Valorization of Capsicum annuum seed extract as an antifungal against Botrytis cinerea

Botrytis cinerea Pers., the causal agent of gray mold, is an airborne pathogen that causes signi�cant damage to tomato crops worldwide at all development stages and post-harvest. In this study, the aqueous extract of Capsicum annuum seeds was screened for its phytochemical constituents and assessed at various concentrations (10, 20, 30, and 60%) for antifungal activity in vitro. Selected biochemical, pathological, agronomical, physicochemical, and morphometrical traits were investigated to determine the effectiveness of applying the aqueous seed extract and salicylic acid either separately or in combination to tomato seeds and fruits in vivo. Phytochemical screening of the aqueous seed extract showed the presence of 2, 2-diphenyl-1-picrylhydrazyl, phenolic and �avonoid contents, quinic acid, protocatechuic acid, syringic acid, p-coumaric acid, trans-ferulic acid, rutin, quercetin-3-o-rhamonosic, kaempferol, naringenin, and apigenin at various concentrations. The �ndings suggested that the aqueous extract at a concentration of 60% was most e�cient in vitro where mycelial growth was < 3.8 mm, mycelial growth inhibition was > 52%, and mycelial growth rate of < 1.05 mm/h. In vivo, the combined treatments of tomato seeds produced the greatest reduction in gray mold damage (disease severity index 8.67%) and the most favorable growth parameters of seedlings were chlorophyll a > 1.50 mg/g.f.Wt.; chlorophyll b > 1.76 mg/g.f. Wt.; total chlorophyll content > 3.26 mg/g.f.Wt.; seedling fresh weight > 0.43 g; seedling length > 12.43 cm, respectively. Combined preventive treatment applied to tomato fruits inoculated with B. cinerea resulted in the lowest disease severity (percentage of fruit area covered by gray mold < 33.33%; disease severity index < 46.67%) and the most favorable physicochemical attributes (water content < 98.28%; juice yield > 53.35%; pH < 3.59; titratable acidity > 1.37 g/10 ml juice; Brix degree > 4.73; nitrate content < 383.33 mg/kg; electrical conductivity < 2.47 mS/cm) and morphometrical attributes (fruit �rmness > 3.03). The combined treatments resulted in the strongest activity of peroxidase (> 4.162 units/mg/min), ascorbate peroxidase (> 31.66 µmol/mg/min), and malondialdehyde (> 3.90 µmol/g) on the tomato fruits. The aqueous extract of C. annuum seeds combined with salicylic acid had positive effects in terms of inhibiting B. cinerea and is thus a promising and environmentally friendly alternative substitute for chemical fungicides towards sustainable agriculture under climate change


Introduction
Tomato (Solanum lycopersicum L.), a member of the Solanaceae family, is the second-most-important vegetable crop after potato (Solanum tuberosum L.) worldwide, is commonly cultivated for edible fruits that are used fresh or processed (Quinet et al., 2019).As of 2019, there were some 4.85 million hectares of tomato plants producing 182.3 million tons of fruit worldwide (Quinet et al., 2019).S. lycopersicum contains health-promoting compounds, including phytosterols, carotenoids, monounsaturated fatty acids, essential amino acids, proteins, vitamins, and minerals that may play important roles in preventing or combating cancer, diabetes, and cardiovascular and neurological diseases as well as slowing or reversing the effects of aging (Ali et al., 2021).
Tunisia is the 16th-largest producer of tomatoes worldwide, with more than 1.3 million tons harvested in 2018 (about 70% of which was destined for processing), and the industry plays a signi cant role in the country's socioeconomic life (GIFruit, 2018).
Tomato crops are affected by various diseases, among which the gray mold caused by Botrytis cinerea Pers.(teleomorph: Botryotinia fuckeliana) is one of the most destructive (Deng et al., 2022).This Ascomycota causes signi cant economic losses pre-and post-harvest when climatic conditions are favorable (i.e., low temperatures and humid conditions) (Bahammou et al., 2017).The symptoms of gray mold disease include velvety, gray-brown mold on stems and leaves and soft rot with whitish "ghost spots" (small 3-8 mm rings) on the fruit surface that lead to serious damage during all development stages through storage and result in regrettable losses in total yields (Yfanti et al., 2021).Moreover, the disease is responsible for approximately $10-100 million in economic losses in tomato production worldwide annually, decreasing yields by as much as 60% ( Several control approaches based on the integration of various management strategies for this airborne fungus have been documented, including efforts to prevent appressorium formation and inhibit spore germination (Bahammou et al., 2017;Li and Zou, 2017).Chemical fungicides are the most common tactic that Tunisian farmers employ to reduce gray mold losses since they offer the quickest means to obtain successful results (Deng et al., 2022).However, fungicide treatments may be hazardous to human and animal health and may contribute to ecological pollution.Further, B. cinerea has developed some level of resistance to many fungicides, including hydroxyanilide, pyrimethanil, procymidone, diethofencarb, fenhexamid, benzamide, dicarboximides, and anilinopyrimidines (Mosbach et al., 2017;Adnan et al., 2019).
Foliar treatments of tomatoes (leaves and fruits) with salicylic acid and aqueous extracts (from the seeds, roots, leaves, and bulbs of many botanical plants) represent a promising environmentally friendly alternative for achieving the total or partial reduction of mycelial growth and destruction of B. cinerea spores (Bahammou et al., 2017;Ullah et al., 2019).Several researchers have found Capsicum annuum extracts to be effective as a bio-fungicide against many phytopathogens, decreasing disease severity and stimulating plant defenses (Shayan et al., 2013;Silva et al., 2017;Simo et al., 2019).Chemical analysis of the aqueous extract of C. annuum seeds shows a pro le rich in avonoids, phenolic compounds, alkaloids, saponins, triterpenoids, terpenoids, phlobatannins, and tannins that have antifungal activity su cient to delay or suppress the action of many phytopathogens (Zimmer et al., 2012;Echave et al., 2020).The promoter effect of salicylic acid may be attributable to its regulation of biochemical and physiological processes (i.e., the biosynthesis and availability of organic foods, growth regulation, differentiation, the division and elongation of cells, photosynthesis, the movement of mineral nutrients, and ion uptake) in ways that stimulate plant growth and defenses (El-Shafey et al., 2020; Sampedro-Guerrero et al., 2022).Thus, much effort has been devoted to enhancing the activities of these treatments by combining them (Ullah et al., 2019;Sampedro-Guerrero et al., 2022).The aim of the current study was to complete a phytochemical analysis of the aqueous seed extract of Capsicum annuum.The antifungal activities of the extract against B. cinerea have been studied by assessing their effects on mycelial growth under laboratory conditions, but we investigated the effectiveness of preventive treatments with the aqueous seed extract and salicylic acid (applied separately and in combination) by evaluating the development of gray mold on tomato fruits and seedlings in vivo.We considered a range of attributes-biochemical (peroxidase, polyphenol oxidase, catalase, ascorbate peroxidase, total phenolic content, total protein content, and malondialdehyde), pathological (disease severity index, resistance level, and percentage of fruit area covered by gray mold), agronomical (chlorophyll a, chlorophyll b, total chlorophyll content, fresh weight of seedlings, and seedling length), physicochemical (water content, juice yield, pH, titratable acidity, Brix degree, nitrate content, and electrical conductivity), and morphometrical (fruit rmness and color density).We performed these analyses on treated (aqueous seed extract and/or salicylic acid) and untreated healthy tomato seeds and fruits and compared the results with those of the positive control.We focused on tomato because it is one of the most economically important crops in Tunisia, and gray mold damages all parts of the plant, especially leaves and fruits, even post-harvest.

Preparation of the aqueous seed extract
Capsicum annuum seeds were washed thoroughly in tap water, air-dried, and used to prepare the extract by mixing 10 grams of crushed seeds with 100 ml of sterile distilled water.The mixture was squeezed through four folds of ne tissue, and the extracts were centrifuged at 3,000 rpm for 20 min.The supernatants were ltered using Whatman lter paper, and the ltrate was collected in Erlenmeyer asks (250 ml) (Matrood and Rhouma, 2021;Yaseen et al., 2021).

Total phenolic content
The total phenolic content was extracted according to the method of Folin-Ciocalteu: 100 µl of the aqueous seed extract was added to 2 ml of saturated sodium carbonate and 100 µl of Folin, vortexed, and allowed to stand for 30 min.The optical density was determined at a wavelength of 750 nm using a spectrophotometer (Apolonio-Rodríguez et al., 2017).

Total avonoid content
The total avonoid content was evaluated with the aluminum chloride colorimetric assay: 400 µl of the aqueous seed extract was mixed with 120 µl of sodium nitrite (5%) followed, after 5 min of incubation, by 120 µl of aluminum chloride (10%).After 6 min, 800 µl of sodium hydroxide (1 mol/L) was added to the mixture.The mixture was allowed to stand for 15 min, and absorbance was measured at 510 nm (Al-Mayahi and Fayadh, 2015).The total phenolic and avonoid content was expressed as mg gallic acid equivalents per gram dry weight (mg GAE/g DM).
DPPH (1, 1-diphenyl-2-picrylhydrazyl) We performed a DPPH assay according to the method described by Echave et al. (2020).The stock reagent solution was prepared by dissolving 7.8 mg of DPPH in 100 ml of ethanol, and 1 ml of DPPH and 1 ml of the aqueous seed extract were vortexed well before being kept in the dark for 30 min at room temperature.The absorbance of the solution was measured at 517 nm using a spectrophotometer.The scavenging activity was calculated according to the formula DPPH (%) = [(Ac-Ae)] × 100, where Ac is the absorbance of the control and Ae is the absorbance of the aqueous seed extract (Echave et al., 2020).

Identi cation and quanti cation of active compounds by HPLC-DAD
The identi cation and quanti cation of active compounds were performed using a Shimadzu UFLC XR system (Shimadzu Corp., Kyoto, Japan) equipped with a SIL-20AXR autosampler, a CTO-20 AC column oven, an LC-20ADXR binary pump, and a diode-array detector (DAD).Chromatographic separation was obtained by HPLC.The mobile phase consisted of formic acid (0.1%) in both acetonitrile (A) and water (B).The injected volume was 5 µl, and the ow rate was kept at 0.5 ml/min.The diode-array detector scanned from 190 to 400 nm (Echave et al., 2020).

In vitro evaluation of the activity of the aqueous seed extract against Botrytis cinerea
The antifungal activity of the aqueous seed extract was evaluated in inhibiting the mycelial growth of B. cinerea in vitro using the poisoned food technique.Thus, 20 ml of each extract concentration was incorporated separately under aseptic conditions into 200 ml of molten potato dextrose agar (PDA) medium.In this experiment, four concentrations (10,20,30, and 60%) of the aqueous seed extract were tested for their e cacy.After 10 ml of poisoned medium was poured into separate sterile Petri dishes, a one-disc plug (Ø 0.5 cm) of B. cinerea (from a 4-day-old culture) was placed in the center of the poisoned medium.A pathogen plug without aqueous extract served as a control treatment.Three replicates (with nine plates/replicate) were conducted for each individual treatment, and the plates were incubated at 28 ± 2°C for 8 days (Matrood and Rhouma, 2021).
Mycelial radial growth (mm) was measured 1, 2, 3, 5, 6, and 8 days after inoculation.The percentage of mycelial growth inhibition (MGI) was assessed 8 days after inoculation according to the formula of Matrood and Rhouma (2021), MGI (%) = (1-Ce/Ct) × 100, where Ce is the radial growth diameter of B. cinerea in the presence of the aqueous seed extract and Ct is the radial growth diameter of the B. cinerea in the absence of the extract.The mycelial growth rate (MGR) was calculated according to the formula of Matrood and Rhouma (2021) In vivo evaluation of the aqueous seed extract and/or salicylic acid on tomato seedlings in the presence of Botrytis cinerea Tomato seeds (cv.Firenze) were sterilized by treatment with ethanol, washed with sterile distilled water twice, and dried at room temperature.The seeds were treated separately with the aqueous seed extract (at a concentration of 60%), 0.001M salicylic acid, or a combination of the aqueous seed extract (at a concentration of 60%) and salicylic acid.This assay was carried out by dipping the seeds into a ask containing each of the aforementioned treatments for 30 min; 24 h later, the tomato seeds were inoculated with the pathogen (10 6 spores/ml).Two controls were performed, one by dipping the tomato seeds in pure water (negative control) and the other by exposing them to the pathogen only (positive control).The treated seeds were placed in Fahraeus medium at an average of seven seeds per Petri plate and subsequently incubated for 21 days in a growth chamber with a 12 h/12 h day/night photoperiod at 27°C.The experimental design was a randomized complete block arranged in 3 blocks of 10 Petri plates each.The entire experiment was repeated twice (Brito et al., 2021).
At the end of the experiment (21 days of incubation), the tomato seedlings were carefully removed from the Petri dishes.The symptoms of gray mold were scored using a scale from 0 to 4 such that 0 = healthy leaves, 1 = approximately 1-25% of the tomato leaves infected, 2 = approximately 26-50% of the tomato leaves infected, 3 = approximately 51-75% of the tomato leaves infected, and 4 = more than of 75% of the tomato leaves infected (Brito et al. 2021).Using McKinney's formula, we calculated the disease severity index (DSI) as a percentage, (Σvn)/(NV) × 100, where v represents the numeric value of the disease index scale, n is the number of seedlings assigned to the disease index scale, N is the total number of the seedlings, and V is the numeric value of the highest disease index scale (Matrood and Rhouma, 2021).
In addition, the chlorophyll content was evaluated using the method described by El-Shafey et al., (2020).Thus, 0.5 g of tomato leaves were homogenized in 10 ml of 80% cold acetone (-10°C) and centrifuged at 14,000 rpm for 5 min, and the mixture was then assessed spectrophotometrically at 663 and 645 nm.The chlorophyll content data was calculated using the following equations, which yield the content of chlorophyll a (Chl a), chlorophyll b (Chl b), and total chlorophyll (Chl T) expressed in mg/ g fresh weight: We evaluated other agricultural measurements of the seedlings' fresh weight (FWS; g) and length (SL; cm) as well.DSI, Chl a, Chl b, Chl T, SL, and FWS were evaluated on 30 seedlings per treatment and per block (3 blocks).
In vivo evaluation of the aqueous seed extract and/or salicylic acid on tomato fruits in the presence of Botrytis cinerea Healthy tomato fruits (cv.Firenze) at the ripening stage and of uniform size were harvested from the eld of the Regional Centre of Agricultural Research of Sidi Bouzid, Tunisia, and immediately transported to the laboratory.The selected fruits were rinsed with sterile distilled water, immersed in sodium hypochlorite solution (2.5%) for 3 min, washed twice with sterile distilled water, and dried under a sterile ow cabinet.The fruits were wounded (5 mm deep) on the blossom end with a sterile needle, and 20 µL of each treatment was pipetted onto each wound site.After 2 h, the tomato fruits were inoculated with 20 µl of a spore suspension of Botrytis cinerea (10 6 spores/ml).This experiment consisted of ve treatments: T1, the negative control (fruits treated only with 20 µl of sterile distilled water); T2, the positive control (fruits treated only with 20 µl of B. cinerea spore suspension); T3, the aqueous seed extract (at a concentration of 60%); T4, 0.001M salicylic acid; and T5, salicylic acid + the aqueous seed extract (at a concentration of 60%).An average of six treated fruits were placed in plastic containers on sterile wet paper.The containers were enclosed in a plastic bag to maintain high humidity (> 90%) and subsequently incubated for 7 days in a growth chamber with an 8 h/16 h day/night photoperiod at 21°C.The experimental design was a randomized complete block and was arranged in 3 blocks of 10 containers each, and the entire experiment was repeated twice (Brito et al. 2021).
After 7 days, a set of pathological, morphometrical, physicochemical, and biochemical attributes was investigated to determine the antifungal activity of the aforementioned treatments on tomato fruits.The percentage of fruit area (PFA) covered by gray mold was estimated using the formula PFA (%) = (LAP/TLA) x 100, where LAP represents the fruit area covered by gray mold and TLA represents the total fruit area (Rhouma et al. 2021).The disease severity index (DSI) and resistance level of each treatment were also assessed.
The morphometric quality of the fruits was analyzed based on two parameters, rmness and color density.The rmness was determined according to the method of Geasa and Hassan (2022) using an FT-327 penetrometer with a precision of 0.1 kg, with assessments in 3 locations around the equatorial region.The color density of the fruits was measured using a Konica Minolta CR10 colorimeter.The device ran according to the L*a*b* color space system, generally known as CIELAB, created by the International Commission on Illumination in 1976.The readings were obtained on the sidewall of the fruits.The expression of color was determined using three values: brightness (L), variance from red to green (a), and variance from blue to yellow (b) (Hajlaoui and Hajlaoui, 2021).The (a/b) value is an essential parameter in color analysis, re ecting the color quality as well as the proportion of the presence of red (i.e., the ratio increases in step with the percentage of red color) (El-Shafey et al., 2020).
The physicochemical measurements that we performed on the tomato fruits included the water content, juice yield, pH, titratable acidity, soluble sugar content (Brix degree), nitrate content, and electrical conductivity.The water content of the fruit was measured as the difference between the weight of the fresh fruit, determined directly after harvesting using a scale, and the weight of the dry fruit determined after drying in a drying oven at 100°C for 24 hours.The equation is WC (%) = (FW-FS)/(FW) × 100, where WC is water content, FW is the fresh weight of the fruit, and FS is the dry weight.The fruits were weighed, cut with a knife, and then crushed in a Black and Decker electric orange press.Once the extraction was complete, the juice obtained was weighed and the juice content was determined using the equation JC (%) = (weight of juice)/(fresh weight of fruit) × 100, where JC is the juice content, FF is the fresh weight of the fruit, and FS is the dry weight.Once we nished the determination of JC, the extracted juice was used to determine the pH by immersing the electrode of an electronic pH meter (Hanna type HI 9125, USA) in tomato juice for 1-2 min (Fratianni et al., 2020).The TSS was measured and expressed in degrees Brix by pouring 1-2 drops of tomato juice onto the prism of a digital pocket refractometer (type Atago pal-, Japan).The prism of the refractometer was cleaned with distilled water and dried between samples (Hajlaoui and Hajlaoui, 2021).The electrical conductivity and nitrate content of the juice were determined using a portable conductivity meter (Hanna type HI 99301, USA) and sensor (LAQUA-twin, Germany), respectively.The titration technique served to evaluate the titratable acidity (TA) of the juice (to pH 8.1 with 0.1 M NaOH).The TA was estimated as g of citric acid (AC)/10 ml of juice using the formula TA = k×a×f, where TA is titratable acidity de ned as g AC/10 ml juice, K is a factor for citric acid conversion equal to 0.64, f is the 0.1M NaOH solution factor equal to 1, and a is the volume of 0.1M sodium hydroxide solution used expressed in ml (Hajlaoui and Hajlaoui, 2021).
To understand the biochemical changes in the tomato fruits treated preventively with salicylic acid and aqueous seed extract (separately and in combination), we assessed a set of biochemical parameters.For assessment of the enzyme activities, fruit extraction was performed by homogenizing 1 g of sample fruit in extraction buffer (50 mM phosphate buffer + 0.2 mM EDTA + 1 mM PMSF [pH = 7]) and centrifugation at 12,000 × g for 10 min at 4°C.The supernatant was collected, transferred to Eppendorf tubes, and assayed for enzyme activity.The peroxidase activity was determined according to the method of Yfanti et al., (2021), and a decline in absorbance was found at 420 nm.The catalase activity was examined according to the method of Kasmi et al. (2017), and the absorbance was examined at 240 nm.The method of Gholamnezhad (2019) was employed to determine the polyphenol oxidase (PPO) activity, and the absorbance was examined at 408 nm.The total phenolic content was assessed using the Folin-Ciocalteu method, and the absorbance was examined at 760 nm (Kasmi et al., 2017).The malondialdehyde (MDA) content was determined according to the method of Gholamnezhad et al. (2016), and the absorbance was examined at 532 and 600 nm.The total protein content was calculated according to the method described by Gholamnezhad (2019).The ascorbate peroxidase (APX) activity was evaluated according to the method of Fratianni et al. (2020), and the absorbance was examined at 290 nm.

Statistical analysis
We performed the statistical analysis using the mean values of the replicates.The data were analyzed by ANOVA using SPSS version 20.0 statistical software (SPSS, SAS Institute, USA).Duncan's Multiple Range Test served to check the homogeneity of variances and normality as well as the differences among the treatments.All of the statistical tests were performed at a signi cance level of 5% (P ≤ 0.05).A principal component analysis (PCA) was also performed to assess the relationships among the correlated parameters in a smaller subset using XLSTAT-2020 software.

Determination of total phenolic content, total avonoids, and antioxidant activities
The analysis of the aqueous Capsicum annuum seed extract revealed the presence of antioxidant and bioactive compounds.The total phenolic content (TPC) of the aqueous seed extract was 8.83 mg gallic acid equivalent/g of dry weight.The total avonoid content was 10.87 mg gallic equivalent/g of dry weight.The extract also contains a relatively high percentage of DPPH (71.47%) (Table 1).Quinic acid (183.788ppm) was the primary ingredient in the investigated extract, followed by quercetin (107.900ppm) and protocatechuic acid (66.653 ppm), while the concentrations of apigenin, p-coumaric, and trans-ferulic acid were the lowest, with values of 0.081, 0.448, and 0.649 ppm, respectively (Table 1).The data are the average of 3 samples of the aqueous seed extract per replicate (with 3 replicates).

Means±standard error
In vitro evaluation of the activity of the aqueous seed extract against Botrytis cinerea The data presented in Figs.<link rid=" g1">1</link>-a and 1-b show clearly that the treatments had a strong effect (P < 0.01) on the mycelial growth of B. cinerea after 7 days of incubation using the poison food technique.A signi cant decrease in mycelial growth was noted as the concentration of the aqueous seed extract increased, with the greatest reduction occurring at the highest concentration (60%).The various treatments with the aqueous seed extract were associated with signi cant inhibitions in mycelial growth, which varied from 11.32% (at the 10% concentration) to 52.20% (at the 60% concentration) (Fig. 1-a).The aqueous seed extract at a 60% concentration showed the lowest growth rate, with a decrease in the mycelial growth rate of 1.05 mm/h (control = 2.54 mm/h).However, the lowest growth rate was recorded at a concentration of 10% (2.10 mm/h) (Fig. 1-b).
Statistical analysis revealed highly signi cant differences (P < 0.01) between the concentrations of the aqueous seed extract and the sampling moments on the one hand and their interactions (concentrations * sampling moments) on the other (Table 2).The highest values for the mycelial growth of Botrytis cinerea were recorded at the last sampling moment.Generally, the concentration of the Capsicum annuum aqueous extract correlated negatively with the mycelial growth values at all sampling moments (Table 2).The data thus obtained revealed that the aqueous extract at a 60% concentration reduced mycelial growth signi cantly, with the values ranging from 1.18 mm (after 1 day of incubation) to 3.80 mm (after 8 days of incubation).However, the lowest values were at a 10% concentration, 1.73 and 7.05 mm after 1 and 8 days of incubation, respectively (Table 2).The values for the untreated plates were 1.96 and 7.95 mm after 1 and 8 days of incubation, respectively (Table 2).The small letters compare various concentrations at the same sampling moment.
The capital letters compare the same concentration at various sampling moments.
The data are the average of 9 Petri dishes per replicate (with 3 replicates).

Means ± standard error
These results indicate that aqueous C. annuum extract at a concentration of 60% was the most effective treatment under laboratory conditions; the assays described in the following discussion served to con rm them.
In vivo evaluation of the effect of the aqueous seed extract and/or salicylic acid on tomato seedlings in the presence of Botrytis cinerea The DSI, Chl a, Chl b, Chl T, FWS, and SL values of the tomato seedlings showed that the effectiveness of the preventive applications of the aqueous seed extract and/or salicylic acid differed signi cantly (P < 0.01) (Table 3).The treatment of the tomato seeds with the aqueous Capsicum annuum extract and/or salicylic acid in the presence of B. cinerea reduced the DSI signi cantly.The combination of the aqueous seed extract and salicylic acid had the best results, with a decrease in the DSI of 8.67%, followed by treatment with the aqueous extract, with a decrease in the DSI of 13% (positive control = 94.33%)(Table 3).The level of resistance to gray mold indicated that only the treatment of tomato seeds with the aqueous extract applied separately or in combination with salicylic acid, was effective against B. cinerea (Table 3).

Means ± standard error
As Table 3 shows, the treatment effect on Chl a was greater when the tomato seeds were treated separately with the aqueous extract (2.03 mg/g fresh weight) or salicylic acid (2.05 mg/g fresh weight) (positive control = 1.87 mg/g fresh weight; negative control = 5.67 mg/g fresh weight).However, the combination of the treatments increased the Chl b and Chl T content in the seedlings by 1.76 and 3.26 mg/g fresh weight, respectively.
The tomato seeds treated separately or in combination with the aqueous extract and salicylic acid showed the greatest seedling length, with values ranging from 11.43 cm (aqueous extract) to 12.43 cm (salicylic acid + aqueous seed extract) (positive control = 6.63 cm; negative control = 13.63 cm) (Table 3).
The PCA revealed a clear difference between the effects provided by treatments (seed extract aqueous, salicylic acid, seed extract aqueous + salicylic acid, and the two controls) in terms of the DSI, Chl a, Chl b, Chl T, FWS, and SL of the tomato seedlings, explaining 82.47% of the total variance (F1 and F2) (Fig. 2).The rst principal component, PC1, explained 64.20% of the total variance.The second, PC2, explained 18.27% of the total variance and had a positive correlation with Chl a and Chl T and a negative correction with FWS and SL (Fig. 2).The results of correction obtained revealed that Chl T and Chl a correlated strongly, with an r-value of 0.947.The FWS and SL of the seedlings likewise correlated strongly, with an r-value of 0.841.However, the DSI correlated negatively with FWS (r = -0.902)and SL (r = -0.938)(Table S1).
In vivo evaluation of the effect of the aqueous seed extract and/or salicylic acid treatments on tomato fruits in the presence of Botrytis cinerea As Table 4 shows, the various treatments were associated with signi cant reductions in the PFA covered by gray mold and with the DSI (P < 0.01).The treatment of the tomato with only the aqueous seed extract thus proved effective against B. cinerea.In fact, the PFA and DSI decreased dramatically in the presence of the extract, by an average of 23.33% (positive control = 86.67%)and 22.09% (positive control = 93.33%),respectively (Table 4).

Means ± standard error
The levels of resistance to gray mold assessed based on the DSI and observed on the tomato fruits after treatment with the aqueous seed extract and/or salicylic acid varied (Table 5).The treatment with the aqueous extract thus protected the fruits, reducing their susceptibility to disease compared with the positive controls, which were highly susceptible to gray mold (Table 4).

Means ± standard error
The analysis showed signi cant differences in the effectiveness of the aqueous extract and/or salicylic acid against B. cinerea (P < 0.01) in terms of fruit rmness (FF), pH, electrical conductivity (EC), water content (WC), juice yield (JY), titratable acidity (TA), soluble sugar content (°Brix), color density (L), and nitrate content (NC), whereas no difference (P ≥ 0.05) was observed in the color density (a/b) (Table 5).Generally, the application of the aqueous extract and/or salicylic acid produced excellent results against B. cinerea with respect to the morphometric parameters of the tomato fruits assessed, with the improvements at times exceeding 50% compared with the controls (Table 5).
Among the treatments, the combination of the aqueous extract and salicylic acid increased the rmness of the fruit (3.03) in comparison with the negative control (2.97) (positive control = 0).The lowest values of pH and WC were registered on fruits treated with the aqueous extract + salicylic acid (pH = 3.59; WC = 98.28%) and on the untreated fruits (pH = 3.57; WC = 98.34%).
Likewise, the aqueous extract (1.98 mS/cm) and salicylic acid (1.75 mS/cm) recorded the lowest values of electrical conductivity (positive control = 2.63 mS/cm; negative control = 2.11 mS/cm).The most effective treatments of the fruits in terms of juice yield were the separate treatments with the aqueous extract (53.52%) and salicylic acid (53.85%) (Positive control = 30.62%;negative control = 43.41%).The combination of the aqueous extract and salicylic acid produced the best results in terms of enhancing TA (1.37 g/10 ml juice) (positive control = 0.89 g/10 ml juice; negative control = 1.16 g/10 ml juice).Fruits inoculated by B. cinerea and treated separately with salicylic acid (Brix = 5.20) or in combination with the aqueous extract (Brix = 4.73) showed an improvement in sugar content (positive control = 4.80; negative control = 6.37).The aqueous extract applied separately or in combination with salicylic acid showed the lowest NC, 373.33 and 383.33 mg/kg, respectively (positive control = 793.33mg/kg; negative control = 453.33mg/kg).However, the statistical analysis revealed no signi cant difference (P ≥ 0.05) in color density between the positive and negative controls (Table 5).
With respect to biochemical characterization, the PPO, catalase (CAT), peroxidase (POX) and APX activities, total phenolic content (TPC), total protein content (TP), and MDA differed dramatically between the treatments (P < 0.01) (Table 6).The fruits treated with the aqueous extract + salicylic acid (4.162 units/mg/min) demonstrated higher peroxidase activity against gray mold compared with the positive (3.555 units/mg/min) and negative (3.958 units/mg/min) controls, followed by the aqueous extract treatments (3.775 units/mg/min) (Table 6).The fruits treated with the aqueous extract and those left untreated showed the lowest PPO activity (11.08 and 11.01 units/mg/min, respectively) and the highest CAT activity (40.20 and 39.96 µmol H 2 O 2 /mg protein, respectively) (positive control: PPO = 18.01 units/mg/min; CAT = 38.12µmol H 2 O 2 /mg protein) (Table 6).The aqueous extract and salicylic acid signi cantly increased the APX activity, with values of 36.17 and 41.94 µmol/mg/min, respectively (positive control = 19.11µmol/mg/min; negative control = 36.59µmol/mg/min) (Table 6).The results presented in Table 6 indicate that the TPC was greater when the fruits were treated with salicylic acid, with a value of 4.42 µg/g.However, B. cinerea showed strong resistance to the combination of the aqueous extract and salicylic acid (2.63 µg/g).The treatments reduced the TPC in comparison with the positive control (36.36 mg/g), with values of 11.71 mg/g for salicylic acid and 12.87 mg/g for the aqueous extract (Table 6).The same result was obtained for MDA: the decrease was greater for the fruits treated with the aqueous extract (2.47 µmol/g), followed by the salicylic acid treatments (2.99 µmol/g) (positive control = 5.68 µmol/g) (Table 6).

Discussion
Tunisian farmers favor the use of chemical fungicides to prevent or limit the losses to their tomato harvests caused by gray mold because this approach delivers results quickly.However, numerous studies have demonstrated that chemical control methods are neither economical nor adequate and harm the environment and human and animal health (Deng et al. 2022).At the same time, the processing of C. annuum for harissa and hrous generates a large amount of seed waste that can be di cult for manufacturers to dispose of.This material can be used to create an aqueous extract that, recent research suggests, shows promise as an environmentally friendly fungicide.
Analyses of the aqueous extract from seeds of C. annuum have identi ed a wide range of phytochemical compounds.).The increase in plant growth may be related to the presence of vitamins, nutrients, or other compounds in the aqueous extracts that promote the production of plant hormones (Al-Mayahi et al., 2015).The promoter effect of salicylic acid may be attributable to its effect as a bioregulator of biochemical and physiological processes (the biosynthesis and availability of organic foods, cell differentiation, cell division, cell elongation, the movement of mineral nutrients, and ion uptake), leading to increased plant growth rmness, ethylene production, and total soluble solids content.The activity of defense-related enzymes may, then, indicate the resistance of tomato fruit to B. cinerea, with an increase in their activity and accumulation depending on the treatments and the physiological condition of the plant.A series of morphological and biochemical changes trigger the synthesis of defense chemicals against plant phytopathogens that suppress or retard their development (Gholamnezhad et al., 2016;Gholamnezhad, 2019).The fungistatic activity of the aqueous extract of C. annuum seeds against B. cinerea reported in the present study could be attributable to the presence of avonoids, phenolic compounds, alkaloids, saponins, triterpenoids, terpenoids, phlobatannins, and/or tannins (Zimmer 2012;Echave et al., 2020).
Chl a = 12.41 × absorbance 663 − 2.59 × absorbance 645 Chl b = 22.90 × absorbance 645 − 4.68 × absorbance 663 Chl T = Chl a + Chl b (El-Shafey et al., 2019; El-Shafey et al., 2020).El-Shafey et al. (2019, 2020) found that a combination of salicylic acid and aqueous extract functioned as a plant growth promoter by enhancing rhizosphere mineral transfer, photosynthesis, ion homeostasis processes, and the production of growth hormones.El-Saadony et al. (2017) and El-Shafey et al. (2020) demonstrated that the foliar treatment of P. sativum with salicylic acid combined with botanical extracts had a positive effect on photosynthetic pigments (Chl a, b, and T) in plants infected with Botrytis spp., possible by increasing the levels of endogenous cytokinins (Fratianni et al., 2020) that stimulate growth and chloroplast differentiation.In the present study, among the treatments tested, the combination of the aqueous extract of C. annuum seeds and salicylic acid resulted in the greatest reductions in the intensity of gray mold and the strongest biochemical, physicochemical, and morphometrical reactions on the tomato fruits.Kasmi et al. (2017) evaluated the e cacy of aromatic and medicinal plants (Melissa o cinalis, Cymbopogon citratus, Thymus vulgaris, and Lavandula o cinalis) against fruit gray mold and found that aqueous extracts of C. citratus signi cantly decreased the disease incidence on tomato fruit.Brito et al. (2021) found that the application of the aqueous T. vulgaris extract and phycobiliproteins reduced disease intensity by more than 76%.Du et al. (2019) found 1-methylcyclopropene to be effective against B. cinerea infection of tomato as measured by the levels of decay and fruit

Table 1
Phytochemical screening of total polyphenol content, total avonoid content, antioxidant capacity, and content of phenolic constituents in aqueous seed extract of Capsicum annuum

Table 2
(10,20,30 the aqueous seed extract of Capsicum annuum at various concentrations(10,20,30, and 60%) on the mycelial growth a Duncan's Multiple Range Test; the values followed by the various superscripts differ signi cantly at P ≤ 0.05.b probabilities associated with individual F tests

Table 3
Effect of preventive treatments using the aqueous seed extract of Capsicum annuum and/or salicylic acid on the disease severity index, chlorophyll a, chlorophyll b, total chlorophyll content, fresh weight, and the length of tomato seedlings in the presence of Botrytis cinerea under laboratory conditions a Duncan's Multiple Range Test; the values followed by the various superscripts differ signi cantly at P ≤ 0.05.b Probabilities associated with individual F tests DSI: Disease Severity Index; Chl a: chlorophyll a; Chl b: chlorophyll b; Chl T: Total chlorophyll content; FWS: fresh weight of seedlings; SL: seedling length; ER: extremely resistant; R: resistant; S: susceptible; HS: highly susceptible; Nd: not determined Data are the average of 30 tomato seedlings per treatment and per block (3 blocks).

Table 4
Effect of preventive treatments using the aqueous seed extract of Capsicum annuum and/or salicylic acid on the percentage of fruit area covered by grey mold and the disease severity index on tomato fruits inoculated with Botrytis cinerea under laboratory conditions Duncan's Multiple Range Test; the values followed by the various superscripts differ signi cantly at P ≤ 0.05.
a b Probabilities associated with individual F tests PFA: percentage of fruit area covered by grey mold; DSI: disease severity index; ER: extremely resistant; R: resistant; S: Susceptible; HS: highly susceptible; Nd: not determined Data are the average of 30 tomato fruits per treatment and per block (3 blocks).

Table 5
Effect of preventive treatments using the aqueous seed extract of Capsicum annuum and/or salicylic acid on the morphometric parameters of tomato fruits inoculated with Botrytis cinerea under laboratory conditions a Duncan's Multiple Range Test, values followed by various superscripts differ signi cantly at P ≤ 0.05.b Probabilities associated with individual F tests Data are the average of 30 tomato fruits per treatment and per block (3 blocks).

Table 6
Effect of preventive treatments of the aqueous seed extract of Capsicum annuum and/or salicylic acid on catalase and peroxidase activities, polyphenol-oxidase, ascorbate peroxidase, total phenolic content, total protein content, and malondialdehyde in tomato fruits inoculated with Botrytis cinerea under laboratory conditions Multiple Range Test, values followed by various superscripts differ signi cantly at P ≤ 0.05.
a Duncan's Gholamnezhad (2019))2019)ls, and avonoids in C. annuum reported here differed from those reported byZimmer et al. (2012).These differences may be attributable to the geo-climatic conditions, extraction and analytical methods, plant maturity, and/or botanical variety(Echave et al., 2020).The concentrations of polyphenols (quercetin and quinic acid) that we observed differ from those reported by Fratianni et al.(2020), who found that Capsicum spp.contained mainly gallic and chlorogenic acid,Wahyuni etal.(2013),foundhighconcentrations of ascorbic acid, and Materska and Perucka (2005), who found high concentrations of quercetin-3-O-l-rhamnoside, sinapoyl, and feruloyl glycosides.The quanti cation of the polyphenol pro le of the aqueous extract of C. baccatum seeds by Zimmer et al. (2012), however, showed relatively high concentrations in quercetin, ascorbic acid, and capsaicin, and Zamljen et al. (2021) found relatively high concentrations of quinic acid in an extract of C. annuum.Regarding the in vitro biological control of B. cinerea, the aqueous extract of C. annuum seeds at a 60% concentration appeared to be the most effective of the treatments tested, being associated with mycelial growth of less than 3.8 mm (after 8 incubation days) and decreases in the mycelial inhibition growth (52.20%) and mycelial growth rate (1.05 mm/h).Little information is available about the effectiveness of the aqueous seed extract against B. cinerea under laboratory conditions.Silva et al. (2017)found it to have the strongest antifungal activity against Colletotrichum spp.under laboratory conditions, andMaracahipes et al. (2019)found that it inhibited the growth of the phytopathogens Fusarium lateritium, F. solani, F. oxysporum and Colletotrichum gloeosporioides.The latter researchers observed abnormal growth of the hyphae and clustering of cells in microscopic analysis.Simo et al. (2019)found that the aqueous extract of Capsicum spp.signi cantly inhibited completely the growth of Phytophthora megakarya, with a reduction of 100%.Likewise, other researchers have reported that the seed extract of Capsicum spp.displayed potent activity against Aspergillus niger, A. avus, Rhizopus spp., and Penicillium spp.(Soumya and Nair, 2012), Saccharomyces cerevisiae, Candida tropicalis, C. albicans, C. guilliermondii, C. parapsilosis, Kluyveromyces marxiannus, Pichia membranifaciens(Diz et al. 2006), Citrobacter spp., Bacillus spp., Micrococcus spp., and Pseudomonas spp., with an inhibition rate of greater than 90% under laboratory conditions (Shayan and Saeidi, 2013).Several aqueous extracts have been shown to reduce the mycelial growth of B. cinerea.Thus, El-Khateeb et al. (2013) noted that aqueous leaf extracts of Vitis vinifera, Zizyphus spina-christi, Punica granatum, and Ficus carica decreased the mycelial growth and spore germination of B. cinerea.Bahammou et al. (2015)found that the aqueous extracts of the botanical plants Asteriscus imbricatus, Pulicaria mauritanica, Lavandula dentata, and Globularia alypum inhibited completely the growth of B. cinerea at 1,000 and 2,000 ppm.Zakari et al. (2016)reported that the extent of inhibition at the highest concentration (80%) showed that Vernonia amygdalina (55%) and Azadirachta indica (57%) were more effective on B. cinerea.combination between the aqueous seed extract and salicylic acid has been found to decrease signi cantly the DSI of the pathogenicity of B. cinerea and to stimulate the growth of tomato seedlings, but there have been no studies about treatments of tomato seed with this combination.Ha et al. (2022)reported that the treatment of Rosa spp.leaves with nanosilver and salicylic acid reduced disease incidence, and El-Shafey et al. (2020) reported that salicylic acid and aqueous extracts of botanical plants greatly diminished the severity of chocolate spot disease caused by Botrytis fabae when applied to V. faba in the eld.On tomato, Li and Zou (2017) found that salicylic acid and calcium ions reduced the severity of gray mold disease and the mycelial growth of B. cinerea, possibly because of the stronger defense responses of the plants increases in the production of superoxide anion phenylalanine ammonia-lyase activity and hydrogen peroxide and in the expression of pathogenesis-related protein genes to many phytopathogens(Li and Zou, 2017).Salicylic acid has antifungal effects, acting as a decoupling agent of organelle membranes (Da Rocha Neto et al., 2015), and decreases the fungal development of several pathogens (including B. cinerea as well as Eutypa lata, Alternaria alternata, Phytophthora infestans, Fusarium oxysporum, Penicillium expansum, and Geotrichum candidum) in the greenhouse and in the eld by displacing the hydroxyl group on the aromatic ring of the salicylic acid structure (Da Rocha Neto et al., 2015; Sampedro-Guerrero et al., 2022).The nding that the treatment of tomato plants with the aqueous extracts decreased the severity of the disease caused by inoculations of B. cinerea and stimulated plant defenses is consistent with the ndings of previous researchers (Yfanti et al., 2021).Al-Masri et al. (2015) and Bahammou et al. (2017) assessed the effectiveness of some botanical plants against gray mold on tomatoes and found that the aqueous extracts of Cystoseira tamariscifolia, Inula viscose, and Bifurcaria bifurcate signi cantly decreased the incidence of the disease.Similarly,Gholamnezhad (2019)found that foliar spraying of the aqueous extracts of Azadirachta indica seeds and Ferula assa-foetida leaves drastically decreased the severity of the disease caused by B. cinerea.In fact, the foliar application of the aqueous extracts and salicylic acid as biological control agents against B. cinerea not only reduced the severity of the disease but also improved plant growth in this study.EI-Awadi et al. (2017) and El-Saadony et al. (2017) reported that spraying Beta vulgaris and Pisum sativum with salicylic acid and the aqueous extract of Allium sativum increased plant growth.These results are consistent with the work of Hanafy et al. (2012), who found that the aqueous extract of A. sativum combined with salicylic acid stimulated the vegetative and root biomass of Sche era arboricola.El-Shafey et al. (2020) demonstrated similarly that the foliar application of botanical extracts (from A. sativum cloves, A. cepa bulbs, and Eucalyptus spp.leaves) combined with salicylic acid signi cantly increased the fresh and dry weights of Vicia faba plants in the presence of Botrytis fabae compared with untreated controls.Foliar treatment with salicylic acid and aqueous extract signi cantly improved the fruit yield and vegetative growth of Capsicum annuum (Ibrahim et al. 2019), Solanum lycopersicum (Ullah et al. 2019)d faba (El-Shafey et al. 2020) inoculated with Botrytis spp.Similar studies have previously shown that salicylic acid combined with aqueous extract from some botanical plants promote plant height (El-Shafey et al., 2019; El-Shafey et al., 2020 Apolonio-Rodríguez et al. (2017) found that the aqueous leaf extracts of wild grapevines and citrus displayed the highest fungal activity of the extracts that they tested against B. cinerea.Similar results with other aqueous extracts have been reported by Piesik et al. (2015).More recently, Ahmadu et al. (2021) reported that the seed extract of Moringa oleifera treatment at concentrations of 5 mg/ml (100%) and 10 mg/ml (98.10%) reduced the spore germination and colony diameter of B. cinerea.The