Content area
Background
Nitrogen (N) deposition has become a major driving factor affecting the balance of terrestrial ecosystems, changing the soil environment, element balance and species coexistence relationships, driving changes in biodiversity and ecosystem structure and function. Human-induced nitrogen input leads to a high NH4+/ NO3- ratio in soil. However, relatively few studies have investigated the effects of different nitrogen sources on forest plant-microbial symbionts. In this study, the effects of four nitrogen sources, N free, NH4Cl, L-glutamic acid, and Na(NO3)2 (N-, NH4+-N, Org-N, and NO3--N) on four fungal species, Suillus granulatus (Sg), Pisolithus tinctorius (Pt), Pleotrichocladium opacum (Po), and Pseudopyrenochaeta sp. (Ps), which were isolated from the roots of Pinus tabulaeformis, were studied in vitro. The effects of inoculation with the four fungi on the growth performance, nutrient uptake and nitrogen metabolism-related enzymes of Pinus tabuliformis under different nitrogen source conditions were subsequently studied.
Results
The biomass and N concentration of the Sg and Po strains were the highest under the NO3--N treatment, while the biomass and N concentration of the Pt and Ps strains were significantly greater under the NH4+-N and NO3--N treatments than under the Org-N and N- treatments. All four fungi could effectively colonize the roots of P. tabuliformis and formed a symbiotic relationship with it. Under all nitrogen conditions, the inoculation of the four fungi had positive effects on the growth, root development and nutrient concentration of the P. tabuliformis seedlings. Under the Org-N and NO3--N treatments, the nitrate reductase (NR) activity of the inoculated plants was significantly greater than that of the noninoculated control (CK) plants. Under all nitrogen conditions, the glutamine synthetase (GS) activity of the inoculated plants was significantly greater than that of the CK plants.
Conclusions
The four fungi can establish good symbiotic relationships with P. tabuliformis seedlings and promote their growth and development under different nitrogen source treatments.
Introduction
With human activities, nitrogen deposition increases gradually, long-term nitrogen inputs can lead to changes in species structure and there are differences in the responses of different ecosystems to nitrogen deposition [1,2,3]. Ammonium N is currently the dominant form of N deposition, but the deposition rates of nitrate N are increasing [4]. However, there is limited research on the impact of N forms on plant-fungal interactions. It has been shown that organic N favors mycorrhizal symbiosis, whereas mineral N has a detrimental effect on plants, and that alterations in belowground microbial communities and their functions are closely related to changes in the components of the N source [5]. Some plant symbiotic fungi play a positive role in forest ecosystems by forming reciprocal symbiotic relationships with their host plants and providing water and inorganic nutrients to their hosts, and changes in their communities may also affect the growth of their host plants [6]. Therefore, studying the effects of different nitrogen forms on fungi and their symbionts will help to reveal plant growth strategies and ecosystem stability maintenance mechanisms in the context of global climate change.
In China, more than 30% of ectomycorrhizal fungi (ECMFs) have been identified in pine forests [7], and the main hosts studied in the northern region are P. tabulaeformis, Larix gmelinii and Pinus sylvestris [8]. Zhang et al. [9] reported that association with all three ECMFs could promote the shoot and root growth of P. tabulaeformis, increase the proportion of large roots, improve root vitality, and increase survival rates three months after inoculation. Nitrogen uptake by mycorrhizal fungi is an important pathway for plant nitrogen uptake [10]. 75% of the total nitrogen in the plant root system comes from uptake by the extraradical mycelia of the ectomycorrhizal fungi, which are symbiotically associated [11]. ECMF inoculation has been shown to enhance Populus tremula × tremuloide ammonia N absorption [12]. Under conditions of low NH4+-N and NO3--N availability, ECMF can enable plants to utilize otherwise inaccessible N sources [13]. However, different ECMFs have different preferences for the form of nitrogen source. Moreover, Termitornyces albuminosus is more sensitive to the variability of nitrogen sources, and its ability to utilize nitrate N and organic N is superior to that of ammonium N [14]. Nitrogen is an important nutrient element limiting the growth and reproduction of fungi, so experiments are needed to determine the optimum nitrogen conditions for the growth of different ECMFs and to provide theoretical guidance for the practical use of these strains.
The ecological significance of dark septate endophytes (DSEs), which are a broad group of fungi with the same wide distribution as ECMFs, cannot be underestimated [15, 16]. Studies have shown that DSEs can also form symbiotic relationships with most plants and that DSEs have functions similar to those of mycorrhizal fungi. The presence of a large number of extraroot mycelia of DSEs in the rhizosphere environment can enhance the absorption of host plants to mineral elements in the surrounding environment, such as by promoting the absorption and transport of N and P by host plants [17,18,19], significantly increasing the biomass of host plants [20, 21], improving the photosynthetic efficiency of host plants, promoting the metabolism of host plants, and changing the rhizosphere microenvironment of host plants [22,23,24]. The positive effects of inoculating plants with DSEs are more evident in plants supplied with organic sources of N and P than in those supplied with inorganic sources [25]. Fully understanding and mastering the biological characteristics of strains, so that strains and hosts form a good symbiotic relationship, is a prerequisite for the function of DSEs. The response of strains to nitrogen sources is fundamental in the study of their biological properties. At present, studies identifying a suitable nitrogen source for DSEs are rare, and further discussion and in-depth studies are needed to establish a foundation for the application of DSEs.
Pinus tabulaeformis plantations are crucial forest ecosystems in northern China. As the dominant tree species for greening and afforestation in northern China, P. tabulaeformis is also a typical mycorrhizal tree species [26]. However, due to the slow growth of P. tabulaeformis seedlings and the long seedling raising period, artificial vegetation construction methods that promote and accelerate the growth of P. tabulaeformis plants are urgently needed. It has been widely reported that plant symbiotic fungi including mycorrhizal fungi and endophytic fungi can improve host growth and stress resistance [27]. It is highly important to explore efficient growth-promoting fungal resources and construct an excellent fungus-tree combination system for P. tabulaeformis forestation. At the same time, globally, the ongoing deposition of high levels of anthropogenic N, primarily in mineral form, poses a major ecological threat [5]. An increase in global nitrogen deposition and the deposition ratio of nitrate N will inevitably impact and alter plant symbiotic fungi, thereby directly influencing the growth of the host plant P. tabulaeformis. This raises the question of how an unbalanced N cycle and different nitrogen forms impact plant‒microbe symbiosis. The beneficial associations of plant‒microbe symbioses in response to different nitrogen forms remain poorly understood. The strength of nitrogen metabolism is extremely important for plant growth and development. NR and GS are closely related to the assimilation, absorption, and utilization of nitrogen by plants, which can improve the nitrogen use efficiency of plants [28]. Therefore, in this study, we conducted two experiments using four strains isolated from P. tabulaeformis to test (1) their growth performance in vitro under different nitrogen sources and (2) the effects of inoculation with these four strains on the growth and nitrogen metabolism of P. tabulaeformis plants under different nitrogen source conditions.
Materials and methods
Experimental material
The four fungal strains used in this study were two ECMFs, Suillus granulatus (OK335188.1) and Pisolithus tinctorius (PP531177.1), and two DSEs, Pleotrichocladium opacum (OK335191.1) and Pseudopyrenochaeta sp. (OK335190.1), which were isolated from the roots of P. tabulaeformis in the Wuling Mountain Nature Reserve, Hebei Province (117°17′–117°35′E, 40°29′–40°38′N) and preserved in the Laboratory of Garden Plant Ecology, Hebei Agricultural University, China. Preliminary experiments on the nitrogen concentration stress tolerance of seven fungi were conducted previously, and these four dominant fungi were selected for the nitrogen source stress experiments by analysing and comparing the growth and nutritional indicators of the seven fungi. The collection of the roots of P. tabulaeformis was authorized by the local agricultural administration. The identification of these four fungi was conducted through examination of their morphological characteristics and phylogenetic analyses of nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequences (Fig. S1, 2). These fungal isolates were cultured on potato dextrose agar (PDA) plates and maintained at 27 °C.
P. tabulaeformis seeds were collected from the same mother tree in the P. tabulaeformis forest in Wuling Mountain Nature Reserve and identified by the researchers of our group. We obtained permission from local authorities to collect the seeds, which were then stored at the College of Landscape Architecture, Hebei Agricultural University. The P. tabulaeformis seeds were disinfected with 0.5% KMnO4 solution for 1 h, followed by rinsing with sterile water 3–4 times. The seeds were subsequently soaked in warm water at 40℃ for one day and then placed on filter paper in a Petri dish. Finally, the seeds were placed in a constant-temperature incubator at 25 °C to promote germination [29]. After germination, the seedlings were transplanted into a pot (with a diameter of 15 cm and a height of 18 cm) at a density of three plants per pot. The pots were filled with 2 kg of autoclaved substrate, which consisted of sand and soil (sourced from the campus of Hebei Agricultural University) in an equal volume ratio (1:1) [30]. The seedlings were cultivated in the greenhouse of Hebei Agricultural University.
Effects of the amendment of different nitrogen sources on fungi in vitro
Nitrogen-free Modified Melin-Norkrans (MMN) medium was utilized as the basal medium. Three different nitrogen sources, NH4Cl, L-glutamic acid and Na(NO3)2, were individually supplemented, and the control group (N-) did not receive any additional nitrogen (N-, NH4+-N, Org-N, and NO3--N). The nitrogen concentration in the medium was 0.053 g·L− 1 for each nitrogen source [30]. After the fungi were cultured in MMN solid media for 20 days, the colony diameter was measured. The fungi were incubated in MMN liquid media on a constant-temperature shaking bed for 20 days, after which the mycelial biomass and nitrogen concentration were determined [31].
Pot experiment of P. tabulaeformis
The pot experiment was performed in a greenhouse using a completely randomized design in a 5 × 4 factorial arrangement with fungal inoculation treatment (noninoculated control (CK), Sg, Pt, Po and Ps) and nitrogen source amendment treatment (N-, NH4+-N, Org-N, and NO3--N) as the variables. Each treatment was replicated five times, for a total of 100 experimental pots. An Erlenmeyer flask containing 150 ml of MMN liquid medium was inoculated with three 6 mm fungal mycelia discs, and the flask was placed on a constant-temperature shaking bed for oscillation culture to obtain fungal inoculums. The plants were inoculated with 40 ml of fungal inoculum, while the CK plants were treated with an equal amount of sterile MMN liquid media [32].
Forty days after inoculation, the mycorrhizal infection rate was detected via microscopic examination and the staining methods of Phillips and Hayman [33]. After the formation of mycorrhizae, different nitrogen source amendment treatments were applied. The different nitrogen source treatments were controlled by the addition of modified Hoagland nutrient solution [34], with NH₄Cl, L-glutamic acid, and Na(NO3)2 as the three nitrogen sources and the nitrogen concentration in each treatment was 40 kg·hm− 2·yr− 1 [30]. The control group (N-) did not receive any additional nitrogen and there were four different nitrogen source treatments (N-, NH4+-N, Org-N, and NO3--N).
Sample collection and determination
The ectomycorrhizal root colonization rates were estimated by microscopic examination of mycorrhizal and nonmycorrhizal root tips in the seedling roots, and the percentage of root tips with obvious ectomycorrhizal structures was calculated for each treatment [35]. The colonization rates of dark septate endophytic fungi were estimated by trypan blue staining and microscopic examination of the fungal structures within the roots, and the percentage of root segments with fungal structures in each treatment was calculated [33].
The plants were harvested after 10 months of treatment and the plant height and ground diameter were measured with a Vernier calliper. The roots of the plants were separated from the aboveground parts, placed on trays and scanned by a scanner (EPSON Perfection V800 Photo, Japan). The total root length, root surface area, root volume and average root diameter were measured by a WinRHIZO image analysis system [36]. The aboveground and underground parts of the plants were put into envelopes, subjected to a temperature of 105 °C for 20 min and then dried at 80 °C to constant weight to determine the shoot biomass and root biomass.
The mycorrhizal growth response (MGR) of P. tabulaeformis was calculated according to the following equation [37]: MGR (%) = 100 × (M – NMmean)/NMmean), where M is the plant total dry weight in the given replicate of the fungal inoculation treatment and NMmean is the mean total dry weight in the corresponding noninoculated treatment. A positive MGR indicated that plant growth was promoted by fungi, and negative values indicated that plant growth was suppressed by fungi.
The dried shoots and roots were ground into powder and passed through a 2 mm sieve. The powder (approximately 0.5 g) was heated and digested in a digestion oven until it became colorless and transparent. The N concentrations of the plants were analysed by the Kjeldahl method, and the P concentrations were measured by the molybdenum-antimony colorimetric method [38].
The nitrate reductase (NR) activity of the needles was determined by an in vitro method [39]. The glutamine synthetase (GS) activity of the needles was quantified by a spectrophotometric method [40].
Statistical analysis
Data analysis was performed with R language (version 4.3.3) using two-way analysis of variance (ANOVA). Differences were considered to be significant at p < 0.05. Two-way ANOVA was performed to analyse the effects of the root symbiotic fungal species (F) and the different N source amendment treatments (N) on the growth and physiological characteristics of the four fungi and P. tabulaeformis. The data are expressed as the means with standard errors. The values presented in the figures and tables represent the means of at least three independent replicates.
Results
Experiment 1
Colony diameter of the four fungi
The different nitrogen sources had different effects on the colony diameters of the four fungi (Figs. 1 and 2). The colony diameters of the Sg, Po and Ps strains were greatest under the NO3--N treatment (Fig. 1A). The colony diameter of the Pt strain under the NH4+-N treatment displayed higher values than that under the other treatments (Fig. 1A). The colony diameter of the four strains was the smallest in the N- treated group, which was lower than that in the other three nitrogen source treatment groups (Fig. 1A). The colony morphology of the four fungi differed under different nitrogen source treatments (Fig. 2). Under the N- treatment, only the colonies of Po strain were large but sparse. The four fungi grew better under the NH4+-N and NO3--N treatments, and the colonies were large and dense in texture.
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Biomass of the four fungi
The effects of the different nitrogen sources on the biomass of the four fungi were similar to those on colony diameter. Under the NO3--N treatment, the biomasses of the Sg, Po and Ps strains were significantly greater than those under the other treatments (Fig. 1B). The biomass of the Pt strain under the NH4+-N and NO3--N treatments was greater than that under the other treatments (Fig. 1B). The biomass accumulation of the two dark septate endophyte strains, Po and Ps, was greater than that of the two ectomycorrhizal fungal strains, Sg and Pt, under all nitrogen source treatments.
Nitrogen concentration of the four fungi
The interaction between fungal species and nitrogen sources significantly affected the nitrogen concentration (Table S1). The N concentrations of the Sg and Po strains reached their highest values under the NO3--N treatment. The N concentrations of the Pt and Ps strains were greatest under the NH4+-N treatment (Fig. 1C). In addition to the N- treatment, the N concentration of the two ectomycorrhizal fungal strains was lower than that of the two dark septate endophyte strains.
Experiment 2
Growth performance of P. tabulaeformis
After inoculation, both Sg and Pt infected the roots of P. tabuliformis plants and established ectomycorrhizae. The root tip system of P. tabuliformis plants inoculated with Sg was enlarged, as indicated by the presence of a circular shield and an elongated bar (Fig. 3B-D). The root tip system of P. tabuliformis plants inoculated with Pt formed bifurcated branches and clustered branches (Fig. 3F-H). In the cortical intercellular space of the seedling roots inoculated with Po and Ps, coarse mycelia with obvious septa could be observed (Fig. 3I, J), exhibiting varying septa spacings. Simultaneously, after the mycelia invaded the root cells, the cell wall thickened, expanded, became tightly stacked and formed different shapes of cell clusters, resulting in the formation of a microsclerotium structure (Fig. 3K, L). The roots of the noninoculated plants had none of the above characteristics (Fig. 3A, E). The results of two-way ANOVA of the inoculation treatments showed that the colonization rates of all the treatments reached a maximum under the NO3--N treatment, and the colonization rates of Pt, Po and Ps were greater than 80%, which was significantly greater than that of Sg (Fig. 4A).
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Under all nitrogen source treatments, the growth indices of the Pt-, Po- and Ps-inoculated plants significantly increased compared to those of the noninoculated plants. Under the NH4+ -N treatments, the growth indices of the Pt-inoculated plants were significantly greater than those of the Sg-inoculated plants (Fig. 5). There was a significant interaction effect between fungal inoculation and nitrogen source amendment on the mycorrhizal growth response of the seedlings (Table S2). Under all nitrogen source treatments, the values of the mycorrhizal growth response were positive, indicating that inoculation had a positive effect on the growth of P. tabulaeformis seedlings (Fig. 4B). Under the N- and NO3--N treatments, the mycorrhizal growth responses of the Po-inoculated plants were greater than those of the other inoculated plants and were significantly greater than those of the Sg-inoculated plants.
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Root morphology and development of P. tabulaeformis
The total root length, root volume and average root diameter of the Pt-inoculated plants were greatest under the NH4+-N treatment and were significantly greater than those of the noninoculated plants (Fig. 6). The root surface area, root volume and average root diameter of the Sg-, Po- and Ps-inoculated plants were greatest under the NO3 --N treatment. Under the N- treatment, the total root length, root surface area, root volume and average root diameter of the Pt- and Po-inoculated plants were significantly greater than those of the noninoculated plants, and the average root diameter of the Ps-inoculated plants was significantly greater than that of the noninoculated plants.
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Nutrient uptake of P. tabulaeformis
The N: P ratios of the Pt, Po and Ps plants were significantly greater than those of the CK plants (Fig. 7). Under the N- treatment, the concentrations of N and P in the Po-inoculated plants were significantly greater than those in the noninoculated plants (Fig. 7). Under the NH4+-N treatment, the concentrations of N and P in the Pt- and Ps-inoculated seedlings were significantly greater than those in the noninoculated seedlings. Under the Org-N treatment, the N concentrations of the Pt-, Po- and Ps-inoculated plants and the P concentrations of the Po-inoculated plants displayed higher values than those of the noninoculated plants. Under the NO3--N treatment, the N concentrations of the Pt-, Po- and Ps-inoculated plants and the P concentrations of the Po- and Ps-inoculated plants were significantly greater than those of the noninoculated plants.
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Nitrogen metabolism-related enzyme activities of P. tabulaeformis
The NR activities of needles of seedlings reached their maximum under the NO3--N treatment, whereas the GS activities of needles of seedlings were maximal under the NH4+-N treatment (Fig. 8). Under the N- and NH4+-N treatments, the NR activities of the Pt-, Po- and Ps-inoculated plants significantly increased compared to those of the noninoculated plants (Fig. 8A). Under the Org-N and NO3--N treatments, the NR activities of all the inoculated plants were significantly greater than those of the noninoculated plants. Under all nitrogen source treatments, the GS activity levels of all the inoculated plants were greater than those of the noninoculated plants (Fig. 8B).
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Correlation analysis
Correlation analysis of different inoculation treatments on the growth and physiological indexes of P. tabuliformis under nitrogen source conditions showed positive correlations between growth performance and nutrient parameters and nitrogen metabolism-related enzyme activities (Fig. 9). The root indicators of Pt-inoculated seedlings were significantly positively correlated (p < 0.05) with NR activity and GS activity (Fig. 9B). The plant height, ground diameter, root biomass and total biomass of Po- and Ps-inoculated seedling showed significant positive correlations (p < 0.05) with shoot N concentration and root N concentration (Fig. 9C, D). These results indicate that the nitrogen metabolism-related enzyme activities of P. tabulaeformis significantly affect root morphology and development. Po-and Ps-inoculated seedlings have a high nutrient uptake capacity, which promotes growth and biomass accumulation.
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PCA analysis
PCA was used to assess the similarity among different inoculation treatments and the relationships between the indicators, including growth performance, root morphology and development, nutrient composition and nitrogen metabolism-related enzyme activities (Fig. 10). The first principal component (PC1) and the second principal component (PC2) explained 78.7% and 7.8% of the variance, respectively. There were significant differences between the control and inoculated samples under nitrogen source treatments (Fig. 10). The results suggest that inoculation treatments can regulate root development and alter root architecture, thereby enhancing plant uptake of nutrients from the soil and ultimately affecting biomass accumulation.
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Discussion
Due to their different physiological characteristics and ecological environments, different fungi have different nitrogen requirements, and the uptake of different nitrogen sources varies among different fungal species. It has been reported that most ECMFs absorb and utilize NH4+-N, which is less able to absorb and utilize NO3--N [5]. However, the research by Liao et al. [41] showed that the growth of three strains of Lactarius deliciosus supplied with NO3--N was greater than that of those supplied with NH4+-N, and the NR activities significantly improved, indicating that the former had a greater effect on promoting their growth. In this study, Pt and Ps strains could absorb NO3--N and NH4+-N, possibly because when the nitrate reductase activity in the strain is high, the mycelium preferentially absorbs NO3--N. However, in the late growth stage of the mycelium, the presence of methionine in the strain can inhibit the synthesis of other amino acids and affect the nitrate reductase activity, causing the strain to absorb NH4+-N in the late growth stage [42].
Usually, the stronger the mycorrhizal infection ability of plant roots is, the more favourable the plant growth is [43]. Therefore, the mycorrhizal infection ability of plant roots can reflect their nutrient utilization status [44]. In this study, the mycorrhizal infection ability of Po and Ps was similar to the absorption ability of the two fungi under pure culture conditions for different nitrogen sources. This suggests that different fungi have different absorption rates of different nitrogen sources, which may lead to their mycorrhizal infection ability being similarly affected. In addition, a recent study has shown that inorganic N availability alters host receptivity to ECMFs [45]. These results thus suggest that the beneficial activities of root-associated microbes are conditional upon the different N forms present. The results of this study showed that the root fungal inoculation promoted the increase in plant height, ground diameter and biomass. Our observations were consistent with those of previous studies [46, 47]. Plants can absorb nitrate N and ammonium N from the soil via the mycelia. After nitrate N is absorbed by mycorrhizal epiphytic mycelia, mycorrhizal fungi secrete nitrate reductase and nitrite reductase, which generally reduce nitrate N to form NH4+. NH4+ combines with carbohydrates to form various amino acids, which are subsequently transported to the host plant for use, promoting the absorption of nitrogen nutrients in the soil by the host plant and thereby promoting the growth of the host plant.
Root length, surface area and volume are important indices for measuring the distribution range of seedling roots in soil, and the average root diameter is an important index for measuring root absorption [48]. In the present study, inoculation promoted the growth of roots, but the effects of different fungal strains differed. This finding is consistent with previous research results on the improvement of plant root indices by mycorrhizal fungi [49, 50]. The reason may be that after plant roots form mycorrhizae, the extraroot mycelia can form a strong network structure to promote the indirect absorption of water and mineral nutrients, greatly improving the ability of the roots to absorb water and mineral nutrients [51]. The aboveground parts obtain sufficient nutrients and water, improving primary productivity, increasing the amount of carbohydrates allocated to the roots, promoting the development of forked branches, and improving space occupancy ability by increasing secondary branches so that they can effectively obtain water and nutrients.
The mycorrhizal symbionts that form between mycorrhizal fungi and plants significantly affect the absorption of nitrogen and phosphorus nutrients by plants [52]. It has been found that P. tabulaeformis plants inoculated with ECMFs have greater biomass and greater N, P and K concentrations than nonmycorrhizal plants [29]. This study also revealed that the N concentrations in the inoculated plants were greater than those in the noninoculated plants and that the shoot N concentration was higher than root N concentration in inoculated plants. These findings show that root fungal inoculation helps plants absorb nitrogen and that nutrients are preferentially supplied to organs with vigorous metabolism. The aboveground part is the organ for photosynthesis and the most vigorous part of the plant body. Thus, the shoot nitrogen concentration was greater than the root nitrogen concentration. Although it has been established that fungi play a key role in the N nutrition of their host trees. Little is known about the molecular mechanisms associated with the utilization of different N forms in fungal-plant symbioses. The transfer of NH4+ from fungal hyphae into the symbiotic interface has been found to be mediated by fungal plasma membrane transporters, followed by uptake by plant ammonium transport proteins localized at the root cell plasma membrane [53]. The N: P ratio can intuitively reflect the growth of plants restricted by N or P to some extent. Studies have shown that the average N: P thresholds of terrestrial plants are 12 and 13, respectively. An N: P ratio < 12 indicates that plant growth is mainly restricted by N [54,55,56]. In this study, the root N: P values of the Pt-, Po- and Ps-inoculated plants were significantly greater than those of the noninoculated plants, but the mean N: P ratios were all less than 12, indicating that the growth of the P. tabulaeformis plants in this study was restricted by N and that the inoculation treatment could alleviate this restriction to a certain extent. It was also found that when nutrient limitation changes, the root system and microorganisms may adjust the production of relevant enzymes to ensure that plants and microorganisms balance the dynamic equilibrium between nutrient demand and supply from the surrounding environment [57].
Different nitrogen forms have important effects on nitrogen metabolism in plants. Ammonium N can increase the activity of glutamine synthetase [58]. The results of this study also showed that exogenous ammonium N application increased the activity of glutamine synthetase in P. tabulaeformis seedlings, while nitrate N application increased the activity of nitrate reductase more than ammonium N application. In the present study, the nitrate reductase and glutamine synthetase activities of the Pt-, Po- and Ps-inoculated plants under all the nitrogen source treatments were significantly greater than those of the noninoculated plants, indicating that the inoculation treatment improved the activities of nitrogen metabolism-related enzymes and thus enhanced the nitrogen assimilation ability of the plants. Khan et al. [59] also reported that mycorrhizal fungi could increase the activities of multiple nitrogen assimilation enzymes in Leymus chinensis, which was beneficial to the nitrogen assimilation process of the plant. In conclusion, root fungal inoculation can promote the growth and nitrogen use efficiency of P. tabulaeformis plants. The application of fungi has certain significance for promoting plant growth and alleviating the environmental pollution caused by nitrogen loss, which provides a solution to the problems of nitrogen oversaturation and land nutrition imbalance caused by the current excessive use of nitrogen fertilizer.
Conclusions
The four fungi isolated from P. tabulaeformis differ in their absorption and utilization of different nitrogen sources under pure culture in vitro, but inoculation with these four fungi can improve growth, root development, nutrient absorption and enhance the activity of nitrogen metabolism-related enzymes in host plants under different nitrogen sources treatments. Thus, symbiotic fungi can directly affect the growth of plants in the short term and may affect the carbon and nutrient cycles of ecosystems in the long term. Therefore, with global environmental changes, it is urgent to study the response of symbiotic fungi to environmental changes and their impact on the surrounding microenvironment. However, other biochemical indexes of the fungi were not evaluated in this study and the interaction mechanism of fungal plant symbiosis has not been revealed at the molecular level.
Future studies should adopt more effective methods, such as amplification and sequencing, to study the effects of symbiotic fungi on plants and nutrient cycling from the perspective of the mechanism involved.
Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.
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