Fungistatic

The fungistatic effect of various concentrations of the extract was observed as inhibition of mycelial growth. However, re-culturing the mycelial growth following the re-inoculation into the fresh medium resulted in the re-growth of the fungal mycelium in the new medium, implying that the alga extract could only inhibit the growth of the fungi (fungistatic).

In the study of fungicidal or fungistatic effects of extracts at concentrations that completely inhibited mycelium growth, the re-cultures resulting from the transfer of the fungi-containing ring to the PDA culture medium showed that the fungi reappeared and grew after 5–7 days on the fresh culture medium. The results revealed that *P. oryzae* and *A. niger* started to grow after 5 days and *P. expanstum* and *B. cinerea* started to grow after 7 days. During the study period (7 days), the growth of mycelium in the re-cultures of the control fungi was completely evident from the second day, and it was much higher (up to 90%) at the end of 7 days than the re-cultures performed at concentrations in which mycelium growth was completely inhibited. These results indicate that the algae extract only inhibited the growth of fungi and fungicidal properties were observed in none of the extract concentrations.

## **4. Discussion**

The results obtained from the GC-MS analysis of this study specify that *G. persica* showed a large number of bioactive compounds with antioxidant, antibacterial, and antifungal properties. There are various reports about the compounds derived from macroalgae as a potential source of biochemical and medicinal properties including antibacterial [57], antifungal [58], antiviral [59], antioxidant [60], and anti-inflammation activities [61].

The results of the present study revealed that the extract of *G. persica* had a high potential to inhibit the mycelial growth of plant pathogenic fungi. Previous research findings have indicated that alga extracts have agents for the biological control of the growth of hyphae and germination, an increase in intracellular holes (vacuolization), and the disruption of the functioning of fungal cells [62]. On the other hand, research on different species of *Gracilaria* has revealed this genus' antibacterial and antifungal activities. In this regard, Singh and Raadha [63] studied the *G. corticata* extract. They found that this

species, at a rate of 1000 μL, could inhibit the growth of human pathogenic bacteria and fungi, including *Salmonella typhimurium*, *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans*, and was a natural source of antibiotics. Dayuti [64] also reported the antibacterial activity of *G. verrucosa* against *S. typhimurium* and *E. coli*. A study on the antifungal and antibacterial properties of the *G. confervoides* extract showed that 100 μL of the *G. confervoid* extract could prevent the aerial mycelial growth of the cucumber pathogens *Rhizoctonia solani* and *Macrophomina phaseolinae* [58]. This is consistent with our findings as to the fungistatic activity of the *G. persica* extract. In an experiment, Kolanjinathan and Stella [65] found that *G. corticata* could inhibit the growth of *Aspergillus flavus*, *A. fumigatus*, and *A. niger*, as well as the human pathogen *albicans*. The minimum inhibitory rate of this extract was estimated at 2–16 mg/mL. This is in agreemen<sup>t</sup> with our findings as to the antifungal property of *G. persica* in inhibiting the growth of *A. niger*.

The results of HPLC analysis in our study showed that *G. persica* is rich in polyphenolic compounds. It is now well established that the antifungal activity of alga extracts may be related to the presence of phytochemicals, e.g., tannins and phenols. Phenolic compounds are likely to influence the growth and metabolism of fungi [66]. Sea resources are the most enormous remaining reservoirs of natural molecules, which are assessed for therapeutic activities and provide valuable ideas for developing new medications against cancer, microbial infections, and inflammations [67]. Although terrestrial biodiversity constitutes the basis of the pharmaceutical industry, oceans have rich biodiversity and can produce commercially invaluable modern compounds. A comparison of our findings with those of other studies confirms that *G. persica* is a rich source of biological components.

Some phytocompounds, which combine alkaloids, flavonoids, and saturated and unsaturated fatty acids, have antimicrobial, anti-inflammation, anti-cancer, anti-coagulant, and anti-arrhythmic activities. According to the results of GC-MS, most of the compounds detected in the methanolic extract of *G. persica* (36 out of the 50 detected components) are compounds whose antimicrobial and antifungal activities have already been documented. These interpretations corroborate the findings of current research (Table 1).

The results of HPLC revealed that *G. persica* had invaluable phenolic compounds, e.g., rosmarinic acid and quercetin, implying its antioxidant effect. It has been reported that the difference in plant extracts' biological activity depends on their constituents; therefore, a single compound may be responsible for an extract's effects alone or in synergy with other compounds [68].

Polyphenols are a remarkable group of plant metabolites that have an efficient antimicrobial performance. A number of studies have postulated about the interaction of the synergy of polyphenols with antibiotics against microbial resistance, e.g., epigallocatechin gallate of green tea [69], tellimagrandin I, and rugosin B of *Rosa floribunda* 'Dubline Bay' (aka 'MACdub') [70], and the synergy of rosmarinic acid and antibiotics against methicillinresistant *Staphylococcus aureus* [71]. Likewise, the antimicrobial effect of quercetin as the second dominant phenolic compound in *G. persica* against *Escherichia coli*, *Staphylococcus aureus*, and *Pseudomonas fluorescens* [72], and the antifungal effects of quercetin and rutin against *Cryptococcus* spp. [73] have been documented. It has been reported that these compounds can change the structure of the cell membrane, thereby destroying the plasma membrane integrity in fungal cells and increasing its permeability, which results in an increase in the K+ outflow from the cytoplasm of fungal cells. These effects may cause the polarity of the membrane to be lost by altering ion transport, or they may reduce energy production (ATP) by altering the membrane structure through impairing glucose uptake or inhibiting the enzymes involved in oxidative stress or phosphorylation precursor. The increase in cytoplasm membrane permeability ultimately leads to cell death due to the dispersion and loss of the cell pH gradient, a decline in the ATP level, and the failure of the proton driving force. Indeed, nutrient uptake, nucleic acid synthesis, and ATP*ase* activity sections are most damaged in the tissues of the fungi [74,75].

The experimental evidence of current research regarding the examination of polyphenolic compounds in *G. persica* showed that quercetin was the highest among the flavonoid

compounds. The possible activity of flavonoids may also be involved in mitochondrial damage and ROS production by inducing the transcription factors related to apoptosis and increasing the level of proptose proteins [76]. Hwang et al. [77] revealed that flavonoids disturbed the performance of mitochondria in the *C. albicans* strain by increasing the ROS level. The elevated intracellular ROS level and the disturbed mitochondrial performance play a significant role in apoptosis induction [78,79]. Quercetin has two aromatic rings in its structure and can penetrate the phospholipid membrane [80], where it damages DNA by inducing oxidative damage and, finally, cause cell death by apoptosis, which is an irreversible process [81].

## **5. Conclusions**

Herbal medicines play a significant role in human health and are an inspiring source of new medicinal compounds. It can be concluded from the results of the present study that *G. persica* has a high potential to be used in the pharmaceutical industry to improve health owing to its different compounds with antimicrobial activity.

The inhibitory effect of the alga on the growth of plant pathogenic fungi *in vitro* is apparent from our experimental evidence. Thus, this alga might be used to produce an environmentally friendly, reliable, and economic antifungal agen<sup>t</sup> to control *B. cinerea*, *P. oryzae*, *A. niger*, and *P. expansum*. This can be a high potential alternative to highly toxic chemical fungicides in plant disease management. The fungicidal efficacy still has to be shown in vivo. Above all, our results sugges<sup>t</sup> that the algae in the Persian Gulf and Oman Sea can be a rich source of different macroalgae species with unique antimicrobial activities, which may have various applications in agriculture and the control of plant disease in the future.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/plants10091781/s1, Figure S1: The effect of different concentrations of the *G. persica* extract (control, 400, and 1000 μL) on the mycelial growth of *B. cinerea* and *P. expansum.* Figure S2: The effect of different concentrations of the *G. persica* extract (control, 400, and 1000 μL) on the mycelial growth of *P. oryzae* and *A. niger.*

**Author Contributions:** Writing—original draft, methodology, and formal analysis, L.P.; conceptualization, supervision, and project administration, S.S.M.; writing—editing and proofreading and formal analysis, R.Z.S. and H.A.E.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by RMC, Universiti Teknologi Malaysia (UTM), Malaysia through gran<sup>t</sup> No. R.J130000.7609.4C336 and R.J130000.7609.4C359.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** H.A.E. would like to thank RMC, Universiti Teknologi Malaysia (UTM), Malaysia for financial support through gran<sup>t</sup> No. R.J130000.7609.4C336 and R.J130000.7609.4C359.

**Conflicts of Interest:** The authors declare no conflict of interest.
