AMF uses Funneliformis mosseae (Fm) (BGC HK01) and Glomus etunicatum (Ge) (BGC GZ03C), the inoculant is a mixture of alfalfa plant roots, mycorrhizal fungal spores and extrarhizosphere mycelium, fungal spore density of 20~30 piece·g^-1, purchased from Qingdao Agricultural Mycorrhizal Research Institute, China. AMF can form a good symbiotic relationship with the roots of most terrestrial plants, construct a root network in the soil, promote the absorption and utilization of nutrients by host plants, and thus plant growth can be promoted. Due to the drought and high light intensity in Xinjiang, WL366HQ was selected as the test plant for alfalfa varieties. The variety has a fall dormancy level of 5, high yield and cold tolerance. It can better adapt to the local environment in Xinjiang and is widely planted in Xinjiang.

Because the climatic conditions in Xinjiang are dry, the precipitation is scarce, and the soil is mostly gray desert soil, the soil used for potting is gray desert soil, which is collected from the 2nd Experimental Station of Shihezi University (44°18′N, 86°03′E), and the soil is naturally dried and sieved through 0.5 cm to remove stones and plant roots from the soil. The physical and chemical properties of the soil are shown in Table 1 , the soil is sterilized at 121°C high temperature and humid heat for 2 h, and then naturally cooled for backup, and the potting substrate is sterilized soil and perlite 3:1 (V:V) mixed to prevent soil compaction.

The experiment was designed with two factors of complete randomization, with two factors: fungi application and phosphorus application. Fungi application was set at 4 levels: 10 g of Funneliformis mosseae (Fm, T₁), 10 g of Glomus etunicatum (Ge, T₂), 5 g of Funneliformis mosseae and 5 g of Glomus etunicatum (Fm×Ge, T₃), with no fungi as the control (T₀). Four more phosphorus levels were set under each application condition: P₂O₅ 0 (P₀), 50 (P₁), 100 (P₂) and 150 (P₃) mg·kg^-1, and a total of 16 treatments were performed with 10 replicates of each treatment, for a total of 160 pots, and the pots were randomly placed.

The potting experiment was carried out in 2021-2022 in the experimental park of the College of Agriculture, Shihezi University (44°18′N, 86°03′E), and the experiment used pots with a size of 23 cm×17 cm×15 cm (pot mouth diameter× pot floor diameter × pot height), each pot containing about 3 kg of soil substrate, and the pots were disinfected with 75% alcohol before sowing. High-quality alfalfa seeds of uniform size and full grains were selected, sterilized with 10% H₂O₂ for 10 min, and then rinsed repeatedly with distilled water, and sown on May 1, 2021, with 10 seeds evenly distributed per pot. After the alfalfa three-leaf stage, five robust and uniform alfalfa seedlings were retained. In order to simulate the natural growth conditions of alfalfa, pots were placed in sunny places, and phosphorus-free Hoagland ‘s nutrient solution and equal amount of water were applied weekly during alfalfa seedlings. The phosphate fertilizer used was monoammonium phosphate (containing P₂O₅ 52%, N 12.2%), with its main component being P₂O₅, which has good water solubility and can be better absorbed and utilized by alfalfa, because the phosphate fertilizer had a small amount of N, so a certain amount of urea (containing N 46%) was supplemented under different phosphorus gradients in this experiment to make the N content under different phosphorus gradients consistent to offset the influence of nitrogen content in phosphate fertilizer on the experiment results. The fertilizer is applied with water droplets using the drip irrigation method of fertilizer with water, and the specific fertilization time is June 23, August 1, 2021, and April 16 and June 7, 2022. Alfalfa is harvested at the beginning of flowering (10%), on July 14, August 23, 2021, May 15, and June 30, 2022. Due to the insufficient environmental conditions for the first measurement of photosynthetic gas exchange parameters, the indicators were measured in the second stubble.

In pots, three pots of alfalfa plants with uniform growth in the second stubble of the two years were selected, and the aerial part of the alfalfa plant was cut 2 cm from the surface of the potted soil for weighing, and the fresh weight was recorded. The fresh sample of alfalfa was dried at 105°C for 30 min, and then dried to constant weight at 65°C, the moisture content was determined and the dry matter yield was converted, the calculation formula was as follows: Dry matter yield = plant fresh weight × (1-moisture content), where moisture content refers to the calculation based on plant fresh weight.

Chlorophyll is responsible for capturing light energy and driving the photosynthetic process. It is the most important pigment in photosynthesis. Chlorophyll a and chlorophyll b in fresh alfalfa leaves were extracted by absolute ethanol method (Cui and Yang, 2011), and the absorbance values were determined by spectrophotometer, and the total chlorophyll a, chlorophyll b and chlorophyll content in alfalfa leaves were calculated.

Under cloudless conditions with sufficient light, the photosynthetic gas exchange parameters of well-grown alfalfa leaves were randomly determined by measuring the first flowering stage (10%) of the second stubble of two-year potted alfalfa on August 20, 2021 and June 27, 2022 at 11:00~13:00 a.m. The healthy intact and fully expanded leaflets in the middle of the third triple compound leaf of alfalfa plants from top to bottom were selected, and the photosynthetically active radiation (PAR) was controlled at about 1500 μmol·m^-2·s^-1, and each treated sample was measured in 3 replicates for analysis and averaged. Specific photosynthetic gas exchange parameters include net photosynthetic rate (P_n, which represents the rate of carbon dioxide absorption and assimilation during photosynthesis, μmol·m^-2·s^-1), stomatal conductance (G_s, which is the degree of stomatal opening, mmol·m^-2·s^-1), transpiration rate (T_r, which represents the amount of water transpiration per unit leaf area in a certain period of time, mmol·m^-2·s^-1), intercellular CO₂ concentration (C_i, which reflects the utilization efficiency of CO₂ by plants, μmol·mol^-1), light use efficiency (LUE, which is an index to measure the light use efficiency and production level of crops in a certain area, mmol·mol^-1) and alfalfa leaf instantaneous water use efficiency (WUE, which reflects the water utilization capacity of plant leaves, μmol·mmol^-1), in which the light energy use efficiency and alfalfa leaf instantaneous water use efficiency are calculated by formulas, the specific calculation formula is as follows: LUE=P_n/PAR; WUE=P_n/T_r.

Microsoft Excel 2010 was used for data processing, DPS 7.05 software (Data Processing System, China) was used for statistical analysis of data, two-factor experiment in a completely randomized design was used to statistically analyze the interaction between fungus treatment (T), phosphorus treatment (P) and fungus phosphorus (T×P), multiple comparisons were made by Duncan’s new complex polarity difference method, and the mapping software used Origin 2021 (OriginLabOriginPro, USA).

Because the performance of each treatment is different in different indexes, it is not comprehensive to evaluate the optimal bacterial phosphorus treatment with any single index (Zhang et al., 2017a). The membership function analysis method is used to comprehensively evaluate the optimal treatment, and the specific formula is: UX(+) = (X_ij-X_imin)/(X_imax-X_imin); UX(-) =1-UX(+), where X_ij is the measurement value of the jth index in the ith treatment; X_imax and X_imin are the maximum and minimum values of the jth indicator in all treatments, respectively. UX(+) is the value of the positive indicator membership function, and UX(-) is the value of the negative indicator membership function. The range of membership function values is 0-1.

According to the test results, dry matter yield of alfalfa exhibited an increase at lower P doses (P₀-P₂) and a decrease at higher doses (P₂-P₃) within the same fungal treatment condition ( Figure 1 ). Except for P₁ treatment significantly larger than P₀, P₂ and P₃ in 2022 (p < 0.05), the dry matter yield of alfalfa treated with phosphorus treatment reached the highest value in P₂ treatment, which was 25.87 g·pot^-1 in 2021, which was 58.2% higher than that of T₀P₀ treatment. In 2022, it would be 26.68 g·pot^-1, which is 59.35% higher than that of T₀P₀ treatment. The dry matter yield of alfalfa under P₂ treatment exhibited a statistically significant increase compared to other treatments (p < 0.05).

When the phosphorus treatment was 100 mg·kg^-1, the dry matter yield of double inoculated Fm and Ge alfalfa exhibited a statistically significant increase compared to no fungus treatment (p < 0.05), and the maximum value of T₃ treatment was reached under P₂ treatment. The effect of simultaneous inoculation of Fm and Ge was significantly better than that of single inoculation (p < 0.05). The dry matter yield of alfalfa under the treatment of fungus (T), phosphorus treatment (P) and fungus phosphorus interaction (T×P) in two years showed extremely significant differences (p < 0.01).

There were obvious AMF × phosphorus interactions for chlorophyll a, chlorophyll b, and total chlorophyll contents. According to the test results, chlorophyll a, chlorophyll b, and total chlorophyll contents demonstrated an increase at lower P doses (P₀-P₂) and a decrease at higher doses (P₂-P₃) within the same fungal treatment condition ( Figure 2 ). Except for the T₀ treatment in 2021, the chlorophyll b content of alfalfa was significantly higher in the P₃ treatment (p < 0.05), the chlorophyll a and total chlorophyll content was significantly higher in the P₃ treatment (p < 0.05) under T₀ condition in 2022, the chlorophyll b content was significantly higher in the P₁ treatment (p < 0.05). The chlorophyll a, chlorophyll b and total chlorophyll contents of alfalfa was the highest when most of the phosphorus treatment was 100 mg·kg^-1, and the P₂ treatment exhibited a statistically significant increase compared to the P₀ treatment (p < 0.05).

Under the condition of double inoculation of Fm and Ge fungal (T₃), the chlorophyll a, chlorophyll b and total chlorophyll contents of alfalfa at the phosphorus treatment of 100 mg·kg^-1 (P₂) in 2021 and 2022 exhibited a statistically significant increase compared to the P₀ treatment (p < 0.05). In 2021, the total chlorophyll content, chlorophyll a content and chlorophyll b content of alfalfa treated with T, P treatment and T×P showed extremely significant differences (p < 0.01), and in 2022, the chlorophyll a and total chlorophyll content of alfalfa between T, P treatment and T×P treatment showed extremely significant differences (p < 0.01). In 2022, the chlorophyll b content T and P treatment showed significant differences (p < 0.05).

Effects of two years of different phosphorus treatments on photosynthetic gas exchange parameters of alfalfa ( Figure 3 ).

Pearson correlation analysis showed that except for the instantaneous water use efficiency of alfalfa leaves and intercellular CO₂ concentration, the other indexes were extremely significantly positively correlated each other (p < 0.01) ( Figure 5 ). Alfalfa leaves were extremely positively correlated with net photosynthetic rate and light use efficiency (p < 0.01), while alfalfa leaf water use efficiency was positively correlated with dry matter yield, chlorophyll a, chlorophyll b, total chlorophyll content and stomatal conductance (p < 0.05), but not significantly positively correlated with transpiration rate (p > 0.05). Intercellular CO₂ concentration (C_i) was extremely negatively correlated with dry matter yield, chlorophyll a content, chlorophyll b content, total chlorophyll content, leaf net photosynthetic rate, stomatal conductance and light use efficiency (p < 0.01), and intercellular CO₂ concentration was negatively correlated with transpiration rate and instantaneous water use efficiency of alfalfa leaves (p < 0.05).

In order to avoid one-sidedness in the analysis of a single index and evaluate the optimal phosphorus and fungal application treatment (Sun et al., 2019), the membership function analysis evaluated eight key indicators of alfalfa performance ( Table 2 ). The net photosynthetic rate, transpiration rate, stomatal conductance, light energy use efficiency and instantaneous water use efficiency of alfalfa leaves were all positive indicators, while the intercellular CO₂ concentration was negative. The top three treatments in terms of comprehensive ranking were T₃P₂, T₃P₁, and T₁P₂.

Alfalfa, as a perennial legume forage, has always been known for its high quality and high yield. Dry matter yield is one of the important representatives of alfalfa production performance index. The intricate relationship between phosphorus application and AMF inoculation significantly shapes alfalfa’s dry matter yield. Studies have shown that phosphorus application and AMF inoculation can significantly increase the phosphorus use efficiency and leaf chlorophyll content of alfalfa, thereby increasing the aboveground biomass of plants (Mickky et al., 2018). The results showed that, with the increase of phosphorus application, the dry matter yield of alfalfa increased at the lower dose of P (P₀-P₂), while it decreased at the higher dose of P (P₂-P₃), and the application of phosphorus fertilizer in alfalfa was better than that without phosphorus fertilizer (p < 0.05). When the amount of phosphorus (P₂O₅) reached 150 mg·kg^-1, the dry matter yield of alfalfa decreased, which may be due to the high concentration of phosphorus, which caused a significant decrease in the diversity of AMF in the mycorrhizal community of alfalfa root and outside the root, and the colonization rate also decreased, which affected plant growth and reduced plant dry matter yield (Hinsinger, 2001). Therefore, the appropriate phosphorus application level is helpful to increase the yield.

In addition, high concentrations of phosphorus were found to damage the branches, vesicles and root fungal activity of maize (Zea mays L.) roots after AMF inoculation and phosphorus application (Chen et al., 2015.). In this study, under the condition of phosphorus application of 100 mg·kg^-1, Fm × Ge treatment was significantly higher than that of the control group (p < 0.05). It can be seen that inoculation of the two fungi has a significant positive effect on the growth of alfalfa, the main reason may be that AMF inoculation can significantly reduce the content of phosphorus phytate in alfalfa plants, and the arbuscular mycorrhizal fungal mycelium can absorb phosphorus from the soil and transfer it to the roots of alfalfa (Sun et al., 2022). Studies have shown that inoculation of sweet potatoes (Ipomoea batatas (L.) Lam.) with arbuscular mycorrhizal fungi is able to exhibit higher levels of photosynthetic pigments, enhance chlorophyll fluorescence and net photosynthetic rate under dehydration stress (Yooyongwech et al., 2014), resulting in better growth and yield traits (Yooyongwech et al., 2016). Therefore, the increase in the dry matter yield of alfalfa is due to the accumulation of photosynthetic products and the increase of available phosphorus content in the soil.

Photosynthesis is the basis for the formation of organic matter, oxygen and dry matter yield in plants, and improving plant photosynthetic efficiency and chlorophyll content is of great significance for increasing the accumulation of plant dry matter yield and improving the maintenance of plant nutritional quality (Sun et al., 2022). Phosphorus has a very close relationship with the photosynthesis of plants. Studies have shown that ATP and enzymes in plant photosynthesis require the participation of phosphorus, and the increase of phosphorus in plants is beneficial to the progress of plant photosynthesis, and the assimilation of CO₂ is directly related to the growth, development and photosynthesis of plants (Balestrini et al., 2020; Valkov et al., 2020). The results showed that, with the increase of phosphorus application, the net photosynthetic rate, transpiration rate and stomatal conductance of alfalfa increased at the lower dose of P (P₀-P₂), while it decreased at the higher dose of P (P₂-P₃), which may be because when the phosphorus content in the soil is insufficient for the phosphorus requirement of plants, the low phosphorus content will affect the opening and closing of the stomata in the leaves of plants, limit the regeneration of ribulose diphosphatase in alfalfa leaves (Zhang et al., 2016), and hinder the synthesis of ribulose diphosphate, which in turn affects the progress of alfalfa photosynthesis (Qi et al., 2013), photosynthesis is limited by the regeneration ability of RuBP in Calvin cycle. Rubisco is the rate-limiting enzyme of photosynthetic carbon assimilation, and its activity directly affects the photosynthetic rate of light saturation(Qi et al., 2013) and the application of phosphate fertilizer helps to improve the photosynthesis of alfalfa. When the phosphorus application rate reached 150 mg·kg^-1, the excess phosphorus impaired the photosynthesis process of alfalfa plants, which in turn reduced the net photosynthetic rate of alfalfa (Song et al., 2021). Therefore, reasonable fertilization strategies can help improve plant photosynthesis.

The results showed that when the amount of double fungi and phosphorus application was 100 mg·kg^-1, the light use efficiency and the instantaneous water use efficiency of leaves of alfalfa increased at the lower dose of P (P₀-P₂), while it decreased at the higher dose of P (P₂-P₃), and the net photosynthetic rate of leaves was significantly positively correlated with the light use efficiency and instantaneous water use efficiency (p < 0.05), which may be because phosphorus application and double fungus can improve the drought resistance of alfalfa. The lower the decrease of net photosynthetic rate, the less damage to photosynthetic organs in plant leaves, the higher the activity of photosynthetic cells, and the stronger the drought resistance of plants. Water use efficiency is an important symbol to judge the drought resistance of plants, which can reflect the water consumption and water use ability of plants. The higher the water use efficiency is, the stronger the adaptability of plants to drought is (Tang et al., 2023). Studies have shown that plants reduce chlorophyll content and stomatal conductance during drought stress, which affects plant photosynthetic capacity, which in turn reduces biomass (Naylor et al., 2018). Therefore, the appropriate bacterial fertilizer strategy is helpful to improve the drought resistance of plants.

Chlorophyll content is a key factor affecting the photosynthetic efficiency of alfalfa, and participates in the process of absorption, transfer and conversion of light energy (Zhu et al., 2017). Therefore, the chlorophyll content of plants plays an important role in their growth. The results showed that AMF inoculation of Robinia spp. seedlings significantly improved leaf area, carboxylation efficiency, chlorophyll content, net photosynthetic rate and effective photochemical efficiency of PSII (Mo et al., 2016), thereby maximizing their biomass (Zhu et al., 2014). Arbuscular mycorrhizal fungi can modulate the source-library relationship by enhancing the exchange of carbohydrates and mineral nutrients with the plant host, thereby stimulating the plant host to increase the rate of photosynthesis and increasing the carbon demand of arbuscular mycorrhizal fungi (Zhao et al., 2022b). Therefore, inoculation of arbuscular mycorrhizal fungi helps to increase chlorophyll content, promote photosynthesis, and thus increase crop yield.

Determining the best bacterial phosphorus treatment for the agricultural environment will help to improve the economic benefits of alfalfa. The effects of inoculation of Fm and Ge on dry matter yield, chlorophyll content and photosynthesis of alfalfa were different, and the membership function analysis and evaluation method was to comprehensively evaluate the performance of different treatments on each index and screen out the optimal phosphorus sterilization treatment among all treatments (Sun et al., 2019). In this study, the optimal combination of different treatments was ordered by T₃P₂ treatment ( Table 2 ), indicating that when the phosphorus application rate was 100 mg·kg^-1, the photosynthetic efficiency and dry matter yield of mixed inoculation of Funneliformis mosseae and Glomus etunicatum were maximized. Simultaneous inoculation of two fungi under suitable phosphorus application conditions can effectively promote the uptake of available phosphorus in the soil by plants, and then increase the dry matter yield of alfalfa.