Mycorrhiza DOI 10.1007/s00572-013-0546-3

ORIGINAL PAPER

Evidence that arbuscular mycorrhizal and phosphate-solubilizing fungi alleviate NaCl stress in the halophyte Kosteletzkya virginica : nutrient uptake and ion distribution within root tissues Huan Shi Zhang & Feng Fei Qin & Pei Qin & Shao Ming Pan

Received: 18 July 2013 / Accepted: 20 November 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract The effects of an arbuscular mycorrhizal (AM) fungus, Glomus mosseae, and a phosphate-solubilizing microorganism (PSM), Mortierella sp., and their interactions, on nutrient (N, P and K) uptake and the ionic composition of different root tissues of the halophyte Kosteletzkya virginica (L.), cultured with or without NaCl, were evaluated. Plant biomass, AM colonization and PSM populations were also assessed. Salt stress adversely affected plant nutrient acquisition, especially root P and K, resulting in an important reduction in shoot dry biomass. Inoculation of the AM fungus or/ and PSM strongly promoted AM colonization, PSM populations, plant dry biomass, root/shoot dry weight ratio and nutrient uptake by K. virginica, regardless of salinity level. Ion accumulation in root tissues was inhibited by salt stress. However, dual inoculation of the AM fungus and PSM significantly enhanced ion (e.g., Na+, Cl−, K+, Ca2+, Mg2+) accumulation in different root tissues, and maintained lower Na+/K+ and Ca2+/Mg2+ ratios and a higher Na+/Ca2+ ratio, compared to non-inoculated plants under 100 mM NaCl conditions. Correlation coefficient analysis demonstrated that plant (shoot or root) dry biomass correlated positively with plant nutrient uptake and ion (e.g., Na+, K+, Mg2+ and Cl−) concentrations of different root tissues, and correlated H. S. Zhang : F. F. Qin : P. Qin (*) Halophyte Research Laboratory, Nanjing University, 22 Hankou Road, Nanjing, 210093, China e-mail: [email protected] S. M. Pan (*) School of Geographic and Oceanographic Sciences, Nanjing University, Nanjing, 210093, China e-mail: [email protected] H. S. Zhang Nanjing Institute for Comprehensive Utilization of Wild Plants, Nanjing, 210042, China

negatively with Na+/K+ ratios in the epidermis and cortex. Simultaneously, root/shoot dry weight ratio correlated positively with Na+/Ca2+ ratios in most root tissues. These findings suggest that combined AM fungus and PSM inoculation alleviates the deleterious effects of salt on plant growth by enabling greater nutrient (e.g., P, N and K) absorption, higher accumulation of Na+, K+, Mg2+ and Cl− in different root tissues, and maintenance of lower root Na+/K+ and higher Na+/Ca2+ ratios when salinity is within acceptable limits. Keywords Glomus mosseae . Phosphate-solubilizing Mortierella sp . Salt stress . Nutrient uptake . Tissue ion accumulation . Na+/K+ ratio

Introduction Saline soils occupy 8 % of the earth's land surface and represent a major limiting factor for plant growth and biomass production (Pitman and Läuchli 2004; Hajiboland 2013). The availability of solutions to saline stress is of utmost importance in saline soil agriculture. The use of plant growthpromoting microorganisms is an economical and environmentally friendly approach to counteracting the adverse effects of salt stress. Arbuscular mycorrhizal (AM) fungi have been shown to occur widely in saline environments (Wang and Liu 2001; Yamato et al. 2008), and inoculated plants grow better than non-inoculated plants under a variety of salinity stress conditions (Asghari et al. 2005; Daei et al. 2009; Garg and Manchanda 2009; Estrada et al. 2013). In addition, some phosphate-solubilizing microorganisms (PSM) can solubilize P precipitated with Ca2+, Mg2+ and Zn2+ ions in salt-stressed soil (Grattan and Grieve 1999) and so enhance the amount of available phosphate in saline alkaline soils (Srividya et al.

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2010). The released P, which cannot be transferred to the roots by PSM, may potentially be taken up by the external AM mycelium. In this context, the simultaneous application of PSM and AM fungi has been shown to stimulate plant growth more than inoculation of either microorganism alone when the soil is P-deficient (Zaidi and Khan 2006; Babu and Reddy 2011), especially in a saline soil (Zhang et al. 2011b). The improved growth of mycorrhizal plants under saline conditions is often explained by a better P nutrition and the fact that a larger plant biomass results in the dilution of toxic ions (Al-Karaki and Hammad 2001; Daei et al. 2009; Wu et al. 2010; Campanelli et al. 2012). Also, several studies have demonstrated a synergistic effect of PSM and AM fungi in promoting nutrient uptake, especially P, by plants (Omar 1998; Osorio and Habte 2001; Zaidi and Khan 2006; Osorio and Habte 2013). However, there is no available information regarding the impact of combined inoculation with AM fungi and PSM on the nutrient uptake of plants under salt stress. Elevated Na+ and Cl− levels in the soil solution prevent root uptake of other nutrients by interfering with various transporters in the root plasma membrane, such as K+-selective ion channels, and inhibit root growth through osmotic effects of Na+ (Wild 1988). Therefore, high concentrations of Na+ and Cl− ions in the soil solution may depress nutrition-related activities of roots and produce extreme ratios of Na+/K+, Na+/Ca2+ and Ca2+/Mg2+ in plant tissues. Recent studies have shown that reducing Na+ uptake is the most efficient approach to controlling Na+ accumulation in most crops (Estrada et al. 2013). The ability of plants to maintain a high cytosolic K+/ Na+ ratio is likely to be one of the key determinants of plant salt tolerance (Hajiboland 2013). Hammer et al. (2011) indicated that AM fungi can selectively take up elements such as K and Ca, which act as osmotic equivalents as they avoid the uptake of toxic Na. Cantrell and Linderman (2001) showed that in mycorrhizal plants, Na+ may be kept inside root cell vacuoles and intraradical fungal hyphae, and may not be allocated to the shoots. According to Manchanda and Garg (2007), plant roots are exposed to a range of soil microorganisms with which they have a variety of interactions. However, the effects of combined inoculation with AM fungi and PSM on ionic balance within root tissues under saline conditions remain unclear. Kosteletzkya virginica (L.) Presl. is a perennial herbaceous halophyte of the Malvaceae family that is native to the brackish marshes of the mid-Atlantic and southeastern United States. It was introduced into Northern Jiangsu, China, by the Halophyte Research Laboratory of Nanjing University in 1993 as a species with the potential to improve saline soils in order to develop saline agriculture. Ruan et al. (2005) reported that in saline soil, the concentration of Na+ in the organs (roots, stems, leaves) of K. virginica decreased greatly a few years after its planting, which may have caused an increase in K+/Na+ and Ca2+/Na+ ratios. These authors suggested that

such changes could mitigate salt stress in the growth habitat of K. virginica. Under short-term experimental conditions, a significant increase in Na+ and decrease in K+ and Ca2+ has been observed to occur simultaneously in K. virginica tissues exposed to 100 mM NaCl (Ghanem et al. 2010). In a former study, Zhang et al. (2011b) reported that inoculation with PSM and AM fungi could significantly increase K. virginica growth at different salinity levels but the effect of co-inoculation with the two soil microorganisms on ion uptake in K. virginica under salt stress is unknown. Therefore, the aims of the present study were to evaluate the effects of an AM fungus and PSM on (1) N, P and K uptake by K. virginica plants under salt stress, and (2) K+, Na+, Cl−, Ca2+ and Mg2+ distribution within different root tissues of the salt-treated plants.

Materials and methods Experimental design To study the effects of the inoculation of the AM fungus, Glomus mosseae , and the PSM, Mortierella sp., on K. virginica under salt stress, an experiment was conducted using a completely randomized design with four inoculation treatments (AMF, PSM, AMF+PSM, control [CK])×two salinity treatments (0 and 100 mM NaCl) with three replicate pots per treatment. AM fungal and PSM inocula G. mosseae (Nicol. and Gerd.), chosen for the present study for its greater ability to alleviate saline stress (Porras-Soriano et al. 2009), was obtained from the Bank of Glomales in China. The inocula, which consisted of spores (2,830 spores per 100 g−1 soil), hyphae, and colonized root fragments, were collected from a 6-month-old pot culture of G. mosseae grown on sorghum in sterile sandy soil. The Mortierella sp. was isolated from the topsoil (0~10 cm) samples of a Spartina alterniflora community in North Jiangsu province. It had been previously identified as a phosphate-solubilizing fungus that could significantly enhance available P concentrations when cultured in the presence of 100 mM NaCl (Zhang et al., 2011b). Liquid PSM inoculum was prepared by adding 3 ml sterile water to a test tube slant with the fungus then pouring the mixture into 50 ml Martin culture medium (K2HPO4, 1 g; MgSO4⋅7H2O, 0.5 g; NaCl, 11.5 g; peptone, 5 g; glucose, 10 g; gelose, 10 g; 1/ 30000 Bengal red water solution, 100 ml; demineralized water, 900 ml) to which 1.15 % NaCl was added. The PSM was cultured on a shaker for 96 h at 180 rpm and 28 °C. The solution ultimately contained 1.9×105 colony forming units (CFU) ml−1 and was stored at 4 °C until use. In addition,

Mycorrhiza

portions of the AM fungal and PSM inocula were autoclaved three times at 121 °C for 90 min for use as control treatments. Plant culture conditions The test soil, which was collected from Nanjing University botanical garden, had the following characteristics: pH 7.8; EC, 0.03 dS m−1; organic matter, 4.3 g kg−1; total N, 11.5 mg kg−1; available P, 2.6 mg kg−1; extractable K, 20.1 mg kg−1. The growth substrate, consisting of a mixture of washed sand and soil (1:1, v/v), was sterilized by autoclaving for 1 h at 121 °C twice on 2 consecutive days and then sieved (2 mm). A total of 900 g of the sieved sandy soil substrate was transferred to pots (diameter 15 cm, height 15 cm). The fungal inocula were added to the soil at a depth of 5 cm with the following design: 10 gG. mosseae inoculum and 10 ml sterile Mortierella sp. inoculum for the AMF treatment; 10 g sterile G. mosseae inoculum and 10 ml Mortierella sp. inoculum for the PSM treatment; 10 g G. mosseae inoculum and 10 ml Mortierella sp. inoculum for the AMF+PSM treatment; 10 g sterile G. mosseae inoculum and 10 ml sterile Mortierella sp. inoculum for the CK treatment. Seeds of K. virginica, collected in 2011from Jinhai Agricultural Experimental Farm (Dafeng City, Jiangsu Province, China), were surface-sterilized by soaking in a 5 % NaOCl solution for 10 min and rinsed with sterile distilled water. They were then transferred aseptically to Petri dishes filled with water and incubated for 4 days at 25 °C. One week after germination, three seedlings of uniform size were transferred to each pot that was placed on a 2-cm-deep plate, and plants were grown until April 10, 2012 in a greenhouse under controlled conditions (16 h of 220 μmol m−2 s−1 daylight intensity at 28 °C, 8 h of night at 18 °C, relative humidity kept at 65–85 %). NaCl (Fisher ACS) was added to a Hoagland nutrient solution without P, modified from Hoagland and Arnon (1950), to give final NaCl concentrations of 0 and 100 mM. The salt concentration of 100 mM is within the range of the habitat of K. virginica (Blits and Gallagher 1990). All of the nutrient solutions were prepared using demineralized water (pH 7.38). After irrigation with water for 35 days, the plants were watered with 25 ml of the NaCl solution at 3-day intervals for 27 days. The soil substrate was salinized step-wise to prevent osmotic shock. The leachate was collected and added back to the soil to maintain salinity near the target level. Distilled water was added as necessary to maintain the soil moisture. PSM population and AM fungal colonization assessment Whole plants were extracted from pots on the 30th day after the beginning of the stress period. Soil samples obtained by gentle shaking of the roots were collected in sterilized culture dish and stored at 4 °C until the PSM population analyses

using a 10-fold serial dilution test tube technique (Li et al. 2008). The roots from three plants were cut into 1-cm segments, cleared in 10 % KOH and stained with 0.05 % trypan blue (Phillips nd Hayman 1970). Thirty fragments were examined for AM colonization under a compound microscope (100× magnification) according to McGonigle et al. (1990). Plant biomass and nutrient (N, P, K) measurements At harvest time, two plants per pot (total of six plants per treatment) were separated into roots and shoots which were dried in a forced-air oven at 80 °C for 72 h for biomass determination. The oven-dried samples were ground separately and sieved through a 0.5-mm sieve. A 0.1-g tissue sample was digested in a tri-acid mixture (60 % HClO4/HNO3/ H2SO4 =1:5:0.5). P and N concentrations were determined following the method of Allen (1989). K concentration was determined using an atomic absorption spectrophotometer. Determination of elemental composition of root tissues After washing in demineralized water and quick immersion in liquid nitrogen for cryofixation and sectioning, frozen sections of lateral roots were transferred quickly into a vacuum evaporator (modified Hitachi HUB-5GB) and dried under a 5× 10−4 Torr vacuum for 12 h. The samples were sputter-coated with gold on the sample stage and analyzed in an scanning electron microscope (model S-3000 N; Hitachi HighTechnologies Corporation, Tokyo, Japan) equipped with an energy-dispersive X-ray detector (EDX; Horiba Inc., Kyoto, Japan) at an accelerating voltage of 20 kVand beam current of 60 μA. The working distance from the EDX detector was 13.5 mm. A point analysis was conducted six times for each sample in order to obtain high-resolution images and element content in cells of the epidermis, exodermis, cortex, endodermis, and root stele. Three randomly selected lateral roots per treatment (one lateral root per pot) were analyzed. Elemental composition was determined in six cells per root tissue to give18 replicates for each ion concentration. The results were calculated by expressing the atomic number for a particular element in a given point as a percentage of the total atomic number for all of the elements measured in the root tissues. Statistical analysis All data were statistically analyzed by two-way analysis of variance (ANOVA) using the Statistical Product and Service Solutions (SPSS) software package (SPSS 18.0 for Windows). Probabilities of significance were used to test the significance among treatments and interactions, and means were separated using Duncan's test at p values 0.05). However, under salt stress the highest K concentration in the shoots and the lowest values in the roots were recorded in PSM-treated plants. Co-inoculated plants showed the highest total K concentrations whatever the NaCl level. Ion distribution in different root tissues Sodium There was a decreasing trend in Na+ concentrations from the epidermis to the stele in the roots of all K. virginica plants (Fig. 1a, b). NaCl stress strongly inhibited Na+ accumulation in the different tissues within the roots. However, inoculation treatments enhanced Na+ accumulation in almost all tissues within the roots. The NaCl-treated plants inoculated with both AMF and PSM exhibited the highest Na+ concentrations in the exodermis (Fig. 1b). Potassium Regardless of NaCl level, increased K accumulation and decreased Na+/K+ ratios were observed from the epidermis to the stele (Fig. 1c–f). Dual inoculation had significantly positive effects on K+ concentration compared to noninoculated plants of K. virginica at 0 and 100 mM NaCl (p < 0.05; Fig. 1c). The single inoculation of PSM showed the same effects in the epidermis at 0 mM NaCl (Fig. 1d).

However, dual inoculation maintained lower Na+/K+ ratios in the different root tissues of NaCl-treated plants, except for the stele, compared to other treatments (p< 0.05; Fig. 1f). The inoculation treatments had no significant effects on the Na+/ K+ ratios of the non-NaCl-treated plants (Fig. 1e). Calcium Accumulation of Ca in the different root tissues was inhibited by NaCl stress, which induced significantly higher Na+/Ca2+ ratios than those in unstressed plants (Fig. 2). All inoculation treatments significantly enhanced Ca2+ concentrations in root tissues at the 0 mM NaCl level (p< 0.05; Fig. 2a), but only dual inoculation showed promoting effects on Ca2+ concentrations in the epidermis and exodermis under salt stress (Fig. 2b). Meanwhile, the inoculated plants had higher Na+/ Ca2+ ratios in most of the root tissues compared to those in non-inoculated plants regardless of NaCl level (Fig. 2c, d). Magnesium Salt stress induced decreased Mg2+ accumulation in different tissues of non-inoculated roots of K. virginica (Fig. 3a, b). For plants without NaCl additions, only dual inoculation with the AM fungus and PSM showed promoting effects on Mg2+concentrations compared to those of non-inoculated plants, except for the cortex (p< 0.05; Fig. 3a). Inoculation treatments had a positive effect on Mg2+ accumulation at the 100 mM NaCl level (Fig. 3b). Dual inoculation stimulated Mg2+ accumulation in the epidermis, exodermis, and stele (p < 0.05). Compared to the marked changes in the Ca2+/Mg2+ ratios of plants after single inoculation with one of the

Mycorrhiza

Fig. 1 Effects of Glomus mosseae (AMF) and Mortierella sp. (PSM) inoculation of Kostelelzkya virginica on accumulation of Na+ and K+, as well as Na+/K+ ratios, in the different root tissues at 0 (a, c, and e) and

100 (b, d, and f) mM NaCl levels, as compared to non-inoculated plants (CK). Data are means±SE of 18 replicates

microorganisms, dual inoculated plants exhibited relatively steady Ca2+/Mg2+ ratios at the 0 mM NaCl level (Fig. 3c). In general, under salt stress, plants had lower Ca2+/Mg2+ ratios compared to those in non-NaCl-treated plants (Fig. 3d). At the 100 mM NaCl level, the Ca2+/Mg2+ ratios of the inoculated plants were significantly lower than those of the noninoculated plants except in the stele (Fig. 3d).

p< 0.05) and total K concentrations (p< 0.05 and p< 0.05) in plants. Similar positive relations were observed with growth parameters such as shoot dry weight (p< 0.01 and p< 0.05), root dry weight (p< 0.05 and p< 0.01) and root/shoot dry weight ratio (p< 0.05 and p< 0.05). The relationship between AM colonization levels and PSM populations was also positive (p< 0.05). Moreover, AM colonization levels correlated positively with shoot N concentration and root P concentration (p< 0.05). PSM populations also correlated positively with both P and K concentrations in shoots and root N concentration (p< 0.05). Simultaneously, there was a positive correlation between root dry weight and shoot P, shoot N and root N concentrations (p< 0.05). Shoot dry weight of K. virginica correlated positively with Na+ concentrations in epidermis and exodermis, K+ concentrations in nearly all root tissues, Mg2+ concentrations in epidermis and stele, and Cl− concentration in epidermis. There was a negative correlation with Na+/K+ ratios in epidermis and cortex (p< 0.05; Table 4). Root dry weight correlated positively with K+ concentration in exodermis, cortex and endodermis (P< 0.05) and Mg2+ concentrations in epidermis and stele (p< 0.01), but correlated negatively with Na+/K+ ratio in

Chlorine Salt stress inhibited Cl− accumulation in the roots. All the inoculation treatments significantly improved Cl− accumulation in the roots compared to the non-inoculation treatments regardless of NaCl level (p< 0.05; Fig. 3e, f). Roots inoculated with PSM had the highest accumulation of Cl− in the epidermis, exodermis and cortex at 0 mM NaCl. Correlation coefficient analysis As shown in Table 3, both AM colonization levels and PSM populations of K. virginica roots showed a positive relationship with total P (p< 0.05 and p< 0.05), total N (p< 0.01 and

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Fig. 2 Effects of Glomus mosseae (AMF) and Mortierella sp. (PSM) inoculation of Kostelelzkya virginica on accumulation of Ca2+ and Na+/ Ca2+ ratios in different root tissues at 0 (a and c) and 100 (b and d) mM

NaCl levels, as compared to non-inoculated plants (CK). Data are means ±SE of 18 replicates

epidermis (p< 0.05). Root/shoot dry weight ratio gave a positive correlation with Mg2+ concentration in stele tissues (P< 0.05) and Na+/K+ ratios in the cortex (p< 0.05), endodermis (p< 0.05) and stele (p< 0.01). Both AM colonization and PSM populations showed a significantly positive relationship with Na+ concentrations in exodermis and stele (p< 0.05), K+ concentrations in most root tissues (p< 0.05), Mg2+ concentrations in epidermis and stele (p< 0.05 and p< 0.01) and Cl− concentration in epidermis (p< 0.05). A negative correlation with Na+/K+ ratio was observed in the epidermis (p< 0.05).

mycorrhiza increase the exudation of carbonaceous substances used as a C source by PSM in the rhizosphere soil (Singh and Kapoor 1999). This hypothesis is supported by the results that PSM populations correlated positively with AM colonization and shoot P concentration. Salt stress severely reduced shoot dry weights of K. virginica plants compared to untreated controls. This is in agreement with previously published data (Wild 1988; Pitman and Läuchli 2004; Ghanem et al. 2010; Campanelli et al. 2012; Hajiboland 2013). Salt stress had significantly negative effects on nutrient accumulation in K. virginica plants, and shoot dry weights correlated positively with total P, total N and total K concentrations of plant. These results suggest that the reduction in shoot dry weight may be partly due to inhibition of nutrient absorption. In the present study, interactions between PSM and AMF stimulated dry biomass of K. virginica, regardless of salinity, and both AM colonization and PSM populations correlated positively with total P, total N and total K concentrations in plants, confirming a synergistic effect of PSM and AMF in promoting nutrient uptake (Omar 1998; Osorio and Habte 2001; Zaidi and Khan 2006; Osorio and Habte 2013). This may be due to the inoculated PSM releasing P ions from insoluble P sources in the substrate which, together with other nutrient (i.e., N and K), were taken up by the external AM mycelium (Osorio and Habte 2013). Zandavalli et al. (2004) and Sheng et al. (2009) reported that AM colonization decreased plant root/shoot ratios under saline conditions. However, root dry weights of K. virginica were not influenced by salt stress; the NaCl-treated plants and the inoculated plants exhibited larger root/shoot dry weight ratios compared to non-NaCl-treated plants and non-

Discussion Results from the present study show that salinity decreased the colonization of K. virginica roots by the AM fungus G. mosseae. This is in agreement with earlier reports that salinity can hamper colonization capacity, spore germination and growth of AM fungal hyphae (Al-Karaki and Hammad 2001; Giri et al. 2007; Sheng et al. 2008; Wu et al. 2010; Campanelli et al. 2012). Present results are in accordance with an earlier report that 100 mM NaCl treatment showed a promoting effect on PSM populations associated with K. virginica roots (Zhang et al. 2011b). This is probably due to that fact that the PSM used in the present study was isolated from saline soil samples and was cultured in a medium containing 1.15 % NaCl. The promotion of mycorrhizal colonization and PMS populations of K. virginica roots by the combined inoculation of G. mosseae and PMS may be related release of available P, needed for AM fungal growth, by the PSM population (Toro et al. 1996) and the fact that

Mycorrhiza

Fig. 3 Effects of Glomus mosseae (AMF) and Mortierella sp. (PSM) inoculation of Kostelelzkya virginica on accumulation of Mg2+, Cl− and Ca2+/Mg2+ ratios in different root tissues at 0 (a, c, and e) and 100 (b, d,

and f) mM NaCl levels, as compared to non-inoculated plants (CK). Data are means±SE of 18 replicates

inoculated plants, respectively. This suggests that a larger root/ shoot ratios one of the important mechanisms for the successful establishment of some halophyte plants under severe saline stress (Zhang et al. 2011a) and that inoculation with AM fungi and PSM may stimulate the mechanism related to higher salt tolerance by creating a greater surface area for nutrient uptake. In the present study, K. virginica growth was strongly correlated to P concentrations. However, more P was

distributed to shoots than roots in a same inoculation treatment in the 100 mM NaCl treatment. Salt stress has greater inhibitory effects on shoot growth than on root growth; the transfer of more P to the shoots may reduce the negative effects of Na+ and Cl− ions by maintaining vacuolar membrane integrity, which facilitates compartmentalization within vacuoles as well as selective ion intake (Rinaldelli and Mancuso, 1996). P ions precipitate with Ca2+ ions in saline soils and become one of the least available of essential nutrients (Grattan and

Table 3 Correlation coefficients between nutrient content and growth parameters, AM colonization and PSM populations (n =48) P

N

K

AM

PSM

Shoot

Root

Total

Shoot

Root

Total

Shoot

Root

Total

Shoot Root Root/shoot

0.55 0.79* 0.74*

0.73* 0.54 0.042

0.94** 0.97** 0.38

0.78* 0.81* 0.31

0.41 0.75* 0.81*

0.96** 0.95** 0.29

0.38 0.54 0.55

0.56 0.26 0.46

0.96** 0.89** 0.24

0.84** 0.78* 0.80*

0.79* 0.91** 0.73*

AM PSM

0.49 0.85*

0.79* 0.41

0.81* 0.81*

0.89** 0.69

0.60 0.76*

0.89** 0.82*

0.13 0.79*

0.50 0.13

0.77* 0.79*

1 0.75*

0.75* 1

AM AM colonization, PSM PSM populations Significant at *p< 0.05 and **p< 0.01

Mycorrhiza Table 4 Correlation coefficients between ion concentrations in different root tissues and growth parameters, AM colonization and PSM populations (n =48) Ion

Tissue Shoot

Root

Root/shoot AM

PSM

Na+

Ep Ex Co En St Ep Ex Co En

0.78* 0.74* 0.13 0.17 0.43 0.73* 0.77* 0.80* 0.78*

0.59 0.62 0.03 0.27 0.57 0.61 0.75* 0.78* 0.88*

−0.11 −0.04 −0.07 0.34 0.45 −0.01 −0.25 −0.20 0.01

0.48 0.77* −0.04 −0.06 0.71* 0.84* 0.79* 0.82* 0.54

0.46 0.83* −0.17 0.27 0.79* 0.88* 0.74* 0.70* 0.70*

St Ep Ex Co En St Ep Ex Co En St Ep Ex Co En St Ep Ex

0.40 0.28 0.47 0.18 0.44 0.29 0.73* 0.46 −0.23 0.49 0.77* 0.71* 0.49 0.23 0.43 −0.21 −0.76* −0.37

0.40 0.09 0.15 −0.09 0.13 0.04 0.85** 0.60 −0.03 0.54 0.92** 0.39 0.37 0.12 0.20 −0.05 −0.78* −0.18

0.04 −0.29 −0.49 −0.45 −0.43 −0.35 0.57 0.34 0.36 0.29 0.74* −0.07 −0.09 −0.07 −0.24 0.19 −0.17 0.34

0.80* 0.15 0.37 0.14 0.37 0.27 0.75* 0.40 −0.01 0.29 0.70* 0.81* 0.10 0.01 0.28 0.05 −0.83* −0.45

0.54 0.07 0.12 −0.22 −0.04 −0.12 0.93** 0.72* −0.14 0.40 0.86** 0.72* 0.29 −0.06 −0.11 0.08 −0.72* −0.34

Co En St Ep Ex Co En St Ep Ex Co En St

−0.73* −0.34 0.27 −0.25 −0.21 −0.28 −0.41 0.10 −0.20 −0.19 0.50 −0.20 0.05

−0.36 −0.12 0.42 0.08 0.12 0.08 −0.03 0.52 −0.42 −0.08 0.42 −0.18 0.07

0.14 0.43 0.45 0.64 0.66 0.71* 0.71* 0.88** −0.47 0.13 −0.03 −0.02 0.08

−0.34 −0.32 −0.74* 0.04 0.03 −0.21 −0.24 0.09 0.04 −0.17 0.42 −0.40 −0.18

−0.75* −0.11 0.32 0.00 0.04 0.03 0.01 0.51 −0.44 0.10 0.47 −0.07 0.01

K+

Ca2+

Mg2+

Cl−

Na+/K+

Na+/Ca2+

Ca2+/Mg2+

Ep epidermis, Ex exodermis, Co cortex, En endodermis, St Stele, AM AM colonization, PSM PSM populations Significant at *p< 0.05 and **p< 0.01

Grieve 1999). Salt stress induces reduced P uptake and concentrations in plant tissues, resulting in reduced plant growth (Giri et al. 2007). A similar phenomenon was found

concerning the P contents of K. virginica roots in the present study. Exposure to the 100 mM NaCl stress level severely limited root P accumulation and shoot dry weight. This is further corroborated by the results that shoot dry weight correlated positively with root P concentration. Osorio and Habte (2001, 2013) reported that soil inoculated with PSM or an AM fungus alone did not influence the P content of plants. However, results from the present study clearly show that both single and combined inoculation of G. mosseae and PSM mediated greater P absorption, evidenced by significantly enhanced P contents of the roots and shoots of K. virginica under salt stress. As mentioned above, this could be because the PSM used in this study was able to solubilize insoluble inorganic or organic phosphate by acidification of the salinized soil substrate (Zhang et al. 2011b). On the other hand, extensive hyphal development of the AM fungus could increase the PSM population in the saline habitat and facilitate P uptake by the plants (Ruiz-Lozano and Azcón 2000). Improved P absorption may also favour the enzymatic antioxidant defense system to alleviate negative osmotic stress induced by saline conditions (Daei et al. 2009; Wu et al. 2010; Campanelli et al. 2012). K. virginica growth was also positively correlated with N concentration of the plants. As for P, shoots accumulated more N than roots. Frechill et al. (2001) indicated that salinity interferes with N acquisition and utilization, inducing decreased plant growth. However, in the present study, N concentrations in the shoots were not significant affected by 100 mM NaCl, while N concentrations in the roots showed an increasing trend. This may represent an important mechanism for this halophyte to cope with severe salt stress. Single inoculation of G. mosseae and dual inoculation with PSM greatly enhanced N accumulation in roots and shoots of K. virginica at both 0 and 100 mM NaCl levels. Garg and Chandel (2011) also recorded higher accumulation of N in mycorrhizal Cicer arietinum (L.) than in non-mycorrhizal plants. Such effects have been attributed to the extraradical mycelia of AM fungi taking up both nitrate and ammonium and translocating N as arginine to the plant (Cruz et al. 2004, 2007; Govindarajulu et al. 2005; Guether et al. 2009). Moreover, AM fungi and PSM may promote urease activity in soil so catalyzing the hydrolysis of urea to release ammonia or ammonium ions (Zhang et al. 2011b). Studies have reported that improved N nutrition may help to reduce the toxic effects of Na ions by reducing their uptake and this may indirectly help in maintaining the chlorophyll content of the plant and also proline concentration (Neto et al. 2006). In the present study, K. virginica growth was also correlated positively with total K concentration in plants. A large number of studies have demonstrated that salinity reduces K+ uptake and accumulation due to Na+ competing with K+ for entry sites at the root plasma membrane for ingression into the symplast (Grattan and Grieve 1999). In the present study,

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salt stress reduced root K contents compared to non-NaCltreated plants, partly due to the reduced plant biomass under salt stress. Shoot contents of K+ were, on the contrary, enhanced by 100 mM NaCl which may reflect part of the general mechanism of salt tolerance of K. virginica. Single inoculation with an AM fungus and dual inoculation with PSM could, as Abdel-Fattah and Asrar (2012) also pointed out, significantly promote K+ accumulation in roots and shoots compared to those of non-inoculated plants. This may be due to AM roots exploring a greater volume of soil via the extraradical mycelium, enhanced root exudation or changes in rhizosphere pH that increase K availability (Marschner 2012). PSMtreated plants, however, showed the lowest K+ concentrations in roots under salt stress. The reason for this is unclear but mass multiplication of Mortierella sp. may have induced competition with roots for K+ uptake. Observations on ionic distribution within different root tissues of K. virginica give further insight into mechanisms of salt stress alleviation in this halophyte. Since Na+ and K+ have similar physiological properties, Na+ directly competes with K+ for binding sites that are essential for various metabolic functions (Evelin et al. 2009). High Na+/K+ ratios disrupt various metabolic processes in the cytoplasm (Grattan and Grieve 1999) so that maintenance of a high cytosolic K+/Na+ ratio is a key feature of plant salt tolerance. Na+ from soil enters the xylem stream of plant roots and is transported to the shoots with the transpiration flow (Gilliham and Tester 2005). In the present study, NaCl-treated plants showed lower Na+ concentrations in the different root tissues compared to those of nonNaCl-treated plants, which may reflect a mechanism of prevention of Na+ entry into the root as a strategy of K. virginica to resist salt stress. In addition, Na+ concentrations decreased from the epidermis to the stele in of K. virginica root tissues, which suggests that Na+ may have been sequestrated in the root and translocation prevented to shoot tissues. Ruan et al. (2005), in fact, reported that Na+ concentrations decreased from the roots to the stems to the leaves of K. virginica. According to Evelin et al. (2012), the presence of an AM fungus preventing excess uptake of Na+ by root tissues but enhancing K+ absorption under saline conditions may help in maintaining a high K + /Na + (low Na + /K + ) ratio, thus preventing the disruption of various enzymatic processes and inhibition of protein synthesis. However, the present study on K. virginica showed that inoculation with G. mosseae and PSM could enhance Na+ accumulation in each root tissue type, especially in the dual inoculation treatment, which is in line with previous findings that AM fungi sometimes enhance plant Na+ uptake (Allen and Cunningham 1983; Mardukhi et al. 2011). Moreover, both AM colonization and PSM populations showed a positive relationship with Na+ concentrations in exodermis and stele, and shoot dry weight correlated positively with Na+ concentrations in epidermis and exodermis. In accordance with reports by Giri et al. (2007) and Ruiz-

Lozano et al. (2012) that Na+ concentrations probably increase in mycorrhizal plants with increasing salinity levels up to a certain level and subsequently decrease at higher salt concentrations, G. mosseae alone and with PSM may stimulate Na+ uptake by K. virginica when tissue contents are within acceptable limits. Cantrell and Lindermann (2001) suggested that Na+ might be retained in intraradical AM fungal structures (arbuscules, vesicles), and not allocated to the shoots. However, further research is required to understand the mechanisms of this phenomenon since mycorrhiza formation in K. virginica enhanced Na+ concentrations in the stele which is not colonized by AM fungi. The promoting effects of PSM alone on Na+ accumulation may be related to improved P nutrition and plant growth, which helped improve the uptake of univalent cations. Furthermore, some Mortierella species have been found growing as endophytes in the healthy cortex of plant roots (Kageyama et al. 2008; Osorio and Habte 2013). If the PSM tested in the present study can colonize K. virginica roots, it may accumulate Na+ as it tolerates saline soil and 1.15 % NaCl in culture. The increased K+ accumulation from the epidermis to the stele of K. virginica roots at two salinity levels may result in the transfer of higher amounts of K+ to shoot tissues, enhancing plant growth. The positive correlation of plant dry weight with K+ concentrations in nearly all root tissues supports this hypothesis. Salinity treatment significantly reduced K accumulation in root tissues and was associated with a reduction in plant growth. However, inoculation of K. virginica with both G. mosseae and PSM significantly promoted K+ accumulation in the different root tissues under salt stress conditions, resulting in decreased Na+/K+ ratios in the epidermis, cortex, and endodermis. Higher K+ accumulation by mycorrhizal plants in saline soil could be beneficial if higher K+/Na+ (lower Na+/K+) ratios are maintained and could influence the ionic balance of the cytoplasm or the Na+ efflux from plants (Giri et al. 2007). In the absence of salt stress, inoculation with the two fungi maintained relatively higher Na+/K+ ratios in K. virginica roots, and plants showed highest root and shoot dry weights, compared with non-inoculated plants. These findings indicate that higher Na+/K+ ratios (within limited levels) had positive effects on the growth of K. virginica under non-NaCltreated conditions. However, negative correlations were observed between shoot and root dry weights and Na+/K+ ratios in the epidermis, between AM colonization and Na+/K+ ratios in the epidermis and stele, and between PSM populations and Na+/K+ ratios in the epidermis and cortex. Overall, these results suggest that the capacity of plants to maintain a lower cytosolic Na+/K+ ratio is one of the key determinants of salt tolerance in plants inoculated with an AM fungus and/or PSM (Hajiboland 2013). Salt stress has been reported to inhibit Ca2+ uptake and transport in roots, inducing a higher Na+/Ca2+ ratio (Grattan and Grieve 1999; Evelin et al. 2012; Hajiboland 2013). A

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similar phenomenon was observed in K. virginica where salt stress sharply decreased Ca2+ accumulation and resulted in higher Na+/Ca2+ ratios in root tissues. Ca2+ participates in important processes that preserve the structural and functional integrity of plant membranes, stabilize cell wall structures, and regulate ion transport and selectivity (Maathuis and Amtmann 1999). Although Evelin et al. (2012) reported that higher Ca2+ uptake and Ca2+/Na+ ratios in mycorrhizal plants may mitigate NaCl-induced ionic imbalances, present results with K. virginica showed that neither G. mosseae nor PSM inoculation had significant effects on Ca2+ accumulation in root tissues at 100 mM NaCl, except for that of dual inoculation on Ca2+ concentrations in the epidermis and exodermis. This finding is in agreement with the results of Giri et al. (2004). It is likely that, under salt stress, single inoculation of AM fungi or PSM have no important role in the transport of Ca2+ to the plant roots whilst inoculation with both fungi may have mediated a relevant mechanism for stimulating Ca2+ uptake and transport. Regardless of salinity level, the higher Na+/Ca2+ ratios in root tissues of inoculated K. virginica plants was due to increased Na+ concentrations and decreased Ca2+ accumulation. Although there was no significant correlation between root Ca2+ concentration and plant dry weight, root/shoot dry weight ratio exhibited a positive correlation with Na+/Ca2+ ratiosin the cortex, endodermis and stele. Reduction in Mg2+ uptake may reduce chlorophyll concentrations and photosynthesis in leaves, resulting in a reduction in plant growth (Giri and Mukerji 2004; Sheng et al. 2008). Giri et al. (2003) reported that negative effects on Mg2+ uptake by Acacia auriculiformis imparted by NaCl could be alleviated by mycorrhiza. Similarly, in the present study, inoculation with G. mosseae and PSM significantly affected salt stress-induced decreases in Mg2+ accumulation in different tissues of K. virginica roots, and both AM colonization and PSM populations showed a positive relationship with Mg2+ concentrations in the epidermis and stele. Plant dry weight correlated positively with Mg2+ concentrations in the epidermis and stele. These results suggest that AMF and PSM reduce the antagonistic effect of Na+ (Giri and Mukerji 2004) by helping in the absorption of Mg2+ from the saline soil. Ca2+ is strongly competitive with Mg2+; binding sites on the root plasma membrane appear to have less affinity for highly hydrated Mg2+ than for Ca2+ and salt stress induces lower Ca2+/Mg2+ ratios in plants (Marschner 1995). It has been reported that the mycorrhizal plants maintain higher Ca2+/ Mg2+ ratios in their tissues than their non-mycorrhizal counterparts (Evelin et al. 2012). This was not the case for K. virginica root tissues, except for the epidermis. Furthermore, not only salt stress reduced Ca2+/Mg2+ ratios in root tissues of K. virginica, but also inoculation with G. mosseae and PSM, except for the stele, when compared to non-inoculatedplantsin100 mM NaCl. This maybe

attributable to increased Mg2+ concentrations in the inoculated root tissues as discussed above. Also, the relatively low level of radiation used in this study may have reduced photosynthesis and the need for Mg2+ in shoots, so that more Mg2+ accumulated in roots. Under saline conditions, root cells take up Cl− from the soil solution, resulting in high tissue concentrations that can be toxic to crop plants and may restrict agriculture in saline regions (Xu et al. 2000). In the present study, Cl− accumulation was inhibited by salt stress in all the root tissues of K. virginica and this may contribute to this halophyte's salt tolerance. In an earlier report, mycorrhizal plants were able to reduce the uptake of Cl− (Zuccarini and Okurowska 2008) whilst in the present study, root tissue Cl− concentrations were on the contrary increased by fungal inoculation at both 0 and 100 mM NaCl level. These findings are concurrent with those of Graham and Syversten (1984) who suggested that the carbon drain imposed by mycorrhizal hyphae on plants could enhance the translocation of highly mobile anions like Cl− from the soil (Graham and Syversten 1984). Alternatively, enhanced uptake of essential nutrients like P, N, K, Ca and Mg by mycorrhizal plants grown in saline soil may alleviate the deleterious effects of Na+ and Cl− ions by maintaining vacuolar membrane integrity (Giri et al. 2007; Evelin et al. 2012). In conclusion, all of the above results indicate that co-inoculation with an AM fungus and PSM increases salt tolerance of K. virginica and improves nutrient uptake and translocation under both salt-stressed and unstressed conditions. In non-inoculated plants, 100 mM NaCl decreased nutrient acquisition (root P and K), led to lower accumulation of important ions (i.e., Na+, K+, Ca2+, Mg2+ and Cl−) in different root tissues, and induced higher ionic ratios (Na+/K+ and Na+/Ca2+) and a lower Ca2+/Mg2+ ratio, resulting in an important reduction in shoot dry biomass. The combined inoculation of G. mosseae and PSM alleviated the deleterious effects of 100 mM NaCl on K. virginica and stimulated plant growth principally by enhancing nutrient absorption (P, N and K), increasing K+ and Mg2+ accumulation and maintaining lower Na+/K+ and higher Na+/Ca2+ ratios in each root tissue type, compared to non-inoculated plants. Some researchers have reported that improved salt resistance in plants induced by AM fungi is attributable to physiological processes rather than nutrient uptake (Ruiz-Lozano et al. 2012). This will be the area of future investigations. Acknowledgements This research was supported by a China Postdoctoral Science Foundation funded project (2012 M511728) and National Science Foundation of China (31370533). We are grateful to two

Mycorrhiza anonymous referees for their constructive critical review of a previous draft of this paper.

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