Abstract

Introduction

Bacterial diseases represent some recalcitrance and difficulties to manage the pest problems affecting commercial vegetable production. Spraying with antibiotics and pesticides, usually suggested controlling bacterial diseases, have never been satisfactory. Antibiotics and synthetic pesticides are forbidden in many countries because of their exerting a negative impact such as high and acute toxicity, long degradation periods and accumulation in the food chain. Therefore, losses from bacterial diseases can be substantial all over the world. Plant diseases caused by bacteria are factors in several plant family members (Aktar et al., 2009[1]). In addition; bacterial diseases caused by pathovars of Xanthomonas also result in a decreased yield of harvest. Pathovars of Xanthomonas are reported to have developed resistance to several antibiotics such as kanamycin, ampicillin, penicillin, and streptomycin (Rodriguez et al., 1997[18]). This seriously hinders the management of diseases of crops and agricultural products (Satish et al., 1999[19]). Besides, control of the disease is difficult, often requiring expensive and complex integrated pest management (IPM), including the use of contamination-free seeds, sanitization practices, and the use of chemicals (Araujo et al., 2003[2]). Naturally occurring biologically active plant products such as plant-based essential oils and organic extracts, pure compounds could be a source of new eco-friendly pesticides to serve as templates for new and more effective compounds in controlling plant pathogenic microorganisms. Furthermore, bio-pesticides have been suggested as effective substitutes for chemical pesticides (Gan-Mor and Matthews, 2003[9]).

Poncirus trifoliata Rafin (Rutaceae), also known as trifoliate orange, is a close relative to the Citrus trees. It is a decidious or semi-decidious shrub, a native of China and Korea, and is also known as the Korean bitter orange. Traditionally, trifoliata oranges (P. trifoliata) have been widely used in folk medicine as a remedy for gastritis, dysentery, inflammation, digestive ulcers, etc. A scientific investigation into the health-maintaining properties of trifoliate orange fruit has revealed its anti-inflammatory, antibacterial and anti-anaphylactic activities (Kim et al., 1999[11]). In Korea, fruit extracts of P. trifoliate are used in some over-the-counter drugs for the treatment of a variety of gastrointestinal (GI) disorders (Lee et al., 2005[13]). Another researcher reported that poncirus fruit is a potent antileukaemic agent by promoting apoptosis of cancer cells (Yi et al., 2005[23]). Several compounds such as poncirin, coumarins, auraptine, hesperidin and naringin have been identified from poncirus fruits (Avula et al., 2005[3]). Previously, we reported the chemical compositions of seed essential oil of Poncirus trifoliata Rafin by GC-MS and evaluated antibacterial potential of the oil and organic seed extracts of P. trifoliata Rafin against foodborne pathogens (Rahman et al., 2009[16]). Further, we have previously reported on the isolation, characterization and anti-listerial potential of naturally occurring limonin and imperatorin from P. trifoliata seed (Rahman et al., 2012[17]). However, there is no report available in the literature on antibacterial properties especially on Xanthomonas spp. including morphological changes using volatile essential oil, isolated natural compounds of P. trifoliata seeds.

Considering the deleterious effects of synthetic antibiotics and pesticides on life supporting systems, there is an urgent need to search for alternative approaches for the management of plant pathogenic microorganisms. Therefore, to find out the environmentally friendly alternatives to chemical pesticides we investigated the role of secondary metabolites (essential oil and compounds limonin and imperatorin) of P. trifoliata as an antibacterial potential on Xanthomonas spp. in vitro.

Materials and Methods

Results

In vitro antibacterial effect of seed essential oil of P. trifoliata against phytopathogenic Xanthomonas spp.

Seed essential oil also showed potent in vitro inhibitory effect against plant pathogenic bacteria of Xanthomonas spp. At the concentrations of 5, 10 and 15 μl/disc of 1:5 (v/v) dilution of seed essential oil with methanol, the oil exhibited a potent inhibitory effect against all four strains of Xanthomonas spp. such as X. campestris pv. compestris KC94-17-XCC, X. campestris pv. vesicatoria YK93-4-XCV, X. oryzae pv. oryzae KX019-XCO and Xanthomonas sp. SK12 as a diameter of inhibition zones of 13.1~20.0, 14.0~22.1, 16.2~21.0 and 15.2~21.2 mm, respectively along with their MIC values ranging from 62.5 to 125 μg/ml (Table 1(Tab. 1)).

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Table 1

In vitro antibacterial effect of seed essential oil of Poncirus trifoliata Rafin against phytopathogenic Xanthomonas spp.

In vitro antibacterial activities of limonin and imperatorin against Xanthomonas spp.

Limonin and imperatorin exhibited potent antibacterial activity against phytopathogenic bacteria of Xanthomonas spp. (MIC value: 15.62~62.5 μg/ml) as shown in Table 2. Bacterium Xanthomonas spp. SK12 was found to be the most sensitive organisms toward limonin and imperatorin (MIC: 15.62 μg/ml for each), while X. campestris pv. vesicatoria YK93-4-XCV was the most sensitive to imperatorin (MIC: 15.62 μg/ml) (Table 2(Tab. 2)).

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Table 2

In vitro antibacterial effect of limonin and imperatorin on phytopathogenic Xanthomonas spp.

Scanning electron microscopic (SEM) studies on Xanthomonas sp. SK12 using compounds limonin and imperatorin

On the basis of lower MIC values (15.62 μg/ml) of both limonin and imperatorin on X. spp. SK12, the effect of these compounds (MIC) on the morphology of this bacterium was observed by scanning electron microscopy (SEM) and the results showed the detrimental effect of these compounds on the cell morphology of X. sp. SK12 (Figure 1(Fig. 1)). Control cells in the absence of limonin and imperatorin showed a regular, smooth surface as shown in Figure 1 (A1-B1)(Fig. 1). In contrast, cells inoculated with limonin and imperatorin at MIC value (15.62 μg/ml) revealed severe detrimental effect on the morphology of cell membranes, showing disruption, pore formation and analysis of the membranes integrity, as shown in Figure 1 (A2-A3 & B2-B3)(Fig. 1).

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Figure 1

Effect of limonin and imperatorin on morphological changes on Xanthomonas sp. SK12. Morphological damages of Xanthomonas sp. SK12 treated with limonin and imperatorin (MIC 15.62 µg/ml) by scanning electron microscopy (SEM). A1: Control (limonin 0 µg/ml); A2 and A3: Treated with limonin (15.62 µg/ml) showing distorted cell, pore fomation and lysis of cell. B1: Control (imperatorin 0 µg/ml); B2 and B3: Treated with imperatorin (15.62 µg/ml) showing distorted cell, pore fomation and lysis of cell.

Discussion

The increasing social and economic implications caused by plant pathogenic bacteria means there is a constant striving to produce safer foods (crops, vegetables and fruits) and to develop new antibacterial agents. In general, plant-derived essential oils are considered as non-phytotoxic compounds and potentially effective against pathogenic bacteria (Vasinauskiene et al., 2006[22]; Basim and Basim, 2003[4]). In recent years, the use of antimicrobial compounds such as essential oils extracts and natural compounds are one of the first choices after outbreaks of bacterial plant diseases. Besides, interest has been generated in the development of safer antibacterial agents to control plant pathogenic bacteria in agriculture which also include essential oils and extracts (Deena and Thoppil, 2000[8]).

Essential oils, which are odorous and volatile products of plant secondary metabolism, have wide applications to control pathogenic bacteria (Ozturk and Ercisli, 2007[15]). Various publications have documented the antimicrobial activities of essential oils and plant extracts (Saxena et al., 2012[20]; Shafia et al., 2002[21]; Bougatsos et al., 2004[5]; Jirovetz et al., 2006[10]). Mono- and sesqueterpenes, which are phenolic in nature, have potential antimicrobial activities. It seems reasonable to assume that their antimicrobial mode of action might be related to the phenolic compounds present (Cakir et al., 2004[6]). Most of the studies on the mechanism of phenolic compounds have focused on their effects on cellular membranes. Actually, phenolic compounds not only attack cell walls and cell membranes, thereby affecting their permeability and release of intracellular constituents (e.g. ribose, Na glutamate) but also interfere with membrane functions (electron transport, nutrient uptake, protein, nucleic acid synthesis and enzyme activity). Thus, active phenolic compounds present in the oil of P. trifoliata might have several invasive targets which could lead to the inhibition of plant pathogenic bacteria.

In our study, it has become clear that seed essential oil strongly inhibited in vitro the growth of Xanthomonas spp. Bacterial leaf blight caused by X. oryzae pv. oryzae has been reported the most serious disease of rice in South East Asia, particularly since the widespread cultivation of dwarf high-yielding cultivars (Zhou et al., 2013[24]).

Compounds limonin and imperatorin also showed a great potential of antibacterial activity against plant pathogenic bacteria of Xanthomonas spp. In this study, Xanthomonas sp. SK12 was found to be the most sensitive organism to both limonin and imperatorin. SEM study also revealed the detrimental effect of limonin and imperatorin on the cell morphology of the tested organism such as Xanthomonas sp. SK12. The results of this study would be worthy as an important bio-control approach to inhibit such severe plant pathogenic bacteria.

Notes

Prof. Md. Atiqur Rahman and Prof. Sun Chul Kang (Phone: +82-53-850-6553; FAX: +82-53-850-6559, eMail: sckang@daegu.ac.kr) contributed equally as corresponding authors.

References