The Effect of Some Wild Grown Plant Extracts and Essential Oils on Pectobacterium betavasculorum: The Causative Agent of Bacterial Soft Rot and Vascular Wilt of Sugar Beet

The bacterial soft rot and vascular wilt of sugar beet are the major diseases of sugar crops globally induced by Pectobacterium betavasculorum and P. carotovorum subsp. carotovorum (Pcc). The control of this bacterial disease is a severe problem, and only a few copper-based chemical bactericides are available for this disease. Because of the limitations of chemicals to control plant bacterial pathogens, the essential oils and extracts have been considered one of the best alternative strategies for their control. In this study, twenty-seven essential oils and twenty-nine plant extracts were extracted and evaluated for their antibacterial activities against Pectobacterium betavasculorum isolate C3, using the agar diffusion method at 0.01%, 0.1%, and 100% (v/v). Pure Pimpinella anisum L. oil exhibited the most anti-bacterial activity among three different concentrations of essential oils and extracts, followed by Thymus vulgaris L. oil and Rosa multiflora Thunb. extract. The efficacy of effective essential oils and extracts on Ic1 cultivar of sugar beet seeds germination and seedling growth in vivo also were tested. The seed germination of the Ic1 cultivar was inhibited at all the concentrations of essential oils used. Only extracts of Rosa multiflora Thunb., Brassica oleracea L., Lactuca serriola L., Salvia rosmarinus Spenn., Syzygium aromaticum (L.) Merr. and L.M.Perry, Eucalyptus globulus Labill., and essential oils of Ocmium basilicum L., Pimpinella anisum L., and Mentha× piperita L.L. in 0.1% concentration had no inhibition on seed germination and could improve seedling growth. This is the first report of the antibacterial activity of essential oils and extracts on Pectobacterium betavasculorum.


Introduction
Bacterial sugar beet soft rot disease caused mainly by Pectobacterium betavasculorum and P. carotovorum subsp. carotovorum (Pcc) (formerly Erwinia carotovora ssp. carotovora). The genus Pectobacterium is an important Gram-negative plant pathogen that belongs to the family Enterobacteriaceae. This genus causes severe losses of many commercial crops, including sugar cane, in the field and during storage [1][2][3]. It is proven that Pectobacterium betavasculorum (Pb) is the causal agent of sugar beet soft rot and vascular wilt disease in Iran [4]. The control of bacterial diseases is a severe problem in plant production. Only a few chemical bactericides are available for management using copper-based bactericidal substances or different antibiotics [5][6][7]. It is shown that chemical bactericides have limitations such as general toxicity, the development of resistant bacterial strains, and negative effects on the environment and the yield because of accumulation in the food chain and long degradation periods. Different plant compounds have been tested for antimicrobial activity as an alternative strategy for plant protection against pathogens, including bacteria. Essential oils, as odorous and volatile products of the secondary metabolism of aromatic plants, normally formed in special cells or groups of cells, could be used as antimicrobial agents in the control of the disease caused by bacteria [8,9] and were proved to be better substitutes for synthetic antibiotics as they have an easily biodegradable nature [10]. Their antimicrobial activity is assigned to small terpenoids and phenolic compounds [11]. The inhibitory effect of some essential oils has been demonstrated on some plant pathogenic bacteria, including Clavibacter michiganensis subsp. michiganensis [12,13], Pectobacterium carotovorum [14][15][16][17][18][19][20], Pseudomonas mariginalis pv. mariginalis, Erwinia chrysantemi [21] Ralstonia solanacearum [14], Xantomonas campestris pv. malvacearum, Pseudomonas syringae pv. tomato [13], Pseudomonas syringae pv. syringae, Xanthomonas arboricola pv. pruni [22], Xanthomonas oryzae, Xanthomonas malvacearum [23], Erwinia amylovora [19,[24][25][26], Dickeya solani, Agrobacterium tumefaciens [24][25][26], Pseudomonas aeruginosa [27], Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola [28], Pseudomnas putida and Pseudomonas fluorescens [29]. Plant extracts from different organs of plants, including roots, barks, seeds, shoots, leaves, and fruits, were studied for anti-bacterial abilities against plant pathogenic bacteria [30][31][32][33][34][35][36]. The interest in studying the antimicrobial activities of essential oils and plant extracts for plant disease control, especially bacterial diseases, has recently increased [23]. The present work aimed to evaluate the antibacterial activities of twenty-seven essential oils and twenty-nine plant extracts on Pectobacterium betavasculorum; the causal agent of bacterial soft rot and vascular wilt of sugar beet in Iran. We also tested the efficacy of these essential oils and extracts on seed germination and stem and root growth of sugar beet seedlings. The detailed information on the anti-bacterial effect of these essential oils and extracts on, Pectobacterium betavasculurum is scarce.

Essential Oils
The essential oil of all 27 air-dried plants (100 g) was extracted by hydro-distillation for 4-6 h using a Clevenger apparatus [37]. The distillate oils were dried over anhydrous sodium sulfate (Na 2 SO 4 ) and stored in closed dark vials at 4 • C. This extract was serially diluted with sterilized distilled water. The plants were used for extraction are summarized in Table 1. One-hundred grams of all the 29 plant leaves were powdered and extracted separately in ethanol by soxhlet extraction [38]. Extraction was done for 18-24 h. Ethanol was evaporated in a water bath. A 5 g of each plant was soaked in 95 mL distilled water for 1 h at room temperature for aqueous extracts. Each extract was serially diluted with sterilized water. The plants were used for extraction are summarized in Table 2. Leaves were used to extract almost all plants, and flowers and leaves were used for Robinia pseudocacacia L. and Satureja hortensis L. All the plant materials used in the present study were kindly supplied by the Medicinal and Aromatic Plants collection of the Department of Horticulture, College of Agriculture, Urmia University, Urmia, Iran.

Bacterial Strain
Pectobacterium betavasculorum strain C3, the causal agent of sugar beet soft rot and vascular wilt disease in Iran, was obtained from an Iranian collection (Department of Plant Protection, College of Agriculture, Kerman University, Kerman, Iran). Bacterial culture was maintained on Luria-Bertani (LB) agar medium at 4 • C.

In Vitro Antibacterial Activity
The well diffusion method determined the in vitro antibacterial activity of the essential oils and plant extracts. Petri plates with 15 mL of nutrient agar (0.5% peptone, 0.3% beef extract/yeast extract, 1.5% bacteriological agar, 0.5% NaCl, Merck) were inoculated with 1.5 × 10 7 CFU/mL of 24 h suspensions of Pectobacterium betavasculorum, grown on nutrient agar. Wells were cut into the agar and filled with 20 µL of each essential oil or extract. The plates were placed for 2 h at 4 • C to allow the diffusion of essential oils and extracts and cultivatedfor 24-48 h at 26 • C. After cultivation, the distinct growth inhibition zone around the wells was measured in mm. The antibacterial activity was assessed by measuring the diameter of the inhibition zone in mm. The experiments were performed in four replicates and 0.01% (10 −2 ), 0.1% (10 −1 ), and 100% solutions of the essential oils and extracts in absolute ethanol [29][30][31].

In Vivo Test 2.4.1. On Seed Germination
Sugar beet seeds of Ic 1 cultivar susceptible to Pectobacterium betavasculorum were disinfested by emerging in 1% NaOH for 5 min, then rinsed and washed in sterilized water and air-dried at 24 • C according to [31]. Disinfected seeds were soaked for 30 s individually in two concentrations of oils and extracts (100% and 0.1) and then laid between two wet sterile Whatman filter papers in a Petri dish for 10 days at 26 • C, and 500 µL water was added to each petri dish daily. Petri dishes were checked daily to remove the germinated seeds and determine the negative effect of the oils and extracts on seed germination. This experiment was done in three replicates with 30 seeds for each replicate.
In preliminary experiments, it was observed that several used essential oils and extracts caused phytotoxicity and inhibited seeds germination. To remove these negative adverse effects and statistical analysis with low errors, the negative efficacy of the essential oils and extractswas measured.

On Stem and Root Growth Rate of Seedling
After surface sterilization, sugar beet seeds of the Ic1 cultivar were soaked in essential oils and extracts for 30 s. For soil infestation, Pectobacterium betavasculorum (1 × 10 1 cfu/g) was added and mixed thoroughly to ensure an equal distribution of Pectobacterium betavasculorum inoculum. Infested soil was then filled in sterile glass containers and irrigated daily for one week before sowing. Seeds were sown in infested soil and kept at 28-30 • C and relative humidity between 70-90% until the end of the experiment. Approximately five weeks after sowing, the seedling stem and root growth rate were measured. This experiment was done in four replicates. All tests were laid in Completely Randomized Design (CRD) with four replicates. Glass containers filled with sterilized soil and sterilized seeds were used as a negative control, and glass containers filled with Pectobacterium betavasculorum-infested soil and sterilized seeds were used as a positive control.

Statistical Analysis
All the experiments were performed in triplicates, Data was statistically analyzed, and mean values were calculated. Data were subjected to Analysis of Variance (ANOVA) using an SAS 2012 model (SAS Version 9.2., SAS Institute, Cary, NC, USA). Effects were declared significant or non-significant at (p ≤ 0.01) level of probability for those parameters where the ANOVA was significant using least significant difference (LSD) at 1% level of probability (p ≤ 0.01) according to Duncan's multiple range test.

In Vivo Test
All used essential oils and extracts had phytotoxicity on seeds at pure concentration, and no seed was germinated at this concentration. The statistical analysis showed a significant difference between treatments with control (p ≤ 0.01). At 10 −1 concentration, essential oils of Ocimum basilicum L., Pimpinella anisum L., and Mentha× piperita L. had 21.34, 19.78, and 15.52% negative efficacy on seed germination, respectively. Other essential oils inhibited seed germination completely (Table 4). Among plant extracts, Rosa multiflora Thunb., Brassica oleracea L., Lactuca serriola L., Salvia rosmarinus Spenn., Syzigium aromaticum, and Eucalyptus globulus had 74.1, 72.2, 70.3, 66.5, 56.05, and 56.05% negative efficacy on seed germination, respectively. So only these essential oils and extracts were used to measure the growth rate of seedling stem and root in the next step. They had a significant difference in seedling growth than the positive control (p ≤ 0.01). There was no statistically significant difference between these essential oils and extracts to improve the seedling's root and stem growth (Table 5, Figure 1).

Discussion
The various essential oils and extracts exhibited varying degrees the C3 isolate of Pectobacterium betavasculorum pathogen, the causal a vascular wilt of sugar beet in Iran [4], using the well diffusion met study, anise (Pimpinella anisum L.) essential oil had the most poten vitro test (P ≤ 0.01). Thyme (Tymus vulgaris L.) could inhibit Pectobact growth in vitro, but in vivo test, it was phytotoxic and had negative

Discussion
The various essential oils and extracts exhibited varying degrees of inhibition against the C3 isolate of Pectobacterium betavasculorum pathogen, the causal agent of soft rot and vascular wilt of sugar beet in Iran [4], using the well diffusion method in vitro. In our study, anise (Pimpinella anisum L.) essential oil had the most potent inhibitory effect in vitro test (p ≤ 0.01). Thyme (Tymus vulgaris L.) could inhibit Pectobacterium betavasculorum growth in vitro, but in vivo test, it was phytotoxic and had negative efficacy on seed germination. Some researchers reported the antibacterial activity of thyme on Clavibacter michiganensis subsp. michiganensis [12], Pectobacterium carotovorum [14][15][16], Pseudomonas mariginalis pv. mariginalis, Erwinia chrysantemi [22], Xanthomonas vesicatoria [39], Pseudomonas syringae pv. tomato [40], and Ralstonia solanacearum [14]. It has stronger antimicrobial properties as its oil possesses more phenolics, including carvacrol, eugenol, and thymol. They act through the Quorum sensing machinery to inhibit specific virulence determinants, including biofilm formation and plant cell wall degrading enzymes (PCWDEs) [41]. The results of the present study are in agreement with the findings of earlier researchers that some essential oils, including thyme oil, are highly phytotoxic, especially on seed germination [42][43][44]. Among other tested essential oils, peppermint (Mentha× piperita L.) and sweet basil (Ocimum basilicum L.) had good inhibitory effects in the in vitro test. Also, they had a positive effect on seed germination and seedling growth under in vivo tests. Rosa multiflora Thunb. extract had a good inhibitory effect on Pectobacterium betavasculorum in vitro test and seed germination in vivo. To our knowledge, this is the first study to provide data on the evaluation of essential oils and plant extracts against Pectobacterium betavasculorum. According to the in vitro and in vivo tests, anise (Pimpinella anisum L.), peppermint (Mentha× piperita L.) and sweet basil (Ocimum basilicum L.) essential oils and Rosa (Rosa multiflora Thunb.), cabbage (Brassica oleracea L.), prickly lettuce (Lactuca serriola L.), Rosemary (Salvia rosmarinus Spenn.), clove (Syzigium aromaticum) and Eucalyptus (Eucalyptus globulus Labill.) extracts have a strong anti-bacterial activity on Pectobacterium betavasculorum. Rosemary essential oil has been tested on Clavibacter michiganensis subsp. Michiganensis [12], Ralstonia solanacearum [14], Pseudomonas mariginalis pv. mariginalis, Erwinia chrysantemi [20], and Pectobacterium carotovorum [14,16], but no report on its extract anti-bacterial activity exists. This study revealed that essential oils and extracts of these plants have anti-bacterial activities and can be used to control Pectobacterium betavasculorum. The treatment with these essential oils and extracts of sugar beet seeds artificially contaminated with Pectobacterium betavasculorum confirmed the efficacy determined in the in vitro experiments. The anti-bacterial activity of these plants may be attributed to various antimicrobial phytochemicals [45,46] and so should be studied clearly. This study has shown that some plants effectively inhibit Pectobacterium betavasculorum growth. Most of the plants here were tested for their antibacterial activity first time. The antimicrobial activity evaluated in this work could be attributed to different compounds in variable amounts in plant essential oils and extracts. These compounds should be studied in detail. It is proven that the assayed antimicrobial activity of the plant species depends on the botanical species, age, part of the plant studied, and the solvent used for the extraction procedures [46]. Further analyses on the concentrations and combinations of these essential oils and extracts are required to obtain an anti-bacterial effect on Pectobacterium betavasculorum with minimum undesirable effects on seed germination and seedling growth. They should also be used in field studies to learn their effectiveness in field conditions. Therefore, more research on the activity of these plants against the other plant pathogenic bacteria and other plant pathogens would be of great value. These findings persuaded us to continue screening more plant species for their anti-bacterial activity and their compounds in agriculture.In conclusion, this research evaluates the effectiveness of anise (Pimpinella anisum L.), peppermint (Mentha× piperita L.) and sweet basil (Ocimum basilicum L.) essential oils, and Rosa (Rosa multiflora Thunb.), cabbage (Brassica oleracea L.), prickly lettuce (Lactuca serriola L.), Rosemary (Salvia rosmarinus Spenn.), clove (Syzigium aromaticum) and Eucalyptus (Eucalyptus globulus Labill.) extracts on Pectobacterium betavasculorum; the cusal agent of bacterial soft rot and vascular wilt of sugar beet in Iran. This research would augment the growing body of plant pathogenic Pectobacterium research and its utility in management studies of this pathogen using natural and non-chemical materials.