Monoterpenoids and Their Synthesized Brominate Derivatives as Eco-Friendly Measures to Control Some Plant Pathogenic Fungi and Bacteria

Background and Objective: Phyto-pathogenic fungi or bacteria are one of the most important causes that decrease food and cash crops. Chemical pesticides cause damage to the environment. Thus, globally, there are attempts to provide eco-friendly products. Therefore, this work aimed to find alternative natural products for managing serious diseases (fungi and bacteria). Materials and Methods: Fungicidal and bactericidal of four monoterpenoids, as well as their synthesized brominated (Br) derivatives were investigated against six fungi and four bacteria in vitro . The synthesized brominated derivatives were confirmed by a mass spectrometer and GC/MS. Results: The soil and air-borne fungi were very sensitive to chlorothymol followed by thymol and carvacrol, respectively. However, the synthesized brominate derivatives such as bromocarvacrol were the most effective treatment against all the tested fungal strains, while, bromoegunol was a less effective treatment. Concerning the antibacterial activity, the tested monoterpenoids exhibited variable degrees of antibacterial against all of the tested bacterial strains and the conversion of monoterpenoids into bromo-derivatives enhanced the bactericidal activity. Conclusion: The chlorothymol (monoterpene) recorded the highest fungicidal activity against soil and air-born fungi. For bacterial activity, results exhibited that chlorothymol was the most effective monoterpene against bacteria. All the bromo-derivatives showed high activity against the most of tested fungi and bacteria.


INTRODUCTION
Biopesticides are considered a unique solution to the problem of agrochemicals in crop protection.It reaches the target pest leaving the remaining 99.9% to enter the environment to cause hazards to nontarget organisms including humans 1 .In the last two decades, scientists all over the world tried to minimize the use of synthetic pesticides for the management of plant pathogens, insects, acari and weeds to avoid environmental pollution hazards.Besides, the targeted pathogen, pesticides may also kill various beneficial organisms.The increasing incidence of resistance among pathogens towards synthetic chemicals is also a cause of serious concern 2 .Major food and cash crops are attacked by certain pathogens especially fungal diseases which cause approximately 20% reductions in the yield 3,4 .
The discovery of antimicrobial compounds from plant sources is a safe route to a new generation characterized by eco-friendly, low toxicity, selectivity, highest efficacy and biodegradability [5][6][7] .
Search extensively for new biologically active terpenoids as a potential source for agrochemicals.Monoterpenes together with sesquiterpenes and diterpenes form the majority of essential oil.Monoterpenes are unsaturated hydrocarbons and some are oxygenated derivatives such as alcohol, ketones, carboxylic acid and phenol 8,9 .Chemical modification of natural monoterpenoids to various derivatives has been reported to result in modification of biological activity [10][11][12][13][14] .Therefore, this work was one of several attempts that have been conducted to find alternative natural products which are safe, biodegradable and eco-friendly for managing serious pests (fungi and bacteria) which attack crop plants.

MATERIALS AND METHODS
In this investigation, the evaluated monoterpenoids and/or their synthesized brominated derivatives were implemented under laboratory conditions in 2021 at the Fungicide Bioassay Laboratory, Department of Pesticide Chemistry and Technology, Faculty of Agriculture, University of Alexandria, Alexandria, Egypt.

General procedure for bromination of tested monoterpenoids:
The desired monoterpenoids (0.01 mol) thymol, eugenol, chlorothymol and carvacrol were dissolved in carbon disulfide (25 mL) in a round bottom flask fitted with a reflux condenser and pressure equivalent-dropping funnel.Bromine (0.01 mol) was dissolved in carbon disulfide (16 mL) and then added dropwise to a cooled flask (below 5°C) after the reaction mixture was stirred at room temperature for 1hr to complete (Scheme 1) and then monitored by thin layer chromatography (TLC with aluminum plate of 25, silica gel coated with fluorescent indicator F254, TLC size is 20×20 cm, MERCK, Germany).The reaction was quenched with water and extracted three times with methylene chloride (30 mL each).The organic layer was washed three times with water (100 mL) and dried over anhydrous sodium sulfate (50 g).The organic solvent was removed using a rotary evaporator under a high vacuum (brand name: Kryqcn, Model: RE501, rotating speed: 120 rpm, vacuum power: 0.098 mpa, China).The product was identified by a mass spectrometer (HP model, MS-5988) and GC/MS (Shimadzu QP2010) and Melting points were measured with capillary melting point apparatus model 1002-USA, UV spectra were recorded on a UV Spectrophotometer model UV-1601 SHIMADZU shown in Table 1 and 2  The antifungal activity of tested compounds was investigated by using the radial growth technique method 15 .Appropriate volumes of the stock solutions of the tested compounds either in dimethyl sulfoxide (DMSO) for solvent extracts or in distilled water for water extracts were added to molten nutrient agar (potato dextrose agar medium, PDA) to achieve the desired concentration immediately before pouring into the Petri dishes (9.0 cm in diameter) at 40-45°C.
Each concentration was tested in triplicate.Parallel controls were maintained with DMSO mixed with PDA.The discs of mycelial felt (0.5 cm diameter) of the plant pathogenic fungi, taken from 8-day-old cultures on PDA plates, were transferred aseptically to the center of Petri dishes.The treatments were incubated at 25°C in the dark.Colony growth diameter was measured after the fungal growth in the control treatments which completely covered the Petri dishes.Moreover, the percentage of mycelial growth inhibition was calculated according to the following formula 16

Statistical analysis:
The effective concentrations of evaluated compounds that inhibit the fungi mycelia or bacteria growth by 50% (EC 50 ), were conducted by a linear regression method with probit analysis 18 .

Identification of mono terpenoids derivatives:
The reaction of thymol, carvacrol, chlorothymol and eugenol with carbon disulfide and bromine gave bromothymol and bromocarvacrol as crystals, while bromocholorothymol and bromoeugenol as oily compounds.The percentage of the product yield obtained from the reaction was 80, 83, 92 and 90% for Bromothymol, Bromochlorothymol, Bromocarvacrol and Bromoeugenol, respectively.Physical properties and identification of the synthesized monoterpenoidal brominate derivatives showed in Table (1, 2) and Fig. (1-4).-------------------------------- The obtained results indicated that carvacrol, thymol, cholorothymol and eugenol exhibited the highest antifungal activity against phytopathogenic fungi [19][20][21][22] .The activity of thymol, carvacrol, chorothymol and eugenol against fungi may be attributed to containing the OH group in the aromatic ring.Also, the chlorine atom on the chlorothymol is playing an important role in the fungicidal activity against the tested fungal strains 20,23 .Therefore, it's possible to use these monoterpenoids as lead compounds to obtain new friendly fungicides for both humans and the environment.

Fungicidal activity of synthesized monoterpenoidal brominate derivatives against soil and air-born fungi:
The fungicidal activity of brominate monoterpenoid derivatives against soil-born fungi i.e., F. oxysporum, R. solani and P. debrinumwas shown in  14 , who found that halogenated thymol derivatives e.g.chlorothymol, dichlorothymo, monobromothymol and dibromothymol have strong antifungal and antibacterial activities.Also, Chauhan et al. 24 , found that thymol and its derivatives succeeded to inhibit the mycelia growth of Rhizoctonia solani during the in vitro study, while the soil application under greenhouse conditions thymol or its derivatives suppressed the damping-off in cucumber seedlings at a range of 26.67 to 100%.
Several studies reported that the monoterpenes exhibited antifungal and antibacterial activities against a wide range of microorganisms [25][26][27][28] .The results of the current investigation are in agreement with El-Zemity et al. 29 who stated that chlorothymol was effective as a bactericide against Agrobacterium tumefaciens and Erwinia carotovora var carotovora.The activity of chlorothymol may be attributed to the chlorine atom which plays a good role in the bactericidal activity against the tested bacteria.Also, Mahboub and Memmou 30 found that synthesized 6-bromoeugenol or eugenol has antibacterial activity, especially in both positive and negative gram bacteria.Also, thymol derivatives had antibacterial activities with minimum inhibitory concentration values, 40 to 80 μg mLG 1 31 .Thymyl-4-nitrobenzoate recorded good inhibitory action towards plant pathogenic bacteria either as a major constituent or in combination with other antimicrobial agents 32 .Abd-El-Aziz et al. 33 indicated that using the essential oils of caraway (Carum carvi L.) and thyme (Thymus vulgaris L.) exhibited antibacterial efficacy against Agrobacterium tumefaciens in a laboratory study.Moreover, the application of caraway and thyme oils suppressed the gall formation by 63.16 and 89.47%, respectively, on apricot seedlings in pots.

CONCLUSION
From the above results, it could be concluded that chlorothymol (monoterpene) recorded the highest fungicidal activity against the most of soil and air-born fungi.While, the brominated derivatives of monoterpenoids showed that bromocarvacrol was the most effective against all tested fungi, whereas, bromoegunol was the least one.The same trend was recorded with bacteria, where chlorothymol (monoterpene) recorded the highest bactericidal activity.However, the brominated derivatives of monoterpenoids were more effective against all the tested bacteria.Therefore, this study proposed some eco-friendly solutions, but more experiments are needed in the future.

SIGNIFICANCE STATEMENT
Fungi and bacteria are among the most common plant pathogens that lead to losses in agricultural crops, whether during planting or after harvest.Combating these diseases has become costly, especially recently, so it was necessary to search for effective alternatives that can be used safely without any adverse effects.This study was thus conducted to assess the impact of some monoterpenes and their brominate derivatives against soil and air fungi as well as bacteria.Also, results showed that some of the monoterpenes or their derivatives e.g., chlorothymol and bromocarvacrol were effective as fungicides and/or bactericides.

. Scheme 1 :
Scheme 1: Synthesis of the monoterpenoidal brominate derivatives : DC -DT Mycelial growth inhibition = ×100 DC where, DC and DT are the average diameters of the fungal colony of control and treatment, respectively.http://doi.org/10.17311/ajbs.2023.264.274| Page 266 Four phytopathogenic bacteria, Agrobacterium tumefaciens (Crown gall bacteria), Erwinia carotovra subsp.Carotovra (soft rot bacteria), Erwinia amylovora (fire blight bacteria) and Pseudomonas solanacearum were provided by the Department of Plant Pathology, Faculty of Agriculture, Alexandria University, Egypt.The bacterial strains were cultured in a glycerol agar medium.Streptomycin sulfate as a standard bactericide was supplied by El-Nile Company for Chemical Industry and Drugs, Egypt.Determination of minimum inhibitory concentrations (MICs): Tested compounds were dissolved in dimethyl sulfoxide (DMSO).Appropriate volumes of the stock solution were added to nutrient agar and poured into Petri dishes (diameter 9 mm).After solidification, a bacterial culture has grown in a nutrient broth for 18 hrs (approximately 10 8 CFU mLG 1 was planted on four lines per each plate with three replicates from four bacteria species Agrobacterium tumefaciens, Erwinia carotovra, Erwinia amylovora and Pseudomonas salnacearum on the surface of the agar.The inoculum line was allowed to dry before inverting the plates for incubation at 27°C for 24 hrs.The control was nutrient agar with a maximum volume of dimethylsulfoxide added to the treatment.The antibiotic, streptomycin was used for comparison, The MIC was determined as the lowest concentration of the tested compound showing no visible bacterial growth in the agar plates.As recommended by the European Society of Clinical Microbiology and Infection Disease 17 .

Table 1 :
Physical properties of the monoterpenoidal derivatives