**1. Introduction**

Faba bean (*Vicia faba*) is Egypt's most important legume crop and is widely produced throughout the Mediterranean as a protein source for human and animal consumption [1]. The high protein content of faba bean ranges from 25 to 35 percent and has been credited with its nutritional value. The seeds are also high in thiamin, tocopherols, niacin, and folic acid and are notably high in calcium and iron [2]. At the same time, its cultivation increases the amount of nitrogen in the soil [3].

Chocolate spot disease of faba bean is caused by *Botrytis fabae* and *B. cinerea* and is considered the most important disease in the Northern region of the Egyptian Nile Delta [4], which has relatively low temperature and, at the same time, high relative humidity that favor the spread of this disease [5]. The disease causes a severe yield loss reaching 60–80%

**Citation:** Alnefaie, R.M.; EL-Sayed, S.A.; Ramadan, A.A.; Elmezien, A.I.; El-Taher, A.M.; Randhir, T.O.; Bondok, A. Physiological and Anatomical Responses of Faba Bean Plants Infected with Chocolate Spot Disease to Chemical Inducers. *Life* **2023**, *13*, 392. https://doi.org/ 10.3390/life13020392

Academic Editors: Hakim Manghwar and Wajid Zaman

Received: 6 December 2022 Revised: 20 January 2023 Accepted: 23 January 2023 Published: 31 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

among the susceptible cultivars [6]. It decreases the total carbohydrates, nitrogen, nucleic acid, and protein contents of the yielded seeds [7]. Under stress conditions, the production of reactive oxygen species (ROS) (known for signaling intermediates during abiotic and biotic stress conditions) increases and causes plant oxidative stress [8]. In fact, ROS damages cellular membranes in the processes of lipid peroxidation and are also able to cause harmful effects on DNA, proteins, and chlorophyll [9]. Plants produce several major antioxidant enzymes, for example, superoxide dismutase (SOD), which has an essential role in singlet oxygen and scavenging ROS from the cytosol, mitochondria, and chloroplasts in the cell [10].

Utilization of chemical inducers is a new approach in fungal and bacterial infections control within an environmentally friendly defense system in crop plants. These substances induce resistance throughout the signal transduction system, which promotes the production of specific enzymes that catalyze biosynthetic responses to form resistance compounds such as polyphenols, and pathogenesis-related proteins that enhance the plant resistance to pathogens [11]. Salicylic acid (SA) is an endogenous growth regulator that works as a phenolic non-enzymatic antioxidant (a defense mechanism in plants against stress conditions) that helps plants to regulate some physiological activities [12]. The SA also plays a vital role in the plant growth and development, seed germination, pigmentation, photosynthesis, transpiration rate, ion uptake and transport, fruit yield, glycolysis, and induces changes in leaf anatomy and chloroplast ultrastructure [13]. The use of SA significantly reduced chocolate spot disease severity in faba beans caused by *B. fabae* [14,15].

From the anatomical point of view, Cárcamo et al. [16] on *Zea mays*, L., Nour et al. [17] on bean and Gomaa et al. [18] on *Lupinus termis* L., reported that SA minimized the harmful effects of stress conditions. Applying SA improved anatomical measurements of cell wall, epidermis, fiber strands, cortex, xylem and phloem tissues, the parenchymatous area of the pith and vessel diameter, midvein, and leaflet lamina. Benzoic acid (BA) is a natural antioxidant organic acid also considered a biosynthetic precursor of SA [19]. It works as a key intermediate in shikimate and phenyl propanoid pathways. Shikimic acid is a precursor of many alkaloids, aromatic amino acids, and indole derivatives that improve plant growth and productivity [20] and provide plants with abiotic stress tolerance [21].

Moreover, oxalic acid (OA) is crucial in controlling fungal infection [22] since fungal mutants deficient in OA production were non-pathogenic to common bean plants [23]. Decreasing OA accumulation by using fungal mutants or the overexpression of oxalate oxidase leads to ROS generation, allowing the plant to activate some defense responses [24]. At later stages of pathogen infection, OA reduces ROS production [25]. As a result, at an advanced stage of *Sclerotinia sclerotiorum*, the plant antioxidant system most likely plays a role in inhibiting ROS formation [26]. Nicotinic acid (NA), known also as niacin, nicotinamide, and vitamin B3, is a known component of the pyridine dinucleotide coenzymes NADH and NADPH, which are involved in a variety of enzymatic oxidation-reduction events in plant cells [27]. Nicotinamide is a growth-regulating substance that can modify various physiological features of plants in small amounts [28]. Moreover, nicotinamide is a stress-related chemical that causes and controls the activity of the secondary metabolic accumulation process and/or defensive metabolism expressed in plants [29]. Niacin may be utilized to improve stress tolerance in kiwi fruit when exposed to short-term stressful conditions [30]. Furthermore, foliar spray with the niacin solution increased NADPH and NADP+ levels and decreased both O2 − generation and H2O2 content for a short time.

The present study examines the protective effects of selected organic acids and resistance inducers in controlling the chocolate spot disease of faba bean plants and studying their effects on the antioxidant defense system.

#### **2. Materials and Methods**

Chocolate spot disease of faba bean was surveyed at six Egyptian Governorates, namely El-Beheira (Nubaria and Kafr-Eldawar), Kafer El-Sheik (Sakha), Gharbiya (Tanta), Minufiya (Serce-Alian), Sharkia (Zagazig) and Qalubia (Qalub). The severity of the chocolate spot disease of faba bean in Egypt varied by local weather (temperature and humidity) in each Governorate.

The survey was conducted during 2019/2020 and 2020/2021 growing seasons, where the survey was chosen to coincide with the flowering, fruiting, and late fruiting stages of faba bean when the disease reached its peak [31,32]. The survey of the examined sites started at one corner of each field and transected in an M-shaped pattern for approximately 800 paces, stopping at ten equally spaced spots along the way for sampling.

#### *2.1. Studies on the Causal Pathogens*

#### 2.1.1. Isolation of Chocolate Spot Pathogens

Samples of faba bean leaves naturally infected with chocolate spot disease symptoms were collected from the studied locations at the flowering stage. The infected leaves were cut into small pieces (5 mm), each containing a single lesion. The infected tissues were sterilized by soaking in 5% sodium hypochlorite for two minutes, then washed thoroughly several times with sterilized distilled water and dried between two layers of sterilized filter paper. The surface sterilized pieces were transferred onto potato dextrose agar (PDA) plates at the rate of five pieces/plate. All plates were incubated at 20 ± 1 ◦C for 5–7 days. The isolated fungi were purified using the hyphal tip technique [33].

#### 2.1.2. Identification of Isolated Fungi

Isolated fungi were identified as described by Moussa et al. [34] according to their morphological and microscopical characteristics. The identification was carried out at the Department of Mycology, Survey and Identification Unit, Plant Pathology Research Institute, Agriculture Research Center, Giza, Egypt. Pure cultures of each isolate were kept on PDA slants at 4 ◦C for further studies.

#### 2.1.3. Pathogenicity Test

Pathogenicity test was carried out using seven local isolates of *B. fabae*, i.e., (Nubaria, Kafer-Eldawer, Sakha, Tanta, Serce-Alian, Zagazig, and Qalub) under greenhouse conditions.

#### Inoculum Preparation

Isolates of *B. fabae* were grown on leaves of faba bean extract agar media. For spore suspension preparation, the medium was added into sterilized Petri dishes prior to solidification. Then the solidified media in plates were inoculated on equal discs (5 mm) of each test isolate and incubated at a temperature of 20 ◦C ± 1 for a period of 12 days [35] under alternating light (12 h) and darkness (12 h) procedure in automatically incubated to boost the production of spores. For replication, a total of ten plates were used for each isolate. When the incubation period passed, 10 mL of distilled sterilized water was added to the plates and then brushed carefully using a rubber brush. Three layers of cheesecloth were used to filter the suspension in order to eliminate the residues of mycelia. A Spencer Haemacytometer slide was used to count the number of spores/mL in the spore suspension, and then the spores/mL rate was adjusted to approximately 2.5 × <sup>10</sup><sup>5</sup> of *B. fabae.*

#### Plant Preparation

Faba bean susceptible cultivar Giza 429 (*Vicia faba*, L.) used in these experiments were obtained from the Field Crops Research Institute, Agricultural Research Center, Giza, Egypt. Four abiotic inducers (SA, NA, OA, and BA) were obtained from Sigma Company.

Faba bean plants were grown in plastic pots (20 cm), each planted with eight seeds and thinned to five plants/pot with five pots specified for each treatments under greenhouse conditions. After forty-five days from sowing, each group of faba bean plants was sprayed until runoff of abiotic inducers 24 h before inoculation with *B. fabae* at the rates of 1, 3, and 5 mM for salicylic acid (SA), 1, 2, and 3 mM for Nicotinic acid (NA) and Oxalic acid (OA). Furthermore, Benzoic acid (BA) was applied at 0.8, 1.6, and 3.2 mM. All examined

materials were firstly dissolved in 2 mL of 100% dimethyl sulfoxide (DMSO) and then adjusted to the final concentration using sterilized water for each inducer to examine the possibility of alleviating the adverse effects of chocolate spot. The sprayed plants were covered with polyethylene bags for two days before spraying with *B. fabae* spore suspension (2.5 × 105 spores/mL), about 10 mL/each pot. Whereas the control plants were sprayed with 10 mL sterilized water only.

#### Pathogenicity Assessment and Development of Choloate Spot Disease

The inoculated plants were examined for chocolate spot disease infection. The diease severity were recorded after 2, 3, and 5 days of spray *B. fabae* inoculation. This test was done under greenhouse conditions following Bernier et al. [36]. Moreover, four abiotic inducers, SA, NA, OA, and BA, were used as comparison treatments. Each treatment was repeated three times, and the experimental design was a randomized complete block design under open field conditions to investigate their effectiveness against chocolate spot disease severity in faba bean. The inducers were sprayed twice, first at 15 days (at the 1st leaf stage) while the other was at 30 days (at the 6th leaf) from sowing. After that results were recorded with natural infection at 15, 30, and 50 days after the second spray treatment in the two successive seasons (2019/2020 and 2020/2021).

#### Determination of Chocolate Spot Disease Severity

The disease severity was recorded after 2, 3 and 5 days of inoculation using the scale (0–9) using the following equation:

$$\text{Disease severity}^{\circ}\%=\Sigma(\mathbf{n}\times\mathbf{v})/9\text{N}\times100\tag{1}$$

whereas: n = number of plants in every grade, v = numerical grade, N = total number of examined plants, and 9 = maximum disease grade.

#### *2.2. Biochemical Analysis*

Antioxidant activity of some enzymes performed on the tested inducers best concentration (5 mM for SA, 3 mM for both OA and NA and 3.2 mM BA) which were noticiable in disease severity results. Treated and untreated samples were taken before spraying and 6, 12, 24, and 48 h after spraying. A known weight of *vicia faba* leaves which was extracted in 10 mL of 100 mM phosphate buffer (pH 6.8) and kept at 4 ◦C overnight. The extract was centrifuged at 5000 rpm for ten minutes and reserved to assay the activities of enzymes [37].

#### 2.2.1. Peroxidase (POX) Assay

The POX activity was assayed according to [38]. Aliquot of 0.2 mL plant enzyme extract was reacted with 5.8 mL of phosphate buffer (50 mM; pH 7.0), 2.0 mL pyrogallol (20 mM) and 2.0 mL hydrogen peroxide (20 mM). The increase in absorbance was determined within 60 s against a reagent without enzyme at 470 nm using a spectrophotometer. The amount of crude enzyme that converts one mM of hydrogen peroxide in one minute at room temperature equals one unit of enzyme activity.

#### 2.2.2. Polyphenol Oxidase (PPO) Assay

The PPO activity was assayed according to Atrooz [39]. A volume of 2.0 mL extract of plant enzyme was reacted with 1.2 mL of phosphate buffer (pH 6.8) and 0.6 mL catechol (2%). The blank tube has only the substrate and the buffer. Thenafter, all samples incubated for 5 min. and the reaction stopped by adding 1 mL of H2SO4 and the optical density was read at wavelength 430 nm by a spectrophotometer at intervals of 20 min for 100 min. The activity of PPO was expressed as the change in the absorbance of the mixture every 0.5 min. period.

#### 2.2.3. B-1, 3 Glucanase Assay

The method of Abeles and Forrence [40] was used to determine B-1, 3 glucanase activity. Laminarin was used as substrate and dinitro salicylic acid as reagent to measure the reducing sugars. Plant enzyme extract (0.5 mL) was added to 0.5 mL of 0.05 M potassium acetate buffer (pH 5) containing 2% Laminarin. The mixture was incubated for 60 min at 50 ◦C. The reaction was stopped by adding one ml of dinitrosalicylic acid reagent and heating the tubes for 5 min at 100 ◦C. The tubes were cooled and 3 mL of distilled water were added before assay. The optical density was adjusted at 500 nm. B-1, 3 glucanase activity was expressed as mM glucose equivalent released gram fresh weight tissues/60 min.

#### 2.2.4. Chitinase Assay

Twenty five grams of chitin was milled, suspended in 250 mL of 85% phosphoric acid (H3PO4) and stored at 4 ◦C for 24 h, then blended in 2 L of distilled water using a warning blender and the suspension was centrifuged. This washing procedure was repeated twice. The colloidal chitin suspension in the final wash was adjusted to pH 7.0 with 1 N NaOH, separated by centrifugation and the pelted colloidal chitin was store at 4 ◦C. The determination was carried out according to the method of [41]. One mL of 1% colloidal chitin in 0.05 M citrate phosphate buffer (pH 6.6) in a test tube, then one ml of enzyme extract was added and mixed by shaking. Tubes were kept in a water bath at 37 ◦C for 60 min, then cooled and centrifuged before assaying. Reducing sugar was determined by adding 1 mL of supernatant with 1 mL of dintrosalicylic acid and 3 mL distilled water in the test tubes and boiled in water bath for 5 min and then cooled, then determined at 540 nm. Chitinase activity was expressed as mM N-acetyl glucose amine equivalent released gram fresh weight tissue/60 min.
