In vitro and in silico approach of fungal growth inhibition by Trichoderma asperellum HbGT6-07 derived volatile organic compounds

2.1 Fungal isolates and culture conditions

Trichoderma isolates were isolated from agriculture soil in different locations of China (Supplementary Table S1). Soil specimens were put in clean containers, delivered to the laboratory and held at 4 °C until they were used. The sample were prepared 10^-4 serial dilutions in sterilized distilled water (SDW) and 500 µL sample (diluted) was spread on the potato dextrose agar (PDA) media plates and incubated at 20 ± 2 °C for 72 h. The cultivation plates were checked frequently, and each visualized colony was known to be one colony-forming unit (CFU). Various fungal colonies were sub-cultured to PDA plates following the counting of CFU. For this analysis, Botrytis cinerea strains B05.10 and one isolate (A367) of Sclerotinia sclerotiorum were taken. Originally isolated B05.10 and A367 was obtained from grapes (Büttner et al., 1994), and eggplant (Magioli and Mansur, 2005). The experimental cultures of the isolates were developed by shifting the mycelia to PDA plates, and incubated at 20 °C between 5 and 10 days under the 12-h dark-light regime.

2.2 Assay of inhibition of B. cinerea and S. sclerotiorum growth by Trichoderma spp. Through dual culture method

For dual cultures, isolates of Trichoderma were screened for inhibition against B. cinerea B05.10 and S. sclerotiorum A367. The microbes and Trichoderma were cultivated for 5–6 days at a temperature of 20 ± 2 °C on PDA plates. Mycelium agar plug (MAPs, 5 mm in diameter) of the target necrotrophic fungus (B. cinerea and S. sclerotium) collected from the periphery and inserted onto new PDA dishes. After 2 days incubation of Trichoderma spp. the fungal discs were moved aseptically in the center of the target fungi plate and were kept at 25 °C with intermittent light-dark conditions for 10 days and monitored frequently. Triplicates were used in each study, and after 10 days of constant growth of B. cinerea and S. sclerotium colonies, the degree of the invasion was estimated and the control (pure cultures of B. cinerea and S. sclerotium) was compared. The fungal growth inhibition zone formula was estimated by I = (C-T)/C × 100, where C is the growth of mycelium in the control plate, T is the test species growth of mycelium in the inserted plate, and I is the mycelial growth inhibition (Wonglom et al., 2019). Each experiment was done three times, with three replications each time. Finally, the plates where Trichoderma colony completely covered by B. cinerea or S. sclerotium colony surface was selected as a super isolate.

2.3 Biological characterization of isolates T. asperellum HbGT6-07

According to the protocol of Samuels et al. 2002, the phenotypic and cultural properties of T. asperellum HbGT6-07 isolates were analyzed in numerous media viz. czapex dox agar (CDA), carrot potato agar (CPA), minimal media (MM), modified melin norkrans (MMN), malt-200Byeast agar (MYA), potato dextrose agar (PDA), sabouraud dextrose agar (SDA), yeast malt extract agar (YMEA), yeast peptone glucose (YPG), and yeast soluble starch (YSS) (Supplementary Table S2). Mycelial discs of developing isolates T. asperellum HbGT6-07 were inoculated at the edge of the petri plates that included earlier in this section-mentioned media and incubated at 25 ± 2 °C for a week. Colony radius was calculated at 24, 48, and 72 h intervals. The test was replicated three times, and the tests for each isolate were averaged. External characteristics including the presence of pigments, green conidia, odor and colony appearance are also noted. Morphological findings from mycelial growth on PDA plates have been reported (Anees et al., 2010). Thus, every feature was calculated in 3% KOH for each isolate from the water after preliminary soaking. In addition, six Petri-plates containing approximately 10 mL PDA media were used to investigate the optimal growth of T. asperellum HbGT6-07 isolates at different temperatures. 5 mm mycelial discs of pure culture isolates were positioned in the center of the Petri-dishes. The six Petri-plates were then incubated at six different temperatures (10–35 °C) for seven days (Sultana et al., 2018).

2.4 Genomic DNA extraction, PCR amplification and phylogenetic tree analysis

Isolates T. asperellum HbGT6-07 with the strongest inhibition against B. cinerea and S. sclerotiorum growth was further identified through the analysis of its 5.8S rRNA sequence. Genomic DNA were extracted by cetyltrimethylammonium bromide (CTAB) method (Kamaruzzaman et al., 2018) and DNA was dissolved in 50 μL TE buffer fluid to create DNA suspension and quantified using fluorescence of ethidium bromide (Raeder and Broda, 1985). Then polymerase chain reaction (PCR) amplification was done by using the universal primers internal transcribes spacers (ITS1) (3′-TCCGTAGGTGAACCTGCGG-5′) and ITS4 (3′-TCCTCCGCTTATTGATATGC-5′). A total volume of 25 μL of reaction mixture was used for PCR amplification. Every reaction comprises 0.2X PCR buffer, 0.16 mM MgCl₂, 0.01 μM ITS1 (forward primer), 0.01 μM (reverse primer), 0.144 mM dNTP, 0.5 mU/μL Taq polymerase, 2.00 μL template DNA and 14.95 μL PCR water, respectively. The initial denaturation of these reactions was 90 s at 95 °C, followed by 30 cycles of 1 min at 95 °C, 30 s at 55 °C and 1.5 min at 72 °C, with a final extension of 10 min at 72 °C and a final hold of 4 °C. The PCR band were visualized using a 1% agarose gel. PCR products were cloned in E. coli DH5α with vector pMD18-T (You et al., 2016) and sequenced by Wuhan Tianyi Huiyuan Biological Technology Co., Ltd, Hubei, China. The obtained sequence was submitted to the GeneBank to get the accession number. Multiple sequence alignments and comparisons with reference strain for each of the genes were performed through the aid of CLUSTALW and Neighbour-joining method was used to constructed phylogenetic tree topologies by performing bootstrap values of 1000 data sets using MEGA7.0 (Molecular Evolutionary Genetic Analysis) tools (Islam et al., 2020a,b). The corresponding sequence accession numbers was listed in Supplementary Table S3 for constructing phylogenetic tree analyses. The sequence was deposited to the GeneBank under the mentioned accession number: MH280010.

2.5 Preparation of the culture filtrate (CF) of T. asperellum HbGT6-07 isolates

Two blocks of a 7-day-old T. asperellum HbGT6-07 mycelia agar plug (5 mm diameter) was inoculated in a 250 mL conical flasks containing 150 mL of sterilized potato dextrose broth (PDB) and cultured in an electrical shaker for 7 days at 150 rpm and 22 °C. Then, the fermented outcome was centrifuged at room temperature at 10,000 rpm for 10 min to remove the mycelium debris. Obtained supernatant was collected after the pass through a 0.22 µm membrane filter (Millipore Sigma, USA).

2.6 The T. asperellum HbGT6-07 CF influenced the growth and morphology of B. cinerea and S. sclerotiorum

B. cinerea and S. sclerotiorum were separately inoculated on PDA and cultured at 20 °C. Purified CF of T. asperellum HbGT6-07 were used in PDA plate to evaluate the effects on the mycelial radial growth of B. cinerea S. sclerotiorum. In the experiment, the select concentrations of the CF (10%, 6%, 2%, and 1% (V/V)) were mixed with PDA, while sterile dd water was mixed with PDA used as a control (0%). treatment. One three-days-old mycelial agar plug (5 mm in diameter) of B. cinerea was put in the middle of each petri dish and incubated at 20 C. Similarly, S. sclerotiorum mycelial agar plug as used. Each treatment was done with three replications. Five days after incubation, the colony diameter of each dish was calculated in two reverse directions, and the growth inhibition (GI) percentage (% GI) of HbGT6-07 was calculated using the formula % GI = [(mean of colony diameter in control – mean of colony diameter in treatment)/mean of colony diameter in control] × 100 (Hao et al., 2020). This experiment was repeated three times.

2.7 The application of mycelial hyphal fragments (HFs) to inhibit the necrotic diseases lesion

Rapeseed oil plants seeds were planted in plastic pots with organic culture mix including 2%–5% N + P₂O₅ + K₂O (N:P₂O₅:K₂O = 1:1:1, w:w:w:w). The pots were placed in a chamber for plant growth where water was needed. The plants were softened to one seedling per pot at the multiple-true-leaf level (45-d old). Isolates T. asperellum HbGT6-07, B05.10 and A367 were individually cultured at 20 °C below 12 h light-dark intervals on PDA for 5 d, and the subsequent mycelial volumes of every isolates were obtained and mixed in PDB to create HFs mixtures at a density of roughly 3 × 10⁶ HF per mL. T. asperellum HbGT6-07 HF mixture was combined with B05.10 HF suspension comprising hyphal segments (HFB05.10) at density proportions 6:3, 5:5 and 3:6 or coupled with A367 HF mixture also containing hyphal segments (HFA367) at the similar size ratio (Fig. 1) (Sánchez et al., 2019).

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Fig. 1

Hyphal fragments preparation outlines at different treatment ratios (33.33:66.67, 50:50 and 66.67:33.33).

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Every one of such HF mixtures were considered as inoculum in double isolates. They were lifted on the upper portion of the 5 mm diameter size of filter paper discs (FPD) positioned on rapeseed leaves to assist strengthen the inoculum, 25 μL culture on each FPD, single FPD around each leaf, and five plants per inoculum (a total of 12 to 15 leaves per inoculum). HFS^HbGT6-07 acts as negative control, HF^B05.10 or HF^A367 alone used as a positive control. Most of the plants were kept in a humid cabinet at 20 °C for three days in 12-h light-dark conditions. The leaf lesion diameter was estimated on the growth of leaves around each FPD. The following formula was used to calculate the biocontrol efficacy by the treatment of T. asperellum HbGT6-07 (BE^{HbGT6-07 HFs}) hyphal fragments: $$B{E}^{HbGT6-07HFs}=\left\{\left(A{D}^{\mathit{Positive}control}-A{D}^{\mathit{Treatment}HFs}\right)/A{D}^{\mathit{Positive}control}\right\}\times 100\%$$ [Where AD^{Positive control} is the average length of the leaf lesions in the treatment with HF^B05.10 alone or HF^A367 by itself as an inoculum, while AD^{Treatment HFs} is the average length of the leaf lesions in the HF^HbGT6-07 treatment with HF^{B05.10 or A367} treatment at the stated HF ratio]. Repeated the test three more times.

2.8 Effect of mixed culture on sclerotia production

Sterilized carrots were used as a carrier for sclerotia reduction experiment. Briefly, about 75 g carrots were cut into pieces (1.5–2.5 cm in size) and sterilized at 121 °C for 30 min in a 250 mL conical flask. Five actively growing MAPs of B05.10 or A367 were placed inside the conical flask used as control. The mycelial mixtures (5 MAPs of HbGT6-07 + 5 MAPs B05.10) or (5 MAPs of HbGT6-07 + 5 MAPs of A367) were used in two separate treatments. Six conical flasks of each treatment were inoculated at 20 °C for 15 d (Kamaruzzaman et al., 2020). The average number of sclerotia and weight per flask were calculated. This experiment was repeated three times.

2.9 Antifungal volatiles production by T. asperellum HbGT6-07

In order to check the antifungal volatiles activity of the T. asperellum HbGT6-07 isolates, Dish-within-Dish (DwD) sets method was performed where includes one pair of the dish, a small inside dish (6 cm in diameter) and a big outside dish (16 cm in diameter) (Kamaruzzaman et al., 2020). For this study, B. cinerea isolate B05.10 was selected as a fungal target to evaluate the antimicrobial efficacy of the volatiles generated by T. asperellum HbGT6-07. There were three DwD sets for the three treatments. Autoclaved wheat grain (AWG) in a 250 mL Erlenmeyer flask containing 100 g AWG was inoculated with four MAPs of T. asperellum HbGT6-07 or B05.10 or A367 (20 °C) worked as a source of VOCs. In the case of B05.10, blank AWG (50 g) alone in the internal dish and 5 mm mycelial agar plug (MAP) of B05.10 on the external dish was defined as the first DwD set and considered as a negative control. The second DwD set, T. asperellum HbGT6-07 and AWG (50 g) together in the inner dish and 5 mm MAP of B05.10 on in the outer dish was known as target treatment. The third DwD set was B05.10 and AWG (50 g) together in the inner dish and 5 mm MAP of B05.10 on in the outer dish was known as B05.10/B05.10 act a second negative control, that were chosen to remove the effect of T. asperellum HbGT6-07 O₂ consumption and/or CO₂ development on the radial formation of B05.10 in the outer plates in the inside plates. The three DwD groups were first developed by examining the inner dishes, where the B05.10 was left empty, inoculated with T. asperellum HbGT6-07 (one MAP each plate), or seeded with B05.10 (one MAP for each plate), and put in an incubator at 20 °C at 12 h light-dark intervals for 20 d. Next, B05.10, one MAP for every dish at a length of 5.5 cm from the internal dish was seeded on the external dishes (Fig. 2). Same techniques have been employed to establish the DwD sets against S. sclerotiorum A367 to recognize antimicrobial behavior of T. asperellum HbGT6-07 volatile components (Fig. 2). The experimental set up were incubated at 20 °C under 12 h light-dark conditions for 5 d and used as the volatile origin in the subsequent DwD sets: fresh AWG/A367 (a negative control), HbGT6-07/A367 AWG culture, and A367 AWG culture (another negative control). Simultaneously with the loading of the fresh AWG, the AWG culture of HbGT6-07 and the AWG culture of A367 in the internal dishes, A367 MAPs from a 3-day PDA culture (20 °C) were incubated in the external dishes. All of the DwD sets were covered separately with parafilms (Parafilm M, Chicago, USA) and held at 20 °C for the next 20 d. Diameters of the B05.10 or A367 colony were calculated in each inner dish. Meanwhile, the regular growth rate, colony length, and sclerotia generated in the B05.10 or A367 colony were recorded and measured in each outer platter. The procedure was replicated once in each repetition, with three replicates per test.

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Fig. 2

Diagram of detection of antifungal activity through the production of volatile organic compounds using dish within dish method (DwD).

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2.10 Gas chromatography/Mass spectrometry (GC-MS) analyses

Using the GC-MS experiment, the chemical elements of the T. asperellum HbGT6-07 VOCs have been quantified. T. asperellum HbGT6-07 was cultivated in a 250 mL Erlenmeyer sterile flask including 100 mL PDA with 6 pcs MAPs. The mycelial cultures were placed in a 25 °C incubator for 7 days. An initial experiment revealed that the emission of VOCs has achieved the plateau under favorable environments in 7 days. The T. asperellum HbGT6-07 VOCs were obtained in the flask for 20 min at 40 °C via solid-phase micro-extraction (SPME) fiber assemblies (Superco, PA, USA) (Kamaruzzaman et al., 2021; Oszako et al., 2021). The fiber (2 cm, 50/30 µm divinylbenzene-DVD) was placed straightly into the TRACE™ GC Ultra (TRACE-DSQ II) (Thermo Electron Corporation, USA) GC intel splitless mode. The desorption time was 5 min and the deported compounds were segregated on a DB-5 MS capillary column (30 m × 0.25 μm × 0.25 mm) through the following operating program. Initially, the oven temperature was kept at 50 °C for 3 min. The column temperatures were slowly raised at 10 °C/min from 50 °C to 180 °C and increased to 240 °C at 4 °C/min, then retained for 5 min. Total running time was 30 min. Helium gas (99.99%) as used as a carrier with a flow rate of 1.0 mL/min. The ionising power was set at 70 eV with an acquiring range of 50 to 800 m/z and 1 scan/s scan rate. The temperature of the ion source was 230 °C, and the transfer axis was adjusted at 280 °C. Data acquisition and processing were performed with Thermo Scientific Mass Frontier software system. Based on the comparison of their comparable retention time and their mass spectra, the chemical components were classified with those in the NIST07 database (National Institute of Standard and Technology). Individual peak compositions as a relative percentage of total peak area were recorded. In the meantime, it also obtained and classified the VOCs released from non-inoculated sterilized AWG. Finally, the VOCs appearing in the SPME extract from the non-inoculated AWG were removed during computation. This experiment was repeated three times.

2.11 Ligand preparation

The ligand molecules from T. asperellum HbGT6-07 were retrieved from the PubChem database (Kim et al., 2016) as 3D sdf format. The compounds were further energy minimized in Avogadro software using the mmff94 force fields with the steepest gradient approaches (Hanwell et al., 2012).

2.12 Protein preparation

The protein structure of Aspergillus oryzae, Saccharomyces cerevisiae and Candida albicans (PDB ID: 4MAI, 4WMZ and 5TZ1) were extracted from the Protein databank (Berman et al., 2000). The crystal structure contains heteroatoms, water, and ligand molecules. These molecules were cleared in the Pymol software package (DeLano, 2002). Later, the cleaned structures were minimized in YASARA software (Land and Humble, 2018) by AMBER14 force field (Case et al. 2014).

2.13 Molecular docking

The molecular docking is an effective tool for computer aided drug designing to screen the hit molecules from a large compound’s datasets. The AutoDock Vina (Trott and Olson, 2010) tools was used for conducting the molecular docking simulations. The ligands and protein molecules were converted to PDBQT format (Cosconati et al., 2010; Morris et al., 2008). The center of the box size and grid box for 4MAI, 4WMZ, 5TZ1 were (X:33.08, Y:54.01, Z:12.26), (X:39.10, Y:43.97, Z:41.00), and (X:21.95, Y:13.28, Z:20.10), (X:87.70, Y:79.08, Z:64.37), and, (X:62.57, Y:66.77, Z:2.65), (X:66.60, Y:55.04, Z:66.97) Å respectively. Finally, the docking calculations was conducted in Pyrx (Dallakyan and Olson, 2015) and best compounds were screened based on binding affinity. The docked poses and interactions were analyzed with the aid of Discovery Studio (Biovia, 2017), and Pymol software (DeLano, 2002).

2.14 Molecular dynamics

The molecular dynamics simulation was conducted in YASARA dynamics tools (Land and Humble, 2018) where AMBER14 force field (Case et al., 2014) was used. The complex was initially optimized and hydrogen bond network was oriented. The cubic simulation cell was created where TIP3P or Transferable intermolecular potential 3 points was used for the solvation with a periodic boundary condition and solvent density was set as 0.997 gL^-1. The pKa or acid dissociation constant value was calculated for the amino acid present in the protein. The SCWRL algorithms was applied to maintain the correct protonation state of the amino acid. The physiological condition of the simulation system was maintained at pH 7.4 with the addition of 0.9% NaCl at 298 K temperature. The energy of each system was minimized with the steepest gradient approaches by simulated annealing method (Krieger and Vriend, 2015). The time step of the simulation system was fixed as 1.25 fs. The Particle Mesh Ewalds method was applied to calculate the long-range electrostatic interactions with a cut off was set as 8.0 Å (Krieger et al., 2006). Following the Berendsen thermostat and constant pressure, the simulation was run for 50 ns and simulation trajectories were saved after every 100 ps. The simulation trajectories were used to analysis the root mean square derivation (RMSD), solvent accessible surface area (SASA), radius of gyration (Rg) and hydrogen bond (Hb) (Bappy et al., 2020; Islam et al., 2020a,b; Khan et al., 2020; Mahmud et al., 2021, 2020a, 2020b, 2019; Munia et al., 2021; Pramanik et al., 2021).

2.15 Statistical analysis

The procedure of Analysis of Variance (ANOVA) in SAS software (SAS ver. 8.0, NC, USA) was used to examine data from bioassays and the effect of culture filtrate. The least significant difference (LSD) test was used to calculate the inhibition assay, dry weight of mycelium, disease lesion diameter, treatments of the VOCs of, and control at α = 0.05 (Kamaruzzaman et al., 2021). Data linked to independent control with the Student's t-test (P < 0.01 or 0.05).

3.1 Isolation and screening of isolates T. asperellum HbGT6-07

A total of 16 Trichoderma isolates were isolated from various agricultural field in a different province of China. The initial screening for effective Trichoderma isolates revealed the percentage of inhibition range at 3 days-after-inoculation (DAI) 69.52–81.43% and at 6 DAI for B. cinerea B05.10 (Supplementary Fig. S1 (A) Table S4), Table S3). On the other hand, the percentage of inhibition range at 3 DAI 69.52–81.43% and at 6 DAI for S. sclerotiorum A367 (Supplementary Fig. S1 (B), Table S4). Among them, T. asperellum HbGT6-07 potentially reduced the growth of B. cinerea B05.10 and S. sclerotiorum A367, respectively. Besides the inhibition of fungal growth, T. asperellum HbGT6-07 suppressed the conidial production of B. cinerea B05.10 and S. sclerotiorum A367 in the dual culture assay plates.

3.2 Colony morphology and molecular identification of isolates T. asperellum HbGT6-07

The morphological investigations such as mycelium growth and sporulation ability was tested on ten different culture medium. However, after 7 days of incubation, we observed a higher significant phenotype in PDA, SDA, and CPA medium, such as rough, bright green spores (spores color), white mycelium (mycelium color) whereas YPG did not produce a similar margin of mycelium. Based on the findings, we speculated that the aforementioned medium is far more useful than other medium (Fig. S2). In addition, to find out the optimum temperature for the growth of the pathogen, the isolate T. asperellum HbGT6-07 was grown at different temperatures on potato dextrose agar medium. After 10 DAI, the average mycelial growth, colony diameter, and the number of spores per dish were recorded. From the analysis presented in Supplementary Fig. S3, it can be concluded that the growth of the fungi was better at the temperature range of 20–30 °C. However, the maximum average dry weight was observed at 30 °C.

Sequencing reactions performed with ITS1/ITS4 primer pairs which amplified the fragments of −600 bp (Supplementary Fig. S4). Following an evolutionary analysis of the ITS sequences, NCBI BLAST showed that this new strain exhibits the maximum similarity with T. asperellum T-17 (KC884774) followed by T. hamatum (KC884761), T. koningiopsis (KC884790), T. atroviride (KC884770), T. hatzianum (KC884786), and T. saturnisporum (KC884818), with Protocrea pallida (NR_111329) used as an outgroup (Fig. 3). We identified the strain to be T. asperellum along with the morphological characteristic and called it T. asperellum HbGT6-07.

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Fig. 3

Phylogenetic tree depending on the ITS region of the genomic rDNA gene of 2 isolates and 11 representative strains of Trichoderma. The Neighbor-Joining (NJ) method was done through MEGA7 where bootstrap values (n = 1000) higher than 50% are visible at the internodes in the tree. As the outer group, Protocrea pallida CBS299.78 strain was used.

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3.3 The T. asperellum HbGT6-07 CFs influenced the growth of B. cinerea and S. sclerotiorum

B. cinerea and S. sclerotiorum isolates were cultured on potato dextrose agar (PDA) media with CF of T. asperellum HbGT6-07, and PDA plus dd H₂O was used as the control. In case of B. cinerea, the radial colony size on PDA that amended 1% (V/V) CF were significantly lesser as compared with colonies grown on control treatment. The inhibition percentages of T. asperellum HbGT6-07 CF to B. cinerea significantly (P < 0.01) increased with an increase in concentration of T. asperellum HbGT6-07 CF (Fig. 4A). Among of the applied concentrations (10%, 6%, 2%, and 1% (V/V) of CF, CF at a concentration of 10% showed 93% growth inhibition to B. cinerea, and an inhibition rate of 29% was recorded when treated with culture filtrate at a concentration of 1%. On the other hand, the GI percentage of T. asperellum HbGT6-07 CF to S. sclerotiorum significantly (P < 0.01) increased with an increase in concentration of T. asperellum HbGT6-07 CF (Fig. 4B). Concentration of 10% showed 91% growth inhibition to S. sclerotiorum, and an inhibition rate of 17% was found when treated with CF at a concentration of 1%.

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Fig. 4

Inhibition percentages of HbGT6-07 CF to B. cinerea and HbGT6-07 CF to S. sclerotiorum.

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3.4 Efficacy of the VOCs of T. asperellum HbGT6-07 in suppression of B. cinerea and S. sclerotiorum

The dish within dish method was used to detect the antifungal activity of T. asperellum HbGT6-07 against B. cinerea and S. sclerotiorum through the production of antifungal volatiles. In the double dish sets of blank AWG/B05.10 and B05/B05 (inner/outer dishes), isolate B05.10 in the external dishes grew and produce sclerotia on PDA with average colony diameters bigger than 14.5 cm (Fig. 5A-B). Notably, mycelial growth reduction was observed during the treatments. Initially, all outside B05.10 culture grew normally (average 12 mm) up to 4 days but at 5th day HbGT6-07/B05.10 treatment reduced growth rate about 1 mm while AWG and B05.10/B05.10 treatment growth rate 10 and 8 mm/day, respectively (Fig. 5C). B05.10 colonies sclerotia were obtained in the with average sclerotia yields up to 33 and 28 sclerotia per dish, respectively in blank AWG/B05 and B05/B05. In comparison, in the DwD sets of HbGT6-07/B05.10, isolate B05.10 grew in the outer plates, but developed limited colonies with an average colony size of 4.1 cm without apparent sporulation or sclerotia (Fig. 5D).

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Fig. 5

Effect of volatile organic compounds of T. asperellum HbGT6-07 isolate on B. cinerea B05.10. (A) The initial efficacy of VOCs of isolate T. asperellum HbGT6-07, B. cinerea B05.10 and AWG designated as control (CK). (B) Indicates colony diameter of AWG+HbGT6-07 and AWG+B05.10 isolates compare to treated and non-treated dishes CK (AWG). (C) Culture growth rate after 5 days. (D) The limited number of sclerotia were developed with an average colony size on the dish of isolates AWG+HbGT6-07.

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The DwD sets of HbGT6-07/A367 demonstrated a related antifungal impact of volatiles from the AWG cultures of HbGT6-07 on mycelial growth and sclerotial development by S. sclerotiorum A367. In the DwD sets of blank AWG/A367 and A367/A367, isolate A367 in the outer plates developed rapidly, colonised the whole outer plates at 20 °C for 15 d incubation, and generated sclerotia with yield potential up to 53 and 42 sclerotia per dish. Besides, in the HbGT6-07/A367 DwD sets, isolate A367 in the outer dishes developed gradually, forming modest colonies with an average size of 7.6 cm after 20 °C incubation (Fig. 6A-D).

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Fig. 6

Effect of volatile organic compounds of T. asperellum HbGT6-07 isolate on S. sclerotiorum A367. (A) The initial efficacy of VOCs of isolate T. asperellum HbGT6-07, S. sclerotiorum A367. (B) Indicates colony diameter of AWG+HbGT6-07 and AWG+A367 isolates compare to treated and non-treated dishes CK (AWG). (C) Culture growth rate after 5 days. (D) The limited number of sclerotia were developed with an average colony size on the dish of isolates AWG+HbGT6-07.

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3.5 Biocontrol of disease suppression by T. asperellum HbGT6-07

The findings of the antimicrobial analysis on detached rapeseed leaves indicated that T. asperellum HbGT6-07 hyphal mixtures were successful in suppressing lesion extension responsible by B. cinerea and S. sclerotiorum (Fig. 7). In the negative control, the hyphal mixtures of T. asperellum HbGT6-07 alone as inoculum, no noticeable exposure or mild infection with development of small leaf lesions (<0.5 mm in size) were recorded on the leaves at 3 day post-inoculation (dpi) under 20 °C. Furthermore, the serious outbreak was reported on the leaf tissue in the positive control with hyphal components of B05.10 alone as inoculum, and broad necrotic leaf lesions were developed with an average lesion size of up to 23 mm. The hyphal fragments of T. asperellum HbGT6-07 and B05.10 isolates with three biocontrol treatments at the ratios of 33.33:66.67, 50:50 and 66.67:33.33 (HbGT6-07: B05.10) as inoculum, the diameter of leaf lesion were reduced by 43%, 65% and 97%, respectively, associated with the treatment of positive control (Fig. 7 left, Supplementary Fig. S5A).

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Fig. 7

Biocontrol assay of disease suppression by isolate HbGT6-07 on rapeseed leaves. The hyphal fragments (HbGT6-07:B05.10) with the treatment level at ratios of 33.33:66.67, 50:50 and 66.67:33.33 was reduced the leaf lesion diameter of 43, 65 and 97% where HbGT6-07:A367 fragments moderately reduced lesion diameter of 16, 9 and 55%, respectively.

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Equal inhibitory action on rape seed leaves was detected from available hyphal fragments of T. asperellum HbGT6-07 toward invasion with S. sclerotiorum A367. While the positive treatment group with the available hyphal mixtures of A367 alone as inoculum responsible for more prominent leaf lesions with 27 mm of average lesion diameter at 3 dpi (20 °C), the biological control action with viable hyphal fragments of HbGT6-07 and A367 at the ratios of 33.33:66.67, 50:50 and 66.67:33.33 (HbGT6-07:A367) as inoculum caused small leaf lesions with average lesion diameters of 16, 9 and 0.6 mm, respectively. The effectiveness of biocontrol strategy of these three treatments was as high as 40%, 66% and 94% respectively, compared to positive control treatment (Fig. 7 right, Supplementary Fig. S5B).

3.6 Reduction of sclerotia formation under mixed culture condition

The carrot blocks were the perfect substrates for sclerotia production by B. cinerea and S. sclerotiorum. In the mixed culture condition, the number of sclerotia production and the weight of sclerotia were significantly reduced in the co-cultured by the action of Trichoderma isolates HbGT6-07 (Fig. 8A). Results from this mixed culture assay exhibited that the isolate T. asperellum HbGT6-07 was significantly (P < 0.01) reduced the sclerotia production turned into zero as compared to control for B05.10 and A367, respectively (Fig. 8B). Moreover, the dry weight of sclerotia data also revealed that isolates T. asperellum HbGT6-07 caused complete inhibition of sclerotia production (Fig. 8C).

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Fig. 8

Suppression of sclerotia formation with two different culture conditions. (A) (HbGT6-07+B05.10 and HbGT6-07+A367). (B) Mixed culture showed significant reduction of sclerotia as compare to for B05.10 and A367. (C) The dry weight data of sclerotia production revealed HbGT6-07 isolates completely inhibit the sclerotia production.

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3.7 GC/MS profiling of T. asperellum HbGT6-07 VOCs

The mass spectra structural information of the VOCs was evaluated via the data in the NIST Mass Spectral Search Program (version 2.2). Results of GC-MS analysis identified 32 compounds in the T. asperellum HbGT6-07. The molecular weight (MW), name of the compound (NoC), chemical formula (CF), retention time (RT) and relative peak area (RPA) were given in Table 1 and illustrated in Fig. 9. These compounds were into classes of alkane (R-H), alcohols (R-OH), aldehydes (R-CHO), alkene (R = ), amines (R-NH₂), benzene (R-C₆H₆), and ketone (R-CO) (Supplementary Fig. S6). 2-Ethylhexanal (C₈H₁₆O) seemed to be the enormous compound with 18.8% relative peak area (RPA), accompanied by Octan-3-one (C₈H₁₆O) with the RPA value of 11.2% and Octan-2-one (C₈H₁₆O) with the RPA value of 5.6%. The other 18 molecules, such as 1-octen-3-ol, were less common, with the RPA levels between 0.9% and 4.2%.

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Fig. 9

GC-MS profiling of volatile organic compounds emitted by the isolates T. asperellum HbGT6-07 and fresh AWG. The values on the peaks refer to the retention time for the volatiles listed in Table 1. The VOCs with low quantity or untraceable levels were not shown.

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3.8 Molecular docking

The lytic polysaccharide monooxygenases from A. oryzae and the volatile compounds from T. asperellum HbGT6-07 were docked to understand their binding interactions. Two volatile compounds from T. asperellum HbGT6-07, Butylated hydroxytolune, Beta-Cedrene showed higher binding affinity than other compounds in molecular docking. The Butylated hydroxytolune had −5.3 Kcal/mol energy in docking study and it forms one hydrogen bond at Met174 positions and one alkyl bond at Val176, and two pi-alkyl interactions at Phe27 and Trp81 position (Fig. 10B). The Beta-Cedrene had comparatively higher binding affinity (−5.7 Kcal/mol) than Butylated hydroxytolune and it formed three alkyl bonds at Phe27, Val176, and Val179 positions whereas one electrostatic bond at Lys77 and two hydrogen bonds at Gly24 and Asn180 positions respectively (Fig. 10A).

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Fig. 10

The molecular docking and non-bonded interactions of the volatile compounds against fungal proteins. The figure was generated with the aid of Discovery studio and Pymol software package. Here, (A) interactions between lytic polysaccharide monooxygenases from Aspergillus oryzae and Butylated hydroxytolune. (B) Interactions, between lytic polysaccharide monooxygenases from Aspergillus oryzae and Beta-Cedrene. (C) Lanosterol 14-alpha-Demethylase from Saccharomyces cerevisiae and Beta-Cedrene, (D) Lanosterol 14-alpha-Demethylase from Saccharomyces cerevisiae and Butylated hydroxytolune, (E) sterol 14 alpha-demethylase from Candida albicans and alpha-bergamotene, (F) sterol 14 alpha-demethylase from Candida albicans and Beta-Cedrene.

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Moreover, the Lanosterol 14-alpha-Demethylase from Saccharomyces cerevisiae were docked against the volatile compounds from T. asperellum HbGT6-07. In this docking study, the two previous hits from lytic polysaccharide monooxygenases from A. oryzae showed better affinity than other compounds. The Beta-Cedrene had −8.0 Kcal/mol binding energy while docked against the Lanosterol 14-alpha-Demethylase and Butylated hydroxytolune had −6.8 Kcal/mol affinity. The Beta-Cedrene formed five hydrogen bonds at Ala69, Val242, Arg98, Met509, and Ser382, two alkyl bonds at Leu95, Pro238 positions, one amide pi interactions at Leu96 residues, four pi-alkyl interactions at Tyr72, Phe241, His381, and Phe384 (Fig. 10C). The Butylated hydroxytolune had one hydrogen bond at Phe506, one Pi-Pi-T-shaped interactions at Tyr72, two alkyl bonds at Leu95, Met509, and two Pi-alkyl bonds at Phe241, Phe384 positions (Fig. 10D).

Finally, the sterol 14 alpha-demethylase from the C. albicans were also docked where alpha-bergamotene (−7.5 Kcal/mol) and beta-cedrene (−7.4 Kcal/mol) had higher affinity. The where alpha-bergamotene formed three alkyl bonds at Leu87, Pro230, Met508 whereas four pi-alkyl interactions at Tyr64, Phe233, His377 and Phe380 residues (Fig. 10F). The beta-cedrene formed four hydrogen bonds at Glu194, Pro193, Tyr221, Ser222 and two alkyl bonds at Ala218, Phe198, and two Pi-alkyl bonds at Phe213 and Ile197 residues (Fig. 10E).

3.9 Molecular dynamics

The molecular dynamics simulation was conducted to find out the structural stability and flexible nature of the docked complexes (Table 2). The RMSD of the c-alpha atoms were assessed from the simulation trajectories. From the Fig. 11 it can be observed that Butylated hydroxytoluene and Beta Cedrene along with lytic polysaccharide monooxygenases from A. oryzae complexes were initially unstable as they exhibit higher RMSD. However, the both complexes were stable and showed less flexible nature for the rest of the simulation time as lesser degree of deviations were found for RMSD. Moreover, the SASA were explored to understand the deviation in the complexes surface volume. The Butylated hydroxytoluene had initially stable SASA profile, as they did not change the protein volume, therefore, after 40 ns this complex lowered it SASA profile, which indicates the shrunken nature of the protein complex. The Beta Cedrene had constant SASA profile which specifies the stable nature of the protein volume. The radius of gyration of the simulated systems were analyzed to understand the mobile nature of the complexes. The two complexes; Butylated hydroxytoluene and Beta Cedrene were stable in Rg profile and did not fluctuate much. Furthermore, the hydrogen bond of the simulated systems was analyzed as they play an important role in specifying the stable nature of the protein-ligand complexes. Therefore, the hydrogen bond pattern of the both systems were stable and did not fluctuates much.

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Fig. 11

The molecular dynamics simulation of the docked complex of the volatile compounds and lytic polysaccharide monooxygenases from Aspergillus oryzae; (A) root mean square deviation of the c-alpha atoms of the docked complex (B) solvent accessible surface area to evaluate the change in the protein surface area, (C) radius of gyration to explore the compactness of the complex, (D) hydrogen bond.

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Therefore, Lanosterol 14-alpha-Demethylase from S. cerevisiae and Beta Cedrene, Butylated hydroxytoluene complexes were also analyzed in the simulating environment (Fig. 12). Both complexes initially rise RMSD profile although the degree of deviation was lesser. The Beta Cedrene had comparatively lower RMSD than Butylated hydroxytoluene, which indicates the more stable nature of this complex. After 20 ns, both complexes exhibit unchanging RMSD value until the 50 ns time. The SASA of the both complexes were unstable at the beginning phases but after 10 ns time the protein, volumes were relatively rigid and stable. The Rg profile of the both complexes were stable until 20 ns but it lowered its Rg value after then. Both complexes had lower Rg profile comparted to the beginning phases which indicates more stiff nature of the complexes. The hydrogen bond of the complexes was stable till 50 ns simulation time.

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Fig. 12

The molecular dynamics simulation study of the volatile compounds and Lanosterol 14-alpha-Demethylase from Saccharomyces cerevisiae; (A) RMSD, (B) SASA, (C) Radius of Gyration and (D) Hydrogen Bond.

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Moreover, sterol 14 alpha-demethylase from the C. albicans and two hit ligands; alpha-bergamotene and beta-cedrene complexes were simulated at the atomistic conditions (Fig. 13). The RMSD profile from both complexes rises at the starting point to till 15 ns. After then, both complexes had straight RMSD line and did not changes too much which corelates with structural rigidity. The SASA profile from both two complexes were different from each other where alpha-bergamotene had higher SASA profile, which indicates the expansion of the protein surface area. The beta-cedrene complexes had lowered SASA profile that indicates the truncation of the protein volumes. The Rg and hydrogen bond pattern of the both complexes were relatively stable in the simulating environment, which correlates with the complex’s stability and less flexible nature.

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Fig. 13

The molecular dynamics simulation of the volatile compounds and sterol 14 alpha-demethylase from Candida albicans; (A) RMSD, (B) SASA, (C) Radius of Gyration and (D) Hydrogen Bond.

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