Academia Journal of Agricultural Research 4(11): 667-674, November 2016 DOI: 10.15413/ajar.2016.0118 ISSN: 2315-7739 ©2016 Academia Publishing

Research Paper Various aspects of essential oils application for pathogens controlling in Strawberry in vitro culture Accepted 7th March 2016 ABSTRACT

Mina Taghizadeh, Mousa Solgi and Iman Shahrjerdi Department of Horticultural Engineering, Faculty of Agriculture and Natural Resources, Arak University, Arak 38156-8-8349, Iran. *Corresponding author e-mail: [email protected]. Tel: (+98) 8632222314. Fax: (+98)8612761007.

Development of procedures by using essential oils to establish free pathogens media could be as one of the best protocol for the plant micropropagation. Thus, for sterilization of strawberry tissue culture, the essential oils (eugenol, carvacrol, and thymol) were tested. Essential oils were added to the medium at concentrations of 0.01, 0.02, 0.04, 0.5, 1, 2.5, and 5% in the first experiment, respectively. Treated media supplemented with essential oils were compared with the control without autoclaving medium. In the second experiment, explants were sterilized by 0.5% combination essential oils at different times (5, 10, and 15 min). Stock plants spraying with combination of essential oils were applied at greenhouse during third experiment. Results showed that, sterile conditions of medium were obtained by 100% free fungi at 0.01% and free bacteria at 0.5% concentration from each essential oil. Combination of essential oils for 5 min in leave disinfestation provided 100% free from fungi and 50% free from bacteria contamination. Stock plants spraying with eugenol plus thymol decreased 75% bacteria and 84% fungi contaminations when compared with control. So, the use of essential oils in in vitro culture could have prospective future to control contaminations. Key words: Eugenol, carvacrol, thymol, disinfection, in vitro culture.

INTRODUCTION During the various stages of in vitro culture, plant tissues may be placed in the stresses that have never faced in their natural habitat. These stresses have considerable consequences on expression of plant gnome which can cause new response on tissue culture. In total, stresses in tissue culture are undesirable; although, some stresses may have beneficial effects. The most obvious disorders in in vitro culture condition are included in plant pathogens, hyperhydrisity vitrification, browning, shoot-tip necrosis, ethylene, habituation, and somaclonal variation (Ruffoni and Savona, 2013; Bairu and kane, 2011). In vitro pathogens are one of the most serious problems in micropropagation process. Commonly, more microorganisms that are problematic for in vitro culture do not create serious problem in outdoor or in in vivo culture.

However, when plant materials are removed from the farm and used for tissue culture, they cause infection and stop in vitro culture process at the early stages, despite the use of disinfection procedures. The existence of nutrient elements that make bacteria and fungi grow up quickly is the reason of this action. So, disinfection stage is the first step in plant tissue culture (Cassells, 2000; Debergh and Vanderschaeghe, 1991). The materials that are used for disinfection against microorganisms are poisonous for microorganisms, while non-poisonous for plant tissues. Some of these materials are fungicides, sodium hypochlorite, calcium hypochlorite, ethyl alcohol, silver nitrate, mercuric chloride, and antibiotics (Perez-Tornero et al., 1999). Meristem culture, thermotherapy, and chemotherapy are common methods

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for removal of virus infection in many of plant species. But, sometimes internal infections are not removed even with these techniques. In total, an ideal disinfectant material should be effective and low concentration. Obtaining plant materials without pathogen for in vitro culture is so difficult and if explants are not disinfected, they will be contaminated until to 95% (Torres, 1989; Kumar, 2001). Nowadays, there are many reports about the effects of essential oils substances in food and agricultural industry. Antimicrobial properties of essential oils may be used to increase vase life of crops, such as fruits, vegetables, and cut flowers (Solgi and Ghorbanpour, 2014). Studies on the use of essential oils in plant in vitro culture are rarely available. Taghizade and Solgi (2014) reported that disinfection by essential oils of thymol and carvacrol at 100 to 200 mg/L concentrations are effective on bacterial and fungal infections, respectively on nodal explants of Cynodondactylon species. Antimicrobial activity of carvacrol and thymol are depended on concentration and time of treatments, too. Gouran et al. (2013) used the Lavender essential oil at concentrations of 250 to 1000 ppm in order to disinfect grape leaf explants for 1 and 4 h and they have observed that the concentration of 1000 ppm could remove the infections totally. Deein et al. (2013) studied the effect of some essence of medicinal plants on sterilizing micropropagation medium of Chrysantemum ‘Moneymaker Improved’, and reported that betel oil, clove oil, cinnamon oil, lavender oil, holy basil oil, lemon oil, tea tree oil, and turmeric oil in medium are effective for sterilizing medium. Strawberry (Fragaria × ananassa (Duch)) is one of the most important fruits all over the world and is cultivated in temperate and subtropical climate. Good fragrance, taste, and nutrition value make this fruit popular (Debnath and Teixeira da Silva, 2007). Although, strawberry was propagated by runners, but this method is not suitable, because of fungal diseases risk. Also, strawberry could be propagated by seeds. This method can only be used in varieties that have no runners or in breeding programs. Additionally, seed of strawberry needs 6 to 7 weeks of stratification and special treatments. Moreover, asexual methods produce a limited number of seedlings and this method completely depended on season (Wilson et al., 1973). Thus, creating quick and efficient methods for vegetative propagation like in vitro culture are always attractive. Plant tissue culture is a very useful technology for plants propagation. Although, more farmers can not establish a plant tissue culture laboratory, because of high production cost. The expensive equipment especially equipment that can be used for autoclave operation and disinfection is one of the most important problem. Thus, development of methods that use chemicals or essential oils to remove infections and eliminate autoclaving procedure for the establishment of pathogen-free medium could be the best protocol for in vitro culture of plants. Due to the economic value and availability of environmental condition in various parts of

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Iran to expand the cultivation of strawberry, this research was performed to optimize disinfection medium and explant of strawberry with plant essential oils, such as eugenol, thymol, and carvacrol. MATERIALS AND METHODS The "Gaviota" variety of strawberry grown in greenhouse with standard condition was used in this research. Explants were derived from the young leaves. At the first, leaves were washed under running water by a drop of dish washing soap for 20 min. Sterilization operation carried out by 50% of commercial bleach (Golrang home bleach with 2.5% active material for 5 min for explants that have need of common disinfection). Murashige and Skoog (MS) medium was used for in vitro culture of explants and supplemented with 30 g/L sucrose and 5.5 g/L agar (Sigma Aldrich Company). The medium was complemented with 0.5 mg/L 2,4-D and 4 mg/L BA plant growth regulators. The pH of the medium was adjusted to 5.8. Explants were cut at about 1×1 cm and then adaxial surface of leaf were cultured on the medium. Cultured plates were sealed with parafilm and were kept in controlled growth chamber with temperature of 23±0.1°C and 16 h light and 7 h dark condition. The eugenol, carvacrol and thymoloils (Sigma Aldrich Company) were used in this study. These compounds were solved at 60 to 70°C distilled water on heater stirrer and then were added to the medium or explant disinfection solution. This research was made in three experiments in term of various applications of essential oils in order to reduce outside and/or inside infections. After data assessment culture turned to percent and then analyzed by software's of SAS and ANOVA. Experiment 1: Use of various essential oils in medium culture Eugenol, carvacrol, and thymol at concentrations of 0, 0.01, 0.02, 0.04, 0.5, 2.5, and 5% were added to the medium. Any methods were used for sterilization of equipment, plates and explants, in this experiment. Culturing was done in laboratory environment and outside of laminar air flow cabinet, too. There were three replications in each treatment with four explants in each plate. This experiment was done as factorial in completely randomized design with two factors of essential oil type (3 levels) and concentration (7 levels). Cultures were evaluated for explants browning and bacterial or fungal infections after 7 days. Means were compared by Duncan’s Multiple Range Test at P = 0.05. Experiment 2: Using essential oils for explants disinfecting The effect of essential oils to remove contamination tissue

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A

669

B

C D

Figure 1.Traits evaluated at strawberry explants with various applications of essential oils inin vitrocondition (a) Initiation callus in explant (b) Browning explant (c) Fungi infection (d) Bacteria infection.

was evaluated before cultivation, in this experiment. The combinations of eugenol+carvacrol, eugenol+thymol, carvacrol + thymol, and eugenol+carvacrol+ thymol at concentration of 0.5% for 5, 10 and 15 min were used for this purpose. Explants were soaked in distilled water for 15 min and then were cultured as control. This experiment was done as a factorial with two factors of essential oil combination and disinfection exposure in completely randomized design (CRD) by three replications and means were compared by Duncan’s Multiple Range Test (P ≤ 0.05). Experiment 3: Application of essential oils spraying on mother stocks for pathogen controlling A field experiment as factorial was carried out to evaluate the effects of combination of essential oils on controlling mother stock microorganisms and to reduce contamination populations in in vitro culture, based on

completely randomized design with three replications in green house. The mother stocks were sprayed with combinations of eugenol +carvacrol, eugenol +thymol, carvacrol + thymol, and eugenol +carvacrol+ thymol and then leaves explants were cultured without disinfection (treated in distilled water for 5 min) and with disinfection by 0.5% of eugenol for 5 min. Control plants had no essential oils foliar application and explants were taken and cultured directly. RESULTS Results of this experiment was evaluated according to the necrosis of explants, browning symptoms, bacterial infection (based on existence of white, milky yellow or pink gelatin patches), fungal infection (base on black or white mycelium colonies) and callus production (Figure 1) in different treatments.

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Table 1. Interaction effects of essential oils and concentration in medium for sterilization of strawberry leaf explants.

Essential oils

Eugenol

Carvacrol

Browning (%)

Bacterial contamination (%)

Fungal contamination (%)

0

0e

91.6a

83a

0.01

71.6c

83.3a

0b

a

0b

b

0.02

85

0.04

100a

16.6d

0b

0.5

100a

0d

0b

a

d

83.3

0b

1

100

2.5

100a

0d

0b

a

d

0b

5

100

0

0e

0 0

91.6a a

50

bc

83a 0b

0.01

100

0.02

100a

0.04

100

a

0.5

100a

0d

0b

a

d

0b 0b

33.3cd 25

100

2.5

100a

0d

a

d

100 e

0

0

0.01

40d

0

cd

1 5

Thymol

Traits

Concentrations (%)

0

91.6 a

0b 0b

0b a

83a

100a

0b

ab

0b

0.02

100

75

0.04

100a

16.6d

0b

0.5

100a

0d

0b

a

d

0b

1

100

2.5

100a

0d

0b

a

d

0b

5

100

0 0

Mean values followed by different letters are significantly different.

Experiment 1: Use of various essential oils in medium culture

with 0.5% concentration of all type of essential oils (Table 1).

Results showed that all the essential oils inhibited the growth of in vitro contaminations from bacteria and fungi at 0.01 and 0.5% concentrations, respectively. According to assessment of cultures in various concentrations, cultures which contain essential oils, the medium never contaminated with increasing essential oils concentrations while the source of infection related to the explants. The percentage of bacterial infection in medium with carvacrol was lesser than other oils in lower concentrations (0.01 to 0.04%). However, addition of essential oils increased the necrosis of explants (Table 1). Nevertheless, there was a significant interaction between the concentration essential oils and the phytotoxicity symptoms. The least amount of browning explants was observed in explants treated by 0.01% thymol. No bacterial contamination was observed

Experiment 2: Use of essential oils for explants disinfecting A primary experiment was carried out on the application of three essential oils of eugenol, carvacrol, and thymol for explant disinfection. Results indicated that the use of essential oils was not successful for disinfecting explants (data not shown). So, the authors tried to use the combination of essential oils at 0.5% concentration for disinfecting explants in this experiment. The results showed that the concentration of 5% from eugenol+ carvacrol, eugenol+thymol, and carvacrol + thymol treatment have no signs of explant necrosis. Browning increased when the time of disinfection was more than 5 min. So, explants died completely in each

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Table 2. Interaction effects of essential oils and disinfection time on sterilization of strawberry leaf explants in in vitro culture.

Essential oils

Traits Bacterial contamination (%) Fungal contamination (%) 100a 100a b 75 0b

Disinfection time (min)

Browning (%)

Control

0 5

0f 0f

Eugenol + Carvacrol

10 15 5

100a 100a 0f

66.6b 16.6def 41.6bcde

0b 0b 16.6b

Thymol + Carvacrol

10 15 5

100a 100a 0f

25cdef 0f 58.3bc

0b 0b 16.6b

Eugenol + Thymol

10 15 5

86.6c 93.3b 50d

50bcd 8.3ef 50bcd

0b 0b 0b

Eugenol + Carvacrol + Thymol

10 15

86.6c 100a

25cdef 8.3ef

0b 0b

Mean values followed by different letters are significantly different.

treatment which contained carvacroal. Bacterial infection reduced by increasing time of disinfection, and was significant in comparison with control. In 15 min disinfection treatment with combination of essential oils, bacterial contamination was less than 20% and no bacterial infection was seen in 15 min treatment by using eugenol + thymol. Most of the treatments could decrease fungal contamination comparison with control and no fungal pathogens were observed in 15 and 10 min time (Table 2). Experiment 3: Foliar application of essential oils on mother stocks plants Because of non-efficiency of foliar application on stock plant with three essential oils alone (data not shown), their composition for pathogens controlling were used in the following experiments. There was no adverse effect in the foliar application of mother stocks in the greenhouse on tissues health when compared with previous experiments. When carvacrol was used as a component of essential mixture, the explants browning phenomenal increased in comparison with those without carvacrol, among different composition of essential oils. The highest level of explants necrosis (20%) was observed from derived plants stock sprayed by eugenol+carvacrol disinfected with eugenol. Foliar application with thymol+carvacrol and eugenol+ carvacrol+ thymol treatments increased browning (8 to 10%) in disinfected explants and/or not disinfected explants when compared with control. The stock spraying with essential oils

mixture causes the control of bacterial explants infection in particular when explants are compared with eugenol. The minimum amount of bacterial infection were seen in disinfected explants from stock plants sprayed with eugenol+thymol and eugenol+thymol+carvacrol and the rate of bacterial infection was reduced to 75% comparison with control. The spraying of mixture of essential oils was useful for controlling the spread of fungal contamination and all compositions extremely limited fungal infection especially when explants was disinfected by eugenol before culturing. All explants were taken from sprayed stocks and then disinfected had fungal infections of 75 to 100 less than controls (Table 3). DISCUSSION Results of this study showed that existence of essential oils in the medium was useful to control of all kinds of contaminations that can grow in in vitro culture condition. Sterile conditions of medium were obtained (100%) by using eugenol, carvacrol or thymol at 0.5% concentration without any autoclaving of medium and vessels. Prevention and controll of microbial infection in plant tissue culture producers are the main factors for success. Contamination caused by microorganisms is the most important reason for destroying plants in in vitro culture. Surface disinfection is one of the most important stages to prevent bacterial and fungal infection on explants to improve the way and research for new disinfecting material would be an outstanding achievement. Removing

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Table 3. Interaction effects of essential oils and Disinfection method on sterilization of strawberry leaf explants in in vitro culture.

Essential oils

Disinfection method

Traits

Browning )%(

Bacterial contamination (%) Control

c

Fungal contamination (%)

a

100a

-

0

Non -disinfection

0c

75ab

58.3b

Disinfection

20a

41.6bc

16.6c

Non -disinfection

8.3b

66.6ab

25c

Disinfection

10b

50bc

0c

Non -disinfection

0c

50bc

25c

Disinfection

0c

25c

16.6c

Non -disinfection

8.3b

66.6ab

25c

Disinfection

10b

25c

8.3c

Ojinol + Carvacrol

Thymol + Carvacrol

Ojinol + Thymol

Ojinol + Carvacrol + Thymol

100

Mean values followed by different letters are significantly different.

all of tiny creatures like bacteria, fungi and spores in medium is sterilization operation. Many factors, such as microorganisms, type and concentration materials and time of treatment are effective on chemical disinfections. Nowadays, resistance of bacteria and fungi against bactericide and fungicide limited the sterilization of plant materials and medium. Most of the chemical antibacterial materials are poisonous to living tissues. Today, there is great interest for the use of new materials that are safe and effective. This study clearly revealed the efficient bactericidal and fungicidal action of carvacerol, thymol, and eugenol. Other studies have shown that essentials oils like carvacerol, thymol, eugenol, limonin, and borneol may be a suitable replacement for destroying resistance microbials. These are natural components that are safe for human health and also are compatible with environment. Anti-bacterial activity of essentials oils such as betel (Hoque et al., 2011), ginger (Kamazeri et al., 2012), Hindi clove (Joshi et al., 2011), Lavender (Hui et al., 2010), lemon (Kirbaslar et al., 2009), and turmeric (Allawi et al., 2009) has been reported against some food pathogens. Several studies reported that eugenol, thymol, and carvacerol as thymus extract are the most effective antimicrobial and antioxidant material. Thymol and carvacerol due to their phenol compound and hydroxyl group act on bacterial cell wall and its components. An important feature of carvacerol and thymol is hydrophobicity role which enables them to penetrate to lipid membrane of cell wall and bacterial mitochondria, attach to membrane protein, release lipopolysaccharides, and at least cause impairment

in membrane permeability and structure; finally, loss of cell contents and release of molecules and vital ions leading to microbial death. Kind of essential oils, concentration, and time of treatment are important in their antimicrobial characteristic. Knobloch et al. (1989) reported that difference of fungicidal of essential oils is depended on the solubility in water and lipophilic properties. Fungicidal activity of essential oils has great relationship with their concentration. Carvacroal has a phenolic structure and its antimicrobial activity has been proved on bacteria, fungi, and yeast (Botelho et al., 2007; Martinez-Romerro et al., 2007; Yahyazadeh et al., 2008). Also, thymol is one of the bactericidal and fungicidal products used for plant diseases effectively (Braga et al., 2008; Yahyazadeh et al., 2008; Olasupo et al., 2007; Svircev et al., 2007). There isa little report about the use of essential oils for disinfecting in vitro explants. Various usages of essential oils in order to disinfecting explants or medium were effective on fungal infection rather than bacterial infection in this study. Taghizadeh and Solgi (2015) used thymol and carvacroal for disinfection of Cynodon dactylon species explants and found that fungal infection is controlled by these essential oils. Also, Deen et al. (2013) examined the effect of essential oils as disinfecting materials on sterilization of MS medium and growth of chrysantimum internodes in in vitro. They introduced extract of cinnamon, lavender, lemon, etc., for disinfection of medium instead of autoclave based on complete elimination of fungal and bacterial contamination which is similar to the

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findings of this study. Control of in vitro growth of pathogens had inverse relationship with browning of leaf explants of strawberries and release of phenol and explants death during experiment. It may be related to the nature of essential oils and Rosacea family. Strawberry produce lots of phenol compound and stress can increase them. During explant isolation and cutting tissues, phenolic compounds oxidized, because of polyphenol oxidase activity which exists in plastids and causes browning of tissue. The products of this oxidation stop enzyme activity and cause necrosis of tissue, lack of explant establishment in medium, and ultimately death of explant (Hussain et al., 2007). Similarly, Goran et al. (2013) reported grape explants necrosis due to high concentration of essential oil in medium. While these compounds controlled contaminations which correspond with the results of this research. Extreme sensitivity of herbaceous plants tissue to essential oils may prevent its application in disinfection protocols. Nevertheless, use of antioxidant compounds with essential oils in the medium could have resolved to adverse effects. CONCLUSIONS Generally, use of combination of essential oils for disinfection of explants, had better effects to controlling in vitro contamination, so that disinfection with compounds of eugenol+thymol+ carvacrol for 5 min would control 50% of bacterial contamination and 100% fungal contamination (however browning phenomenon occurred for 50%) in all the experiments, which is the most accepted treatment. Spraying of stock plants in greenhouse with eugenol+thymol and then disinfection of explant with 0.5% of eugenol decreased bacterial infection (to 75%) and fungal infection (to 84%) without browning of explants after culturing. Limitation of this method was the production of phenolic compound in strawberry explants that cause browning explants and if this problem is resolved, it is an excellent alternative for disinfection method to medium, vessels autoclaving, disinfection of explants, and use of laminar air flow. ACKNOWLEDGEMENT The authors are grateful to Arak University for supporting this work under research No: 92/13496.

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Cite this article as: Taghizadeh M, Solgi M, Shahrjerdi I (2016). Various aspects of essential oils application for pathogens controlling in Strawberry in vitro culture. Acad. J. Agric. Res. 4(11): 667-674. Submit your manuscript at http://www.academiapublishing.org/journals/ajar