Microbes in Applied Research

Determination of chemical composition and antibacterial properties of essential oil of Mentha longifolia ssp. longifolia against phytopathogenic bacteria 1,*

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Derya Yanmis , Arzu Gormez , Sedat Bozari , Furkan Orhan , Medine Gulluce , 1 4 Guleray Agar and Fikrettin Sahin 1

Ataturk University, Faculty of Science, Biology Department, TR-25240 Erzurum, Turkey Department of Plant Protection, Faculty of Agriculture, Ataturk University, Erzurum 25240, Turkey 3 Mus Alparslan University, Faculty of Arts and Science, Department of Biology, Mus 49100, Turkey 4 Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering, 34755 Kayışdağı, Istanbul, Turkey

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In the present study, the chemical composition of the essential oil (EO) of Mentha longifolia L. Hudson ssp. longifolia (ML) and the antibacterial activity of the EO against phtopathogenic bacteria were determined. The EO of ML that grown in Eastern Anatolia was isolated by the hydrodistillation method and analyzed by GC-MS. According to GC analysis of the chemical composition of the EO of ML, it consisted of 12 identified components. Major components of the oil were cis-Piperitone epoxide (26.52%-GC), Piperitenone oxide (26.40%) and Pulegone (15.66%). The antibacterial activity of the EO was also tested against 20 phtopathogenic bacteria. In general, the oil had antibacterial activity at a wide spectrum on the growth of phtopathogenic bacteria. In conclusion, the results revealed that the EO of ML have significant antibacterial activity, and the findings of the present study are valuable for further investigations focusing on controlling plant pathogenic bacteria that cause crop loss. Keywords Antibacterial activity; Mentha longifolia L; Hudson ssp. Longifolia; biopesticide.

1. Introduction The number of plant diseases caused by phtopathogenic bacteria is continuously growing. These bacteria can cause crop loss and serious economic losses in agriculture [1, 2]. At the present day, rapid and effective control of plant diseases caused by bacteria is mostly carried out by using chemical compounds and antibiotics. On the other hand, it was revealed that the chemical compounds and antibiotics have many harmful effects on environment and all the organisms because of their slow biodegradation in the environment and their toxic residues in the agricultural products [3, 4, 2]. Furthermore, in recent years, multiple drug/chemical resistance in pathogenic microorganisms has developed due to indiscriminate use of commercial antimicrobial drugs/chemicals commonly used in the treatment of infectious diseases [5, 6]. Therefore, recently the number of the researches on natural antibacterial compounds like plant extracts and essential oils that have a rich source of bioactive chemicals against plant pathogenic bacteria are increasing [7, 8]. In the present study, we investigated an alternative biopesticide against phytopathogenic bacteria that is an essential oil of Mentha longifolia commonly used as food additives constituting a rich source of bioactive chemicals. Mentha widely used in food, flavour, cosmetic and pharmaceutical industries is a medicinal and aromatic plant belonging to the Lamiaceae [9]. Mentha ssp. has been used as a folk remedy for treatment of nausea, bronchitis, flatulence, anorexia, ulcerative colitis, and liver complaints due to its antinflammatory, carminative, antiemetic, diaphoretic, antispasmodic, analgesic, stimulant, emmenagogue, anticatharrhal, antimutagenic and antioxidant activities [10]. In our work, we aimed to determine chemical composition of hydrodistilled EO of a member of Mentha genus, Mentha longifolia by GC-MS system and its antibacterial (biopesticide) potential of the EO which have not been tested against this many phytopathogenic bacteria in previous.

2. Materials and Methods ML was collected during the flowering stage in July 2009, from Erzurum. The identification of plant materials was confirmed by a plant taxonomist, Assoc. Prof. Dr. Yusuf KAYA, in the Department of Biology, Ataturk University, Erzurum, Turkey. Plant samples were dried in a canopy room. The aerial parts (leaves, flowers and steams) of the plant were powdered with blender and then subjected to water distillation for 2-3 h in a Clevenger-type apparatus (Thermal Laboratory Equipment, TURKEY). The chemical composition of EO analyses was performed through GC-MS. All the bacterial strains were isolated from some fruits and vegetables exhibiting typical bacterial disease symptoms on their respective host plants and identified by using a microbial 531

identification system (MIS) (1). Antimicrobial tests were carried out by disc diffusion assay using 100 µl of suspension containing 108 cfu/ml of bacteria spread on tryptic soy agar (TSA) medium by a sterile swab according to Murray et al. (1995). The minimal inhibition concentration (MIC) values were also studied for the bacteria determined as sensitive to the EO in disc diffusion assay according to Zgoda and Porter, (2001).

3. Results The EO of the MLwas tested against 20 plant pathogenic bacterial strains which were shown in Table1.


Table 1. Plant pathogenic bacterial species used in the study Bacterium

Host

Agrobacterium tumefaciens Bacillus pumilus Clavibacter michiganensis subsp. michiganensis Enterobacter intermedius Erwinia caratovora caratovora Erwinia chrysanthemi Pseudomonas cichorii Pseudomonas corrugata Pseudomonas fluorescens Pseudomonas syringae pv. syringae Pseudomonas syringae pv. syringae Pseudomonas syringae pv. syringae Pseudomonas syringae pv. syringae Pseudomonas syringae pv. phaseolicola Pseudomonas syringae pv. pisi Pseudomonas syringae pv. tabaci Pseudomonas syringae pv. tomato Ralstonia solanacearum Xanthomonas axonopodis pv. campestris Xanthomonas vesicatoria

Apricot Apricot Tomato Cherry Tomato Apricot Peach Tomato Apricot Cherry Apricot Apricot Apricot Beans Peach Apricot Cherry Apricot Pepper Tomato

Strain No AA-685 AA-479 AA-703 AA-184 AA-687 AA-58 AA-234 AA-684 AA-616 AA-218 AA-637 AA-638 AA-647 AA-652 AA-237 AA-704 AA-220 AA-116 AA-705 AA-683

Antibacterial activity of ML against the number of bacteria is shown in Table 2. The EO showed antibacterial activity against all tested bacterial strains (7-45 mm inhibation zone). MIC values showed by the EO of ML were in the range of 7.81 - 62.5 µl/ml (Table 2). Bacillus pumilus, Enterobacter intermedius, Pseudomonas cichorii, Pseudomonas fluorescens, Pseudomonas syringae pv.syringae (from apricot), Pseudomonas syringae pv. phaseolicola, Pseudomonas syringae pv. pisi, Pseudomonas syringae pv. tabaci, Pseudomonas syringae pv. tomato, Ralstonia solanacearum, Xanthomonas axonopodis pv. campestris and Xanthomonas vesicatoria were determined the most sensitive microorganism (MIC value 7.81) against to EO. The other sensitive microorganisms were Erwinia caratovora caratovora and Erwinia chrysanthemi (MIC value 15.63); Pseudomonas syringae pv. syringae isolated from cherry, Agrobacterium tumefaciens and Pseudomonas corrugata (MIC value 31.25). MIC values of Clavibacter michiganensis subsp. michiganensis were in a higher level than the others (MIC value 62.5). Antibiotics used as the positive control showed important inhibition zone (10-30 mm). DMSO used as the negative control didn’t show any inhibition zone against the bacteria. The chemical composition of ML EO was analyzed by GC–MS, leading to comparison of the relative retention times and the mass spectra of oil components with those of authentic samples and mass spectra from data library, as shown in Table 3.

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Table 2. Antibacterial activities of the essential oil of ML Concentration 500 µl

250 µl

125 µl

62.5 µl

31.25 µl

15.63 µl

7.81 µl

15 16 11

11 14 9

10 12 8

9 10 7

9 9 -

8 -

7 -

31.25 7.81 62.5

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Positive control Standart antibiotic discs 29 (SCF) 23 (OFX) 26 (SCF)

16 15 18 36 15 25 19

13 13 15 27 13 23 13

12 11 12 19 11 21 12

10 10 11 15 11 13 9

9 9 9 12 8 10 8

8 8 8 11 9 -

7 9 7 -

7.81 15.63 15.63 7.81 31.25 7.81 31.25

-

26 (SCF) 30 (OFX) 25 (SCF) 25 (OFX) 26 (OFX) 10 (OFX) 25 (OFX)

26 25 26 24

25 24 24 22

21 19 20 19

14 13 15 15

11 9 9 12

9 9 8 9

7 7 7 7

7.81 7.81 7.81 7.81

-

20 (OFX) 20 (OFX) 20 (OFX) 24 (OFX)

23 29 26 45 17

19 25 18 43 15

15 22 14 40 12

13 18 11 16 10

11 11 9 14 9

10 9 8 11 8

9 7 7 9 8

7.81 7.81 7.81 7.81 7.81

-

24 (OFX) 23 (OFX) 24 (OFX) 22 (SCF) 20 (SCF)

15

13

11

11

9

8

8

7.81

-

21 (SCF)


Bacterium

Agrobacterium tumefaciens Bacillus pumilus Clavibacter michiganensis ssp. michiganensis Enterobacter intermedius Erwinia caratovora caratovora Erwinia chrysanthemi Pseudomonas cichorii Pseudomonas corrugata Pseudomonas fluorescens Pseudomonas syringae pv. syringae*** Pseudomonas syringae pv. syringae Pseudomonas syringae pv. syringae Pseudomonas syringae pv. syringae Pseudomonas syringae pv. phaseolicola Pseudomonas syringae pv. pisi Pseudomonas syringae pv. tabaci Pseudomonas syringae pv. tomato Ralstonia solanacearum Xanthomonas axonopodis pv. campestris Xanthomonas vesicatoria

MIC

Negative control DMSO

*DMSO; Dimethyl sulfoxide (%10), ** OFX, ofloxacin (10 /g/disc); SCF, sulbactam (30 /g/disc) + cefoperazone (75 /g) (105 /g/disc) were used as positive reference standart antibiotic discs (oxoid) *** from different host (cherry).

Table 3. Essential oil content of ML

No

RI

RT

Components

(%)

Identificitation methods

1 2 3 4 5 6 7 8 9 10 11 12

1159 1165 1197 1234 1255 1296 1345 1369 1419 1460 1574 1579

20.74 21.17 22.88 24.77 25.64 27.55 29.49 30.54 32.27 33.81 39.40 39.60

Menthone Isomenthone cis-Dihyro carvone Pulegone cis-Piperitone epoxide Carvacrol Piperitenone ȕ Piperitenone oxide Į β-Caryophyllene α-Humulene Spathulenol Caryophyllene oxide

1.17 2.89 1.37 15.66 26.52 2.16 13.57 26.40 3.89 0.53 1.16 2.37

GC, MS, RI GC, MS, RI GC, MS, RI GC, MS, RI GC, MS, RI GC, MS, RI GC, MS, RI GC, MS, RI GC, MS, RI GC, MS, RI MS, RI GC, MS, RI

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4. Discussion There are many literatures concerning the determination of chemical compositions and antimicrobial activities of the EOs of various Mentha species, as well as their applications in various commercial preparations, mainly as antimicrobial and antioxidant agents [14, 15, 16, 17, 10, 18, 19, 20, 21, 22, 23]. It was reported that the extracts or EO of Mentha longifolia had strong antibacterial activity against many bacterial species [16, 17, 10, 21, 22]. Our data confirmed the previous findings. The results showed that the EO of ML had a great potential antimicrobial activity against all the tested bacterial species. According to the literature, gram+ bacteria are known more sensitive to plant oils and extracts than gram- bacteria [24]. However, the results of our research showed that EO of ML has strong antimicrobial activity against both of the gram – and gram + bacteria. In addition, we observed that P. s. pv. syringae strains had not equal sensitivity to the EO. P. s. pv. syringae strain isolated from cherry was more resistant than P. s. pv. syringae strains isolated from apricot. It is thought that the sensitivity can be caused by the differences in host, virulent of pathogens, toxins produced by these pathogens. Gulluce et al. (2007) tested the EO of ML against many different microorganisms including only a few plant pathogenic bacteria. But, according to our knowledge, the EO obtained from Mentha longifolia has never been studied against this many phtopathogenic bacteria. Therefore, this is the first study showed that EO of ML has antibacterial activity against phytopathogenic bacteria and owns a potential to be used as a biopesticide. The current study showed that the average of inhibition zones of the EO were effective as much as positive control antibiotics even at low concentrations. This result may be explained by the high content of cis-piperitone epoxide (26.52%), piperitenone oxide (26.40%), pulegone (15.66%) and piperitenone (13.57%) in the EO of ML analyzed in our study. Recent studies have shown that there was a strong correlation between major constituents and antibacterial activities of plant EOs [25, 26, 18, 10]. GC/MS analysis resulted in the identification of 12 compounds representing 97.7% of the oil. The determined compounds and their amounts are cis-piperitone epoxide (26.52%), piperitenone oxide (26.40%), pulegone (15.66%) and piperitenone (13.57%). It was known that the main constituents of EO of M. longifolia were piperitone oxide or piperitenone oxide [27]. Our results generally confirmed the findings of previous studies. According to previous studies, the EOs of ML has been found to differ qualitatively and quantitatively. It was showed that the EOs of Mentha species are rich in oxygenated monoterpenes, in spite of the difference between the major compounds [28, 29, 30, 16,]. Based on these results, it is possible to conclude that some major components are same, but their amounts are different as compared to our results. These differences might have been derived from local, climatic, seasonal and experimental factors [31, 17]. This is the first study that the EO of ML possessing antibacterial activity was evaluated against phytopathogen microorganisms. In conclusion, the antibacterial activity of the EO against plant pathogenic bacteria is considerable strong. Thus, it is thought that the findings of the present study are valuable for further investigations focusing on a new biopesticide discovery. In this way, the development of natural antimicrobial agents will be able to help to decrease the negative environmental effects such as toxic residues of synthetic components. However, a further study in vivo condition is also necessary to confirm the safety and toxicity of these compounds against the agricultural plants.

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