of each partner were not resolved. Dual culture plate assays showed minimal antagonism: a transient inhibition zone appeared around T. asperellum at day three but vanished by day ten, and no inhibition was observed between T. harzianum and the bacteria on either medium. Fermentation broth tests, in which filtered spent culture fluids from each microbe were tested against the others, confirmed the absence of inhibitory interactions across all pairings. Under the tested conditions, these organisms simply get along.

The implications reach well beyond the laboratory bench. The biostimulant industry faces a persistent constraint: a lack of standardisation in product composition, which makes consistency and quality control difficult. The discriminant metabolites identified here, from tryptophan and sphingosine to tryptamine and linoleoyl ethanolamide, provide candidate chemical markers that could be used to distinguish Trichoderma-based materials, monitor compositional consistency across production batches and support evidence-based strain selection. Such markers would need validation across additional strains, formulations and independent sample sets before deployment, but they represent a concrete step towards chemically defined, quality-assured microbial products.

The authors are equally candid about the limits of their work. Metabolomic profiling was performed on powdered fungal biomass rather than freshly harvested cultures, so processing and storage may have influenced the stability of some metabolites through degradation or oxidation, and the profiles reported represent the chemistry of the stored materials under the conditions investigated. The methanol extraction, while effective for polar and semi-polar compounds, may have missed highly hydrophobic metabolites that complementary biphasic extraction would capture. Pathway mapping relied on an Aspergillus niger reference library because a Trichoderma-specific KEGG library was not available at the time of analysis. And crucially, no plant experiments were conducted, so the proposed links between indole metabolites, lipid profiles and plant growth promotion remain literature-supported hypotheses rather than demonstrated effects.

Future research, the team argues, should integrate metabolomic profiling with controlled plant experiments to test whether the observed chemical differences translate into differential biostimulant effects in living crops. Metabolomic investigation of Trichoderma-Bacillus co-cultures could reveal chemical exchanges invisible when each organism is studied alone, and compatibility studies should standardise inocula by viable cell and spore concentrations rather than mass, employing species-specific quantification to distinguish additive, synergistic and antagonistic responses. If those steps are taken, the chemical blueprint assembled here could become the foundation for rationally designed microbial consortia, products built not on trial and error but on a molecular understanding of who plays well with whom in the underground economy of the rhizosphere.

Subject of Research: Comparative metabolomic profiling of Trichoderma species and their compatibility with a Bacillus consortium for microbial biostimulant development

Article Title: The chemical blueprint of Trichoderma for next-generation microbial biostimulants

Article References: Mudau, T., Tshehlane, L. P., Ncube, K. T., & Tugizimana, F. (2026). The chemical blueprint of Trichoderma for next-generation microbial biostimulants. Metabolomics, 22(5), Article 166. https://doi.org/10.1007/s11306-026-02531-4

Image Credits: AI Generated

DOI: 10.1007/s11306-026-02531-4

Keywords: Trichoderma, Bacillus, metabolomics, biostimulants, rhizosphere, LC-MS, sphingolipids, indole-3-acetic acid, microbial consortia, sustainable agriculture, plant growth promotion, molecular networking