Growth and nutrient content of Echinacea purpurea as affected by the combination of phosphorus with arbuscular mycorrhizal fungus and Pseudomonas florescent bacterium under different irrigation regimes

Journal of Environmental Management 231 (2019) 182–188

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Research article

Growth and nutrient content of Echinacea purpurea as affected by the combination of phosphorus with arbuscular mycorrhizal fungus and Pseudomonas florescent bacterium under different irrigation regimes

T

Mahmood Attarzadeha, Hamidreza Balouchib,∗, Majid Rajaiec, Mohsen Movahhedi Dehnavib, Amin Salehib a b c

PhD. student, Agronomy, and Plant Breeding Department, Yasouj University, Yasouj, Iran Associate Professor, Agronomy, and Plant Breeding Department, Yasouj University, Yasouj, Iran Soil and Water Research Department, Fars Agricultural and Natural Resources Research and Education Center, AREEO, Shiraz, Iran

A R T I C LE I N FO

A B S T R A C T

Keywords: Drought Growth-promoting bacteria Leaf nutrients Manganese Medicinal plants Zinc

The excessive use of chemical fertilizers has caused many environmental problems and threatens the health of the human communities at the global level. However, the use of some beneficial soil microorganisms in addition to supplying nutrients to plants helps protect the environment. In order to achieve this goal, the effects of different irrigation regimes and application of phosphorus (P) fertilizer, with mycorrhizal arbuscular fungus (AMF) or Pseudomonas fluorescens bacterium (PFB), were studied on the growth and nutrients of Echinacea purpurea. The main factor included soil irrigation after 25, 50 and 75% of soil moisture depletion and a subfactor of P supplied in six levels (100% chemical P, 50% P + AMF, AMF, 50% P + PFB, PFB and a control test without P fertilizer). Results showed that an increase in drought intensity reduced the absorption of nutrients and relative water content (RWC), while ion leakage increased in the leaf of E. purpurea. The AMF had a more clear effect on the N, Cu, Mn, and Fe, but PFB was more effective in an increase of Zn. With the use of PFB in the second harvest, the amount of leaf and root Zn was increased by 30.39% and 31.88%, respectively. Although 100% chemical P could increase more P concentration in the root, the combination of P fertilizer with AMF transferred more P from root to leaf. In the first and second harvest, a combination of P with PFB respectively increased the plant biological yield by 10.77% and 17.33% as compared to control. Vegetative traits, Mn, and Zn illustrated a significant increase in the second harvest. Finally, the results showed successful coexistence of biofertilizers with E. purpurea in increasing the content of nutrients, improving water absorption, and reducing the adverse effects of drought stress.

1. Introduction Echinacea purpurea (L.) Moench. is a perennial herbaceous plant which belongs to the Asteraceae family and natively comes from North America (Flagel et al., 2008). All organs of this plant contain valuable materials that have made it an important medicinal herb, leading to its spread through most parts of the world and its wide cultivation in Europe and the United States. In the pharmaceutical industry, the three major species of plant used are E. angustifolia (DC.) Hell., E. pallida (Nutt.) Nutt., and E. purpurea (L.) Moench. (Barnes et al., 2005; Barrett, 2003). The effective ingredients of E. purpurea play an important role in

improving the body's immune system with antifungal, antiviral and antibacterial properties. That's why this plant is used to treat many diseases (Birt et al., 2008). Phosphorus (P) is one of the essential nutrient elements, having a key function in the metabolism of carbohydrates and in the energy transfer system in plants. P is a vital constituent of the DNA, RNA, ATP, and phospholipids, meaning its deficiency can lead to a reduction of many metabolic processes. This includes cell division and development, respiration, and photosynthesis (Sims and Sharpley, 2005). Chemical fertilizers generally provided the plants P requirements. However, a large amount of P in fertilizers may become insoluble and lose their

Abbreviations: P, Phosphorus; AMF, Mycorrhizal arbuscular fungus; PFB, Pseudomonas fluorescens bacterium; RWC, Relative water content; Zn, Zinc; Cu, Copper; Mn, manganese; N, Nitrogen ∗ Corresponding author. E-mail addresses: [email protected] (M. Attarzadeh), [email protected] (H. Balouchi), [email protected] (M. Rajaie), [email protected] (M. Movahhedi Dehnavi), [email protected] (A. Salehi). https://doi.org/10.1016/j.jenvman.2018.10.040 Received 17 June 2018; Received in revised form 10 October 2018; Accepted 12 October 2018 0301-4797/ © 2018 Elsevier Ltd. All rights reserved.

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Fig. 1. The climate conditions for the seasonal patterns from May 2016 to September 2017 identified. (National Meteorological organization, Iran).

The environmental problems and concerns have been globally noticed due to the excessive use of chemical fertilizers in agricultural lands, recently. In other words, the global approach to the production of medicinal plants is towards the use of sustainable agricultural practices and the application of management methods such as the use of biofertilizers to reduce environmental hazards. According to the stated facts, bio-fertilizers have a positive effect on drought stress alleviation, dissolving insoluble phosphates in soil and facilitating their absorption by the plant. Therefore, the aim of this investigation was elucidate the vegetative growth and nutrient content of E. purpurea, as affected by the combination of phosphorus with mycorrhizal arbuscular fungus (AMF) and Pseudomonas florescent bacterium (PFB) as one of the growth-promoting rhizobacteria, under different irrigation regimes.

plant availability after entrance into the soil (Adhya et al., 2015). In calcareous soils that have evolved under arid and semi-arid climatic conditions, the presence of calcium carbonate, high pH, small amounts of organic matter, and soil dryness may cause the plant's available P to be less than the amount required for optimal growth of most crops. The utilization of chemical P fertilizers, which is one of the ways to compensate for this element deficiency, is not very efficient in calcareous and alkaline soils, meaning the efficiency of P fertilizers in these soils does not exceed 20% (Spinks and Barber, 1947; Tisdale et al., 1993). In addition to the consumption of chemical fertilizers, an alternate method for the provision of P required by plants is the utilization of biological resources. Research conducted in different parts of the world shows that inoculation of bio-fertilizers can increase the plant availability of soil P (Zahir et al., 2006). Many researchers' findings confirm that bio-fertilizers can increase plant access to nutrients and improve plant growth through the synergistic and exacerbated effects that they produce (Sharma and Sharma, 2002). In many investigations related to sustainable agriculture, the existence of a synergistic relationship has been reported between microorganisms, such as Arbuscular mycorrhiza fungi (AMF), and a variety of plants, such as Asteraceae family, such that the plant P content and growth increased by inoculation of this fungus (Baum et al., 2015). As it was stated by Joner et al. (2000) and Koide and Kabir (2000), AMF can produce phosphatases to facilitate hydrolyze phosphorus from natural P compounds. AMF is perhaps the most extensive plant symbiosis and is formed by approximately 80% of plant species (Smith and Read, 2010). Considerable evidence suggests that AMF increases the tolerance of host plants under drought stress conditions (Lazcano et al., 2014). In fact, the coexistence of AMF with host plants not only improves water absorption through the fungus hyphae to the plant root, alleviating the adverse effects of drought stress (Augé et al., 2007) but also increases the absorption of plant required nutrients (Omirou et al., 2013). Furthermore, this coexistence recuperates root hydraulic conductivity, plant gas exchange (Bárzana et al., 2014), osmotic (Aroca et al., 2007), and antioxidant (Bompadre et al., 2014) regulation. In addition to AMF symbiosis, growth-promoting rhizobacteria can also promote root system development, increase water holding capacity, and improve the expression of genes responsible for creating resistance to environmental stresses (Ortiz et al., 2015). The possible explanation for the mechanisms of plant growth improvement induced by rhizobacteria include satisfying the plant nitrogen (N) requirement by N fixation, dissolving low soluble phosphates and zinc, supplying iron (Fe) with the production of microbial siderophores, producing phytohormones such as auxins, cytokinins and gibberellins, and reducing the intensity of ethylene in host plant roots (Vurukonda et al., 2016).

2. Material and methods 2.1. Experimental design and treatments A field split-plot in time and space experiment was conducted in a randomized complete block design with three replications in Fars Province of Iran (29024‫ ׳‬N, 54015‫ ׳‬E and 1370 m elevation from sea level). Single planting was done in late 2016 and two harvests were performed during the growing seasons of 2016 and 2017. The climate conditions for the seasonal patterns from May 2016 to September 2017 identified (Fig. 1). The main factor included irrigation regimes at three levels (irrigation after 25, 50 and 75% of soil moisture depletion to regain field capacity moisture) and a sub-factor of phosphorus (P) supply in six levels (100% P requirement from the source of triple superphosphate, 50% P requirement + AMF, AMF, 50% P requirement + PFB, PFB and a control test without P fertilizer). At the beginning of the experiment, a compound of the surface (0–30 cm) soil samples was collected, air-dried, passed through a 2-mm sieve and the soil physical and chemical properties were measured (Table 1). Echinacea purpurea seeds usually have little vegetative power and, due to their slow early growth, indirect cultivation is always used for their production (Finnerty and Zajicek, 1992). Therefore, the seeds of E. purpurea were first cultivated under a suitable environment in a nursery. Origin of primary population seeds was Germany (Jelitto Staudensamen GmbH, Schwarmstedt, Germany). After planting of the seeds and turning to 90-day seedlings, they were transplanted to the experimental field in late May 2016. During the plant growth, weeding operation was carried out on the plots by hand. Each experimental plot consisted of five 3-m long rows, at 50 cm distance from each other, and 20 cm distance between the plants within rows (Omidbaigi, 2002). The distances between the main and sub-plots and the distance between the 183

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Table 1 Physical and chemical properties of experimental field soil. Depth

Texture

pH −1

(cm) 0–30

EC (dS m

Loam Silty

)

2.0

TNV

O.C

N

32

0.91

0.09

Mn

Zn

Cu

8

196

3.4

3.1

2.9

2.6

model), whereas the amount of Fe, Zn, Cu, and Mn was determined using a Shimadzu-AA 6400 atomic absorption device (Madison, 1971). 2.4. Measurement of physiological and vegetative traits Physiological and vegetative characteristics were measured for two harvest time (in November 2016 and September 2017) at the 50% flowering stage. Leaf relative water content (RWC) was calculated in plants as follows: (Fresh weight − Dry weight)/(Turgid weight − Dry weight) × 100 (Weatherley, 1950) and Ion leakage were determined as described by Sairam et al. (2009). To determine the plant height, 10 plants in the middle line of each plot were randomly selected and, after the necessary measurements, their mean values were calculated. For biological yield and leaf area index, plants in an area equal to one square meter were selected and cut from a height of 15 cm from the ground (Omidbaigi, 2002). Leaf area was measured by a leaf area meter (CI-202, USA model), and Leaf area index was calculated by dividing the leaf area on the ground surface occupied by it (Arshad et al., 2016). 2.5. Statistical analysis Analysis of data variances was performed using SAS software version 9.1. Bartlett test was performed on all studied traits. When the variance error of traits in two consecutive years were pairwise homogeneous, data was performed in a compound analysis. If the interaction was significant, slicing and mean comparison was done using the Duncan's multiple range test. Figures were also drawn using the Excel software. The tables of variance analysis were not shown in the manuscript but were publicized in supplementary data. It should be noted that the results of mean comparison were only presented for the traits that the effect of experimental factors on them was statistically significant. If interactions were significant, means comparison of interactions were merely described in results.

2.2. Moisture treatments application All plots were uniformly irrigated after transplanting. The second and third irrigation was performed based on 25% moisture depletion treatment for all treatments. After the third irrigation, the exertion of irrigation treatments was initiated. Irrigation treatments were applied based on the soil water depletion at a depth of root development. The application of irrigation treatments was made by the weighing method, through repeated soil sampling from the depth of root development in the middle of each plot. Drip irrigation was carried out by an electric pump and tape. The crop water requirement (Ig) was calculated according to equation (1) (Sánchez et al., 1998).

(θfc − θpwp) × t × ρ × D × A100 Ea

Fe

mg kg

blocks were 2, 1 and 2 m respectively. Application of biological and chemical fertilizers was done based on the utilized treatments after preparing the ground for planting. The bacterial suspension of PFB (P169 strain) was prepared as discussed by Burd et al. (2000). The roots of seedlings were soaked in bacterial suspension for 20 min and then transplanted on the field. In addition to the initial inoculation of seedlings, 5 ml of inoculum was injected into the soil of rhizosphere at a depth of 5 cm (Ghorchiani et al., 2013). The spores of AMF used in this study were Rhizophagus irregularis. The inoculum was supplied by Soil Biology Research Division, the Institute of Soil and Water Research, Tehran, Iran, propagated on sorghum root environment. The resulting inoculum is a mixture of spores, hyphae, inoculated roots of sorghum, and sand (Schenck and Perez, 1990). The inoculum contained 120 spores per gram of soil. AMF treatments received 5 g of fresh inoculum per plant at the time of planting. To supply 100% of plant P requirement from a chemical source, 100 kg ha−1 of triple superphosphate (Berti et al., 2001; Omidbaigi, 2002) was used. For plant N requirement urea fertilizer was applied at the total rate of 150 kg ha−1 in three equal splits during the plant's six-leaf stage, stemming, and flowering (Omidbaigi, 2002). Similarly, 100 kg ha−1 of potassium sulfate was uniformly placed under the lines of planting to fulfill plant potassium requirement. Similar amounts of chemical and biological fertilizers were added to the soil of plants root after the first harvest.

Ig =

K −1

(%) 7.8

P

3. Results 3.1. Leaf and root N, and P content

(1) The effect of irrigation management and phosphorus sources on N of leaf and root E. purpurea was significant, the increasing intensity of drought stress caused a decrease in the N of the plant (Table 2), meaning the amount of N reduction in irrigation regime of 75% compared to 25% moisture depletion was 22.28% and 29.30%, respectively. On the other hand, the highest N (2.21% and 2.01%, respectively) was found in the combination of P fertilizer with AMF, which of course had no significant difference with the individual inoculation of AMF (Table 2). While leaf P was only affected by P sources, the effects of irrigation regimes, P sources, and harvest time on root P were significant (Table 2). The increasing intensity of drought stress caused a decrease in root P content (Table 2). The amount of root P in irrigation regimes of 25 and 50% moisture depletion had no major differences (Table 2). The highest amount of root P (0.21%) was found in complete supply of plant P by chemical fertilizer. The treatment of P chemical fertilizer with AMF and PFB significantly increased root P by 15.8% and 22.2%, respectively, as compared to the inoculation only of these two biologic fertilizers. The highest content of leaf P was obtained from the

where θfc and θpwp are the soil moisture content at field capacity and wilting point respectively, t is soil moisture depletion percentage, ρ is a soil bulk density, D is the root development depth, A is plot area, and Ea is irrigation water efficiency, which was considered an average of 90% (Tafteh and Sepaskhah, 2012). 2.3. Measurement of nutrients concentration Nutrient content in the plant leaf and root were measured for two harvest time (in November 2016 and September 2017) at the 50% flowering stage. Plant N were measured using a Kjeldahl method and equipment (device model of V40) (Lang, 1958). In order to measure the root nutrients, in each harvest a planting line was considered for root analysis. Measurement of P, Fe, zinc (Zn), copper (Cu) and manganese (Mn) was carried out in the extract obtained from the dissolution of plant ash in 2 N of hydrochloric acid. P concentration was determined using the vanadium phosphomolybdate colorimetric method at the wavelength of 420 nm by a spectrophotometer device (Vis 2100 184

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Table 2 Effect of irrigation regimes and P sources on leaf N and Cu and root N, P and Fe in Echinacea purpurea.

Table 3 Effect of irrigation regimes and P sources on relative water content (RWC), plant height, leaf number and leaf area index (LAI) of Echinacea purpurea.

Irrigation regimes (Soil moisture depletion)

Leaf N (%)

Root N (%)

Root P (%)

Leaf Cu (mg kg−1)

Root Fe (mg kg−1)

Irrigation regimes (Soil moisture depletion)

RWC (%)

Plant height (cm)

Leaf number (number plant−1)

LAI

25% 50% 75% P Sources 100% P 50% P + AMF AMF 50% P + PFB PFB Control

2.28 a 2.03 b 1.76 c

2.15 a 1.84 ab 1.52 b

0.18 a 0.17 a 0.14 b

17.18 a 16.52 a 13.68 b

107.52 a 96.03 ab 85.83 b

75.58 a 64.13 b 52.44 c

57.38 a 50.58 b 36.75 c

58.00 a 49.69 b 43.63 b

2.55 a 2.14 ab 1.57 b

1.95 b 2.21 a 2.19 a 2.02 b 2.01 b 1.76 c

1.69 c 2.01 a 2.01 a 1.93 ab 1.82 b 1.56 d

0.21 a 0.19 b 0.16 d 0.18 c 0.14 e 0.11 f

14.43 b 16.88 a 17.03 a 16.31 a 16.67 a 13.45 b

88.12 b 103.59 a 108.41 a 98.75 a 98.85 a 81.02 b

25% 50% 75% P Sources 100% P 50% P + AMF AMF 50% P + PFB PFB Control

63.08 bc 69.66 a 66.60 ab 64.40 abc 62.53 bc 58.02 c

48.94 b 54.61 a 52.55 ab 49.16 b 44.55 c 39.61 d

53.44 ab 58.16 a 53.00 ab 50.66 bc 46.00 cd 41.38 d

2.28 a 2.32 a 2.24 ab 2.06 bc 1.88 cd 1.75 d

Means in each column followed by the same letters have no significant difference on the basis of Duncan's multiple range test at 5% error probability. (AMF: Mycorrhizal arbuscular fungus; PFB: Pseudomonas fluorescens bacterium).

Means in each column and treatment followed by the same letters have no significant difference on the basis of Duncan's multiple range test at 5% error probability. (LAI: Leaf area index; RWC: Relative water content; AMF: Mycorrhizal arbuscular fungus; PFB: Pseudomonas fluorescens bacterium).

combination of P fertilizer with AMF, which of course had no considerable difference in the utilization of triple superphosphate (Fig. 2). Also, the P of root in the second harvest was higher than in the first harvest (Table 4).

Table 4 Effect of harvest time on root P, leaf Fe, ion leakage, leaf number and leaf area index (LAI) of Echinacea purpurea. Harvest time

Root P (%)

Leaf Fe (mg kg−1)

Ion leakage (%)

Leaf number plant−1

LAI

1 (2016) 2 (2017)

0.15 b 0.18 a

136.72 a 118.30 b

56.02 a 46.35 b

46.35 b 54.53 a

1.96 b 2.21 a

3.2. Leaf and root Cu, Mn, Zn, and Fe content The increasing intensity of drought stress caused a decrease in leaf Cu (Table 2). The highest amount of Cu in leaf was observed in AMF treatment which was 21.02% higher than control, which had no significant difference in comparison with the combined use of chemical P fertilizer with PFB and AMF or the only inoculation of PFB (Table 2). In the combined use of chemical P fertilizer with AMF, the amount of root Cu was respectively increased by 14.5 and 20.3% as compared to 100% P requirement from chemical source and control test (Fig. 2). The least leaf Mn content was observed in 75% moisture depletion regime which showed a reduction of 26.0% in comparison to 25% moisture depletion (Fig. 3). The interaction of P sources and harvest time demonstrated that the highest amount of Mn was observed in AMF treatment in both harvests, which had no significant difference with combined use of chemical P fertilizer and AMF, except for root Mn. In the second harvest, the amount of leaf and root Mn increased by 35.3 and 28.5%, respectively, as compared to the control (Table 5). Root Fe content in 75% soil moisture depletion was decreased by 20% rather than 25% moisture depletion (Table 2). The highest amount of root Fe (108.4 mg kg−1) was observed at AMF treatment, which observed a 25.3 and 18.7% increase compared to control and 100% chemical P requirement, respectively. This had no significant difference in comparison with the combined use of chemical P fertilizer with PFB, and AMF or the individual inoculation of PFB (Table 2). Additionally,

Means in each column followed by the same letters have no significant difference on the basis of Duncan's multiple range test at 5% error probability. (LAI: Leaf area index).

the Fe of leaf in the first harvest was higher than the second harvest time (Table 4). With increasing intensity of drought stress, and in each irrigation regime, AMF, and PFB could improve the leaf Fe, but the effect of AMF was more conspicuous (Table 6). The effect of irrigation regimes, P sources, and harvest time on the amount of leaf Zn, along with the effect of P sources and harvest time on the amount of root Zn, was significant. Also, the amount of leaf and root Zn was influenced by the interaction of P sources and harvest time. The increasing intensity of drought stress caused a decrease in leaf Zn (Fig. 3). In the first harvest, the highest amount of leaf and root Zn (38.6 and 28.5 mg kg−1 respectively) were observed in PFB treatment, which had no significant difference in comparison with the combined use of chemical P fertilizer with PFB (Table 5). A similar trend was observed in the second harvest, such that in PFB treatment, Zn was increased by 30.4%, and 31.9% respectively (Table 5).

Fig. 2. Effect of different sources of phosphorus on leaf phosphorous and root copper of Echinacea purpurea. The same letters on each column have no significant difference on the basis of Duncan's multiple range test at 5% error probability. (Error bars represent ± standard error; N = 18). 185

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Fig. 3. Effect of irrigation regimes on manganese and zinc leaf of Echinacea purpurea. Soil moisture depletion: (SMD). The same letters on each column have no significant difference on the basis of Duncan's multiple range test at 5% error probability. (Error bars represent ± standard error; N = 36). Table 5 Mean comparison of harvest time and P sources interactions on leaf and root Mn, leaf and root Zn, and biological yield of Echinacea purpurea. Harvest time

P Sources

Leaf Mn (mg kg−1)

Root Mn (mg kg−1)

Leaf Zn (mg kg−1)

Root Zn (mg kg−1)

Biological yield (g m−1)

1 (2016)

100% P 50% P + AMF AMF 50% P + PFB PFB Control 100% P 50% P + AMF AMF 50% P + PFB PFB Control

34.96 c 46.77 ab

13.80 c 17.40 ab

28.24 e 34.43 c

284.3 a 283.0 a

50.44 a 43.15 b

18.08 a 15.18 bc

35.67 bc 37.41 ab

19.26 d 23.05 bc 23.87 b 27.20 a

42.84 b 34.22 c 39.93 c 57.88 a

14.41 c 12.78 c 31.75 d 40.25 b

38.64 a 31.72 d 31.77 e 36.72 cd

28.50 a 21.89 c 22.34 e 26.60 cd

254.3 bc 242.8 c 403.2 b 429.2 a

67.55 a 54.76 b

47.56 a 37.23 bc

39.77 c 45.22 b

29.33 c 33.31 b

381.7 bc 372.7 c

59.68 b 43.67 c

40.49 b 33.96 cd

48.52 a 33.77 de

36.38 a 24.78 de

339.0 d 308.1 e

2 (2017)

Table 6 Mean comparison of interaction between irrigation regimes and P sources on leaf Fe and ion leakage of Echinacea purpurea. Irrigation regimes (Soil moisture depletion)

P Sources

25%

100% P 50% P + AMF 50% P + PFB Control 100% P 50% P + AMF 50% P + PFB Control 100% P 50% P + AMF 50% P + PFB Control

263.5 abc 270.6 ab 50%

75%

Means slicing by P sources in each harvest time followed by the same letters have no significant difference on the basis of Duncan's multiple range test at 5% error probability. (AMF: Mycorrhizal arbuscular fungus; PFB: Pseudomonas fluorescens bacterium).

AMF PFB

AMF PFB

AMF PFB

Leaf Fe (mg kg−1)

Ion leakage (%)

127.61 b 146.00 a 148.41 a 140.18 a 148.57 a 123.18 b 107.32 b 141.66 a 148.25 a 130.50 a 131.68 a 108.05 b 102.28 c 134.75 a 139.83 a 112.90 b 112.26 b 91.76 d

35.51 a 37.26 a 37.98 a 38.04 a 39.88 a 39.70 a 52.39 a 46.50 a 47.23 a 47.67 a 49.27 a 53.62 a 75.84 a 56.56 c 56.23 c 63.21 b 66.19 b 78.27 a

Means slicing by P sources in each irrigation regime in followed by the same letters have no significant difference on the basis of Duncan's multiple range test at 5% error probability. (AMF: Mycorrhizal arbuscular fungus; PFB: Pseudomonas fluorescens bacterium).

3.3. Leaf relative water content and ion leakage The increasing intensity of drought stress caused a decrease in leaf relative water content (Table 3). The highest amount of leaf relative water content (69.7%) was obtained in the combined use of chemical P fertilizer and AMF, which did not show any significant difference in comparison with the chemical P fertilizer combination with PFB and solitary inoculation of AMF. The lowest leaf relative water content (58.02%) was observed in control treatment (Table 3). The ion leakage in the second harvest demonstrated a decrease compared to the first harvest (Table 4). Water stress intensification caused an increase in leaf ion leakage. Although in 25 and 50% soil moisture depletion, ion leakage had no significant variation among the applied fertilizer treatments (Table 6), in 75% soil moisture depletion, ion leakage was decreased by inoculation of AMF or its combination with chemical P fertilizer. In addition, PFB after AMF had a high ability to reduce leaf ion leakage compared to control (Table 6).

Fig. 4. Effect of irrigation regimes on biological yield of Echinacea purpurea in each harvest time. The same letters on each column have no significant difference on the basis of Duncan's multiple range test at 5% error probability. (Error bars represent ± standard error; N = 18).

3.4. Vegetative traits The biological yield was affected by irrigation regimes, P sources, 186

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and Abidi, 2010). The present study demonstrated that E. purpurea inoculation with AMF and PFB reduced the adverse effects of drought stress. The higher leaf relative water content and reduction of ion leakage, in plants inoculated with AMF and PFB, compared to non-inoculated ones confirms this claim. It is also probable that better uptake of nutrients in inoculated plants will provide better conditions for water absorption. By improving the root development and providing a more suitable absorption surface for nutrients, P fertilizer resources has led to an increase in the relative water content and the reduction of leaf ion leakage in E. purpurea. The water and nutrients limitation can lead to the production of reactive oxygen species, which may cause cellular damage by influencing plant physiological processes (Zgallai et al., 2005). Nutrients are capable of preserving carbon dioxide fixation, photosynthesis, and protecting chloroplast under stressed conditions (Han and Lee, 2005). Through the control of stomatal conductance and the improvement of osmotic adjustment, the inoculation of plants with some microorganisms maintains the leaf relative water content under drought stress. This strategy can be considered as a successful method to improve plant growth under drought environments (Ortiz et al., 2015). Other researchers have also reported similar results in improving water and nutrient absorption in plants inoculated with AMF (Smith and Read, 2010). Zhao et al. (2015) stated that plant inoculation with AMF improves root development and progression, which, in consequence, is efficient in water absorption, transfer, and conductance in the leaves. It seems that the reduction of ion leakage in second harvest than the first one can be due to the difference in weather conditions during the implementation of the experiment or the adaptation of the plant to the environmental conditions. In the present study, AMF and PFB proved more effectual in helping inoculated plants to increase plant nutrients, improve leaf relative water content, and decrease the ion leakage. These compounding effects caused an increase in growth indices. Also more biological yield in the second harvest can be attributed to the improvement of soil rhizosphere conditions by AMF. Bio-fertilizers have also been reported to develop the plant growth through the absorption of nutrients and increase of photosynthesis productions (Han and Lee, 2005). Also, it has also been reported that bio-fertilizers can affect plant growth and development through the production of herbal stimulating hormones such as auxin, cytokinin, and gibberellin (Lucy et al., 2004). Han and Lee (2005) reported that biological compounds could increase photosynthetic activity and carbon dioxide fixation through morphological changes such as an increase in leaf area index. On the other words, it seems that an increase in the leaf area index, leaf number and biological yield in second harvest than the first one is due to the root development and the increased plant rootstocks.

harvest time and the interaction of harvest time with irrigation regimes and P sources. An increase in the intensity of drought stress caused a major decrease in biological yield (Fig. 4). In first and second harvest the lowest biological yield was observed at 75% soil moisture depletion, which respectively explained a decrease of 28.4%, and 30.1% as compared with 25% soil moisture depletion (Fig. 4). On the other hand, in the first harvest, the highest biological yield (284.3 gm−2) was observed in 100% chemical P treatment, which showed a significant difference for the inoculation of PFB and control (Table 5). In the second harvest, the highest biological yield was observed in the combined use of chemical P with AMF (Table 5). In both harvests, biological yield was improved by application of chemical P with PFB as compared to control (Table 5). The effect of irrigation regimes and P sources on plant height was significant. The lowest plant height was observed in 75% moisture depletion, which decreased by 35.9% compared with 25% moisture depletion (Table 3). Application of chemical P with AMF resulted in the highest plant height, which had no significant difference with the only inoculation of AMF (Table 3). Chemical P, application of chemical P with PFB, and solitary inoculation of PFB increased plant height when compared with control (Table 3). The lowest leaf number and leaf area index were observed at 75% moisture depletion, which in comparison with 25% moisture depletion, reduced 24.8% and 38.4%, respectively. The highest leaf number and leaf area index of E. purpurea were obtained where the chemical P was inoculated with AMF, but this treatment had no significant difference in comparison with single application of chemical P and AMF. Also, PFB could increase leaf number and leaf area index compared to control (Table 3). Leaf number and leaf area index in the second harvest compared to the first harvest increased 15.0 and 11.3%, respectively (Table 4). 4. Discussion Inoculation of E. purpurea with AMF and PFB improved the uptake of P. Kim et al. (1998) reported that phosphate solubilizing bacteria usually convert insoluble organic and inorganic forms of P to absorbable ones through the secretion of phosphatase enzymes and organic acids. In addition, the vast hyphal network of AMF efficiently expands into the root zone of the plant; led it right to the entry plant-available phosphorus (Elbon and Whalen, 2015). Application of triple superphosphate with P solubilizing microorganisms increased the availability of soil P, P uptake, and plant growth (Panhwar et al., 2012). The higher concentration of root P in second harvest than the first one can be attributed to the increased plant strength in second harvest through the increase of rootstocks, further root development and thus the better absorption of P. AMF, and to a lesser extent PFB, had a good ability to increase the N in the leaves and roots of E. purpurea. Moreover, application of P fertilizer, in comparison with control, significantly increased plant N. Literature review shows that the increase in N uptake in AMF inoculated plants can be attributed to the effect of P (Azcón et al., 2003). Smith and Read (2010) reported that plant N increased by P application. It can be stated that P fertilizers have resulted in the efficient uptake of nutrients, such as N, through the improvement of root growth and absorption surface. AMF, and to a lesser extent PFB, could improve the Cu, Mn, Zn, and Fe in E. purpurea. In fact, AMF and PFB have increased the transfer and uptake of these elements through the secretion of siderophores and chelation (Caris et al., 1998; Chang and Yang, 2009). PFB could dissolve the low-soluble compounds, such as zinc, by producing organic acids and reducing soil acidity (Saravanan et al., 2007). But, the application of P fertilizer by an antagonistic effect reduced the Mn and Zn of the plant. Literature review shows that increased P in soil decreases the solubility of micronutrients and reduces the concentration of these elements in potato and wheat (Hopkins and Ellsworth, 2003; Mishra

5. Conclusion In general, the results proved that an increase in drought intensity hurt vegetative traits, nutrient concentration, leaf relative water content, and ion leakage of E. purpurea. Echinacea purpurea inoculation with AMF and PFB increased the concentration of N, P, Cu, Mn, Fe and Zn in plant and alleviated the adverse effects of drought stress. Among AMF and PFB used in this study, AMF had a more conspicuous effect on the concentration of N, Cu, Mn, and Fe, but PFB was more effective in increasing leaf and root Zn than AMF. Although, 100% chemical P treatment could increase P in the root, the combination of P fertilizer and AMF transferred more P from root to leaf and resulted in higher P in the leaf. Sole utilization of AMF and PFB, or their combination with P fertilizer, were efficient in increasing the relative water content and reducing ion leakage and thus, increased plant height, leaf area index, and biological yield. Finally, the results of this study confirmed the successful coexistence of bio-fertilizers with E. purpurea in increasing the concentration of nutrients, promoting water absorption, improving 187

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growth, and reducing the adverse effects of drought stress.

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