Punica granatum L. extract contributes to phytopathogens control and enhances Eruca vesicaria (L.) Cav. germination in vitro and in vivo

The study aimed to investigate antimicrobial activity of the hydroal­ coholic crude extract from the fruit peel of Punica granatum (Pp) and punicala­ gin compound (Pg) on phytopathogenic bacterial isolates and its potential use as a sustainable alternative in treatment of vegetable seeds. The antimicrobial activity in vitro was tested by agar well diffusion assay and through viability tests in liquid medium. In vivo treatment with Pp was tested on Eruca vesicaria seeds infected with Xanthomonas campestris pv. campestris. Pp induced the formation of large inhibition zones to the growth of the tested pathogens (35.33 mm ­ 6.66 mm), with dose­dependent effect. Viability tests confirmed the antimicrobial activity of the Pp on X. campestris pv. campestris and P. caro‐ tovorum subsp. carotovorum with minimum inhibitory concentration (MIC) of 125 μg/mL. Punicalagin compound presented MIC of the 31.25 μg/mL. The seed treatment with Pp indicated control of pathogen­induced symptoms in seedlings of the E. vesicaria and positive effect in seed germination, emergence and in stomatal functionality. The results indicate strong potential of the extract from the fruit peel of P. granatum and Punicalagin for formulating botanical pesticides for plant disease control. (*) Corresponding author: macielmcg@gmail.com Citation: DE SOUSA SILVA S., ALVES P.C.S., COUTINHO D.F., LUZ T.R.S.A., FONTOURA G.M.G., BERRETTA A.A., GONÇALVES MACIEL M.C., 2021 ­ Punica grana‐ tum L. extract contributes to phytopathogens control and enhances Eruca vesicaria (L.) Cav. germination in vitro and in vivo. ­ Adv. Hort. Sci., 35(3): 217­231. Copyright: © 2021 De Sousa Silva S., Alves P.C.S., Coutinho D.F., Luz T.R.S.A., Fontoura G.M.G., Berretta A.A., Gonçalves Maciel M.C. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 20 March 2021 Accepted for publication 26 May 2021 AHS Advances in Horticultural Science Adv. Hort. Sci., 2021 35(3): 217­231

Abstract: The study aimed to investigate antimicrobial activity of the hydroal coholic crude extract from the fruit peel of Punica granatum (Pp) and punicala gin compound (Pg) on phytopathogenic bacterial isolates and its potential use as a sustainable alternative in treatment of vegetable seeds. The antimicrobial activity in vitro was tested by agar well diffusion assay and through viability tests in liquid medium. In vivo treatment with Pp was tested on Eruca vesicaria seeds infected with Xanthomonas campestris pv. campestris. Pp induced the formation of large inhibition zones to the growth of the tested pathogens (35.33 mm 6.66 mm), with dosedependent effect. Viability tests confirmed the antimicrobial activity of the Pp on X. campestris pv. campestris and P. carotovorum subsp. carotovorum with minimum inhibitory concentration (MIC) of 125 μg/mL. Punicalagin compound presented MIC of the 31.25 μg/mL. The seed treatment with Pp indicated control of pathogeninduced symptoms in seedlings of the E. vesicaria and positive effect in seed germination, emergence and in stomatal functionality. The results indicate strong potential of the extract from the fruit peel of P. granatum and Punicalagin for formulating botanical pesticides for plant disease control.

Introduction
According to Food and Agriculture Organization of the United Nations estimates, by 2050 the world population should exceed 9.5 billion inhabitants, rais ing the demand for food by up to 60% (FAO, 2016). Plants account for 80% of food ingested in the human diet, providing affordable, safe and nutritious resources for a healthy life. However, pests and dis eases pose a threat to food security, due to damage caused to crops that compromises access to food and rises product prices (FAO, 2017).
Phytopathogenic bacteria causes a large number of different plant diseases, some of which are devas tating to agricultural crops (Van Der Wolf and De Boer, 2015). Ralstonia solanacearum (Smith) (Yabuuchi et al., 1995) stands out as one of most destructive pathogens due to the rapid development of wilting symptoms and death of host plants (Yuliar et al., 2015). The pathogen affects a large range of host plants, comprising almost 450 species from 54 different botanical families (Allen et al., 2005).
Direct losses in important crops are estimated in 0 to 91% (tomatoes) and in 33 to 90% in potatoes (Elphinstone, 2005). The bacterium Xanthomonas campestris pv. campestris (Pammel) Dowson is a vas cular (Ryan et al., 2011) and seedborn (Griesbach et al., 2003) pathogen which is distributed worldwide. Infested seeds may emerge in young seedlings infect ed by the pores on the margin of the cotyledons. This pathogen causes black rot disease, which seriously affects Brassicaceae (Cruciferous) crops (Vicente and Holub, 2013), important food items grown worldwide (Gupta et al., 2013). Finally, Pectobacterium is widely studied softrot bacterial pathogen causing infections in potato crops and stored tubers, reducing the pro duction and quality of tubers (Adeolu et al., 2016). Ralstonia solanacearum, X. campestris and P. carotovorum have been included among the 10 most important bacterial pathogens of the plants accord ing to their economic and scientific impact (Mansfield et al., 2012).
Control of bacterial diseases in conventional agri culture often uses fastacting synthetic pesticides and antimicrobials (Kotan et al., 2014). According to national phytosanitary pesticide database (AGROFIT, 2016), substances unsafe to the environment like kasugamycin, cuprous oxide, copper hydroxide and 'extremely toxics' like benzalkonium chloride were registered for control of soft rot P. carotovorum subsp. carotovorum in potatoes. Pesticides indicated for control of bacterial wilt caused by R. solanacearum like Bismerthiazol and Thiodiazole cop per have shown low efficacy, high phytotoxicity, harmful environmental effects and bacterial resis tance development (Yang and Bao, 2017).
Cultural practices like the use of pathogenfree seeds is recommended to prevent black rot disease in crops (Chitarra et al., 2002). If pathogenfree seed is not available, seed should be treated to eliminate the bacteria. However, seed treatments do not always eliminate 100% of bacteria on or in the seed, and may adversely affect seed germination and vigor (Celetti and Callow, 2002).
The need to reduce chemical pesticide use in crops, associated with demands for healthy food and development of sustainable agriculture, has driven research for natural compounds with low impact on the environment and on people health (Jiménez Reyes et al., 2019). The secondary metabolism of the plants produces many bioactive compounds that pro vide protection against pests and pathogens (Borges et al., 2018). Unlike synthetic pesticides, natural com pounds exhibit rapid biodegradation after use in the field (Soberón et al., 2014), little or no phytotoxicity, abundant sources and low costs, since they come from a renewable source (Zheng et al., 2016). Thus, medicinal plant uses with antimicrobial activity can be considered an effective component in the inte grated management against phytopathogens (Khan et al., 2020).
Punica granatum L. (Pomegranate) is a plant of the Lythraceae family, native from central Asia (northern India to Iran), nowadays cultivated in sever al parts of the world, including Africa and America (ViudaMartos et al., 2010;Erkan and Dogan, 2018). The fruit of Pomegranate (called balausta) is a pulp berry formed by a thick and leathery skin with vari able color depending on the variety. The seeds are a reproductive structure that present a fleshy outer testa called sarcotesta where the juice is extracted (Melgarejo et al., 2020). The production, marketing and consumption of pomegranate fruit have increased rapidly throughout the world in recent years, mainly due to greater awareness of their healthpromoting attributes (Selcuk and Erkan, 2015).
The peel of P. granatum represents 30 to 40% of the fruit, being usually discarded as waste during industrial processing for the production of pome granate juice (Gullon et al., 2016). However, this part of the fruit is rich in phenolic acids, tannins (such as punicalin and punicalagin) and flavonoids with vari ous biological functions, including activity against pathogenic microorganisms (Dey et al., 2012;Türkyılmaz et al., 2013). Punicalagin compound is an important bioactive agent found in pomegranate fruit peel, with antioxidant, antimicrobial, antiviral and immunosuppressive activity. The compound belongs to the ellagitannin family which includes other tannins such as punicalin and gallic acid, char acterized by good water solubility (Akhtar et al., 2015).
In the last years several studies have evidenced the antimicrobial activity of the pomegranate extract against many species of the plant pathogenic fungi suggesting high potential source of natural antifungal agents (Mohamad and Khalil, 2015;Balah and Nowra, 2016;Elsherbiny et al., 2016;Li Destri Nicosia et al., 2016;Rongai et al., 2017;Karm, 2019;El Khetabi et al., 2020). However, few studies have investigated the antimicrobial activity of the pome granate extract against phytopathogenic bacteria (Quattrucci et al., 2013;Farag et al., 2015;Khaleel et al., 2016). Khaleel et al. (2016) have indicated in vitro antimicrobial activity of the ethyl acetate pomegran ate peel extract against R. solanacearum, P. carotovorum subsp. carotovorum and X. gardineri and Farag et al. (2015) highlighted notable in vitro antimi crobial activities from the methanol pomegranate peel extract against variety of temperate climate (race 3, biovar 2) of the R. solanacearum. Despite this, the knowledge about the antimicrobial activity of the hidroalcoholic extract of P. granantum and iso late compounds against these pathogens is scarce. Thus, this study aimed to investigate the in vitro antimicrobial activity of hydroalcoholic crude extract from the fruit peel of the P. granatum and Punicalagin compound on isolates of phytopathogen ic bacteria. The potential of the extract for natural control of X. campestris pv. campestris as an sustain able alternative for treatment of vegetable seeds was assayed.

Bacterial Isolates
The bacterial isolates were provided by the collec tion of the Chemical characterization of the hydroalcoholic crude extract from the fruit peel of the P. granatum Total phenolic compounds. For determination of total phenolics an analytical curve of tannic acid (SigmaAldrich) was carried out. Pomegranate extract was prepared in 50 mL volumetric flask using water as solvent. The samples were homogenized and, the flasks were brought to the ultrasonic bath for 30 min utes. A 0.5 mL aliquot was transferred to another 50 mL flask where 2.5 mL of FolinDenis reagent and 5.0 mL of 29% sodium carbonate were added. The sam ples were protected from the light and the readings were performed after 30 minutes in a UVVis spec trophotometer at 760 nm (Fernandes et al., 2018). All samples were analyzed in triplicate.
Ellagic acid. Ellagic acid (EA) was acquired from Fluka (95.0%, Batch BCBN4398V). The High Performance Liquid Chromatography (HPLC) grade methanol was supplied by J.T. Baker (Mexico City, Mexico), and purified water was obtained using a MilliQ Direct Q5 filter system (Millipore, Bedford, USA). The analytical grade acetic acid was purchased from Synth (Labsynth, Diadema, Brazil).
To determine the ellagic acid content (EAC), the extracts previously diluted in methanol were proper ly homogenate using a vortex and then remained for 30 minutes in ultrasound bath. The solution was fil tered and subjected to HPLC analysis (Shimadzu apparatus equipped with a CBM controller, LC20AT quaternary pump, a SPDM 20A diodearray detector and auto sampler, Shimadzu LC solution software, version 1.21 SP1) using a 100 mm x 2.6 mm Shim pack ODS C18 column.
The mobile phase used for ellagic acid was methanol and acetic acid aqueous solution 2% using a elution gradient (07 min, 2072.5% v/v methanol, 77.5 min, 72.595% v/v methanol, 7.58.5 min. 95% v/v methanol, 8.59 min 9520% v/v methanol, 910 min 20% v/v methanol) with a flow rate of 1.0 mL min −1 , and oven temperature of 25°C. The eluted samples were detected by UV detector at 254 nm. Calibration curve was constructed by plotting the peak area (y) against concentration in μg mL −1 of standard solutions (x). The standard equation obtained from the curve was used for quantification of ellagic acid as mg/g extract of sample. All assays were carried out in triplicates and the ellagic acid quantification was reported.

Determination of antimicrobial activity
The Pp was assayed for antibacterial activity at different concentrations using a standard agarwell diffusion assay (CLSI, 2012). Suspensions of bacteria strains (1.5 x 10 8 CFU/mL) were spread using swabs over the 523 Kado & Hesket agar media in sterilized Petri dishes. Then, wells with a diameter of 6 mm were punched aseptically and 25 μL of Pp at different concentrations were introduced into each well (100, 50, 25, 12.5, 6.25 and 3.125 mg/mL, solubilized in an isotonic phosphate buffered saline (PBS). All plates were incubated at 28°C for 48 hours. Measures of the zones around the wells (mm) were recorded as inhibition zone for Pp. Streptomycin sulfate (500 µg/ml, P. carotovorum subsp. carotovorum) (Pachupate and Kininge, 2013) and oxytetracyclin hydrochloride (Terramicin ® ) (30 μg/mL, X. campestris pv. campestris and R. solanacearum) (Santos et al., 2008) were used as positive control. The isotonic phosphate buffered saline (PBS) was used as negative control. All tests were performed in six replicates.
Minimum inhibitory concentrations (MICs) were performed in 96well microplates (Eloff, 1998) using serial dilutions of Pp (500; 250; 125; 62.5; 31.25 μg/mL) and Pg (250; 125; 62.5; 31.25 μg/mL). Hundred microliters of Pp or Pg diluted in liquid cul ture medium and the tested microorganism suspen sions (1.5 x 10 5 colonyforms unity CFU/well). After incubation (28°C for 24 h), the content of each well was sown in Petri dishes with agar culture medium. The Petri dishes were incubated for 48 hours at 28°C to account for the colonyforms unity (CFU). To indi cate viable bacteria cells in the microplate, 10 μL of thiazolyl blue (tetrazolium salt 3(4.5dimethylthia zol2yl)2.5diphenyltetrazolium bromide) reagent were added to the microplate wells and incubated at 28°C for 13 h (Mosmann, 1983). The color change produced in reaction was measured in a spectropho tometer (540 nm) and the values were correlated to the viable bacteria cells in the microplate. MIC was measured as the lowest concentration necessary to inhibit growth of the tested pathogen. Minimum Bactericidal Concentration (MBC) was considered as the minimum concentration in which no growth was visually observed in Petri dishes with solid medium, with 99.99% of eradication of the initial inoculum (De Nova et al., 2019). The concentration of the Pp and Pg that inhibited the growth of half of the inoculum was estimated as the inhibitory concentration 50 (IC 50 ) (Soothill et al., 1992), represented as Log (inhibitor) versus normalized absorbance (%) (dose response inhibition model). Streptomycin sulfate (500 μg/mL) and oxytetracycline hydrochloride (30 μg/mL) were used as positive control and isotonic phosphate buffered saline (PBS). All tests were per formed in four replicates.

Effect of Hydroalcoholic crude extract from the fruit peel of the P. granatum on X. campestris pv. campestris control in seeds
Seeds of the Eruca vesicaria (L.) Cav. (Feltrin ® , cultivated arugula variety, germination from 7 to 10 days) were purchased from a local market. Seeds were disinfected and coated with pathogenic bacte ria according standard protocol (Kotan et al., 2014). Seeds coated with pathogen were directly soaked in treatments consisting in: 1 Pp suspension (500 µg/mL or 250 µg/mL) or 2 association between Pp (500 µg/mL or 250 µg/mL) and antibiotic (strepto mycin sulfate, 500 µg/ml) for 3 hours. The seeds were left to dry on sterile Whatman filter paper sheets overnight in laminar flow hood. The seeds were sown in plastic pots containing garden soil and sand (1:1) totaling thirty seeds per treatment (ten seeds/pot). Other part of the seeds was transferred to Petri dishes with Whatman paper filter placed on the bottom (moistened with 10 mL of sd. H 2 O) total ing thirty seeds per treatment (ten seeds/plate). The percentage of germination and seedling emergence was determined 1012 days after sowing. E. vesicaria seedlings were assessed 18 days after emergence to determine the appearance of symptoms of disease (Vicente and Holub, 2013) and survival rate. After this, the seedlings were removed from the substrate for assessment of the effect of the extract in growth promotion. Antibiotic (streptomycin sulfate at 500 µg/ml), disinfected seeds infected with pathogen, and sterilized seeds not infected with pathogen (healthy) were used as controls. All tests were per formed in triplicates.

Anatomical analysis
Eruca vesicaria (L.) Cav. seedling samples leaves were preserved in fixative solution FAA (Formaldehyde, Glacial Acetic Acid, 95% EtOH) (Johansen, 1940). To prepare the samples, leaves were sectioned in transverse and paradermic sections using disposable razors. Leaf diaphanization was per formed according to the standardized technique (Kraus and Arduin, 1997) and stained with safranin and Astra blue solutions, both at 0.5%. Semiperma nent slides were analyzed with optical microscope to visualize the adaxial epidermis, abaxial epidermis, pal isade parenchyma, spongy parenchyma, stomatal density and stomatal morphology.

Statistical analysis
Results were expressed as the mean ± standard deviations. To determine difference between sam ples, oneway ANOVA followed by Tukey post hoc test and Student's t test were performed at p<0.05. Inhibitory concentration 50 (IC 50 ) was performed by Nonlinear Regression analysis (doseresponse inhibi tion model) with 95% profile likelihood. All analyses were performed in GraphPad Prism ® v. 8.0 software.

Chemical characterization of the hydroalcoholic crude extract from the fruit peel of the P. granatum
The results demonstrated that Pp possessed 6.34 mg/g of ellagic acid and 0.83 g/g of total phenolic as tannic acid. HPLC chromatogram was performed focused on ellagic acid, and the fingerprint is present ed in figure 1.
In vitro antimicrobial activity of the hydroalcoholic crude extract from the fruit peel of the P. granatum Hydroalcoholic crude extract from the fruit peel of the P. granatum (Pp) was tested for its antimicrobial properties against phytopathogenic bacteria. Pp pro duced bacterial growth inhibition zones for all three investigated isolates (Table 1 and Fig. 2). The highest mean values of inhibition zones were verified for R. solanacearum, followed by X. campestris pv. campestris and P. carotovorum subsp. carotovorum.
There was an increase of the inhibition zone pro duced as the increase of the P. granatum extract con  (Fig. 3 AE and 4 AE) and Pg (Fig. 3 BE and 4 BE). Were made tests with different con centrations of the Pp (500 to 31.25 μg/mL) to verify cell viability of the bacteria in comparision with nega tive control and antibiotic. Bacteria in the negative control remained with high celular viability. In the groups treated with Pp in the highest concentrations (500 and 125 μg/mL) there was a reduction of the cell viability of the both bacteria in relation to the negative control (Fig. 3 AC and 4 AC). The antimicro bial effect of the Pp in concentrations of 500 and 250 μg/mL for X. campestris pv. campestris was similar to antibiotic and different of the negative control (Fig. 4  AB). Concerning to P. carotovorum subsp. carotovorum, the antimicrobial effect of the Pp in concentra tion of 500 μg/mL was similar to antibiotic and diffe rent of the negative control (Fig. 3 A).The smallest concentrations of the Pp (62.5 and 31.25 μg/mL) did not produce any antimicrobial effect in cell viability for these both pathogens (Fig. 3 DE and 4 DE). Antimicrobial activity of the Pg against the investi gated pathogens was higher than action of the Pp. The lowest concentration of the Pg that inhibits bac terial growth (MIC) to both P. carotovorum subsp. carotovorum and X. campestris pv. campestris was 31.25 μg/mL ( Fig. 3E and 4E). Punicalagin compound at the highest tested concentration (250 μg/mL) showed antibioticlike antimicrobial activity, in terms of the cellular viability of the both pathogens ( Fig. 3B  and 4B). The observation of bacterial growth in cul ture plates with agar medium indicated that Pg in this concentration (250 μg/mL) may present bacterio static action for P. carotovorum subsp. carotovorum (Fig. 3B) or bactericidal action to X. campestris pv. campestris (Fig. 4B).

In vivo antimicrobial and biostimulant activity of the hydroalcoholic crude extract from the fruit peel of the P. granatum in E. vesicaria seeds infected by X. campestris pv. campestris
The most effective concentrations of the Pp in microdilution assays (500 μg/mL and 250 μg/mL) were tested for the control of the X. campestris pv. campestris in E. vesicaria seeds. Infected and untreated seeds (negative control) presented a lower emergence percentage compared to healthy seeds. On the other hand, treatment of infected seeds with Pp (500 μg/mL) promoted an increase of the 15% in the percentage of emergence in relation to the nega tive control (Table 2).
In addition, E. vesicaria seedlings treated with Pp did not develop main symptom of black rot disease caused by X. campestris pv. campestris (the "V" chlorotic lesion in the margin of the leaflet). This symptom was verified in seedlings of infected and untreated seeds. There was no phytotoxic effect of the Pp in seedlings development. Seedlings of the E. vesicaria treated with highest concentration of the Pp (500 μg/mL) showed a biggest growth length of radicle (Fig. 6). Treatment with streptomycin sulfate crude or associated with Pp resulted in seedlings with chlorosis symptom (yellowish leaves) (Fig. 7).
The results of this study showed different seedling survival rates of the E. vesicaria according to each treatment (Fig. 8AD). Infected and untreated seedlings (negative control) showed an abrupt drop in the percentage of survival at the 7th day after emergence. Seedlings treated with Pp at the highest concentration (500 μg/mL) showed slowly decrease    8A). On the 13 th day after emergence, the survival rates of the seedlings treated with Pp (500 μg/mL and 250 μg/mL) were 50% and 42%, respectively, versus 20% of the negative control (Fig. 8B). Association between Pp (500 μg/mL and 250 μg/mL) and antibiotic also promoted higher survival rates in relation to negative control in the same period (61% and 61.9%, respectively) (Fig. 8B). After 16 days of seedling emergence there were similar rates for treatment with Pp (500 μg/mL and 250 μg/mL) and treatment with association between Pp and antibiot ic. These percentages remained high (36%38%) in relation to negative control (15%) (Fig. 8D). Anatomical analyses of E. vesicaria seedling leaves indicated differentiation of mesophilic structures, especially in relation to the palisade parenchyma (Fig.  9AF). Uninfected (healthy) seedlings presented well preserved anatomical structures (Fig. 9A). Seedlings of the infected and untreated seeds group (negative control) showed some alterations in mesophilic tis sue, especially in relation to incomplete differentia tion of palisade parenchyma, when compared to healthy plants (Fig. 9B). Seedlings in the group previ ously treated with streptomycin sulfate presented altered palisade parenchyma cells in a more rounded shape (Fig. 9C). Seedlings treated with Pp at a con centration of 500 μg/mL showed clear differentiation of mesophilic structures, with wellstructured pal isade parenchyma (Fig. 9D). Seedlings treated with Pp at a concentration of 250 μg/mL did not present clear differentiation of mesophilic elements (Fig. 9E).
Appearance of stomata of seedlings in different treatments with Pp was represented in figure 10 (A F). From these images, the mean values of the fol hydroalcoholic extract (Pp) in survival rates of Eruca vesicaria L. (Cav.) seedlings germinated in pots. Clean (health)= seeds uninfected with X. campestris pv. campestris; Ctrl = infected and untrated seeds; Ctrl+= seeds infected and treated with streptomycin sulfate; Pp 500 µg/mL and Pp 250 µg/mL= Seeds infected and treated with Punica granatum L. hydroalcoholic extract (Pp); Pp 500 µg/mL + Atb or Pp 250 µg/mL + Atb= seeds infected and treated with association between Punica granatum L. hydroalcoholic extract (Pp) and streptomycin sulfate. A) total survival rates; B) survival rates in thirteenth day; C) survival rates in fourteenth day; D) survival rates in sixteenth day. Means with (*) are different from the negative control by the Student's t test at p<0.05. Fig. 9 Cross sections of the mesophyl of Eruca vesicaria L. (Cav.) seedlings infected with the X. campestris pv. campestris submitted to different treatments. A) Uninfected seedlings; B) seedlings infected with X. campestris pv. campestris and untrated; C) seedlings infected and trea ted with streptomycin sulfate; D) Seedlings infected and treated with Punica granatum L. hydroalcoholic extract (Pp) (500 µg/mL); E) Seedlings infected and treated with P. granatum L. hydroalcoholic extract (Pp) (250 µg/mL); F) Seedlings infected and treated with association between P. granatum L. hydroalcoholic extract (Pp) (500 µg/mL) and streptomycin sulfate. Epad= adaxial epidermis; Abed= abaxial epidermis; Lp= lacunous parenchyma; Pp= palisade parenchyma; Vb= vascular bundle.
lowing parameters were taken: polar diameter (PD), equatorial diameter (QD), stomatal functionality (FUN), area (A) and stomatal density (SD) ( Table 3). Seedlings of the group treated with Pp (500 μg/mL) showed highest values of PD, FUN and A (μm 2 ) in comparison to negative and positive control (Table  3). Stomatal functionality of the group treated with Pp (500 μg/mL) was similar to that of healthy seedlings. The highest mean of SD was verified for the group treated with Pp at a concentration of 250 μg/mL, being statistically similar to the clean group. Seedlings of the infected and untreated group showed smallest values of PD, FUN and SD. Small val ues of PD and FUN were observed too in seedlings treated with antibiotic (Table 3).

Discussion and Conclusions
In the present study, the effective antimicrobial action of the P. granatum fruit peel extract and its isolated punicalagin compound on phytopathogenic bacteria (R. solanacearum, P. carotovorum subsp. carotovorum and X. campestris pv. campestris), listed among the ten most important species in scientific and economic aspects worldwide (Mansfield et al., 2012), was shown . The wide zones of inhibition of bacterial growth in agardiffusion tests and marked reduction in the percentage of the cell viability in broth microdilution assays indicate high potential of pomegranate extract in the control of these phy topathogens.
Pomegranate peel has substantial amounts of phenolic compounds, such as hydrolysable tannins (punicalin, punicalagin, ellagic acid, and gallic acid), flavonoids (anthocyanins and catechins), and nutri ) seedlings submitted to different treatments. A) Uninfected seeds; B) seeds infected with X. campestris pv. campestris and untrated; C) seeds infected and treated with streptomycin sulfate; D) seeds infec ted and treated with Punica granatum L. hydroalcoho lic extract (Pp) at concentration of 500 µg/mL; E) seeds infected and treated with P. granatum L. hydroalcoho lic extract (Pp) at concentration of 250 µg/mL; F) seeds infected and treated with association between P. granatum L. hydroalcoholic extract (Pp) (500 µg/mL) and streptomycin sulfate. St= stomata; Ep= epidermis. ents, which are responsible for its biological activity (Magangana et al., 2020). The fruit peel has high antioxidant and antimicrobial activities and may be used as an excellent natural additive for food preser vation and for quality enhancement. The healthpro moting benefits of pomegranate peel have prompted the food industry to focus on pomegranatepeelcon taining food preparations, which include nutraceuti cals, phenolicenriched diets, and food supplements (Opara et al., 2009;Fawole et al., 2012). The Pp showed both bacteriostatic (P. carotovorum subsp. carotovorum,) and bactericidal effect (X. campestris pv. campestris). Bactericidal effect is desirable in order to inhibit the emergence of resis tant bacterial strains and toxicity (Soberón et al., 2014). The study results support literature data on the antimicrobial action of the ethanolic P. granatum fruit peel extract against phytopathogenic bacteria Pseudomonas syringae pv. tomato, the cause of bac terial spot disease in tomatoes, with bacterial growth inhibition zones of 526 mm and dosedependent effect (Quattrucci et al., 2013). Additionally, the results agreement with studies that reported antibac terial action of the methanolic pomegranate peel extract against R. solanacearum, with growth inhibi tion zone of the 13.9 mm (50 mg/mL), and ethyl acetate extract against R. solanacearum, P. carotovorum subsp. carotovorum and X. gardneri with inhibi tion zone of 8.522.75 mm (concentrations of 25200 mg/mL) (Farag et al., 2015;Khaleel et al., 2016). Studies with extracts from other parts of the plant (leaf and seed) have shown more discrete antimicro bial activity on R. solanacearum and X. campestris (Hassan et al., 2009;Uma et al., 2012).
Antimicrobial activity of the P. granatum fruit peel extract against Gramnegative and Grampositive bacteria has been correlated with the presence of polyphenolic compounds in it, mainly punicalagin (Gullon et al., 2016). A relatively high amount of polyphenols (867 mg/g) was detected in a pomegran ate peel extract preparation, especially the ellagitan nin punicalagin (296 mg/g), with antimicrobial action of the extract on isolates of S. aureus, Escherichia coli, Aspergillus niger and Saccharomyces cerevisiae (Ibrahium, 2010). The antimicrobial mechanism of action of polyphenols seems to be related to the direct action of these compounds on the bacterial cell wall by formation of complexes with wall pro teins, causing lysis (Akhtar et al., 2015). The interac tion of these compounds with sulfhydryl groups of extracellular microbial proteins results in inhibition of protein activity (Dey et al., 2012).
The impressive antimicrobial action of punicalagin on P. carotovorum subsp. carotovorum and X. campestris pv. campestris verified in the present study suggests that this molecule may be an essential component in the biological activity of P. granatum fruit peel extract against phytopathogenic bacteria. Several studies prove antimicrobial activity of the punicalagin compound against isolates of clinical importance such as Staphylococcus aureus (MIC 250 μg/mL ) and methicillinresistant Staphylococcus aureus (MRSA) (Xu et al., 2017;Mun et al., 2018). According to Xu et al. (2017) punicalagin compound has direct action on cell membrane disruption, increased K + ion flow and inhibition of biofilm forma tion in S. aureus. To the best of our knowledge, this is the first report of the antibacterial activities of the isolated compound punicalagin against plant patho genic bacteria. Further investigations may elucidate the mechanism of action of this compound on phy topathogenic bacteria.
Data from the in vivo assays of this study indicat ed that Pp is effective in control of X. campestris pv. campestris in E. sativa seeds. Black rot infection caus es tissue necrosis, premature leaf fall, atrophied growth and death of young plants (Vicente and Holub, 2013). In the present study, treatment with Pp in the highest concentration reduced incidence of disease symptoms and promoted a high survival rates of seedlings in comparison to infected and untreated group. Additionally, the association between Pp and antibiotic resulted in percentages of seedling survival above that observed in treatment with antibiotic alone (16 th day of observation), indi cating probable synergistic interaction between treatments. P. granatum extract produced no harm ful effect on germination, emergence or seedling development of the E. sativa. This is in agreement with a study that proved the effective action of treat ment with natural plant extract (Origanum onites) in the control of Clavibacter michiganensis ssp. michiganensis, Xamthomonas axonopodies pv. vesicatoria and Xanthomonas campestris pv. vitians in seeds of tomato and lettuce, without affecting seedling germi nation and growth (Kotan et al., 2014). Treatment of the seeds with hot water (50°C for 2030 min) has been the most effective treatment for seedborn blackrot control. However, treatments do not always eliminate 100% of the bacteria and may adversely affect seed germination and vigor (Celetti and Callow, 2002). Natural plant extracts, like pomegranate fruit peel extract, may represent a good alternative to control of seed born phythopatogens ensuring seed viability after treatment.
Treatment of E. vesicaria seeds with streptomycin sulfate, despite being effective in controlling X. campestris pv. campestris, demonstrated toxic effect for seedlings, evidenced by the yellowish aspect of the leaves (chlorosis). This result corroborates the findings of Napoles et al. (1991) for treatment of Brassica oleracea seeds with the same antibiotic (500 ppm for 1 h). The streptomycin is associated with several phytotoxic effects like blocking chlorophyll synthesis, especially in younger leaves, inhibition of methionine and phosphate absorption, production of photosynthetic process changes and enzymatic inhi bition (Falkiner, 1990). In addition, treatment of seeds with streptomycin resulted in severe reduction in the ratio between the polar and equatorial diame ters (stomatal functionality). However, treatment with Pp (500 μg/ml) keep values of stomatal func tionality (FUN) similar to the healthy seedlings. The highest ratio of FUN indicates stomata with more elliptic morphology, a mechanism of the drought tol erant plants to keep the water present in its interior at a maximum as a response to its hydric state (Melo et al., 2014).
Treatments with Pp indicated antagonistic values of stomatal density (SD) and stomatal area (A). The parameters SD and A can directly affect mechanisms such as photosynthesis, transpiration and efficient water use in plants ( Lawson and Blatt, 2014). The increase in SD coupled with reduction in A may result in the optimization of gas exchange (Franks et al., 2009). This pattern was observed in seedlings treated with Pp at a concentration of 250 μg/ml and similarly in the health group. On the other hand, the reduction in SD may represent a more conservative water use (Bertolino et al., 2019). This pattern was verified in seedlings treated with Pp at a concentration of 500 μg/ml. Since there is no damage to CO 2 fixation or plant cooling, this reduction in water loss can be advantageous in environments with low water avail ability (Bertolino et al., 2019). Thus, the Pp can influ ence both pathogen control in seeds and the physio logical characteristics of the plant through changes in stomatal patterns.
In conclusion, hydroalcoholic crude extract from the fruit peel of the P. granatum (Pp) demonstrated high potential for control of the phytopathogenic bacteria R. solanacearum, X. campestris pv. campestris and P. carotovorum subsp. carotovorum.
Punicalagin compound potentiated antimicrobial activity on these pathogens, corroborating studies that relate the abundance of the phenolic com pounds (flavonoids and tannins) with the antibacteri al activity of pomegranate fruit peel. Pp extract was effective to control seedborn pathogen X. campestris pv. campestris in seeds of the E. vesicaria and pro moted several beneficial effects to seedlings with no phytotoxic effect. Moreover, association between Pp and antibiotic indicated probable synergistic interac tion between treatments (16th day of observation) potencializing the seedling survival over the observed in relation of the antibiotic utilizing in isolated way.
The investigation of antimicrobial activity of the Pp and isolate compounds like Punicalagin repre sents a promising path regarding the biotechnologi cal development of botanical pesticides that ensure quality and safe of the food crop production.