Effect of antimicrobial peptides and monoterpenes on control of fire blight

Aim of study: Antimicrobial peptides and monoterpenes are safe compounds that have been used for control of many plant diseases. Herein, the effects of two recombinant antibacterial peptides (AMPs) were compared with two monoterpenes for control of Erwinia amylovora directly or via induction of plant defense enzyme guaiacol peroxidase (GPOD).Area of study: The experiments were performed at the Ferdowsi University of Mashhad (Iran).Material and methods: The central composite design (CCD) method was used to study the effect of mixing the compounds and copper compound (Nordox) in controlling the pathogen. The resistance level was studied on shoots of tolerant (‘Dargazi’) and semi-susceptible (‘Spadona’) pear cultivars treated with the antibacterial compounds.Main results: Thanatin and 1,8-cineole showed the highest and lowest antibacterial effects. All treatments reduced E. amylovora pathogenicity on blossom. The CCD analysis revealed that the best reduction in colony number obtained by mixing Lfc, thanatin, thymol, 1,8-cineole and Nordox at concentrations of 32, 16, 24, 250 and 250 μg/mL. Thymol and 1,8-cineole at 500 μg/mL decreased disease severity significantly compared to that of AMPs. The level of GPOD enzyme in ‘Dargazi’ was higher than in ‘Spadona’. All treatments increased the GPOD levels in both cultivars. Furthermore, resistance level and GPOD ratio were negatively correlated.Research highlights: Antimicrobial peptides showed better effect on growth inhibition of E. amylovora than monoterpenes. Mixing of these peptides and monoterpens at special dosage enhanced their antimicrobial efficacy against E. amylovora; that could represent a new method in control of fire blight disease.


Introduction
The fire blight disease with the bacterial agent Erwinia amylovora (Burrill) Winslow et al. (1920) is undoubtedly one of the most vicious diseases of pome fruit trees worldwide imposing heavy and irreparable damage each year (Gusberti et al., 2015). This disease is the most destruc-tive bacterial disease, particularly on pears and apples, the two most economically important members of Rosaceae (Emeriewen et al., 2019). The main symptoms of fire blight infection are burnt-like appearance of infected tissues, bacterial ooze released from the infected tissues, shepherd's crook, wilting, and water soaked appearance (Awais Khan et al., 2011). In the last two centuries, this pathogen has been spread globally and, consequently, E. amylovora has been classified as a quarantine organism in some countries, where it is subject to phytosanitary legislation. Recently, it has been included in the top 10 plant pathogenic bacteria (Mansfield et al., 2012;Piqué et al., 2015). The regular use of chemicals poses a challenge to the control of this disease, which can be performed by integrated management based on using copper compounds and antibiotics (Cabrefiga & Montesinos, 2017). Application of copper compounds during blooming has been recommended for the disease control, but these chemicals sometimes cause damage on blossoms and fruits (Steiner, 2000;Karami-Osboo et al., 2010;Akhlaghi et al., 2018). The use of antibiotics is prohibited in many countries due to the risk of developing resistance in the target and non-target populations of bacteria (Iacobellis et al., 2005). Nevertheless, developing new active compounds with low phytotoxicity, decreased environmental effect and broad spectrum of activity is still essential (Cabrefiga & Montesinos, 2017). There are small peptides and proteins in the nature that exhibit antimicrobial activity. Antimicrobial peptides (AMPs) are emerging as a novel class of compounds for preventing plant diseases (Cabrefiga & Montesinos, 2017). AMPs are capable of being natural antibiotics created by animals, plants, protozoa, fungi, and bacteria (Biswaro et al., 2018). The antimicrobial role of this group of compounds is well recognized and their application in the agriculture has been discovered. In recent years developments in the synthesis and screening of AMPs are crucial to the antimicrobial use of these compounds (Marcos et al., 2008). The lack of AMPs in natural resources, along with extraction and purification costs, prompted scientists to use the recombinant DNA technology for achieving these valuable bio-products. Due to advances in genetic engineering, the AMPs can be produced in a cost-effective manner compared to natural resources and industrial biochemical production (Li, 2011).
Lactoferricin (Lfc) and lactoferrampin (Lfp) are two AMPs, which are the primary components of lactoferrin in milk. Lactoferrin is known as iron-binding glycoprotein with 80-kDa molecular weight; it has several biological activities, such as defense against pathogens, regulating the iron uptake, controlling the immune system, and promoting cellular growth (Fukuta et al., 2012). It has been shown that the antimicrobial effect of Lfc+Lfp is more potent than that of Lfp or Lfc alone (Wang et al., 2016). The thanatin peptide is also derived from the spined soldier bug (Podisus maculiventris S.) insect that possesses antimicrobial properties. Thanatin contains two amino acids, including two cysteine residues, forming a disulfide bridge (Mandard et al., 1998). It is the first inducible insect peptide having diverse activities against bacteria and fungi at various concentrations . Former research attempted to develop new antimicrobial agent paving the way for identifying peptides with activity against some plant pathogenic bacteria, including Erwinia amylovora, Pseudomonas syringae pv. syringae (van Hall) and Xanthomonas axonopodis pv. vesicatoria (Doidge) (Cabrefiga & Montesinos, 2017).
In addition to the antimicrobial influence of AMPs for bacterial control, other alternative ways like applying active ingredients in essential oils (EOs) have been proposed as well. Currently, there is a new trend to look for antimicrobial compounds from medicinal plants. These sources may include EOs and plant extracts. An important feature of EOs and their active ingredients are hydrophobicity enabling them to partition with the lipids present in the cell membrane of bacteria making them more permeable by unsettling the cell structures (Chouhan et al., 2017). In general, more phenolic compounds in the EO lead to higher antimicrobial properties. Thymol is a well-known component of many EOs. A greater quantity of this monoterpene is found in Lamiaceae plants, including Thymus spp., Monarda spp., and Origanum spp. (Salehi et al., 2017). Thymol and carvacrol are similar in terms of construction, but they differ in terms of the position of the hydroxyl group of the phenolic ring. Eucalyptol, also known as 1.8-cineole, is a monoterpene found in the EOs of some herbs such as eucalyptus, daphne, cardamom, rosemary, and sage (Brown et al., 2017). This compound has antioxidant, antimicrobial and anti-inflammatory properties and its antimicrobial effects on gram negative and gram positive bacteria have been shown (Hassanzadeh, 2005;Ji et al., 2005).
Fire blight disease severity ranges from widespread damage on a highly susceptible host to limited symptoms, or the lack of symptoms on a resistant host (Viljevac et al., 2009). The symptom severity is a result of different plant resistance levels that could be associated with the histochemical and/or biochemical variations such as accumulation of reactive oxygen species (ROS) and activity of antioxidative enzymes (Viljevac et al., 2009). It is believed that ROS accumulation is one of the first defense responses to the bacterial infection (Mittler, 2002;Viljevac et al., 2009). Several kinds of ROS such as superoxide anion, hydrogen peroxide, and hydroxyl radical have direct antimicrobial activities, which lead to decreased pathogen viability. However, the improved ROS production could also result in the oxidative damage of pigments, proteins, nucleic acids and lipids (Mandal et al., 2008;Viljevac et al., 2009).
Host plants have developed effective protective mechanisms against oxidative stress using enzymatic and non-enzymatic antioxidants for balancing ROS levels.
The main enzymatic antioxidants involved in response to pathogen attack are peroxidases (ascorbate peroxidase and guaiacol peroxidase (GPOD)), superoxide dismutase and catalase. Considering the potential antimicrobial effects of the above-mentioned compounds, the present study seeks to examine the inhibitory effects of these Effect of antimicrobial peptides and monoterpenes on control of fire blight compounds against E. amylovora. In this study, antibacterial effects of two recombinant antimicrobial peptides (Lfc+Lfp and thanatin) and two monoterpenes (thymol and 1,8-cineole) against E. amylovora were investigated. Many studies have explored diverse aspects of these compounds on human microorganisms. But scant attention has been paid to the effects of these compounds on plant pathogens. In addition, the level of GPOD enzyme changes in two pear cultivars, tolerant 'Dargazi' and semi-susceptible 'Spadona', under treatment with four antibacterial compounds was assayed.

Bacterial strain and culture conditions
The highly virulent E. amylovora isolate used in this study was obtained from the microbial collection of the Dept. of Plant Prot., Ferdowsi Univ. Mashhad, Iran. This strain was isolated from a garden of pear trees in Mashhad, Khorasan-Razavi province, Iran (Akhlaghi et al., 2018). When it was needed, fresh bacterial culture was prepared in Luria-Bertani (LB) broth medium (Liofilchem, Teramo, Italy, 25 g/L) overnight at 28 °C. The concentration of bacterial cells was adjusted to an optical density (OD) of 0.2 (108 CFU/mL) at 600 nm on LB broth (Bellemann et al., 1994).

Disk diffusion assay
Fresh culture of the bacterium in LB broth medium with a concentration of 10 8 CFU/mL was distributed on the surface of nutrient agar (NA) (Quelab, Montreal, Canada, 22 g/L) medium. After 15 min, 10 µL of each compound in final concentration of 250, 500, 1000, 1500 µg/mL were loaded over sterile blank paper discs (6 mm). Subsequently, plates were incubated at 28 °C for 48 h and the diameters of bacterial growth inhibition zones around the discs were measured. The experiments were performed in triplicate (Sokovic & Van Griensven, 2006).

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
MIC and MBC for each compounds were determined in 96-well plates containing NA medium as described previously (Mirzaei-Najafgholi et al., 2017). These assays were carried out in triplicate in three independent experiments.

In vitro flower inoculation assay
Pear flowers (Pyrus communis cv. 'Spadona') at balloon stage (a day before opening) were transferred into a sterile 10% sucrose solution. Fresh culture at the concentration of 10 7 to 10 8 bacteria was made overnight in a sterile 0.05 M Na/KPi buffer containing 0.03% Tween 20 (pH=7). Four microliters of bacterial suspension was placed on the cup (hypanthium) of each flower. The bacteria were sprayed after 4 h of treatment with the antibacterial compounds at the MIC concentration. Na/KPi buffer treatment (without bacteria) in hypanthium and water spray (negative control) and Na/KPi buffer treatment (with bacteria) and water spray (positive control) were used. The flower was incubated at 24 to 26 °C with a humidity of 80%. After 4 days, disease symptoms were investigated on the flowers) Fischer et al., 2012). Four types of marks were used for grading: A, flowers were healthy and without color change; B, color change in hypanthium and darkening of the tissue; C, tissue and color degradation in the hypanthium, style and filament; and D, blackened whole flower, ooze production, spreading symptoms to pedicle (Lelliott & Stead, 1987;Gerami et al., 2013).

Experimental design and optimization for mixing the compounds
In this study, response surface methodology (RSM) was used to optimize mixing of the antimicrobial compounds with the copper compound (Nordox cuprous oxide (Cu2O)). The RSM is an advantageous method to investigate the impact of several variables on the responses by changing them concurrently and performing a restricted number of tests. No studies employing the experimental design and optimization modeling approach for mixing the AMPs, monoterpene and copper compounds have been reported in the literature, so far. The central composite design (CCD) is an effective design for sequential experimentation letting a sensible amount of information to test the lack of fit while excluding a remarkably large number of design points. The CCD method was used to determine the mixing ratio of different antibacterial compounds. Therefore, 43 mixing states of the four antibacterial compounds were obtained with Nordox. Subsequently, concentrations (1/2 MIC, MIC, 3/2 MIC) of Lfc+Lfp 16, 32 and 47 µg/mL, thanatin and thymol 8, 16, and 24 µg/mL, 1,8-cineole 125, 250 and 375 µg/mL, Nordox 250, 500 and 750 µg/mL were prepared. The mixing method of the tested materials was used in the LB broth medium in the microplate. For this purpose, the concentrations were prepared and mixed in equal volumes (30 µL) in the LB broth and 10 μL of bacteria were added to each well with the concentration of 10 8 CFU/mL. After 24 h, 100 μL of bacterial cultures (concentration of 10 -3 CFU/mL) was distributed on the surface of NA medium. The number of colonies grown after incubation (24 h) was counted (Demirel & Kayan, 2012).

Disease severity on tolerant and semi-susceptible pear cultivars treated with compounds
Two-year-old pear (Pyrus communis) trees cv. 'Spadona' and 'Dargazi' were used for greenhouse experiments. These cultivars were obtained from Horticultural Science Research Institute (HSRI), Karaj, Iran. All tests were done on pears in the active growth phase of terminal shoots. The trees were kept to grow in separate pots in a greenhouse at 22 to 25 ºC under humidity and natural photoperiod. Pear seedlings used in this assay included plants sprayed with distilled water and inoculated with E. amylovora by placing 50 µL of the bacterial suspension on the shoots using a syringe (10 8 CFU/mL) (zero time). Plants were sprayed with antibacterial compounds (24 h before inoculation) in the 125, 250 and 500 µg/mL concentration and inoculated with the pathogen. Disease severity (DS) was monitored 16 days after inoculation and the symptoms were recorded. Total shoot and lesion lengths were measured for each shoot. Shoot susceptibility to fire blight was computed as follows: DS=(Length of blighted shoot/Total shoot length) × 100. The experiment was carried out with three replications (Bell et al., 2004;Ozrenk et al., 2011).

Activity of guaiacol peroxidase in two pear cultivars
The experiment was performed as described by Sklodowska et al. (2011) with some modifications. Two-yearold pear (cvs. 'Spadona' and 'Dargazi') seedlings were obtained from HSRI, Karaj, Iran. The growth conditions of seedlings and inoculations were similar to those of the previous stage. Pear seedlings used in this assay include: control plants sprayed with distilled water, plants sprayed with distilled water and shoots were punctured by a needle connected to a syringe containing the bacterial suspension (10 8 CFU/mL), at the axil of the third leaf below the shoot tip (zero time). Plants sprayed with antibacterial compounds (24 h before inoculation) in the 500 µg/mL concentration and inoculated with E. amylovora. Guaiacol peroxidase activity was measured at 0, 1, 3, 5 and 7 days after inoculation.
Collection and preparation of leaf samples as well as determination of GPOD activity and protein content were done as described earlier (Kar & Mishra, 1976;Viljevac et al., 2009). To extract the enzyme, three replicates were obtained from the control and infected leaves. Each sample included one leaf. After removing the main veins and adding polyvinyl pyrrolidone, leaf tissue was macerated into fine powder with liquid nitrogen using the pestle and mortar. Leaf powder (500 mg) was extracted with 3 mL of 100 mM potassium phosphate buffer, pH=6.8. After centrifugation for 15 min at 15000 g and 4 °C, the supernatant was taken for the GPOD assays.
Determination of the GPOD (EC 1.11.1.7) activity was done as described by Plewa et al. (1991) and . The GPOD activity was calculated by observing the increased absorbance at 470 nm over 2 min. The GPOD activity was measured using the guaiacol precursor and measuring tetraguaiacol absorption rate. In this method, the complex reaction consisted of 1.4 mL of 50 mM phosphate buffer (pH=7), 800 μL of guaiacol 2%, 0.5 mL of 1% hydrogen peroxide and 200 μL of the extracted solution.

Statistical analysis
The ANOVA test was conducted on a factorial experiment based on a completely randomized design with three replications. After ensuring the normal distribution of the data, ANOVA was performed using SPSS (V22.0). The means were compared using the Duncan's multiple rang test at 5% probability level. The charts were drawn by Excel 2016. To correlate the disease severity and GPOD activity levels and avoid any possible errors, the ratios were calculated between values obtained for control and treated plants. The Pearson correlation coefficient between the data obtained from the GPOD activity assay and disease Effect of antimicrobial peptides and monoterpenes on control of fire blight severity was calculated using SPSS (V22.0) software during three days.

Screening antibacterial activity of various compounds against E. amylovora
The results of analyzing the antibacterial effects of AMPs and monoterpene compounds suggest their potential antibacterial effect against E. amylovora (Table 1). Regarding the inhibition zone diameter, thanatin peptide had the highest antibacterial activity at 1500 µg/mL against E. amylovora with 16.17 mm inhibition zone. The Lfc peptide and thymol with 12.67 and 12.33 mm diameter inhibition zones were placed in subsequent positions. The lowest inhibition was observed for the 1,8-cineole compound, where the mean diameter of inhibition hole was 8.67 mm (similar to the Nordox). AMPs had less antibacterial effect than thymol at concentrations of 250 and 500 µg/mL. Improved antibacterial properties of compounds were directly related to the increased concentrations. According to the results of antibacterial activity screening, the AMPs had stronger antimicrobial effect than that of monoterpene compounds.

Determination of MIC and MBC
Microplate dilution is an accurate and sensitive method for determining the antimicrobial effects of various compounds. The MIC results revealed that thanatin and thymol at concentration of 15.62 μg/mL restricted the growth of E. amylovora and the MBC was 31.5 μg/mL. The highest levels of MIC were obtained for Nordox and 1,8-cineole at 250 μg/mL and 500 μg/mL, respectively. The highest amount of MBC was obtained at 1000 μg/mL for the Nordox as a copper compound (Table 2).

In vitro flower inoculation assay
Considering the effect of antimicrobial compounds on pear flowers, all compounds were capable of slowing down the disease progress compared to the control (Type D) as shown in Fig. 1. The lowest disease symptoms were observed on the flowers treated with both of thanatin (type B) and thymol (type B). The highest level of disease symptom was observed on flowers treated with the 1,8-cineole (Type C). The results of this test suggested that, unlike AMPs, the two monoterpene compounds used in this study had phytotoxicity on flower petals (Table 3).

Optimization analysis for mixing the antibacterial compounds
The results of mixing the antibacterial compounds were compared using a completely randomized design with the CCD method (  reduction of bacterial colony number in NA (Table 4).
Other results also showed that the mixing ratio of 32, 8, 16, 250 and 750 µg/mL, (seventeenth row, 104 colonies) had the least effect on Lfc+Lfp, thanatin, thymol, 1,8-cineole and Nordox respectively. The antibacterial effect of AMPs and copper compound during mixing of all treatments was more than monoterpenes (Table 4).

Effect of antimicrobial compounds on protecting the shoots of pear cultivars against E. amylovora
The results of disease severity assay revealed that the percentage of damage caused by E. amylovora on cv. 'Dargazi' (tolerant) (21.87%) was lower than in 'Spadona' (semi-susceptible) (58.85%). Application of all antibacterial compounds tested on 'Spadona' and 'Dargazi' reduced the DS compared to the Nordox treatment and control (Fig. 2). The results indicate that the Nordox was not significantly different in reducing the DS in comparison with the control (water spray) in the three concentrations tested. The results of DS and concentration showed that in three concentrations (125, 250 and 500 µg/mL) Effect of antimicrobial peptides and monoterpenes on control of fire blight used in this assay, the maximum DS reduction was observed at concentrations of 500 µg/mL. The maximum reduction of DS on 'Spadona' was obtained using the 1,8-cineole (16.05%) and thymol (18.88%) at concentrations of 500 µg/mL (Fig. 2). 1,8-cineole and thymol had a greater role in reducing the percentage of necrosis on the shoot and there was no statistically significant difference in the in-vivo effect of two tested AMPs on this cultivar at 500 µg/mL. The maximum reduction of DS on cv. 'Dargazi' was obtained using the thymol (7.71%) and 1,8-cineole (9.05%) at concentrations of 500 µg/mL (Fig. 2). There was no statistically significant difference with the application of 250 µg/mL concentrations of each treatment on 'Dargazi' except thymol. However, the differences between the effect of Lfc+Lfp, thanatin and 1,8-cineole on 'Dargazi' were not significant compared to Nordox treatment. The least effect on reducing the DS was associated with the application of the lowest concentrations (125 µg/ mL) of each compound, which was more pronounced for AMPs, although they did not significantly differ. Other results revealed the fact that there is a direct correlation between concentration of antibacterial compounds and reduction in the percentage of shoot necrosis.

Guaiacol peroxidase activity in two pear cultivars
The results of GPOD activity assay showed that the primary level of enzyme in 'Dargazi' (tolerant) was higher than the enzyme level in 'Spadona' (semi-susceptible). Effect of antimicrobial peptides and monoterpenes on control of fire blight Moreover, the GPOD activity pattern was not similar in the two cultivars (Fig. 3). Generally, in 'Dargazi' the enzyme level showed an increasing trend up to 3 days after inoculation (DAI), and then decreased. In 'Spadona' an increasing trend was observed for the GPOD activity at all five time points (Fig. 3). In 'Dargazi', the control treatment (water spray) showed a constant trend, while the treatment with bacteria tested showed a rising trend (up to 3 DAI, the enzyme level showed an increasing trend, followed by a decreasing one). The GPOD activity increased until 3 DAI, then showed a decreasing trend until the 5 DAI, and the enzyme level increased again until the 7 th day (Fig. 3). In 'Dargazi' all antibacterial compounds increased the level of GPOD over a period of three days. The highest level of the enzyme was obtained by treatment with 1,8-cineole (Fig. 3d) and thymol (Fig. 3c). The rate of GPOD activity under Lfc+Lfp (Fig.  3a) and thanatin (Fig. 3b) treatments was lower than that of the plants treated with 1,8-cineole and thymol. However, the enzyme level in the plants treated with thanatin was close to the bacterial treatment at 5 DAI, but in case of Lfc+Lfp, a lower decrease in the level of enzyme was observed at 5 DAI (Fig. 3). In 'Spadona' the control treatment (water spray) had a constant trend, while treatment with bacteria showed an increasing trend until 7 DAI. In this cultivar, all antibacterial compounds increased the level of GPOD for three to seven days. The highest level of enzyme activity was observed in the plants treated with 1,8-cineole (Fig 3d) and thymol (Fig. 3c) over a period of 5 to 7 days. Like in 'Dargazi', the activity of GPOD enzyme in plants treated with Lfc and thanatin was at the lowest level during the time period investigated compared to 1,8-cineole and thymol and did not show considerable differences with Nordox and control (Fig. 3).

Discussion
Fire blight causes serious damage to pome fruit trees in Iran and other countries, so it is necessary to combat E. amylovora bacteria to increase yield. In addition to antibiotics, copper compounds and other biological controlling products have also been applied to control fire blight; nevertheless, applying these materials is restricted due to their unpredictable control effects and application of copper compounds can lead to phytotoxicity (Patel et al., 2017). On the other hand, it is difficult to assess the effect of copper compounds on fire blight and the ineffectiveness of other chemical compounds in controlling the disease due to the resistance or tolerance of microorganisms (Al-Daoude et al., 2009;Ivanović et al., 2016).
Every year, approximately 2.5 million tons of pesticides are used on crops worldwide to fight plant disease (Koul et al., 2008;Ben Kaab et al., 2019). Natural products derived from plants and AMPs have no harmful effects of chemical compounds on human health and the environment. In addition to medical applications, the AMPs can also be used in plant protection. The reduced yield, quality, and safety of agricultural products caused by plant diseases can be overcome using AMPs (Holaskova et al., 2015). Large amounts of high purity peptides are required for basic research and clinical trials. In comparison with the process of separation from natural sources and chemical synthesis, the recombinant expression method suggests an effective and economic means to produce peptide in high yield. In this regard, using recombinant AMPs (naturally occurring in camel milk and insect), we showed that they have unique antibacterial effect on E. amylovora.
To date, several studies have been performed on thanatin. For instance, the transgenic rice (Oryza sativa L.) encoded thanatin peptide was resistant to blast disease (Magnaporthe grisea M.E. Barr) in field evaluations (Imamura et al., 2010). Other study reported that the transgenic Arabidopsis thaliana encoded thanatin was resistant to the phytopathogens (Wu et al., 2013). Great antifungal activity of the AMP was observed at μg level under both in vitro and in vivo conditions against five fungal phytopathogens . Although no comprehensive research has been conducted on the anti E. amylovora role of thanatin, this study showed that the peptide possesses high antimicrobial activity similar to that of previous studies. The antimicrobial effect of Lfc peptide on plant pathogenic bacteria has been evaluated in Lfc of cow milk against Xanthomonas campestris pv. phaseoli (Smith) Constantin et al., Ralstonia solanacearum (Smith) Yabuuchi et al., and Pseudomonas syringae pv. phaseolicola (van Hall) (Fukuta et al., 2012). Tanhaeian et al. (2018) studied the effect of lactoferrin+lactopharmin on 14 plant pathogenic bacteria, including E. amylovora, and found that the MIC for the bacterium is 1.31 μg/mL. Compared to previous studies, the results of this study showed that the MIC for the E. amylovora was higher (62.5 μg/mL), which is probably due to the type of E. amylovora isolate, which was highly virulent. Other researchers reported that the antibacterial activity of a recombinant chimeric peptide is significantly stronger than its non-recombinant peptide (Bolscher et al., 2009;Tang et al., 2012). The results of this study revealed that, contrary to previous studies, thanatin (a non-recombinant chimeric peptide) has more antibacterial properties than the Lfc peptide (a recombinant chimeric peptide). It seems that the role of the N-terminal tail of recombinant peptides in antibacterial activities is more important than the fusion of two peptides. Considering that peptide is edible and has high durability (temperature resistance of 90 °C), the use of this peptide to control bacterial pathogens can be considered as a new strategy in the biological control. As an alternative approach for preventing the spread of plant diseases, it is possible to use natural compounds of plants as a source of new pesticides (Yumlembam & Borkar, 2014). Thymol is generally used as a general disinfectant or additive in cosmetics, food industry, or medicine.
In agriculture, the effect of thymol against microorganisms and insects has been depicted (Calvet et al., 2001;Ji et al., 2005). However, there are few studies about the use of thymol in greenhouses and farms. Applying thymol as a fumigant in laboratory and greenhouse conditions leads to a significant reduction in the population of Ralstonia solanacearum bacteria in the soil (Ji et al., 2005). The analysis of thymol effect in this study showed its antibacterial effect on E. amylovora, which is consistent with the results of previous studies in the control of gram-negative and gram-positive bacteria (Hassanzadeh, 2005;Ji et al., 2005). The study showed that thymol has potential to control E. amylovora. However, further research is required to investigate the possibility of direct application of thymol on flowers due to the sensitivity of tissue, the possibility of damage to pollination process and formation of fruits. The 1,8-cineole is used as a component of some aromatic plants such as coriander, oregano, rosemary, thyme and ginger (Van Vuuren & Viljoen, 2007). The results of this study, consistent with other studies, revealed the antibacterial effects of 1,8-cineole against E. amylovora. The antibacterial effect of 1,8-cineole on flowers and fruits was lower than thymol, which is related to the structural differences between the two compounds. Identical to thymol, 1,8-cineole also had improved effects on reducing the disease on the fruits compared to the blossom, and showed a phytotoxicity rate lower than that of thymol. Although reduction in disease symptoms on pear blossoms treated with 1,8-cineole was not clear (because of the phytotoxicity of this compound on the blossoms), but 1,8-cineole inhibited the disease progress of E. amylovora on pear branches better than other tested compounds when used at 500 µg/mL. In natural conditions, initial infection of pear trees occurred by epiphytic populations of E. amylovora that become established on the stigmas of flowers. The pathogen spreads from stigmas to the hypanthium in surface moisture and invades through the nectarthodes (Thomson, 1986). Therefore, application of copper compounds or antibiotics either before or immediately after the pathogen reaches to the pear flowers or shoots would be very useful because of direct contact of applied materials with the pathogen. Once E. amylovora enters the host xylem or cortical parenchyma and spreads in the endophytic phase of pathogenesis (Koczan et al., 2009), external control methods become ineffective. Plant resistance inducers and biological control agents are potential alternatives to antibiotics and copper (Johnson & Temple, 2013). The two tested AMPs, Lfc+Lfp and thanatin, reduced disease symptoms caused by E. amylovora on pear flowers and did not show phytotoxicity, while the two monoterpenes used in this study, specially 1,8-cineole, showed high phytotoxicity. However, 1,8-cineole and thymol reduced disease symptoms on branches. Therefore, we suppose these compounds could be new alternatives for management of fire blight disease in epiphytic or endophytic growth phase of E. amylovora.
Another part of this study focused on applying the RSM and finding an applicable approximating function for the ratio of mixing antibacterial compounds (antimi-crobial peptides and monoterpene) with a copper compound (Nordox) for finding the most effective antibacterial MIC (1/2 MIC, MIC, 3/2 MIC) concentration. RSM is a type of mathematical and statistical method to design experiments, create models, assess the relative significance of several independent variables, and determine the optimum conditions for necessary responses (Zhang & Zheng, 2009;Demirel & Kayan, 2012). The two most common designs widely applied in RSM are the CCD and the Box-Behnken design. The CCD is perfect for sequential experimentation paving the way for a sensible amount of information to test lack of fit while excluding remarkably considerable number of design points (Somayajula et al., 2011;Demirel & Kayan, 2012). In this study, the mixing conditions of tested compounds were determined using the CCD method. Using the mentioned statistical method was better than other methods due to reduced number of experiments and the simultaneous analysis of the parameters. More studies have been done on the mixing of antimicrobial compounds and toxins, antibiotics, essential oils and plant extracts against various pathogens (Langeveld et al., 2014). In another study, the synergistic effects of piperacillin antibiotic with cinnamon essential oil and chloramphenicol antibiotic with oil of savory were assayed against E. coli (Bassolé & Juliani, 2012). However, the effects of mixing between new antimicrobials agents and copper compounds were not assayed and discussion on comparing the antibiotic, essential oils and plant extracts with poison was important. Various biotic and abiotic stresses activate a series of reactions leading to resistance in plants. Applying protective agents and chemical activators of plant defense system for decreasing the disease damage are novel methods favored by researchers in recent years. Plants are capable of increasing their resistance against plant pathogens agent. This phenomenon is known as induced resistance activated by some microorganisms, natural or artificial chemicals, or by lesions. The GPOD activity assay in this study showed that in tolerant and semi-susceptible cultivars, the GPOD activity was significantly different. Ebadi et al. (2014) found that in resistant cultivars such as 'Dargazi' and 'Harrow Sweet', the GPOD level reached to its maximum level at 3 DAI. But in the susceptible cultivars 'Bartlett' and 'Mohhamdali', the GPOD activity peaked later at 6 DAI, followed by a decreasing trend until 12 DAI, which is in agreement with the results of the present study. It seems that antioxidant activity (such as peroxidase and catalase) in early days after inoculation of bacteria in resistant pear cultivars causes a hypersensitive response. In resistant cultivars, the hypersensitive response (which is a mechanism, used by plants, to prevent the spread of infection) is a local, defensive, and quick reaction leading to the cell death in the infected area inhibiting the spread of bacteria in the host tissue. As the results of this study showed, the tested antibacterial compounds play a role in the induction of resistance, which is more evident in monoterpene compounds. In susceptible or semi-susceptible cultivars, the free radicals are very high after the bacterial attack, but the defense system was unable to decompose them due to the lower level of antioxidant enzymes and the bacteria developed more rapidly inside the plant (Ebadi et al., 2014). The use of antibacterial compounds, especially monoterpenes, in this study showed that like some natural and artificial chemical compounds, monoterpenes could play an important role in increasing the antioxidant enzymes, such as GPOD. So far, the mechanism of action of monoterpenes compounds and AMPs in induction of resistance was not fully understood, but it seems that they activate some of the defense related mechanisms in plants.
The correlation coefficient is one of the most often used statistical tools for analyzing the associations among the traits. Pearson's correlation coefficient is a statistical method of quantifying the association, or "coherence", between two variables. Therefore, it is a very popular tool to analyze data many scientific disciplines like biology (Soares et al., 2011). The results of this study revealed that the GPOD enzyme has a negative correlation with disease severity (-0.701, p=0.0001). This study is the first report on the use of antibacterial compounds, such as 1,8-cineole, thymol and two AMPs (thanatin and Lfc) on blooms, fruits and seedlings of pear. In this study, for the first time, statistical methods for mixing new antibacterial compounds with common copper compounds were used to control fire blight. It seems that the antibacterial compounds used in this study, in addition to their antibacterial role, can also play a role in activating defense responses in the pear tree. If these materials provide a higher level of protection against the fire blight pathogen before the induction of resistance using their antimicrobial properties, the effective control of the disease will occur. The results of this research could be helpful for applying novel alternative compounds in fire blight disease management.