- Email: [email protected]
Arbuscular mycorrhizae alleviate mild to moderate water stress and improve essential oil yield in thyme
Author’s Accepted Manuscript Arbuscular mycorrhizae alleviate mild to moderate water stress and improve essential oil yield in thyme Ali Abdollahi Arpanahi, Mohammad Feizian www.elsevier.com
PII: DOI: Reference:
S2452-2198(18)30131-9 https://doi.org/10.1016/j.rhisph.2018.10.003 RHISPH129
To appear in: Rhizosphere Received date: 15 October 2018 Revised date: 25 October 2018 Accepted date: 26 October 2018 Cite this article as: Ali Abdollahi Arpanahi and Mohammad Feizian, Arbuscular mycorrhizae alleviate mild to moderate water stress and improve essential oil yield in thyme, Rhizosphere, https://doi.org/10.1016/j.rhisph.2018.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Arbuscular mycorrhizae alleviate mild to moderate water stress and improve essential oil yield in thyme Ali Abdollahi Arpanahi1, Mohammad Feizian2*
1. Soil Sciences department, Agriculture Faculty, Lorestan University, Khorramabad, Iran. Email: [email protected] 2. *Corresponding Author; Soil Sciences department, Agriculture Faculty, Lorestan University, PO Box: 68151-44316, Khorramabad, Iran. Email: [email protected]
Abstract In order to study the effects of arbuscular mycorrhizal fungi and water stress on Thymus Vulgaris L. essential oil composition, an experiment was designed in full factorial randomized pattern with three replications. Factors consisted of four irrigation levels and two different inocula. Results showed that water stress decreased growth parameters of T. vulgaris but inoculation with AMF alleviated adverse effects of water stress. Result showed that when we used AMF with mildmoderate water stress, there is an increase in essential oil content but at severe water-stress essential oil content decreased. Interaction of AMF and water stress had significant effects on most components of T. vulgaris essential oil.
Keywords: Thymus Vulgaris L., Essential Oils, Arbuscular Mycorrhizal Fungi, Drought Stress
Thymus vulgaris L. (Lamiaceae) commonly known as thyme, is one of the well-known medicinal and aromatic plants from the mint family used in pharmaceutical, cosmetic, and food industries, for its aromatic and medicinal properties (Nabavi et al. 2015; Lorens-Molina et al., 2016). The aims of the present study was to assess the interactions of AMF inoculation and water stress in terms of the growth parameters and oil composition of T. vulgaris L. Arbuscular mycorrhizal fungi (AMF) inoculation is a natural way to stimulate plant growth and promoting essential oils in aromatic and medicinal herbs and spices (Rydlova et al., 2105). Under water-stress, AMF inoculation improve plant growth by increasing root length, leaf area, plant biomass, and nutrient uptake, improving soil structure, soil-borne pathogen protection (Abdel Latef et al., 2016). Production of secondary metabolites such as essential oils (from here on simply referred to as oil), flavonoids and phenolics in aromatic and medicinal plants, could be improved by inoculation with AMF (Karagiannidis et al., 2011; Urcoviche et al., 2015). The effect of water deficiency on improving the oil composition and yield of several aromatic and medicinal plants are known (Table 1). Some of these studies reported alleviation of water stress with various compounds, including applications of salicylic acid, proline, jasmonate, chitosan, and ethephon. Only four studied
1
the mycorrhizal effect on water stress and essential oil composition (Amiri et al. 2015; Gheisari et al. 2017; Amiri et al. 2017; Fadaee et al. 2018). The experiment was a complete factorial in randomized pattern design with three replications in Shahrekord, Chaharmahal & Bakhtiari province, Southwest Iran. The first factor included four irrigation levels, well-watered (A: absence of stress); irrigation after depletion of 20-25% from field capacity (L: low stress); irrigation after depletion of 35-40% from field capacity (M: moderate stress); and irrigation after depletion of 55-60% from FC (S: severe stress). The second factor included two AMF treatments (M: Rhizophagus intraradices, Funneliformis mosseae) and one without AMF (C: control). For oil isolation, 30 g powdered plant material was subjected to hydro-distillation (500 ml dH2O) for 3 h using a Clevenger-type apparatus. For GC-MS analysis, a Shimadzu model GC-17A (Kyoto Japan) gas chromatograph coupled to a Shimadzu Quadruple-MS model QP5050 mass spectrometer was used. Mycorrhization and amount of mycorrhizal root infection were determined (Amiri et al. 2017). Root and Shoot growth. Our results indicate that Interaction of AMF inoculation and water stress was significant in root and shoot parameters (p ≤ 0.01). In comparison with control treatments, inoculations with AMF increased these factors under water stress (Table2). Decrease of T. vulgaris growth parameters under water stress in this study were consistent with previous work (AlaviSamani et al. 2013, 2015; Bahreininejad et al. 2013; Emami-Bistgani et al. 2017). Hazzoumi et al. (2015) showed that under water stress, growth of basil (Ocimum basilicum) increased with mycorrhiza inoculation. We also know that shoot biomass of T. vulgaris increases with AMF inoculation in a fungal species and plant variety dependent manner (Tarraf et al. 2015). Although water stress decreased growth parameters of T. vulgaris, inoculation with AMF alleviate to some extent the adverse effects of water stress and this is more obvious in root traits. Our results of root mycorrhization are in agreement with those of Bahadori et al. (2013) who reported that percent root colonization was significantly higher in inoculated T. daenensis, with AMF plants becoming more larger compared to non-inoculated plants. Oil yield. Interaction of AMF inoculation and water stress was significant for oil and mycorrhization (p ≤ 0.01) and by increasing water stress, oil content and mycorrhization increased, but decreased at the highest level of water stress (Table3). There are several reports that show water stress increase oil content in thyme (Amiri et al. 2017; Alavi-Samani et al., 2013, 2015; Bahreininejad et al., 2013; Askary et al., 2018). Emami Bistgani (2017) reported that oil concentration of thyme was maximized when plants were grown under stress condition, and oil yield was highest when plants experienced a moderate water stress, unlike Taraf et al. (2015) who reported AMF colonization in T. vulgaris L. has no impact on oil accumulation. Here AMF increased T. vulgaris oil content significantly. Similar results were obtained in other medicinal plants (Hazzoumi et al. 2105; Rydlova et al. 2015). Overall, the results of different experiments show that a low degree of water stress increases oil synthesis and accumulation in aromatic or medicinal herbs. Consistent with other publications, we found that when we used AMF with low to moderate water stress there is an increase in oil content, but at severe water stress yield and oil content decreased. GC–MS analysis resulted in the identification of 30 constituents of the oil composition including PCymene, Thymol, Carvacrol, Linanool, Camphene, Myrcene, Gamma Terpinene, Alpha Thujene, and 1-Octen-3-ol. Together these composed the bulk of the oil (76.72% - 87.19%), of total oils (Table4). Interaction of AMF inoculation and water stress showed that Thymol, an important constituent of T. vulgaris oil, was not significantly affected by AMF and water stress. However, the highest percentage of Thymol was extracted from non-stressed plants with AMF inoculation. Carvacrol, another 2
important constituent of T. vulgaris oil, increased with increasing water stress but AMF decreased the rate of increase (Table4). Alavi-Samani et al. (2013; 2015), Ghasemi Pirbalouti et al. (2014) and Askary et al. (2018) reported that levels of irrigation had a significant effects on the main T. vulgaris components. Emami Bistgani et al. (2017) in a study reported that the highest percentage of Thymol was obtained from moderate stress and well-watered treatments and the highest amount of Carvacrol was extracted from moderate or severe stress conditions. It have been reported that AMF inoculation treatments significantly increased the concentration of Thymol in T. daenensis plants with respect to the control plants (Bahadori et al. 2013). By summarizing above reported and with respect to different results, as Ghasemi Pirbalouti et al. (2013) stated in their report, T. vulgaris essential oils and its’ chemical compositions are strongly affected by environmental conditions and agronomic management practices and AMF inoculation in water stress conditions could ameliorate negative effects of water shortage. In conclusion, our results showed that AMF inoculation together with low-moderate water stress improve Thymus vulgaris herb morphological traits, oil yield and oil accumulation. These results are consistent with similar conclusions from studies with other aromatic and medicinal herbs. Based on these results, we can recommend that in semiarid regions, oil yield can be improved by appropriate AMF species inoculation, combined with mild water stress, for highest economic benefit of extracted oils including Thymol and Carvacrol, for pharmaceutical, therapeutic and culinary purposes. The positive changes induced by mild water stress can be used as an agronomic strategy for improving yield of specific bioactive compounds in aromatic and medicinal herbs.
References Abdel Latef A.A.H., Hashem A., Rasool S., Abd_Allah E.F., Alqarawi A.A, Egamberdieva D., Jan S., Anjum N. A. and Ahmad P., 2016. Arbuscular Mycorrhizal Symbiosis and Abiotic Stress in Plants: A Review. J. Plant Biol. 59:407-426. DOI 10.1007/s12374-016-0237-7 Alavi-Samani, S.M., Ghasemi Pirbalouti, A., Ataei Kachuei M., Hamedi B., 2013. The influence of reduced irrigation on herbage, essential oil yield and quality of Thymus vulgaris and Thymus daenensis. Int. J. Herb. Med. 4 (3), 109-113. Alavi-Samani, S.M., Kachouei, M.A., Pirbalouti, A.G., 2015. Growth, yield, chemical composition, and antioxidant activity of essential oils from two thyme species under foliar application of Jasmonic acid and water deficit conditions. Horticult. Environ. Biotechnol. 56 (4), 411–420. Amiri R., Nikbakht A., Etemadi N., 2015. Alleviation of drought stress on rose geranium [Pelargonium graveolens (L.) Herit.] in terms of antioxidant activity and secondary metabolites by mycorrhizal inoculation. Sci. Hortic. DOI: 10.1016/j.scienta.2015.09.062 Amiri, R., Nikbakht, A., Rahimmalek, M., Hosseini, H., 2017. Variation in the Essential Oil Composition, Antioxidant Capacity, and Physiological Characteristics of Pelargonium graveolens L. Inoculated with Two Species of Mycorrhizal Fungi Under Water Deficit Conditions. J. Plant Growth Regul. 36 (2), 502-515. Askary, M., Behdani, M.A., Parsa, S., Mahmoodi, S., Jamialahmadi, M., 2018. Water stress and manure application affect the quantity and quality of essential oil of Thymus daenensis and Thymus vulgaris. Ind. Crops Prod. 111, 336–344. Bahadori, F., Sharifi Ashorabadi, E., Mirza, M., Matinizade, M., Abdosi, V., 2013. Improved Growth, Essential Oil Yield and Quality in Thymus daenensis Celak on Mycorrhizal and Plant Growth Promoting Rhizobacteria Inoculation. Intl. J. Agron. Plant Prod. 4 (12), 3384-3391. Bahreininejad, B., Razmjoo, J., Mirza, M., 2013. Influence of water stress on morphophysiological and phytochemical traits in Thymus daenensis. Int. J. Plant Prod. 7 (1), 151–166.
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Emami Bistgani, Z., Siadat, S.A., Bakhshandeh, A., Ghasemi Pirbalouti, A., Hashemi. M., 2017. Morpho-physiological and phytochemical traits of (Thymus daenensis Celak.) in response to deficit irrigation and chitosan application. Acta Physiol. Plant 39 (231), 1-13. Fadaee E., Parvizi Y., Gerdakane M., Khan-Ahmadi M., 2108. The effects of mycorhiza (glomus mosseae and glomus intraradiceae) and phosphorus on growth and phytochemical traits of dracocephalum moldavica L. under drought stress. Journal of Medicinal Plants 17(66), 100-112. Ghasemi Pirbalouti, A., Hashemi, M., Taherian Ghahfarokhi, F., 2013. Essential oil and chemical compositions of wild and cultivated Thymus daenensis Celak and Thymus vulgaris L. Ind. Crops Prod. 48, 43–48. Ghasemi Pirbalouti, A., Samani, M.R., Hashemi, M., Zeinali. H., 2014. Salicylic acid affects growth, essential oil and chemical compositions of thyme (Thymus daenensis Celak.) under reduced irrigation. Plant Growth Regul. 72, 289-301. Gheisari Zardak, S., Movahhedi Dehnavi, M., Salehi, M., and Gholamhoseini, M. 2017. Responses of field grown fennel (Foeniculum vulgare Mill.) to different mycorrhiza species under varying intensities of water stress. J. Appl. Res. Med. Aromat. Plants 5, 16-25. Hazzoumi, Z., Moustakime, Y., Elharchli, E.H., Amrani Joutei, K., 2105. Effect of arbuscular mycorrhizal fungi (AMF) and water stress on growth, phenolic compounds, glandular hairs, and yield of essential oil in basil (Ocimum gratissimum L). Chemical and Biological Technologies in Agriculture 2:10. DOI 10.1186/s40538-015-0035-3 Karagiannidis, N., Thomidis, T., Lazari, D., Panou-Filotheou, E., Karagiannidou, C., 2011. Effect of three Greek arbuscular mycorrhizal fungi in improving the growth, nutrient concentration, and production of oils of oregano and mint plants. Sci. Hortic. 129, 329–334. Llorens-Molina J. A., Vacas, S., 2016. Effect of water stress on essential oil composition of Thymus vulgaris L. (Chemotype 1, 8-cineole) from wild populations of Eastern Iberian Peninsula, J. Essent. Oil Res., DOI: 10.1080/10412905.2016.1211561 Nabavi S.M., Marchese A., Izadi M., Curti V., Dagli M., Nabavi S.F., 2015. Plants belonging to the genus Thymus as antibacterial agents: From farm to pharmacy. Food Chem. 173, 339–347 Rydlová J., Jelínková, M., Dušek, K., Dušková, E., Vosátka, M., Püschel, D., 2015. Arbuscular mycorrhiza differentially affects synthesis of essential oils in coriander and dill. Mycorrhiza. DOI 10.1007/s00572-015-0652-5 Tarraf W., Claudia R., De Cillis, F., Tagarelli, A., Tedone, L., De Mastro, G., 2015. Effects of mycorrhiza on growth and essential oil production in selected aromatic plants. Ital. J. Agron. 10, 633. Urcoviche, R.C., Gazim, Z.C., Dragunski, D.C., Barcellos, F.G., Alberton, O., 2015. Plant growth and oil content of Mentha crispa inoculated with arbuscular mycorrhizal fungi under different levels of phosphorus. Ind. Crops Prod. 67, 103–107. doi:10.1016/j.indcrop.2015.01.016
Table 1. Recent publications presenting water-stress effects in aromatic and medicinal plants essential oils. Paper DOI
Author
Plant Latin name (common name)
Stressors (water and others if applicable)
-
Anderson et al. 2016
Calendula officinalis (Marigold)
Water
-
Fadaee et al. 2018
Dracocephalum moldavica L. (Moldavian dragonhead)
Water
10.1016/j.rhisph.2018.02.001
Zardak et al. 2018
Foeniculum vulgare L. (Fennel)
Water
10.1016/j.indcrop.2017.10.020
Gholami Zali et al. 2108
Foeniculum vulgare Mill. (Fennel)
Water
4
10.1016/j.jarmap.2016.09.004
Gheisari et al. 2017
Foeniculum vulgare Mill. (Fennel)
Water
10.1007/s13199-016-0466-z
Amiri et al. 2017
Pelargonium graveolens L. (Rose geranium)
Water
10.1016/j.scienta.2015.09.062
Amiri et al. 2015
Pelargonium graveolens L. (Rose geranium)
Water
10.1016/j.indcrop.2018.03.021
Vosoughi et al. 2018
Salvia officinalis L. (Sage)
Water
-
Miranshahi B., Sayyari 2105
Satureja hortensis (Summer savory)
Water
10.1016/j.cj.2017.04.003
Emami et al. 2017
Thymus daenensis Celak (Thyme)
Water
10.1080/14786419.2018.1460843
Hassan et al. 2018
Thymus Vulgaris L. (Thyme)
Water
10.1016/j.plaphy.2017.12.007
Khalil et al. 2018
Thymus Vulgaris L. (Thyme)
Water
-
Biswasa et al. 2015
Various
Salt, water, metals
Table2. Effects of mycorrhiza inoculation and water stress on root and shoot growth.
FW:
Treatment
Root FW (gr)
Root DW (gr)
Shoot FW (gr)
Shoot DW (gr)
CA
101.31 ± 2.69 b
30.35 ± 0.85 b
51.64 ± 0.55 b
27.08 ± 0.13 a
CL
67.25 ± 2.07 d
19.58 ± 1.25 e
48.35 ± 0.62 c
23.93 ± 0.24 c
CM
77.72 ± 0.80 c
23.58 ± 0.53 c
37.65 ± 0.99 g
19.47 ± 0.46 e
CS
34.88 ± 1.58 g
11.61 ± 0.31 g
31.52 ± 0.47 h
18.09 ± 0.39 f
MA
185.08 ± 8.33 a
69.32 ± 1.58 a
53.62 ± 1.26 a
26.26 ± 0.35 b
ML
35.82 ± 1.55 fg
12.70 ± 0.83 fg
39.66 ± 1.05 f
20.19 ± 0.70 e
MM
45.94 ± 1.52 e
13.64 ± 0.22 f
41.52 ± 0.78 e
22.00 ± 0.64 d
MS
41.54 ± 2.02 ef
21.83 ± 0.26 d
44.97 ± 0.75 d
21.96 ± 0.57 d
ANOVA
p ≤ 0.01
p ≤ 0.01
p ≤ 0.01
p ≤ 0.01
fresh weight, DW: dry weight, C: control, first M: mycorrhiza inoculation, A: absence of water stress, L: low water stress, second M: medium water stress, S: severe water stress, Means (±SD) accompanied by the same letter do not differ significantly at P ≤ 0.05 and P ≤ 0.01 by Duncan’s multiple range test
5
Table3. Water stress effect on mycorrhiza and oil yield in T. vulgaris Interaction CA
CL
CM
CS
Mycorrhizaton (%) Essence (ml/100 g DM)
0.81 ± 0.02 cd
0.92 ± 0.02 b
0.78 ± 0.02 d
0.91 ± 0.02 b
MA
ML
MM
MS
ANOVA
48.28 ± 0.50 e
54.71 ± 1.00 c
83.85 ± 3.22 a
51.74 ± 0.91 d
p ≤ 0.01
0.83 ± 0.03 c
0.83 ± 0.03 b
0.99 ± 0.39 a
0.90 ± 0.33 b
p ≤ 0.01
C: control without mycorrhiza, first M: mycorrhiza inoculation, A: absence of water stress, L: low water stress, second M: moderate water stress, S: severe water stress; n.s: not significant, Means (±SD) accompanied by the same letter do not differ significantly at P ≤ 0.01 by Duncan’s multiple range test
Table4. Effects of mycorrhiza inoculation, water stress level, and their interaction on oil composition. RI
Inoculation
Compound
1-Octen-3ol
Linalool Thymol Carvacrol
CL
CM
CS
MA
ML
MM
MS
1.99 ± 0.07 bc 1.31 ± 0.01 b 1.77 ± 0.02 c 3.88 ± 0.22 ab 24.31 ± 0.95 bc 12.82 ± 1.03 b 4.56 ± 0.37 a 31.22 ± 2.58 2.32 ± 0.50 c
1.57 ± 0.01 d
2.13 ± 0.10 ab
p≤ 0.01
0.92 ± 0.02 d 1.62 ± 0.04 d 2.73 ± 0.34 c
1.34 ± 0.01 a 2.06 ± 0.04 b 4.26 ± 0.13 a
p≤ 0.01 p≤ 0.01 p≤ 0.01
17.39 ± 4.70 d 9.89 ± 1.05 d
27.30 ± 1.05 ab
p≤ 0.01
16.60 ± 0.91 a
p≤ 0.01
3.44 ± 0.49 b 30.09 ± 2.49 15.81 ± 1.05 b
2.93 ± 0.72 b 26.06 ± 1.73 2.23 ± 0.30 c
p≤ 0.01 n.s
p≤ 0.01
2.30 ± 0.16 a
1.57 ± 0.50 d
1.43 ± 0.01 d
1.12 ± 0.19 e
1.93 ± 0.05 c
953
p ≤ 0.01
978
n.s
991
p ≤ 0.01
p≤ 0.01 p≤ 0.01 p≤ 0.01
1.35 ± 0.01 a 2.64 ± 0.03 a 4.26 ± 0.25 a
0.88 ± 0.02 e 1.49 ± 0.03 e 2.98 ± 0.03 c
0.94 ± 0.02 d 1.36 ± 0.02 f 2.85 ± 0.38 c
0.65 ± 0.01 f 1.09 ± 0.05 g 2.11 ± 0.13 d
1.14 ± 0.02 c 1.04 ± 0.04 g 3.70 ± 0.27 b
1026
p ≤ 0.01
n.s
1062
p ≤ 0.01
p≤ 0.01
1098
n.s n.s
1298
p ≤ 0.01
20.96 ± 1.32 c 12.33 ± 0.71 bc 3.45 ± 0.37 b 27.90 ± 1.97 5.82 ± 1.53 b
23.90 ± 1.15 bc 10.01 ± 1.80 d 2.56 ± 0.47 b 28.26 ± 2.06 16.41 ± 1.02 b
17.47 ± 0.80 d 10.59 ± 1.46 cd -
1290
p≤ 0.01 n.s
16.36 ± 0.80 d 12.45 ± 1.00 bc 4.58 ± 0.55 a 28.41 ± 2.22 3.97 ± 1.00 c
28.48 ± 0.63 a 13.73 ± 1.09 b 3.55 ± 0.45 b 30.61 ± 2.67 3.41 ± 0.51 c
P-Cymene
p≤ 0.01
ANOVA
CA
p ≤ 0.01
Myrcene
γTerpinene
Interaction %
931 α-Thujene Camphene
Water Stress
31.72 ± 2.05 20.66 ± 2.26 a
p≤ 0.01
RI: retention indices determined on GC-MS capillary column, C: control, first M: mycorrhiza inoculation, A: absence of
water stress, L: low water stress, second M: medium water stress, S: severe water stress, n.s: not significant, Means (±SD) accompanied by the same letter do not differ significantly at P ≤ 0.01 by Duncan’s multiple range test
6
PII: DOI: Reference:
S2452-2198(18)30131-9 https://doi.org/10.1016/j.rhisph.2018.10.003 RHISPH129
To appear in: Rhizosphere Received date: 15 October 2018 Revised date: 25 October 2018 Accepted date: 26 October 2018 Cite this article as: Ali Abdollahi Arpanahi and Mohammad Feizian, Arbuscular mycorrhizae alleviate mild to moderate water stress and improve essential oil yield in thyme, Rhizosphere, https://doi.org/10.1016/j.rhisph.2018.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Arbuscular mycorrhizae alleviate mild to moderate water stress and improve essential oil yield in thyme Ali Abdollahi Arpanahi1, Mohammad Feizian2*
1. Soil Sciences department, Agriculture Faculty, Lorestan University, Khorramabad, Iran. Email: [email protected] 2. *Corresponding Author; Soil Sciences department, Agriculture Faculty, Lorestan University, PO Box: 68151-44316, Khorramabad, Iran. Email: [email protected]
Abstract In order to study the effects of arbuscular mycorrhizal fungi and water stress on Thymus Vulgaris L. essential oil composition, an experiment was designed in full factorial randomized pattern with three replications. Factors consisted of four irrigation levels and two different inocula. Results showed that water stress decreased growth parameters of T. vulgaris but inoculation with AMF alleviated adverse effects of water stress. Result showed that when we used AMF with mildmoderate water stress, there is an increase in essential oil content but at severe water-stress essential oil content decreased. Interaction of AMF and water stress had significant effects on most components of T. vulgaris essential oil.
Keywords: Thymus Vulgaris L., Essential Oils, Arbuscular Mycorrhizal Fungi, Drought Stress
Thymus vulgaris L. (Lamiaceae) commonly known as thyme, is one of the well-known medicinal and aromatic plants from the mint family used in pharmaceutical, cosmetic, and food industries, for its aromatic and medicinal properties (Nabavi et al. 2015; Lorens-Molina et al., 2016). The aims of the present study was to assess the interactions of AMF inoculation and water stress in terms of the growth parameters and oil composition of T. vulgaris L. Arbuscular mycorrhizal fungi (AMF) inoculation is a natural way to stimulate plant growth and promoting essential oils in aromatic and medicinal herbs and spices (Rydlova et al., 2105). Under water-stress, AMF inoculation improve plant growth by increasing root length, leaf area, plant biomass, and nutrient uptake, improving soil structure, soil-borne pathogen protection (Abdel Latef et al., 2016). Production of secondary metabolites such as essential oils (from here on simply referred to as oil), flavonoids and phenolics in aromatic and medicinal plants, could be improved by inoculation with AMF (Karagiannidis et al., 2011; Urcoviche et al., 2015). The effect of water deficiency on improving the oil composition and yield of several aromatic and medicinal plants are known (Table 1). Some of these studies reported alleviation of water stress with various compounds, including applications of salicylic acid, proline, jasmonate, chitosan, and ethephon. Only four studied
1
the mycorrhizal effect on water stress and essential oil composition (Amiri et al. 2015; Gheisari et al. 2017; Amiri et al. 2017; Fadaee et al. 2018). The experiment was a complete factorial in randomized pattern design with three replications in Shahrekord, Chaharmahal & Bakhtiari province, Southwest Iran. The first factor included four irrigation levels, well-watered (A: absence of stress); irrigation after depletion of 20-25% from field capacity (L: low stress); irrigation after depletion of 35-40% from field capacity (M: moderate stress); and irrigation after depletion of 55-60% from FC (S: severe stress). The second factor included two AMF treatments (M: Rhizophagus intraradices, Funneliformis mosseae) and one without AMF (C: control). For oil isolation, 30 g powdered plant material was subjected to hydro-distillation (500 ml dH2O) for 3 h using a Clevenger-type apparatus. For GC-MS analysis, a Shimadzu model GC-17A (Kyoto Japan) gas chromatograph coupled to a Shimadzu Quadruple-MS model QP5050 mass spectrometer was used. Mycorrhization and amount of mycorrhizal root infection were determined (Amiri et al. 2017). Root and Shoot growth. Our results indicate that Interaction of AMF inoculation and water stress was significant in root and shoot parameters (p ≤ 0.01). In comparison with control treatments, inoculations with AMF increased these factors under water stress (Table2). Decrease of T. vulgaris growth parameters under water stress in this study were consistent with previous work (AlaviSamani et al. 2013, 2015; Bahreininejad et al. 2013; Emami-Bistgani et al. 2017). Hazzoumi et al. (2015) showed that under water stress, growth of basil (Ocimum basilicum) increased with mycorrhiza inoculation. We also know that shoot biomass of T. vulgaris increases with AMF inoculation in a fungal species and plant variety dependent manner (Tarraf et al. 2015). Although water stress decreased growth parameters of T. vulgaris, inoculation with AMF alleviate to some extent the adverse effects of water stress and this is more obvious in root traits. Our results of root mycorrhization are in agreement with those of Bahadori et al. (2013) who reported that percent root colonization was significantly higher in inoculated T. daenensis, with AMF plants becoming more larger compared to non-inoculated plants. Oil yield. Interaction of AMF inoculation and water stress was significant for oil and mycorrhization (p ≤ 0.01) and by increasing water stress, oil content and mycorrhization increased, but decreased at the highest level of water stress (Table3). There are several reports that show water stress increase oil content in thyme (Amiri et al. 2017; Alavi-Samani et al., 2013, 2015; Bahreininejad et al., 2013; Askary et al., 2018). Emami Bistgani (2017) reported that oil concentration of thyme was maximized when plants were grown under stress condition, and oil yield was highest when plants experienced a moderate water stress, unlike Taraf et al. (2015) who reported AMF colonization in T. vulgaris L. has no impact on oil accumulation. Here AMF increased T. vulgaris oil content significantly. Similar results were obtained in other medicinal plants (Hazzoumi et al. 2105; Rydlova et al. 2015). Overall, the results of different experiments show that a low degree of water stress increases oil synthesis and accumulation in aromatic or medicinal herbs. Consistent with other publications, we found that when we used AMF with low to moderate water stress there is an increase in oil content, but at severe water stress yield and oil content decreased. GC–MS analysis resulted in the identification of 30 constituents of the oil composition including PCymene, Thymol, Carvacrol, Linanool, Camphene, Myrcene, Gamma Terpinene, Alpha Thujene, and 1-Octen-3-ol. Together these composed the bulk of the oil (76.72% - 87.19%), of total oils (Table4). Interaction of AMF inoculation and water stress showed that Thymol, an important constituent of T. vulgaris oil, was not significantly affected by AMF and water stress. However, the highest percentage of Thymol was extracted from non-stressed plants with AMF inoculation. Carvacrol, another 2
important constituent of T. vulgaris oil, increased with increasing water stress but AMF decreased the rate of increase (Table4). Alavi-Samani et al. (2013; 2015), Ghasemi Pirbalouti et al. (2014) and Askary et al. (2018) reported that levels of irrigation had a significant effects on the main T. vulgaris components. Emami Bistgani et al. (2017) in a study reported that the highest percentage of Thymol was obtained from moderate stress and well-watered treatments and the highest amount of Carvacrol was extracted from moderate or severe stress conditions. It have been reported that AMF inoculation treatments significantly increased the concentration of Thymol in T. daenensis plants with respect to the control plants (Bahadori et al. 2013). By summarizing above reported and with respect to different results, as Ghasemi Pirbalouti et al. (2013) stated in their report, T. vulgaris essential oils and its’ chemical compositions are strongly affected by environmental conditions and agronomic management practices and AMF inoculation in water stress conditions could ameliorate negative effects of water shortage. In conclusion, our results showed that AMF inoculation together with low-moderate water stress improve Thymus vulgaris herb morphological traits, oil yield and oil accumulation. These results are consistent with similar conclusions from studies with other aromatic and medicinal herbs. Based on these results, we can recommend that in semiarid regions, oil yield can be improved by appropriate AMF species inoculation, combined with mild water stress, for highest economic benefit of extracted oils including Thymol and Carvacrol, for pharmaceutical, therapeutic and culinary purposes. The positive changes induced by mild water stress can be used as an agronomic strategy for improving yield of specific bioactive compounds in aromatic and medicinal herbs.
References Abdel Latef A.A.H., Hashem A., Rasool S., Abd_Allah E.F., Alqarawi A.A, Egamberdieva D., Jan S., Anjum N. A. and Ahmad P., 2016. Arbuscular Mycorrhizal Symbiosis and Abiotic Stress in Plants: A Review. J. Plant Biol. 59:407-426. DOI 10.1007/s12374-016-0237-7 Alavi-Samani, S.M., Ghasemi Pirbalouti, A., Ataei Kachuei M., Hamedi B., 2013. The influence of reduced irrigation on herbage, essential oil yield and quality of Thymus vulgaris and Thymus daenensis. Int. J. Herb. Med. 4 (3), 109-113. Alavi-Samani, S.M., Kachouei, M.A., Pirbalouti, A.G., 2015. Growth, yield, chemical composition, and antioxidant activity of essential oils from two thyme species under foliar application of Jasmonic acid and water deficit conditions. Horticult. Environ. Biotechnol. 56 (4), 411–420. Amiri R., Nikbakht A., Etemadi N., 2015. Alleviation of drought stress on rose geranium [Pelargonium graveolens (L.) Herit.] in terms of antioxidant activity and secondary metabolites by mycorrhizal inoculation. Sci. Hortic. DOI: 10.1016/j.scienta.2015.09.062 Amiri, R., Nikbakht, A., Rahimmalek, M., Hosseini, H., 2017. Variation in the Essential Oil Composition, Antioxidant Capacity, and Physiological Characteristics of Pelargonium graveolens L. Inoculated with Two Species of Mycorrhizal Fungi Under Water Deficit Conditions. J. Plant Growth Regul. 36 (2), 502-515. Askary, M., Behdani, M.A., Parsa, S., Mahmoodi, S., Jamialahmadi, M., 2018. Water stress and manure application affect the quantity and quality of essential oil of Thymus daenensis and Thymus vulgaris. Ind. Crops Prod. 111, 336–344. Bahadori, F., Sharifi Ashorabadi, E., Mirza, M., Matinizade, M., Abdosi, V., 2013. Improved Growth, Essential Oil Yield and Quality in Thymus daenensis Celak on Mycorrhizal and Plant Growth Promoting Rhizobacteria Inoculation. Intl. J. Agron. Plant Prod. 4 (12), 3384-3391. Bahreininejad, B., Razmjoo, J., Mirza, M., 2013. Influence of water stress on morphophysiological and phytochemical traits in Thymus daenensis. Int. J. Plant Prod. 7 (1), 151–166.
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Emami Bistgani, Z., Siadat, S.A., Bakhshandeh, A., Ghasemi Pirbalouti, A., Hashemi. M., 2017. Morpho-physiological and phytochemical traits of (Thymus daenensis Celak.) in response to deficit irrigation and chitosan application. Acta Physiol. Plant 39 (231), 1-13. Fadaee E., Parvizi Y., Gerdakane M., Khan-Ahmadi M., 2108. The effects of mycorhiza (glomus mosseae and glomus intraradiceae) and phosphorus on growth and phytochemical traits of dracocephalum moldavica L. under drought stress. Journal of Medicinal Plants 17(66), 100-112. Ghasemi Pirbalouti, A., Hashemi, M., Taherian Ghahfarokhi, F., 2013. Essential oil and chemical compositions of wild and cultivated Thymus daenensis Celak and Thymus vulgaris L. Ind. Crops Prod. 48, 43–48. Ghasemi Pirbalouti, A., Samani, M.R., Hashemi, M., Zeinali. H., 2014. Salicylic acid affects growth, essential oil and chemical compositions of thyme (Thymus daenensis Celak.) under reduced irrigation. Plant Growth Regul. 72, 289-301. Gheisari Zardak, S., Movahhedi Dehnavi, M., Salehi, M., and Gholamhoseini, M. 2017. Responses of field grown fennel (Foeniculum vulgare Mill.) to different mycorrhiza species under varying intensities of water stress. J. Appl. Res. Med. Aromat. Plants 5, 16-25. Hazzoumi, Z., Moustakime, Y., Elharchli, E.H., Amrani Joutei, K., 2105. Effect of arbuscular mycorrhizal fungi (AMF) and water stress on growth, phenolic compounds, glandular hairs, and yield of essential oil in basil (Ocimum gratissimum L). Chemical and Biological Technologies in Agriculture 2:10. DOI 10.1186/s40538-015-0035-3 Karagiannidis, N., Thomidis, T., Lazari, D., Panou-Filotheou, E., Karagiannidou, C., 2011. Effect of three Greek arbuscular mycorrhizal fungi in improving the growth, nutrient concentration, and production of oils of oregano and mint plants. Sci. Hortic. 129, 329–334. Llorens-Molina J. A., Vacas, S., 2016. Effect of water stress on essential oil composition of Thymus vulgaris L. (Chemotype 1, 8-cineole) from wild populations of Eastern Iberian Peninsula, J. Essent. Oil Res., DOI: 10.1080/10412905.2016.1211561 Nabavi S.M., Marchese A., Izadi M., Curti V., Dagli M., Nabavi S.F., 2015. Plants belonging to the genus Thymus as antibacterial agents: From farm to pharmacy. Food Chem. 173, 339–347 Rydlová J., Jelínková, M., Dušek, K., Dušková, E., Vosátka, M., Püschel, D., 2015. Arbuscular mycorrhiza differentially affects synthesis of essential oils in coriander and dill. Mycorrhiza. DOI 10.1007/s00572-015-0652-5 Tarraf W., Claudia R., De Cillis, F., Tagarelli, A., Tedone, L., De Mastro, G., 2015. Effects of mycorrhiza on growth and essential oil production in selected aromatic plants. Ital. J. Agron. 10, 633. Urcoviche, R.C., Gazim, Z.C., Dragunski, D.C., Barcellos, F.G., Alberton, O., 2015. Plant growth and oil content of Mentha crispa inoculated with arbuscular mycorrhizal fungi under different levels of phosphorus. Ind. Crops Prod. 67, 103–107. doi:10.1016/j.indcrop.2015.01.016
Table 1. Recent publications presenting water-stress effects in aromatic and medicinal plants essential oils. Paper DOI
Author
Plant Latin name (common name)
Stressors (water and others if applicable)
-
Anderson et al. 2016
Calendula officinalis (Marigold)
Water
-
Fadaee et al. 2018
Dracocephalum moldavica L. (Moldavian dragonhead)
Water
10.1016/j.rhisph.2018.02.001
Zardak et al. 2018
Foeniculum vulgare L. (Fennel)
Water
10.1016/j.indcrop.2017.10.020
Gholami Zali et al. 2108
Foeniculum vulgare Mill. (Fennel)
Water
4
10.1016/j.jarmap.2016.09.004
Gheisari et al. 2017
Foeniculum vulgare Mill. (Fennel)
Water
10.1007/s13199-016-0466-z
Amiri et al. 2017
Pelargonium graveolens L. (Rose geranium)
Water
10.1016/j.scienta.2015.09.062
Amiri et al. 2015
Pelargonium graveolens L. (Rose geranium)
Water
10.1016/j.indcrop.2018.03.021
Vosoughi et al. 2018
Salvia officinalis L. (Sage)
Water
-
Miranshahi B., Sayyari 2105
Satureja hortensis (Summer savory)
Water
10.1016/j.cj.2017.04.003
Emami et al. 2017
Thymus daenensis Celak (Thyme)
Water
10.1080/14786419.2018.1460843
Hassan et al. 2018
Thymus Vulgaris L. (Thyme)
Water
10.1016/j.plaphy.2017.12.007
Khalil et al. 2018
Thymus Vulgaris L. (Thyme)
Water
-
Biswasa et al. 2015
Various
Salt, water, metals
Table2. Effects of mycorrhiza inoculation and water stress on root and shoot growth.
FW:
Treatment
Root FW (gr)
Root DW (gr)
Shoot FW (gr)
Shoot DW (gr)
CA
101.31 ± 2.69 b
30.35 ± 0.85 b
51.64 ± 0.55 b
27.08 ± 0.13 a
CL
67.25 ± 2.07 d
19.58 ± 1.25 e
48.35 ± 0.62 c
23.93 ± 0.24 c
CM
77.72 ± 0.80 c
23.58 ± 0.53 c
37.65 ± 0.99 g
19.47 ± 0.46 e
CS
34.88 ± 1.58 g
11.61 ± 0.31 g
31.52 ± 0.47 h
18.09 ± 0.39 f
MA
185.08 ± 8.33 a
69.32 ± 1.58 a
53.62 ± 1.26 a
26.26 ± 0.35 b
ML
35.82 ± 1.55 fg
12.70 ± 0.83 fg
39.66 ± 1.05 f
20.19 ± 0.70 e
MM
45.94 ± 1.52 e
13.64 ± 0.22 f
41.52 ± 0.78 e
22.00 ± 0.64 d
MS
41.54 ± 2.02 ef
21.83 ± 0.26 d
44.97 ± 0.75 d
21.96 ± 0.57 d
ANOVA
p ≤ 0.01
p ≤ 0.01
p ≤ 0.01
p ≤ 0.01
fresh weight, DW: dry weight, C: control, first M: mycorrhiza inoculation, A: absence of water stress, L: low water stress, second M: medium water stress, S: severe water stress, Means (±SD) accompanied by the same letter do not differ significantly at P ≤ 0.05 and P ≤ 0.01 by Duncan’s multiple range test
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Table3. Water stress effect on mycorrhiza and oil yield in T. vulgaris Interaction CA
CL
CM
CS
Mycorrhizaton (%) Essence (ml/100 g DM)
0.81 ± 0.02 cd
0.92 ± 0.02 b
0.78 ± 0.02 d
0.91 ± 0.02 b
MA
ML
MM
MS
ANOVA
48.28 ± 0.50 e
54.71 ± 1.00 c
83.85 ± 3.22 a
51.74 ± 0.91 d
p ≤ 0.01
0.83 ± 0.03 c
0.83 ± 0.03 b
0.99 ± 0.39 a
0.90 ± 0.33 b
p ≤ 0.01
C: control without mycorrhiza, first M: mycorrhiza inoculation, A: absence of water stress, L: low water stress, second M: moderate water stress, S: severe water stress; n.s: not significant, Means (±SD) accompanied by the same letter do not differ significantly at P ≤ 0.01 by Duncan’s multiple range test
Table4. Effects of mycorrhiza inoculation, water stress level, and their interaction on oil composition. RI
Inoculation
Compound
1-Octen-3ol
Linalool Thymol Carvacrol
CL
CM
CS
MA
ML
MM
MS
1.99 ± 0.07 bc 1.31 ± 0.01 b 1.77 ± 0.02 c 3.88 ± 0.22 ab 24.31 ± 0.95 bc 12.82 ± 1.03 b 4.56 ± 0.37 a 31.22 ± 2.58 2.32 ± 0.50 c
1.57 ± 0.01 d
2.13 ± 0.10 ab
p≤ 0.01
0.92 ± 0.02 d 1.62 ± 0.04 d 2.73 ± 0.34 c
1.34 ± 0.01 a 2.06 ± 0.04 b 4.26 ± 0.13 a
p≤ 0.01 p≤ 0.01 p≤ 0.01
17.39 ± 4.70 d 9.89 ± 1.05 d
27.30 ± 1.05 ab
p≤ 0.01
16.60 ± 0.91 a
p≤ 0.01
3.44 ± 0.49 b 30.09 ± 2.49 15.81 ± 1.05 b
2.93 ± 0.72 b 26.06 ± 1.73 2.23 ± 0.30 c
p≤ 0.01 n.s
p≤ 0.01
2.30 ± 0.16 a
1.57 ± 0.50 d
1.43 ± 0.01 d
1.12 ± 0.19 e
1.93 ± 0.05 c
953
p ≤ 0.01
978
n.s
991
p ≤ 0.01
p≤ 0.01 p≤ 0.01 p≤ 0.01
1.35 ± 0.01 a 2.64 ± 0.03 a 4.26 ± 0.25 a
0.88 ± 0.02 e 1.49 ± 0.03 e 2.98 ± 0.03 c
0.94 ± 0.02 d 1.36 ± 0.02 f 2.85 ± 0.38 c
0.65 ± 0.01 f 1.09 ± 0.05 g 2.11 ± 0.13 d
1.14 ± 0.02 c 1.04 ± 0.04 g 3.70 ± 0.27 b
1026
p ≤ 0.01
n.s
1062
p ≤ 0.01
p≤ 0.01
1098
n.s n.s
1298
p ≤ 0.01
20.96 ± 1.32 c 12.33 ± 0.71 bc 3.45 ± 0.37 b 27.90 ± 1.97 5.82 ± 1.53 b
23.90 ± 1.15 bc 10.01 ± 1.80 d 2.56 ± 0.47 b 28.26 ± 2.06 16.41 ± 1.02 b
17.47 ± 0.80 d 10.59 ± 1.46 cd -
1290
p≤ 0.01 n.s
16.36 ± 0.80 d 12.45 ± 1.00 bc 4.58 ± 0.55 a 28.41 ± 2.22 3.97 ± 1.00 c
28.48 ± 0.63 a 13.73 ± 1.09 b 3.55 ± 0.45 b 30.61 ± 2.67 3.41 ± 0.51 c
P-Cymene
p≤ 0.01
ANOVA
CA
p ≤ 0.01
Myrcene
γTerpinene
Interaction %
931 α-Thujene Camphene
Water Stress
31.72 ± 2.05 20.66 ± 2.26 a
p≤ 0.01
RI: retention indices determined on GC-MS capillary column, C: control, first M: mycorrhiza inoculation, A: absence of
water stress, L: low water stress, second M: medium water stress, S: severe water stress, n.s: not significant, Means (±SD) accompanied by the same letter do not differ significantly at P ≤ 0.01 by Duncan’s multiple range test
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