*3.9. Characterization of Synthesized Nanoparticles*

The UV-visible absorption spectra was observed within the range of 372–374 nm. The obtained results matched with that of the UV–vis spectra of ZnO NPs for olive leaves (*Olea europaea*), chamomile (Figure 4c).

[52] (Figure 4a).

flower (*Matricaria chamomilla* L.) and red tomato fruit (*Lycopersicon esculentum* M.) and showed strong absorption bands at 384, 380 and 386 nm respectively according to Ogunyemi et al. [52] (Figure 4a). the agglomerated small particles of ZnO. The high resolution TEM image shows the well-defined crystal planes. The SAED patterns are well matched with the (hkl) values corresponding to the prominent peaks of the PXRD profiles (Figure 9a–d).

The formed shapes of ZnO NPs were displayed in SEM images with different surface morphology. The elements involved in the formation of nanoparticles were subjected for the EDAX analysis to know the qualitative difference as well as the quantitative difference. The analysis revealed the highest proportion of zinc (50.36%) in nanoparticles and oxygen (49.64%) in all the

TEM and SAED patterns correspond to ZnO compounds were obtained. The TEM image shows

*J. Fungi* **2020**, *6*, x FOR PEER REVIEW 12 of 18

The UV-visible absorption spectra was observed within the range of 372–374 nm. The obtained results matched with that of the UV–vis spectra of ZnO NPs for olive leaves (*Olea europaea*), chamomile flower (*Matricaria chamomilla* L.) and red tomato fruit (*Lycopersicon esculentum* M.) and showed strong absorption bands at 384, 380 and 386 nm respectively according to Ogunyemi et al.

The FTIR spectrum showed the absorption at 400 cm −1 to 600 cm −1 which further confirms the presence and formation of ZnO nanoparticles by using *Trichoderma* spp. Similar results were observed from nanoparticles synthesized by the biological method using plant extracts Ogunyemi et al. [52] At 700 °C, the obtained fungal secondary metabolites were converted to their respective oxides, leading to the formation of ZnO NPs. This implies that most of the compounds present in the sample do not have a high thermal stability. Hence there were no other vibration modes detected in

The biosynthesized ZnO NPs PXRD patterns showed noticeable peaks and it was well-matched to JCPDS No. 75-576. Similarly, nanoparticles from *Trichoderma* spp. are synthesized with ZnO. The biosynthesized ZnO-NPs of the crystalline structure was confirmed by stiff and narrow diffraction peaks with no significant variance in the diffraction peaks, suggesting that the crystalline product was free of impurities. Similarly, Lakshmeesha et al. [30] reported the green synthesis of *Nerium oleander* ZnO-NPs with no impurities in the obtained crystalline product. The size of the present study's crystalline particles of green synthesized ZnO-NPs was calculated using Scherrer's formula, which was within a range of 12–35 nm. Accordingly, Dobrucka and Dlugaszewska [53] reported the biosynthesis of ZnO nanoparticles using *Trifolium pratense*, with a hexagonal wurtzite shape and the sharp peaks calculated using Scherrer's formula were 60–70 nm according to Murali et al. [54]

the FT-IR spectra as shown in (Figure 4b) other than ZnO NPs.

*harzianum* (PGT4). The FTIR spectrum showed the absorption at 400 cm <sup>−</sup><sup>1</sup> to 600 cm <sup>−</sup><sup>1</sup> which further confirms the presence and formation of ZnO nanoparticles by using *Trichoderma* spp. Similar results were observed from nanoparticles synthesized by the biological method using plant extracts Ogunyemi et al. [52] At 700 ◦C, the obtained fungal secondary metabolites were converted to their respective oxides, leading to the formation of ZnO NPs. This implies that most of the compounds present in the sample do not have a high thermal stability. Hence there were no other vibration modes detected in the FT-IR spectra as shown in (Figure 4b) other than ZnO NPs.

The biosynthesized ZnO NPs PXRD patterns showed noticeable peaks and it was well-matched to JCPDS No. 75-576. Similarly, nanoparticles from *Trichoderma* spp. are synthesized with ZnO. The biosynthesized ZnO-NPs of the crystalline structure was confirmed by stiff and narrow diffraction peaks with no significant variance in the diffraction peaks, suggesting that the crystalline product was free of impurities. Similarly, Lakshmeesha et al. [30] reported the green synthesis of *Nerium oleander* ZnO-NPs with no impurities in the obtained crystalline product. The size of the present study's crystalline particles of green synthesized ZnO-NPs was calculated using Scherrer's formula, which was within a range of 12–35 nm. Accordingly, Dobrucka and Dlugaszewska [53] reported the biosynthesis of ZnO nanoparticles using *Trifolium pratense*, with a hexagonal wurtzite shape and the sharp peaks calculated using Scherrer's formula were 60–70 nm according to Murali et al. [54] (Figure 4c).

The formed shapes of ZnO NPs were displayed in SEM images with different surface morphology. The elements involved in the formation of nanoparticles were subjected for the EDAX analysis to know the qualitative difference as well as the quantitative difference. The analysis revealed the highest proportion of zinc (50.36%) in nanoparticles and oxygen (49.64%) in all the synthesized nanoparticles according to Prasad et al. [55] (Figures 5–8).

analysis.

*J. Fungi* **2020**, *6*, x FOR PEER REVIEW 13 of 18

*J. Fungi* **2020**, *6*, x FOR PEER REVIEW 13 of 18

*J. Fungi* **2020**, *6*, x FOR PEER REVIEW 13 of 18

*J. Fungi* **2020**, *6*, x FOR PEER REVIEW 13 of 18

**Figure 5.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma harzianum* (PGT4) at lower (**a**) and higher magnification (**b**); (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 5.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma harzianum* (PGT4) at lower (**a**) and higher magnification (**b**); (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 5.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma harzianum* (PGT4) at lower (**a**) and higher magnification (**b**); (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 5.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma harzianum* (PGT4) at lower (**a**) and higher magnification (**b**); (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 5.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma harzianum* (PGT4) at lower (**a**) and higher magnification (**b**); (**c**) represents energy-dispersive X-ray spectroscopy (EDAX)

**Figure 6.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma reesei* (PGT5) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 6.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma reesei* (PGT5) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 6.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma reesei* (PGT5) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 6.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma reesei* (PGT5) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 6.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma reesei* (PGT5) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis.

**Figure 7.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma reesei* (PGT13) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 7.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma reesei* (PGT13) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 7.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma reesei* (PGT13) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 7.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma reesei* (PGT13) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 7.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma reesei* (PGT13) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis.

**Figure 8.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma* spp. co-culture of (PGTA) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. (PGTA) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. (PGTA) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 8.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma* spp. co-culture of (PGTA) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis. **Figure 8.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma* spp. co-culture of (PGTA) at lower (**a**) and higher (**b**) magnification; (**c**) represents energy-dispersive X-ray spectroscopy (EDAX) analysis.

**Figure 8.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma* spp. co-culture of

**Figure 8.** SEM image of zinc oxide nanoparticles synthesized from *Trichoderma* spp. co-culture of

TEM and SAED patterns correspond to ZnO compounds were obtained. The TEM image shows the agglomerated small particles of ZnO. The high resolution TEM image shows the well-defined crystal planes. The SAED patterns are well matched with the (hkl) values corresponding to the prominent peaks of the PXRD profiles (Figure 9a–d). *J. Fungi* **2020**, *6*, x FOR PEER REVIEW 14 of 18

**Figure 9.** TEM micrographs (at different magnifications) and SAED patterns of Zno NPs synthesized from (**a**) *Trichoderma harzianum* (PGT4); (**b**) *Trichoderma reesei* (PGT5); (**c**) *Trichoderma reesei* (PGT13); and (**d**) *Trichoderma* spp. co-culture (PGTA). **Figure 9.** TEM micrographs (at different magnifications) and SAED patterns of Zno NPs synthesized from (**a**) *Trichoderma harzianum* (PGT4); (**b**) *Trichoderma reesei* (PGT5); (**c**) *Trichoderma reesei* (PGT13); and (**d**) *Trichoderma* spp. co-culture (PGTA).

#### *3.10. Antibacterial Activity 3.10. Antibacterial Activity*

concentrations.

(Concentration expressed in µg/mL).

The antibacterial activity of ZnO NPs has been evaluated by measuring the zone of inhibition around the disc. The antibacterial activity of biosynthesized ZnO NPs was tested by an agar disc diffusion method, which was placed on the pre-swabbed Mueller-Hinton agar plate. The zone of inhibition is represented in Figure S2 and tabulated in Table 7. Further MIC values were determined for the biosynthesized ZnO NPs by the 96 well plate method, which is tabulated in Table 7 and is represented in Figure S3. The pronounced antibacterial activity of ZnO NPs can be due to its relatively small size and high surface-to-volume ratio. The present study clearly signifies the potentiality of ZnO NPs as antibacterial agents against Xoo (Figure S3) (Table 7). Our results correlated with the results of Ogunyemi et al. [52], where the zone of inhibition was recorded and the antibacterial activity of ZnO NPs was checked against Xoo when used in different The antibacterial activity of ZnO NPs has been evaluated by measuring the zone of inhibition around the disc. The antibacterial activity of biosynthesized ZnO NPs was tested by an agar disc diffusion method, which was placed on the pre-swabbed Mueller-Hinton agar plate. The zone of inhibition is represented in Figure S2 and tabulated in Table 7. Further MIC values were determined for the biosynthesized ZnO NPs by the 96 well plate method, which is tabulated in Table 7 and is represented in Figure S3. The pronounced antibacterial activity of ZnO NPs can be due to its relativelysmall size and high surface-to-volume ratio. The present study clearly signifies the potentiality of ZnO NPs as antibacterial agents against Xoo (Figure S3) (Table 7). Our results correlated with the results of Ogunyemi et al. [52], where the zone of inhibition was recorded and the antibacterial activity of ZnO NPs was checked against Xoo when used in different concentrations.

**ZnO NPs Disc Diffusion Values (in mm) MIC Values (µg/mL)**

PGT 4 00 50

PGT13 00 50 PGTA 15.67 ± 0.33 25 Positive 13.67 ± 0.33 25 Negative 00 00

**Table 7.** Evaluation of the bactericidal activity of biosynthesized zinc oxide nanoparticles from different species of *Trichoderma* against different strains of *Xanthomonas oryzae* pv. *oryzae* (Xoo)


**Table 7.** Evaluation of the bactericidal activity of biosynthesized zinc oxide nanoparticles from different species of *Trichoderma* against different strains of *Xanthomonas oryzae* pv. *oryzae* (Xoo) (Concentration expressed in µg/mL).
