Biodiversity of the beneficial soil-borne fungi steered by *Trichoderma*-amended biofertilizers stimulates plant production

The study addresses how to maximize and mechanistically explain the positive effects of soil-borne probiotics on crop growth and yield in field conditions. While soils and their microbiota are vital for plant productivity, steering the soil microbiome to enhance beneficial functions remains challenging. Biodiversity is often linked to productivity and stability, yet specific microbial taxa can disproportionately drive plant growth and disease suppression. Introducing exogenous microorganisms via bio-organic fertilizers can deliver direct functional traits and indirectly reshape native microbiomes through competition, antagonism, or plant-mediated effects. Trichoderma spp. are widely used beneficial fungi that enhance plant growth and stress resistance, but field-scale ecological mechanisms remain unclear. This study tests whether Trichoderma-amended bio-organic fertilizers promote cabbage yield by altering the soil fungal community, with a particular focus on the diversity and density of beneficial fungal taxa.

The paper synthesizes literature showing: (1) soil microbes underpin plant growth and ecosystem services; (2) biodiversity can enhance ecosystem productivity and stability, though specific keystone taxa may largely determine functions like nutrient cycling, productivity, and pathogen suppression; (3) microbial interactions (competition, antagonism, cross-feeding) modulate community functionality; (4) exogenous inoculants can improve multifunctionality directly (e.g., N fixation, mineralization, biocontrol) and indirectly by reshaping native communities; (5) bio-organic fertilizers can stimulate indigenous beneficial microbes and disease suppressiveness; (6) Trichoderma spp. enhance plant growth and stress tolerance, but mechanisms in field conditions are insufficiently resolved. Prior work also links fertilization regimes and soil C/N to microbial diversity and life-history strategies, and highlights that diversity of beneficial phylotypes may better predict productivity than overall diversity.

Field experiment: An 8-season Chinese cabbage–wild cabbage rotation field trial (randomized complete block, 3 replicates) was conducted at Nanjing Institute of Vegetable Science, Hengxi, Nanjing, China (31°43′N, 118°46′E), starting September 2015. Six treatments: CK (no fertilizer), CF (chemical fertilizer: urea, superphosphate, potassium sulfate), SOF (sterilized organic fertilizer), SOF+T (sterilized organic fertilizer inoculated with Trichoderma guizhouense NJAU4742), OF (non-sterilized organic fertilizer), OF+T (non-sterilized organic fertilizer inoculated with T. guizhouense NJAU4742). Organic fertilizer comprised liquid amino acids (animal carcasses) plus pig manure compost; key properties: organic matter 457.1 g kg−1, water 28%, total N 14.8 g kg−1, P 25.2 g kg−1, K 20.1 g kg−1. Sterilization used Co-60 γ-ray irradiation. Equal nutrients across CF, SOF/OF, and bio-organic treatments were applied one week before planting each season; standard agronomy was used. T. guizhouense NJAU4742 inoculation density in OF+T and SOF+T was 5.0 × 10^7 CFU g−1. Cabbage yields were measured; sampling occurred June 2019 during harvest. Soil sampling and DNA: For each plot, five 0–15 cm cores combined into one composite bulk soil; 18 samples (6 treatments × 3 replicates) were sieved (2 mm) and stored at −80°C. DNA was extracted from 0.5 g soil (PowerSoil kit) and quantified. qPCR: Strain-specific TaqMan assay (ITS1 S/ITS1 R; probe ITS TM-037 FAM/TAMRA) quantified T. guizhouense NJAU4742 on ABI 7500; results in log10 gene copies. Amplicon sequencing: ITS1 region amplified with ITS1F/ITS2 universal primers and sequenced (Illumina Nova6000, 250 bp PE). Reads processed with standard pipeline; OTUs clustered at 97% using USEARCH; chimeras removed; taxonomy assigned with RDP classifier against UNITE database, yielding 4,629 OTUs. Culturing: Culturable fungi isolated from OF+T and OF soils on Rose Bengal Agar. Serial dilutions (10−1 to 10−4) plated; colonies purified on PDA at 28°C. ITS (ITS1/ITS4) amplified and sequenced; identities via NCBI BLAST; phylogeny with MEGA7 and iTOL. Greenhouse experiments:

• Experiment 1: Tested growth-promotion of selected isolates belonging to potentially beneficial groups: 12 Aspergillus, 9–10 Trichoderma, 4 Penicillium, 3 Fusarium. Cabbage seedlings in 150 mL pots with sterilized substrate (vermiculite:seedling substrate:quartz sand = 4:1:2), inoculated at 1 × 10^4 spores g−1; 6 replicates; 25°C, 80% RH, 16h light/8h dark; harvest at 15 days; fresh weight measured.
• Experiment 2: Co-inoculation of T. guizhouense NJAU4742 with individual beneficial isolates (Trichoderma sp. t102, Aspergillus sp. t35, Penicillium sp. 45, Fusarium sp. 53) vs single inoculants. Total density 1 × 10^4 spores g−1; two-strain mixes at 0.5 × 10^4 each; same growth conditions; 6 replicates; 15 days.
• Experiment 3: Biodiversity-function test of microbial richness: communities of 1, 2, 3, or 4 strains from Aspergillus sp. t35, Trichoderma sp. t102, Penicillium sp. 45, Fusarium sp. 53 (15 combinations). Total inoculum 1 × 10^4 spores g−1; conditions as above; 6 replicates; 15 days.
• Experiment 4: Population density test: single strains at 1 × 10^3, 1 × 10^4, or 1 × 10^5 spores g−1; same conditions; 6 replicates; 15 days. Statistics: In field data, t-test compared bio-organic (SOF+T, OF+T) vs organic (SOF, OF) yields; Tukey tests for among-treatment differences in yield and alpha diversity. Alpha diversity (richness, Shannon) via R vegan. Beta diversity via NMDS (Bray-Curtis); ADONIS tested factors. Multiple regression trees (MRT) assessed treatment drivers. Spearman correlations (FDR<0.05) between OTU relative abundance and yield identified potentially beneficial fungi. Network analysis between T. guizhouense NJAU4742 and beneficial genera used Spearman |r|>0.6, p<0.05; visualized in Gephi. Plant growth promotion reported as percent increase in fresh weight vs control. Random forest (rfPermute) assessed importance of richness and densities; linear regressions related richness/density to biomass.

• Yield: All fertilizers increased cabbage yield vs control (Tukey, P<0.05). Bio-organic treatments (SOF+T, OF+T) significantly increased yield, with OF+T highest vs OF, SOF, and CF (Tukey, P<0.05). Bio-organic group (SOF+T, OF+T) > organic group (SOF, OF) (t-test, P<0.05).
• Fungal communities: Alpha diversity similar across CK, CF, OF, SOF+T, OF+T; SOF had significantly lower richness than OF+T (Tukey, P<0.05) and tended to reduce richness and diversity vs OF, SOF+T, OF+T. NMDS/ADONIS showed significant compositional differences among treatments (R2=0.47, P<0.001). MRT identified T. guizhouense NJAU4742 application as primary driver, then fertilizer type; OF+T and SOF+T communities clustered together and differed from others.
• Potentially beneficial fungi: 88 OTUs showed positive correlation with yield (FDR<0.05). Enriched counts by treatment: CF 31, SOF 50, OF 33, SOF+T 73, OF+T 87. OF+T and SOF+T had the largest number and cumulative relative abundance of beneficial OTUs. Richness (P<0.001) and composition (P<0.01) of beneficial fungi, but not overall fungal richness/composition (P>0.05), positively correlated with yield.
• Taxonomy and network: Beneficial OTUs assigned to 39 genera, mostly Ascomycota; higher abundance and variety in OF+T and SOF+T, with OF+T highest. Network of beneficial genera with T. guizhouense had 40 nodes and 188 edges, all positive, indicating coexistence.
• Culture-based validation: Many Aspergillus, Penicillium, and Trichoderma isolates promoted cabbage growth in greenhouse. Four isolates with strong effects selected: Trichoderma sp. t102, Aspergillus sp. t35, Penicillium sp. 45, Fusarium sp. 53.
• Co-inoculation: T. guizhouense NJAU4742 combined with each selected isolate increased biomass beyond single strains; the NJAU4742 + Penicillium sp. 45 pair performed best.
• Richness and density effects: Increasing community richness (1→4 strains) and inoculum density (10^3→10^5 CFU g−1 soil) generally increased plant biomass. Some 2–3 strain communities matched 4-strain performance; a 3-strain mix (Aspergillus sp. t35 + Trichoderma sp. t102 + Fusarium sp. 53) increased fresh weight by 36.49% ± 13.23% (mean ± SD). Random forest identified community richness and population density as key drivers of growth promotion.

The study demonstrates that Trichoderma-amended bio-organic fertilizers enhance cabbage yield primarily by reshaping soil fungal communities to favor a richer and denser set of beneficial phylotypes. This addresses the central question of maximizing probiotic effects and clarifies that indirect stimulation of native beneficial fungi complements the direct effects of the inoculant. Field amplicon profiling showed that treatments with T. guizhouense NJAU4742 converge to similar fungal communities distinct from other fertilizer regimes, with higher numbers and abundances of yield-associated OTUs. Culture and greenhouse tests corroborated these patterns, showing that selected beneficial isolates promote plant growth and that co-inoculation with NJAU4742 further amplifies effects, highlighting positive interactions among beneficial taxa. Importantly, yield correlated with the biodiversity of beneficial phylotypes rather than overall fungal diversity, emphasizing that functional subsets of the microbiome drive productivity. Potential mechanisms include niche creation, altered plant exudation, resource competition, and changes in soil C/N associated with fertilization regimes. Collectively, the findings underscore that managing the biodiversity and density of beneficial fungi through bio-organic fertilization is a promising strategy to improve crop performance.

Trichoderma-amended bio-organic fertilizers significantly promote cabbage growth and yield in a long-term field rotation by shifting soil fungal community composition toward higher biodiversity and density of beneficial taxa. The study integrates field sequencing, culturing, and greenhouse experiments to show that beneficial phylotype diversity, not overall diversity, predicts yield, and that co-inoculation strategies can synergistically enhance plant growth. These results provide a basis for microbiome-informed fertilization practices aimed at stimulating resident beneficial fungi. Future research should examine how different crop types modulate these microbiome responses and delineate the mechanistic plant–microbe and microbe–microbe interactions underlying the observed effects.

The work was conducted within a specific cabbage–wild cabbage rotation at a single site and timepoint sampling, which may limit generalizability across crops, soils, and climates. Mechanistic pathways were inferred from community shifts and correlations rather than direct functional measurements. The authors note the need to investigate how different crop types mediate the stimulation of native beneficial fungi by Trichoderma-amended bio-organic fertilizers and to further resolve the mechanistic dialogue among microorganisms and with plants.