Introduction

Given that plants are confined to their growing locations, they are typically unable to avoid unpredictable and unfavourable changes in their environment (Bussotti and Pollastrini 2021). Acid rain (AR) has become a growing environmental issue worldwide, and ongoing global changes are likely to enhance the severity and exposure of plants to this problems in the coming decades (Andrade et al. 2020; Liu et al. 2019). In China, over the past three decades, more than 30% of the land area has suffered from AR, particularly in southern China (Liang et al. 2016; Wei et al. 2017; Zhang et al. 2007). Zhejiang Province is an economically developed region in southeastern China, and in recent years, with the rapid growth of the economy and the increasing intensity of fuel consumption, 91.3% of the county-level cities received AR pollution in 2016, with sulfuric AR being the main type of AR (Zhejiang Ecology and Environment Bureau 2016). Furthermore, economic losses associated with AR in China are estimated to reach 110 billion RMB each year (Bao et al. 2020; Singh and Agrawal 2008; Wei et al. 2017). Therefore, AR has aroused widespread public concern, with tree damage and loss of particular concern because of their vulnerability to this form of pollution (Larssen et al. 1999; Li et al. 2021; Liu et al. 2019).

AR usually has adverse effects on a range of soil properties, microorganisms and microbial processes, and plant morphological traits, which can subsequently affect plant physiological functioning (Bao et al. 2020; Gilani et al. 2020; Rodriguez-Sanchez et al. 2020; Singh and Agrawal 2008). Compared to other plant organs, leaves are much more sensitive to AR (Macaulay and Enahoro 2015). For example, as one important group of leaf functional traits, photosynthetic parameters are considered the best indicators for understanding and evaluating the effects of AR on plant performance (Wang et al. 2017). Nevertheless, the photosynthetic responses of plant species to acidic conditions differ with the intensity of AR. For example, the photosynthetic activity of Platanus occidentalis is decreased at pH 2.0 (Singh and Agrawal 2008), and the chlorophyll content and maximum quantum efficiency of PSII photochemistry (Fv/Fm) are reduced in Camellia sinensis at pH 3.5 (Zhang et al. 2020). For soybean (Glycine max), chlorophyll content and net photosynthesis rate are impeded at pH 4.5 (Wen et al. 2011). All of these effects decrease plant production. Effort is currently being made to reduce such deleterious effects using appropriate measures, such as liming, and adopting policy measures to control the emission of acid precursors (i.e., SO2 and NOX). However, these approaches are often either expensive or unpractical (Rengel 2003; Singh and Agrawal 2008). Among the available biotechnological methods, generating mutualistic symbioses between plants and arbuscular mycorrhizal fungi (AMF) has proved effective under acidic conditions (Aguilera et al. 2015; Vosátka and Dodd 1998).

It is generally accepted that AMF can form mycorrhizal associations with approximately 80–90% vascular plant species, which facilitate mineral nutrient acquisition (such as N or P) from the soil. In return, the host plants provide carbon compounds for fungal development (Albornoz et al. 2020; Grilli et al. 2014; Smith and Read 2008). There is strong evidence that AMF can benefit plants by stimulating mineral nutrient absorption, enhancing photosynthesis, improving soil properties, and increasing tolerance to abiotic stresses (Al-Karaki 2006; Anwar et al. 2019; Li et al. 2021; Ruiz-Lozano et al. 2012). It has been reported that AMF can survive in acidic soils with pH values ranging from 2.7 to 9.2, while pH tolerance appears to vary between different fungal isolates (Clark 1997; Date et al. 1995; Liu et al. 2020; Rohyadi 2008). Some studies have shown that AMF can stimulate the growth of grasses and crop species under acidic conditions (Medeiros et al. 1994; Rohyadi 2008; Vosátka and Dodd 1998); however, few studies have been conducted on trees. Furthermore, while promoting nutrient acquisition is the prominent effect of AMF on plants under unfavorable conditions, Albornoz et al. (2020) recommended that non-nutritional mycorrhizal efficiency deserves more attention. Indeed, the photo-physiological mechanisms underlying the beneficial mycorrhizal effects on tree species subject to AR remain poorly understood.

Zelkova serrata (Thunb.) Makino, a Class II protected tree species in China, is a culturally and economically valuable indigenous species in Zhejiang Province, widely used in the construction, horticultural, shipbuilding, and pharmaceutical industries (Wang et al. 2019). It has been reported that Z. serrata can form mycorrhizal associations with enhanced photosynthetic ability under stressful conditions, and frequent AR events have become a severe limiting factor for the survival and growth of Z. serrata (Jiang et al. 2012; Wang et al. 2019; Zhu et al. 2018). Therefore, this study was conducted based on the hypothesis that AMF can mediate the adverse effects of AR on the performance of Z. serrata. Furthermore, previous studies suggest that the efficacy of mycorrhizal fungi in alleviating plant stress is usually stress-dependent (van der Heijden and Sanders 2003) and varies with AMF species (Amanifar and Toghranegar 2020). Therefore, we also hypothesized that the protective effects of AMF under acidic conditions may vary with the intensity of AR and fungal species. Here, we describe a factorial experiment investigating the effects of various AMF types on the growth and photosynthesis of Z. serrata seedlings under simulated AR conditions. In doing so, we sought to better understand the potential application of AMF in vegetation restoration and afforestation programs.

Materials and methods

Plant materials, AMF inoculum, and soil

Z. serrata seeds were obtained from Richu Seeds Co., Ltd., China. After being surface sterilized with 5% sodium hypochlorite for 10 min and rinsed with distilled water, the seeds were sown in autoclaved river sand in a growth chamber set to a 19/15 °C day/night regime with a 12/12 h photoperiod. After emergence, 100 seedlings of similar size were selected for the following treatments.

Two broad-spectrum mycorrhizal fungal species, Diversispora versiformis (P. Karst.) Oehl, G. A. Silva and Sieverd (BGC GD01C) and Rhizophagus irregularis (N.C. Schenck & G.S. Sm.) C. Walker & A. Schüßler (BGC BJ09) were provided by the Glomales Germplasm Bank of the Beijing Municipal Academy of Agriculture and Forestry Science in China. D. versiformis and R. irregularis were originally isolated from Eremochloa ciliaris rhizosphere in Shaoguan, Guangdong Province, southern China, and from tomato plants in Langfang, Hebei Province, northern China, respectively. Moreover, D. versiformis and R. irregularis are acidic- and non-acidic-tolerant fungi, respectively. These two AMF were propagated individually using Sorghum bicolor as trap plants in plastic pots with sterilized fine sand as the substrate (Qiu et al. 2020). After five months of culturing, the fungal inoculum collected from the host rhizosphere consisted of spores, hyphae, sand, and colonized root fragments. Both types of AMF contained approximately 210 spores per 10 g of soil and were subjected to the highest possible number test.

The soil used in the experiment was composed of a mixture of field soil and peat (3:1, w/w), which was sterilized with γ-irradiation at a dose of 25 kGy (McNamara et al. 2003). The sterilized growth medium had the following properties: organic matter = 22.1 mg g−1, total N = 0.85 mg g−1, Olson P = 0.42 mg g−1, and pH = 5.6 (water:soil = 5:1).

Experimental design

The experiment was conducted using a full factorial design consisting of 12 factorial combinations of AR regimes and AMF regimes. Three AR regimes (pH 5.6, 4.0, or 2.5) and four AMF inoculation regimes (inoculated with sterilized inoculum or R. irregularis and D. versiformis, either alone or the combination) were tested, with five replicates for each treatment combination. On May 11, 2018, the seedlings were moved into a greenhouse located at the Pingshan Research Station of Zhejiang A & F University (30°15 N, 119°43 E) for acclimation. Nine days later, 60 seedlings of similar size were selected and transplanted into plastic pots (20.5  × 21  × 16.5 cm; one seedling per pot). These pots contained 4 kg of sterilized soil medium. The seedlings were replaced with new, healthy ones if they died within one month. Immediately before transplanting, the corresponding inoculum was placed below the roots of the seedlings. The AMF-inoculated plants (AM plants) received 40 g of R. irregularis, D. versiformis, or the combined inoculum comprising an equal proportion of the two AMF taxa per plant. Non-mycorrhizal plants (NM plants) were provided with the same amount of autoclaved mixed inoculum with 40 mL of soil extract to compensate for the differences in soil microflora (Evelin et al. 2012). To avoid acid shock during fungal establishment, we began the AR treatment on June 20, 2018. To simulate the AR status in most regions of Zhejiang Province, a stock acid solution was prepared with H2SO4 and HNO3 at a ratio of 8:1 (Zhang et al. 2007). Furthermore, in southeastern China, the pH of AR varies between 4.5 and 3.5, reaching as low as 2.95 in some cities (Cao et al. 2009; Huang et al. 2008; Niu et al. 2017; Zhang et al. 2007). Therefore, the stock acid solution was subsequently diluted to pH 5.6, 4.0, and 2.5 with distilled water (the average pH was approximately 6.8). According to the mean annual precipitation in Hangzhou, Zhejiang Province (Song et al. 2015) and the surface area of the plant pots, the daily spraying amount from June to December was calculated. Thus, 398 mL of the corresponding acid solution was applied to each pot every two days. The pot positions were changed every two weeks to reduce edge effects. The experiment ended on December 26, 2018, lasting for more than six months. During the experiment, the average temperature and relative humidity in the greenhouse were 28.2 °C and 68.3%, respectively, and the mid-day photosynthetic photon flux density was approximately 950 μmol m−2 s−1.

Determination of leaf chlorophyll fluorescence and photosynthetic parameters

Before harvesting, chlorophyll fluorescence parameters (Fv/Fm, the maximum quantum yield of PSII; Yield, the actual photosynthetic quantum yield; qN, non-photochemical quenching; and qP, photochemical quenching) were measured using a portable chlorophyll fluorometer (PAM-2500 WALZ, Effeltrich, Germany) following the saturation pulse method as described by Wang et al. (2019). Four plants from each treatment combination and three fully expanded leaves on the upper part of the plants were randomly selected for chlorophyll fluorescence measurements. Before measurement, the leaves were dark-adapted for 30 min, and then the determination was made at 1-h intervals between 9:00 am and 14:00 pm.

Subsequently, photosynthetic parameters (A, leaf net photosynthetic rate; gs, stomatal conductance; and E, transpiration rate) were determined for the same leaves as used for the chlorophyll fluorescence measurements using a LI-6400 portable photosynthesis system (LI-COR, Inc., Lincoln, USA). During these measurements, the leaf temperature was set at 27 °C in line with the mean ambient temperature and the relative humidity of the leaf chamber was set at 70%. Measurements were taken with a photosynthesis active radiation of 1200 μmol m−2 s−1, an airflow of 500 μmol s−1, and an ambient CO2 concentration of 400 μmol mol−1 (León-Sánchez et al. 2016). Instantaneous water use efficiency (WUE) was also calculated as A/E.

Measurements of leaf area and plant biomass

At the end of the experiment, one fully expanded fresh leaf was selected randomly from each replicate pot. These leaves were then scanned (Epson V330, Japan) and their leaf areas (LA) determined using Image J software (1.44p; National Institutes of Health, Bethesda, MD, USA) (Wang et al. 2018). Specific leaf area (SLA) was calculated as the leaf area divided by the dry biomass of each leaf. Subsequently, the plants were rinsed with distilled water three times and separated into roots and shoots. The dry weights of the roots and shoots were then weighed after oven-drying at 70 °C for 48 h.

Determination of mycorrhizal colonization and fungal activity

After harvesting, 3 g of root samples were taken randomly from three plants in each treatment combination. The root samples from each replicate pot were then evenly divided into three subsamples; the first subsample was stained with adjusted trypan blue (Wang et al. 2018) and microscopically examined for mycorrhizal colonization (hyphae or vehicle) using the gridline intersect method (Giovannetti and Mosse 1980); the second and third subsamples were used to measure succinate dehydrogenase (SDH) activity (Zhao et al. 1997) and alkaline phosphatase (ALP) (Tisserant et al. 1993) in the hyphae.

Statistical analysis

To quantify the mycorrhizal efficiency, mycorrhizal growth response (MGR) values were calculated as follows (Johnson et al. 2015):

$${\text{MGR}} = \ln \left( {{{{\text{DW}}_{{{\text{AMF}}}} } \mathord{\left/ {\vphantom {{{\text{DW}}_{{{\text{AMF}}}} } {\overline{{{\text{DW}}_{{{\text{non}} - {\text{AMF}}}} }} }}} \right. \kern-\nulldelimiterspace} {\overline{{{\text{DW}}_{{{\text{non}} - {\text{AMF}}}} }} }}} \right)$$
(1)

where \({\text{DW}}_{{{\text{AMF}}}}\) and \(\overline{{{\text{DW}}_{{\text{non - AMF}}} }}\) represent the total dry weight of the mycorrhizal plants and mean dry weight of the non-mycorrhizal plants under the same pH conditions (n = 5), respectively.

The acid tolerance index (ATI) of each plant was determined as follows (He et al. 2019):

$${\text{ATI}} = 1 - {\raise0.7ex\hbox{${\left( {B_{i,\max } - B_{i} } \right)}$} \!\mathord{\left/ {\vphantom {{\left( {B_{i,\max } - B_{i} } \right)} {B_{i,\max } }}}\right.\kern-\nulldelimiterspace} \!\lower0.7ex\hbox{${B_{i,\max } }$}}$$
(2)

where Bi,max is the largest shoot biomass of the five plants at pH 5.6 with identical AMF inoculation, and Bi is the shoot biomass of each of the five plants at pH 4.0 and 2.5, respectively (i.e., the stressful pH conditions applied in our study). In this experiment, target species with ATI values of 0.3 < ATI < 0.6 and ATI > 0.6 were considered moderately acid-sensitive and acid-tolerant, respectively.

A two-way ANOVA (SPSS 23.0; SPSS Inc., Chicago, IL, USA) was performed to estimate the responses of different plant parameters to the AR and AMF treatments. Before analysis, all data were subjected to Levene’s tests to check for equality of variance, and the Shapiro–Wilk test was used to test for normality. When the interactive effects of AR and AMF were significant (P < 0.05), the Fisher’s least significant difference test was applied to compare differences between the treatments. Otherwise, pairwise trait relationships between all the measured Z. serrata parameters were assessed via Pearson’s correlation analysis across the 12 treatment combinations in the R 4.0.3 statistical platform (http://www.R-project.org/).

Results

Mycorrhizal colonization and AMF activity

The percentage of root colonization was significantly higher in the mycorrhizal (AM) plants compared to the non-mycorrhizal (NM) plants (Fig. 1a, Fig. S1). The maximum percentage colonization (60%) was observed in the roots of the combined inoculated Z. serrata seedlings at pH 5.6. The root colonization of the combined inoculated plants tended to slowly decrease, while colonization of the plants inoculated with R. irregularis initially increased and then typically decreased under the harsher acidic conditions. In contrast, the mycorrhizal colonization of the D. versiformis-inoculated plants drastically increased with increasing AR intensity. Therefore, D. versiformis was notably more tolerant of the acidic experimental conditions. Non-inoculated seedlings also exhibited a small degree of root colonization (ranging from 1.33 to 2.46%), but there were no differences between the pH levels. Mycorrhizal colonization was significantly dependent on the fungal species as well as the interaction of the AR treatment and AMF, but not by AR alone (Table 1).

Fig. 1
figure 1

Effects of arbuscular mycorrhizal fungi on mycorrhizal coloniztion (a), succinate dehydrogenase (SDH) (b) and alkaline phosphatase (ALP) (c) of Zelkova serrata under sulfuric acid rain (SAR) with pH 2.5, 4.0 and 5.6. NM, Ri, Dv and Ri + Dv represent the four AMF treatments: inoculation with sterilized mycorrhizal fungi, with Rhizophagus irregularis, Diversispora versiformis, either alone or the combination, respectively. Values are presented as the means ± SE (n = 3). Two-way ANOVA is performed to compare the effects of acid rain and mycorrhizal inoculation and their interactions on plants. Different letters indicate a significant difference according to LSD at P < 0.05

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Table 1 F values of Two-way ANOVA for the effects of acid rain (AR), arbuscular mycorrhizal fungi (AMF) and their interactive effects on the parameters of Zelkova serrata
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Both AR and AMF had significant effects on SDH (Table 1). Specifically, at lower pH values (pH 4.0 and 2.5), the SDH activity of the hyphae in the AM plants significantly increased, with the exception of the D. versiformis-inoculated plants (Fig. 1b). The highest proportion of SDH-active hyphae was recorded in the plants inoculated with the combined fungi at all pH values. The ALP activity of the hyphae in the AM plants significantly increased as pH decreased, specifically in the combined inoculated plants, in which approximately 57% of the hyphae showed ALP activity under the most acidic treatment (Fig. 1c). Moreover, the proportions of metabolically active (SDH and ALP) hyphae in the D. versiformis-inoculated and NM plants were consistent across all pH values.

Plant growth

AR alone significantly decreased the total dry weight of the NM plants. In comparison, the total dry weights of the AM plants were significantly higher, with the highest values recorded for the R. irregularis-inoculated plants at pH 5.6 and the D. versiformis-inoculated plants at lower pH values (pH 4.0 and 2.5) (Fig. 2a). AR alone had no effect on the root:shoot ratio, although the effects of AMF on the ratio depending on the pH and AMF species (Fig. 2b). Significant interactive effects of AR and AMF were detected on total dry weight and the root:shoot ratio (Table 1).

Fig. 2
figure 2

Effects of arbuscular mycorrhizal fungi on total dry weight (a) and root: shoot ratio (b) of Zelkova serrata under sulfuric acid rain (SAR) with pH 2.5, 4.0 and 5.6. NM, Ri, Dv and Ri + Dv represent the four AMF treatments: inoculation with sterilized mycorrhizal fungi, with Rhizophagus irregularis, Diversispora versiformis, either alone or the combination, respectively. Values are presented as the means ± SE (n = 5). Two-way ANOVA is performed to compare the effects of acid rain and mycorrhizal inoculation and their interactions on plants. Different letters indicate a significant difference according to LSD at P < 0.05

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Leaf photosynthetic parameters and chlorophyll fluorescence

AR, AMF, and their interaction had significant effects on A and gs; additionally, AR alone and the interaction between AR and AMF had significant effects on E and WUE (Table 1). AR alone had negative effects on A, gs, and WUE (Fig. 3). There were no differences in A between the AM and NM plants at pH 5.6, whereas under the lower pH conditions (pH 4.0 and 2.5), the A of the AM plants was significantly higher than that of the NM plants, with the highest values observed for the D. versiformis-inoculated plants (Fig. 3a). Except for the E of the plants with mixed inoculation at pH 4.0, AMF inoculation had no positive effects on gs, E, and WUE at any pH level (Fig. 3b–d).

Fig. 3
figure 3

Effects of arbuscular mycorrhizal fungus on net photosynthetic rate (A) (a), stomatal conductance (gs) (b), transpiration rate (E) (c) and instantaneous water-use efficiency (WUE) (d) of Zelkova serrata under sulfuric acid rain (SAR) with pH 2.5, 4.0 and 5.6. NM, Ri, Dv and Ri + Dv represent the four AMF treatments: inoculation with sterilized mycorrhizal fungi, with Rhizophagus irregularis, Diversispora versiformis, either alone or the combination, respectively. Values are presented as the means ± SE (n = 4). Two-way ANOVA is performed to compare the effects of acid rain and mycorrhizal inoculation and their interactions on plants. Different letters indicate a significant difference according to LSD at P < 0.05

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AR alone exhibited no effect on Fv/Fm but had a slight effect on qN; AMF alone had no effect on these two variables (Table 1; Fig. 4a, b). With an increase in AR intensity, the Yield and qP of the plants initially decreased and then frequently increased. At pH 5.6, the Yield and qP of the AM plants were lower than for the NM plants; at lower pH values (pH 4.0 and 2.5), although there were no differences in Yield and qP between the AM and NM plants, the highest values were detected in the D. versiformis-inoculated plants. Significant interactive effects of AR and AMF were detected in the case of Fv/Fm, Yield, and qP (Table 1).

Fig. 4
figure 4

Effects of arbuscular mycorrhizal fungus on the maximum quantum yield of PSII (Fv/Fm) (a), non-photochemical quenching (qN) (b), the actural photosynthetic quantum yield (Yield) (c), and photochemical quenching (qP) (d) of Zelkova serrata under sulfuric acid rain (SAR) with pH 2.5, 4.0 and 5.6. NM, Ri, Dv and Ri + Dv represent the four AMF treatments: inoculation with sterilized mycorrhizal fungi, with Rhizophagus irregularis, Diversispora versiformis, either alone or the combination, respectively. Values are presented as the means ± SE (n = 4). Two-way ANOVA is performed to compare the effects of acid rain and mycorrhizal inoculation and their interactions on plants. Different letters indicate a significant difference according to LSD at P < 0.05

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Mycorrhizal efficiency and acid tolerance

AR alone did not affect ATI, whereas AMF treatment had a significant effect in this regard (Table 1). The ATI values of the plants inoculated with D. versiformis, R. irregularis, and the mixture of the two were 175.6, 115.1, and 84.7% higher at pH 4.0, and 121, 63.4, and 127.6% higher at pH 2.5, respectively, compared to those of the NM plants (Fig. 5a). Mycorrhizal efficiencies (MGR) also varied with pH and AMF species (Fig. 5b). Specifically, at pH 5.6 and 4.0, the MGR values of D. versiformis and R. irregularis alone were higher than those of the mixture of the two, while under the most acidic condition (pH 2.5), the MGR value of D. versiformis was higher than those with the other two mycorrhizal inoculations. Furthermore, MGR was negatively correlated with A, gs and WUE, whereas ATI was positively correlated with A (Fig. S2).

Fig. 5
figure 5

Effects of arbuscular mycorrhizal fungi on acid-tolerance index (ATI) (a) and mycorrhizal growth response (MGR) (b) of Zelkova serrata under sulfuric acid rain (SAR) with pH 2.5, 4.0 and 5.6. NM, Ri, Dv and Ri + Dv represent the four AMF treatments: inoculation with sterilized mycorrhizal fungi, with Rhizophagus irregularis, Diversispora versiformis, either alone or the combination, respectively. Values represent means ± SE (n = 5). Two-way ANOVA is performed to compare the effects of acid rain and mycorrhizal inoculation and their interactions on plants. Different letters indicate a significant difference according to LSD at P < 0.05

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Discussion

Our results show that AR had negative effects on A, gs, E, and total dry weight, whereas AMF inoculation, especially with D. versiformis, positively influenced ALP, SDH, total dry weight, A, and acid tolerance under acidic conditions compared to the NM controls. Nevertheless, mycorrhizal efficiencies varied with the intensity of AR. These observations support our hypotheses that AMF can mediate the detrimental effects of AR on Z. serrata seedlings, and mycorrhizal efficiencies varied with different AR levels and with different AMF isolates.

Biomass can be an appropriate indicator of plant growth and development under AR stress, which reflects differences in resource capture and biomass production (Dovrat et al. 2019; Liu et al. 2019). In our study, AR alone substantially decreased the total dry weight of NM plants (Fig. 2a), and we found that pH 4.0 was critical for the growth of Z. serrata seedlings compared to pH 3.0 in the case of rice (Oryza sativa) and pH 4.5 for soybean (Glycine max) (Liang et al. 2020). Inoculation with all three of the AMF treatments (R. irregularis, D. versiformis, and in combination) significantly increased the total biomass of Z. serrata seedlings under acidic conditions (i.e., pH 4.0 and 2.5). Such mycorrhizal efficiency conforms with observations of 24 tropical forage legumes and grasses inoculated with a combination of Glomus manihotis, Acaulospora longula, and Entrophospora colombiana at pH 4.36 (Saif 1987), Calamagrostis villosa inoculated with A. tuberculata at pH 3.2 (Vosátka and Dodd 1998), S. bicolor inoculated with G. deserticola at pH 4.5 (Raju et al. 1988), and Torreya grandis inoculated with mycorrhizal fungi at pH 4.0 (Xia et al. 2021). In contrast, mycorrhizal inoculation was found to have no effect on the biomass accumulation of Deschampsia flexuosa grown under acidic conditions (Vosátka and Dodd 1998). Thus, mycorrhizal benefits depend on the fungal taxa, the host plant species, and the intensity of AR. Additionally, the AMF inoculation in our study negatively affected biomass allocation to roots (Fig. 2b), which conforms to the responses observed in 14 other host plant species (Saif 1987) but is contrary to previous observations of cowpea plants (Rohyadi et al. 2004). Dovrat et al. (2019) postulated that variations in biomass partitioning could reflect species adaption and adjustment to environmental perturbations. Such a response indicates that mycorrhizal fungi prefer the development of shoots rather than roots, thereby potentially protecting shoots from acid deposition.

Photosynthesis is a primary process affected by AR (Du et al. 2017). We observed significant reductions in the A, gs, and E of the non-mycorrhizal Z. serrata plants as the intensity of AR was increased (Fig. 3), which is in accordance with previous studies on Pinus massoniana (Tong and Zhang 2014) and rice (da Fonseca et al. 2020). Jiao et al. (2017) suggested that photosynthetic activity could be regulated by stomatal factors (i.e., conductance and stomatal behaviors) as well as non-stomatal factors. This is also supported by our observation that stomatal factors, such as lower gs, could be related to reductions in A. However, the AM plants had higher A, gs, and E values than the NM plants in the lower pH treatments, which is consistent with the observations under salt stress made by Ruiz-Lozano et al. (2012) and Wu et al. (2010). The mycorrhizal-induced enhancement in photosynthetic ability could modulate the damage to photosynthetic organelles incurred by over-reduction of the reaction centers in the Photosystem II of plants subjected to AR stress (Ruiz-Lozano et al. 2012). Furthermore, this photosynthetic improvement would increase carbon assimilation rates, enhance its availability for plant growth and fungal development, and, thereby, improve plant acid tolerance as seen from the positive relationship between ATI and A (Fig. S2). However, in our experiment, mycorrhizal efficacy was negatively correlated with photosynthetic capability (Fig. S2), suggesting that some other mechanisms was responsible for the observed responses. Clark (1997) suggested that the enhanced uptake of some commonly deficient minerals (P, Ca, Mg, and K) could be responsible for the beneficial effects of mycorrhizal fungi in plants grown under acidic conditions, which has been verified for T. grandis under acidic conditions (Xia et al. 2021). Thus, the importance of AMF in buffering the detrimental effects of AR on Z. serrata plants warrants further examination.

Based on our observations, mycorrhizal efficacy is expected to vary with the intensity of AR, which is in disagreement with the previous theoretical models that predict that the magnitude of positive effects of neighbors on a target species will be higher under harsh abiotic stresses (Bertness and Callaway 1994). Raju et al. (1988) and Medeiros et al. (1994) reported similar mycorrhizal effectiveness under AR stress in sorghum. Nevertheless, other studies report that mycorrhizal benefits are enhanced under more acidic conditions (Chen and Lei 2019; Rohyadi 2008; Xia et al. 2021). Furthermore, in our experiment, plant parameters responded differently even when inoculated with the same fungal species under AR stress. For example, the total dry weight of the D. versiformis-inoculated plants was constant across all pH levels, whereas the photosynthetic capabilities (e.g., A and gs) of these plants decreased at lower pH values (Figs. 2, 3). As Maestre et al. (2005) suggest such departures from theoretical predictions can be induced by local environmental conditions, meaning that the variability in the estimators of plant performance can occur within target plant species.

Moreover, there were differences in mycorrhizal efficiencies between the AMF isolates, with D. versiformis proving more effective than both R. irregularis alone and their use in combination. It has been suggested that AR can have negative effects on the germination of spores, germ tube growth, the development of extra-radical mycelia, and mycorrhizal viability (expressed by SDH and ALP activities in hyphae) following colonization of the host plant (Liu et al. 2020; Vosátka and Dodd 1998; Vosátka et al. 1999). These effects are dependent on host preferences, pH tolerance, and functional diversity (Aguilera et al. 2015). For example, the optimal pH for R. irregularis is approximatly 5.0 (Medeiros et al. 1994), compared to that ranging from pH 3.8 to 8.0 for D. versiformis (Sieverding 1991). In our experiment, D. versiformis was isolated from acid soils and R. irregularis was isolated from agricultural soils with neutral pH, indicating that the former was more tolerant of acidic conditions. Smith et al. (2004) argued that such functional differences between fungal isolates may reflect stress-specific adaption mechanisms, such as variations in P uptake. Our results indicated that individual mycorrhizal species, i.e., D. versiformis in this case, provide a greater degree of acid tolerance in Z. serrata plants than when occurring in combination (i.e., with R. irregularis). Similarly, Xia et al. (2021) found that individually, R. irregularis had a more beneficial effect on T. grandis than in combination with Funneliformis mosseae. These observations provide empirical evidence for functional redundancy, as suggested by Gosling et al. (2016), although there is some evidence of enhanced nutrient uptake and plant growth as a result of functional complementarity of dual inoculation (Jansa et al. 2008; Koide 2000). Such contradictory resluts may be attributed to variations in the physiology of the different fungal isolates and host plant species or the experimental procedures and conditions employed (Jakobsen et al. 1992).

Conclusions

This study demonstrates that AR negatively affects the growth and photosynthetic characteristics of Z. serrata seedlings, while these detrimental effects were mitigated by AMF inoculation, especially in the case of D. versiformis. Furthermore, the magnitude of these effects varied with AR intensity and the type of AMF. Therefore, our observations indicate that AMF inoculation could offer a viable management strategy in areas subject to AR pollution, although two factors require further consideration. First, our experiment was conducted under greenhouse conditions, thus eliminating plant competitive effects. Second, mycorrhizal efficacy is affected by several environmental factors, and nitric AR is becoming more common than sulfuric acid deposition in some areas. Therefore, field trials are now required to examine the efficacy of mycorrhizal inoculation of Z. serrata under different AR conditions (both sulfuric and nitric types, and with different intensities) to further inform the development of AR mitigation strategies.