Colonization by arbuscular mycorrhizal fungi improves salinity tolerance of eucalyptus (Eucalyptus camaldulensis) seedlings

Soil salinity affects soil quality and reduces plant performance. Arbuscular mycorrhizal fungi (AMF) can enhance the tolerance of plants under salinity stress. Cultivation of eucalyptus (Eucalyptus camaldulensis), which exhibits high water use efficiency, is possible in saline areas to produce raw materials for the pulp industry. We determined the effects of arbuscular mycorrhizal fungi (AMF) on the growth and survival of eucalyptus seedlings under saline conditions. Three different clones of eucalyptus seedlings were pre-inoculated with three salt-tolerant AMF species, namely Glomus sp.2, Gigaspora albida and G. decipiens, and without pre-inoculation. The seedlings were grown in a greenhouse for 45 days. They were then transferred to individual pots, filled with field soil and subsequently treated with NaCl solution until electro-conductivity (EC) reached 10, 15 and 20 dS m−1. They were watered for 90 days under nursery conditions. The results show that increased salinity levels reduced plant performance, fractional AMF root colonization, spore number, and eucalypt K/Na ratio. AMF significantly increased chlorophyll and decreased leaf proline concentrations by more than 50% and 20% respectively and increased the K/Na ratio three- to six-fold compared with non-inoculated plants. Pre-inoculation with AMF before outplanting also improved plant performance by more than 30% under salinity stress compared to non-inoculated plants. We conclude that AMF can alleviate the negative impacts of salinity on plant physiological and biochemical parameters.

Experimental design. The eucalyptus cuttings were transplanted into individual pots that were filled with 20 kg field soil, with the following properties: pH 4.87, EC 5.72 dS m −1 , soil organic matter 3.5 g kg −1 , total N 195 mg kg −1 , total P 50 mg kg −1 , total K 5,950 mg kg −1 , exchangeable Ca 100 mg kg −1 and Na 464 mg kg −1 . The experiment was a 3 × 3 × 4 complete factorial experiment in a randomized complete block design (RCBD) with three salinity levels (10, 15 and 20 dS m −1 ), three eucalyptus clones (H4, H8 and P6) and four AMF treatments (Glomus sp.2 KKU-BH-001, G. albida KKU-BP-001, G. decipiens KKU-BP-002, and a treatment without AMF pre-inoculation). Each treatment had three replicates. After fourteen days, to avoid plant shock from salinity, 5% of NaCl solution was gradually added to the soil every seven days to increase the initial EC from 5.72 (0% NaCl) to 10, 15 and 20 dS m −1 , respectively. All eucalyptus cuttings were watered with 1,000 mL distilled water every three days, and excess water in saucer was reused in order to maintain salinity. Every six days before watering the pots we took soil samples to check the EC. Assessment of plant and fungal performance parameters was conducted at 90 days.
Assessment of plant and fungal parameters. Plant fresh and dry weight (g), and plant height (cm) were measured. Eucalyptus roots were scanned by an Epson scanner V700 PHOTO and analyzed with WIN-RHIZO Pro2004a (REGENT Instruments Inc., Qc, Canada). We assessed root length and root diameter and calculated on that basis specific root length, root surface area, and root tissue density.
Mycorrhizal root colonization was determined after staining with acetic glycerin solution with trypan blue and scoring root fragments with the method proposed by Trouvelot et al. 17,18 . Spore density (number of spores g −1 dry soil) was observed after sucrose centrifugation 19 .
Plant nutrient analysis. Plant N concentration was determined after digestion by the Kjeldahl method and analyzed by the FLA method 20 , while plant P and K concentrations were determined by the wet oxidation method 21 and Na concentration determined by flame photometer 22 .
Leaf relative water content (LRWC). Leaf disc samples (10 mm diameter) were punched from each plant after 90 days to determine the tolerance of mycorrhizal and non-mycorrhizal plants at each salinity level. We calculated LRWC using the following equation 23 : Leaf chlorophyll concentration. Leaf chlorophyll concentration (chlorophyll a, chlorophyll b, and total chlorophyll) was determined by the method described by Arnon 24 . Fresh leaves (0.5 g) were ground with 20 mL of 80% acetone. The homogenate was then centrifuged at 4,000 rpm for 15 min. The supernatant was read using a spectrophotometer (Thermo Scientific GENESYS 10S UV/Vis Spectrophotometer, model EW-02654-22) at absorbance readings at 645 (A645) and 663 (A663) nm. The chlorophyll content was calculated using the following formulae: Proline concentration. Proline concentrations were determined using the method described by Bates et al. 25 . Fresh leaves (0.5 g) were homogenized in 10 mL of 3% sulfosalicylic acid and then sieved through Whatman's No. 1 filter paper. Then 2 mL filtrate solution were mixed with 2 mL of acid-ninhydrin and glacial acetic acid in a test tube, respectively. The reaction mixture test tubes were placed in a water bath at 100 °C for 1 h and then placed in ice to stop the reaction. The mixture was extracted by 4 mL toluene and the chromophore containing the toluene was separated to measure absorbance of 520 nm using a Thermo Scientific GENESYS 10S UV/ Vis Spectrophotometer (model EW-02654-22). The calculated proline concentration was then compared with the proline standard.
Statistical analysis. The treatment effects and the interactions were tested by three-way analysis of variance (ANOVA) using the Statistix program version 8.0. All data complied with the ANOVA assumptions of homoscedasticity and normality. Means were compared between treatments using Tukey's Honestly Significant Difference (HSD) at a 0.05 probability level.

Results and discussion
Results of the analysis of variance are provided in Table 1. In almost all cases, salinity and AMF were significant sources of variation. Interactions between AMF and clone were significant sources of variation (except for leaf P concentration), indicating species-specific AMF responses on different eucalyptus clones. Eucalyptus clone and the other interactions were significant sources of variation for a number of parameters as well.   www.nature.com/scientificreports/ AMF colonization and spore density. Control plants (plants that were not pre-inoculated) were also colonized by AMF, which was caused by the experiment, which, after pre-inoculation or not in sterilized soil, was executed in non-sterile field soil, however, colonization levels were much lower than in the pre-inoculated seedlings. Spore density and fractional root colonization significantly (P ≤ 0.05) declined with increasing salinity levels ( Table 2). Mycorrhizal colonization and spore density were very significantly correlated (r = 0.64; n = 36; P < 0.001). The significant interaction between AMF and salinity level for both parameters (Table 1) indicated that the protective effect of pre-inoculation diminished at higher salinity levels. The interaction between AMF and eucalyptus clone was also significant for fractional root colonization, suggesting species-specific responses to different eucalyptus clones. Root colonization and spore densities with clone H4 and H8 were highest with G. albida, while eucalyptus clone P6 showed highest spore densities and root colonization with Glomus sp.2. AMF are generally characterized as showing little or no host specificity, however plant species or plant variety-specific responses to individual species of AMF have been observed before 26,27 . Our results are consistent with earlier studies that showed that salinity inhibited spore germination, suppressed the growth of hyphae after initial infection, and reduced the number of arbuscules 28-31 . Plant performance. Both AMF and salinity were significant sources of variation for root and shoot biomass, whereas clone was only a significant source of variation for root parameters. The interaction between AMF and clone was also significant, again demonstrating AMF species-specific responses in combination with different clones (Table 1). Salinity decreased plant performance parameters, with a larger effect at higher salinity levels, whereas pre-inoculated plants produced more biomass than control plants. At all salinity levels, plants preinoculated with G. albida usually showed higher biomass than plants pre-inoculated with the other AMF species (Table 3). However, at the salinity level of 15 dS m −1 , eucalyptus clone P6 pre-inoculated with Glomus sp.2, was significantly heavier than when pre-inoculated with the other AMF species. These data fit with the selectivity of the different AMF for different clones as assessed by fractional root colonization and spore density. Negative effects of salinity have been reported for many glycophytes, such as Allium cepa L., Medicago sativa L., Triticum aestivum L. and Hordeum vulgare L. 28,29,32 and the alleviation of these negative effects of salinity by AMF, and plant and fungal species specificity with respect to this protective effect has also regularly been reported 31,33-35 . Leaf relative water content (LRWC) was also significantly affected by salinity (S), AMF, eucalyptus clone (C), and the interaction of AMF × C and S × C (Table 1). Salinity reduced, but mycorrhizal plant increased LRWC. Again, eucalyptus clone H8 that was pre-inoculated with G. albida and clone P6 pre-inoculated with Glomus sp.2, showed the highest positive mycorrhizal effect (Table 3). There are several reasons why the AMF plants have a higher LRWC, (1) AMF roots have higher hydraulic conductivity at low water potential 36 ; (2) AMF induce alterations to the root system 37 ; (3) mycorrhizal plants have higher stomatal conductance 38 ; (4) AMF accumulate solutes and improve plant osmotic adjustment 39 , and (5) improved water relation by AMF hyphae 40 .
Root length and root surface area were both significantly affected by salinity level, AMF and interaction of AMF × C. In the case of root surface area, the interaction of S × C was also significant (Table 1). Root length was significantly positively correlated with LRWC. Root diameter showed a significant negative correlation with root length, specific root length, and root tissue density (Table 4). Salinity reduced, and pre-inoculation with mycorrhiza increased, root length and root surface area (Table 3). Seedlings pre-inoculated with Glomus sp.2 had larger root diameter than control seedlings and seedlings pre-inoculated by both Gigaspora species, an effect described before 41 and likely due to hormonal effects. Table 2. Effect of salinity on AMF spore density (spore number g −1 dry soil; SD) and intensity of root colonization (I) of three eucalyptus clones (H4, H8, P6) pre-inoculated with various species of AMF after 90 days of cultivation at three salinity levels. AMF1; Glomus sp.2, AMF2; G. albida, AMF3; G. decipiens, and control (C); without pre-inoculation. Values followed by different letters, per salinity level and clone, are significantly different (P ≤ 0.05) by HSD.      Table 4. Correlations between eucalyptus root architecture and leaf relative water content. **and ns significant at P ≤ 0.01 and non-significant probability levels, respectively. www.nature.com/scientificreports/ Plant nutrient concentration. AMF, salinity, and eucalyptus clone were all significant sources of variation, and many interactions were significant as well (Table 1). Especially the interaction of AMF × S was significant for N, Na and the K/Na ratio, but not for P and K. Concentrations of N, P and K in plant shoots decreased with high salinity, while those of Na increased. The mycorrhizal effect on lowering Na concentrations was stronger than the mycorrhizal effect in increasing K concentrations; in combination, pre-inoculation with AMF increased the K/Na ratio three-to sixfold. Pre-inoculation with AMF increased leaf nutrient concentrations compared to the non-inoculated control across all salinity levels. Eucalyptus clones H4 and H8 benefitted most when preinoculated with G. albida, showing higher N, P, K, and lower Na concentrations than the control whereas P6 was positive when pre-inoculated with Glomus sp.2 (Table 5). Many studies have reported that increasing salinity levels lowered N and K concentrations, for example in pepper (Piper nigrum L.), olive (Olea europaea L.), peanut (Arachis hypogaea L.) and faba bean (Vicia faba L.) [42][43][44][45] . High concentrations of K can maintain K/Na ratio and photosynthetic rate. Higher phosphorus (P) uptake in all pre-inoculated plants is consistent with the major role of AMF in extending the depletion zone of P in the rhizosphere and increasing P uptake. Both a higher-affinity uptake system and a lower threshold concentration for absorption by AMF than by plant roots are major mechanisms of higher P uptake 46,47 . Table 5. Influence of different salinity levels on nitrogen (N), phosphorus (P), potassium (K) and sodium (Na) concentrations and K/Na mass ratio in eucalyptus shoot tissue. AMF1; Glomus sp.2, AMF2; G. albida, AMF3; G. decipiens, and control (C); without pre-inoculation. Values followed by different letters, per salinity level and clone, are significantly different (P ≤ 0.05) by HSD.

Salinity levels
Treatments N (mg kg −1 ) P (mg kg −1 ) K (mg kg −1 ) Na (mg kg −1 ) K/Na ratio for plant photosynthetic capacity, was significantly affected by all three main factors (salinity, AMF, eucalyptus clone) and by all two-way and three-way interactions (Table 1). Salinity significantly reduced leaf chlorophyll concentration ( Fig. 1) likely caused by repression of specific enzymes of the photosynthesis system and reduction of nutrient uptake such as Magnesium (Mg) and Nitrogen (N) for chlorophyll biosynthesis 48,49 . Mycorrhiza significantly increased leaf chlorophyll concentration. This result is likely due to enhanced nutrient uptake and reduced Na concentrations in the plants, resulting in overall higher photosynthetic capability 50 . In some combinations of eucalyptus clone and AMF species, there was a major effect when increasing salinity levels from 10-15 dS m −1 , whereas in other combinations a major decline was observed only when salinity increased from 15 to 20 dS m −1 . Due to the fact that two-way and three-way interactions were significant, other patterns were difficult to explain. Eucalyptus clones H4 and H8 pre-inoculated with G. albida had higher chlorophyll concentration compared to other AMF treatments, while eucalyptus clone P6 pre-inoculated with Glomus sp.2 had higher leaf chlorophyll concentration than the other AMF treatments.
Leaf proline concentration. The accumulation of free amino acid, proline-reported modifications induced by water and salt stress, and an exogenous application of proline could play an important role in enhancing plant stress tolerance 3,49 . In saline conditions, plants can accumulate proline as a protective osmolyte, maintain an osmotic balance, stabilize proteins and membranes, protect plants against free radical-induced damage, and maintain appropriate NADP + /NADPH ratios 51,52 . Our study resulted that leaf proline concentrations were significantly affected by all main factors (AMF, S, C) and all two-way and three-way interactions (Table 1). Proline concentrations increased with increasing salinity and were lower in AMF pre-inoculated seedlings compared with control plants, At the lowest salinity level there were significant differences between varieties, with H8 showing lowest proline concentration and H4 showing highest concentrations. With increasing salinity levels, the differences between the eucalyptus clones attenuated. Clones H4 and H8 pre-inoculated with G. albida and P6 pre-inoculated with Glomus sp.2 had significantly lower proline concentrations across all salinity levels (Fig. 2). Proline concentrations were negatively correlated with the concentrations of chlorophyll a, chlorophyll b, and total chlorophyll (Table 6). Apparently, higher nutrient uptake, LRWC, and chlorophyll content due to the mycorrhizal symbiosis constitute an alternative way to alleviate salt stress without increasing proline production. Many authors have reported that proline concentrations increased in AMF plants compared to non-AMF  www.nature.com/scientificreports/