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Salinity stress and PGPR effects on essential oil changes in Rosmarinus officinalis L.
Agriculture & Food Securityvolume 8, Article number: 2 (2019)
Medicinal plant species have been used by the ancestors around the world since ancient times. Rosmarinus officinalis is one of the most used medicinal plants, which belongs to the family Lamiaceae. To investigate the effects of different levels of salinity stress along with the induction of bacterial growth stimulation on the amount of essential oil composition in R. officinalis, an experiment was conducted in a randomized complete block design with 12 treatments and five replications. Salinity treatments included 0 (control), 2.5 (T1), 5 (T2), 7.5 (T3), 10 (T4) and 12.5 (T5) NaCl g/L, and the bacterium was pseudomonas fluorescence.
The percentage of essential oils showed a significant relationship with increasing salinity either alone or in composition with plant growth-promoting rhizobacteria (PGPR) inoculation treatments and it increased with increasing salinity levels to treatment 4 (T4, 10 g/L NaCl) but decreased with further increases in salinity levels in treatments without using PGPR and it was constant in treatment with using PGPR. Phellandrene, one of the main compounds of essential oils, showed a trend like the whole amount of essential oils in both group of treatments.
Abiotic and biotic factors may influence the different mechanisms and limit the interactions between plant and beneficial bacteria, resulting in less-than-acceptable performance in plant growth promotion and management of diseases. In this context, the results revealed that the application of PGPRs can help improve the essential oil yield in R. officinalis even in salinity conditions.
Medicinal plants species and aromatic plants have been used by the ancestors around the world since ancient times . Rosmarinus officinalis L. (rosemary) belongs to the family Labiatae or Lamiaceae and occurs as a shrub, under the shrub or herbaceous . It is a dense aromatic plant with dark green lavender-like leaves and is a native of the Mediterranean region. The flowering tops and the rosemary leaves mainly contain flavonoids, phenolic acids, especially rosmarinic acid (choleretic activities), and an essential oil (containing pinene, camphene, cineole, borneol and camphor) to which it must have stimulatory effects . Rosemary oils have been widely used for centuries as an ingredient in cosmetics, soaps, perfumes, deodorants, both for flavoring and for preservation of food products , and they have also many therapeutics and help the distribution of drugs and antiseptics . Rosemary is used for treating different diseases in traditional medicine, including depression, insomniac and arthritic pains [28, 47].
According to Beattie , bacteria that reduce the incidence or severity of plant diseases are often referred to as biocontrol agents, whereas those that exhibit antagonistic activity toward a pathogen are defined as antagonists. The idea of using bacteria to sustain land productive for future generations is not new, and the utilization of bacteria to stimulate plant growth in agriculture has been practiced for millennia. There is increasing evidence that beneficial microbes can enhance plants’ tolerance to adverse environmental stresses such as salinity stress , drought stress , weed infestation , nutrient deficiency and heavy metal contaminations . PGPR, as biocontrol agents, can act through various mechanisms, regardless of their role in direct growth promotion, such as by the known production of auxin phytohormone , a decrease in plant ethylene levels  or nitrogen-fixing associated with roots . Studies on the effect of salinity and PGPR on a plant have been neglected. Therefore, the present study aims to determine the changes in amount and performance of essential oils under the salinity stress either alone or in combination with PGPR bacteria in R. officinalis.
Materials and methods
To investigate the effects of different levels of salinity stress along with the induction of bacterial growth stimulation on the amount of essential oils composition of R. officinalis, an experiment was conducted in a completely randomized block design with 12 treatments and five replications in the greenhouse of the Natural Resources Faculty at the University of Kashan. Salinity treatments in this experiment included 0 (control), 2.5 (T1), 5 (T2), 7.5 (T3), 10 (T4) and 12.5 (T5) gram NaCl per liter. A fresh culture of bacteria was used to prepare the suspension or inoculum. The bacterium in this experiment was Pseudomonas fluorescens, a strain of CHAO; nutrient broth medium was required to prepare the CHAO suspension. In the Soil Laboratory of the University of Tehran, the pure bacteria of CHAO were cultured in a solid nutrient agar medium and were located at a laboratory normal temperature for 36–48 h. Then, several lobes were removed from the new bacteria and cultivated in Nb (nutrient broth) fluid medium for 48 h on a shaker at 150–250 rpm and then were centrifuged at 4500 g for 10 min. The white cells of bacteria accumulated at the bottom of tubes were removed from the nutrient environment in this way, and they were mixed with distilled water. Some of the bacterial suspensions were placed into the spectrophotometer (Model 2100-UV) at 600 nm with the absorption of one (OD600 = 1) resulted in a concentration of 109 cfu/ml . The cuttings of R. officinalis were transferred to plastic pots after 6 months when they were deployed and rooted. Soil characteristics were examined before starting the treatments presented in Table 1. Then, all plants (in the PGPR and salinity treatments) were inoculated with PGPR growth-stimulating bacteria and after that salinity stress was applied to the plants. The time of stress lasted 4 months in salinity stress either alone or in combination with PGPR. For all treatments, all planted seedlings were harvested and aerial parts of seedlings were dried and powdered as standard. The essential oils were separately extracted by the Clevenger device. Isolation and identification of rosemary essential oil compounds were performed by gas chromatography–mass spectrometry (GC/MS) machine in the laboratory. Statistical analyses were performed with mean comparison of Duncan’s multiple range method using SPSS software version 24.0.
Results and discussion
Based on the analysis of variance, the percentage of essential oils in R. officinalis showed a significant relationship (p < 0.01) with increasing salinity either alone or in composition with PGPR inoculation treatments (Table 2). The results showed that the amount of essential oils increases with increasing salinity levels to treatment 4 (T4, 10 g/L NaCl) but decreased with further increases in salinity levels (Table 3, Figs. 1 and 2). These results were confirmed by Ghorbani et al.  studying Nitraria schoberi and Panahi et al.  studying Salsola orientalis, explaining that the moderate salinity levels can improve the growth parameters and the plant will be injured by increasing salinity levels.
In the other side, the results of treatments along with PGPR inoculation showed that the amount of essential oils increases with increasing salinity levels to treatment 4 (T4, 10 g/L NaCl) and it is constant with further increases in salinity levels (Table 3, Figs. 1 and 2). The highest amount of essential oils in R. officinalis is 0.882 and 0.784 in treatment 4 (T4) without using PGPR and in treatment 4 and 5 (T4, T5) with using PGPR, respectively (Table 3). The synergistic effects of combined inoculation of PGPRs have also been reported in various medicinal and aromatic plants (MAPs), for example in Azadirachta indica , Saracaasoca , Phyllanthus amarus , Alpinia galanga and Coleus amboinicus , Ocimum basilicum , Calendula officinalis  and Silybum marianum . Beneficial rhizosphere bacteria are of two general types, those forming a symbiotic relationship with the plant and those that are free-living in the soil and root [5, 7, 27]. On the other hand, various PGPR strains have been also proven to be able to increase nutrient availability in the rhizosphere .
Based on the analysis of variance, the percentage of main compounds of essential oils in R. officinalis showed a significant relationship (p < 0.01) with increasing salinity either alone or in composition with PGPR inoculation treatments (Tables 4 and 5). The results showed that the amount of phellandrene content increases with increasing salinity levels to treatment 4 (T4, 10 g/L NaCl) but decreased with further increases in salinity levels (Fig. 3a) in treatments without PGPR inoculation. But in another group (treatments with both salinity and PGPR inoculation), the results showed an increasing trend in phellandrene content with increasing salinity levels (Fig. 3b). The highest amount of phellandrene is 57.48 in treatment 5 (T5, 12.5 g NaCl per liter) with using PGPR inoculation. Trends of other compounds are shown in Fig. 3 and Tables 4 and 5. Nevertheless, the application of PGPR has specifically shown a significant positive effect on essential oil production in R. officinalis.
Dehydration, salinity, low- and high-temperature stresses and other abiotic stresses lead to metabolic toxicity, generation of ROS, membrane disorganization, prevention of photosynthesis, reduced nutrient acquisition and altered hormones levels . Accumulation of osmoprotectants, production of superoxide radical scavenging mechanisms, exclusion or compartmentation of ions by the efficient transporter and symporter systems and production of specific enzymes involved in the regulation of plant hormones are among the mechanisms that plants have evolved for adaptation to abiotic stresses [12, 29, 37, 40, 41]. Similar to these, findings of PGPR have been reported by some other workers [19, 26].
Essential oil yield can be increased by plant in association with mycorrhiza and humic substances, which benefit root ramification, improving water absorption and phosphorus uptake. Furthermore, they can also influence the chemical composition of EOs [10, 22]. Adesemoye and Kloepper  compiled the benefits derivable from plant–PGPR interactions to include the following: improvements in seed germination rate, root development, yield, leaf area, shoot and root weights, chlorophyll content, protein content, hydraulic activity and nutrient uptake (including phosphorus and nitrogen). The bacteria, with their physiological adaptation and genetic potential for increased tolerance to drought, increasing salt concentration and high temperatures, could improve plant production in degraded sites [30, 45] (Tables 6 and 7).
The plant growth-promoting microorganisms were found to have a great potential for use as bioinoculants to increase production of medicinal and aromatic plants . The literature clearly demonstrates that PGPR induces plant growth and development through their numerous direct and indirect mechanisms of action . In this work, synthesis of herbal organs for essential oils of R. officinalis was described. Optical properties were established as strongly dependent on the application of PGPR. In this context, our results revealed that the application of PGPRs can help to improve the essential oil yield in R. officinalis even in the salinity conditions. The essential oils represent an important part of the folk medicine for their medicinal properties such as the antioxidant activity . Notably, abiotic and biotic factors may influence the different mechanisms and limit the interactions between plant and beneficial bacteria, resulting in less-than-acceptable performance in plant growth promotion and management of diseases [16, 17]. Finally, it can be stated that PGPR can help to improve the essential oil yield in normal and salinity conditions, but further investigations are needed to evaluate its performance in different conditions and under multi-stress situations.
plant growth-promoting rhizobacteria
gas chromatography–mass spectrometry
gram per liter
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