*2.3. Anatomical Studies*

Samples were prepared for anatomical investigation according to the method proposed by Nassar and El-Sahhar [44]. One a square centimeter of the terminal leaflet was removed, and it was dehydrated in a succession of solutions with ethyl alcohol concentrations ranging from 50% to 100%. The samples were then embedded in paraffin wax (the melting point of paraffin wax range is 58–62 ◦C using xylol as a solvent). Sections were cut at a thickness of 15 microns using a rotary microtome and then mounted on slides using egg albumin as an adhesive agent. The slides were subjected to a declining sequence of ethyl alcohol solutions ranging from 100% to 50% ethyl alcohol concentrations. The anatomical characters (Each value represents five sections with five readings each) of faba been leaflet including the upper epidermal layer (μm), lower epidermal layer (μm), palisade tissue thickness (μm), spongy tissue thickness (μm), length of the vascular bundle (μm), and width of the vascular bundle (μm) While in the anatomical study, the percentage (%) was calculated to show the increase or decrease attributed to the control.

#### *2.4. Statistical Analysis*

A significant differences test among means of five replicates was performed at a signifi cance level of *p* < 0.05 using the LSD (Least Significant Difference) test [45] using the SPSS software. However, under open field conditions, three replicates were used to compare mean differences.

#### **3. Results**

#### *3.1. Severity of Chocolate Spot Disease*

Four abiotic inducers, including SA, NA, OA, and BA were investigated for their effects on the severity of chocolate spot disease on faba bean plants grown in greenhouse conditions (Figure 1).

**Figure 1.** Effect of spraying faba bean plants with varying concentrations of organic acids on the severity of chocolate spot disease under greenhouse conditions. Letters a to k represent significant levels (*p* < 0.05), i.e., a treatment with the letter "a" is significantly different from "b", and "b" is significantly different from "c", and so forth. If two treatments have the same letter, they are not significantly different from each other.

The presented results indicated that all tested abiotic inducers clearly decreased disease severity. There was a positive correlation between the reduction of disease severity and increments in abiotic inducer concentrations. Plants treated with salicylic, nicotinic, oxalic, and benzoic acids reduced chocolate spot disease severity by 83.2, 76.6, 74.2, and 72.8%, respectively, at the highest used concentration.

#### *3.2. Development of Chocolate Spot Disease*

Effects of four abiotic inducers (SA, OA, NA, and BA) on chocolate spot disease severity in faba bean, compared to the control, after 15, 30, and 50 days of treatment with inducers under open field conditions are illustrated in Figure 2.

Results showed that spraying faba bean plants with the tested abiotic inducers were able to manage chocolate spot disease using the tested treatments during the two studied seasons. The result indicated that disease severity increased by increasing the plant age. Whereas among abiotic inducers, salicylic acid at a concentration of 5 mM gave the highest reduction through the two seasons by 81.73 and 79.59%, respectively, followed by nicotinic acid at 3 mM concentrations that led to 73.99 and 71.32% reduction, respectively. Benzoic acid at the concentration of 3.2 mM resulted in the lowest reduction in the two seasons by 60.70 and 56.98%, respectively.

#### *3.3. Antioxidant Enzymes Activity*

The effect of selected chemical inducers (SA: 5 mM, OA: 3 mM, BA: 3.2 mM, and NA: 3 mM) in addition to the untreated control on the activity of enzymes (Peroxidase, polyphenol oxidase, β-1, 3-glucanase, and chitinase enzymes) were presented in Figure 3. It is worth mentioning that the antioxidant activity was increased with increasing the action time of the content of all tested enzymes till 24 h and then decreased compared to the corresponding control. The healthy faba bean leaves recorded significantly (*p* < 0.05) the lowest value of all antioxidant action compared to the infected plants with *B. fabae*.

**Figure 2.** Effect of spraying faba bean with various concentrations of organic acids on the development of chocolate spot disease in field conditions in the two successive seasons. Letters a to k represent significant levels (*p* < 0.05), i.e., a treatment with the letter "a" is significantly different from "b", and "b" is significantly different from "c", and so forth. If two treatments have the same letter, they are not significantly different from each other.

**Figure 3.** Activity of peroxidase (Unit/g fresh wt./min) in faba bean leaves infested with *B. fabae* plants treated with chemical inducers. LSD 5% for healthy and infected plants at 0, 6, 12, 24, and 48 h are 0.012, 0.012, 0.021, 0.004, 0.018, 0.005, 0.00.016, 0.010, and 0.021, respectively. Letters a to k represent significant levels (*p* < 0.05), i.e., a treatment with the letter "a" is significantly different from "b", and "b" is significantly different from "c", and so forth. If two treatments have the same letter, they are not significantly different from each other.

#### 3.3.1. Peroxidase Activity (POX)

The obtained values of peroxidase presented in Figure 3 showed a gradual increase with time intervals 0, 6, 12, 24, and 48 h in chemical inducers treated faba bean plants. Foliar spray of faba bean infested with *Botrytis fabae* with various organic acids resulted in a significant increase (*p* < 0.05) of peroxidase activity compared with the untreated control. The maximum increase in peroxidase activity was recorded after 24 h for all treatments. The most pronounced increase was obtained with SA, followed by NA, OA, and BA. Also, peroxidase activity in healthy plants recorded a reduced value compared with infected plants with time intervals in all treatments.

#### 3.3.2. Polyphenol Oxidase (PPO) Activity

Data presented in Figure 4 indicated that treating faba bean plants infected with *B. fabae* as a foliar treatment with different abiotic inducers resulted in an increase of polyphenol oxidase compared with the untreated infected control.

**Figure 4.** Activity of polyphenol oxidase in faba bean leaves infested with *B. fabae* plants treated with chemical inducers. LSD (5%) for infected and healthy plants at 0, 6, 12, 24, and 48 h are 0.009, 0.008, 0.062, 0.044, 0.099, 0.010, 0.039, 0.011, 0.014, and 0.037, respectively. Letters a to k represent significant levels (*p* < 0.05), i.e., a treatment with the letter "a" is significantly different from "b", and "b" is significantly different from "c", and so forth. If two treatments have the same letter, they are not significantly different from each other.

#### 3.3.3. β-1,3-Glucanase Activity

Data presented in Figure 5 showed a significant increase in the β-1, 3-glucanase activity in all treatments during the examination periods compared to the control in both infected and healthy plants. Some treatments recorded the highest increase in β-1, 3-glucanase activity after 12 h, while other treatments recorded the highest values after 24 h. For infected faba bean plants, OA and SA caused the maximum increase in β-1, 3-glucanase after 12 h, followed by BA. Meanwhile, SA recorded the maximum increase after 24 h, followed by OA then BA came in the third order. The increase of β-1, 3-glucanase activity was more than two-fold of the untreated control. In the case of healthy faba bean plants, all chemical inducers produced the highest increase in β-1, 3-glucanase activity after 24 h, where OA came in the first order, followed by NA, then SA, and BA treatments. After that, the activity reduced slightly at 48 h but was still higher than the untreated control.

### 3.3.4. Chitinase Activity

A perusal of data in Figure 6 showed that the infected and healthy faba bean foliar treated with different abiotic inducers was associated with increased chitinase activity compared with the untreated corresponding control. All treatments increased significantly (*p* < 0.05) chitinase activity. Salicylic acid recorded the maximum increase in this concern after 24 h of both infected and healthy plants, followed by benzoic and oxalic acids, respectively. Meanwhile, nicotinic acid recorded the lowest value compared to untreated plants (control).

**Figure 5.** Activity of β-1,3-glucanases in faba bean leaves infested with *B. fabae* treated with chemical inducers. LSD (5%) for infected and healthy plants at 0, 6, 12, 24, and 48 h are 0.096, 0.096, 0.104, 0.061, 0.122, 0.099, 0.083, 0.012, 0.011, and 0.060, respectively. Letters a to k represent significant levels (*p* < 0.05), i.e., a treatment with the letter "a" is significantly different from "b", and "b" is significantly different from "c", and so forth. If two treatments have the same letter, they are not significantly different from each other.

**Figure 6.** Activity of chitinase in leaves of faba bean infested with *B. fabae* plants treated with chemical inducers. LSD 5% for infected and healthy plants at 0, 6, 12, 24, and 48 h are 0.011, 0.011, 0.120, 0.101, 0.103, 0.066, 0.079, 0.086, 0.105, and 0.049, respectively. Letters a to k represent significant levels (*p* < 0.05), i.e., a treatment with the letter "a" is significantly different from "b", and "b" is significantly different from "c", and so forth. If two treatments have the same letter, they are not significantly different from each other.

#### *3.4. Protein Electrophoretic Banding Patterns*

A banding pattern of the soluble proteins in infected and healthy leaves of faba bean that belongs to the cultivar Giza 429 infected with *B. fabae* and treated with selected chemical inducers. The inducers included salicylic acid (5 mM), oxalic acid (3 mM), benzoic acid (3.2 mM) and nicotinic acid (3 mM), via SDS-PAGE as presented in Table 1. A total of 27 polypeptides of faba bean leaves displayed heterogeneity compared to control. The infected and treated plants with some chemical inducers with molecular weights (MWs) ranged from 35 to 320 kDa after 24 and 48 h of application and 43 kDa in response to oxalic

and benzoic acids only after 24 h from the application. Meanwhile, after 48 h from the application, data showed a new protein polypeptide bands appeared at Mwt. of 172, 104, 59, 44, and 35 kDa due to all organic acid treatments (tested chemical inducers) compared to the control plants. The number if protein bands increased due to the salicylic, nicotinic, oxalic, and benzoic acid treatments, which recorded the numbers of 8, 9, 16, and 14 bands after 24 h from the application. Meanwhile, the protein bands 11, 11, 13, and 11 were recorded after 48 h from the application. The most pronounced increases were observed in response to oxalic acid treatment, which recorded 16 bands after 24 h and 13 after 48 h of application, followed by benzoic acid, which recorded 14 and 11 bands, respectively, after 24 and 48 h. It is evident from the obtained data that all the treatments of faba bean plants with organic acids cause the appearance of polypeptide protein bands with Mwt. of 59 and 51 kDa.


**Table 1.** Separation of soluble proteins (SDS-PAGE) in faba bean leaves treated with various chemical inducers against *B. fabae*.

+ = Presence of band and − = Absence of band.

Data in Figure 7 showed the appearance of a new band at Mwt. of 176, 85, 65, 59, 51, and 41 in response to NA, OA, and BA, and also 172, 130, and 77 kDa.

**Figure 7.** Separation of soluble proteins (SDS-PAGE) in faba bean plant leaves treated with chemical inducers against *B. fabae*.

The infected faba bean plants with *Botrytis fabae* induced a *de novo* synthesis of new polypeptide bands that appeared at molecular weights 297, 104, 66, 59, and 43 kDa after 24 h of application and also at 104, 59, and 35 kDa after 48 h of application. At the same time, there is a disappearance of 3 protein bands with molecular weight 98, 78, and 66 after 24 h of application, and after 48 h, only one band (OA treatment) was absent at Mwt 137 kDa.

Lane M: Marker, (1) Healthy control after 24 h from the application, (2) Infected control after 24 h from application, (3) Salicylic acid after 24 h from the application, (4) Nicotinic acid after 24 h from the application, (5) Oxalic acid after 24 h from the application, (6) Benzoic acid after 24 h from application, (7) Healthy control after 48 h from the application, (8) Infected control after 48 h from the application, (9) Salicylic acid after 48 h from the application, (10) Nicotinic acid after 48 h from the application, (11) Oxalic acid after 48 h from application and (12) Benzoic acid after 48 h from the application.

#### *3.5. Anatomical Characteristics*

Microscopically counts and measurements of specific histological characteristics in transverse-sections through the blade of mature leaflet of faba been plant, benzoic, and salicylic acids presented in Figures 8 and 9a–c. The SA (Figure 9c) recorded the highest value with an increase in leaf thickness by (+30.1%) more than the control (Figure 9a). Such increase in leaf thickness corresponds with the enhancement recorded on the upper and lower epidermal layer thickness as well as palisade and spongy tissues by +42.8, +33.3, +28.0 and +28.5%, respectively more than the control. Such response also resulted in a clear appearance and arrangement of spongy and palisade tissue parenchymatous cells as compared to the control. However, foliar applications with BA (Figure 9b) recorded the lowest value with increased leaf thickness by (+15.8%) more than the control. Such increase in leaf thickness is related to the increment recorded in the upper and lower epidermal layer thickness as well as palisade and spongy tissues by (+28.5%, +22.2%, +12.0%, and +17.8%) respectively, more than the control.

This effect is associated with vast intercellular spaces between both palisade and spongy tissues parenchymatous cells. Furthermore, parenchymatous cells of upper and lower epidermal layers, as well as palisade and spongy tissues, are bigger in size and rounded in shape under treatment with SA. Clearly, Figures 8 and 9a–c revealed that foliar application with SA recorded the highest value with increased thickness of the midrib zone by (+38.9%) more than the control. Such an increase in midrib zone thickness corresponds with the enhancement recorded on the length and width of the vascular bundle as well

as the diameter of xylem vessels by +29.1, +21.4, and +45.4%, respectively, more than the control.

**Figure 8.** Effect of foliar application with chemical inducers on the anatomical characteristics of faba bean (*Vicia faba* L.) terminal leaflet under chocolate spot disease stress.

**Figure 9.** Transverse-sections through blade of the terminal leaflets of faba bean (*Vicia faba* L.) under chocolate spot disease: (**a**) Untreated plant (Control), (**b**) Plant treated with benzoic acid and (**c**) Plant treated with salicylic acid. Abbreviations: Le = Lower epidermis; P = Palisade tissue; St = Spongy tissue Ue = Upper epidermis and Vb = Vascular bundle.

In addition, the area occupied by collenchymatous cells behind the main vascular bundle is occupied by larger sizes and more layers of collenchymatous cells. Such effect corresponds with clear development and differentiation of the main vascular bundle's elements, especially xylem vessel elements. Also, metaxylem vessels also changed from a rounded shape, as recorded in the control, to an elongated shape in the treated plants. This response is mainly due to the increment that occurred in the area occupied by vascular bundle elements, which appeared in the area coupled by xylem vessels, cambium, and phloem as compared to the untreated plants.

#### **4. Discussion**

#### *4.1. Severity of Chocolate Spot Disease*

The greenhouse experimental results indicated that pre-treated faba bean plants with abiotic inducers foliar treatment resulted in a significant reduction in disease severity of *B. fabae* compared with untreated control. Using bioagent isolates revealed antagonistic activity against *B*. *fabae* due to the production of protease, lipase, IAA, and ammonia. Moreover, bioagents release tricalcium phosphate (TCP) which promote multiple plant growth characteristics [46]. Abiotic inducers; SA, NA, OA, and BA gave a reduction of 83.2, 76.6, 74.2, and 72.8% at the highest concentration of each treatment. Among all foliar treatments, SA spray had the highest reduction of 83.2% at 5 mM of 5-day treatment. A second highest reduction of 80.9% was observed at a 3-day treatment with SA with 5 mM concentration. This was followed by NA application that reduced disease severity by 76.7% and 76.6% in 3-day and 5-day, respectively. The third highest reduction in severity was observed with OA application that reduced the severity by 75.8% and 74.2% in 3-day and 5-day treatments, respectively. While the fourth highest reduction was obtained with BA application at 5 mM, which reduced the disease severity by 72.8% and 71.5% in 5-day and 3-day treatments, respectively.

This agrees with Metwaly [47], who studied the evaluation of some chemical inducers, i.e., ascorbic, citric, salicylic acids, and calcium chloride (as a nutrient salt) to control faba bean chocolate spot disease. The study concluded that all tested organic acids significantly reduced the in vitro mycelial growth of the pathogenic fungus (*B. fabae*), and complete growth inhibition was recorded when 2500 ppm concentration was applied [47]. SA was the most effective one, followed by ascorbic acid. Plant treatment with organic acids and calcium chloride under greenhouse and/or field conditions led to a significant effect on controlling the disease and increasing phenols content as well as the activity of oxidative enzymes [6]. The results also agreed with the results of salicylic, citric, ascorbic, and oxalic acids as effective chemical inducers.

Under field conditions, plants treated with SA significantly reduced the disease severity in comparison with the control treatment. SA was highly efficient in controlling the fungal disease compared to other treatments that provided limited partial protection. In this regard, Thakur et al. [48] explained that chemical inducers are elicitors' compounds that activate plant chemical defense. Activation of a variety of biosynthetic pathways was observed in treated plants, which depended upon the used compound. Commonly, the studied elicitors include SA, methyl salicylate, benzothiadiazole, BA, and chitosan, with a key role in phenolic acid production as well as activation of several plant enzymes involved in defense mechanisms. Also, Zian et al. [49] observed that using SA led to a significant decrease in the root rot and wilt diseases of lupine. They observed that the SA leads to an increase in the activity of chitinase, β-1, 3-glucanase, peroxidase, and polyphenoloxidase. Morever, the induced resistance caused by some abiotic (BTH) and/or biotic inducers might provide a practical, eco-friendly management approach to chocolate spot disease when they are combined with suitable agronomic practices [50]. In their study, applying 0.3 and 0.5 mM benzothiadiazole as the foliar treatment significantly reduced the severity of chocolate spot disease.

#### *4.2. Antioxidant Enzymes Activity*

The antioxidant activities by enzymes, such as peroxidase, polyphenol oxidase, β-1, 3-glucanase, and chitinase in the leaves of faba bean plants increased significantly under biotic stress (Figure 3a–d). In this regard, Gholami et al. [51] reported that these antioxidant enzymes were thought to be part of a preservation system that reduced oxidative damage caused by the increased formation of reactive oxygen species (ROS) due to biotic and abiotic stress. Higher content of H2O2 is detoxified at stress conditions through catalase and glutathione peroxidase [52].

Interestingly, the antioxidant system in plants includes enzymes that scavenge ROS, such as ascorbate peroxidase, catalase, peroxidase, and superoxide dismutase [26]. Sairam

and Srivastava [53] determined that plants having higher levels of either inducible or constitutive enzymes showed to be more resistant to oxidative harmful effect. OA appears to have a biphasic effect on ROS metabolism since it suppresses ROS buildup at first and activates their generation at later phases of pathogen infection [25]. Application of bioagents was characterized by the expression of cyclolipopeptides (cLP) genes, encoding for induced resistance factors, as well as the production of lipopeptides, indoleacetic acid, siderophores, hydrocyanic acid, and extracellular enzymes such as amylase, protease, pectinase, and cellulase [54].

Pretreatment of faba bean plant (infected and healthy plants) with the studied natural organic acids improved stress tolerance by increasing the tested antioxidant enzyme activities compared to control. Furthermore, Sharma and Dubey [55] observed that a viable protection against oxidative damage was produced by this antioxidant system. This enhances active oxygen species lifetime in the cellular environment. The defense mechanism of plant β-1, 3-glucanases is directly through suppressing ROS buildup and/or hydrolyzing the fungal pathogen cell walls along with chitinase isozymes. Laboratory studies showed that fungal pathogens were directly affected by β-1, 3-glucanases through deteriorating b-1, 3/1, 6-glucans. Also the chitinases produced their effects via the C1 and C4 bond that are consecutive N-acetylglucosamines of chitins in the cell walls of fungi [56]. The plants treated with salicylic acid increased the activity of enzymes such as catalase, peroxidase, superoxide dismutase, ascorbate peroxidase, and glutathione reductase. [57]. Peroxidase belongs to oxido-reductase enzymes and contributes to oxidation-reduction reactions.

Concerning the effect of benzoic acid on the antioxidant enzyme system, a similar result was obtained by Zhang et al. [58] on tomato seedlings and Amist and Singh [59] on wheat seedlings, who found that the increasing antioxidant enzymes seem to protect plants from oxidative stress. Hassanein et al. [60] found that 100 ppm of nicotinamide (either soaking or spraying) increased the content of antioxidant enzymes (Superoxide dismutase, peroxidase, and catalase) and decreased lipid peroxidation of *Zea mays* plants. Also, in this regard exogenous application of methyl jasmonate increased the activity of antioxidant enzymes, improved photosynthetic pigments and PSII efficiency. This resulted in enhanced growth of pea plants under Cd stress. The improved traits included increments in the fresh and dry weights of shoots and roots. Thus, the mitigating effect of methyl jasmonate was due to its role in cellular redox balance and photosynthetic mechanism of plants under Cd stress conditions [54].

### *4.3. Changes in Protein Electrophoretic Patterns*

The data clearly demonstrated that, in general, after 24 h from the application, there was an absence of some polypeptide bands at molecular weights 117 in response to salicylic acid and 98, 78, and 66 kDa in response to nicotinic acid treatments. In this respect, the disappearance of protein bands and also the synthesis of a new group of soluble proteins were observed by many authors due to biotic and abiotic stress. The absence of some bands may result from inherited effects of infection with *B. fabea*, which explains the basis of the mutational event on the regulatory genes that prevent or attenuate transcription [61].

It is obvious from the obtained results (Figure 4 and Table 1) that all treatments of faba bean plant with organic acids cause the appearance of the polypeptide protein band with Mwt. Of 59 and 51 kDa. In this regard, Spreitzer and Savucci [62] discovered that the 51 kDa band, that might be associated with Ribulose-1, 5-bisphosphate carboxylase activase (Rubisco activase), was elevated. By promoting the separation of firmly bound sugar-phosphates from Rubisco in an ATP-dependent mechanism, this enzyme could modify the Ribulose-1 activity, 5-bisphosphate carboxylase/oxygenase (Rubisco), a crucial enzyme involved in the initiation of photosynthetic and photorespiratory carbon metabolic processes. Enhancing Rubisco's capability has important implications for plant productivity and resource use efficiency [63]. Moreover, these total proteins have osmoprotectant functions and protective effects on cellular structures [64].

Amino acids are primary metabolites that play essential roles in plant immunity against many pathogens. The variation in plant tissues amino acid quantity may determine the chance of environment for the pathogenic attackers like fungi, bacteria, and viruses. This finally strengthens plant defense to resist pathogenic attack effectively or surrender before vigorous infection [65]. Morphological and structural barriers, chemical substances, proteins, and enzymes are all examples of biotic stress protection mechanisms. By preserving products and giving them strength and stiffness, they confer tolerance or resistance to biotic stressors. In addition, amino acids serve an important function in stress response and secondary metabolism in plants [66]. For scavenging ROS resulting from biotic stress (infection with *B. fabae*), the plant has a protective system against the devastating oxidative reaction. The protective system includes osmoprotectants (total soluble protein) and antioxidative enzymes, which are known to play a crucial role in defense mechanisms, and all of them are proteins and can appear as protein bands. In this regard, Ramadan et al. [67] on flax plant found that benzoic acid increased total soluble protein and free amino acids. Also, Mahgoob and Talaat [68] found that foliar application of nicotinic acid at 50 mg/L significantly increased total protein contents of rose geranium.

The appearance of *de novo* synthesis of new polypeptides band and/or increase the band density 77, 59, 51 and 41 may represent an antioxidant enzyme (chitinase and β-1, 3-glucanase) which is considered as defense proteins to protect the plant from pathogens. In ripe cherimoya fruits (*Annona cherimola* Mill.), Goni et al. [69] found a highly expressed constitutive chitinase (at 27 kDa), as well as the creation of a unique acidic chitinase (at 26 kDa) and a 1, 3-glucanase (51 kDa). Likewise, a new basic chitinase at 33 kDa was observed in the post-ripening stage of fruits that were stimulated, as well as another basic constitutive β-1, 3-glucanase at the molecular weight of 76 kDa. In vitro, extracts of these acidic and basic proteins suppressed the growth of *B. cinerea*, a necrotrophic fungal pathogen in grape leaves. At the berries' harvest stage, defense proteins were dominant, particularly various chitinase and β-1, 3-glucanase isoforms that support fruit ripening. Also, Shukry [70] explained that the hydrin is defined at the protein band with Mwt. of 40 kDa. These proteins play a preventative role during water stress through their function as ion traps in dehydrating cells and insulating ions as their concentration increases.
