Biochemical response and interactions between arbuscular mycorrhizal fungi and plant growth promoting rhizobacteria during establishment and stimulating growth of Arizona cypress (Cupressus arizonica G.) under drought stress

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Biochemical response and interactions between arbuscular mycorrhizal fungi and plant growth promoting rhizobacteria during establishment and stimulating growth of Arizona cypress (Cupressus arizonica G.) under drought stress Hamed Aalipoura,*, Ali Nikbakhta, Nematollah Etemadia, Farhad Rejalib, Mohsen Soleimanic a b c

Department of Horticulture, College of Agriculture, Isfahan University of Technology, 84156-83111, Isfahan, Iran Soil and Water Research Institute, P.O. Box: 31758-311, Imam Khomeini Blv., Meshkindasht, Karaj, Iran Department of Natural Resources, Isfahan University of Technology, 8415683111, Isfahan, Iran

A R T I C LE I N FO

A B S T R A C T

Keywords: Antioxidant enzymes Coniferous tree Water stress Funneliformis mosseae Pseudomonas fluorescens Rhizophagus intraradices

Arizona cypress is a major urban landscape tree many of which have declined in the recent years. Although the cause for this decline is complicated, drought is generally thought to be a leading factor. This study was designed to investigate the effects of artificial inoculation of seedlings of a major urban forest tree, Arizona cypress (Cupressus arizonica Green) with beneficial microorganisms under the water stress. We conducted this research with three factors comprising arbuscular mycorrhizal fungi (AMF) inoculation with Rhizophagus irregularis or Funneliformis mosseae or a combination of the both fungi species (CF), bacterial inoculation with Pseudomonas fluorescens (PF) and non-inoculated controls, and two levels of irrigation including well-watered (WW) and severe-water deficiency (SWD) using three replications of each treatment. The results clearly showed that SWD adversely affected root colonization, morphological parameters, relative water content, and soluble carbohydrate content. However, antioxidant enzyme contents (i.e. catalase, superoxide dismutase, glutathione peroxidase, ascorbate peroxidase), hydrogen peroxide, malondialdehyde, and proline increased as a result of water stress. Moreover, AMF-inoculated plants grew better than non-inoculated plants under SWD conditions. Dualinoculated plants with CF and PF inoculation accumulated more ascorbate peroxidase and glutathione peroxidase than plants with merely PF or AMF inoculation under SWD conditions. Inoculated plants significantly decreased the water deficit-induced hydrogen peroxide and malondialdehyde in Arizona cypress leaves. In conclusion, the symbiotic association between PF and AMF can alleviate water-deficit damage and improve water stress tolerance in the Arizona cypress.

1. Introduction Arizona cypress (Cupressus arizonica G.) is a coniferous tree native to the southwestern United States, and in Mexico. It is planted as an ornamental tree and can be highly appropriate for reforestation. Because Arizona cypress can tolerate hard conditions and harsh sites, it has spread to vast areas of the world (Ismail et al., 2014). The species was also introduced in Iran in 1954 firstly as an ornamental tree and then for reforestation programs (Emami et al., 2010). However, tree decline has been a serious shortcoming of this precious tree in many parts of Iran. Tree decline is defined as a general reduction in the vigor, growth rate, and productivity of tree, leading eventually to the death of the tree (Zhu and Li, 2007). The reasons behind the decline are not well

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understood, although drought is generally considered to be a leading factor (Amoroso et al., 2015). Drought is one of the main environmental factors that adversely affect plant growth and biomass production. The common symptoms observed in plants after encountering water stress include a hindrance to growth, a reduction in the rate of photosynthesis, an acceleration in the aging of the leaves, and etc. (Sohrabi et al., 2012). Water stress tolerance includes multiple traits and is regulated by numerous mechanisms (Hadiarto and Tran, 2010). Plants under water stresses accumulate proline as an osmolyte in their tissues, contributing to increasing the osmotic pressure and improving the water uptake (Cardoso Filho et al., 2017). Also, water stress can decrease the amount of the total soluble sugar in plants, the outcome of which is a reduction in the photosynthesis rate (Goicoechea et al., 2005).

Corresponding author. E-mail address: [email protected] (H. Aalipour).

https://doi.org/10.1016/j.scienta.2019.108923 Received 5 June 2018; Received in revised form 21 August 2019; Accepted 5 October 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Hamed Aalipour, et al., Scientia Horticulturae, https://doi.org/10.1016/j.scienta.2019.108923

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2.2. Experimental design and treatments

Moreover, exposure to the drought stress is often associated with an increase in the levels of reactive oxygen species (ROS) (Bray, 2004). Some of these ROS act as free radicals in the cell. Free radicals initiate with destructive processes, such as chlorophyll decomposition, lipid peroxidation, or protein oxidation, in the event of inefficiency in the plants’ defense system. The antioxidant defense system consists of a number of hydrophilic (ascorbate and glutathione) and lipophilic (alpha-tocopherol) antioxidants and a number of enzymes (such as catalase, superoxide dismutase, and peroxidase). These enzymes can neutralize the effects of ROS (Tausz et al., 2001; Amiri et al., 2015). Hence, discerning the mechanisms of each plant’s tolerance to drought conditions is an extremely important issue in environmental and survival studies (Bray, 2004). Meanwhile, efforts have been made to develop novel technics to overcome water-deficit stresses. The employment of arbuscular mycorrhizal fungi (AMF) is one of the most promising tools to boost water uptake, to improve soil properties, and to enhance phosphorus nutrition (Auge et al., 2011; Amiri et al., 2015). AMF can positively influence plants through symbiosis with plant roots. This mutual symbiosis is found in more than 80% of vascular plants (Smith and Read, 2008). Water transfer in plants can be affected by changes in environmental conditions, such as mycorrhizal symbiosis, which can increase the movement of water by physiological and biochemical changes in the root system (Siemens and Zwiazek, 2004). Also, several studies have reported that the application of bacteria and fungi have beneficial effects on plant growth and health, which can be employed in soil amelioration programs (Rincon et al., 2005). Among beneficial soil bacteria, one group is distinctive, namely the plant growth-promoting rhizobacterium (PGPR) that can facilitate plant growth and development directly or indirectly by providing mineral nutrients and phytohormones (Chanway, 1997). PGPR, including Pseudomonas fluorescens, has been applied to prevent phytopathogens (biocontrol) and certainly has great potential for the growth of forest trees (Chanway, 1997; Backer et al., 2018). In fact, it has been demonstrated that the effects of AMF on tree roots can be enhanced using P. fluorescens (Aalipour et al., 2019). However, this bacterial association may have both positive and negative impacts (Rincon et al., 2005). According to the information at hand, knowledge regarding the effects of inoculated beneficial fungi and bacteria on water-stress responses of Arizona cypress is highly limited. Finding an appropriate method to reduce drought stresses is one of the major concerns of agricultural researchers. The objective of this experiment was to study the effects of the application of P. fluorescens and mycorrhizal fungi on the Arizona Cypress seedlings inducing resistance under water stress. Moreover, we were trying to shed a light on the biochemical responses and the mechanisms behind the induced dual association.

This study was carried out as a factorial experiment based on a completely randomized design (CRD), with three factors, including AMF inoculation, Pseudomonas fluorescens inoculation, and irrigation levels with three replications of each treatment (each replication was consisted of 3 plants). As far as AMF treatments are concerned, it consisted of non-inoculated control plants (NM), plants inoculated with Rhizophagus irregularis (Ri) or Funneliformis mosseae (Fm), and a combination of the both fungi species (CF). Bacterial inoculation comprised the inoculation with P. fluorescens (PF) strain P12 or non- P. fluorescens (NPF) seedlings served as control. For the irrigation levels, we used either 100% or 50% field capacity, denoted as well watered (WW) or severe water deficit (SWD) treatment, respectively. Arizona cypress seeds were sown into the pots. The seeds were obtained from a maternal base (one tree) in the middle of a cultivated forest in IUT. The forest was a pure stand of Arizona cypress, containing around two thousands of 30-year-old trees. The AMF and P. fluorescens were provided by the National Soil and Water Research Center (Karaj, Iran). Prior to the seedling transplanting, 60 g of the AMF inocula (3000–3600 spores per pot), with a known spore density (approximately 50–60 spores per g), was mixed with the growing media. Also, in order to simulate equal microflora in all NM treatments to AMF treatments, the filtrate of the mycorrhizal inoculum (obtained from both fungi without any spores) was added into the NM treatments. Four milliliters of a bacterial suspension (107 CFU ml−1) was poured on the top of each pot immediately after sowing; while sterile distilled water was poured into the control pots. WW plants were irrigated up to pot capacity. The potential of soil water for scheduling irrigation treatments was monitored using a tensiometer (type Soilmoisture 2710 ARL; Soilmoisture Equipment Corp., USA) at 20-cm depth. As soon as the soil water potential reached −10 kPa, seedlings were watered to the pot capacity. SWD treatment received 50% of the amount of water applied to the WW treatment, and water regimes were applied for 5 months.

2.3. Morphological measurements and AMF colonization After plants grew in the presence of the microorganisms and under different irrigation regimes, we harvested the Arizona cypress seedlings (all seedlings grew well watered during 13 months after sowing, and then water regimes were applied for 5 months before the harvest), and measured morphological parameters. For determination of shoot and root dry weight, we air dried samples, then stored in paper bags before drying them in a forced-air oven at 65 °C to a constant weight before weighing. To determine AMF colonization of roots, we first washed root systems and free of soil:sand mixture before clearing them with KOH (10% w/v) at 95 °C for 1 h. Next, we acidified roots with HCl (1% w/v) for 1 h, then stained them with 0.05 % w/v trypan blue in lactoglycerol (8:1:1 lactic acid, glycerol and water) for 20 min Phillips and Hayman (1970). Finally, we quantified percentage of mycorrhizal colonization of roots using the gridline intersection method of Giovannetti and Mosse (1980).

2. Materials and methods 2.1. Experimental site and soil properties This experiment was conducted at the campus facility of the Department of Horticulture at Isfahan University of Technology (IUT), Isfahan, Iran (32°39′ N, 51°40′ E; 1590 m) during 2015-2017. The plastic pots (dimensions: 33-cm diameter top, 25.5-cm diameter base, and 30-cm pot depth) contained an autoclaved growing mixture (120 °C, 2 h) of soil: sand (2:1, v/v). The soil was autoclaved four weeks before the beginning of the experiment to permit the release of the toxic compound produced during autoclaving. The characteristics of the original soil were a clay loam texture (28% clay, 42% silt, and 30% sand), pH of 7.7, an electrical conductivity of 0.97 dS m−1, and limestone equal to 35.1% (CaCO3). The soil contained 8.27 g kg−1 organic matter, 30 mg kg−1 N (total), 120 mg kg−1 K (exchangeable), 6.2 mg kg−1 P, 8.5 mg kg−1 Fe, 0.7 mg kg−1 Zn, and 96 mg kg−1 Mg (available or extractable).

2.4. Relative water content We determined relative water content (RWC) according to the method developed by Ritchie et al. (1990). About 0.1 g of the leaf sample was cut into smaller pieces and weighed (W1). Then the leaf samples were saturated in 10-mL deionized water for 24 h at 4 °C and weighed to determine the turgid weight (W2). Finally leaf samples were dried at 80 °C for 24 h, and the dry weight was recorded (W3). RWC was determined using Ritchie et al. (1990) formula.

2

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et al., 2000). We measured and calculated hydrogen peroxide (H2O2) using the method proposed by Velikova et al. (2000). Leaf samples (0.2 g FW) were homogenized in 5 mL of 0.1% (w/v) TCA. The extract was centrifuged at 14,000 g for 20 min; 0.5 mL of the supernatant was added to 0.5 mL of 10-mM potassium phosphate buffer with the pH of 7.0, and 1.0 mL of 1-M KI solution. The optical absorption of the supernatant was measured by the spectrophotometer (UV-160A UV–vis Recording Spectrophotometer; Shimadzu, Tokyo, Japan) at 390 nm to determine the H2O2 content (Є = 0.28 μM−1. cm−1). The result was expressed as micromoles per gram of the fresh weight.

2.5. Proline content and total soluble sugars assay Leaf samples (0.2 g) were homogenized in 10 mL of 3% (w/v) sulfosalicylic acid and was centrifuged at 6000 RCF for 20 min at 4 °C, according to the procedure used by Bates et al. (1973) with minor modifications. 1 mL of this homogeny solution reacted with acid-ninhydrin and 1 mL of glacial acetic acid in a tube for 1 h at 100 °C; the reaction was torn up in an ice bath and then extracted with 2 mL of toluene. It was kept at room temperature to stabilize. Finally we measured proline content by a spectrophotometer (UV-160A, Shimadzu, Tokyo, Japan) at 520 nm. We also measured total soluble sugar according to the phenol–sulfuric acid method (Dubios et al., 1956).

2.8. Statistical analysis 2.6. Enzyme activity assay Data were analyzed in terms of normality, and if needed, prior to the analysis, Log-transformation was used to make data conform to normality using Shapiro–Wilk test. For pot means, data were subjected to three-way ANOVA so as to balance the data. Next, where the treatment was statistically significant at P < 0.05, Duncan’s multiple range tests were run to detect the specific differences. SAS statistical software version 9.1 (SAS Institute, Cary, NC, USA) was used to do the statistical analysis.

To measure the specific activity of the antioxidant enzymes, 100 mg of the fresh leaves samples was combined with 1 mL buffer (1% polyvinylpyrrolidone, 0.5% triton X100 and 100 mM K-phosphate buffer (pH 7.0). The homogenate was centrifuged at 15,000 g at 4 °C for 20 min. Then, we assayed the antioxidant enzymes activity or content as follows: Catalase (CAT) activity was calculated using the modified version of the method proposed by Aebi (1984). The given activity was followed by the spectrophotometric detection of H2O2 decomposition at 240 nm. For this purpose, 2.95 mL of the reaction buffer, consisting of a 50 mM K-phosphate buffer (pH 7.0) and 15 mM H2O2, was mixed with 0.05 mL of the enzyme extract. The specific activity of the CAT enzyme was calculated by dividing the amount of CAT activity on the protein extracted by the Bradford method (1976). The results were expressed as the relative enzyme activity per mg of protein. Ascorbate peroxidase (APX) activity was measured using the method proposed by Nakano and Asada with some modofications (1981). 2.95 mL of the reaction buffer (50 mM K-phosphate buffer (pH 7.0), 5 mM ascorbate (AsA), 0.5 mM H2O2, and enzyme extract in a final volume of 0.05 mL was started by the addition of H2O2, and the activity was measured by observing the decrease in the absorbance at 290 nm for 2 min using a spectrophotometer (UV-160A UV–vis Recording Spectrophotometer; Shimadzu, Tokyo, Japan). The activity of glutathione peroxidases (GPX) was assayed based on the method described by Rao et al. (1996) using H2O2 as a substrate. The oxidation of NADPH was recorded at 470 nm for 2 min, and the activity was measured by dividing the amount of GPX activity on the total protein content. The activity of superoxide dismutase (SOD) was measured using the modified version of the method described by Giannopolitis and Ries (1977). Firstly, we added 50 μl of the extracted sample to 3 mL of the reaction buffer (containing 50 mM phosphate buffer (7.8 ppm), 75 nm EDTA, 13 mM methionine, 63 μm nitroblue tetrazolium) and then 1.3 μm riboflavin was added to the solution. The samples were exposed to light for 15 min and their absorbance was measured at 560 nm using the spectrophotometer. 50% color reduction was considered one unit (U) of SOD activity; the activity was expressed as units per mg of the total protein content. The control reaction mixture had no crude enzyme extract, while blank contained the same reaction mixture, but was placed in the dark.

3. Results 3.1. Morphological parameters and root colonization The analysis of variance (Table 1) showed that AMF and irrigation levels significantly affected most of the growth parameters, including plant height, root length, and shoot and root biomass. Plant height and root length were not significantly affected by PF. SWD treatment reduced plant height (23%), root length (49%), root biomass (38%) and shoot biomass (30%) of the Arizona cypress plants compared with WW treatment. The results clearly indicated that inoculated plants by either AMF or PF significantly improved Arizona cypress growth performance (Table 1). Furthermore, plant height (except for Fm species) and root length were higher in mycorrhizal inoculated plants compared to the NM plants under SWD conditions (Table 3). Root colonization significantly decreased under SWD treatment compared to WW treatment (except for Fm species in NPF treatment). It was also increased by the PF activity (by 30%) (Table 1). No root colonization was observed in the NM plants. The root colonization percentage (regardless of the NM treatment) reached the peak in CF (60.4%) in the presence of PF in WW conditions, and the lowest colonization (21.1%) was recorded in the treatment involving Ri species under NPF and SWD conditions (Table 2). 3.2. Relative water content SWD negatively influenced the RWC in the plants (Table 1). RWC significantly (P < 0.05) decreased under SWD condition compared to the WW treatment (by 20%). AMF inoculation resulted in an increase in the RWC in Arizona cypress plants compared to the NM plants (Table 1). Under SWD conditions, RWC increased in all inoculated AMF treatments in comparison with NM plants (Table 3). RWC was not significantly affected by PF (Table 1).

2.7. Malondialdehyde and hydrogen peroxide assays 3.3. Proline content and total soluble sugar In order to determine the content of malondialdehyde (MDA) in the leaves, 0.5 g of leaf tissues was homogenized in 5 mL of 0.1% (w/v) trichloroacetic acid (TCA) for 10 min and then was centrifuged at 12,000 g. 1 mL of conventional solution was mixed with 4 mL of thiobarbituric acid (TBA) (0.5% of TBA in 20%). Then the reaction mixture was placed in a hot bath at 95 °C for 30 min. Finally the mixture was centrifuged at 12,000 g for 15 min and the amount of MDA was subsequently read by the spectrophotometer at 532 and 600 nm (Velikova

Table 1 shows that the proline content significantly increased under SWD condition compared to WW treatment (by 122 %). The table also indicates that single inoculation with the AMF species significantly increased the proline content of Arizona cypress compared with the non-inoculated controls. The proline content increased in all AMF inoculated plants (except for Ri species in NPF treatment) in comparison with the NM ones facing SWD conditions (Table 2). The maximum 3

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Table 1 Effect of irrigation regime, mycorrhizal and bacteria inoculation on arbuscular mycorrhizal (AM) colonization, relative water content (RWC), proline (Pr), Total soluble sugars (TSS), plant height (PH), root length (RL), shoot biomass (SB), root biomass (RB), SOD (superoxide dismutase), CAT (catalase), GPX (glutathione peroxidase), APX (ascorbate peroxidase) activities and MDA (malondialdehyde) and H2O2 (hydrogen peroxide) contents of Arizona cypress seedlings. Treatments Irrigation regime WW SWD AMF status NM Ri Fm CF Bacteria status NPF PF Significance AMF IR PF IR × AMF AMF × PF IR × PF IR × AMF × PF Treatments

Irrigation regime WW SWD AMF status NM Ri Fm CF Bacteria status NPF PF Significance IR AMF PF IR × AMF IR × PF AMF × PF IR × AMF × PF

AM (%)

PH (cm)

RL (cm)

SB (g plant−1)

RB (g plant−1)

RWC (%)

Pr (μmol g−1 FW)

TSS (mg g−FW)

30.1a 21.6b

21.8a 16.9b

24.9a 12.7b

2.33a 1.64b

1.98a 1.29b

86.8a 69.3b

0.223b 0.495a

0.803a 0.587b

0c 33.3b 31.4b 38.7a

17.1c 19.3b 18.8b 22.3a

16.8b 17.4b 20.3a 20.6a

1.56c 2.08ab 2.05b 2.24a

1.22c 1.71b 1.69b 1.91a

75.9b 78.6a 78.9a 78.8a

0.262c 0.326b 0.401a 0.447a

0.662b 0.682b 0.687b 0.748a

19.4b 25.3a

19.5a 19.3a

19.3a 18.3a

1.78b 2.18a

1.47b 1.79a

77.7a 77.4a

0.355a 0.362a

0.780a 0.610b

** ** * ** ** * **

** ** ns ** ns ns ns

** ** ns ** ns ns ns

** ** ** ** ** ** **

** ** ** ** ** * **

* ** ns ** * ns ns

** ** ns ns ns ns *

** ** * * ns ns ns

CAT

GPX (μmol/min/mg protein)

APX

SOD (U/mg protein)

MDA (μmol/ g−1 FW)

H2O2

1.47b 1.67a

0.092b 0.100a

9.96b 49.59a

29.0b 89.5a

0.180b 0.302a

6.57b 20.9a

1.53a 1.61a 1.50a 3.21a

0.068c 0.083b 0.080b 0.152a

25.3c 31.6ab 27.7bc 34.3a

38.3b 64.8a 64.2a 69.7a

0.290a 0.228b 0.217b 0.225b

24. 2a 10.0b 9.69b 11.1b

1.46b 1.68a

0.73b 0.118a

27.5b 31. 9a

50.0b 68.4a

0.257a 0.224b

13.2a 14.3a

* ns * ns ns ns ns

** ** ** ** ** ** **

** ** ** ** ns * **

** ** ** ** ** ns ns

** ** * ** ns ns **

** * ns ** * ns ns

Means (n = 9) followed by similar letters within each column under each specific treatment do not express significant diffrences at P < 0.05 according to LSD test. IR Irrigation regime, WW 100% field capacity, SWD 50% field capacity, AMF arbuscular mycorrhizal fungi, Ri Rhizophagus irregularis, Fm Funneliformis mosseae, CF combination of both fungi, NM, non-mycorrhizal, PF Pseudomonas fluorescens, NPF non- P. fluorescens. ns not significant *P < 0.05, **P < 0.01.

species) caused a significant increase in the CAT activity in the shoots of Arizona cypress compared with the control plants (Table 1). Also, APX activity demonstrated a significant increase in the PF plants compared to the NPF plants (by 16%). Under SWD conditions, co-inoculation with AMF and PF or individul inoculation with AMF resulted in a significant increase in the APX activity compared with the control plants (Table 2). The APX and GPX activities almost showed the same trend (Table 1). SWD conditions promoted the GPX activity compared to WW treatment (by 398%). The GPX activity demonstrated a significant increase in the PF plants compared to the NPF plants (by 38%). The dualinoculated plants with the CF and PF under SWD conditions showed the highest GPX activity (Table 2). CF was more successful in increasing the GPX activity under SWD conditions regardless of PF treatment. The SOD activity significantly (P < 0.01) increased under SWD conditions compared to the WW conditions (by 208%). Also, PF inoculated plants demonstrated a higher SOD activity (by 37%) than noninoculated ones. The same trend was observed in case of AMF inoculated plants in comparison to the NM plants (Table 1). The maximum SOD activity was recorded in the plants inoculated with CF under SWD conditions (Table 3).

proline content was obtained in plants inoculated with the CF in the absence of PF under SWD conditions (Table 2). As far as total soluble sugar (TSS) is concerned, the results indicates a significantl (P < 0.05) decrease under induced by SWD when compared to well-watered plants by 27%. Inoculation with CF resulted in an increase in the TSS compared to the control plants. Our results showed that single inoculation with PF reduced the TSS (by 22%) compared with the NPF plants (Table 1). The highest TSS was obtained in the CF plants under WW conditions, and in contrast, the least amount was obtained in the control plants under SWD conditions (Table 3). 3.4. Enzyme activity and content An elevated CAT activity observed in Arizona cypress plants under SWD conditions compared to the WW plants (Table 1). Also, single inoculation with PF enhanced the CAT activity in comparison with the NPF plants (by13%). No significant difference was observed between AMF plants and NM plants (Table 1). Similarly the APX activity increased under SWD treatment compared to WW treatment (by 9%). AMF inoculations (except for Fm 4

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Table 2 Interaction effects of irrigation regime × AMF × bacteria on arbuscular mycorrhizal (AM) colonization, shoot biomass (SB), root biomass (RB), proline (Pr), glutathione peroxidase (GPX), ascorbate peroxidase (APX) activities and MDA (malondialdehyde) content of Arizona cypress seedlings. RB (g plant−1)

Irrigation regime

AMF status

Bacteria status

AM (%)

WW

NM Ri Fm CF NM Gi Gm CF

PF

0± 48.9 46.8 60.4 0± 26.3 25.2 33.3

0.0h ± 3b ± 4.8b ± 4.4a 0.0h ± 2.5ef ± 1.6fg ± 3.2cd

1.59 2.13 2.33 2.78 1.85 1.76 1.59 1.80

NM Ri Fm CF NM Ri Fm Cf

PF

0± 36.6 30.8 34.7 0± 21.1 22.9 26.4

0.0h ± 2.4c ± 2.8de ± 1.7de 0.0h ± 2.4g ± 4.8fg ± 4.2ef

0.733 ± 0.07f 1.61 ± 0.10de 1.37 ± 0.15e 1.74 ± 0.06d 0.647 ± 0.06f 1.34 ± 0.07e 1.51 ± 0.14de 1.33 ± 0.21e

SWD

NPF

NPF

± ± ± ± ± ± ± ±

0.11de 0.07bc 0.48b 0.49a 0.12cd 0.15d 0.25de 0.11cd

SB (g plant−1)

Pr (μmol g−1 FW)

GPX (μmol/min/mg protein)

APX (μmol/min/ mg protein)

0.21c−e 0.27b 0.2b 0.39a 0.22c 0.21cd 0.22c−f 0.19c−f

0.321 0.225 0.199 0.178 0.241 0.202 0.306 0.111

± ± ± ± ± ± ± ±

0.02de 0.01fg 0.02gh 0.02gh 0.03e−g 0.04gh 0.02ef 0.01h

0.290 ± 0.01j 0.767 ± 0.02f−h 0.830 ± 0.01e−g 1.54 ± 0.03c 0.292 ± 0.0j 0.677 ± 0.02g−i 0.870 ± 0.0ef 1.00 ± 0.01de

5± 6.33 5.67 9.33 6.33 8.33 7.33 6.66

1.5g 2.5g 1.5g 2.5g 3g 2g 2.1g

0.180 0.143 0.163 0.160 0.173 0.193 0.233 0.190

± ± ± ± ± ± ± ±

0.01e−g 0.05g 0.01fg 0.02fg 0.02fg 0.02c−g 0.02b−g 0.02d−g

1.16 ± 0.14g 1.95 ± 0.19c−f 1.73 ± 0.15e−f 2.14 ± 0.22c 0.917 ± 0.09g 1.74 ± 0.17d−f 1.87 ± 0.17c−f 1.62 ± 0.18f

0.323 0.475 0.560 0.561 0.436 0.401 0.540 0.665

± ± ± ± ± ± ± ±

0.02de 0.02bc 0.01b 0.01b 0.06c 0.02cd 0.01b 0.01a

1.06 ± 0.01d 1.47 ± 0.02c 0.897 ± 0.01d−f 2.14 ± 0.01a 0.600 ± 0.01hi 0.547 ± 0.02i 0.613 ± 0.01hi 1.74 ± 0.0b

26.2 ± 2.1f 50.3 ± 6c 53 ± 10c 74 ± 7.9a 31.3 ± 8.6ef 36.3 ± 6de 44.7 ± 9.7d 64 ± 7.5b

0.297 0.290 0.273 0.287 0.510 0.240 0.240 0.277

± ± ± ± ± ± ± ±

0.07b 0.01b 0.02b−e 0.02bc 0.19a 0.02b−f 0.03b−f 0.02b−d

1.99 2.54 2.68 3.25 2.17 2.10 1.92 1.95

± ± ± ± ± ± ± ±

2g ± ± ± ± ± ± ±

MDA (μmol g−1 FW)

Means ( ± SD, n = 9) followed by similar letters within each column do not express significant diffrences at P < 0.05 according to LSD test. WW 100% field capacity, SWD 50% field capacity, AMF arbuscular mycorrhizal fungi, Ri Rhizophagus irregularis, Fm Funneliformis mosseae, CF combination of both fungi, NM, non-mycorrhizal, PF Pseudomonas fluorescens, NPF non- P. fluorescens.

3.5. MDA content and H2O2

4. Discussion

The MDA content were significantly affected by AMF, PF and irrigation levels (Table 1). The MDA content under SWD conditions increased by 68% compared to those in WW conditions (Table 1). As far as inoculation status is concerned, MDA significantly decreased by 8% in PF plants compared to the NPF plants. Moreover, co-inoculation with AMF and PF or individul inoculation caused a clear decline in the MDA content compared with the control plants (Table 1). The highest MDA content was recorded in the NM plants grown under SWD conditions (Table 2). H2O2 content tended to show the same pattern as MDA content as far as irrigation regime is concerned. It means, it increased by 218% under SWD conditions compared to the WW conditions (Table 1). AMF inoculation resulted in a lower accumulation of the H2O2 content compared with the control plants in the shoots of Arizona cypress, while the application of PF did not follow the same pattern. Altough AMF inoculation tended to be not effective under WW condition, but it quiet influenced the H2O2 accumulation and decreased it under SWD conditions (Table 3). Once more the maximum H2O2 content was accumulated in the NM plants under SWD conditions (Table 3).

The present study evaluated the effectiveness of AMF and/or PF inoculation in Arizona cypress seedlings under water stress conditions. The results showed that Arizona cypress growth was increased under SWD conditions when inoculated with AMF or co-inoculation with AMF and PF (Table 2). Many researchers have reported that AMF can improve the plant tolerance to the water deficit (Amiri et al., 2015, 2017; Rahimzadeh and Pirzad, 2017; Bowles et al., 2017). It is partly because of this fact that the mycorrhizal roots can increase water conductivity (Siemens and Zwiazek, 2004). There are contradictory reports on the dual inoculation effect of AMF and PGPR. However, there are some reports supporting the beneficial results obtained in the current study for instance, Nadeem et al. (2014), indicates that the presence of PGPR and AMF in the root zone is effective for improving the activities of both communities. These synergistic interactions between PGPR and AMF can improve plant growth and productivity under water stress conditions and decrease the negative influences of the stress on plant growth and development (Nadeem et al., 2014). In this study, positive effects of AMF and PGPR on the biomass of the Arizona cypress seedlings were observed under SWD treatments. Similarly, the same results were reported regarding flax and tomato seedlings (Gamalero et al., 2004; Rahimzadeh and Pirzad, 2017). Our results showed that the drought conditions not only disrupt the normal biology of the seedlings, but also

Table 3 Interaction effects of irrigation regime × AMF on plant height (PH), root length (RL), relative water content (RWC), Total soluble sugars (TSS), SOD (superoxide dismutase) activity and H2O2 (hydrogen peroxide) contents of Arizona cypress seedlings. Irrigation regime

AMF status

PH (cm)

RL (cm)

WW

NM Ri Fm CF

18.3 21.2 22.5 25.2

± ± ± ±

0.8cd 0.98b 1.2 b 1a

25.0 25.6 27.2 21.2

SWD

NM Ri Fm CF

15.2 17.3 15.8 19.2

± ± ± ±

1f 0.9de 2.4ef 1.2c

8.7 ± 3d 15.5 ± 2.9c 13.5 ± 2.3c 13.2 ± 1.47c

± ± ± ±

RWC (%) 1.4a 1.6a 2.1a 3.2b

TSS (mg g−1 FW)

SOD (U/mg protein) 16.7 29.8 31.8 37.6

88.5 87.2 85.9 85.7

± ± ± ±

3.8a 2.5a 2.3a 1.7a

0.787 ± 0.07b 0.748 ± 0.09bc 0.838 ± 0.15ab 1.01 ± 0.1a

63.2 70.0 71.9 72.1

± ± ± ±

2c 1.9b 4.8b 3.7b

0.525 0.578 0.658 0.587

± ± ± ±

0.09d 0.11cd 0.18b−d 0.14cd

± ± ± ±

5.8e 12d 7.6d 16d

60 ± 4.8c 98.5 ± 22ab 81.8 ± 5.6b 107 ± 15a

H2O2 (μmol g−1 FW) 12.3 3.63 4.55 5.72

± ± ± ±

2d 1.4d 1.3d 1.5d

36.0 ± 1a 16.46 ± 2.3b 14.8 ± 0.97bc 16.6 ± 2.7b

Means ( ± SD, n = 9) followed by similar letters within each column do not express significant diffrences at P < 0.05 according to LSD test. WW 100% field capacity, SWD 50% field capacity, AMF arbuscular mycorrhizal fungi, Ri Rhizophagus irregularis, Fm Funneliformis mosseae, CF combination of both fungi, NM, non-mycorrhizal. 5

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(2017). Increasing the soluble sugar in the AMF-inoculated plants was also reported by Rahimzadeh and Pirzad (2017). Accordingly, the accumulation of soluble sugar might improve the Arizona cypress seedlings tolerance to the unfavorable impacts of drought stresses. PGPR inoculation accompanied by the AMF can improve the plant growth and nutrition (Vafadar et al., 2014), water stress tolerance (Rahimzadeh and Pirzad, 2017), and finally, plant defense responses (Hakeem et al., 2016). This suggested that the application of PGPR under stress conditions could improve the AMF- assisted synergism to host plants. Nevertheless, the inoculation of plants with AMF showed to be more beneficial for improving the water content. Many researchers have reported that AMF can improve water content in the host plants (Meddich et al., 2015; Essahibi et al., 2017; Huang et al., 2017), indicating that mycorrhizal inoculation could improve the RWC; this feature was important for developing plants under water-stress conditions, especially harsh sites. The results of the present study clearly showed the beneficial effects of AMF inoculation in protecting plant cells through enhancement of antioxidant enzymes systems. However, plants try to cope with the challenge through several mechanisms, mainly production or activation of antioxidant enzymes (Mathimaran et al., 2017). It is well documented that, the activity of these enzymes changes following the plant under drought stress (Sohrabi et al., 2012). Furthermore, the dual-inoculated seedlings tended to produce and accumulate H2O2 and MDA, than non-inoculated ones, thereby increasing the water stress tolerance. These results were in line with those reported by Fouad et al (2014) concerning olive (Olea europaea) plants. Moreover, plants response to the environmental stress by producing antioxidant enzymes, such as CAT, SOD, APX, and GPX is well documented (Pandey et al., 2017; Choudhury et al., 2017; Mathimaran et al., 2017). Furthermore, maintaining an efficient antioxidant system can help plants to protect themselves against the harmful impacts of the ROS. The report of Xun et al. (2015) also indicates that although PGPR and AMF separately increased the oat (Avena sativa) growth, the highest antioxidant activities were observed in the dual-inoculated treatment helping the plant to resist severe conditions. The dual inoculation of PGPR and AMF could be very useful for improving water status under water stress conditions (Nadeem et al., 2014). The performance of dual inoculation depends on mycorrhizal species. Positive effects have been reported regarding dual inoculation of AMF and PGPR. The synergistic impacts of both microorganisms can also be described by numerous mechanisms (Nadeem et al., 2014). To the best of the authors’ knowledge, few analogous findings have been presented concerning the interaction effects of dual inoculation (AMF-PGPR communication) on the growth responses of Arizona cypress under water stresses.

impose harmful effects on the activity of microorganisms. The adverse impacts of a drought condition on microorganism responses can be avoided partly by the employment of a mixture of the two groups (AMF and PGPR) (Nadeem et al., 2014). For example, dual inoculation of tomato (Lycopersicon esculentum) with AMF and PGPR synergistically affected biomass and improved drought stress tolerance (Calvo-Polanco et al., 2016). Moreover, in a classic study by Meyer and Linderman (1986), they found a significant enhancement in the shoot and root biomass when both PGPR and AMF were present. AMF colonization of the seedlings roots improved sharply under dual inoculation strategy. These results are supported by Visen et al. (2017) report, showing that simultaneous inoculation by PGPR and AMF in litchi tree (Litchi chinensis S.) resulted in the highest root colonization. The mechanisms behind this phenomena seems to be related the PGPR capability to produce cell wall-degrading enzymes which finally facilitate the establishing of AMF (Visen et al., 2017). Moreover, the attachment of PGPR to the plant root or the AMF could alter the cell-wall features to enhance the acceptance of roots or promote the formation of a mechanical link between the AMF and plant (Aspray et al., 2006). Furthermore, PGPR releases a variety of compounds to the soil such as the secondary metabolites which in turn can increase the establishment of AMF in the rhizosphere (Nadeem et al., 2014; Visen et al., 2017). In the present research, individual inoculation with AMF under SWD conditions alleviates the adverse effects of drought on plant growth in terms of shoot and root biomass compared with the NM plants (Table 2). Nevertheless, the species of AMF did not follow the same pattern. CF turned out to be more advantageous than both single application of the species or NM as far as the above-ground growth of the seedling is concerned (Table 1). These findings were consistent with those reported by Amiri et al. (2017) regarding the rose-scented geranium (Pelargonium graveolens L.) plants treated with CF. Van der Heijden et al. (1998) describe that, this effect could be the result of added beneficial effect of each single AMF species. It was reported that the responses of each AMF species could be different, even in the same site and cultural practice (Giovannetti et al., 2010; Aalipour et al., 2019). This fact is a result of the ability of AM species to acclimate to water stress conditions (Nasim, 2010). In our study CF was the most efficient method to colonize the roots. This fact is more important considering that under SWD, CF could fulfill this job when came with a dual inoculation accompanied by PF. This interaction and the mechanisms behind it on water management have mostly been ignored. In this study, the growth parameters increased in AMF, PF and dual-inoculated seedlings under SWD treatments. It was also revealed that mycorrhizal fungi were more effective when applied with other microorganisms such as PGPR. For example, Rahimzadeh and Pirzad (2017) reported the positive effects of dual-inoculated Pseudomonas putida and AMF on the root colonization and the growth of flax (Linum usitatissimum L.). However, this symbiotic effect seems to be at least partially controlled by the plant species as we could not track such an effect on rye grass (Lolium perenne) with the same microorganisms species (Zarganian and Nikbakht, 2015). The accumulation of proline under SWD conditions (Table 2) was likely a general response of Arizona cypress to water-stress conditions as an osmotic regulator. The proline content was higher in the dualinoculated plants than in separate inoculated plants. In contrast, the lower proline concentrations in the dual-inoculated seedlings under complete irrigation showed that this species was less affected by the osmotic stress and required lower proline concentrations compared with non-inoculated plants. These results were in agreement with those reported by Rahimzadeh and Pirzad (2017). In our experiment, we detected a decline in the soluble carbohydrate when Arizona cypress seedlings were subject to the water stress; this trait could be improved with the application of AMF. An Increase in the total soluble sugar in the AMF-inoculated seedlings was probably related to the enhanced carbon fixation and improved water content as reported by Amiri et al.

5. Conclusion The results of this study demonstrated that the dual-inoculated (AMF and PF) plants enhanced the plant growth parameters of Arizona cypress but only under WW conditions, while under SWD it is quite indifferent to inoculate with only AMF or dual-inoculation. Based on the results, co-inoculation with AMF and PF or individul inoculation with AMF could improve the water stress tolerance by alleviating the oxidative damage, such as H2O2 and MDA, increasing the enzymatic antioxidants. Moreover, the osmotic adjustment mechanism employing, but not restricted to proline, plays an important role in Arizona cypress to maintain its water content under the water-stress conditions. Finally, these results suggested that the application of beneficial microorganisms can present a great potential for increasing the water stress tolerance in the seedlings of the trees, which could be an appropriate method for compensating the negative effects of the water-deficit. Acknowledgement The authors appreciate the contributions of the Isfahan University of 6

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Technology for its scientific and financial support.

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