*3.6. Trichoderma–Trichoderma Interactions through Co-Culture*

The co-culture of selected *Trichoderma* spp. isolates showed the production of secondary metabolites on incubation, showing growth compatibility when grown on solid media. However, no dual cultures were found to be incompatible. Our findings co-related with the results of that of the development of secondary metabolites on a laboratory scale; fungal isolates containing the largest areas of secondary metabolite accumulation on mono and dual cultures on semi-solid media were used by Ortuno et al. [41] (Figure 1).

used by Ortuno et al. [41] (Figure 1).

*J. Fungi* **2020**, *6*, x FOR PEER REVIEW 8 of 18

PGT18 17.67 ijk ± 0.577 14.67 cd ± 0.577 18.33 jk ± 0.577 PGT19 19.67 lmn ± 0.577 18.33 jk ± 0.577 18.00 jk ± 0.000 PGT20 19.67 lmn ± o.577 18.33 jk ± 0.577 19.67 lmn ± 0.577 PGT21 17.33 hij ± 0.577 18.33 jk ± 0.577 18.33 jk ± 0.577 PGT22 19.67 lmno ± 0.577 21.67 pqr ± 0.577 18.33 jk ± 0.577 PGT23 18.33 jk ± 0.577 19.67 lmno ± 0.577 19.67 lmno ± 0.577 PGT24 20.67 nop ± 0.577 19.67 lmn ± 0.577 20.00 mno ± 0.000

± 0.577 24.67 <sup>v</sup>

± 0.577 25.00 <sup>v</sup>

± 0.577 26.00 <sup>v</sup>

Multiple Range Test indicated by different alphabets in the superscripts at *p* < 0.05.

0 <sup>a</sup>

The mean values are recorded and the values are not significantly different according to Duncan's

The co-culture of selected *Trichoderma* spp. isolates showed the production of secondary metabolites on incubation, showing growth compatibility when grown on solid media. However, no dual cultures were found to be incompatible. Our findings co-related with the results of that of the development of secondary metabolites on a laboratory scale; fungal isolates containing the largest

PGT34 21.00 opq ± 0.000 21.67 pqr ± 0.577 19.67 lmno ± 0.577 PGT35 22.67 st ± 0.577 20.00 mn ± 0.000 20.67 nop ± 0.000

± 0.000 21.67 pqr ± 0.577 14.67 cd ± 0.577

± 0.577 18.33 jk ± 0.577 15.67 ef ± 0.577

± 0.000 18.00 jk ± 0.000 15.00 de ± 0.000

± 0.000 18.33 jk ± 0.577 15.00 de ± 0.000

± 0.577 17.67 ijk ± 0.577 19.67 lmn ± 0.577

± 0.577 20.67 nop ± 0.577

± 0.577 21.00 opq ± 0.000

± 0.000 21.67 pqr ± 0.577

± 0.577 23.00 uv ± 0.000

± 0.000 25.00 rs ± 0.000

0 <sup>a</sup>

PGT25 14.00 <sup>c</sup>

PGT26 12.33 <sup>b</sup>

PGT27 11.00 <sup>a</sup>

PGT28 11.00 <sup>a</sup>

PGT29 12.33 <sup>b</sup>

PGT30 24.67 <sup>v</sup>

PGT32 24.67 <sup>v</sup>

Positive 25.00 <sup>v</sup>

Negative 0 <sup>a</sup>

*3.6. Trichoderma–Trichoderma Interactions through Co-Culture* 

PGT31 22.67 st ± 0.577 23.67 <sup>u</sup>

PGT33 22.67 st ± 0.577 25.33 <sup>v</sup>

**Figure 1.** Co-culture of *Trichoderma* spp. plates showing the compatibility: 4—*Trichoderma harzianum*, 5—*Trichoderma reesei* and 13—*Trichoderma reesei.* **Figure 1.** Co-culture of *Trichoderma* spp. plates showing the compatibility: 4—*Trichoderma harzianum*, 5—*Trichoderma reesei* and 13—*Trichoderma reesei*.

#### *3.7. Identification of Secondary Metabolite Compounds by GC-MS Analysis 3.7. Identification of Secondary Metabolite Compounds by GC-MS Analysis*

The ethyl acetate extract of the monocultures of *T. harzianum* (PGT4), *T. reesei* (PGT5) and *T. reesei* (PGT13) and co-cultures of Sample A (PGT4, PGT5, PGT13) were analyzed by GC-MS and the analysis has led to the identification of different compounds present in mono and co-culture of *Trichoderma* spp. Co-cultivation of beneficial fungi can stimulate the synthesis of novel secondary metabolites rather than in monocultures. The significant compounds that were found in the ethyl acetate solvent extract for PGT4 (Table 2), PGT5 (Table 3) and PGT13 (Table 4) and Sample A (Table 5). Gas chromatographic (GC) methods are usually done for the determination of volatile fungal The ethyl acetate extract of the monocultures of *T. harzianum* (PGT4), *T. reesei* (PGT5) and *T. reesei* (PGT13) and co-cultures of Sample A (PGT4, PGT5, PGT13) were analyzed by GC-MS and the analysis has led to the identification of different compounds present in mono and co-culture of *Trichoderma* spp. Co-cultivation of beneficial fungi can stimulate the synthesis of novel secondary metabolites rather than in monocultures. The significant compounds that were found in the ethyl acetate solvent extract for PGT4 (Table 2), PGT5 (Table 3) and PGT13 (Table 4) and Sample A (Table 5). Gas chromatographic (GC) methods are usually done for the determination of volatile fungal metabolites for different fungi such as *Aspergillus, Fusarium, Mucor, Penicillium* and *Trichoderma* (Siddiquee et al. [51] (Figure 2)).


**Table 2.** List of compounds of *Trichoderma harzianum* (PGT4) detected by Gas Chromatography Mass Spectroscopy (GC-MS). RT—Retention Time.


**Table 3.** List of compounds of *Trichoderma reesei* (PGT5) detected by GC-MS. RT—Retention Time.

**Table 4.** List of compounds of *Trichoderma reesei* (PGT13) detected by GC-MS. RT—Retention Time.



**Table 5.** List of compounds of *Trichoderma* spp. co-culture (PGTA) detected by GC-MS. RT—Retention Time.

**Figure 2.** (**a**–**d**) Gas Chromatography Mass Spectroscopy (GC-MS) of compounds identified from secondary metabolite crude extracts of (**a**) *T. harzianum* (PGT4), (**b**) *T. reesei* (PGT5), (**c**) *T. reesei* (PGT13) and (**d**) co-culture of *Trichoderma* spp. (PGTA). **Figure 2.** (**a**–**d**) Gas Chromatography Mass Spectroscopy (GC-MS) of compounds identified from secondary metabolite crude extracts of (**a**) *T. harzianum* (PGT4), (**b**) *T. reesei* (PGT5), (**c**) *T. reesei* (PGT13) and (**d**) co-culture of *Trichoderma* spp. (PGTA).

**Table 2.** List of compounds of *Trichoderma harzianum* (PGT4) detected by Gas Chromatography Mass Spectroscopy (GC-MS). RT—Retention Time. **SL. No. RT (mins) Name of the Compound**  *Trichoderma harzianum* **(PGT4) Molecular Formula Molecular Weight**  1 10.70 4-Propylbenzaldehyde C10H12O 148 2 13.37 6-Pentyl-2H-pyran-2-one C10H14O2 166 Interpretation of Mass Spectrum Gas Chromatography Mass Spectroscopy (GC-MS) was carried out using the database of the National Institute of Standard and Technology (NIST) with more than 62,000 patterns. The unknown spectrum of components was compared with that of the known components contained in the NIST library. The name, molecular weight and structure of the components of the test material were ascertained.

3 14.02 2,4-Di-tert-butylphenol C14H22O 206

5 16.55 Trimethyl-3,4-undecadiene-2,10-dione C14H22O2 222

8 21.44 Phthalic acid, diisobutyl ester C16H22O4 278 9 22.36 Dibutyl phthalate C16H22O4 278 10 22.77 Phthalic acid, butyl 2-pentyl ester C17H24O4 292 11 23.38 Phthalic acid, 6-ethyl-3-octyl butyl ester C22H34O4 362 12 24.17 Dibutyl phthalate C16H22O4 278 13 31.79 3-Ethyl-3-hydroxyandrostan-17-one C21H34O2 318 14 32.44 Mono(2-ethylhexyl) phthalate C16H22O4 278 15 35.87 Digitoxin C41H64O13 764

#### *3.8. In Vitro Screening of PGPR Trichoderma spp. Co-Culture Secondary Metabolites for Its Antibacterial Activity by Agar Well Di*ff*usion Method against Xanthomonas oryzae* pv. *oryzae* (Xoo) *3.8. In Vitro Screening of PGPR Trichoderma spp. Co-Culture Secondary Metabolites for Its Antibacterial Activity by Agar Well Diffusion Method against Xanthomonas oryzae* pv. *oryzae* (Xoo)

15 36.61 Ledene oxide-(II) C15H24O 220

In our study, we analyzed the effects of growing the fungal cultures in single cultures or in combination cultures of *T. harzianum* (PGT4), *T. reesei* (PGT5) and one more culture of *T. reesei* (PGT13) for the production of fungal secondary metabolites in liquid media. In our study, we analyzed the effects of growing the fungal cultures in single cultures or in combination cultures of *T. harzianum* (PGT4), *T. reesei* (PGT5) and one more culture of *T. reesei*

*J. Fungi* **2020**, *6*, x FOR PEER REVIEW 11 of 18

10 26.95 1-Hydroxy-4-methylanthra-9, 10-quinone C15H10O3 238 11 29.29 10,12-Pentacosadiynoic acid C25H42O2 374 12 30.35 4-(2-Oxiranyl)-9H-fluoren-9-ol C15H12O2 224 13 32.47 9-t-Butyltricyclo[4.2.1.1(2,5)]decane-9,10-diol C14H24O2 224

The zone of inhibition was found to range from 26 mm to 29 mm in diameter for the co-culture of (PGTA) followed by *T. harzianum* (PGT4) and *T*. *reesei* (PGT13) ranging from 23 mm to 26 mm in diameter, followed by a mono culture of *T. reesei* (PGT13). The zone of inhibition for *T. reesei* (PGT5) and *T. reesei* (PGT13) ranged from 20 mm to 26 mm. The zone of inhibition was found to be highest in the co-culture rather than in the mono cultures. Our findings co-related with the results of *T. harzianum* M10 and *T. pinophilus* F36CF on the production of fungal secondary metabolites in the liquid culture both in single and combined treatment by Vinale et al. [22] (Figure 3) (Table 6). (PGT13) for the production of fungal secondary metabolites in liquid media. The zone of inhibition was found to range from 26 mm to 29 mm in diameter for the co-culture of (PGTA) followed by *T. harzianum* (PGT4) and *T*. *reesei* (PGT13) ranging from 23 mm to 26 mm in diameter, followed by a mono culture of *T. reesei* (PGT13). The zone of inhibition for *T. reesei* (PGT5) and *T. reesei* (PGT13) ranged from 20 mm to 26 mm. The zone of inhibition was found to be highest in the co-culture rather than in the mono cultures. Our findings co-related with the results of *T. harzianum* M10 and *T. pinophilus* F36CF on the production of fungal secondary metabolites in the liquid culture both in single and combined treatment by Vinale et al. [22] (Figure 3) (Table 6).

**Figure 3.** Screening of *Trichoderma* spp. co-culture secondary metabolites for its antibacterial activity against plant pathogen *Xanthomonas oryzae* pv. *oryzae* (Xoo); 8SB, 9SB and 21 represent strains MBXoo69, MBXoo70 and MBXoo53, respectively. **Figure 3.** Screening of *Trichoderma* spp. co-culture secondary metabolites for its antibacterial activity against plant pathogen *Xanthomonas oryzae* pv. *oryzae* (Xoo); 8SB, 9SB and 21 represent strains MBXoo69, MBXoo70 and MBXoo53, respectively.



*3.9. Characterization of Synthesized Nanoparticles*  The mean values are recorded and the values are not significantly different according to Duncan's Multiple Range Test indicated by different alphabets in the superscripts at *p* < 0.05.
