Soil samples was collected from rhizosphere of a banana plantation without symptom of banana fusarium wilt for more than ten years in Lingao County, Hainan Province, China (19°45’3 “N, 109°55’17 “E). The collected samples were transported to the laboratory in a sterile plastic bag and stored at 4°C. Trichoderma were isolated from soil samples by a gradient dilution method (Rahman et al., 2023). Briefly, 5 g of fresh soil was added to 45 mL of sterile distilled water, incubated at 28°C with shaking at 180 r/min for 30 min, and then diluted with sterile water to 10^-1~10^-3. 100 µL of diluted soil suspension was poured onto the Rose Bengal Agar (RBA) (5.0 g peptone, 10.0 g glucose, 1.0 g dipotassium hydro gen phosphate, 0.5 g magnesium sulfate, 0.033 g Bengal Red, 0.1 g chloramphenicol, 20.0 g agar in one liter water) was spread on the plate using an applicator. Subsequently, the plates were placed at 28°C and incubated for 3-5 days. Single colony on the selection medium was transferred to the potato dextrose agar medium (PDA) for purification. The purified strains were stored at 4°C for the following study.

The isolate with the highest antifungal activity against Foc TR4 was screened by a dual culture method (Tian et al., 2016). Mycelial discs (5 mm in diameter) of Trichoderma were placed on the edge of PDA plates and the same size mycelial discs of Foc TR4 were placed on the opposite side. A mycelial disc of Foc TR4 alone was used as a control. Plates were incubated at 28°C for 7 days. The radius of Foc TR 4 colonies on the dual culture plates was determined. The inhibition rate (MI) was calculated using the following equation: MI = [(R₁-R₂)/R₁] ×100, where R₁ and R₂ represented the radius of Foc TR4 colonies in the control and the treatment groups, respectively. Based on inhibition rate against Foc TR4, an isolate, labeled as strain N4-3, with strong antifungal activity was selected for the following study.

Mycelial discs of strain N4-3 from actively growing colonies were placed on PDA plates, incubated at 28°C for 48-72 h. Morphological characteristics of mycelium and spores were observed using biomicroscope (CellcutPlus, MMI, Germany) and scanning electron microscope (SEM, SIGMA Field Emission Scanning Electron Microscope). The physiological and biochemical parameters of strain N4-3 were determined, including resistance to pH and utilization of carbon and nitrogen sources (Wei et al., 2020).

DNA from fresh mycelium was extracted using the Fungal Genome Rapid Extraction Kit (Aidlab, China). The primers for RNA polymerase II subunit (rpb2) were fRPB2-5f (GAYGAYMGWGATCAYTTYGG) and fRPB2-7cr (CCCATRGCTTGTYYRCCCAT). The primers for translation elongation factor 1 α(tef1) were EF1 (ATGGGTAAGGARGACAAGAC) and EF2 (GGARGTACCAGTSATCATGTT) (Liu et al., 1999; Kothe et al., 2016; Cai and Druzhinina, 2021). The amplified PCR products were sequenced using a Sanger-based automated sequencer (Applied Biosystems). Sequences were edited by DNAMAN. Reference sequences were analyzed by nucleotide BLAST searches in the GenBank database ( Supplementary Table 1 ). Phylogenetic trees were constructed by using two locus combinations of the TEF1 and RPB2 datasets. The gene sequences were aligned with MAFFT and the generated sequences were edited by Gblocks to remove ambiguously aligned positions and divergent regions prior to phylogenetic analysis. Two locus data were pooled and analyzed using the maximum likelihood method in MEGA Version 7.0.

Genomic DNA was extracted from strain N4-3. Libraries were constructed using the Hieff NGS^® MaxUp II DNA Library Preparation Kit for Illumina^® and quantified using the Thermo Qubit 4.0 Fluorescence Quantification Instrument Q33226 (ThermoFisher). Sequencing was performed on the Illumina High-Throughput Sequencing Platform (HiSeq). Quality of raw sequencing data was assayed by FastQC. Sequence correction was performed using PrInSeS-G to correct for editing errors and insertion deletion of small fragments during splicing. Genetic components such as genes, tRNAs, rRNAs were predicted using GeneMark, etc. The repetitive sequences were identified using Repeat Masker (Koren et al., 2017). CRISPR prediction analysis was performed using CRT. Gene protein sequences were aligned with multiple databases such as CDD, KOG, COG, NR, NT, PFAM, Swissprot and TrEMBL. GO and KEGG annotation was analyzed according to the previous methods (Wei et al., 2020).

To evaluate the broad-spectrum antifungal activity of strain N4-3, ten phytopathogenic fungi were selected including C. gloeosporioides (ATCC 58222), C. gloeosporioides (ATCC16330), C. fragariae (ATCC 58718), C. lunata (ATCC 42011), F. graminearum Sehw (ATCC MYA-4620), C. musae (ATCC 96167), F. oxysporum f. sp. cucumerinum (ATCC 204378), C. acutatum (ATCC56815), C. gloeosporioides (ATCC MY A-456), and C. fallax (ATCC 38579). A 5 mm mycelial discs of Trichoderma were placed on the edge of PDA plates. The same size discs of various pathogenic fungi were placed on the opposite side. The pathogenic mycelial discs alone were used as a control. All experiments were repeated in triplicate.

The dynamics of strain N4-3 and Foc TR4 interactions was observed using a dual culture method (Damodaran et al., 2020). The discs were punched at the colonial edge of Trichoderma and Foc TR4, respectively, and placed on the PDA plates at 28°C for incubation. The growth was recorded at 12 h intervals. After successful fungal superparasitism on the pathogen, the dual culture areas were observed using the UItra-depth-of-field Three-dimensional Microscope system (VHX-600, Kerns). Mycelial region of Foc TR4 on the dual culture plate were selected and fixed in 2.5% of glutaraldehyde for 12 h. The samples were dehydrated with a gradient ethanol (30, 50, 70, 80, 90, 95, 100%) for 2-3 min each time. The samples were freeze-dried, and then observed by scanning electron microscopy (SEM).

The metabolites of strain N4-3 were extracted according to Zhang et al. (Zhang et al., 2021). Briefly, strain N4-3 was incubated in PDB at 28°C and 180 r/min for 7 days. The culture broth was mixed with an equal volume of anhydrous ethanol and shaken at 150 rpm for 3 days. After filtration through the Whatman No.1 filter, the extract was evaporated using a rotary evaporator. The dried extracts were dissolved into saturated solution using sterile water. The extracts solution was used to test antimicrobial activity by an agar diffusion method.

Antifungal activity of volatile organic compounds produced by strain N4-3 was tested according to Rajani et al. (Rajani et al., 2021). Mycelial discs (5 mm in diameter) of strain N4-3 and Foc TR4 were picked from the edge of colonies and placed on two separate plates containing PDA. The two plates were placed against each other. To prevent cross-contamination, a sterile cellophane layer was added between the two petri dishes. Only mycelial discs of Foc TR4 were used as a control. The inhibition rate was measured and calculated after 7 days.

Strain N4-3 was incubated in the PDB medium at 28°C and 180 r/min with shaking for 7 days. The culture solution was filtered through filter paper to collect the filtrate. 100 mL of the filtrate was then added to a 250 mL conical flask. Ammonium sulfate was added to prepare a mixed solution with varying saturations (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%) and left overnight at 4°C (Xu et al., 2022). At least three replicates of each experiment were performed. The mixture was then centrifuged at 10,000 r/min for 10 min at 4°C, and the supernatant was discarded. The precipitates were suspended in 2 mL of phosphate buffer (pH 7.2-7.4) to obtain crude enzyme solution for chitinase and β-1,3-glucanase.

The crude enzyme solution was freeze-dried to powder. Different concentrations (400, 200, 100, 50 and 25 mg/mL) of crude enzyme were prepared using PBS. Inhibition efficiency was tested as described by Mohiddin et al. (2021). After filtering through a membrane to remove bacteria, 200 µL of the crude enzyme solution were evenly poured to the PDA medium and inoculated with Foc TR4 mycelial blocks. The crude enzyme solution after high-temperature inactivation was used as a control. At least three replicates were set for each concentration. The inhibition efficiency was calculated by measuring the colony diameter after 5 days. A toxicity regression equation was established as a linear regression using a least squares method. The EC₅₀ value was calculated from the toxicity regression equation (Wei et al., 2020).

The germination rate of Foc TR4 spores was measured as the description of Li et al. (2021) with a minor modification. Firstly, 20 µL of spore suspension (1.0 × 10⁶ CFU/mL) was mixed with an equal volume of crude enzyme solution at a final concentration of 1× EC₅₀. The mixture was then added to a concave slide. The same concentration of heat-inactivated crude enzyme solution was used as a control. The spores were incubated at 28°C for 24 h, and 100 spores on each slide were observed using a light microscope. The spore germination rate was used to evaluate the inhibition efficiency. All experiments were performed in triplicates.

The mycelial discs of Foc TR4 were inoculated in the center of the PDA plate, and four sterilized coverslips were placed equidistantly around the plate. The plate was incubated at 28°C until the mycelia of Foc TR4 covering the whole coverslips. Subsequently, 100 µL of crude enzyme solution (1×EC₅₀) filtered through a syringe filter and covered with mycelia on the coverslips. The plates were gently shaken to ensure that the enzyme solution covered the entire slide. Two coverslips per plate were treated with the crude enzyme solution. Other two coverslips were treated with the same concentration of heat-inactivated crude enzyme solution as a control. After one day of incubation, the morphological changes of the mycelia were observed under a light microscope.

To prepare a spore suspension of Foc TR4, 100 µL of spore suspension (1.0 × 10⁶ CFU/mL) was pipetted onto a coverslip, followed by the addition of 100 µL of crude enzyme solution (2 × EC₅₀) (Chen et al., 2018). The same concentration of heat-inactivated crude enzyme solution was used as a control. The spores were incubated at 28°C for 24 h. After fixation, changes of spore morphology were observed using SEM.

Mycelial discs were inoculated at the center of PDA plates. After two days at 28°C, the peripheral mycelia of colonies were treated with a crude enzyme solution (1 × EC₅₀). The control group was treated with the same concentration of heat-inactivated crude enzyme solution. After one day of incubation, samples were prepared according to Yun et al. (2021). The ultrastructure of Foc TR4 cells was observed with using a transmission electron microscope (TEM, JEM-1400Flash, Hitachi, Ltd., Tokyo, Japan).

Banana seedlings (Musa acuminata L. AAA genotype cv. Cavendish) with the height of 8-10 cm were transplanted into plastic pots (8 cm × 8 cm) containing 1000 g of sterilized soil. The plants were then placed in a greenhouse at 28°C and a relative humidity of 70-80%. Foc TR4 expressing the GFP gene (GFP-Foc TR4) was provided by the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, Haikou, China. Three treatments were set, including Blank (sterile water), Control (positive control, inoculated with GFP-Foc TR4) and Treatment (1.0 × 10⁷ CFU/mL of strain N4-3 fermentation broth and 1.0 × 10⁷ CFU/mL of GFP-Foc TR4). 100 mL of mixture was added to the roots of banana seedlings (Jing et al., 2020). Banana seedlings were treated at 7 days intervals. Roots were selected at 60 days post inoculation (dpi) and sections were made using a manual method (Jing et al., 2020). Foc TR4 infection was observed with a laser scanning confocal microscope (ZEISS, LSM800, Germany). The maximum photosynthetic efficiency, relative chlorophyll content, leaf area, leaf thickness, stem thickness, plant height, fresh weight and index of photosynthetic system II were also measured. Inter-root soil was collected for Foc TR4 quantification as our previous description (Chen et al., 2018). The number of Foc TR4 colony-forming units (CFU/g) was determined using the inter-root soil suspension gradient dilution method. Each experiment was replicated in triplicate. Ten plants were used in each replication.

The data were analyzed using the SPSS software (Version 23.0). All experiments were performed in triplicate. Significant difference (p < 0.05) was determined using the Duncan’s multiple extreme difference test. The results obtained from three independent experiments were expressed as a mean ± standard deviation.

To screen Trichoderma spp. exhibiting antifungal activity against Foc TR4, the rhizosphere soil was selected from banana plantations without disease symptom for ten years. A double incubation experiment was performed. Nine members of Trichoderma spp. were obtained and incubated with Foc TR4 at 28°C for 7 days. The colonial radius of Foc TR4 was measured in both the treatment and control groups ( Figure 1A ). The inhibition efficiency of each strain was calculated. The results showed that strain N4-3 exhibited the highest inhibition activity at 72.37 ± 1.67%, whereas N1-4 displayed the lowest inhibition efficiency at 37.29 ± 2.98%. The inhibition rate of strain N4-3 was significantly higher than that of other strains ( Figure 1B ). Therefore, strain N4-3 was selected for the following experiment.

After 48 h of growth on the PDA medium, strain N4-3 exhibited abundant aerial mycelia. The conidiophores were densely packed and appeared white or green in color ( Figure 2A ). The conidia were ellipsoidal in shape and grew along the main axis, with terminal thick-walled conidia that were globose to subglobose in shape ( Figure 2A ). Strain N4-3 produced a yellow pigment on the PDA medium ( Supplementary Figure 1C ). The results of physiological and biochemical tests indicated that strain N4-3 exhibited tolerance to pH ranging from 5 to 9. Additionally, strain N4-3 demonstrated the ability to utilize all 16 tested carbon sources. Among the tested nitrogen sources, strain N4-3 could utilize eight sources, except for L-arginine ( Supplementary Table 2 ).

The sequencing of PCR products was assembled using DNAMAN software. After alignment using MAFFT and editing with Gblocks, the length of tef1 and rpb2 were 1127 bp and 1074 bp, respectively. To analyze the evolutionary relationship of strain N4-3, tef1 and rpb2 were used for constructing the phylogenetic tree by alignment of the selected 25 type strains. The results revealed that strain N4-3 clustered with Trichoderma parareesei CP55_3, forming a distinct evolutionary branch with 87% of ML-BS ( Figure 2B ). Combining with the morphological characteristics, strain N4-3 was identified as Trichoderma parareesei.

By sequencing and assembling, the genome size of strain N4-3 was 32,339,192 bp. The GC content was 54% and repetitive sequences accounted for 2.08%. A total of 9,271 genes were predicted for the strain N4-3 genome, with an average sequence length of 1,522 bp ( Figure 3A ; Supplementary Table 3 ). Among these genes, 6,320 (68.17%), 4,806 (51.84%), and 3261 (35.17%) were annotated using GO, KOG and KEGG, respectively ( Supplementary Table 4 ). The annotated genes were classified according to the GO terms in the three categories of Biological Process, Cellular Component and Molecular Function, ( Figure 3B ). In KOG, the pathways with high number of annotated genes were translation, ribosome structure and biogenesis (6.39%), followed by amino acid transport and metabolism (5.31%) and energy production and transformation (4.97%) ( Figure 3C ). The metabolic pathways of KEGG were classified based on the linkage between KO and pathway. The pathways were divided into five branches: cellular processes, environmental information processing, genetic information pathways, metabolism and organismal systems ( Figure 3D ).

Ten phytopathogenic fungi were selected to test the broad-spectrum antagonistic potential of strain N4-3. The antifungal activity was measured after one week of dual incubation. Strain N4-3 exhibited different inhibition ability against different pathogenic fungi ( Figure 4 ). By constrast, strain N4-3 had a strong inhibitory activity on the mycelial growth of C. lunata (ATCC 42011), C. acutatum (ATCC56815) and C. fallax (ATCC 38579), resulting in a reduction in colony growth diameters of 75.45 ± 0.91, 75.45 ± 0.91, and 75.17 ± 3.33, respectively. The least inhibitory activity was observed against C. gloeosporioides (ATCC MYA-456), with a reduction in colony growth diameter of 64.40 ± 3.58 ( Figure 4 ). These results indicated that strain N4-3 possessed a broad-spectrum antagonistic ability against phytopathogenic fungi.

We further detected the dynamic interaction between strain N4-3 and Foc TR4. Observation was recorded at 12-hour intervals during the dual incubation. The results showed that two strains started to come into contact at 48 h and show inhibition circles at 60 h ( Figure 5A ). Numerous distinct clusters of green Trichoderma spores appeared in the colony area of Foc TR4 on 10th day. The parasitic area was observed using a stereomicroscope. Mycelia and spores of strain N4-3 attached to the mycelia of Foc TR4, resulting in the lysis of the pathogenic fungal mycelia ( Figure 5B ). Additionally, a large number of Trichoderma spores were observed in the pathogenic area, and gaps were also found in the pathogenic fungal mycelia and spores ( Figure 5B ). Therefore, strain N4-3 was able to destroy the complete structure of mycelia and spores of Foc TR4.

We further evaluate whether crude enzyme solution had a disruptive effect on the morphological structure of Foc TR4. In the control group, the mycelial and spore morphology of the pathogenic fungi remained intact and had a smooth surface. SEM was used to observe the morphological changes of the Foc TR4 spores. After treatment with1×EC₅₀ of crude enzymes, the mycelia of Foc TR4 was broken ( Figure 7 ). The spores exhibited significant deformation and fragmentation, with leakage of cell contents. These observations were consistent with the results obtained from dual cultures ( Figures 5B , 7 ). SEM analysis also demonstrated intact cell walls in the normal growth of Foc TR4 mycelia. By contrast, cell wall of the treated group became thinner and defective. A number of intracellular vesicles and collapse of organelles were observed in Foc TR4 after treatment with crude enzyme solution ( Figure 7 ). These results suggested that the enzymes secreted by strain N4-3 possessed antifungal activity, ultimately leading to deformation, cell wall degradation, organelle collapse and leakage of cell contents of Foc TR4.

To assess the biocontrol efficiency of strain N4-3, a pot cultivation experiment was conducted. At 60 dpi, symptoms of leaf yellowing and wilting were observed in the control plants, while no obvious disease symptom was detected in the banana plants treated with strain N4-3. Comparing with the control group, the disease index decreased from 62.50 ± 6.78% to 17.50 ± 3.12% after strain N4-3 treatment. The biocontrol efficiency was 72.00% ( Figures 8A , 8B ). Infection of GFP-Foc TR4 on banana roots was observed using the laser scanning confocal microscopy. The results showed a strong green fluorescence was observed in the banana roots of the control group, while no obvious signal was detected in the treated group. Additionally, the roots of the control group exhibited black decayed symptom due to Foc TR4 infection ( Figure 8A ). Statistical analysis showed that the number of pathogens in the rhizosphere soil was 1.43 × 10⁵ CFU/g in the control group, while the rhizosphere soil inoculated with strain N4-3 contained 1.41 × 10⁴ CFU/g ( Figure 8C ). These results indicated that application of strain N4-3 effectively reduced the number of pathogenic fungi in the rhizosphere soil, leading to a silght symptom of Foc TR4 infection.

Furthermore, several agronomic characteristics of banana plants were measured. At 60 dpi, the SPAD in the banana leaves of the treated group (52.12 ± 5.33) was higher than that of the control (26.88 ± 7.51) and the blank group (34.79 ± 6.22) ( Figure 9A ). Although there was no significant increase in leaf thickness between the control group (1.09 ± 0.08 mm) and the blank group (1.18 ± 0.11 mm) ( Figure 9C ), the leaf area after strain N4-3 application was 1.75-fold than that of the blank group (138.69 cm²) ( Figure 9B ). Moreover, the maximum photosynthetic efficiency of the leaves was also measured using the chlorophyll fluorometer. The maximum photosynthetic efficiency was 0.81 in the treated group, 0.77 in the blank group and 0.74 in control group ( Supplementary Figure 2 ). These results indicated that strain N4-3 can improve photosynthesis in banana plants. Additionally, the fresh weight of the banana plants was obviously improved by strain N4-3. Compared to fresh weight in the blank group (89.90 ± 5.31 g) and the control group (64.76 ± 19.13 g) ( Figure 9D ), a significant increase was found in the treatment group (106.03 ± 6.40 g). Similar changes were observed in plant height and stem thickness ( Figures 9E , 9F ). Hence, strain N4-3 not only improved the resistance of banana plants to Foc TR4, but also enhanced photosynthetic capacity and plant growth.