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Investigation of PGPR on antioxidant activity of essential oil and microelement contents of sweet basil.

Introduction

Plant growth promoting rhizobacteria (PGPR) are root associated bacteria representing many different genera and species that colonize the rhizosphere, rhizoplane and improve plant growth when artificially introduced onto seeds, seed pieces, roots, or into soil. PGPR (plant-growth promoting rhizobacteria) or PGPF (plant-growth promoting fungi) enhance plant growth through numerous mechanisms including the protection of roots against infection by minor and major pathogens [1], enhancing the availability of nutrients to the host plant, lowering of the ethylene level within the plant or by the enhanced production of stimulatory compounds, such as plant growth regulators [2]. Different PGPR including associative bacteria such as Azospirillum, Bacillus, Pseudomonas and Enterobacter have been used for their beneficial effects on plant growth [3,4]. Many arketable biofertilizers are mainly based on plant growth promoting rhizobacteria (PGPR) that exert beneficial effects on plant development often related to the increment of nutrient availability to host plant [5].

Basil belongs to the genus Ocimum, derived from the Greek ozo which means to smell, in reference to the strong odors of the species within the genus [6]. Basil has been used as a folk remedy for an enormous number of ailments, including boredom, cancer, convulsion, deafness, diarrhea, epilepsy, gout, hiccup, impotency, insanity, nausea, sore throat, toothaches, and whooping cough. Basil has been reported in herbal publications as an insect repellent. [7] [recently the potential uses of O. basilicum essential oil, particularly as antimicrobial and antioxidant agents have also been investigated [8,9,10,11,12]. The O. basilicum essential oils exhibited a wide and varying array of chemical compounds, depending on variations in chemotypes, leaf and flower colors, aroma and origin of the plants [13,14]. It was reported that the leafy parts of basil had tonic, antiseptic [15] and insecticidal properties [16]. It is also known the leaves of basil are suitable for the treatment of pain and cough [17].] The preservative effect of many plant spices and herbs suggests the presence of antioxidative and antimicrobial constituents in their tissues [18]. Many medicinal plants contain large amounts of antioxidants other than vitamin C, vitamin E, and carotenoids [19]. Many herb spices, especially those belonging to the Lamiaceae family, such as sage, oregano, and thyme, show strong antioxidant activity [18]. The genus Ocimum, a member of the Lamiaceae family, contains between 50 and 150 species of herbs and shrubs [20]. A number of phenolic compounds with strong antioxidant activity have been identified in these plant extracts [21].

The purpose of this study was to evaluate the effect of inoculating Ocimum basilicum L. root with PGPR (Pseudomonas putida strain 41, Azotobacter chroococcum and Azosprillum lipoferum) on Ocimum basilicum L. antioxidant activity and microelements content. The results can be beneficial to farmers and people who focus on nutritional values of Ocimumbasilicum L.

Materials and methods

Microorganisms

Applied PGPR contained Pseudomonas putida strain 41, Azotobacter chroococcum strain 5 and A. lipoferum strain OF. To provide microbial inoculants, PGPR were inoculated in nutrient broth medium. Each bacterium was then removed at the end of logarithmic growth phase, and was asepticcally transferred to plastic containers containing sterile perlite and was then mixed well. PGPR concentration was adjusted to 1 x 108 (CFU/gr) in all inoculants.

Growth condition and plant materials

This study was conducted in experimental glasshouse of Islamic Azad University, Firoozabad Branch (28[degrees]35' N, 52[degrees]40' E; 1327 m above sea level). Ocimum basilicum L. seeds were inoculated and sown in a soil field, mixed with waterworn sand and peat (1/3, v/v each of them), in pots containing 7 kg of the mentioned (mixed) soil. Seven PGPR treatments were considered (Pseudomonas putida, Azotobacter chroococcum, A. lipoferum, P. putida + A. chroococcum,P. putida + A. lipoferum, A. chroococcum + A. lipoferum and P. putida + A. chroococcum + A. lipoferum). A non-inoculated treatment was set as control and N fertilizer was added to all the treatments according to the soil test. At full bloom, the plant herbage was cut 10 cm above the soil surface. Microelement contents were determined according to the lab manual of The Soil and Water Institute of Iran. The physicochemical properties of the soil were also determined (Table 1).

Isolation of Essential Oils

50 g sample of dried shoots was subjected to four-hours of hydro distillation using a Clevenger-type apparatus. The obtained essential oils were dried over anhydrous sodium sulphate

Determination of Antioxidant Activity of Essential Oils with the 2,2'-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Method

The antioxidant activity of essential oils was measured in terms of hydrogen donating or radical scavenging ability, using the stable radical DPPH (22).50 mL of methanolic solution of the essential oil (concentrations 20 g/L) was put into a cuvette, and 2 mL of 6 x [10.sup.-5] mol [L.sup.-1] methanolic solution of DPPH was added. Absorbance measurements commenced immediately. The decrease in absorbance at517 nm was determined with spectrophotometer after 1 h for all samples. Methanol was used to zero the spectrophotometer. Percent inhibition of the DPPH radical by the samples was calculated according to the formula of Yen & Duh [23].

% inhibition = (([A.sub.C(o)] - [A.sub.A(t)]) / [A.sub.C(o)]) x 100

where [A.sub.C(o)]) is the absorbance of the control at t = 0 min and [A.sub.A(t)] is the absorbance of the antioxidant at t = 1 h.

Statistical analyses

Experiment was conducted based on a randomized complete block design (RCBD) with four replications. Means were compared with Duncan's new multiple range test (DNMRT) in SAS software for windows.

Results and discussion

The results showed that the PGPR have the capacity to increase Ocimum basilicum microelement contents. Data explained significantly differences between used PGPR treatments on antioxidant activity of essential oil, Fe, Zn, Mn and Cu contents (Table 2). Maximum antioxidant activity of essential oil was observed in the Pseudomonas + Azotobacter + Azosprillum treatment (78%) which was significantly different when compared to other treatments. Minimum antioxidant activity was obtained in control treatment (58.15%). Pseudomonas + Azosprillum treatment (65.60%) had the lower antioxidant activity of essential oil compared to the other PGPR treatments (Fig. 1). It was reported that the usage of PGPR increased antioxidant activity of tomato fruit [24].

Maximum Zn content was showed in Azotobacter + Azosprillum treatment (110 ppm) which had significantly difference than the other treatments. Data explained it was no significantly difference between apply Azosprillum treatment (84.40 ppm) and Pseudomonas + Azotobacter treatment (82.26 ppm). Between PGPR treatments, Pseudomonas + Azosprillum treatment (81.18 ppm) had minimum of Zn content (Fig. 2).

Maximum Fe, Mn and Cu contents were obtained in Pseudomonas + Azotobacter + Azosprillum treatment (respectly 1466.73ppm, 141.52ppm and 30.67ppm) which had significantly difference than the other treatments. At all treatment, minimum of Fe, Mn and Cu contents were found in control treatment (respectly 905.55ppm, 90.50ppm and 17.60ppm). Data showed Pseudomonas + Azosprillum treatment and Pseudomonas + Azotobacter treatment had minimum Fe and Cu content compared to the other PGPR treatment.(Fig. 3, Fig. 4, Fig. 5)

[FIGURE 1 OMITTED]

Based on the results, it was showed a synergistic effect between apply of Pseudomonas + Azotobacter + Azosprillum and Azotobacter + Azosprillum PGPR and an antagonism effect between usage of Pseudomonas + Azotobacter and Pseudomonas + Azosprillum PGPR on microelement contents and antioxidant activity of essential oil at treatments.

Many studies showed that bacterial inoculation significantly increased nutrient contents of plants.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Increased nutrient uptake by plants inoculated with plant growth promoting bacteria has been attributed to the production of plant growth regulators at the root interface, which stimulated root development and resulted in better absorption of water and nutrients from the soil [25,26,4]. Different PGPR including associative bacteria such as Azospirillum, Bacillus, Pseudomonas, Entero bacter have been used for their beneficial effects on plant growth [27; 28]. Abbass and Okon, [29] explained that Azotobacter has positive effect on plant growth. Synergistic effects of com-bined inoculation of PGPRs have also been reported in various crops, for examples tomatoes [30], potatoes 31], rice [32], sugar beet and barley [33].

Acknowledgments

I would like to express my most gratitude to laboratory personals of The Soil and Water Institute of Iran, above all Dr. Kazem khavazi and Mrs Hemmati and also Dear Colleague Dr.Shahram sharafzadeh, Dr. Mahdi Zare , Dr.Omid Alizadeh and Dr. Hasan Zare Neirizi for their help.

References

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[27.] Kloepper, J.W. and C.J. Beauchamp, 1992. A review of issues related to measuring of plant roots by bacteria. Canadian Journal of Microbiology, 38: 1219-1232.

[28.] Hoflich, G., W. Wiehe, G. Kuhn, 1994. Plant growth stimulation with symbiotic and associative rhizosphere microorganisms. Experientia, 50: 897-905.

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[33.] Cakmakci, R., F. Kantar and O.F. Algur, 1999. Sugar beet and barley yields in relation to Bacillus polymyxa and Bacillus megaterium var. phosphaticum inoculation. J. Plant Nutr. Soil Sci., 162: 437-442.

Kourosh Ordookhani

Islamic Azad University, Firoozabad Branch, Iran

Corresponding Author

Kourosh Ordookhani, Islamic Azad University, Firoozabad Branch, Iran

E-mail: kordookhani@hotmail.com

Tel: +989179114470 Fax: +987116277937.
Table 1: Physicochemical properties of soil used

Parameter                                 value

Electrical conductivity ([dsm.sup.-1])    0.83
PH of Saturated soil solution             7.43
Clay (%)                                  14
Silt (%)                                  32
Sand (%)                                  54
Nitrogen (%)                              0.037
Organic carbon (%)                        0.41
Phosphorous(ppm)                          7
Potassium(ppm)                            292
Iron(ppm)                                 4.11
Zinc(ppm)                                 1.02

Table 2: Variance analysis of shoot fresh weight, shoot dry weight,
root fresh weight, root dry weight, shoot height, N,P,K content and
essential oil yield in sweet basil.

SOV           Degree of      Antioxidant      Fe (ppm)
              Freedom(df)    activity (%))

Block         3              0.625ns          2470.30ns
Treatments    7              159.02 **        97616.93 **
C.V%                         1.38             7.22

SOV           Mn (ppm)     Zn           Cu

Block         1.07ns       5.67ns       0.12ns
Treatments    949.85 **    739.47 **    65.31 **
C.V%          2.39         3.67         3.69

Ns, ** are levels of significance (not significant, p<0.01
respectively)

Sov: sources of varition
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Title Annotation:Original Article
Author:Ordookhani, Kourosh
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7IRAN
Date:May 1, 2011
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