Trichoderma-amended biofertilizer stimulates soil resident Aspergillus population for joint plant growth promotion

The study addresses how Trichoderma-amended bio-organic fertilizer (BF) enhances crop yield and whether this effect is driven by changes in soil microbiota—particularly fungi—rather than physicochemical soil properties. Building on evidence that plant growth-promoting microbes (PGPMs) can enhance crop performance, the authors note that mechanisms underlying successful biofertilizer applications remain unclear, especially the direct effects of inoculated fungi versus indirect effects via resident soil communities. The objectives were: (1) to determine whether yield enhancement by BF is due to biological versus physicochemical factors; (2) to assess the relative roles of fungal versus bacterial community changes induced by PGPF in improving plant performance; and (3) to identify key microbial taxa responsible and confirm their functions. The authors hypothesized that Trichoderma-based BF affects crop growth primarily by regulating soil fungi, and that yield increases result from combined actions of Trichoderma and recruited indigenous microorganisms rather than Trichoderma alone.

Prior work has established multiple mechanisms for PGPRs and PGPFs in promoting plant growth, including phytohormone production, nutrient solubilization, mineralization, induced systemic resistance, and pathogen suppression. Bio-organic fertilizers can reshape soil microbiomes and enhance soil functionality, but disentangling direct effects of introduced PGPFs from indirect effects mediated through resident microbiota is challenging. Previous studies showed PGPR-enriched BF can stimulate beneficial microbial groups and induce soil suppressiveness against pathogens like Fusarium. Trichoderma spp. have been linked to enhanced plant growth via mycoparasitism, antibiotic production, induced resistance, and nutrient supply. However, the extent to which Trichoderma-based BF recruits and synergizes with indigenous fungal taxa to drive yield gains has been less explored.

Field/greenhouse experiment: A three-year field experiment (two plantings per year; six seasons total) previously established four treatments (Ctrl, CF, OF, BF). This study focused on OF versus Trichoderma-amended BF (containing Trichoderma guizhouense NJAU 4742) to probe mechanisms behind yield differences. Treatments were arranged in a randomized complete block design with three replicates. Pot experiment 1 (biological vs physicochemical effects): Soils from OF and BF plots (third season) were used to grow cucumber and Arabidopsis in pots. Treatments: OFS (OF soil), BFS (BF soil), SOFS (γ-sterilized OF soil), SBFS (γ-sterilized BF soil). Cucumber was grown in greenhouse in 1000 g dry soil; Arabidopsis (Col-0) in growth chamber in 40 g dry soil; fresh weight measured after three and four weeks, respectively. Pot experiment 2 (bacterial vs fungal contributions via soil slurries): Sterilized quartz sand:vermiculite (2:1, w/w) was amended with soil slurries prepared from OF or BF soils. Treatments (4 replicates each): CK (sterile Hoagland solution only); OFSS and BFSS (unfiltered soil slurries prepared by mixing 50 g soil with 250 mL Hoagland solution, shaking 1 h, centrifuging 569 g for 5 min); fungi-free fractions (OFSS-FF, BFSS-FF) obtained by further centrifugation (20510 g, 15 min) and filtration through 3 μm membranes; microbe-free fractions (OFSS-MF, BFSS-MF) filtered through 0.45 μm membranes. Cucumber and Arabidopsis were planted; cucumber bulk and rhizosphere samples from OFSS and BFSS were collected for sequencing. Fungal isolation and identification: Seventy fungal isolates were obtained from each of OF and BF soils (PDA + chloramphenicol) from the third season. Morphological characterization and ITS sequencing (primers ITS1/ITS4) were used for identification; sequences aligned with MUSCLE/MEGA and BLASTn against GenBank to assign species-level matches. Representative Aspergillus isolates closely matching responsive OTUs were selected: BF21 (A. tamarii; 99% identity to OTU23) and BF68 (A. niger; 99% identity to OTU89). Three Trichoderma harzianum isolates were also recovered from BF soil. Strain inoculation pot test: Seven treatments in OF soil (500 g DW per pot; 10^6 CFU g^-1 per fungus where applicable): OFS (no inoculum), BF68 (A. niger), BF21 (A. tamarii), 4742 (T. guizhouense NJAU 4742), BF68+4742 (1:1), BF21+4742 (1:1), and BFS (field BF soil, no added strains). One cucumber seedling per pot; harvest at 15 days for fresh weight. Molecular and community analyses: DNA extraction and Illumina MiSeq sequencing of bacterial and fungal communities as per prior protocols. Trichoderma guizhouense NJAU 4742 quantified by strain-specific TaqMan qPCR. Community metrics included alpha diversity (Shannon, Chao1, Pielou) and beta diversity (Bray–Curtis PCoA). PERMANOVA tested effects of season and treatment. Generalized linear models evaluated contributions of diversity and structure to yield. Random forest (5000 trees) identified fungal OTUs associated with yield (variable importance via % increase in MSE; model significance via rfPermute/A3 packages). Statistical tests included t-tests and ANOVA with Tukey post hoc in SPSS.

• Yield effects: In the first season, no significant yield difference between BF and OF (P>0.05). From the second season onward, BF significantly increased cucumber yields over OF by 8.7%, 16.7%, 14.8%, 19.1%, 13.1%, and 20.7% across seasons 1–6, respectively (t-test, P<0.05 from season 2).
• Biological vs physicochemical drivers: In pot tests with field soils, BFS plants had significantly greater fresh weight than OFS for cucumber and Arabidopsis; this difference disappeared after γ-sterilization (SBFS vs SOFS), indicating a biological rather than physicochemical basis for BF effects.
• Community responses: Alpha diversity (Shannon, Chao1, Pielou) of bacteria and fungi was not significantly affected by treatments. Beta diversity: Seasons significantly affected bacteria (R2=0.435, P<0.001) and fungi (R2=0.583, P<0.001). Treatment significantly affected fungal community structure (R2=0.072, P<0.05) but not bacterial (R2=0.026, P=0.468) in field soils. In pot slurries, treatments affected fungi (R2=0.44, P<0.001) more than bacteria (R2≈0.12, P=0.22).
• Yield drivers: Generalized linear models indicated fungal community structure explained the largest proportion of yield variation (81.5%), compared to fungal diversity (15.8%), bacterial diversity (2.37%), and bacterial structure (9.98%).
• Slurry transfer: Only the fungal-containing slurry from BF (BFSS) significantly promoted cucumber and Arabidopsis growth; bacterial-only (FF) and microbe-free (MF) fractions did not differ significantly from controls, indicating fungal components drive growth promotion.
• Key taxa: Random forest linked yield to specific fungal OTUs, highlighting Trichoderma (OTU52, OTU57) and Aspergillus (OTU23, OTU89) as top predictors (model R²=0.526, P<0.001). qPCR and sequencing showed higher absolute and relative abundance of Trichoderma and higher relative abundance of Aspergillus in BF vs OF.
• Culture-based results: Among 70 isolates per treatment, Aspergillus dominated both soils; A. niger was most common (31/40 OF; 29/53 BF among Aspergillus). A. tamarii was enriched in BF (17 isolates) vs OF (4). Trichoderma harzianum was isolated only from BF (3 isolates). Fusarium was more common in OF (5 isolates) than BF (1), consistent with sequencing showing lower Fusarium relative abundance under BF.
• Inoculation assays: A. niger BF68 and T. guizhouense 4742 each significantly promoted cucumber growth vs OFS, with no additive effect when co-inoculated. A. tamarii BF21 alone did not promote growth; however, BF21+4742 co-inoculation significantly enhanced growth beyond either alone and matched the promotion observed with BFS (field BF soil), evidencing synergy between Trichoderma and A. tamarii.

The findings support that Trichoderma-amended bio-organic fertilizer enhances crop yield primarily through biological mechanisms, specifically by reshaping the soil fungal community rather than altering soil physicochemistry. Community analyses and slurry transfers demonstrated that fungal, not bacterial, components underpin the observed growth promotion. Random forest and isolation data identified Trichoderma and Aspergillus as key contributors, with BF enriching both groups while reducing Fusarium. Functional assays revealed that while Trichoderma and A. niger can directly promote plant growth, A. tamarii contributes via synergistic interaction with Trichoderma, achieving growth comparable to the whole BF-treated soil microbiome. These results indicate that biofertilizer efficacy stems from both direct actions of the inoculated biocontrol fungus and indirect recruitment/activation of beneficial indigenous fungi, expanding the known mechanisms of Trichoderma-mediated plant growth promotion. This has implications for designing biofertilizers to leverage cooperative interactions within resident fungal communities.

Trichoderma-amended bio-organic fertilizer progressively increased cucumber yield by modulating the soil fungal community. Fungal community structure was the principal driver of yield variation, and transferring fungal (but not bacterial) fractions recapitulated growth promotion. BF enriched Trichoderma and Aspergillus populations; A. tamarii did not promote growth alone but synergized with T. guizhouense NJAU 4742 to reproduce the full growth-promoting effect of BF soil. Effective plant growth promotion thus arises from both direct effects of the introduced Trichoderma strain and indirect stimulation of cooperative indigenous fungi. Future work should elucidate molecular mechanisms underlying these synergisms, assess generality across crops and soils, and integrate multi-strain consortia design into biofertilizer development for sustainable agriculture.