The AMF colonization rate is an important indicator of whether AMF have established a symbiotic relationship with host plants. It can measure the ecological adaptability of AMF and, to a certain extent, also determines plant growth and stress resistance. The results of this study showed that the low phosphorus stress treatments (inoculated or not inoculated with AMF) increased the AMF colonization rate of S. davidii compared with NAM. AMF inoculation significantly increased the AMF colonization rate of the roots of the S. davidii seedlings, which is similar to the findings in Faidherbia albida by Hailemariam et al.. Mycorrhizal plants grown under low phosphorus stress are more responsive and dependent on AMF. Therefore, in a low phosphorus environment, inoculation of AMF can facilitate a good symbiotic relationship with plant roots, thereby enhancing the survival of plants under stress.
3.1 Effects of AMF on the growth mechanism of S. davidii seedlings under low phosphorus stress
AMF are obligate symbiotic fungi, and their main function is to provide mineral elements, especially phosphorus. Phosphorus is an essential mineral element for plants, accounting for 0.2% of the dry weight of plant cells, so plant cell growth requires a large amount of phosphorus. Phosphorus deficiency in soil is the main limiting factor for plant growth, and plant roots are the link between the soil and the plant itself and are the most important organ plant absorption of water and nutrients from the soil environment. A good root system is a prerequisite for plants to adapt to low phosphorus stress. During the process of sensing the changes in nutrients in the environment, roots can produce morphological and physiological changes to cope with environmental stress. Leaves are plant vegetative organs, and their function is to carry out photosynthesis to synthesize organic matter and to facilitate transpiration, providing the root system with the power to absorb water and mineral nutrients from the outside world. From this experiment, without AMF inoculation, root dry weight, root-shoot ratio, total root length, root surface area, root tip number, and root hair number showed an upward trend with the aggravation of low-phosphorus stress. The total root length, root surface area, root tip number and root hair number all reached maximum values under the P0.25 treatment, with levels significantly higher than those under the P0.5 treatment. The plant height and aboveground dry weight decreased gradually with the intensification of low-phosphorus stress. Ting et al found that the phosphorus-efficient Fagopyrum tataricum variety had higher root vigor, root biomass and more developed root systems, which was consistent with the results of this study. This is because under low phosphorus stress conditions, to obtain the phosphorus nutrient elements needed for growth, S. davidii transports more carbohydrates to the roots, increases the biomass of underground roots, increases the root-shoot ratio, promotes root growth, and forms a well-developed root system by increasing root length, total root surface area, and number of root hairs, guaranteeing effective phosphorus absorption to plants. Compared with NAM, inoculation with AMF significantly reduced the root dry weight, root-shoot ratio, total root length, root surface area, root tip number and root hair number under the P0.25 treatment and significantly increased the total root length, root tip number, plant height, growth rate, and aboveground biomass of S. davidii under the P0 treatment. After inoculation with AMF, S. davidii roots formed symbiosis with AMF, and the roots provided nutrients for the mycorrhizal hyphae, which reduced the growth of their own root system and reduced the root biomass; however, with the continuous reduction in phosphorus treatment concentration, AMF can increase total root length and root tip number to increase nutrient uptake to maintain the high biomass of S. davidii under low phosphorus stress, ultimately increasing the growth rate. The promoting effect of AMF on aboveground biomass accumulation and plant height formation under low phosphorus stress was similar to the research results in other plants.
3.3 Effects of AMF on the physiological mechanisms of S. davidii seedlings under low phosphorus stress
Chlorophyll a and chlorophyll b are the most important pigments involved in photosynthesis and have the functions of absorbing, transmitting and transforming light energy. Within a certain range, the chlorophyll content is proportional to the photosynthetic rate, which directly reflects the level of plant photosynthetic capacity. In this experiment, under NAM, low-phosphorus stress reduced the contents of chlorophyll a, chlorophyll b and total chlorophyll in the leaves of S. davidii. Compared with NAM, inoculation with AMF significantly increased chlorophyll a and total chlorophyll contents in the P0.25 and P0 treatments, similar to the findings in Chili by Elahi et al.. Studies have shown that the increase in chlorophyll content may be related to the absorption of phosphorus and magnesium by AMF. In our study, inoculation with AMF significantly increased the leaf phosphorus content in the P0 treatment compared with that in NAM. More phosphorus and chlorophyll contents in leaves provide the basis for maintaining higher photosynthetic capacity.
Plants show a series of physiological adaptation mechanisms under low phosphorus to adapt to these adverse environmental conditions. In this study, when AMF was not inoculated, the contents of proline, soluble sugar and soluble protein in roots increased first and then decreased with the increase in phosphorus stress intensity, which shows that under low phosphorus stress, the osmotic potential of the plant under stress is reduced by these substances, the osmotic potential of the cell is maintained, and the cell is protected so that the plant can adapt to the adversity; however, if the intensity of low phosphorus stress is too high, these osmotic regulators will be destroyed, and their regulatory capacity will be reduced. Compared with NAM, inoculation with AMF increased the osmotic regulators, such as proline, soluble sugar and soluble protein, in roots to a certain extent, all of which were significantly increased under the P0 treatment. This is similar to previous study results. This indicates that AMF symbiosis can induce changes in the secondary metabolism of S. davidii under low phosphorus stress, increase the biosynthesis of phytochemicals and increase the content of osmo-regulatory substances.
Acid phosphatase is an enzyme induced by plant roots according to the amount of external phosphorus. When external phosphorus is deficient, the activity of acid phosphatase in plants increases, thereby increasing the effective phosphorus concentration in the rhizosphere. In this study, when AMF was not inoculated, the acid phosphatase in the roots was significantly increased under the low phosphorus environment and reached the maximum value under the P0 treatment, which is similar to the findings in soybean by NADIRA et al.. Compared with NAM, inoculation with AMF significantly increased the acid phosphatase activity in roots (P0 and P0.5 treatments), possibly because of the symbiotic colonization between AMF and plant roots in root cortex cells to obtain the required carbohydrates; at the same time, mineral nutrients such as N, P, and K can also be transferred from the soil to the root cortex and secrete phosphatases from organophosphorus compounds to hydrolyze phosphate.
Superoxide dismutase, peroxidase and catalase are key enzymes involved in plant stress resistance in the protective enzyme system; they can scavenge the oxygen free radicals generated by the disturbance in plant tissues through oxidation, thereby reducing damage to plants and protecting plants. In this study, under NAM, the activities of superoxide dismutase, peroxidase and catalase in S. davidii roots increased to a certain extent under low phosphorus stress; in particular, catalase and peroxidase activities continued to rise, which is similar to the findings in wheat by Wang et al.. Compared with NAM, AMF inoculation significantly increased superoxide dismutase (P0.25 and P0 treatment), oxidase (P0.25 treatment) and catalase activities (P0.5 and P0. 25 treatment), which shows that the symbiosis between AMF and S. davidii can improve the activity of protective enzymes under low-phosphorus stress, enhance the adaptation to a low-phosphorus environment, and maintain a stable biomass.
Malondialdehyde is the product of membrane lipid peroxidation. When plants are under stress, the accumulation of malondialdehyde increases, which can aggravate cell membrane damage and damage membrane lipids. In this experiment, compared with NAM, inoculation with AMF had no significant effect on malondialdehyde contents in roots because the inoculation of AMF in this study increased the osmotic regulators, such as proline, soluble sugar and soluble protein; moreover, AMF can improve the activities of key protective enzymes in plant stress resistance reactions, such as POD, SOD and CAT, to a certain extent to remove oxygen free radicals in plant tissues and reduce the damage to plants under stress.
3.4 Effects of AMF on the endogenous hormones of S. davidii seedlings under low-phosphorus stress
Endogenous hormones, as important regulators of plant metabolism, are involved in a series of physiological and biochemical processes. In this experiment, when AMF was not inoculated, low phosphorus stress (P0.25 treatment) significantly decreased the contents of BR, GA3 and IAA in leaves and significantly increased the contents of BR, GA3 and IAA in roots. This is because under a low phosphorus environment, plants can induce root structure changes and improve phosphorus utilization efficiency by transporting the accumulated GA3, BR and IAA in leaves to roots. Compared with no inoculation, inoculation with AMF significantly increased leaf IAA contents in the P0.5 and P0 treatments, significantly increased root IAA content in the P0 treatment, significantly increased root GA3 and BR contents under the P0.25 and P0 treatments, and significantly decreased leaf GA3 and BR contents under the P0.5 and P0 treatments. It has been reported that inoculation with AMF significantly increased the content of IAA in roots and leaves of Catalpa bungei C.A.Mey., significantly increased the content of GA3 and BR in roots, and significantly decreased the content of GA3 and BR in leaves to regulate plant growth and improve stress resistance.
3.5 Effects of AMF on the mineral elements of S. davidii seedlings under low-phosphorus stress
Nitrogen is an essential element for plant growth. It is an important part of plant proteins and related enzymes and plays a major role in plant growth. In this study, when AMF were not inoculated, low phosphorus stress significantly decreased the nitrogen content in roots and increased the nitrogen content in stems and leaves, while the changes in nitrogen content in leaves were smaller than those in roots and stems. This is similar to the findings in Zea mays L. by Rafique et al.. This may have been due to the slow growth of plant cells due to the lack of phosphorus, the increase in chlorophyll content, and the increase in nitrogen content in leaves and stems to maintain growth. The fact that low phosphorus stress significantly reduces the nitrogen content of roots is because low phosphorus stress reduces the biomass of plant roots, thereby reducing the ability of plant roots to obtain nitrogen from the outside world. On the other hand, nitrogen is particularly important for plant photosynthesis. Under external stress, to maintain normal growth, plants may choose to transport more nitrogen to the aboveground parts to meet the needs of photosynthesis for nitrogen. Compared with no inoculation, inoculation with AMF significantly increased the nitrogen content in roots (P0.25 and P0 treatments) and decreased the nitrogen content in leaves and stems. This is because AMF can help plant roots obtain nitrogen from the soil, so plants will allocate more carbohydrates to AMF and then obtain more N through AMF, resulting in an increase in the nitrogen content of plant roots, which is similar to the findings in Lolium multiflorum by Liu et al.. In addition, inoculation with AMF increased the adaptation of plants to adversity and promoted the growth of the aerial parts of plants, resulting in a concentration dilution effect, which reduced the nitrogen contents of stems and leaves.
Phosphorus is also one of the essential nutrients for plant growth, and it is the second most important nutrient after nitrogen that limits crop growth. This nutrient is involved in a range of plant processes, such as photosynthesis, respiration, energy production, and nucleic acid biosynthesis, and is a component of some plant structures, such as phospholipids. In this study, when AMF was not inoculated, low phosphorus stress significantly decreased the phosphorus content of leaves and stems (P0 treatment). This is similar to the findings in Zea mays L. by Rafique et al., and occurred because the phosphorus in the plant is obtained from the external environment through the root system. Under phosphorus deficiency, the growth of the plant is inhibited, which reduces the acquisition of phosphorus in the soil by the plant; therefore, the phosphorus content in plant leaves and stems decreases, and when phosphorus is deficient, plant photosynthetic products are preferentially distributed to the underground parts, especially the root tips, to obtain phosphorus, so the phosphorus content of the roots remains unchanged. Compared with NAM, inoculation with AMF significantly increased the phosphorus content of roots and stems (P0.25 and P0 treatments) and significantly increased the phosphorus content of leaves (P0 treatment). Relevant studies have shown that AMF can absorb phosphorus elements within 10 cm of the soil surface through extra root hyphae and then transport the absorbed phosphorus elements to root epidermal cells, where these elements are eventually absorbed by plant cells. AMF can significantly improve the uptake of phosphorus by plants, especially in phosphorus-deficient environments. Under a phosphorus stress environment, the reduction in phosphorus contents in plant leaves and stems will have a significant impact on plant growth, affecting photosynthesis and other physiological processes. Inoculation with AMF can alleviate this adverse effect of phosphorus stress and enhance the adaptation of S. davidii seedlings to phosphorus stress.
The N:P of plant leaves is important for predicting the nutrient limitation of plants. Generally, plants with an N:P ratio less than 14 are in a nitrogen-limited state, and plants with an N:P ratio greater than 14 are in a phosphorus-limited state. Our study showed that without AMF inoculation, the N:P ratio of leaves of S. davidii seedlings under phosphorus stress increased from 17.6 to 18.6, indicating that low phosphorus stress significantly aggravated plant phosphorus limitation. In this study, inoculation with AMF decreased the N:P of leaves, stems and roots compared with those under NAM. Inoculation with AMF can expand the absorption area of plant roots through extraroot hyphae, thereby enhancing the ability to compete for nutrients and reducing the N:P ratio of plants. According to the growth rate hypothesis, living organisms need to make relatively large investments in phosphorus-rich ribosomes and rRNA to support rapid protein synthesis associated with rapid growth. The element stoichiometry leading to fast-growing individuals or taxa would be skewed toward phosphorus, so fast-growing organisms would exhibit lower N:P and C:P ratios. Inoculation with AMF maintained rapid growth under phosphorus stress by reducing the N:P ratio of plants. In addition, our study also found that inoculation with AMF could maintain a constant N:P ratio in leaves under this phosphorus stress. This indicated that the regulation of plant body N:P reached a balance, which could promote the growth of S. davidii seedlings.