Antibacterial Effect of Juglans regia , Citrus sinensis , Vicia faba , and Urtica urens Extracts under In vitro Conditions

. Various agricultural products are known to have anti-microbial, anti-cancer, and 5 anti-inflammatory effects. As we can mention, Walnut ( Juglans regia ) husk, orange ( Citrus 6 sinensis ) peel, Broad bean ( Vicia faba) peel, and Nettle ( Urtica urens ) are proven to have 7 antimicrobial and anticancer actions. Also, plant diseases such as Pectobacterium


INTRODUCTION
Planting fruit trees is a worthwhile experience for both gardeners and commercial growers [1].Anti-microbial effects of different extracts of agricultural products are truly valuable [2].
The genus Juglans includes numerous species which proved to have many benefits.Some extracts hold therapeutically active constituents, for example, polyphenols [3][4].Walnut, Juglans regia, husk, a discarded property of walnut, is found to have a brown pigment called juglone, which is a cause of antimicrobial and anticancer activity [5].Citrus is also known as one of the vital fruits.Orange, Citrus sinensis, is the most ordinary tree fruit in the world, which is grown in tropical and subtropical climates because of the sweet fruit acquired [6].
This very common fruit is proven to have adequate antimicrobial effects [7].Broad been peel, Vicia faba is also another common plant property.It has previously been proven to have some effects on the human body such as reduction of blood sugar and prevention of heart diseases.Its antimicrobial and anticancer effects have also been proven [8].Based on the

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A N U S C R I P T result of some studies, Nettle, Urtica urens, was found to have anti-inflammatory, antiswelling, anti-oxidant, and anti-aging effects altogether [9].
Bacterial plant diseases are known to be the cause of a 10% loss in plant production [10].
Soft rot, a major plant disease is caused by Pectobacterium carotovorum [11].Pectobacterium carotovorum is a main constraint in a wide range of vegetable and flower hosts [11].As well, the Black leg is a severe disease with symptoms the same as soft rot and one of its major hosts is potato.The main responsible bacterium for this disease is Dickeya chrysanthemi [12].Bacterial Wilt is also an epidemic harmful disease for Solanaceae vegetables, which lowers tomato yield by up to 91%.It is caused by a bacterium, called Ralstonia solanacearum.Current illness is known to have a sudden outbreak.It spreads quickly through water and soil throughout the agricultural land, and eventually contaminates most of the plants [13].Both young and old fruit trees such as mango, apple, and hazelnut are at risk of getting bacterial canker, which causes major losses and is mainly caused by Pseudomonoas syringae [14].
Since J. regia husk, C. sinensis peel, V. faba outer peel, and U. urens have shown antibacterial effects on some other bacteria [1][3]- [9] and P. carotovorum, D. chrysanthemi, R. solanacearum, and P. syringae and are known to be a major pathogenic cause of some plant diseases [11]- [14].In this study, we have evaluated their antibacterial activity under in vitro conditions.urens were prepared by macerating 25 g of proper powder into 250 mL of 70% ethanol.The mixture was shaken for 24 h and made into 100 mg/mL concentrates while becoming sterilized by exposure to UV rays.

Antimicrobial assay.
The effect of the ethanolic extracts was observed on the mentioned bacteria, using serial dilution and disc diffusion methods [15] on proper cultural

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A N U S C R I P T media (nutrient agar/Muller-Hinton agar) and each time incubated at 25 °C for 24 h.This study was conducted 3 times.All of the process was done under the maximum possible sterile conditions.

Blank disk diffusion.
To observe the Inhibition zone caused by these extracts 6.4 mm diameter blank disks were soaked into the mentioned extract and each was put in the middle of nutrient agar media, in which studied bacteria were cultured a minute before.Then all the plates were incubated at 25 °C for 24 h.

Well diffusion.
To observe the inhibition zone caused by these extracts, wells were created in the middle of nutrient agar media, in which studied bacteria were cultured a minute before.Then 20 μL of the mentioned extracts were poured into the wells and after that, all the plates were incubated at 25 °C for 24 h.

Minimum inhibitory concentration (MIC).
The MIC of the extracts mentioned was determined by culturing the bacteria in Muller-Hinton broth [16].The culture was standardized by using a standard microbiological 0.5 McFarland standard tube.Possible antimicrobial agents were each serially diluted separately 5 times, 1:1 through Muller-Hinton Broth as a sterile diluent.After the antimicrobial agents were diluted, a volume of standardized inoculum for bacteria was added to each dilution tube (equal to the volume of diluted agent), and a positive test control tube was included to prove microbial growth over the course of incubation and the sterility of media.The tubes, containing microorganisms and antimicrobial agents, were then incubated at 25 °C for 24 h.Because of the tint of antimicrobial agents used, no turbidity or pellet of microorganisms was distinguishable from the media, and therefore all the serially diluted tubes` containing's were cultured in Muller-Hinton agar media and incubated at 25 °C for 24 h.After the incubation, the cultured Muller-Hinton agar plates were observed for microbial growth.The one with less concentrated antimicrobial agent than the last plate in the series, which did not demonstrate growth (MBC), showed less growth and paralleled with the MIC of the antimicrobial agents.

Minimum Bactericidal Concentration (MBC).
The MBC of the antimicrobial agent was determined by culturing the bacteria in Muller-Hinton broth of 0.5 McFarland standard [16].Also, antimicrobial agents were each serially diluted using 1:1 Muller-Hinton Broth as a sterile diluent and inoculated with an equal volume of microorganisms.including a positive

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A N U S C R I P T test control tube to prove microbial growth over the course of incubation and the sterility of media.Then the tubes were incubated at 25 °C for 24 h.All the diluted tubes` containing the most antimicrobial agents concentrated to the least, were cultured in Muller-Hinton agar media and incubated at 25 °C for 24 h.After the incubation, the cultured Muller-Hinton agar plates were observed for microbial growth.The last plate in the series that did not demonstrate growth linked with the MBC of antimicrobial agent.

RESULTS AND DISCUSSION
3.1.Disk and well diffusion.Based on the results mentioned in Tables 1 and 2, J. regia (husk) and V. faba (outer peel) extracts revealed anti-bacterial effect on all four studied bacteria, while C. sinensis (peel) only inhibited the growth of P. syringae and U. urens only had this effect on P. syringae and R. solancearum.expected, the antibacterial effects of plant extracts on the studied pathogens were the same as diffusion methods (Table 3).Considering MIC and MBC values brought, the efficiency of plant extracts were similar except the fact that effectiveness of V. faba (outer peel) on P.
carotovorum and U. urens on R. solancearum was proven to be less.solanacearum, D. chrysanthemi and P. syringae pathogens, respectively [17]- [20].However, anti-biotic resistance has become a worldwide environmental challenge and also some antibiotics are also known to have undesirable effects on chloroplast and nuclear gene expression [21][22].Additionally, fungicides such as captan itself are known to have harmful consequences on soil microbial activity [23].Owing to all these environmental damages, plant extracts are now becoming an alternative to these pesticides [24].
Respectively, based on the previous publications on C. sinensis, J. regia, V. faba and U.
urens, the authors declared their antibacterial activity on some other bacteria [1] [6][8][9].InA C C E P T E D MA N U S C R I P T

Table 1 .
The average and standard deviations of bacterial inhibition zone using disk diffusion.

Table 2 .
The average and standard deviations of bacterial inhibition zone using agar well diffusion.

Table 3 .
MIC and MBC values of the four plant extracts on the studied bacteria.

mg/mL) Juglans regia Citrus sinensis Vicia faba Urtica urens
This experiment aims to control or treat plant diseases such as soft rot and bacterial wilt.Antibiotics and fungi pesticides such as captan, streptomycin, difenoconazole and streptocycline are applied to prevent or treat diseases caused by P. carotovorum, R.