*3.3. Antifungal Activity of AgNPs*

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The result of antifungal activity revealed that the mycelium growth of *M. oryaze* strain Gry was robustly suppressed by AgNPs, shown in (Figure 6). The inhibitory effect on the growth of mycelium increased with the increase of AgNP concentrations. In fact, AgNPs at 10, 20, 30 and 40 µg/mL caused an 18%, 49%, 65% and 88% reduction, respectively, in mycelial diameter. Similarly, previous studies have shown that AgNPs can be used as an antifungal agent to prevent plants from fungal infection [9,10,39–43]. Although there are different hypotheses available, the antimicrobial mechanisms of AgNPs have not yet been clearly defined. The proposed mechanisms were summarized based on the current literature, as follows: attachment of AgNPs to the surface of the cell membrane, altering the lipid bilayer or increasing permeability of the cell membrane, microbial cell intrusion of AgNPs causing damage to intracellular micro organelles (such as mitochondria, vacuoles and ribosomes) and biomolecules including DNA, protein and lipids, and modulation of the intracellular signal transduction method towards apoptosis [44]. ‐ ‐ μ

**Figure 6.** Effect of the biosynthesized AgNPs at four different concentrations (10, 20, 30 and 40 µg/mL) on the mycelial growth of *M. oryaze* strain Gry. The AgNPs at 10, 20, 30 and 40 µg/mL caused an 18%, 49%, 65% and 88% reduction, respectively, in mycelial diameter. Data are a mean value ± standard error of three replicates, and bars with different letters (a–d) are significantly different in LSD test.

The fungal cell wall is a flexible structure that performs several functions in determining the shape of the cell. In addition, the cell wall can protect the fungal cells from environmental stresses, such as pH, temperature and changes in osmolality [9,45]. Therefore, interference by causing harm to the fungal cell wall may lead to loss of content and death. In our results, the hyphae of *M. oryzae* strain Gry that were treated with AgNPs showed abnormal structural, swelling and damage to their cell walls causing some loss of contents. In contrast, the cell walls of *M. oryzae* strain Gry had normal structural characteristics in the absence of the AgNPs (Figure 7). Similarly, the antifungal activity of AgNPs and Cu-NPs have been found on the cell walls of many pathogenic fungi such as *Fusarium graminearum*, *Fusarium oxysporum*, *Fusarium solani* and *Colletotrichum gloeoesporioides* [9,45,46].

The germinated spores of pathogenic fungi are known to perform a major role in colonizing and infecting plants [9,47]. Therefore, inhibition rate of spore germination will significantly reduce the threat of rice fungal pathogens. The biosynthesized AgNPs were able to efficiently inhibit the spore germination and germ tube growth of *M. oryzae* strain Gry, and the antifungal activity increased with the increase in AgNP concentration. In fact, the germination rate of spores was 83%, while the length of the germ tubes was 77.63 µm in the negative control. However, the germination rate of spores was 68%, 53%, 31% and 13%, respectively (Figure 8A), while the germ tubes length was 54.62 µm, 33.73 µm, 21.74 µm and 8.11 µm, respectively, (Figure 8B) in the presence of AgNPs at four different concentrations (10, 20, 30 and 40 µg/mL). Similar results have also been reported in other studies [9,48]. ‐

**Figure 7.** Scanning electron microscopy (SEM) [scale bar = 5 µm] and transmission electron microscopy (TEM) [scale bar = 1 µm] images of *M. oryaze strain Gry* in the absence of the biosynthesized AgNPs; the hyphae showed a normal structural property (**A**,**C**), and in the presence of the biosynthesized AgNPs, it had abnormal structure, swelling and damage to the cell walls' contents (**B**,**D**). μ μ μ μ μ

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 **Figure 8.** Effect of the biosynthesized AgNPs of four different concentrations (10, 20, 30 and 40 µg/mL) on spores' germination rate (**A**) and germ tube growth (**B**). Data are a mean value ± standard error of three replicates, and bars with different letters (a–e) are significantly different in LSD test.

Many fungi form a specialized infection structure called an appressorium that is necessary and required to penetrate the plant cell walls [1,6]. Therefore, appressorium inhibition significantly reduces the risk of rice fungal pathogens. The biosynthesized AgNPs were able to effectively inhibit the appressorium formation and appressorium diameter of *M. oryzae* strain Gry, while the inhibitory effect increased along with the increase in AgNP concentration. In fact, the appressorium formation rate was 83%, while appressorium diameter was 14.00 µm in the negative control. However, the appressorium formation rate was 66%, 34%, 11% and 0%, respectively (Figure 9A), while the appressorium diameter was 11.00 µm, 8.00 µm, 5.38 µm and 0.00 µm, respectively (Figure 9B) in the presence of AgNPs at different concentrations (10, 20, 30 and 40 µg/mL). μ μ μ μ μ μ

 **Figure 9.** Effect of the biosynthesized AgNPs of four different suspensions (10, 20, 30 and 40 µg/mL) on appressorium formation (**A**) and appressorium diameter (**B**). Data are a mean value ± standard error of three replicates, and bars with different letters (a–e) are significantly different in LSD test.
