Hacıoğlu Doğru, Acar, 2019, Biologica Nyssana 10(2) 10 (2) December 2019: 181-187 DOI: 10.5281/zenodo.3600203 Effects of salt stress factors on antimicrobial activity of two Triticum aestivum L. varieties Original Article Nurcihan Hacıoğlu Doğru Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, Çanakkale, Turkey nurcihan.n@gmail.com (corresponding author) Okan Acar Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, Çanakkale, Turkey oacar@comu.edu.tr Received: July 10, 2019 Revised: September 10, 2019 Accepted: September 13, 2019 Abstract: Salinity is one of the most common environmental stress factors that adversely affect plant growth and crop production in cultivated areas worldwide. Herbal or ‘alternative’ medicine is gaining popularity and scientific research about wheat grass as a “functional food” is becoming more available and popular. Wheat grass, Triticum aestivum L. has a long history and is widely used as a health food supplement. It is found to be used as a treatment for minor ailments, and also as a preventive dietary supplement and therapeutic drug. Current study was aimed at evaluate antimicrobial properties of the two varieties of T. aestivum L. [cv. Tosunbey (drought tolerant) and cv. Sultan 95 (drought sensitive)], grown in three different conditions [(1) control; not treated with salt or acetyl salicylic acid; (2) treatment with sea water; (3) sea water and pre-treatment of seeds with acetyl salicylic acid]. The antimicrobial activity of the ethanol extracts of the two varieties of T. aestivum were assayed against Escherichia coli NRRL B-3704, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 13315, Acinetobacter baumanii ATCC 19606, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, S. haemolyticus ATCC 43252 and Candida albicans ATCC 10231 test microorganisms by agar disc diffusion method and broth microdilution method. The results showed that the ethanol extracts from the different treatments showed antimicrobial activities, with the diameters of the inhibition zone ranging from 8 to 13 mm and 2.5 to 20 µg/ mL, respectively. The highest antimicrobial activity was demonstrated against P. aeruginosa ATCC 27853 by the extract of T. aestivum cv. Sultan 95, which grown in sea water and whose seeds were pre-treated with acetyl salicylic acid. Key words: antibacterial activity, T. aestivum cv. Tosunbey, T. aestivum cv. Sultan 95 Apstract: Efekat sonog stresa na antibakterijsku aktivnost dva varijeteta Triticum aestivum L. Salinitet je jedan od najčešćih sredinskih stresnih faktora koji nepovoljno utiču na rast biljaka i produkciju useva u kultivisanim područjima širom sveta. Biljna ili “alternativna” medicina dobija na popularnosti i naučna istraživanja na temu pšenice kao “finkcionalne hrane” postaju sve dostupnija i popularnija. Pšenica, Triticum aestivum L., ima dugu istoriju i veoma je zastupljena kao dodatak ishrani. Utvrđeno je da se koristi kao tretman za blaža oboljenja i kao pre- ventivni dodatak ishrani i terapeutski agens. Ovo istraživanje imalo je za cilj da utvrdi antimikrobni potencijal dva varijeteta T. aestivum L. [cv. Tosunbey (tolerantna na sušu) i cv. Sultan 95 (osetljiva na sušu)], uzgajanih u tri različita uslova [(1) kontrola; netretirana semena; (2) tretman morskom vodom; (3) tret- man morskom vodom i pretretman semena acetil salicilnom kiselinom]. An- timikrobna aktivnost etanolnih ekstrakata dva varijeteta T. aestivum testirana je u odnosu na Escherichia coli NRRL B-3704, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 13315, Acinetobacter baumanii ATCC 19606, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, S. haemo- lyticus ATCC 43252 i Candida albicans ATCC 10231, korišćenjem metoda disk difuzije na agaru i mikrodilucije. Rezultati su pokazali da etanolni ek- strakti iz različitih uslova gajenja poseduju antimikrobnu aktivnost, sa dijamet- rima inhibicionih zona od 8 to 13 mm i minimalnim inhibitornim aktivnostima od 2.5 do 20 µg/mL. Najveća antimikrobna aktivnost, utvrđena u odnosu na P. aeruginosa ATCC 27853, pokazana je od strane ekstrakta T. aestivum cv. Sultan 95, dobijenog od biljaka gajenih u morskoj vodi i čija su semena pretre- tirana acetil salicilnom kiselinom. Ključne reči: antibakterijska aktivnost, T. aestivum cv. Tosunbey, T. aestivum cv. Sultan 95 © 2019 Hacıoğlu Doğru, Acar. 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. 181 13th Symposium on the Flora of Southeastern Serbia and Neighboring Regions Introduction Antibiotic resistance has become a global concern (Westh et al., 2004) and clinical efficiency of many existing antibiotics is being threatened by the emergence of multi-drug resistant pathogens as reported by Bandow et al. (2003). So, there is an urgent need to develop new antimicrobial compounds which are more active against newer and re-emerging infectious diseases (Rojas et al., 1992). Medicinal plants have bioactive compounds which are used for curing of various human diseases and play an important role in healing. Biosynthesis and accumulation of substances such as flavonoids, phenolic acid and anthocyanin enhances the biological activity (antimicrobial, antioxidant, etc.) of plant material (Karakaş and Türker, 2013). Wheat grass, Triticum aestivum L. has a long history and is widely used as a health food supplement. It is found to be utilized as a treatment for minor ailments and serious life-threatening diseases, and as a preventive dietary supplement and therapeutic drug. Salinity is one of the most common environmen- tal stress factors that affect plant growth and crop production in cultivated areas worldwide. Primary saline conditions appear naturally in environment, while anthropogenic activities are responsible for secondary salinity. Increased urbanization and de- forestation are two important human-derived activi- ties for salinity. A number of reports on soil saliniza- tion and their influences on crop productivity, land degradation and ecological disturbances are reported worldwide. Excess amount of salts affected physi- cal, chemical, as well as the biological properties of soils. Plant health in saline soils considerably de- clines owing to poor nutrition, osmotic stress and reduced microbial diversity (Paul and Lade, 2014; Vimal et al., 2018). The response of plants to physiological stress conditions (e.g. salinity) activates genes encod- ing pathogen-related proteins (PRs) (Sehgal and Mohamad, 2018). Therefore, PRs are commonly synthesized in plants not only under biotic stress conditions, but also under abiotic stress conditions (such as osmotic shock, chemical injury, and aging). In fact, there are inducible defense proteins classi- fied into 17 PRs families in plants (Van Loon et al., 2006; Sehgal and Mohamad, 2018). The relationship between antifungal (Schmidt at al., 2019) and anti- microbial resistance (Sharma et al., 2018) has been shown in proteins induced by oxidative stress caused by environmental stresses. In addition, phytoalexins synthesized in response to stresses such as antimi- crobial attack and physical injury in plants (Ejike et al., 2013) have been shown to increase antimicrobial activity in the rice plant in relation to the physiology of chloroplast and mitochondria (Li et al., 2011). Ac- cordingly, it is believed that increases in antimicro- bial activity are a consequence of oxidative stress caused by environmental stresses. In this study, the effects of seawater induced salt stress (24-26% NaCl, 24 h) and salicylic acid (SA) priming combinations were investigated on antimicrobial activity in 21-day-old seedlings of the two wheat varieties [cv. Tosunbey (drought tolerant) and cv. Sultan 95 (drought sensitive)]. Material and methods The plants material: 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). Seawater is taken from the coast of Kepez (Çanakkale), 30 m from the coast, and filtered in the laboratory to remove the particles. Salt concentration of clear seawater is 24-26% (Türkoğlu, 2006) and it was mixed with distilled water to obtain 50% concentration (SW 50%), while full concentration (SW 100%) was also used in experiments. Seedlings were planted in perlite:peat mixture (1:3) containing pots. Plants were watered with Hoagland nutrient solution (100%; Steward, 1983) for 21 days. Seedlings of T. aestivum L. cv. Tosunbey (drought tolerant) and cv. Sultan 95 (drought sensitive) were divided into three groups: (1) control; not treated with salt or acetyl salicylic acid; (2) treatment with 100 ml sea water (50% and 100 %); (3) sea water and pre-treatment of seeds with 1000 mg acetyl salicylic acid (during 2 hours in 2 x 500 mg aspirin tablets solution). Leaf sampling was collected after 24 hours of salt treatment of 21d old plants. Test microorganisms Gram-negative bacteria (Escherichia coli NRRL B-3704, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 13315, Acinetobacter baumanii ATCC 19606), Gram-positive bacteria (Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, Staphylococcus haemolyticus ATCC 43252) and yeast (Candida albicans ATCC 10231) were used for determining the antimicrobial activities of the two T. aestivum varieties. Preparation of extracts for antimicrobial activity 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 182 BIOLOGICA NYSSANA ● 10 (2) December 2019: 181-187 Hacıoğlu Doğru, Acar ● Effects of salt stress factors on antimicrobial activity of two Triticum aestivum L. varieties 183 BIOLOGICA NYSSANA ● 10 (2) December 2019: 181-187 Ta bl e 1. A nt im ic ro bi al a ct iv ity re su lts o f t he tw o va rie tie s of T . a es tiv um e xt ra ct s Te st m ic ro or ga ni sm s 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 C on tr ol P 10 / N Y 10 0 T1 T2 T3 T4 S 1 S 2 S 3 S 4 C on tr ol S T/ N Y 10 0 E . c ol i N R R L B -3 70 4 8. 0 ± 0. 21 11 .0 ± 0. 2 8. 0 ± 0. 03 8. 0 ± 0. 03 12 .0 ± 0. 0 1 11 .0 ± 0. 03 7. 0 ± 0. 0 1 7. 0 ± 0. 0 1 16 .0 20 .0 2. 5 20 .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 ± 0. 11 9. 0 ± 0. 02 7. 0 ± 0. 2 10 .0 ± 0. 01 11 .0 ± 0. 12 7. 0 ± 0. 01 10 .0 ± 0. 02 13 .0 ± 0. 13 8. 0 5. 0 5. 0 20 .0 5. 0 2. 5 20 .0 10 .0 2. 5 1. 0 P. v ul ga ris AT C C 1 33 15 13 .0 ± 0. 01 10 .0 ± 0. 0 1 8. 0 ± 0. 1 11 .0 ± 0. 14 12 .0 ± 0. 12 9. 0 ± 0. 03 9. 0 ± 0. 02 8. 0 ± 0. 01 13 .0 2. 5 5. 0 20 .0 5. 0 2. 5 10 .0 10 .0 20 .0 4. 0 A . b au m an ii AT C C 1 96 06 10 .0 ± 0. 0 1 9. 0 ± 0. 12 8. 0 ± 0. 01 10 .0 ± 0. 12 10 .0 ± 0. 01 7. 0 ± 0. 03 10 .0 ± 0. 04 10 .0 ± 0. 12 12 .0 10 .0 20 .0 20 .0 10 .0 10 .0 20 .0 20 .0 20 .0 2. 0 B . s ub til is AT C C 6 63 3 7. 0 ± 0. 14 8. 0 ± 0. 2 7. 0 ± 0. 12 9. 0 ± 0. 03 11 .0 ± 0. 12 9. 0 ± 0. 03 8. 0 ± 0. 12 10 .0 ± 0. 12 14 .0 20 .0 20 .0 20 .0 5. 0 2. 5 10 .0 20 .0 5. 0 4. 0 S . a ur eu s AT C C 6 53 8P 11 .0 ± 0. 02 9. 0 ± 0. 12 8. 0 ± 0. 04 10 .0 ± 0. 1 10 .0 ± 0. 01 8. 0 ± 0. 01 12 .0 ± 0. 01 7. 0 ± 0. 0 1 15 .0 2. 5 5. 0 20 .0 5. 0 5. 0 20 .0 2. 5 20 .0 4. 0 S . h ae m ol yt ic us AT C C 4 32 52 9. 0 ± 0. 03 10 .0 ± 0. 1 8. 0 ± 0. 12 10 .0 ± 0. 1 11 .0 ± 0. 1 10 .0 ± 0. 2 10 .0 ± 0. 0 1 8. 0 ± 0. 0 1 14 .0 5. 0 5. 0 20 .0 5. 0 5. 0 5. 0 5. 0 20 .0 5. 0 C an di da a lb ic an s AT C C 1 02 31 7. 0 ± 0. 1 8 .0 ± 0. 1 10 ± 0. 1 11 .0 ± 0. 0 1 8. 0 ± 0. 1 7. 0 ± 0. 12 7. 0 ± 0. 03 7. 0 ± 0. 12 16 .0 20 .0 20 .0 10 .0 5. 0 20 .0 20 .0 20 .0 20 .0 2. 5 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 s al in ity s tre ss ; T 4: s al in ity 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 s al in ity s tre ss ; S 4: s al in ity 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 ; N Y 10 0 N ys ta tin 1 00 u g/ di sc Hacıoğlu Doğru, Acar ● Effects of salt stress factors on antimicrobial activity of two Triticum aestivum L. varieties were extracted twice with the same solvent and the extracts were combined. Extracts were stored in a refrigerator (4 °C) until analyzed (Sultana et al., 2007). Screening of antimicrobial activity Disc diffusion assay Empty sterilized antibiotic discs having a diameter of 6 mm (Schleicher and Schull No. 2668, Dassel, Germany) were each impregnated with 50 μL of the extract (10 mg/disc). All the bacteria mentioned above were incubated at 35 ± 0.1 °C for 24 h by inoculation into Nutrient Broth (Difco Laboratories, MI, USA) and the yeast culture studied was incubated in Malt Extract Broth (Difco Laboratories, MI, USA) at 25 ± 0.1 °C for 48 h. An inoculum containing 106 bacterial cells or 108 yeast cells/mL was spread on Mueller Hinton Agar (MHA) (Oxoid Ltd., Hampshire, UK) plates (1 mL inoculum/plate). The discs injected with extracts were placed on the inoculated agar by pressing slightly. Petri dishes were placed at 4 °C for 2 h, afterwards the plate inoculated with the yeast culture was incubated at 25 ± 0.1 °C and bacteria were incubated at 35 ± 0.1 °C for 24 h (Collins et al., 1989). At the end of the period, inhibition zones formed on the medium were measured in millimeters. Studies were performed in triplicate. Treatments with penicillin (P10), and nystatin (NYS30) served as positive controls and treatments with ethanol, without bacterial or fungal materials served as negative controls. Minimum inhibitory concentration assay Minimum Inhibitory Concentration (MIC) was investigated as recommended by instructions of 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 (Teanpaisan et al., 2017). Statistical analysis The results of antimicrobial assay were mean ± SD of the three parallel assays. Results and discussion A total of eight plant extracts were tested against eight test microorganisms. Ethanol was selected as extraction solvent, because it is one of the best sol- vents used for the extraction of antimicrobial sub- stances (Jonathan and Fasidi, 2003). The results showed that the ethanol extracts from the different treatments studied have antimicrobial activities, with the diameters of the inhibition zone 184 ranging from 8 to 13 mm and 2.5 to 20 µg/mL, respectively (Tab. 1). The highest antimicrobial activity was demonstrated against P. aeruginosa ATCC 27853 by the extract of T. aestivum cv. Sultan 95 which grown in sea water and whose seeds were pre-treated with acetyl salicylic acid (S4). However, the disc diffusion method is a qualitative technique and is mainly used for selecting extracts with antimicrobial activity, mostly when diameter zones of inhibition are ≥ 10 mm (Sánchez et al., 2016). So, S. haemolyticus ATCC 43252 was the second most susceptible strain against the tested plants extracts. The same was found when observing the results of micro-dilution method. Although the disc diffusion method is one of the inexpensive and less laborious antimicrobial testing methods currently available, this procedure suffers with the demerit of not always producing reproducible results for plant extracts (Eloff, 1998; Vambe et al., 2018). This is primarily because some constituents do not diffuse readily in polarized agarose gels (Vambe et al., 2018). When the same extracts were screened using microdilution method, five plant extracts, namely T1, T2, S1, S2, S3 demonstrated higher antibacterial activity than the control antibiotic. This discordance observed in the results obtained from these two bioassays could be attributed to several factors (Vambe et al., 2018). It is possible that antibacterial activity was masked in the disk diffusion assay by low diffusion potential. Wheat is a grain containing protein, starch (Vida et al., 2014), fiber, phytochemicals and antioxidant substances which have a very important place in human nutrition (Andersson et al., 2013). While meeting the daily nutrition requirements with protein and starch, it facilitates digestion with fibers and thus reduces the risk of developing colon cancer (Huang et al., 2015). This plant has been also shown to have anti-inflammatory, antioxidant, anti-carcinogenic, immuno-modulatory, laxative, astringent, diuretic, antibacterial and anti-aging properties and is a part of formulations as a highly effective antimicrobial agent (Saha et al., 2018). In the literature there are many investigations about wheat grass antimicrobial activity. Pallavi et al. (2011), tested wheat grass extracts against the Gram-positive bacteria S. aureus, B. subtilis and Gram-negative E. coli, using Amoxicillin as standard. Certain extracts exhibited considerable activity against B. subtilis and moderate activity against S. aureus and E. coli. Ashok (2011) also reported antibacterial activity against E. coli, P. aeruginosa and S. aureus. Antifungal activity was also reported against C. albicans. Sundaresan et al. (2015), tested antimicrobial properties of 7th, 14th, BIOLOGICA NYSSANA ● 10 (2) December 2019: 181-187 Hacıoğlu Doğru, Acar ● Effects of salt stress factors on antimicrobial activity of two Triticum aestivum L. varieties and 21st day wheat grass extracts obtained with five different solvents (water, ethanol, methanol, ethyl acetate and hexane). All these extracts showed antibacterial activity against seven food borne pathogens. Amongst them, hexane extracts from the seven-day-old wheat grass showed maximum antibacterial activity, especially against Yersinia enterocolitica and Listeria monocytogenes. Das et al. (2012) found that acetone (80%) extract of wheatgrass was effective against five foodborne microorganisms, including the fungus Aspergillus niger, a common contaminant of food. There appears to be a lack of published scientific data on the salt stress effect to antimicrobial properties of wheat grass, thereby highlighting the need for continued research. Plants are frequently subjected to a variety of harsh environmental stresses such as scarcity of water, extreme temperatures, high soil salinity, herbivore attack, and pathogen infection diminishing their productivity (Sewelam et al., 2016). Different salt concentrations induce physiological and metabolic changes in plants affecting their seed germination, growth, development, yield and also decreases the rate of respiration and photosynthesis in plants. In addition to the growth and yield, the composition of bioactive compounds present in the aromatic and medicinal plants is affected by salinity (Gil et al., 2002). The increased levels of plant secondary metabolites such as phenols, flavonoids, tannins, alkaloids and others under the influence of increased salt concentrations as a part of defense mechanism have been reported (Kate, 2008). Reactive oxygen species (ROS) are continuously produced under normal environmental conditions in plant cells. Bioactive compounds also trigger metabolic pathways in plant cells and result in a higher ROS accumulation. The common response observed in salt-stressed plants is the generation of ROS, highly reactive and responsible in damaging cell structures, nucleic acids, lipids and proteins. Plants possess medicinal value with anti-inflammatory and antimicrobial activities; acquired resistance to stress induced by ROS is due to the presence of several bio-active compounds (Foyer et al., 1994). In the scientific literature, there are no data on the effect of salt stress on wheat grass antimicrobial activity. Kotagiri et al. (2017) investigated the secondary metabolites and antimicrobial potential of leaf, stem and root ethanol and chloroform extracts of five different Coleus species; C. aromaticus, C. amboinicus, C. barbatus, C. forskohlii and C. zeylanicus subjected to salinity stress. The up-regulation in the content of plant bioactive compounds, along with the antimicrobial activities of ethanol and chloroform extracts under the influence of salinity stress have been observed during the study in Coleus. The leaf, stem and root extracts of all the five Coleus species showed good antimicrobial activity against the tested pathogenic strains. The leaf extracts of Coleus showed higher inhibitory activity compared to the stem and root extracts. Ethanol extracts showed higher antimicrobial activity ranging from 1.5-100.0 mg/ml when compared with the chloroform extracts ranging from 0.97-250 mg/ ml respectively. The leaf, stem and root extracts of Coleus showed effective antimicrobial activity against the pathogenic strains even under saline conditions is due to the up regulation of secondary metabolites which provides a scope of developing novel drugs to treat infectious diseases. Our results have a correlation with the results of Kotagiri et al (2017) about increasing antimicrobial activity under saline stress and pre-treatment of seeds with acetyl salicylic acid conditions, which is probably related to the up-regulation of secondary metabolites which provides a scope for developing novel drugs to treat P. aeruginosa ATCC 27853. Conclusion The screening of the plant extracts for antimicrobial activity has shown that higher plants represent a potential source of new anti- infective agents. Herbal medicines are a valuable and rapidly available resource for primary health care and complementary health care systems. 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