Sustainable utilization of phosphogypsum in agriculture enhanced by mycorrhizal organisms: a new perspective

doi:10.21203/rs.3.rs-8239909/v1

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Research Article

Sustainable utilization of phosphogypsum in agriculture enhanced by mycorrhizal organisms: a new perspective

https://doi.org/10.21203/rs.3.rs-8239909/v1

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Background

Arbuscular mycorrhizal fungi (AMF) could promote plant growth and nutrients absorption, and had a certain resistance to arsenic pollution from soil. Phosphogypsum (PG) could provide the necessary elements for crop growth because of containing nutrients such as phosphorus (P), sulfur (S) et al. but PG contained arsenic (As) also could bright pollution risk to crop growth.This study investigated the synergy of AMF and PG on the growth and nutrient absorption of tobacco (Nicotiana tabacum L.) and evaluated the risk of arsenic (As) accumulation.

Results

Results showed thatindependent of AMF inoculation, PG addition significantly increased shoot biomass in ‘NC297’ plants, significantly increased the plant S concentration and absorbed amount, and the S specific absorption rate of ‘KRK26’ plants. In response to each PG addition level and compared to the uninoculated treatment, FM inoculation significantly increased the P specific absorption rate, GA inoculation significantly increased the P concentration and absorbed amount of ‘KRK26’ plants. Furthermore, GA inoculation significantly decreased the shoot As concentration and uptake, thus significantly increasing the absorption ratio of P to As in ‘KRK26’ shoots.Principal component analysis (PCA) showed soil pH contributed significantly to the two principal components extracted.Pearson correlation analysisindicated that a significant positive correlation between root P uptake,root S uptake and soil available P respectively, and soil available As content was significantly negatively correlated with soil pH, indicating that PG could reduce the bioavailability of arsenic by increasing pH.

Conclusion

When PG addition with AMF inoculation was applied to soils with S and P deficiency, a better effect was found for PG40 addition-GA inoculation combination treatment for promoting plant growth and P and S uptake by ‘KRK26’ plants. And PG could reduce the As bioavailability by increasing pH value. It is conducive to the safety and sustainable development of the agricultural ecological environment.

Mycorrhizalorganisms

Arsenic

Phosphorus

Phosphogypsum (PG)

Sustainable utilization Tobacco

Phosphogypsum (PG) is a by-product of the industrial production of phosphoric acid and is generated at a mass ratio of 5/1. According to incomplete statistics, large quantities of PG are produced worldwide (approximately 300 million metric tons of PG is generated annually)[1]. In China, the annual PG is approximately 80 million tons, and the cumulative stockpile has exceeded 870 million tons, accounting for 14.5% of the global stockpile, but its utilization was still less than half in 2021according to the data released by the China Inorganic Salt Industry Association.China's total PG production is mainly located in the provinces Hubei, Yunnan, Guizhou, Sichuan, with Yunnan province contributing a significant portion [2].However, the produced PG and the resulting in situ emissions not only occupy land resources, but also pollute soil, water, and the general environment. Therefore, these PG-related problems need to be urgently solved.

Research has shown that PG, as an acidic fertilizer, adjusts pH, reducing alkalinity and sodium adsorption on soda-saline soil [3, 4]. A further study reported that PG contains a variety of nutrients such as phosphorus (P), sulphur (S), and others, which are needed for plant growth and have been shown to significantly promote crop growth[5, 6]. In addition, according to a report of the Institute of Soil Science, Chinese Academy of Sciences, PG application could increase the average tobacco yield by 14.6%. Therefore, it may be possible for PG to improve soil nutrient contents. However, PG is strongly acidic and contains a certain amount of harmful substances, such as arsenic (As), which harms both the growth of crops and the safety of agricultural products.

Arbuscular mycorrhizal fungi (AMF) are widespread throughout terrestrial ecology systemsand are rhizosphere microorganisms that can live symbiotically with more than 90% of land plants [7]. At present, more than 80% of terrestrial plants could form mycorrhizal with AMF. Mycorrhizal infection can increase nutrient absorption by the host plant in a greater range around the rhizosphere, and improves the spatial availability of soil nutrients, thus increasing mineral nutrient uptake by the host plant [8, 9]. AMF plays an important role for plant growth, increased yield, and quality [10–12]. Another study showed that mycorrhizal fungi also have the potential to remediate the stress environment around mines and to resist soil pollution caused by metals (such as Cd、Pb、As)[3, 13–15]. Especially, Funneliformismosseae (FM) symbiosis is effective in reducing the As concentration in leaves and roots of adult tobacco plants [16], while Glomus aggregatum (GA) inoculation reduced As toxicity symptoms and improved sunflower plant growth [17], andGlomusmosseae or Rhizophagus irregularis inoculation and phosphorusapplication alleviated the toxicity of As to maize cultivated on an arsenic-contaminated soil, respectively [18, 19].

Tobacco is one of the main economic crops of Yunnan Province, Southwest China, where it has a long history of cultivation. According to national statistical data, Yunnan province implemented a tobacco planting area of 0.468 million hectares by the end of 2011. “Yunyan87” accounted for 21.4% of the tobacco planting area of Yunnan Province in 2004. The study showed that the tobacco variety “NC297” has high ecological adaptability and good quality characteristics at different altitudes [20]. In addition, the quality of the central leaves of the variety KRK26 was as good as that of NC297 and the suction quality of both varieties was identical [20].Furthermore, tobacco variety MRGTH 1 and topping level at 18 leaves gave significantly the highest nicotine content compared to others [21].Therefore, we selected different tobacco varieties planted in Yunnan Province for this study.

AMF can resist metal pollution to some extent. To solve the problem of PG storage and to reduce the accumulation risk of metals in agricultural production, protect the security of the agricultural ecological environment and its sustainable development, we used GA and FM for testing. Tobacco is one of the main economic crops of Yunnan Province. Therefore, we selected three tobacco varieties commonly planted in Yunnan Province as test materials. This study investigated the synergy of PG addition and AMF inoculation on the growth and nutrient uptake of tobacco and the ability to withstand the risk of arsenic pollution. It is expected to provide a new and more effective method for the use of PG in agricultural soils in southern China, especially in those agricultural soils with S and P deficiency in Yunnan Province.

2.1. Experimental time and location

A pot experiment was conducted in the greenhouse of the Yunnan Agricultural University; indoor analysis was completed in the Yunnan Soil Fertilization and Pollution RemediationEngineering Research Center.

2.2. Experimental materials

Soil with S and P deficiency was collected from Jiangchuan County, Yuxi City, Yunnan Province, China. After air drying, the soil for cultivation was passed through a 2-mm sieve. The soil was steamed at 121°C for 2 h and then sealed in plastic bags to avoid microbial contamination.

PG was collected from Kunming phosphate fertilizer enterprises (the ancient town of Kunyang Phosphate Fertilizer Factory) in Kunming City, Yunnan province. The PGs for cultivation were passed through 1 mm mesh sieves, mixed evenly, and then also steamed at 121°C for 2 h.

Soil and PG had the following properties, respectively: pH 7.50, 4.67 (the ratio of soil to CaCl₂ solution was 1:2.5); 12.46 g·kg^− 1, 4.46 g·kg^− 1 of organic matter; 0.58 g·kg^− 1, 4.89 g·kg^− 1 of total P; 5.76 mg·kg^− 1, 25.52 mg·kg^− 1 of available P; 34.65 mg·kg^− 1, 395.84 mg·kg^− 1 of available S; 6.70, 18.57 mg·kg^− 1 of total As. The total As content in soil was extracted via concentrated HNO₃ and HCl at 1:3 (V/V) and determined using an atomic fluorescence spectrometer (AFS-230E, Haiguang Instrumental Co., China) [22]. Soil total P was determined via HF-HClO₄ digestion [23] and molybdenum-blue colorimetry. Soil available P was determined via extraction with sodium bicarbonate followed by the molybdenum-blue method [24].Soil available S was determined via Barium Sulfate Turbidimetry. All results were expressed on an oven-dried soil weight basis by correcting for the water content of the soil (105°C, 24 h).

Three varieties of tobacco (Nicotiana tabacum L.), i.e., ‘NC297, Yunyan87, and KRK26’, were selected as host plants for this experiment. Tobacco seeds were provided from professor Fuzhao Nian, who works at the college of tobacco, Yunnan Agricultural University. Tobacco seeds of uniform quality and without scars were selected via floating seedling. The seedling substrate was sterilized with high pressure. Benomyl with certain concentration was added to the floating solution for one week to inhibit the reproduction of other fungi.

Two strains of AMF (Funneliformismosseae (BGC YN05, FM) and Glomousaggregatum (BGC HEB07C, GA)) were supplied by professor Youshan Wang, who works at the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences. Both strains of AMF did not have a background of As tolerance. They were propagated in pot culture on maize and clover plants grown in sandy soil for 10 weeks. Inoculum from the pot culture comprised of a mixture of spores, mycelium, sandysoil, as well as maize and clover root fragments. Every 10 g of inoculum contained approximately 150 fungal spores.

2.3. Experimental treatment and management

The experiment was designed with three factors: different levels of PG addition was either 0 or 40 g·kg^− 1(PG0, PG40), different varieties of tobacco (NC297, Yunyan 87 and KRK26), and different strains of AMF treatment status (NM, FM and GA), respectively, resulting in a total of 18 treatments, with four replications of each parameter measurement, totaling 72 pots. After adding PG to the soil, the pots were allowed to equilibrate for two weeks before planting. Each inoculated plant received 40 mg FM and GA fungi inoculated and all uninoculated plants were treated with the same amount of inocula that had been autoclaved twice at 121°C for 30 min. 1.5 L plastic pots (one plant per pot) were filled with 1 kg of dry soil and fertilizer was added to the soil foundation in solution (N 60 mg·kg^− 1, P 30 mg·kg^− 1, K 67mg·kg^− 1, Ca 20mg·kg^− 1, Mg 4.5 mg·kg^− 1, Mn 0.92 mg·kg^− 1, Cu 0.54 mg·kg^− 1, Zn 1.24 mg·kg^− 1, and Mo 0.06 mg·kg^− 1) respectively by NH₄NO₃, KH₂PO₄, K₂SO₄, CaCl₂·2H₂O, MgSO₄·7H₂O, MnSO₄·H₂O,CuSO₄·5H₂O, ZnSO₄·7H2O, and (NH4)₆Mo₇O₂₄·4H₂O entry form, balance 1 week. At the cross stage (with four leaves), tobacco plants were carefully transplanted into pots that were arranged in a randomized complete block design. Plants were grown in a sunlit greenhouse with natural light, a day/night temperature of 32°C/22°C, and a relative humidity of 40%–60%. Plants were watered to maintain soil moisture at 60%–70% of water holding capacity by adding deionized water during the experimental period. To ensure adequate tobacco growth, N, K, and other nutrients were added for 30 days the tobacco growth period, for a top dressing (N 30 mg·kg^− 1, K 20 mg·kg^− 1).

2.4. Harvest and analysis

Roots and shoots were harvested separately after 12 weeks of growth. Root samples were carefully washed with tap water followed by deionized water to remove adhering soil and sand particles. Approximately 0.7 g of the sub-samples of fresh roots in each pot were collected and cut into approximately 1 cm pieces.For the determination of root mycorrhizal colonization, the root samples were cleaned in 10% KOH (w/v) and stained with 0.05% (w/v) trypan blue in lactoglycerol [25]. The root infection rates were evaluated via the gridline-intersect method under a microscope [26]. Tissues were weighed after oven drying at 60°C for 72 h and then ground to < 0.25 mm in a stainless mill. Dried sub-samples of roots and shoots were digested by concentrated HNO₃ and HCl at 3:1 (V/V) to analyze As concentration. Determination of P concentration in plants was performed spectrophotometrically via the ammonium–vanadate–molybdate method [27]. Total S concentration in plants was determined via turbidimetry after wet ashing with magnesium nitrate and perchloric acid [28]. The specific absorption rate (SAR) was calculatedbased on unit root biomass ‘mg’ to the corresponding plant nutrient uptake ‘µg’ [27].

2.5.Statistical analysis

All data were analyzed via one-way analysis of variance (ANOVA) with treatment as factor. Mean separation was conducted based on Duncan’s multiple range test, and differences at P < 0.05 were considered statistically significant. All statistical analyses were performed via SPSS22.0 for Windows.

3.1. Mycorrhizal formation and tobacco growth under AMF inoculation and PG addition

No root infection was detected inuninoculated tobacco varieties (Table 1); however, the root colonization rates of inoculated tobaccos all remained below 23%. For identical inoculation conditions, PG addition significantly increased the root infection rates of tobacco ‘NC297’ under FM inoculation treatment (P < 0.001). FM and GA inoculation significantly decreased the root infection rates of tobacco ‘KRK26’ (P < 0.001).

Independent of inoculation, PG addition significantly increased the shoot biomass of tobacco ‘NC297’ and the root length of tobacco ‘KRK26’ (P < 0.001) (Table 1). Moreover, PG additionsignificantly increased the shoot biomass under NM treatment and the root biomass under FM treatment of the tobacco ‘Yunyan87’ (P < 0.001). However, FM inoculation significantly increased the shoot and root biomass of the tobacco ‘Yunyan 87’ (P < 0.001). This result shows that for each PG addition level, GA inoculation significantly increased the root biomass of the tobacco ‘NC297’.

Table 1
The growth and mycorrhizal colonization rate of three kinds of tobacco (NC297, YY87 and KRK26)plants.
Tobacco varieties	PG treatment (g·kg^− 1)	AMF inoculation status	Mycorrhizal infection rate (%)	Biomass(g·pot^− 1)		Root length(m·pot^− 1)
Tobacco varieties	PG treatment (g·kg^− 1)	AMF inoculation status	Mycorrhizal infection rate (%)	Shoot	Root	Root length(m·pot^− 1)
NC297	0	NM	0c	4.15k	2.73g	31.1bc
		FM	14.8b	4.23ij	2.77efg	29.0bcd
		GA	18.6a	4.21jk	2.87bc	28.8bcd
	40	NM	0c	4.29ghi	2.78defg	36.0a
		FM	16.9a	4.31fgh	2.80cdef	26.3de
		GA	16.9a	4.35ef	2.86bc	25.1e
yunyan87	0	NM	0z	4.54d	2.73fg	29.1bcd
		FM	13.0y	4.70bc	2.84bcd	28.4cd
		GA	14.9x	4.74ab	2.86bc	26.1de
	40	NM	0z	4.68c	2.76efg	30.3bc
		FM	13.1y	4.78a	2.95a	26.0de
		GA	12.7y	4.76a	2.75fg	28.4cd
KRK26	0	NM	0ε	4.16k	2.72g	30.2bc
		FM	18.4γ	4.35efg	2.83cde	26.0de
		GA	22.5α	4.30fgh	2.90ab	29.5bc
	40	NM	0ε	4.26hij	2.74fg	37.7a
		FM	14.8δ	4.39e	2.91ab	31.7b
		GA	19.9β	4.34efg	2.87bc	36.0a

NM, FM and GA respectively represent uninoculated, inoculatedFunneliformismosseaeandinoculatedGlomousaggregatumtreatments. PG0 and PG40 referto 0 and 40 gkg^− 1in PG addition levelsrespectively. Different letters show significant differences at P < 0.05 levels and different letters system above the columns indicate there are not significant interaction between AMF inoculation and PG addition.

3.2. P and S uptake by tobaccos plants under AMF inoculation and PG addition

For different varieties of tobacco, independent of inoculation, PG addition significantly increased the shoot P concentration and absorbed amount in tobacco ‘KRK26’ plants (P < 0.001) (Fig. 1(a)-(d)). Under PG0 treatment, compared to NM treatment, FM and GA inoculation significantly increased the shoot P concentration and absorbed amount of all tobacco varieties, and significantly increased the root P concentration and absorbed amount in ‘NC297’ and ‘Yunyan 87’ plants (P < 0.001); under the PG40 treatment, FM and GA inoculation significantly increased the P concentration and absorbed amount in ‘NC297’ and ‘KRK26’ plants. The results showed that FM and GA inoculation achieved the best effect to promote P absorption of tobacco ‘NC297’ (P < 0.001).

For different varieties of tobacco, independent of AMF inoculation, PG addition significantly increased the shoot S concentration and absorbed amount for all tobacco varieties, and significantly increased the root S concentration and absorbed amount in ‘KRK26’ plants (P < 0.001) (Fig. 2(a)-(d)). Under PG0 treatment, compared to NM treatment, FM and GA inoculation significantly increased the shoot S concentration and absorbed amount for all tobacco varieties (P < 0.001); for the PG40 treatment, FM inoculation significantly increased the shoot S concentration and absorbed amount for all tobacco varieties, and FM inoculation significantly increased the S concentration and absorbed amount in ‘Yunyan87’ and ‘KRK26’ plants (P < 0.001).

3.3. As uptake by tobacco plants under AMF inoculation and PG addition

For different varieties of tobacco, under PG0 treatment and in comparison to NM treatment, GA inoculation significantly decreased the shoot As concentration and absorbed amount in ‘NC297’ and ‘KRK26’ plants (P < 0.001) (Fig. 3(a)-(d)); under PG40 treatment, GA inoculation significantly decreased the As concentration and absorbed amount in ‘NC297’ plants and the root As concentration and absorption amount in ‘KRK26’ plants (P < 0.001).

3.4. Uptake ration of P to As in tobacco plants under AMF inoculation and PG addition

Independent of AMF inoculation, PG addition significantly increased the uptake ratio of P to As in ‘KRK26’ shoot and significantly increased the uptake ratio of P to As in ‘KRK26’ roots under GA treatment (P < 0.001) (Fig. 4(a)-(b)). Under PG0 treatment, compared to NM treatment, FM and GA inoculation significantly increased the uptake ratio of P to As in shoots for all tobacco varieties, and FM inoculation significantly increased the uptake ratio of P to As in ‘NC297’ and ‘Yunyan 87’ roots (P < 0.001); for PG40 treatment, GA inoculation significantly increased the uptake ratio of P to Asin ‘NC297’ and ‘KRK26’ plants (P < 0.001).

3.5. SAR of P, S, and As by tobacco plants under AMF inoculation and PG addition

PG addition significantly increased the SAR of P in ‘KRK26’ plants and SAR of S in plants for all tobacco varieties under both FM and GA treatments (P < 0.001) (Table 2). Independent of AMF inoculation, PG addition significantly increased the SAR of As in plants for all tobacco varieties (P < 0.001). Under PG0 treatment, compared to NM treatment, both FM and GA inoculation significantly increased the SAR of P in plants for all tobacco varieties, and FM inoculation significantly increased the SAR of S in ‘Yunyan 87’ and ‘KRK26’ plants (P < 0.001). For PG40 treatment, both FM and GA inoculation significantly increased the SAR of P in ‘NC297’ and ‘KRK26’ plants and FM inoculation also significantly increased the SAR of P in plants for all tobacco varieties, while GA inoculation significantly decreased the SAR of As in ‘NC297’ and ‘KRK26’ plants (P < 0.001).

Table 2
The SAR of P, S and Asby three kinds of tobacco (NC297, YY87 and KRK26) plants.
Tobacco varieties	PG treatment (g·kg^− 1)	AMF inoculation status	SAR(µg·mg^− 1)
Tobacco varieties	PG treatment (g·kg^− 1)	AMF inoculation status	P	S	As
NC297	0	NM	3.89d	5.05efg	0.007ef
		FM	8.49a	5.36ef	0.009bcd
		GA	7.54b	4.67gh	0.008de
	40	NM	5.02c	4.10hi	0.009bc
		FM	7.81b	8.97a	0.009b
		GA	7.62b	6.40c	0.007ef
yunyan87	0	NM	6.70z	3.88i	0.007ef
		FM	10.29x	5.27efg	0.006fg
		GA	8.42y	4.25hi	0.007ef
	40	NM	9.84xy	4.30hi	0.008cd
		FM	11.13x	8.12b	0.012a
		GA	8.89y	5.59de	0.008cd
KRK26	0	NM	2.19γ	3.33j	0.004ij
		FM	3.30β	4.34hi	0.005hi
		GA	3.97β	3.93i	0.004hi
	40	NM	2.97βγ	5.04efg	0.005gh
		FM	6.36α	6.12cd	0.006fg
		GA	5.98α	4.97fg	0.003j

3.6.Principal component analysis of key indicators in tobaccos and soils under AMF inoculation and PG addition

Principal component analysis (PCA) was used to comprehensively analyze all indicators in tobaccos and soils. From the results (Fig. 5A), the first two principal components PC1 and PC2 explained 55.5% of the total variance, where PC1 containedtheinformationofroot biomass, shoot P uptake, root P uptake, shoot S uptake, root S uptake, shoot uptake ration of P to As, root uptake ration of P to As, and soil available As content, PC2 containedtheinformationof root length, shoot biomass, shoot As uptake, root As uptake, soil pH, soil available S content and available P content.SoilpH contributed significantly to the two principal components extracted.

Selecting NC297 tobacco, it can be seen (Fig. 5B) that different biological replicates showed aggregation under the same biological treatment, indicating that the experimental results were good. There was a clear separation between PG additiontreatment and no PG additiontreatment, indicating that the PG addition promotedthe growth and nutrient absorption of NC297 plants. Under different PG treatments, NM, GM, and FM treatments also showed clear separation, indicating that inoculation with AMF couldpromote the growth of NC297.

3.7.Regulation factors analysis of tobacco P, S, and AsuptakeinducedbyAMF inoculation and PG addition

Pearson correlation analysis (Fig. 6) showed a significant positive correlation between root P uptake and soil available P (r = 0.80). Shoot P uptake was highly positively correlated with root uptake ration of P to As (r = 0.75). Root S uptake was strongly correlated with soil available S (r = 0.81) and positively correlated with root biomass (r = 0.56). Shoot S uptake was significantly positively correlated with root biomass (r = 0.76).There was a significant negative correlation between root uptakeration of P to As and Shoot As uptake (r=-0.64). There was a significant negative correlation between root Suptake and shoot As uptake (r=།0.63). Soil available As content was significantly negatively correlated with soil pH value(r=།0.71), indicating that PG could reduce the As bioavailability by increasing pHvalue.

4.1.Regulationof P, S, and Asuptake by plants induced by AMF and phosphogypsum

As indicated by a previous study, AMF directly affect the early stages of Rudbeckia laciniata and Solidago gigantea growth [29]. Furthermore, AMF inoculation could also increase both P and S uptake of the host plant and promote plant growth [30–32].Our study found that except for ‘Yunyan87’ roots under PG40 treatment, both FM and GA inoculationincreasedthe plant P concentration and total absorbed amount for all varieties of tobacco to a certain extent. Except for ‘NC297’ roots under PG0 treatment, FM and GA inoculation increased the plant S concentration and total absorption amount, thus promoting tobacco growth. Zhang et al reported that the shoot S concentration andorganic S concentration significantly increased with the improvement of S level, and AMF could promote the host plant to absorb sulfur nutrition of the external environment, thus improving S nutrition and the quality of onions[33]. Our study also found that the available P content in PGs was about five times higher than in soil, and the available S content was about 11 times higher than in soil. With the addition of external P and S nutrients, the specific absorption rate of P and S increased for ‘Yunyan87’ and ‘KRK26’, thus the shoot concentration and total absorbed amount of S were significantly increased, and the shoot biomass was improved to a certain extent, this is consistent with the previous research conclusions. In addition, pearson correlation analysis showed a significant negative correlation between root S uptake and shoot As uptake (r=-0.63). This result confirmedthat S may have a physiological antagonism with the pollutant As.

A previous study showed significant differences between AMF species in their effect on plant nutrition [34].Other study showed that colonisation by AM fungi can affect DNA methylation levels in their hosts and that plant DNA methylation varies in an age-and tissue-specific manner [35].Our results indicated that FM inoculation significantly increased the concentration and total absorbed amount of S in ‘Yunyan87’ and ‘KRK26’ plants,but GA inoculation only significantly increased the shoot concentration and total absorbed amount of S in ‘KRK26’. In addition, FM and GA inoculation significantly increased the shoot P concentration and total absorbed amount, and the SAR of P in ‘NC297’ and ‘KRK26’; however, no significant increase of the total absorbed amount of P was found in ‘Yunyan 87’. Principal component analysis indicated that the three types of tobacco leaves were treated with PG and AMF respectively. Different biological replicates of the NC297 variety all showed aggregation phenomena, indicating that both the addition of PG and the inoculation of AMF can promote the growth of NC297.From this, different strains of AMF not only promote the absorption of nutrients by host plants, but also have a close relationship with tobacco varieties[36].

Mycorrhizal fungi may play an more important role in protecting plants against arsenic (As) contaminationthan the traditional bioremediation methods [37, 38]. Previous studies indicated that As concentrations in roots and stalks after mycorrhizal treatments were much lower than in the control treatment. Under the stress of As contamination, proper mechanisms employed by AM fungi can protect tobacco against As uptake [39, 40]. The present study leaves it unclear whether AMF function like ericoid mycorrhizal fungi that can protect their host plant against As contamination via reducing arsenate to arsenite and via pumping out the latter. However, clearly, superphosphate interaction with F. mosseae reduced the role of mycorrhizal infection in terms of enhancing P nutrition and reducing the uptake of potentially toxic As into plant parts [41]. Arsenate and phosphate share the same transporters in plants and AMF influence the expressions of Pi transporters in rice [42].The previous study demonstrated that AM fungi enhanced plant As tolerance by improving plant P nutrition, inhibiting As uptake and transforming inorganic As into a less toxic organic form [3].There is no doubt that the improved P nutrition and growth of AM plants considerably “diluted” As in both shoots and roots, due to relatively small hyphal uptake of As compared to P. This “dilution effect” led to extremely high P/As ratios in shoots of ‘KRK26’ plants and roots of ‘NC297’ plants. The much higher P/As ratios in AM plants clearly showed that AM colonization is likely to help host plants extract As from potentially toxic soils. In this study, independent of PG addition, GA inoculation significantly increased the plant concentration and the total absorbed amount of P by tobacco ‘NC297’ and ‘KRK26’ plants. However, different degrees decreased the shoot concentration, total absorption amount of As in ‘NC297’, and ‘KRK26’ plants, thus significantly increasingthe shoot P/As ratios. In addition, pearson correlation analysis showed a significant negative correlation between root uptakeration of P to As and shoot As uptake (r=-0.64). This may be the competitive absorption between P and As for the same transport systems [19].

Zhang et alreported that different AM fungal species had different abilities to make their host tolerant to metals. R. irregularis was more resistant to contaminated soil compared to the other two AM fungal species[43].In this study, compared to the control, FM inoculation generally increased the As concentration and absorption of all tobacco, whereas GA inoculation had the opposite effect. This may be due to the different tolerance of different strains to metals. AM plants directed less As to shoots compared to non-inoculated controls. The study found that the As concentration in different varieties of tobacco were different. Overall, KRK26 absorbed less As than both other tobacco types. This may be due to different resistance of different tobaccos to metals [44].Three tobacco varieties and two AMF strains were selected as advantageous combinations. Our study showed that the same strain did not yield the same effects on P, S, and other nutrient absorption levels of different tobacco varieties. It is well known that AMF could promote the nutrient absorption of mycorrhizal plants and could resist arsenic transfer to the shoots. However, different AMF had different effects on different crops and the influencing factors and mechanism require further research.

4.2.Suggestions for agricultural utilization and management of phosphogypsum

It is even more worth mentioning that according to previous national soil surveys [45], the distribution of available sulfur content in Yunnan's soil is extremely uneven, with approximately 1/3 of the soil lacking sulfur (available sulfur content < 12 mg·kg^− 1). The "Bulletin on the Main Data of the Third National Land Survey of Yunnan Province" released by the People's Government of Yunnan Province in December 2021 shows that the cultivated land area of Yunnan Province is 5.3955 million hectares, among which 4.2254 million hectares are dry land. In this experimental treatment, the application rate of PG was 40 g·kg^− 1. Based on a soil tillage layer thickness of 20 cm and an average bulk density of 1.15 g/cm³, the weight of one hectare of soil was approximately 2,250 tons. From this, it can be estimated that each hectare of cultivated soil can consume 90 tons of PG. If PG were applied to all the sulfur-deficient dryland farmlands in Yunnan Province, it would be possible to solve the problem of nearly 130 million tons of PG reserves. This would not only offset the annual amount of PG produced but also address a portion of the PG accumulation issue (China Inorganic Salt Industry Association). At the same time, it can also increase sulfur and phosphorus nutrients in the soil, promoting crop growth and increasing their yield. Therefore, when the results of this experiment are applied to agricultural production, it not only solves the problem of PG accumulation but also helps to address a series of environmental issues caused by the accumulation of solid waste, promoting the sustainable development of agriculture.

Independent of arbuscular mycorrhizal fungi (AMF) inoculation, phosphogypsum (PG) application significantly promoted plant S uptake and shoot P accumulation for ‘KRK26’ plants, thus improving the tobacco growth to a certain extent.At different levels of PG application and compared to NM treatment, FM inoculation increased P and S uptake for all varieties of tobacco at different degrees, thus improving the plant growth of tobacco ‘Yunyan87’ and ‘KRK26’. GA inoculation also significantly increased the absorption ratio of P to As in ‘NC297’ and ‘KRK26’ plants, and thus improved P uptake. Furthermore, GA inoculation decreased the shoot As concentration and absorption amount in ‘NC297’ and ‘KRK26’ plants at different degrees and inhibited the As toxicity to tobacco shoots. When PG addition with AMF inoculation was applied to soils with S and P deficiency, a better effect was found for PG40 addition-GA inoculation combination treatment for promoting plant growth and P and S uptake by ‘KRK26’ plants. This could reduce the risk of As pollution caused by PG application to agricultural soil to some extent. Principal component analysis (PCA)showed that soil pH contributed significantly to the two principal components extracted. The addition of PG promotes the growth and nutrient absorption of NC297 plants.Pearson correlation analysis showed a significant positive correlation between root P、S uptake and Soil availability. Soil available As content was significantly negatively correlated with soil pH value, indicating that PG could reduce the As bioavailability by increasing pH value.

Author Contributions: Cultivation administration, L.Z. and X.-R.Y.;Test analysis, data curation and Formal analysis, L.Z. and J. L.; Funding acquisition, Y.-S.X. and N.-M.Z.; Methodology, N.-M.Z. and C.-G.Z.; Project administration, X.-R.Y. and Y.-S.X.; Resources and Supervision,C.-G.Z., H.W. and M.-J.Y.; Writing—originaldraftand review & editing, L.Z., Y.-S.X. and X.-R.Y.; All authors have read and agreed to the published versionof the manuscript.

Funding: This work was financially supported by theNational Natural Science Foundation of China(Grant No. NSFC32371672), Yunnan Province Science and Technology Talent and Platform Plan Project (Grant No. 202405AM340004),Henan ProvinceOutstanding Youth Science Fund Project (Grant No.222300420001) and Natural Science Foundation of Henan (Grant No. 242300421102).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Not applicable.

Acknowledgements The authors thank professorYoushan Wang and Fuzhao Nian for their help of providing AMF strains and tobacco seeds.

Conflicts of Interest: The authors declare no conflict of interest.

QinXT CYH, Hu GHW, Liu QS, Xu ZH, Hu J, Zhang B, Luo ZY. Resource utilization and development of phosphogypsum-based materials in civil engineering. J Clean Prod. 2023;387:135858.
CuiY, Bai JD, Chang IS, Wu J. A systematic review of phosphogypsum recycling industry based on the survey data in China - applications, drivers, obstacles, and solutions. Environ Impact Assess Rev. 2024;105:107405.
Li JL, Sun YQ, Jiang XL, Chen BD, Zhang X. Arbuscular mycorrhizal fungi alleviate arsenic toxicity to Medicago sativa by influencing arsenic speciation and partitioning. Ecotoxicol Environ Saf. 2018;157:235–43.
Tian T, Zhang CL, Zhu F, Yuan SX, Guo Y, Xue SG. Effect of phosphogypsum on saline-alkalinity and aggregate stability of bauxite residue. Trans Nonferrous Met Soc China. 2021;31(5):1484–95.
Bouray M, Moir J, Condron L, Lehto N. Impacts of phosphogypsum, soluble fertilizer and lime amendment of acid soils on the bioavailability of phosphorus and sulphur under Lucerne (Medicago sativa). Plants.2020;9(7):883.
Michalovicz L, Lopes MMM, Tormena CA, Dick WA, Vicensi M, Meert L. Soil chemical attributes, nutrient uptake and yield of no-till crops as affected by phosphogypsum doses and parceling in southern Brazil.Archives of. Agron Soil Sci. 2018;65(3):385–99.
Tiwari J, Ma Y, Bauddh K. Arbuscular mycorrhizal fungi: an ecological accelerator of phytoremediation of metal contaminated soils. Arch Agron Soil Sci. 2022;68(2):283–96.
Hart M, Ehret DL, Krumbein A, Leung C, Murch S, Turi C, Franken P. Inoculation with arbuscular mycorrhizal fungi improves the nutritional value of tomatoes. Mycorrhiza. 2014;25(5):359–76.
Mei LL, Yang X, Zhang SQ, Zhang T, Guo JX. Arbuscular mycorrhizal fungi alleviate phosphorus limitation by reducing plant N:P ratios under warming and nitrogen addition in a temperate meadow ecosystem. Sci Total Environ. 2019;686:1129–39.
Gao XP, Guo HH, Zhang Q, Gao HX, Zhang L, Zhang CY, Gou ZY, Liu Y, Wei JM, Chen AY, Chu ZH, Zeng FC. Arbuscular mycorrhizal fungi (AMF) enhanced the growth, yield, fiber quality and phosphorus regulation in upland cotton (Gossypium hirsutumL). Sci Rep. 2020;10(1):2084.
He YM, Fan XM, Zhang GQ, Li B, Li MR, Zu YQ, Zhan FD. Influences of arbuscular mycorrhizal fungi and dark septate endophytes on the growth, nutrition, photosynthesis, and antioxidant physiology of maize. Int J Agric Biology. 2019;22:1071–78.
Zhang SJ, Lehmann A, Zheng WS, You ZY, Rillig M. Arbuscular mycorrhizal fungi increase grain yields: a meta-analysis. New Phytol. 2018;222(1):543–55.
Wu FY, Hu JL, Wu SC, Wong MH. Grain yield and arsenic uptake of upland rice inoculated with arbuscular mycorrhizal fungi in As-spiked soils. Environ Sci Pollut Res. 2013;22(12):8919–26.
Zhang Y, Hu JL, Bai JF, Wang JH, Yin R, Wang JW, Lin XG. Arbuscular mycorrhizal fungi alleviate the heavy metal toxicity on sunflower (Helianthus annuus L.) plants cultivated on a heavily contaminated field soil at a WEEE-recycling site. Sci Total Environ. 2018;628–9:282–90.
Akhtar O, Pandey D, Zoomi I, Singh U, Chaudhary KL, Mishra R, Pandey N. Role of arbuscular mycorrhizal fungi in heavy metals homoeostasis in plants. J Plant Growth Regul. 2024;43(11):3971–85.
Degola F, Fattorini L, Bona E, Sprimuto CT, Argese E, Berta G, Toppi LS. The symbiosis between Nicotiana tabacum and the endomycorrhizal fungus Funneliformismosseae increases the plant glutathione level and decreases leaf cadmium and root arsenic contents. Plant Physiol Biochem. 2015;92:11–8.
Ultra UR, Venecio, Tanaka S, Sakurai K, lwasaki K. Arbuscular mycorrhizal fungus (Glomus aggregatum) influences biotransformation of arsenic in the rhizosphere of sunflower (Helianthus annuus L). Soil Sci Plant Nutr. 2007;53(4):499–508.
Xia YS, Chen BD, Christie P, Smithf A, Wang YS, Li XL. Arsenic uptake by arbuscular mycorrhizal maize (Zea mays L.) grown in an arsenic-contaminated soil with added phosphorus. J Environ Sci. 2007;19(10):1245–51.
Cattani I, Beone GM, Gonnelli C. Influence of rhizophagus irregularis inoculation and phosphorus application on growth and arsenic accumulation in maize (Zea mays L.) cultivated on an arsenic-contaminated soil. Environ Sci Pollut Res. 2015;22:6570–7.
Xu YQ, Chang J, Tian YT, Hu BW, Li DT. Study on ecological adaptability of flue-cured tobacco variety'NC297'in Yunnan Province. Agricultural Sci Technol. 2013;14(7):982–7.
Gediya KM, Panchal J, Patel JN. Influence of nitrogen and topping levels on yield and quality of Bidi tobacco hybrid varieties. Ecol Environ Conserv. 2022;28(4):1798–802.
Phillips JM, Hayman DS. Improved procedurs for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans Br Mycological Soc. 1970;55(1):158–61.
Kara D, Özsavaşçi C, Alkan M. Investigation of suitable digestion methods for the determination of total phosphorus in soils. Talanta. 1997;44(11):2027–32.
Song SH, Wen YJ, Zhang JY, Wang H. Rapid spectrophotometric measurement with a microplate reader for determining phosphorus in NaHCO₃ soil extracts. Microchem J. 2019;146:210–3.
Koske RE, Gemma JN. A modified procedure for staining roots to detect VA mycorrhizas. Mycol Res. 1989;92(4):486–8.
Giovannetti M, Mosse B. An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol. 1980;84(3):489–500.
Azcón R, Ambrosano E, Charest C. Nutrient acquisition in mycorrhizal lettuce plants under different phosphorus and nitrogen concentration. Plant Sci. 2003;165(5):1137–45.
Sharifuddin HAH, Fauziah I, Zaharah AR. Technique of soil testing and plant analysis and their utilization for crop production in malaysia. Commun Soil Sci Plant Anal. 1990;21(13–16):1959–78.
Majewska ML, Rola K, Zubek S. The growth and phosphorus acquisition of invasive plants rudbeckia laciniata and solidago gigantea are enhanced by arbuscular mycorrhizal fungi. Mycorrhiza. 2016;27(2):83–94.
Felföldi Z, Vidican R, Stoian V, Roman LA, Sestras AF, Rusu T, Sestras R. Arbuscular mycorrhizal fungi and fertilization influence yield, growth and root colonization of different tomato genotype. Plants. 2022;11(13):1743.
Saia S, Aissa E, Luziatelli F, Ruzzi M, Colla G, Ficca AG, Cardarelli M, Rouphael Y. Growth-promoting bacteria and arbuscular mycorrhizal fungi differentially benefit tomato and corn depending upon the supplied form of phosphorus. Mycorrhiza. 2019;30(1):133–47.
Wu YJ, Chen CJ, Wang GA. Inoculation with arbuscular mycorrhizal fungi improves plant biomass and nitrogen and phosphorus nutrients: a meta-analysis. BMC Plant Biol. 2024;24(1):960.
Zhang YT, Luo Z, Guo T. Effects of arbuscular mycorrhizal fungi inoculation and sulfur fertilization on growth and quality of onion. J Plant Nutr Fertilizers. 2011;17(5):1283–7.
Taylor A, Pereira N, Thomas B, Pink DAC, Jones JE, Bending GD. Growth and nutritional responses to arbuscular mycorrhizal fungi are dependent on onion genotype and fungal species. Biol Fertil Soils. 2015;51(7):801–13.
Varga S, Soulsbury CD. Arbuscular mycorrhizal fungi change host plant DNA methylation systemically. Plant Biol. 2018;21(2):278–83.
Lyu HQ, Yu AZ, Chai Q, Wang YL, Wang F, Wang PF, Shang YP. Arbuscular mycorrhizal fungi mediate soil N dynamics, mitigating N₂O emissions and N-leaching while promoting crop N uptake in green manure systems. Sci Total Environ. 2024;957:177592.
Crisan I, Balestrini R, Pagliarani C. The current view on heavy metal remediation: The relevance of the plant interaction with arbuscular mycorrhizal fungi. Plant Stress. 2024;12:100439.
Hnini M, Rabeh K, Oubohssaine M. Interactions between beneficial soil microorganisms (PGPR and AMF) and host plants for environmental restoration: A systematic review. Plant Stress. 2024;11:100391.
Hua JF, Lin XG, Yin R, Jiang Q, Shao YF. Effects of arbuscular mycorrhizal fungi inoculation on arsenic accumulation by tobacco (Nicotiana tabacum L). J Environ Sci. 2009;21(9):1214–20.
Zhou HY, Nian FZ, Chen BD, Zhu YG, Yue XR, Zhang NM, Xia YS. Synergistic reduction of arsenic uptake and alleviation of leaf arsenic toxicity in maize (Zea mays L.) by arbuscular mycorrhizal fungi (AMF) and exogenous iron through antioxidant activity. J Fungi. 2023;9(6):677.
Ahmed FRS, Alexander IJ, Mwinyihija M, Killham K. Effect of superphosphate and arbuscular mycorrhizal fungus Glomus mosseae on phosphorus and arsenic uptake in lentil (Lens culinaris L). Water Air Soil Pollution. 2011;221(1):169–82.
Chen XW, Wu FY, Li H, Chan WF, Wu SC, Wong MH. Phosphate transporters expression in rice (Oryza sativa L.) associated with arbuscular mycorrhizal fungi (AMF) colonization under different levels of arsenate stress. Environ Exp Bot. 2013;87:92–9.
Zhang X, Chen B, Ohtomo R. Mycorrhizal effects on growth, P uptake and Cd tolerance of the host plant vary among different AM fungal species. Soil Sci Plant Nutr. 2015;61(2):359–68.
Lin BS, Gao HJ, Lai HM, Li CH, Wang Q. Characterization of heavy metals in soils from typical tobaccocultivatec areas, China. Environ Prog Sustain Energy. 2016;36(2):483–8.
Zhao YY, Zeng FG, Liang HZ, Tang YG, Li MF, Xiang JH, Wen XT. Chromium and vanadium bearing nanominerals and ultra-fine particlesin a super-high-organic-sulfur coal from Ganhe coalmine, YanshanCoalfield, Yunnan, China. Fuel. 2017;203:832–42.

No competing interests reported.

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You are reading this latest preprint version

Sustainable utilization of phosphogypsum in agriculture enhanced by mycorrhizal organisms: a new perspective

Status:

Version 1

Abstract

Background

Results

Conclusion

Figures

1. Introduction

2. Materials and Methods

2.1. Experimental time and location

2.2. Experimental materials

2.3. Experimental treatment and management

2.4. Harvest and analysis

2.5.Statistical analysis

3. Results

3.1. Mycorrhizal formation and tobacco growth under AMF inoculation and PG addition

3.2. P and S uptake by tobaccos plants under AMF inoculation and PG addition

3.3. As uptake by tobacco plants under AMF inoculation and PG addition

3.4. Uptake ration of P to As in tobacco plants under AMF inoculation and PG addition

3.5. SAR of P, S, and As by tobacco plants under AMF inoculation and PG addition

3.6.Principal component analysis of key indicators in tobaccos and soils under AMF inoculation and PG addition

3.7.Regulation factors analysis of tobacco P, S, and AsuptakeinducedbyAMF inoculation and PG addition

4. Discussion

4.1.Regulationof P, S, and Asuptake by plants induced by AMF and phosphogypsum

4.2.Suggestions for agricultural utilization and management of phosphogypsum

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1