A Combined Strategy Using Funneliformis mosseae and Phosphorus Addition for Enhancing Oat Drought Tolerance

Abstract

Arbuscular mycorrhizal fungi (AMF) play a crucial role in the soil–plant interface, yet the combined effects of AMF inoculation and phosphorus (P) addition on soil–plant nitrogen (N) and P, as well as oat grain yield, under drought stress remain unclear. Experiments were conducted during the 2021 and 2022 oat-growing seasons, applying AMF (40 g inoculum per pot; sterilized inoculum as the NAMF control) and P (0, 20, and 40 mg kg⁻¹ soil, designated P0, P1, and P2) under 75% and 55% relative water content. This study found that AMF inoculation at the P1 level significantly improved the AMF colonization rate, grain yield, and partial factor productivity of P (PFP_P) of oat. The grain yield increased by 6.2% (2021) and 9.8% (2022) under drought stress compared to the AMF-free treatment. AMF inoculation and P addition showed interactive effects on soil–plant N and P dynamics, which significantly increased microbial biomass phosphorus (MBP), nitrate N, and the available P content in oat soil. P1AMF significantly increased the total N and P contents under drought stress compared to P1NAMF, with maximum increments of 40.7% (N) and 11.1% (P) in 2021 and 15.4% (N) and 32.3% (P) in 2022. Moreover, the P1AMF treatment significantly improved P recovery efficiency (PRE), achieving a maximum increase of 48.4% across the two-year study. The analysis revealed that soil MBP was the key factor influencing oat grain yield, as well as the total N and P content in oat plants. It was concluded that AMF inoculation with a moderate amount of P addition could effectively regulate soil N and P availability and enhance plant N and P contents, as well as P productivity and use efficiency, thereby improving oat drought tolerance. Soil MBP acted as a vital bridge in the oat soil–plant continuum.

1. Introduction

Oat (Avena sativa L.) is an annual grass belonging to the Poaceae family that includes the naked oat (Avena sativa concv. nuda) and the hulled oat (Avena sativa subsp. sativa) [1]. It is commonly cultivated as a food and forage crop [2]. Geographically, oat is grown in 76 countries across five continents, and China mainly grows large-grain naked oat [3]. The oat-growing regions in China are located at altitudes of 1500 m–2500 m (annual rainfall of approximately 400 mm) and are classified as an arid or semiarid agro-pastoral ecotone [4]. In pursuit of healthy food and the promotion of the Ministry of Agriculture of China’s “Grain-to-feed” program, oat cultivation has gained significant attention. However, drought stress continues to be the primary factor limiting the increase in oat yield.

Phosphorus (P) is a significant limiting factor for yield in both natural and agricultural ecosystems, particularly in low-input plant production systems [5]. The availability of P in soil is typically low, particularly under drought stress [6]. In dryland regions, farmers often apply a significant amount of P fertilizers to enhance plant yield. However, P fertilizers tend to be easily adsorbed and fixed in the soil, resulting in 70–90% loss of the applied P fertilizer due to its unavailability to plants [7]. Therefore, improving soil P use efficiency has become a crucial issue for achieving environmentally sustainable agricultural development.

Microbial interactions play a fundamental role in maintaining plant health and ensuring agricultural sustainability [8,9]. Among the plant-supportive microbes, mycorrhizal fungi are one of the most significant symbiotic associates essential for the sustenance of plant productivity and soil health [10,11]. Arbuscular mycorrhizal fungi (AMF) can establish a mutually beneficial symbiont with the root systems of approximately 80% of higher plants, receiving 10–30% of carbohydrates and fatty acids from the host during symbiosis [12,13]. AMF can establish a dense network of hyphae within plant roots, effectively enhancing the contact area between plants and soil [14]. These hyphae can extend up to 25 cm away from the roots and reach the surrounding soil to acquire essential nutrients and water [15,16]. Studies have shown that the mycorrhizal pathway can contribute to more than half of plants’ nutrient uptake, although this varies strongly with soil nutrient levels [17]. Evidently, AMF are more important to plants under drought stress than under well-watered conditions, and the symbiotic relationship between AMF and plants has great potential for mitigating the effects of drought stress [18,19].

AMF play a pivotal role in promoting plant P uptake, with their hyphal networks contributing to up to 60% of plant P requirements [20]. Studies have shown that AMF can selectively enrich rhizosphere microorganisms carrying P genes, significantly enhancing hyphal alkaline phosphatase activity and thereby improving soil P mobility [21]. The hyphosphere, regarded as the “second genome” of AMF [22], can significantly enhance plant P uptake by regulating the P-cycling functions of the rhizosphere microbiome, and this improvement in P nutrition was a key factor in boosting plant drought resistance [23]. Under specific soil P levels, AMF inoculation was known to have a stronger effect on plants that experienced drought stress than on those that did not [24]. These findings suggest that, under drought stress, AMF could not only promote soil P availability but also exhibit synergistic effects through their interactions with soil P.

AMF can form mycorrhizal symbioses with oat [25,26,27,28]. However, the interplay between AMF and P, along with their combined effects on nitrogen (N) and P uptake, oat yield, and P use efficiency under drought conditions, remains poorly understood. A 2-year experiment was performed to explore the following: (1) Does P addition affect AMF colonization in drought-stressed oat roots? (2) How does the mixed addition of AMF inoculation and P affect oat drought tolerance? (3) Which could be the key soil factors influencing oat grain yield, as well as the plant total N and P content, by AMF inoculation and P addition under drought stress?

2. Materials and Methods

2.1. Experimental Site and Design

Experiments were conducted in the dry shed of Shanxi Agricultural University (37°42′44″ N, 112°57′84″ E) from April to August 2021 and from June to September 2022.

The pot experiments were performed using a completely randomized design (CRD) with three factors. The first factor consisted of two water stress levels: well-watered (WW), with a soil relative water content of 75%, and drought stress (DS), with a soil relative water content of 55%. The second factor included different P addition levels (P₂O₅): P0 = 0 mg kg⁻¹ soil, P1 = 20 mg kg⁻¹ soil, and P2 = 40 mg kg⁻¹ soil. The third factor involved two levels of AMF: 40 g sterilized inoculum per pot (NAMF) and 40 g inoculum per pot (AMF, based on preliminary experimental results). There were 12 treatments: WWP0NAMF (CK), WWP0AMF, WWP1NAMF, WWP1AMF, WWP2NAMF, WWP2AMF, DSP0NAMF, DSP0AMF, DSP1NAMF, DSP1AMF, DSP2NAMF, and DSP2AMF. The experiment was performed with 18 replications per treatment (6 pots for each treatment were used at the jointing, filling, and maturity stages, respectively), totaling 216 pots per year.

Non-porous ceramic pots were used. Each pot was filled with 3.5 kg experimental soil and 0.315 g urea (CH₄N₂O, with a N content of 46%) as a base fertilizer. Full-grain oat seeds were selected, mixed with 80% carbendazim, soaked, and germinated. Thirty seeds of similar size were sown in each pot (18 April 2021; 2 June 2022). For the AMF inoculation treatment, 2/3 of 40 g AMF inoculum was applied to the 6 cm soil layer below the seed bed, and 1/3 was applied to the radicles of germinated oat seeds. The same quality of sterilizing inoculum was applied to each pot as the no inoculum treatment. The oat plants were thinned at the three-leaf stage (10 May 2021; 23 June 2022), retaining 10 plants per pot. Subsequently, we weighed each pot every day at 18:00 (Beijing Standard Time) and added water to each pot to maintain its required field water capacity [29].

2.2. Experimental Materials

The experimental oat variety used in this study was “Bayou No.1”, a mid-maturing naked oat with a growth cycle of approximately 90 days.

The AMF used in this study was Funneliformis mosseae (In our prior experiments, we observed that this strain produced larger spores and exhibited a pronounced ability to infect oat roots under drought stress conditions.), from the Beijing Academy of Agriculture and Forestry Research. The inoculum was developed in pots by cultivating the AMF with clover planted in autoclave-sterilized maifanstone. After four months of growth in a controlled-environment incubator, the shoots of clover were removed from the pots, and the material remaining in the pots was used as the inoculum (i.e., spores, extraradical hyphae, mycorrhizal root segments, and sandy soil). A 10 g quantity of this inoculum contained 150 ± 5 spores.

The experimental soil was collected from the 0–25 cm tillage layer, which is a calcareous cinnamon soil (Chinese Soil Taxonomy) classified as Typic Haplustalfs (USDA Soil Taxonomy). After natural air drying, the soil was sieved (<4 mm) and mixed with river sand (10:1). The soil physiochemical properties measured in 2021 and 2022 are shown in

Table 1. Soil physicochemical properties before oat sowing in 2021 and 2022.

Year	Organic Carbon (g kg−1)	Total Phosphorus (mg kg−1)	Available Phosphorus (mg kg−1)	Alkali-Hydrolyzable Nitrogen (mg kg−1)	pH
2021	10.1	73.5	3.5	49.4	8.1
2022	8.9	64.2	5.7	21.4	8.2

. The soil field capacity was 25.01% and 34.12% (gravimetric water content) in 2021 and 2022, respectively. The soil permanent wilting point planted with oats corresponded to a soil gravimetric water content of 5.44% in 2021 and 5.96% in 2022, representing the absolute threshold of soil moisture utilized. The value of soil gravimetric water content was 13.76% in 2021 and 18.76% in 2022 under drought stress, with a soil relative water content of 55%. Based on these values, the available soil gravimetric water content utilizable by oat was calculated to be 8.32% in 2021 and 12.80% in 2022 under drought stress.

To maintain the actual soil conditions, the experimental soil was not sterilized in 2021. Nevertheless, to eliminate the influence of indigenous AMF, the experimental soil was autoclaved at 121 °C for 2 h after drying in 2022. The sterilized soil was stored in a sealed, sterile container in a dark, temperature-controlled environment (4 °C, 60% relative humidity) to prevent microbial contamination and maintain soil integrity until further use. However, to provide a balanced environment, allowing for a fair assessment of impacts of AMF inoculation on oat plant development, the filtrate of AMF-free treatment was prepared by homogenizing unsterilized soil with sterile water (2:1 v/v water/soil) for 30 min at 200 rpm following the methods of Lendzemo et al. [30]. The soil solution obtained was filtered using a 20 μm sieve, and the filtrate of AMF-free treatment was subsequently quantitatively (250 mL) added to each pot to restore the microorganism’s sterilized experimental soil system as closely as possible.

2.3. Sample Collection and Analyses

2.3.1. Sample Collection

Root, plant, and soil samples were collected destructively at the jointing stage (13 June 2021; 14 July 2022), filling stage (1 July 2021; 12 August 2022), and maturity period (26 July 2021; 6 September 2022). For root sampling, oat plants were carefully removed from the pots, the soil adhering to the roots was gently cleaned, and the roots were preserved in 70% ethanol and stored at 4 °C. The topsoil and bulk soil in the root zone were carefully removed after oat overground plants were cut. The soil attached to the roots in the pots was collected and mixed to create a soil sample. Fresh soil samples were divided into two portions: one for the immediate determination of soil microbial biomass phosphorus (MBP), and the other was stored at −40 °C for the subsequent analysis of the ammonium N and nitrate N contents. The dried soil sample was sieved through a 0.5 mm sieve to remove visible plant residues for soil total P and available P determination. Cut oat overground plants were oven-dried at 105 °C for half an hour to cease metabolic activity and then dried to a constant weight at 80 °C. The plants were then mixed and ground into a powder using Tissuelyzer II cell crusher (QIAGEN, Hilden, DER) to analyze the total N and P content.

2.3.2. Laboratory Analysis

Fresh roots were fixed in 2.5% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.2) for 24 h at 4 °C, then dehydrated through an ethanol series (30–100%). Critical point drying was performed using liquid CO₂. Samples were sputter-coated with gold–palladium (10 nm thickness) and observed under a field-emission scanning electron microscope (SEM) (Thermo Fisher Scientific, Eindhoven, NL, USA) operating at an acceleration voltage of 5.0 kv.

The AMF colonization rate was determined using the acetic acid ink staining method [31], which involves four processes: clearing, acidification, staining, and decolorization. The specific methods were as follows: (1) Tender roots with a diameter of approximately 0.5 mm were placed in a small beaker and cleared in a 60 °C water bath for 20 min after the addition of 20% KOH. (2) The roots were rinsed with running water to remove KOH and then acidified with 5% acetic acid for 5 min. (3) The excess acetic acid was rinsed off, and the roots were immersed in 5% acetic acid ink staining solution and placed in a 60 °C water bath for 15 min. The staining solution was then removed. (4) The roots were soaked in water and kept in a cool place for 3 days, during which frequent changes in soaking water occurred. The roots from each treatment were cut into approximately 1 cm segments, and 30 root segments were randomly selected and placed on three slides for observation under an upright BX53 microscope (Evident, Tokyo, Japan). Finally, the colonization rate of the roots was calculated using the colonization grading intensity method, following the approach described by Trouvelot et al. [32].

The field water-holding capacity was measured using the cutting ring method. The soil ammonium N and nitrate N were extracted with KCI using a SmarChem 200 automatic chemical analyzer (Alliance Instruments, Paris, France). The SOC content was determined using the potassium dichromate–external heating method. Alkali-hydrolyzable N was determined using the alkaline hydrolysis diffusion method. The soil pH was measured in a 1:5 (soil/water) extract using a conductivity meter (Hanna Instruments, Woonsocket, RI, USA). The soil total P was digested with HClO₄-H₂SO₄, and the concentrations were subsequently determined using the Mo-Sb colorimetric method. The soil available P was determined with the Olsen method using the NaHCO₃ extraction–Mo-Sb colorimetric method. The total N and P content of the plants was digested with H₂SO₄-H₂O₂ and determined using Nessler’s colorimetric method and vanadium molybdenum yellow spectrophotometry, respectively [33]. Soil MBP was determined using chloroform fumigation–NaHCO₃ extraction [34] and measured spectrophotometrically at 880 nm. The calculation methods for the soil parameters (ammonium N, nitrate N, total P, and available P) and total N and total P content in plants were based on the protocols established by Thioub et al. [35] and Attarzadeh et al. [18].

The P recovery efficiency (PRE) of oat was calculated by using the formula described by Syers et al. [36]:

$$\mathrm{PRE}={\displaystyle \frac{\mathrm{Plant}\mathrm{P}(\mathrm{P}1/\mathrm{P}2)-\mathrm{Plant}\mathrm{P}0({\mathrm{g}·\mathrm{pot}}^{-1})}{\mathrm{P}\mathrm{addition}\mathrm{rate}({\mathrm{g}·\mathrm{pot}}^{-1})}}$$

(1)

The partial factor productivity of P (PFP_P) of oat was calculated by using the formula presented by Cassman et al. [37]:

$${\mathrm{PFP}}_{\mathrm{P}}={\displaystyle \frac{\mathrm{Grain}\mathrm{yield}({\mathrm{g}·\mathrm{pot}}^{-1})}{\mathrm{P}\mathrm{addition}\mathrm{rate}({\mathrm{g}·\mathrm{po}\mathrm{t}}^{-1})}}$$

(2)

2.4. Statistical Analysis

The AMF colonization rate, MBP, N and P content in soil, N and P content in plants, and oat grain yield were analyzed by one-way analysis of variance (ANOVA) with Duncan’s test using SPSS 26.0 (IBM SPSS Statistics, Armonk, NY, USA). A multivariate linear model (MLM) was used to analyze the interaction effects of water, P, and AMF on the root colonization rate and grain yield. Additionally, OriginPro 2021 (v9.8.0.200; Origin Lab Corporation, Northampton, MA, USA) was utilized for mapping and correlation analysis, while Canoco 5.0 (Microcomputer Power, Ithaca, NY, USA) was employed for redundancy analysis.

3. Results and Analysis

3.1. Colonization of Oat Roots by AMF

The AMF colonization rate of oat roots at the jointing, filling, and maturity stages was significantly affected by the water, P, and AMF treatments in both years. Nonetheless, the interaction effects of the three factors on the AMF colonization rate at the three growth stages were inconsistent (Figure 1). At the same level of P, in 2021, when AMF were inoculated, the colonization rate of oat roots was significantly higher in the DS treatment than in the WW treatment at the three growth stages (Figure 1a). In 2022, under the AMF inoculation treatment, the colonization rate of oat roots was significantly higher in the WW treatment than that in the DS treatment at the three growth stages (Figure 1b). SEM is a technique used to observe and analyze the surface or fracture morphology of plant roots. The hair tissue in the oat roots of the AMF-inoculated plants (Figure 2b,d) was more developed than that in the AMF-free roots (Figure 2a,c). Under the conditions of WW or DS and at the same P level, in 2021, the AMF-inoculated plants exhibited a significantly higher colonization rate of oat roots compared to the AMF-free plants. The increase ranged from 1.8 times to 42.1 times (WW) and 1.8 times to 53.1 times (DS) under the different P levels (Figure 1a and Figure A1). The AMF colonization status of oat roots in each treatment in 2022 is shown in Figure A2. The varying degrees of AMF colonization in the roots of the AMF-inoculated plants are shown in Figure 1b and Figure A2b,d,f,h,j,l.

In 2021, when oat roots were inoculated and treated with P1, the AMF colonization rate at the jointing, filling, and maturity stages was significantly higher than that at P0. The increases in AMF colonization rate at the jointing, filling, and maturity stages were 12.8%, 24.4%, and 16.0% under WW conditions and 13.2%, 10.0%, and 13.0% under DS conditions, respectively. Furthermore, when oat roots were inoculated and applied at P2, the AMF colonization rate at the jointing, filling, and maturity stages was significantly lower than that at P1. The reductions in the AMF colonization rate at the jointing, filling, and maturity stages were 20.8%, 25.4%, and 17.2% under WW conditions and 13.3%, 13.0%, and 19.4% under DS conditions, respectively (Figure 1a). In 2022, the AMF colonization rate at the P1 level was significantly higher than that at the P0 and P2 levels at the filling and maturity stages under WW and AMF inoculation conditions. Similarly, the AMF colonization rate at the P2 level significantly increased by 48.9% (filling stage) and 29.4% (maturity stage) compared to that at the P0 level. However, the AMF colonization rate at the P2 level significantly decreased by 14.3% (filling stage) and 53.7% (maturity stage) compared to that at the P1 level. Under DS and AMF inoculation, the AMF colonization rate at the P1 level was significantly higher than that at the P0 and P2 levels at the filling stage. Specifically, the AMF colonization rate for P1 was significantly higher than that for P0 by 33.3%, whereas the AMF colonization rate for P2 was significantly lower than that for P1 by 62.5% (Figure 1b).

3.2. MBP in Oat Soil

The water, P, and AMF treatments all significantly affected the soil MBP content at the jointing, filling, and maturity stages of oat during the two-year experiment. However, no significant interactive effects were observed among these three factors on the soil MBP content across all growth stages of oat (Figure 3). Under the same P addition level, the soil MBP content in the WW treatment was significantly higher than that in the DS treatment across all oat growth stages, regardless of AMF inoculation. AMF inoculation significantly enhanced the soil MBP content at all P addition levels. Specifically, this increase ranged from 16.3 to 40.0% in 2021 and from 16.5 to 38.4% in 2022 (WW), and this increase reached 23.6–121.4% in 2021 and 36.3–106.0% in 2022 (DS) (Figure 3).

The soil MBP content under the P1 level was significantly higher than that under P0, whereas P2 exhibited a declining trend. Following AMF inoculation, the MBP content in oat soil at the P1 level was significantly elevated compared to P0 across all growth stages in both years. In 2021, the P1 level resulted in 55.6%, 50.1%, and 20.0% (WW) increases in MBP content at the jointing, filling, and maturity stages, respectively. Under DS treatment, the corresponding increases were 54.6%, 37.8%, and 88.9%. Similarly, in 2022, the MBP content under P1 was significantly higher than P0 by 47.6%, 58.7%, and 58.5% (WW) at the jointing, filling, and maturity stages, respectively. Under DS conditions, the increases were 27.2%, 34.9%, and 33.9% (Figure 3).

3.3. Ammonium N and Nitrate N Contents in Oat Soil

The water, P, and AMF treatments all significantly affected the soil ammonium N and nitrate N contents at the jointing, filling, and maturity stages of oat during the two-year experiment (

Table 2. Comparison of the mean effects of arbuscular mycorrhizal fungi (AMF) inoculation in combination with phosphorus (P) addition on ammonium and nitrate nitrogen (N) in oat soil under drought stress.

Year	Treatment		Ammonium N (mg kg−1)			Nitrate N (mg kg−1)
			Jointing Stage	Filling Stage	Maturity Stage	Jointing Stage	Filling Stage	Maturity Stage
2021	Water treatment	WW	7.77 b	6.84 b	6.21 b	7.01 b	10.47 a	7.00 b
		DS	8.92 a	8.31 a	6.97 a	9.18 a	11.65 a	7.87 a
		p-value	0.002	0.001	0.007	<0.001	0.278	0.033
		F	12.42 **	15.87 **	8.86 **	26.51 ***	ns	5.18 *
	P treatment	P0	8.90 a	7.70 a	6.95 a	8.66 a	14.34 a	7.59 a
		P1	8.28 a	7.91 a	6.55 a	8.20 a	9.56 b	8.09 a
		P2	7.86 a	7.11 a	6.28 a	7.42 a	9.27 b	6.62 b
		p-value	0.092	0.382	0.183	0.255	<0.001	0.007
		F	ns	ns	ns	ns	52.39 ***	6.42 **
	AMF treatment	NAMF	8.90 a	7.99 a	6.94 a	8.74 a	11.68 a	7.60 a
		AMF	7.79 b	7.15 a	6.24 b	7.44 b	10.44 a	7.27 a
		p-value	0.003	0.079	0.014 *	0.030	0.253	0.446
		F	11.09 **	ns	7.78	5.35 *	ns	ns
2022	Water treatment	WW	7.76 b	6.92 b	6.06 b	12.28 b	7.03 b	6.38 b
		DS	12.45 a	10.02 a	9.12 a	21.30 a	27.65 a	18.25 a
		p-value	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
		F	145.92 ***	65.36 ***	99.88 ***	79.46 ***	158.25 ***	478.73 ***
	P treatment	P0	9.57 a	8.27 a	7.23 a	18.36 a	20.58 a	13.60 a
		P1	10.39 a	8.90 a	7.72 a	16.67 a	17.82 a	12.04 a
		P2	10.36 a	8.23 a	7.82 a	15.35 a	13.62 a	11.31 a
		p-value	0.782	0.737	0.783	0.532	0.48	0.768
		F	ns	ns	ns	ns	ns	ns
	AMF treatment	NAMF	10.82 a	8.95 a	7.88 a	18.61 a	18.55 a	12.57 a
		AMF	9.39 a	7.99 a	7.29 a	14.98 a	16.13 a	12.06 a
		p-value	0.178	0.21	0.415	0.088	0.609	0.845
		F	ns	ns	ns	ns	ns	ns

Water treatment values indicate the mean (n = 18), P treatment values indicate the mean (n = 12), and AMF treatment values indicate the mean (n = 18). Different letters indicate significant differences between treatments (p < 0.05). ns indicates no significant difference, * indicates significant at the 0.05 level, ** indicates significant at the 0.01 level, and *** indicates significant at the 0.001 level. WW, well-watered; DS, drought stress; P0, 0 mg kg⁻¹ soil; P1, 20 mg kg⁻¹ soil; P2, 40 mg kg⁻¹ soil; NAMF, AMF-free; AMF, AMF inoculation.

and

Table 3. Ammonium and nitrate nitrogen (N) in oat soil under drought stress caused by arbuscular mycorrhizal fungi (AMF) inoculation in combination with phosphorus (P) addition.

Year	Water Treatment	P Treatment	AMF Treatment	Ammonium N (mg kg−1)			Nitrate N (mg kg−1)
				Jointing Stage	Filling Stage	Maturity Stage	Jointing Stage	Filling Stage	Maturity Stage
2021	WW	P0	NAMF	8.76 cd	7.02 de	6.81 cd	7.57 e	14.80 b	6.99 f
			AMF	7.64 fg	6.22 e	6.10 fg	6.45 f	12.29 d	7.59 de
		P1	NAMF	8.13 e	7.17 de	6.32 ef	7.39 e	9.30 h	7.81 cd
			AMF	6.90 h	6.48 e	5.31 h	7.16 e	8.86 j	7.43 ef
		P2	NAMF	8.00 ef	7.39 de	6.66 de	7.59 e	9.04 i	7.07 ef
			AMF	7.21 gh	6.77 de	6.09 fg	5.91 f	8.55 k	5.15 h
	DS	P0	NAMF	10.09 a	9.55 a	7.90 a	11.48 a	16.73 a	7.54 de
			AMF	9.13 bc	8.01 bc	6.98 cd	9.14 c	13.55 c	8.26 b
		P1	NAMF	9.46 b	9.09 ab	7.39 b	9.71 b	10.27 e	8.08b c
			AMF	8.62 d	8.89 ab	7.21 bc	8.56 d	9.83 f	9.04 a
		P2	NAMF	8.96 cd	7.71 cd	6.61 de	8.73 cd	9.94 f	8.10 bc
			AMF	7.26 gh	6.60 de	5.76 gh	7.45 e	9.57 g	6.18 g
	ANOVA	W	p-value	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
		P		<0.001	0.052	<0.001	<0.001	<0.001	<0.001
		A		<0.001	0.005	<0.001	<0.001	<0.001	0.003
		W × P		<0.001	0.006	<0.001	<0.001	<0.001	0.164
		A × W		0.429	0.623	0.483	0.003	0.002	0.018
		A × P		0.458	0.489	0.534	<0.001	<0.001	0.164
		W × A ×P		0.009	0.564	0.022	0.003	<0.001	0.015
2022	WW	P0	NAMF	7.53 g	6.91 e	5.20 g	15.10 e	8.46 e	7.84 d
			AMF	6.63 h	5.53 f	5.48 fg	11.17 h	7.54 ef	7.08 d
		P1	NAMF	8.49 f	7.00 e	5.93 ef	13.67 f	7.76 ef	6.84 de
			AMF	7.34 g	7.25 de	5.84 fg	11.88 gh	6.54 fg	5.48 e
		P2	NAMF	9.00 e	7.73 d	7.51 d	12.46f g	6.59 fg	5.45 e
			AMF	7.58 g	7.10 e	6.39 e	9.43 i	5.29 g	5.59 e
	DS	P0	NAMF	12.32 b	11.47 a	9.89 a	26.60 a	36.29 a	18.67 b
			AMF	11.79 c	9.19 c	8.36 c	20.58 c	30.03 b	20.81 a
		P1	NAMF	13.88 a	11.48 a	10.04 a	22.39 b	30.28 b	18.88 b
			AMF	11.87 b	9.89 b	9.06 b	18.75 c	26.70 c	16.96 c
		P2	NAMF	13.72 c	9.11 c	8.74 bc	21.43 d	21.94 d	17.75b c
			AMF	11.15 c	9.02 c	8.64 bc	18.09 d	20.68 d	16.45 c
	ANOVA	W	p-value	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
		P		<0.001	<0.001	0.002	<0.001	<0.001	<0.001
		A		<0.001	<0.001	<0.001	<0.001	<0.001	0.051
		W × P		0.001	<0.001	<0.001	0.001	<0.001	0.471
		A × W		0.003	0.001	0.026	0.005	<0.001	0.539
		A × P		<0.001	<0.001	0.937	0.002	0.004	0.006
		W × A ×P		0.003	<0.001	0.001	0.200	0.001	0.007

The values indicate the means (n = 3). Different letters in the same column indicate significant differences among treatments (p < 0.05). W, A, and P represent water treatment, AMF, and phosphorus treatment. WW, well-watered; DS, drought stress; P0, 0 mg kg⁻¹ soil; P1, 20 mg kg⁻¹ soil; P2, 40 mg kg⁻¹ soil; NAMF, AMF-free; AMF, AMF inoculation.

). In 2021, the ammonium N content in oat soil at the jointing and filling stages under DS significantly increased by 14.8 and 21.5% compared to that in WW. Additionally, the soil nitrate N content in the DS treatment was significantly higher than that in the WW treatment by 310 and 12.4% at the jointing and maturity stages, respectively. In 2022, the ammonium N and nitrate N contents in oat soil were significantly higher in DS than in WW. The maximum increases in DS compared with WW were 60.4% (ammonium N) and 293.3% (nitrate N) (Table 2).

There was no significant difference in the ammonium N content in oat soil among the different P levels in both years. Nevertheless, in 2021, as the P level increased, there was a significant decrease in the soil nitrate N content at the filling and maturity stages. The soil nitrate N content in the P2 treatment decreased by 35.4% (filling stage) and 12.8% (maturity stage) compared to that in the P0 treatment. Nonetheless, there was no significant effect on the nitrate N content in oat soil in 2022 (Table 2).

In 2021, the ammonium N content in oat soil with AMF inoculation was significantly lower than that in the AMF-free treatment by 14.3% at the jointing stage and 11.2% at the maturity stage. Additionally, in 2022, the soil nitrate N content at the jointing stage was significantly lower in the AMF inoculation treatment than in the AMF-free treatment by 14.9% (Table 2). These results suggest that AMF inoculation could enhance the absorption of ammonium N and nitrate N by oat.

3.4. Total P and Available P Contents in Oat Soil

The water, P, and AMF treatments all significantly affected the soil total P and available P contents at the jointing, filling, and maturity stages of oat during the two-year experiment (

Table 4. The mean comparison of the effects of arbuscular mycorrhizal fungi (AMF) inoculation in combination with phosphorus (P) addition on total and available P in oat soil under drought stress.

Year	Treatments		Total P (mg kg−1)			Available P (mg kg−1)
			Jointing Stage	Filling Stage	Maturity Stage	Jointing Stage	Filling Stage	Maturity Stage
2021	Water treatment	WW	70.34 a	61.79 b	51.84 b	8.18 a	5.66 b	3.77 b
		DS	77.79 a	68.38 a	58.53 a	6.53 b	4.60 a	2.82 a
		p-value	0.057	0.044	0.043	<0.001	0.005	0.007
		F	ns	4.38 *	4.42 *	18.03 ***	9.85 **	8.78 **
	P treatment	P0	62.21 c	56.87c	47.27 b	6.62 b	4.34 b	2.61 b
		P1	73.78 b	64.37 b	53.25 b	7.45 ab	5.08 b	3.12 b
		P2	86.21 a	74.01 a	65.03 a	8.20 a	5.97 a	4.16 a
		p-value	<0.001	<0.001	<0.001	0.010	0.001	<0.001
		F	40.03 ***	17.69 ***	21.05 ***	5.76 *	10.41 **	11.16 ***
	AMF treatment	NAMF	78.85 a	70.53 a	60.29 a	6.89 a	4.73 b	2.95 a
		AMF	69.28 b	59.64 b	50.08 b	7.83 a	5.53 a	3.64 a
		p-value	0.013	<0.001	0.001	0.065	0.040	0.062
		F	6.90 *	15.42 ***	12.45 **	ns	4.77 *	ns
2022	Water treatment	WW	64.86 a	56.33 a	45.19 b	5.40 a	5.10 a	2.11 a
		DS	72.23 a	61.76 a	51.93 a	4.81 a	3.76 a	1.69 a
		p-value	0.091	0.152	0.044	0.157	0.139	0.087
		F	ns	ns	4.59 *	ns	ns	ns
	P treatment	P0	57.82c	49.49c	41.20 b	4.15 b	2.36 b	1.45 b
		P1	69.02 b	59.63 b	49.13 a	5.20 a	3.63 a	1.96 ab
		P2	78.78 a	68.02 a	55.34 a	5.96 a	4.30 a	2.29 a
		p-value	<0.001	<0.001	0.001	<0.001	<0.001	0.01
		F	21.51 ***	25.59 ***	10.90 **	13.15 ***	19.70 ***	5.74 *
	AMF treatment	NAMF	72.80 a	62.85 a	52.72 a	4.55 b	2.95 b	1.50 b
		AMF	64.29 b	55.25 b	44.40 b	5.65 a	3.93 a	2.30 a
		p-value	0.048	0.04	0.01	0.005	0.017	<0.001
		F	4.37 *	4.78 *	7.85 *	9.57 **	6.61 *	19.38 ***

Water treatment values indicate the mean (n = 18), P treatment values indicate the mean (n = 12), and AMF treatment values indicate the mean (n = 18). Different letters indicate significant differences between treatments (p < 0.05). ns indicates no significant difference, * indicates significant at the 0.05 level, ** indicates significant at the 0.01 level, and *** indicates significant at the 0.001 level. WW, well-watered; DS, drought stress; P0, 0 mg kg⁻¹ soil; P1, 20 mg kg⁻¹ soil; P2, 40 mg kg⁻¹ soil; NAMF, AMF-free; AMF, AMF inoculation.

and

Table 5. Total phosphorus (P) and available P in oat soil under drought stress caused by arbuscular mycorrhizal fungi (AMF) inoculation in combination with P addition.

Year	Water Treatment	P Treatment	AMF Treatment	Total P (mg kg−1)			Available P (mg kg−1)
				Jointing Stage	Filling Stage	Maturity Stage	Jointing Stage	Filling Stage	Maturity Stage
2021	WW	P0	NAMF	60.89 g	57.15 f	47.02 f	6.79 f	4.30 j	2.69 g
			AMF	53.47 h	50.31 g	43.29 g	7.70 d	5.21 f	3.81 d
		P1	NAMF	75.78 d	65.52 e	52.99 de	8.10 c	5.43 e	3.47 e
			AMF	65.79 f	55.71 f	45.42 fg	9.09 a	6.02 c	3.76 d
		P2	NAMF	88.48 b	78.91 b	68.51 b	8.28 b	6.17 b	4.13 c
			AMF	77.64 d	63.14 e	53.81 d	9.11 a	6.82 a	4.79 a
	DS	P0	NAMF	70.66 e	64.07 e	54.13 d	5.07 i	3.46 i	1.66 i
			AMF	63.83 f	55.96 f	44.63 fg	6.12 g	4.37 g	2.29 h
		P1	NAMF	82.52 c	71.90 c	63.63 c	5.84 h	3.97 h	2.38 h
			AMF	71.02 e	64.35 e	50.96 e	6.76 f	4.91 f	2.86 f
		P2	NAMF	94.77 a	85.64 a	75.44 a	7.24 e	5.05 e	3.36 e
			AMF	83.95 c	68.35 d	62.37 c	8.17 c	5.85 c	4.35 b
	ANOVA	W	p-value	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
		P		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
		A		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
		W × P		<0.001	<0.001	0.164	<0.001	<0.001	<0.001
		A × W		0.003	0.002	0.018	0.104	<0.001	0.866
		A × P		<0.001	<0.001	<0.001	0.041	<0.001	<0.001
		W × A ×P		0.003	<0.001	0.015	0.045	<0.001	<0.001
2022	WW	P0	NAMF	56.01 h	48.39 h	39.53 h	4.07 g	2.16 h	1.32 f
			AMF	52.60 i	43.34 i	34.17 i	5.38 de	2.96 f	2.03 d
		P1	NAMF	69.26 e	61.20 d	52.04 d	4.84 f	3.45 e	1.61 e
			AMF	60.22 g	53.94 f	40.43 h	5.74 c	4.45 b	2.51 b
		P2	NAMF	81.14 b	70.10 b	56.13 c	5.59 cd	4.26 c	2.03 d
			AMF	69.93 e	61.05 d	48.84 e	6.79 a	5.27 a	3.15 a
	DS	P0	NAMF	63.55 f	55.77 ef	47.12 ef	3.01 h	1.69 i	0.94 g
			AMF	59.11 g	50.48 g	43.98 g	4.12 g	2.61 g	1.52 e
		P1	NAMF	77.64 c	66.89 c	58.37 b	4.60 f	2.61 g	1.52 e
			AMF	68.99 e	56.48 e	45.69 fg	5.63 c	4.00 d	2.22 c
		P2	NAMF	89.20 a	74.75 a	63.13 a	5.21 e	3.54 e	1.61 e
			AMF	74.88 d	66.20 c	53.28 b	6.26 b	4.30b c	2.35 bc
	ANOVA	W	p-value	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
		P		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
		A		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
		W × P		0.121	0.006	0.003	<0.001	<0.001	<0.001
		A × W		0.137	0.177	0.457	0.37	0.114	0.001
		A × P		<0.001	0.001	<0.001	0.078	<0.001	0.003
		W × A ×P		0.225	0.104	0.022	0.226	0.001	0.18

The values indicate the means (n = 3). Different letters in the same column indicate significant differences among treatments (p < 0.05). W, A, and P represent water treatment, AMF, and phosphorus treatment. WW, well-watered; DS, drought stress; P0, 0 mg kg⁻¹ soil; P1, 20 mg kg⁻¹ soil; P2, 40 mg kg⁻¹ soil; NAMF, AMF-free; AMF, AMF inoculation.

). In 2021, the total P content in oat soil under DS was significantly higher than that under WW at the filling and maturity stages, with increases of 10.7% and 12.9%, respectively. Conversely, the available P content in oat soil under WW was significantly higher than that under DS, with the highest increase being 33.7%. In 2022, the total P content in oat soil was significantly higher in the DS treatment than in the WW treatment by 14.9% at the maturity stage. However, the water treatment did not have a significant effect on the available P content in oat soil in 2022 (Table 4).

The total P and available P contents in oat soil increased significantly with increasing the P levels in both years. The maximum increase in total P content in oat soil under P2 treatment was 38.6% (2021) and 37.4% (2022) compared to that under P0 treatment. Similarly, the maximum increase in the available P content in oat soil was 92.6% (2021) and 82.2% (2022) (Table 4).

AMF inoculation significantly reduced the total P content in oat soil in both years. The total P in oat soil in the 2021 and 2022 treatment groups decreased by 12.1%, 15.4%, and 16.9% and by 11.7%, 12.1%, and 15.8%, respectively, at the jointing, filling, and maturity stages, compared to those in the corresponding AMF-free treatment groups (Table 4). These findings indicated that AMF inoculation promoted the uptake of total P in the soil. After AMF inoculation, the available P content in oat soil at the filling stage in 2021 and at the jointing, filling, and maturity stages in 2022 was significantly higher than that in the AMF-free treatment, with increases of 16.9% in 2021 and 24.2%, 33.2%, and 53.3% in 2022, respectively (Table 4). These findings suggested that AMF inoculation could promote the turnover of soil P, thereby increasing the available P content in oat soil. Notably, under drought stress, AMF inoculation combined with P addition (P2) significantly increased the soil available P content in oat, reaching its highest level. The soil available P content in P2 increased by 162.1% and 154.4% in 2021 and 2022, respectively, compared to the other drought treatments (Table 5). This finding confirms the positive role of AMF inoculation combined with P addition in alleviating drought stress and enhancing P availability.

3.5. Total N and Total P Content in Oat Plants

The water, P, and AMF treatments all significantly affected the total N and P content in oat plants at the jointing, filling, and maturity stages of oat during the two-year experiment (

Table 6. The mean comparison of the effects of arbuscular mycorrhizal fungi (AMF) inoculation in combination with phosphorus (P) addition on the total nitrogen (N) and P in oat plants under drought stress.

Year	Treatments		Total N (g kg−1)			Total P (g kg−1)
			Jointing Stage	Filling Stage	Maturity Stage	Jointing Stage	Filling Stage	Maturity Stage
2021	Water treatment	WW	12.86 a	8.57 a	5.39 a	4.46 a	3.58 a	2.81 a
		DS	12.41 a	8.20 a	5.17 a	2.73 b	2.36 b	1.99 b
		p-value	0.685	0.651	0.707	<0.001	<0.001	<0.001
		F	ns	ns	ns	75.77 ***	107.62 ***	42.81 ***
	P treatment	P0	10.41 b	6.42 c	4.11 b	3.28 a	2.80 a	2.19 a
		P1	14.61 a	10.16 a	6.78 a	3.76 a	3.05 a	2.46 a
		P2	12.89 a	5.56 b	4.96 b	3.74 a	3.06 a	2.54 a
		p-value	0.002	<0.001	<0.001	0.571	0.688	0.370
		F	8.86 **	18.83 ***	22.89 ***	ns	ns	ns
	AMF treatment	NAMF	10.80 b	7.27 b	4.61 b	3.31 a	2.78 a	2.22 b
		AMF	14.47 a	9.50 a	5.95 a	3.88 a	3.15 a	2.58 a
		p-value	<0.001	0.003	0.013	0.168	0.193	0.084
		F	24.02 ***	11.35 **	7.25 *	ns	ns	ns
2022	Water treatment	WW	20.48 a	15.99 a	9.48 a	4.12 a	3.02 a	2.18 a
		DS	18.54 b	15.36 a	9.62 a	2.59 b	2.22 b	1.71 b
		p-value	0.013	0.466	0.838	<0.001	<0.001	<0.001
		F	7.27 *	ns	ns	74.05 ***	31.89 ***	35.91 ***
	P treatment	P0	17.90 b	14.80 a	8.40 b	3.09 a	2.47 a	1.80 a
		P1	20.22 a	16.09 a	10.36 a	3.48 a	2.71 a	2.03 a
		P2	20.41 a	16.14 a	9.88 ab	3.49 a	2.68 a	2.00 a
		p-value	0.012	0.358	0.043	0.610	0.621	0.273
		F	5.49 *	ns	3.67 *	ns	ns	ns
	AMF treatment	NAMF	18.62 b	13.84 b	8.30 b	3.01 b	2.32 b	1.81 b
		AMF	20.40 a	17.52 a	10.79 a	3.70 a	2.92 a	2.08 a
		p-value	0.025	<0.001	<0.001	0.056	0.003	0.030
		F	5.75 *	99.82 ***	29.43 ***	ns	10.82 **	5.38 *

Water treatment values indicate the mean (n = 18), P treatment values indicate the mean (n = 12), and AMF treatment values indicate the mean (n = 18). Different letters indicate significant differences between treatments (p < 0.05). ns indicates no significant difference, * indicates significant at the 0.05 level, ** indicates significant at the 0.01 level, and *** indicates significant at the 0.001 level. WW, well-watered; DS, drought stress; P0, 0 mg kg⁻¹ soil; P1, 20 mg kg⁻¹ soil; P2, 40 mg kg⁻¹ soil; NAMF, AMF-free; AMF, AMF inoculation.

and

Table 7. Total nitrogen (N) and phosphorus (P) in oat plants under drought stress caused by arbuscular mycorrhizal fungi (AMF) inoculation in combination with P addition.

Year	Water Treatment	P Treatment	AMF Treatment	Total N (g kg−1)			Total P (g kg−1)
				Jointing Stage	Filling Stage	Maturity Stage	Jointing Stage	Filling Stage	Maturity Stage
2021	WW	P0	NAMF	8.59g	5.39 h	3.63 f	3.67 d	3.18 d	2.28 d
			AMF	12.45 d	8.06 e	4.71 e	4.11 c	3.43 c	2.62 c
		P1	NAMF	12.83 d	9.14 cd	5.80 b	4.19 c	3.42 c	2.68 c
			AMF	16.79 a	11.41 a	7.95 a	5.14 b	3.89 b	3.06 b
		P2	NAMF	11.55 ef	7.67 f	4.73 e	4.13 c	3.34 c	2.69 c
			AMF	14.94 b	9.47 b	5.52 c	5.48 a	4.19 a	3.51 a
	DS	P0	NAMF	8.40 g	5.06 h	3.46 f	2.49 h	2.08 h	1.71 g
			AMF	12.20 de	7.17 g	4.61 e	2.830 f	2.49 ef	2.14 e
		P1	NAMF	12.39 d	8.98 d	5.55 c	2.71g	2.33 g	1.95 f
			AMF	16.41 a	11.11 a	7.81 a	3.01 e	2.58 e	2.15 e
		P2	NAMF	11.02 f	7.36 fg	4.51 e	2.65 g	2.37 fg	1.98 f
			AMF	14.06 c	9.49 bc	5.10 d	2.69 g	2.34 g	1.99 f
	ANOVA	W	p-value	0.003	<0.001	<0.001	<0.001	<0.001	<0.001
		P		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
		A		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
		W × P		0.278	0.063	0.18	<0.001	<0.001	<0.001
		A × W		0.626	0.121	0.94	<0.001	<0.001	<0.001
		A × P		0.047	0.206	<0.001	<0.001	0.261	0.05
		W × A ×P		0.771	0.159	0.246	<0.001	<0.001	<0.001
2022	WW	P0	NAMF	17.11 f	13.36 f	6.97 h	3.54 d	2.50 d	2.04 cd
			AMF	20.38 c	16.94 cd	9.86 e	4.08 b	3.23 b	2.18 b
		P1	NAMF	19.39 de	13.69 f	8.19 g	3.77 cd	2.79 c	2.15 b
			AMF	22.61 b	18.39 b	10.96 bc	4.77 a	3.47 a	2.32 a
		P2	NAMF	19.80 cd	14.60 e	8.84 f	3.78 c	2.68 c	2.09 bc
			AMF	23.58 a	18.98 a	12.04 a	4.78 a	3.47 a	2.31 a
	DS	P0	NAMF	16.58 f	12.52 g	6.94 h	2.20 g	1.89 g	1.30 f
			AMF	17.24 f	16.38 d	9.81 e	2.54 f	2.25 e	1.68 e
		P1	NAMF	19.47 de	14.98 e	10.69 cd	2.36f g	1.98 fg	1.62 e
			AMF	19.40 de	17.29 c	11.60 b	3.02 e	2.62 cd	2.04 cd
		P2	NAMF	19.08 e	13.87 f	8.19 g	2.39 fg	2.10 ef	1.67 e
			AMF	19.18 e	17.12 c	10.49 d	2.99 e	2.50 d	1.94 d
	ANOVA	W	p-value	<0.001	<0.001	0.018	<0.001	<0.001	<0.001
		P		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
		A		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
		W × P		0.003	<0.001	<0.001	0.29	0.761	<0.001
		A × W		<0.001	<0.001	<0.001	0.003	0.001	<0.001
		A × P		0.34	0.44	<0.001	0.004	0.307	0.563
		W × A ×P		0.293	<0.001	<0.001	0.61	0.049	0.067

The values indicate the means (n = 3). Different letters in the same column indicate significant differences among treatments (p < 0.05). W, A, and P represent water treatment, AMF, and phosphorus treatment. WW, well-watered; DS, drought stress; P0, 0 mg kg⁻¹ soil; P1, 20 mg kg⁻¹ soil; P2, 40 mg kg⁻¹ soil; NAMF, AMF-free; AMF, AMF inoculation.

). In 2022, the total N content in oat plants at the jointing stage was significantly (10.8%) higher in the WW treatment than that in the DS treatment. The DS treatment significantly reduced the total P content in oat plants in both years. DS caused maximum reductions in the total P content of oat plants by 40.0% in 2021 and 36.6% in 2022 compared to WW (Table 6).

In 2021, the total N content in oat plants was significantly higher in P1 than that in P0. The maximum increase in total N content in oat plants under P1 was 65.9% compared to P0. This trend continued in 2022, and the total N content in oat plants was 12.9% and 23.8% higher, respectively, in P1 than in P0 at the jointing and maturity stages (Table 6). In contrast, P addition had no significant effect on the total P content in oat plants.

AMF inoculation significantly increased the total N content in oat plants in both years. The total N content in oat plants with AMF inoculation at the jointing, filling, and maturity stages was 34.3%, 30.1%, and 30.4% higher (2021) and 9.7%, 26.8%, and 30.1% higher (2022) (Table 6) compared to that in AMF-free plants. The total P content in oat plants with AMF inoculation at the maturity stage in 2021 and at the jointing, filling, and maturity stages in 2022 was significantly higher than that in the AMF-free plants, with increases of 8.3% in 2021 and 23.3%, 26.1%, and 16.7% in 2022, respectively (Table 6). Additionally, AMF inoculation with P addition (P1) significantly enhanced the total N and P content in oat plants under drought stress. The maximum increase in the total N and total P content under P1AMF reached 40.7% and 11.1% in 2021 and 15.4% and 32.3% in 2022, respectively, compared to P1NAMF (Table 7).

3.6. Oat Grain Yield

The water, AMF, and P treatments had significant effects on oat grain yield in both years (Figure 4). In 2021, there was no interaction effect among the three treatments on oat grain yield (Figure 4a), whereas, in 2022, there was an interaction effect among the three treatments on oat grain yield (Figure 4b). For the same P level in the AMF-free treatment, oat grain yield was significantly lower under DS than that under WW in both years, with reductions of 21.3% and 23.4% (P0), 9.7% and 16.5% (P1), and 10.6% and 14.2% (P2), respectively. For the same P level in the AMF inoculation treatment, oat grain yield was significantly lower in DS than that in WW, with reductions of 20.1% and 26.8% (P0), 13.9% and 17.2% (P1), and 13.5% and 16.3% (P2), respectively (Figure 4).

At the same P level, AMF inoculation exhibited a significantly higher oat grain yield compared to the AMF-free treatment. However, oat grain yield of AMF inoculation under DS conditions was significantly lower than that of the AMF-free treatment under WW conditions in both years, with decreases of 10.8% and 19.2% (P0), 4.1% and 8.4% (P1), and 6.3% and 7.8% (P2), respectively (Figure 4). These results indicated that the difference in oat grain yield was reduced between well-watered and drought-stressed conditions with AMF inoculation.

In 2021, P addition led to significant differences in oat grain yield under WW or DS conditions compared to the AMF-free conditions in the order of P1 > P0 > P2 (WW) and P1 > P2 > P0 (DS). Under drought stress conditions, oat grain yield under the P1AMF treatment increased significantly by 6.2% compared to P1NAMF (Figure 4a). In 2022, P addition also significantly affected the oat grain yield. The order was P1 > P2 > P0 under WW with AMF-free conditions, similar to P1, P2 > P0 under DS with AMF-free conditions. Under drought stress conditions, oat grain yield under the P1AMF treatment increased significantly by 9.8% compared to P1NAMF (Figure 4b).

3.7. P Productivity and Uptake Efficiency

Under both water treatments (WW and DS), the two-year experimental data consistently demonstrated that oat PRE reached the peak under the P1 level combined with AMF inoculation, exhibiting maximum increases of 58.5% (WW) and 1100.0% (DS) in 2021 and 63.6% (WW) and 312.5% (DS) in 2022, compared to other treatments. Under drought stress, the P1AMF treatment improved PRE by a maximum of 48.4% over the two-year study (Figure 5a).

Under both water treatments (WW and DS), the two-year experimental data consistently demonstrated that oat PFP_P was significantly higher at the P1 level than at the P2 level. Specifically, under AMF inoculation conditions, PFP_P of P2 decreased by 54.0% (WW) and 56.7% (DS) in 2021 and by 50.9% (WW) and 50.4% (DS) in 2022, compared to the P1 level. Under drought stress, the P1AMF treatment improved PFP_P by a maximum of 9.8% over the two-year study (Figure 5b).

3.8. Relationship Between Oat Grain Yield and Each Index

Oat grain yield showed significant positive correlations with soil MBP, soil available P, plant total N, and plant total P in both 2021 and 2022, except for the non-significant positive correlation between grain yield and soil available P under the WW treatment in 2021. Moreover, soil MBP was significantly positively correlated with AMF colonization rate, available P, and the plant total N and P content (Figure 6).

Soil factors accounted for 94.3% and 3.7% (Figure 7a), 97.4% and 0.3% (Figure 7b), 96.5% and 1.5% (Figure 7c), and 84.4% and 9.3% (Figure 7d), respectively, of the variation in oat grain yield, as well as in the plant total N and P content on the first and second axes, respectively. In other words, soil factors accounted for 98.0% (Figure 7a), 98.0% (Figure 7b), 98.1% (Figure 7c), and 93.7% (Figure 7d) of the variation in oat grain yield, AMF colonization rate, and the plant total N and P content on the first two axes, respectively. The contribution rates of soil MBP to oat grain yield, as well as the plant total N and P content, reached 88.7%, 77.5%, 56.2%, and 77.4% in 2021WW, 2011DS, 2022WW, and 2022DS, respectively, with significant differences (

Table 8. Importance ranking and significance test results of the soil microbial biomass phosphorus (MBP), ammonium nitrogen (N), nitrate N, total phosphorus (P), and available P in oat soil.

Year	Soil Index	WW				DS
		Importance Ranking	Relative Contribution Rate (%)	F	p-Value	Importance Ranking	Relative Contribution Rate (%)	F	p-Value
2021	MBP	1	88.7	114	0.002	1	77.5	49.9	0.002
	Ammonium N	2	4.2	7.6	0.002	3	1.5	7.4	0.01
	Nitrate N	4	1.5	3.2	0.046	2	20.5	70.3	0.002
	Total P	5	1.4	15.0	0.002	4	0.4	2.4	0.106
	Available P	3	4.1	20.7	0.002	5	<0.1	0.3	0.764
2022	MBP	1	56.2	19.9	0.002	1	77.4	46.7	0.002
	Ammonium N	5	0.8	6.6	0.004	3	5.0	9.6	0.004
	Nitrate N	4	1.0	6.0	0.006	4	2.4	6.5	0.008
	Total P	2	32.1	36.7	0.002	5	1.0	2.9	0.104
	Available P	3	10.0	44.5	0.002	2	14.3	17.4	0.002

WW, well-watered; DS, drought stress.

).

4. Discussion

4.1. AMF Colonization of Oat Roots with P Addition Under Drought Stress

The establishment and sustenance of AMF symbionts require specific carbohydrates and lipids as sources of material and energy [13]. The growth of oat plants remained robust during the filling stage, which could provide additional carbohydrate and lipid nutrients to mycorrhizal fungi to facilitate the survival and reproduction of AMF, further promoting their higher colonization of roots in this study. Comparative analysis revealed that the overall AMF colonization rate in 2021 was 172.03% higher than that in 2022. Notably, distinct soil treatment protocols were implemented between the two experimental years: the 2021 trial maintained the indigenous AMF communities and soil microbial activity in non-sterilized soil, whereas sterilized soil was used in 2022. These findings suggest that niche competition or cooperation between indigenous AMF communities and inoculated strains may generate synergistic effects, thereby enhancing the AMF colonization capacity. This discovery provides novel empirical evidence for understanding the interaction mechanisms within AMF communities.

A study on AMF inoculation demonstrated a significant decline in the colonization rate of Potentilla anserina roots as drought stress duration increased [38]. Similarly, Li et al. [39] observed that, in AMF-inoculated ryegrass, root colonization rates decreased markedly when the soil relative water content dropped from 70% to 30%. The discrepancy between these findings and the results of this study (2021) may stem from variations in plant–AMF interactions under different soil moisture conditions. Zhang et al. [40] reported that, when plants experience drought stress, their roots receive and transmit signals to AMF, and by signal transduction, the AMF promote the formation of arbuscular cells, leading to the continuous colonization of plant roots. This colonization increases the colonization rate and enhances the root morphology of plants, ultimately improving their drought resistance [41]. Oat is known for its strong drought resistance [42]. When subjected to drought stress, oat roots may effectively transmit drought stress signals to AMF and stimulate the formation of mycorrhizal symbioses with their roots, thereby enhancing the colonization rate. However, noteworthily, extreme drought conditions can also have negative effects on plants, such as reducing yield and biomass, limiting the supply of organic nutrients to AMF, and inhibiting mycorrhizal development [43]. These findings also corroborated the assertion of Lee et al. [44] that the sterilization of indigenous soil could have an impact on the colonization of Panax ginseng roots by AMF.

AMF colonization also varies depending on the level of soil P [45]. In this study, under drought stress, the AMF-inoculated plants showed that a high P level (P2) tended to inhibit the rate of AMF colonization in oat roots, whereas a moderate level of P (P1) addition was found to be more conducive to AMF colonization. This observation is similar to the findings of Zhang et al. [46], who observed that, with a P addition of 50 mg kg⁻¹, the colonization rate of AMF and the structure of maize roots were significantly higher than those without P and a higher P addition. Furthermore, Guo et al. [47] reported that the colonization rate of ryegrass roots by AMF at the P level of 10 mg kg⁻¹ was significantly higher than that of the other P treatments. A certain amount of P fertilizer in the soil altered the distribution of benefits between AMF and plants in the symbiotic system. This could result in a decrease in the allocation of materials and energy to AMF [48]. Moreover, under high soil P conditions, the phospholipid composition of the root cell membrane increases, as does the permeability of the cell membrane [49]. This leads to a decrease in the signal substance strigolactone (which induces AMF perception, promotes spore germination, and promotes hyphal branching) via root secretion [50]. Hence, high soil P conditions could affect the AMF colonization rate of plants. Nevertheless, AMF were still able to establish a beneficial association with maize roots regardless of the water treatment conditions under high available P (48.7 mg kg⁻¹), as determined by Qin et al. [51]. Therefore, it is important to clarify the P addition threshold that significantly increases the colonization rate of plant roots for mating plant growth under drought stress.

4.2. AMF and P Addition Enhance N and P Uptake in Oat Plants Under Drought Stress

Within the P addition range of this study, drought stress significantly reduced the soil MBP content during the oat growth stages, while AMF inoculation effectively counteracted this effect by markedly increasing the soil MBP content. As a crucial functional component of soil microbiota, AMF can constitute 5–50% of the total soil microbial biomass [52]. Previous studies have demonstrated that AMF colonization in maize develops extensive extraradical mycelial networks [53]. These hyphae secrete carbon-containing compounds that substantially alter carbon availability in the mycorrhizosphere [54], thereby modulating the soil microbial community composition. This suggests that the expansion of AMF mycelial networks and their stimulatory effects on associated microbes may represent a key mechanism for enhancing the overall soil microbial biomass. Notably, under drought conditions, AMF inoculation increased the maize rhizosphere soil glomalin-related protein content [51] while significantly elevating the soil organic matter levels. These modifications provide critical substrates for microbial proliferation. Therefore, within specific soil P thresholds, AMF inoculation enhances soil microbial biomass through these multifaceted mechanisms. This improvement is ecologically significant for maintaining soil nutrient reservoirs and promoting biogeochemical cycling under drought stress conditions.

The current understanding of the combined effects of AMF inoculation and P addition on soil MBP under drought conditions remains limited. This study demonstrated that, under drought stress, the combination with the addition of AMF and a moderate P level (P1) significantly enhanced soil MBP in oat soil. These findings suggest that, in semiarid agricultural ecosystems, the combination with the addition of AMF and P could effectively alleviate the drought-mediated suppression of soil MBP, potentially restoring it to levels comparable to those of the well-watered without inoculation condition.

Soil nutrients are influenced by soil type, soil parent material, and human activities such as fertilization [55]. The water, P, and AMF treatments strongly influenced the ammonium N, nitrate, total P, and available P content in oat soil (Table 2, Table 3, Table 4 and Table 5). It is important to make full use of nutrients in the soil by artificial regulation to improve plant yield in dry farming regions [56]. In this study, drought stress could restrain the absorption of nutrients in the soil by oat, resulting in an increase in the ammonium N, nitrate, total P, and available P content in oat soil. Previous studies have indicated that drought stress can result in an uneven distribution of nutrients in the soil, exacerbate the accumulation of residual nutrients in farmlands, and notably inhibit the soil N and P cycles [57].

The extraradical hyphae of AMF can extend into the soil beyond the reach of plant roots, playing a significant role in ecosystems through nutrient cycling [58]. Additionally, AMF release phosphatase directly into the soil via hyphae under drought stress. This phosphatase facilitates the conversion of insoluble P in the soil into soluble P, thereby promoting overall P conversion in the soil [59]. Previous research on kapok [60] revealed that AMF inoculation reduced the total P in rhizosphere soil under moderate drought (soil relative water content of 45%) but increased ammonium N and nitrate N under severe drought (soil relative water content of 30%). The results of this study indicate that AMF inoculation showed a significant trend in reducing ammonium N, nitrate N, and total P content in oat soil while significantly increasing the available P content. The discrepancy between the results of this study and previous findings may stem from the regulatory effects of the AMF symbiotic system on plant N uptake pathways. Specifically, AMF may significantly upregulate the expression levels of ammonium transporters (AMTs) and nitrate transporters (NRTs) in oat roots, thereby enhancing the plant’s absorption efficiency of NH₄⁺ and NO₃⁻ from the soil [61]. This could ultimately lead to the rapid depletion of the soil inorganic N pool.

The development of a precise P addition system, integrated with science-based P management strategies, is essential for maximizing P fertilizer use efficiency [62]. Fall et al. [63] revealed that AMF inoculation increased the content of ammonium N, nitrate N, and available P in maize soil when combined with a moderate P level. Ul Haq et al. [64] indicated that the synergistic effect of AMF inoculation and P addition led to significant improvements in soil N and P availability and plant N and P concentrations in wheat, surpassing the effects of P addition alone. In this study, AMF inoculation with P addition significantly increased the nitrate N, total P, and available P content in oat soil. Noteworthily, under drought stress conditions, AMF inoculation at the P0 level resulted in the most significant increase in the available P content in oat rhizosphere soil (Table 5). Previous studies have demonstrated that optimal P supply not only facilitates the efficient transport of available P (e.g., H₂PO₄⁻) through AMF hyphae but also may enhance the hyphae-mediated activation and utilization of fixed P in soil [65]. This dual mechanism could improve plant P acquisition efficiency.

Plants and soil are two subsystems of biogeochemical cycles. The N and P contents in plants can reveal the effectiveness of soil nutrients and their efficiency of uptake by plants [66]. Drought is a recurring global climatic phenomenon and the most common natural disaster in many regions of the world [67], which reduces the nutrient uptake and transfer by plants [68]. In this study, drought stress reduced the total P content in oat plants (Table 6 and Table 7).

As a functional link between plants and soil, AMF can form a network of hyphae resembling cobwebs and play a key role in nutrient absorption, especially for N and P [69,70]. In a study conducted by Li and Zhang [71], ¹⁵N labeling experiments revealed that AMF hyphae could absorb NH₄⁺ from the soil a few centimeters away from the roots of maize plants and transport it to various organs of maize through the root system. In this study, there was a significant coordination effect between AMF and oat plants. After AMF inoculation, the symbiotic relationship between AMF and oat plants led to the formation of numerous hyphae, which could enhance the ability of oat plants to acquire soil N and P.

The synergistic addition of optimal P and AMF inoculation markedly improves the mineral nutrient acquisition [23]. Zhang et al. [72] reported that AMF inoculation and P addition significantly increased the P content in wheat stems, grains, and leaves under water deficit conditions. Under drought stress, AMF inoculation at the P0 level resulted in the most significant increase in the total N and P contents in oat plants (Table 7). Additionally, under drought stress, the PRE of oat in this study reached its peak values at the P1 level when combined with AMF inoculation. This result further validates the concept of “less but better” fertilization with the help of AMF, which balances both ecological and sustainable agricultural development benefits.

4.3. AMF and P Addition Mitigate Drought-Induced Oat Yield Loss

From an agricultural perspective, yield is the most important index for characterizing drought avoidance. Drought negatively affects agricultural production and leads to a reduction of more than 50% in the average global plant yield [73]. The results showed that drought stress significantly reduced oat grain yield compared to well-watered conditions, regardless of AMF inoculation or P addition. A similar observation was made by Li et al. [74], who noted that drought stress significantly reduced oat grain yield under AMF inoculation conditions. Das et al. [75] also confirmed that drought stress reduced the yield of rice under AMF inoculation regardless of the cultivation method. The observed phenomenon may be attributed to the fact that drought stress inhibits the growth-promoting effects of both AMF inoculation and P addition, thereby directly disrupting multiple critical physiological and metabolic processes in plants [76].

Under drought stress, within the experimental range of P levels, AMF inoculation was found to improve oat grain yield, and when P was applied at the P1 level, oat grain yield was higher. Karaki et al. [77] reported that wheat grain yield was higher in mycorrhizal plots than in nonmycorrhizal plots, irrespective of soil moisture. Wang et al. [78] also reported that AMF inoculation can lead to an increase in tomato yield of 2–28% under both mild and severe drought stress conditions. In this study, although AMF inoculation significantly increased oat grain yield under drought stress, it was still different from that of well-watered plants. However, AMF inoculation at the P1 level significantly reduced the difference in oat grain yield. In fact, Smith [79] established that AMF-inoculated plants generally exhibit higher yields than AMF-free plants, primarily due to an increased uptake of P. Additionally, under drought stress, the PFP_P of oat in this study reached its peak values at the P1 level when combined with AMF inoculation. The results indicate that AMF inoculation at moderate P levels demonstrates optimal PFP_P during drought stress. These findings suggest that the addition of optimum P combined with mycorrhizal fertilizer-related AMF could serve as a technical approach to mitigating the negative impact of drought stress on oat in dryland regions.

Correlation analysis revealed that soil MBP showed significant positive correlations with AMF infection rate, soil available P, the plant total N and P content, and oat grain yield. Moreover, the research data also showed that soil MBP acted as the dominant factor regulating oat grain yield and the plant total N and P content. This synergistic effect could primarily stem from complex interaction mechanisms within the “mycorrhiza–microbe–plant” system [23]. Consequently, soil MBP should be further studied under the combined addition of AMF and P under drought stress in scientific research and in the field.

5. Conclusions

Taken together, the findings of this study indicate that inoculation with Funneliformis mosseae combined with a moderate amount of P (P1) significantly increased the AMF colonization rate of oat roots, the soil MBP, and the oat grain yield under drought stress. Notably, oat PFP_P and PRE also peaked at the P1 level, further highlighting the synergistic role of AMF and balanced P addition. Nevertheless, the oat grain yield of AMF-inoculated plants under drought stress still differed from that of well-watered AMF-free plants, highlighting the lingering functional limitations of AMF under drought stress. AMF inoculation in combination with P addition confers drought tolerance upon oat by optimizing the soil–plant N and P levels through soil MBP regulation. However, the current understanding of the effects of AMF inoculation in combination with P addition is predominantly derived from controlled pot experiments. Future studies should validate these findings across field environments at varying spatial scales to establish their agronomic relevance under real-world conditions.