**4. Discussion**

Disease severity was the first guide to govern systemic resistance in treated plants by cyanobacteria. Data presented in this study reported that *F. oxysporum* shows a highly destructive effect on pepper plants that caused typical wilt symptoms with DI 83.33%, similar to earlier studies on the same pathogenic fungus [40]. Using cyanobacterial extract to treat *Fusarium* wilt-infected pepper plants greatly reduced the symptoms of the disease, which is the primary criterion for assessing resistance in the pepper plant. Data showed that foliar spraying with cyanobacteria extracts was more effective than putting them in soil; foliar spraying with *A. platensis* showed high protection against *Fusarium* infection (95%), followed by *D. muscorum* (90%), then *A. oryzae* (69.9%), these results are consistent with [49], which stated that foliar spraying was more effective than soil application, where the nutrients in the foliar spray are absorbed up directly by the leaves of the plant.

Moreover, the results in this study revealed that the highest total phenolic and flavonoid contents were determined in *Arthrospira platensis,* which provided the pepper plant with the highest percentage of protection through foliar spraying. This result may recommend choosing cyanobacterial strains with elevated levels of phenolics and flavonoids to stimulate immune responses in pepper plants against *Fusarium* wilt. The results explained that spraying with cyanobacteria caused an improvement in pepper plant resistance against biotic stresses, including fungal pathogens [49]. Furthermore, antifungal activity of *A. platensis* [50], *D. muscorum* [51], and *A. oryzae* [18] against fungal pathogens were also reported. Reducing symptoms and severity of infection is one of the most important goals of using fungicides, whether chemical or biological [52]. Undoubtedly, applying natural or biological factors such as cyanobacteria is more environmentally friendly. Interestingly, the use of cyanobacteria understudy led to a reduction in symptoms, and a reduction in the severity of infection was reflected positively on the health of plant growth. These results are consistent with several scientific studies [25,53] that confirm that cyanobacteria contain many antioxidants, proteins, vitamins, hormones, and antimicrobials.

The results indicated that *F. oxysporum* hurt all vegetative pepper growth compared to healthy control. *F. oxysporum* minimized shoot length by 46.89%, root length by 48.45%, and several leaves by 45.07%, respectively. Therefore, the infection of the pepper plant with

*F. oxysporum* caused a significant inhibition of all growth parameters, where our findings align with those of a huge number of other researchers [3,54]. Regarding the effect of cyanobacteria, it was observed that infected plants treated with *D. muscorum*, *A. oryzae,* and *A. platensis* in soil and foliar spraying exhibited promising recovery. It was determined that *A. platensis* was the most effective cyanobacteria for recovering plant height by 70.64% and 69.53%, root lengths by 92.94%, 92.94%, and the number of leaves by 76.92%, and 58.97% through the soil and foliar application, respectively. These results agree with a previous study [21], which reported that the application of *A. platensis* improved plant growth through polysaccharides production. The use of cyanobacteria to enhance crop growth has been proposed as a potential executive performance in crop enhancement. [55]. These results align with [35], who found that treating plants with cyanobacteria greatly improved their vegetative growth. The increase in vegetative growth and crop yield with cyanobacteria could mainly be due to the release of plant nutrients like N, P, and K and the excretion of plant growth regulators (auxin, gibberellins), amino acids, and vitamins [56].

After applying cyanobacteria, photosynthetic pigments had become a clear positive indicator of sufficient treatments. By analyzing data from this investigation, it was obvious that chlorophylls a and b (Chl a and b) were severely inhibited in *Fusarium* infected pepper plants by 58.22% and 59.46%, respectively. This reduction in chlorophyll was induced by the production of reactive oxygen species (ROS) after the attack with *Fusarium*, which destroyed chlorophyll contents, preventing the plants from capturing sunlight and reducing photosynthesis [38,57]. Chlorophyll disruption, reduced chlorophyll synthesis, and thylakoid membrane strength are also diminished [58]. On the other hand, the present study showed that the level of carotenoids in pepper plants increased by 70% in response to *Fusarium* infection. These findings are consistent with those of other studies [3,6,39,59,60], which found that the content of carotenoids in plants increased dramatically in response to *Fusarium* infection. In terms of cyanobacteria's beneficial impacts, it was discovered that diseased plants improved after applying *D. muscorum*, *A. oryzae*, and *A. platensis* through various modalities. It was found that foliar and soil application of *A. platensis* was the most effective way to enhance infected plants' Chl a, b, and carotenoids levels. Increased chlorophyll contents in infected plants treated with cyanobacterial strains could be resulted from the higher amount of atmospheric nitrogen assimilation by cyanobacteria then transported to pepper plant tissues [59].

Moreover, cyanobacteria supply decreased ethylene production and chlorophyll, stimulated the synthesis of carotenoids which defend chlorophyll from oxidation, and increased chlorophyll content [60]. These results are in agreement with the results reported in this study. Photosynthetic protection may have been supplied by improved synthesis of carotenoids due to enhancing ROS scavenging [61].

In the current study, the results clearly showed *F. oxysporum* infection caused a major reduction in contents of soluble sugars by 38.50% and soluble protein by 57.13% over the healthy control. Still, it caused proline increment by 11.84% over the healthy control. These results agree with the previous studies [3,62]. The infected pepper plants treated with cyanobacteria showed amelioration in the contents of total carbohydrates, protein, and proline. These results may be explained by the potency of cyanobacteria to secrete complex heteropolymers, polysaccharides, and lipopolysaccharides to induce defense-related gene expression [63,64]. Cyanobacteria-treated plants were capable of fighting against *Fusarium* infection by accumulating more proline, which protects proteins from oxidation [65]. The osmolytes (soluble sugar, proline, and soluble protein) levels in the cyanobacteriatreated plants were significantly higher than those in the infected control plants. Also, oil organic carbon, nutrient absorption, and nitrogen fixation were all improved due to the cyanobacteria treatment [66]. As a result, our findings reveal that osmolyte content significantly increased when infected plants were treated with cyanobacteria, as previously explained [49,62]. This increase in soluble proteins can be explained by activating plant defense systems when pathogens are challenged.

According to the findings of this research, pepper *Fusarium* wilt disease resulted in a rise in phenolics over the healthy control plant. On the other hand, it was observed that infected plants treated with cyanobacteria exhibited a marked increase in phenolics over the infected control plants. The present data indicated that the highest growth in phenolic level was recorded by the infected plants treated with *A. platensis* in the soil treatment. The increased accumulation of phenolics by treatment with cyanobacteria resulted in stress tolerance of the pepper plant against *Fusarium* infection [66,67]. It was observed that cyanobacteria supplementation reduced the generation of H2O2 and lipid peroxidation (MDA) significantly over the infected control plants. Accumulation of H2O2 increased in infected control plants, causing an increase in lipid peroxidation (MDA) over the healthy control plants. Supplementation of infected plants with cyanobacteria reduced the generation of H2O2 over the infected control plants leading to a declined lipid peroxidation (MDA). The data revealed that the most effective cyanobacterial treatment was foliar spraying with *A. platensis*. The findings here are comparable to a previous study conducted by [26], which reported that the application of biological stimulators under stress conditions decreased MDA.

The activity of POD and PPO was assessed to identify enzymes involved in protecting the infected plant. *Fusarium* wilt disease increased the activities of POD and PPO enzymes in the infected control plants over the healthy control plants; however, the application of cyanobacteria through different modes to the infected plants significantly enhanced the PPO and POD enzyme activity over the infected control plants. The recorded data revealed that the maximal increase in the activities of PPO and POD enzymes was detected in the infected pepper plants treated with *A. platensis* and *A. oryzae* through the foliar spraying, respectively. Protective enzymes such as POD and PPO are the most significant in biotic stress response [68]. These enzymes are involved in the early stages of plant resistance to various stressors and the synthesis of phenolic compounds. According to our findings, the antioxidant enzymes in infected plants treated with cyanobacteria in multiple modes, such as soil mode and foliar spraying, significantly increased. The plant displayed distinct strategies for coping with stress by increasing the activity level of some antioxidant enzymes in the cell to maintain a low concentration of reactive oxygen species [26,69].

By detecting the antioxidant isozymes (POD and PPO) by native PAGE, the results showed that a significant part of the plant's response to various stresses is the isozyme substance, which also serves as an important metabolic regulator. Isozyme is a clear indication of the occurrence of resistance, as it plays an important role in mitigating or limiting free radicals that result from oxidative explosions. The findings of this research fully agree with the other scientific reports [70,71]. In the presence of a *Fusarium* wilt disease, cyanobacteria can induce gene expression in infected plants similarly to antioxidant enzymes. It can produce chemicals activating plant immunity, such as phenols and natural hormones [63].

In this study, results highlighted that *Fusarium* infection decreased IAA but increased SA and ABA contents in the infected control plants over the healthy control plants. However, applying cyanobacteria through different modes to the infected plants significantly increased the levels of IAA and SA hormones but decreased ABA concentrations over the infected control plants. Plant hormones are natural organic compounds that affect growth and metabolism, thereby affecting all external manifestations and chemical reactions. These act as chemical signals to activate or inhibit plant growth. When plants are exposed to infections, a considerable modulation in the biosynthesis of hormones occurs, affecting various growth processes [38]. Phytohormones produced by the cyanobacteria have an important function in controlling plant fungal diseases, activating several genes responsible for systemic resistance in plants [49]. The modulation of plant hormone contents is essential in plant defense reactions against *Fusarium* wilt [72]. The SA plays an important role as a plant hormone, as it stimulates growth and works to activate the chemical and synthetic resistance of the plants against any pathogen, increases the absorption of nutrients, and increases the process of photosynthesis [73,74]. Recently, SA has been used externally or

internally as inducers against various plant pathogens [75,76]. SA has been described as a key molecule in the signal transduction pathways of the biological stress response by using cyanobacteria to activate and increase SA, IAA as these hormones act as antimicrobial substances [76,77]. The significance of ABA in plant disease resistance is unclear when compared to that of the plant hormones jasmonic acid and salicylic acid, both of which play key roles in disease resistance [78]. Similarly to our findings, previous research has recorded the accumulation of ABA during the infection of sugar beets by fungi [79].

#### **5. Conclusions**

It is concluded that *Fusarium* wilt generated oxidative destruction and developed into reduced growth and dropped physiological performance. Applying cyanobacteria to *Fusarium* infected pepper plants through soil application or foliar spraying activated the immunity of infected pepper plants. The infected plants treated with cyanobacteria showed enhanced photosynthetic pigments and accumulation of osmoprotectants, phenols, and antioxidant systems that also act as a scavenging tool to remove the excess ROS under *Fusarium* infection. Therefore, it could be applied in agricultural fields through soil or foliar application. As far as we know, this is the first evidence to report that cyanobacteria metabolites influence the isozymes of pepper plants attacked with *Fusarium*, and supplementary molecular findings can provide information on the impact of cyanobacteria on the metabolism of the plant under biotic stress. This study supports the positive application of cyanobacteria in protecting pepper plants under fungal infection; however, further studies are required to unravel actual mechanisms.

**Author Contributions:** Conceptualization, M.S.A., A.M.A. and H.H.S. Methodology, A.M.A., W.A.A., H.H.S., D.A.R. and M.S.S. Software, D.A.R., A.M.A., M.S.S. and M.S.A.; Formal analysis, H.H.S., M.S.A., A.M.A., W.A.A. and M.S.S. Investigation, D.A.R., A.M.A. and M.S.A.; Resources, A.M.A., M.S.A. and H.H.S.; Data Curation, A.M.A. and M.S.A.; Writing—original draft preparation, M.S.A., A.M.A., W.A.A. and H.H.S.; Writing—Review and Editing, A.M.A., D.A.R., H.H.S., M.S.S. and W.A.A.; Supervision, M.S.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** The author would like to thank the Deanship of Scientific Research at Majmaah University for supporting this work under Project Number No. R-2022-231.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the article.

**Acknowledgments:** The authors are grateful to Abd Almoneam Sharaf. Mohamed M. Ali and Amr S. Mohamed for their help during the study, critical reading of the manuscript, and discussions. The authors are also grateful to the Botany & Microbiology Department Faculty of Science, Al-Azhar University 11884 Nasr City, Cairo, Egypt & Botany, and Microbiology Department, Faculty of Science, Cairo University for the technical support offered during this work.

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