Acar, Hacıoğlu Doğru, 2019, Biologica Nyssana 10(2) 10 (2) December 2019: 175-179 DOI: 10.5281/zenodo.3600199 Effects of drought stress factors on antibacterial activity of two Triticum aestivum L. varieties Original Article Okan Acar Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, Çanakkale, Turkey oacar@comu.edu.tr Nurcihan Hacıoğlu Doğru Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, Çanakkale, Turkey nurcihan.n@gmail.com (corresponding author) Received: July 10, 2019 Revised: September 10, 2019 Accepted: September 13, 2019 Abstract: Triticum aestivum L. (Wheat grass), one of the members of Poaceae family, has been considered to be a very efficient therapeutic drug. In the present study, we propose to evaluate antibacterial effects of the two varieties of T. aestivum L. [cv. Tosunbey (drought tolerant) and cv. Sultan 95 (drought-sen- sitive)] which grown in three different stress conditions [(1) drought stress; (2) pre-treatment of seeds with acetyl salicylic acid; (3) drought stress and pre-treatment of seeds with acetyl salicylic acid]. The antibacterial activity of the ethanol extracts was assayed against five pathogens (Pseudomonas aeru- ginosa ATCC 27853, Proteus vulgaris ATCC 13315, Escherichia coli NRRL B-3704, Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633) by agar disc diffusion and micro broth dilution methods. The results showed that the ethanol extracts from the different studied treatments showed antibacterial activities, with the diameters of the inhibition zone ranging from 8 to 15 mm and minimum inhibitory concentration ranging from 2.5 to 20.0 µg/mL, respectively. The highest antibacterial activity, against B. subtilis ATCC 6633, was demonstrated by the extract of T. aestivum cv. Sultan 95, which grown in conditions where drought stress and pre-treatment of seeds with acetyl salicylic acid were combined. Key words: antibacterial activity, T. aestivum cv. Tosunbey, T. aestivum cv. Sultan 95 Apstract: Antibiofilm aktivnost etanolnog ekstrakta Verbascum pinnatifidum Vahl. Triticum aestivum L. (pšenica), jedan od članova familije Poaceae, smatra se za veoma efikasnu terapeutsku drogu. U ovom istraživanju, utvrđivan je an- tibakterijski efekat dva varijeteta T. aestivum L. [cv. Tosunbey (tolerantna na sušu) i cv. Sultan 95 (osetljiva na sušu)] koji su uzgajani u tri različita uslova stresa [(1) sres suše; (2) semena pretretirana acetil salicilnom kiselinom; (3) sres suše i pretretman semena acetil salicilnom kiselinom]. Antibakterijska aktivnost etanolnih ekstrakata ispitivana je u odnosu na pet patogenih vrsta (Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 13315, Es- cherichia coli NRRL B-3704, Staphylococcus aureus ATCC 25923 i Bacil- lus subtilis ATCC 6633) korišćenjem disk difuzije na agaru i mikrodilucione metode. Rezultati su pokazali da etanolni ekstrakti dobijeni iz ispitivanih različitih uslova gajenja poseduju antimikrobnu aktivnost, sa dijametrima in- hibicionih zona koji su varirali od 8 to 15 mm i minimalnim inhibitornim koncentracijama od 2,5 do 20,0 µg/mL. Najveća antibakterijska aktivnost, utvrđena u odnosu na B. subtilis ATCC 6633, pokazana je od strane ekstrakta T. aestivum cv. Sultan 95, koji je rastao u uslovima kombinacije sušnog stresa i pretretmana semena acetil salicilnom kiselinom. Ključne reči: antibakterijska aktivnost, T. aestivum cv. Tosunbey, T. aestivum cv. Sultan 95 Introduction Plants contain numerous of constituents and are valuable sources of new and biologically active molecules having antimicrobial properties that are important for drug designing against diseases (Das et al., 2010; Bhattacharjee and Islam, 2015; Saha et al., 2018). Wheat as one of the members of Gramineae family, has been known for very efficient medical values and several therapeutic drugs that include bran and germ. It is used as a protection against various diseases such as constipation, heart diseases, appendicitis, obesity, diabetes and condition of the colon called diverticulum (Hadjivassiliou et al., © 2019 Acar, Hacıoğlu Doğru. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non- commercially under the same license as the original. 175 13th Symposium on the Flora of Southeastern Serbia and Neighboring Regions 2003; Saha et al., 2018). Triticum aestivum has also been shown to be a rich source of Vitamins A, C, E and B complex, including B12 (Ashok, 2011; Sundaresan et al., 2015). It contains a multitude of minerals like calcium, phosphorus, magnesium, alkaline earth metals, potassium, zinc, boron, and molybdenum. Other compounds that are making this grass therapeutically effective are the indole compounds, choline and laetrile (amygdalin) (Padalia et al., 2011; Sundaresan et al., 2015). Antibiotic resistance has become a global concern (Westh et al., 2004; Saha et al., 2018) and clinical efficiency of many existing antibiotics is being threatened by the emergence of multidrug-resistant pathogens as reported by Saha et al. (2018). So, there is an urgent need to develop new antimicrobial compounds which are more active against novel and re-emerging infectious diseases (Rojas et al., 1992; Saha et al., 2018). Duke and Ayensu (1985) reported that wheat is an easily grown plant in the world and that the young stems had proven effective for the treatment of biliousness, intoxication and removing skin blemishes, antipyretic, antihydrotic and sedative. Triticum aestivum has also been used against cough, sore throat, malaise, spasmic pain, abdominal coldness and constipation (Ashok, 2011). Wheat is also known to have anticancer and antimicrobial properties (Gregorova et al., 2015). Drought is the most critical limiting factor for crop production and is becoming an increasingly severe problem in many regions of the world. Abiotic stress conditions such as drought, salinity, cold stress can induce the synthesis of pathogen-associated proteins (PRs) in plants (Van Loon et al., 2006; Sehgal and Mohamad, 2018). In severe drought conditions, PRs such as chitinase and glucanase have been activated in wheat (Gregorova et al., 2015). In addition, TdLTP4, a PR protein, was found to be higher in wheat variety with salt and drought tolerance (Safi et al., 2015). On the other hand, PRs induced by the effect of salicylic acid (SA) has been shown to have antimicrobial activity in vitro (Van Loon et al., 2006). Also, SA applications have been reported to increase antimicrobial activity in turnip (Thiruvengadam et al., 2016) and sesame plants (Hossein et al., 2016). It is known that antifungal and antimicrobial activity increases due to oxidative stress induced by environmental stresses (Sharma et al., 2018; Schmidt et al., 2019). The relationship between antimicrobial activity and PRs, produced by plants under environmental stress conditions such as defensins (Schmidt et al., 2019) and phytoalexins (Ejike et al., 2013), has been demonstrated earlier (Li et al., 2011). In this study, the effects of drought and salicylic acid (SA) priming combinations were investigated on antibacterial activity in 21-day-old seedlings of two T. aestivum varieties. Material and methods Plant materials The seeds were sterilized by washing with 5% sodium hypochlorite solution for 5 min (1 time) and with sterile distilled water for 2.5 min (3 times). Two tablets of 500 mg aspirin were thoroughly dissolved in 500 ml of distilled water and sterile seeds were kept in this solution for 2 hours for ascorbic acid (ASA) priming. At the end of the period, the seeds were placed on moist filter paper and germinated for three days. Seedlings were planted in perlite:peat mixture (1:3) containing pots. Plants were watered with Hoagland nutrient solution (100%; Steward, 1983) during 21 days in growth chamber (22-24 oC, photoperiod 16/8 day/night). Drought stress application was carried out with water scarcity for 5 days starting from the 21st day. Seedlings of T. aestivum L. cv. Tosunbey (drought tolerant) and cv. Sultan 95 (drought-sensitive) were divided into four groups: (1) control (2) drought stress; (3) pre- treatment of seeds with acetyl salicylic acid; (4) drought stress and pre-treatment of seeds with acetyl salicylic acid. Leaf sampling was done on 21-day old plants. Preparation of plant extracts Air-dried samples of two wheat varieties were grounded into a fine powder in a grinding mill. Pulverized plant samples (1 g) were extracted with 10 mL of 80% ethanol, (1:10 w/v) using an orbital shaker for 8 h at room temperature. The extract was separated from the solids by filtration with Whatman No. 1 filter paper. The remaining solids were extracted twice with the same solvent and extracts combined. Extracts were stored in a refrigerator (4 oC) until analyzed (Sultana et al., 2007). Test Microorganisms Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 13315, Escherichia coli NRRLB-3704), Gram-positive bacteria (Staphylococcus aureus ATCC 6538P, Bacillus subtilis ATCC 6633) were used for determining the antibacterial activities of two T. aestivum varieties. Screening for antimicrobial activities Disc diffusion method was used for qualitative analyses of two T. aestivum species extracts (Collins et al., 1989). Studies were performed in triplicate. Treatments with Penicillin (P10) served as positive controls, respectively and treatments with ethanol without plant extracts served as negative controls. 176 BIOLOGICA NYSSANA ● 10 (2) December 2019: 175-179 Acar, Hacıoğlu Doğru ● Effects of drought stress factors on antibacterial activity of two Triticum aestivum L. varieties Minimum inhibitory concentration assay Minimum Inhibitory Concentration (MIC) was investigated as recommended by the Clinical and Laboratory Standards Institute (CLSI, 2006). The lowest concentration of extracts inhibiting visible growth of each test microorganisms was taken as the MIC. The medium, 0.1% (w/v) Streptomycin (ST), Nystatin (NYS100) and 10% DMSO were used as the non-treated, positive and negative controls, respectively (Teanpasian et al., 2017). Results and discussion Tab. 1 indicated antimicrobial activity of two varieties of T. aestivum plant extracts and the inhibition zones formed by standard antibiotic disks and MIC ratios. The results of antimicrobial testing showed that the ethanol extracts from the different treatments studied showed variable antibacterial activities, with the diameters of the inhibition zone ranging from 8 to 15 mm and 2.5 to 20.0 µg/mL, respectively. The highest antibacterial activity was demonstrated by the extract of T. aestivum cv. Sultan 95, grown in drought conditions (S3 in Tab.1), against B. subtilis ATCC 6633. Triticum aestivum plant varieties used in this study showed higher antibacterial activity against Gram-positive bacteria (B. subtilis ATCC 6633) than Gram-negative test bacteria. The results in present study are parallel to those reported in the previous investigations. In general, the distinctive feature of Gram-negative bacteria is the presence of a double membrane surrounding each bacterial cell. Although all bacteria have an inner cell membrane, Gram- negative bacteria have a unique outer envelope. This outer membrane excludes certain drugs and antibiotics from penetrating the cell, partially accounting for why gram-negative bacteria are generally more resistant to antibiotics than Gram- positive bacteria (Dülger and Dülger, 2018). In the literature there are many investigations about wheat grass antimicrobial activity (Pallavi et al., 2011; Das et al., 2012; Sundaresan et al., 2015). However, there is no data effect of drought stress on wheat grass antibacterial activity in the literature. Drought and salinity conditions can cause oxidative stress due to osmotic stress. This may induce the emergence of PRs such as phytoalexin and defensin (Van Loon et al., 2006; Sehgal and Mohamad, 2018). Our results showed the highest antibacterial activity in drought-sensitive Sultan 95 with SA and drought treatments together. Accordingly, our results are consistent with studies in which severe drought stress activates PRs in wheat (Gregorova et al., 2015), and increases a PR which causes higher fungal resistance in drought-tolerant wheat (Safi et al., 2015). In addition, the increases in 177 BIOLOGICA NYSSANA ● 10 (2) December 2019: 175-179 Acar, Hacıoğlu Doğru ● Effects of drought stress factors on antibacterial activity of two Triticum aestivum L. varieties Ta bl e 1. D is c D iff us io n, M IC ra tio s of th e tw o va rie tie s of T . a es tiv um e xt ra ct s Te st b ac te ri a P la nt e xt ra ct s *D is c D iff us io na (m m ) M IC (µ g/ m L) cv . T os un be y cv . S ul ta n 95 cv . T os un be y cv . S ul ta n 95 T1 T2 T3 T4 S 1 S 2 S 3 S 4 T1 T2 T3 T4 S 1 S 2 S 3 S 4 E .c ol i N R R LB -3 70 4 8. 0 11 .0 10 .0 7. 0 12 .0 11 .0 7. 0 7. 0 16 .0 20 .0 2. 5 10 .0 20 .0 2. 5 2. 5 20 .0 20 .0 4. 0 P. a er ug in os a AT C C 2 78 53 10 .0 9. 0 7. 0 7. 0 11 .0 7. 0 9. 0 9. 0 8. 0 5. 0 5. 0 20 .0 20 .0 2. 5 20 .0 10 .0 2. 5 1. 0 P. v ul ga ris A TC C 1 33 15 13 .0 10 .0 13 .0 8. 0 12 .0 9. 0 11 .0 10 .0 13 .0 2. 5 5. 0 2. 5 20 .0 2. 5 10 .0 10 .0 20 .0 4. 0 S . a ur eu s AT C C 6 53 8P 7. 0 8. 0 7. 0 9. 0 11 .0 9. 0 10 .0 12 .0 14 .0 20 .0 20 .0 20 .0 5. 0 2. 5 10 .0 5. 0 5. 0 4. 0 B . s ub til is A TC C 6 63 3 11 .0 9. 0 10 .0 11 .0 10 .0 8. 0 15 .0 13 .0 15 .0 2. 5 5. 0 10 .0 5. 0 5. 0 20 .0 2. 5 20 .0 4. 0 T: T . a es tiv um c v. T os un be y S : T . a es tiv um c v. S ul ta n 95 T1 : c on tro l; T2 : t re at m en t w ith a ce ty l s al ic yl ic a ci d; T 3: tr ea tm en t w ith d ro ug ht s tre ss ; T 4: d ro ug ht s tre ss a nd p re -tr ea tm en t o f s ee ds w ith a ce ty l s al ic yl ic a ci d S 1: c on tro l; S 2: tr ea tm en t w ith a ce ty l s al ic yl ic a ci d; S 3: tr ea tm en t w ith d ro ug ht s tre ss ; S 4: d ro ug ht s tre ss a nd p re -tr ea tm en t o f s ee ds w ith a ce ty l s al ic yl ic a ci d In hi bi tio n zo ne (m m ); a in cl ud es d ia m et er o f d is k (6 m m ); P 10 = P en ic ill in (1 0 ug /d is c) ; S T: S tre pt om yc in antimicrobial activity by SA priming were similarly shown to increase antimicrobial activity in SA applications in sesame (Hossein et al., 2016) and turnip (Thiruvengadam et al., 2016). As a result, SA and drought application showed high antibacterial activity in drought-sensitive variety. This may indicate different PRs synthesized in this variety compared to the tolerant one. The occurrence of lower antimicrobial activity in the tolerant variety compared to the sensitive variety suggests that some other stress factor is needed due to its tolerance to drought. Conclusion This study was conducted to evaluate the antibacterial activity of two T. aestivum varieties under three different stress conditions. Natural products are important source of new drugs which are having importance in modern medicine. The results pointed that the ethanol extracts from plants treated with combined stress conditions had significant antibacterial activities against P. vulgaris ATCC 13315 (T3) and B. subtilis ATCC 6633 (S3). The need to increase food production and exhaustion of wheat genetic resources has increased interest in alternative approaches for wheat improvement, including the use of different stress conditions. It is well known that abiotic stress leads to a series of morphological, physiological, biochemical, and molecular changes that adversely affect plant growth, productivity and antagonistic activity (Hayat et al., 2013; Ripa et al., 2019). Therefore, selection, screening, and application of the stress-tolerant T. aestivum plant varieties for better farming would considerably facilitate the farming community by overcoming such extreme climate changes. This is the first report on the bioactivities of two varieties of T. aestivum grown in different drought conditions and pre-treatment of seeds with acetyl salicylic acid. This approach may also allow new kind of research in medicinal usage or development of drug research. Acknowledgments. This paper was presented at the Symposium on The Flora of Southeastern Serbia and Neighboring Regions, June, 20-23 2019, Serbia. References Ashok, S.A. 2011: Phytochemical and pharmaco- logical screening of wheat grass juice (Triticum aes- tivum L.). 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Microbial Drug Resistance, 10: 169-76. 179 BIOLOGICA NYSSANA ● 10 (2) December 2019: 175-179 Acar, Hacıoğlu Doğru ● Effects of drought stress factors on antibacterial activity of two Triticum aestivum L. varieties