Arbuscular mycorrhizal fungi increase aleurone layer zinc concentration but reduce overall zinc bioavailability in rice grain

Abstract

Societal Impact Statement

Rice is a critical crop for the delivery of calories and essential micronutrients to the human diet. Biofortification of rice with zinc (Zn) and iron (Fe) aims to combat the health issues associated with “hidden hunger”. Arbuscular mycorrhizal (AM) fungi have been explored for their potential to enhance Zn and Fe uptake in rice products. However, phytate, an anti‐nutritional compound rich in phosphorus (P), reduces the bioavailability of Zn and Fe in cereals. We investigated how AM fungal uptake of Zn, Fe and P interacts to affect bioavailability in rice and found mycorrhizal rice tends to have lower micronutrient bioavailability than non‐mycorrhizal.

Micronutrients such as zinc (Zn) and iron (Fe) play a crucial role in human health. In humans, the ability for micronutrients to be absorbed in the human digestive system (i.e., bioavailability) is hindered by phytate. Accumulation of phytate in the aleurone layer of cereal grains is affected by soil characteristics, particularly the availability of phosphorus (P). Arbuscular mycorrhizal (AM) fungi take up great amounts of P, but also some Zn and Fe into the host plant. In this study, we explored how AM fungi and soil P impact grain gene expression and bioavailability of Zn and Fe in rice under aerobic, controlled‐environment growing conditions. Five rice varieties were grown with or without AM fungal inoculation (Rhizophagus irregularis), under two soil P availabilities. Samples of developing grain were taken for RNAseq analysis. At plant maturity, grain nutrition was examined on a total concentration basis, and specifically in the aleurone layer by Synchrotron x‐ray fluorescence microscopy. The effect of AM fungi on Zn and Fe concentration was positive when only the aleurone layer was considered, but negative for whole grain concentrations. The AM‐colonised plants also accumulated more phytate in their grain compared to the non‐AM plants, leading to lower overall Zn bioavailability. Indirect effects of AM fungi on grain gene expression were small. The impact of AM colonisation on the bioavailability of Zn and Fe in rice was dependent on whether the whole grain, or specifically the aleurone layer, were quantified. This has implications for the consumption of whole vs milled rice products for humans.

INTRODUCTION

Micronutrients such as zinc (Zn) and iron (Fe) play crucial roles in human physiology, including supporting immune function, cognitive development and overall health (Bhattacharya et al., 2016; Cakmak, 2008; Godswill et al., 2020). The deficiency of essential micronutrients in humans, a condition collectively known as “hidden hunger”, is a significant global public health concern, as highlighted by the World Health Organisation (Cakmak, 2008; Hambidge, 2000). Around 30% of the global human population has in-adequate intake of micronutrients in their diet, especially in low- and middle-income countries where crops such as rice (Oryza sativa) is the staple food in the diet (Cakmak, 2008; Cakmak et al., 2017). Consequently, efforts to combat hidden hunger are focused on increasing the total amount and bioavailability of Zn and Fe in staple crops via genetic and agronomic means (Cakmak, 2008; Kumar et al., 2017; Wei et al., 2012).

Rice ranks third in the worldwide production of cereal grains (Statista, 2024) and serves as a staple food source for more than half of the world's population (Lucca et al., 2002; Sperotto et al., 2010). However, rice has inherently low concentrations of Zn and Fe, especially in polished rice, which has a mean of 12 mg kg⁻¹ Zn and 2 mg kg⁻¹ Fe; meanwhile, the recommended dietary intakes are 12–15 mg Zn, and 10–15 mg Fe day⁻¹ (Bouis & Welch, 2010; Welch & Graham, 2004). Furthermore, the nutritional value of cereal products cannot be determined simply through measurement of total Zn and Fe - estimation of the bioavailability of these elements is more relevant to outcomes in terms of human nutrition (Kumar et al., 2017; Meng et al., 2005). The bioavailability of Zn and Fe in cereal products is influenced by factors including chemical speciation, and its localisation within grain structures. Within cereal grains, P is primarily stored as phytic acid (PA), or phytate, which easily forms complexes with Zn and Fe in the aleurone layer of the grain. Plants possess phytase enzymes that can break down PA to access P and other complex elements to support the early growth of the germinating seedling. However, mammalian species do not produce phytases, so the presence of PA in cereal products directly reduces the bioavailability of Zn, Fe and other cations, which in turn diminishes the nutritional quality of the food for mammals (Gupta et al., 2015; Oatway et al., 2001). Therefore, PA is considered an anti-nutritional agent in cereal products, that may contribute to hidden hunger (Gupta et al., 2015; Samtiya et al., 2020). Improving the concentrations of bioavailable Zn and Fe in rice through a multi-pronged genetic and agronomic approach is essential for the future nutrition security of the world's population (Bouis et al., 2011).

There are a number of approaches for the biofortification of Zn and Fe in staple food crops, including conventional breeding and genetic modification techniques to increase the uptake or chelation of Zn and Fe in plants, and/or to reduce the amount of phytate accumulated in grains (Beasley et al., 2022; Colombo et al., 2020; Goudia & Hash, 2015; Gupta et al., 2021; Holm et al., 2002; Sharma et al., 2020). Arbuscular mycorrhizal (AM) fungi provide another avenue for agronomic Zn and Fe biofortification (Dhaliwal et al., 2022; Dhiman et al., 2023). AM fungi colonise the roots of more than 80% of terrestrial plant species including cereals (Hu et al., 2022; Sun et al., 2015; Watts-Williams et al., 2022; Watts-Williams & Cavagnaro, 2018), and AM colonisation can improve grain yield and Zn and Fe nutrition (Ercoli et al., 2017; Lehmann et al., 2014).

Because AM fungi take up P, Zn and Fe in the host plant, this creates a complex situation whereby Zn and/or Fe concentrations may increase in grain, but phytate may also accumulate because of the greater P supply to the plant from AM uptake. The small number of studies that address this complex scenario have reported contrasting results. Ryan et al. (2008) found that soil P fertiliser and AM fungi increased grain PA by 19% in wheat, leading to low Zn and Fe bioavailability in wheat grain. However, Ma et al. (2019) found that AM fungi increased Zn concentrations and bioavailability in wheat (Triticum aestivum L). Similar effects of AM fungi in relation to increased micronutrient bioavailability have been observed in other cereal crops such as durum wheat, maize and sorghum (Subramanian et al., 2013; Tran et al., 2021; Watts-Williams et al., 2022). In particular, Watts-Williams et al. (2022) found that mycorrhizal sorghum plants had greater grain yield and micronutrient concentrations leading to higher Zn and Fe bioavailability compared to non-AM sorghum plants. To our knowledge, no research has been carried out on the effect of AM fungi on grain phytate accumulation in rice.

Here, we explored the effect of AM fungal inoculation on the yield and nutrition of five rice varieties, to identify the impact on the bioavailability and localisation of Zn and Fe; we also report on the grain transcriptome in rice to elicit any indirect effects of AM colonisation on the developing grain. We sought to answer four research questions:

• How does AM inoculation affect the concentration of micronutrients important for human nutrition in rice grain?
• Is the concentration of Zn in rice grain different in the aleurone layer compared with whole grain analysis?
• To what extent does soil P availability interact with AM fungi to affect phytate accumulation in rice?
• Does colonisation by AM fungi or soil P availability induce changes in gene expression in developing rice grain?

MATERIALS AND METHODS

Experiment one

Soil, plants and fungi

The clay loam soil used in this experiment was collected from the Gawler River region of South Australia (Clay & Mineral Sales Pty Ltd, Adelaide). Soil was air-dried and sieved to less than 2 mm to homogenise and remove any coarse debris before autoclaving. Fine sand was steam sterilised and soil was autoclaved twice, air-dried before mixing in a ratio of 1:1 w/w using a cement mixer. This mixture was referred as “soil” thereafter. The soil had a pH of 8.49 (1:5 water) and plant-available (Colwell) P concentration of 8 mg kg⁻¹, DTPA-extractable Zn and Fe concentration of 0.23 mg kg⁻¹ and 7.9 mg kg⁻¹, respectively (Eurofins APAL). Soil P treatments were established by adding KH₂PO₄ solution and mixing through the soil to achieve P concentrations of 0 and 20 mg P kg⁻¹ soil, resulting in two plant-available (Colwell) P concentrations: 8.0 and 19 mg P kg⁻¹, referred to hereafter as low P and high P, respectively. The AM inoculated soils were mixed with the AM fungus Rhizophagus irregularis from a commercial product (Start Up Ultra, Microbe Smart Pty Ltd) at a rate of 1.5 g kg⁻¹ soil (corresponding to approximately 800 spores g⁻¹). Plastic, free-draining pots (0.8 L volume) were then filled with the prepared soils (1.1 kg soil pot⁻¹). The experiment was fully factorial for each P, AM fungal and variety treatment, to a total 20 treatments, each with six biological replicates, giving a total of 120 pots.

Seed of the Australian cultivars was obtained from the New South Wales Department of Primary Industries (Yanco) and consisted of four varieties: Reiziq, Topaz, Sherpa and Langi; Calrose was obtained from the lab of Prof. Alexander Johnson. Seeds were surface-sterilised with 20% sodium hypochlorite solution (NaClO) for 20 minutes, then washed and rinsed with reverse osmosis (RO) water three times. The seeds were soaked in RO water overnight before placing onto wet paper towel, enclosed in a plastic container and incubated in the dark at 28°C for four days. After four days of incubation, seeds were transferred to the lab bench at room temperature for 3 additional days. When the shoots emerged, the seedlings were transplanted to the prepared pots, with two plants per pot. Growing two plants in each pot ensured that there was enough grain sample produced to fulfill all downstream analyses.

Plant growth and harvest

Rice plants were grown in a controlled environment room on the University of Adelaide's Waite Campus, Australia. The conditions were set at 28/20°C day/night temperature, 14/10 hours day/night photoperiod and 60% relative humidity, during the growing period. Plants were watered every second or third day with RO water until just draining. Plants were fertilised with 10 ml of full-strength modified Long-Ashton solution (P omitted) once every ten days (Cavagnaro et al., 2010). Plants were supplemented with 10 mg N per pot in the form of NH₄NO₃ solution at 55 and 70 days after planting (DAP).

Soil cores (15 mm diameter and 11 cm length) were used to collect root subsamples for AM colonisation analysis seven days prior to destructive harvest. Roots were washed free of soil and placed in a tissue cassette then submerged in 50% ethanol solution for at least 24 hours. All plants were destructively harvested between 97 and 125 DAP depending on when the different rice varieties reached maturity. From each plant, two seeds were removed and stored at 4°C for synchrotron-based X-ray fluorescence microscopy (XFM) analysis. Aboveground biomass, including shoot and seeds, was cut at the soil level, weighed for total fresh shoot biomass and then dried at 50°C in an oven for at least 48 hours for shoot dry weight determination. After threshing by hand, grain was collected and dry weight was determined.

Sample analysis

Fresh root subsamples were rinsed of ethanol and submerged in 10% potassium hydroxide (KOH) (w/v) solution for cleaning at room temperature for seven days. Samples were then stained in 5% ink in vinegar solution (Vierheilig et al., 1998). The stained root samples were de-stained in acidified RO water for at least 24 hrs before being stored in 50% glycerol solution for quantification of AM colonisation under a stereomicroscope using the gridline intersect method (Giovannetti & Mosse, 1980).

The dried grain samples were homogenised in a Geno/Grinder 2010 (SPEX SamplePrep.) After that, digest mass sub-samples were cold digested overnight using a 4: 1 (v/v) mix of nitric acid (HNO₃) and hydrogen peroxide (H₂O₂) followed by hot digestion on a heat block for 180 minutes (Zarcinas et al., 1987). The acid digests were then diluted with RO water before being analysed for P, Zn and Fe by inductively coupled plasma optical emission spectroscopy (PerkinElmer - Optical Emission Spectrometer|Avio 200 ICP).

A commercial phytic acid/total phosphorus assay kit (Megazyme, Ireland) was used to examine grain phytic acid content. Firstly, 250 mg of homogenised grain sample was digested with 5 ml of HCl (0.66 M) and then placed in an orbital shaker (40 rpm) for 16 h at room temperature. Following that, the manufacturer's protocol was followed, and the PA content of samples was determined by spectrophotometer at 655 nm wavelength. To estimate the Zn and Fe bioavailability of grain, the molar concentrations of PA, Zn and Fe were calculated, respectively. The molar ratio of PA to Zn or to Fe was used as a proxy for bioavailability (Reddy, 2001). WHO (1996) suggested that the Zn absorption by humans reaches >50% when the PA:Zn ratio is < 5, reduced to 35% absorption when PA:Zn ratio is between 5 and 15, and less than 15% is absorbed at PA:Zn ratio >15. Likewise, a PA:Fe ratio of <1, particularly <0.4, is associated with human Fe absorption of 10–12%, whereas absorption decreases to < 5% when the PA:Fe ratio >1 (Hurrell & Egli, 2010).

Synchrotron X-ray fluorescence microscopy (XFM) to localise grain micronutrients

See Methods S1.

Experiment two: Rice grain RNA sequencing and bioinformatics workflow

See Methods S2.

Statistical analysis

Data from this experiment were analysed using R statistical software v 4.1.1, (Team, 2020). Three-way analysis of variance (ANOVA) was used to analyse shoot dry weights, grain dry weights, grain nutrition and phytate concentrations with Variety, P and Mycorrhiza as treatment factors. In the case of % AM colonisation, % mycorrhizal shoot biomass response (MSBR) and % mycorrhizal grain biomass response (MGBR), a two-way ANOVA was applied, incorporating P and Variety as treatment factors. Following ANOVA, where there was a significant main effect or interaction term, comparisons between treatment means were made using Tukey's honestly significant difference (HSD) post hoc test.

RESULTS

AM fungal root colonisation

There was a significant interaction between rice variety and soil P treatment on the proportion of total root length colonised by AM (p < 0.001). All five rice varieties were highly colonised by AM fungi in the inoculated treatment, and the colonisation varied depending on the interaction between variety and soil P treatment (see Table 1 for ANOVA). While AM colonisation in Sherpa, Topaz and Calrose was higher in the Low P treatment (mean 56.88%) than in the High P treatment (mean 40.92%), the opposite was observed in Reiziq AM plants (Figure 1). In experiment 2, the Nipponbare plants were colonised by R. irregularis more at Low P than at High P.

TABLE 1 Statistical outcomes of two-way ANOVA for mycorrhiza-specific variables.

	Variety	Phosphorus (P)	Variety*P
% mycorrhizal colonisation	<0.01	<0.001	<0.001
% MSBR	<0.001	ns	<0.001
% MGBR	<0.01	<0.001	<0.001

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Shoot and grain biomass

The shoot dry weight (SDW) was significantly influenced by the three-way interaction between Variety, AM fungal inoculation and soil P availability (see Table 2 for ANOVA outcomes). In general, all the rice varieties had greater SDW when grown at High P; mean values ranged from 4.75 g (Sherpa non-AM, Low P) to 8.16 g (Calrose AM, High P) (Figure S1a). Meanwhile, the SDW of Calrose and Reiziq were greater than many of the other varieties, but especially the Topaz plants (Figure S1a). The calculation of mycorrhizal shoot biomass response clearly illustrated the effects of AM inoculation in interaction with Variety and P addition. AM inoculation increased the shoot biomass of all the rice varieties either in Low P or High P treatments except Reiziq plants (Figure S1b). In experiment 2, the SDW of Nipponbare plants increased with soil P addition.

TABLE 2 Statistical outcomes of three-way ANOVA for a range of plant physiological variables.

	Variety	AMF	P	Variety*AMF	Variety*P	AMF*P	Variety*AMF*P
Shoot dry weight	<0.001	<0.05	<0.001	ns	<0.001	ns	<0.001
Grain dry weight	<0.001	<0.01	<0.001	ns	<0.01	<0.05	<0.01
Whole grain P concentration	<0.001	<0.001	<0.001	ns	ns	<0.01	<0.05
Whole grain zinc (Zn) concentration	<0.001	<0.001	ns	ns	<0.05	ns	<0.05
Whole grain iron (Fe) concentration	<0.05	<0.001	ns	ns	<0.01	<0.05	<0.01
Grain aleurone layer Zn concentration	<0.001	<0.001	ns	<0.05	<0.01	ns	<0.001
Grain phytic acid (PA)	<0.001	<0.001	<0.001	ns	ns	<0.001	<0.05
Grain PA: Whole grain Zn molar ratio	<0.001	<0.001	<0.001	<0.001	<0.01	<0.05	<0.001
Grain PA: Whole grain Fe molar ratio	ns	<0.001	<0.001	<0.05	ns	<0.01	<0.01
Grain PA: Grain aleurone layer Zn molar ratio	<0.001	<0.001	<0.001	<0.05	<0.01	<0.05	<0.001

The grain dry weight (GDW) data generally displayed a similar pattern to the SDW data (Figure 2a) with regards to effects of Variety and soil P addition. However, the mycorrhizal grain biomass response shows that AM inoculation affected grain biomass differently to shoot biomass (Figure 2b). At Low P treatment, the GDW of Sherpa was substantially increased by AM fungal inoculation by nearly 30%. AM inoculation also increased GDW in Reiziq and Calrose. In experiment 2, the GDW of Nipponbare plants increased with the addition of soil P, but not AM inoculum.

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Whole grain P, Zn, Fe, phytate and aleurone layer Zn

There was a significant three-way interaction affecting whole grain P concentration and generally, it was higher when the plants were colonised by AM fungi, and when P was added to the soil (Figure S2a). In all rice varieties, the concentration of grain P in the AM-colonised plants was significantly greater that of the non-colonised plants at Low P. Similarly, this trend was also observed in AM-colonised Reiziq, Langi and Sherpa plants under High P treatment. There were also significant three-way interactions for whole grain Zn and Fe concentrations (see Table 2 for ANOVA outcomes). Whole grain Zn concentration was lower in AM plants compared to non-AM plants for Langi and Sherpa (Figure 3a), but there were no AM effects on whole grain Fe concentration, within a variety (Figure S2b). Soil P addition did not affect whole grain Zn or Fe concentrations, except in Langi where the Zn concentration was lower at High P than Low P.

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The Zn concentration in the aleurone layer was significantly affected by the three-way interaction but did not follow the same pattern as for whole-grain Zn concentration (Figure 3b). AM plants had greater Zn concentration in the aleurone layer in Reiziq and Sherpa under Low P treatment and for the Reiziq and Topaz varieties under High P treatment. In contrast, there was no AM effect on the Zn concentration in the aleurone layer of other rice varieties. These results are further illustrated by the XFM scanning images of three replicate grain samples (Figure 4).

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The content of phytate in grain was significantly affected by three-way interaction (Figure 5a). The phytate content was typically higher in the plants in the High P treatment and those colonised by AM fungi, in both cases likely to be a direct consequence of the increased P uptake in those plants (Figure 5a). In the Low P treatment, all the rice varieties experienced around a two-fold increase in phytate content due to AM fungal colonisation. This pattern was also found in the concentration of phytate in grains of Reiziq and Langi AM plants in High P treatment.

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Grain Zn and Fe bioavailability

Since AM fungal inoculation affected the phytate content and, in two varieties, the Zn concentration in rice, the resulting molar ratios of PA:aleurone layer Zn (Zn_a) offer a simplified insight into the complex interactions (Figure 5b). The AM plants had lower or unchanged PA:Zn_a molar ratio in all varieties at Low P treatment, due to the increased Zn concentration in the aleurone layer driven by AM fungal colonisation. Specifically, AM inoculation resulted in a significantly lower PA:Zn_a in Reiziq, Sherpa and Topaz under the high P treatment.

The molar ratios of phytate to whole grain Zn (Zn_g) and Fe (Fe_g) concentrations were also analysed (Figures S3a & S3b). Generally, the estimated bioavailability of Zn and Fe in AM plants was lower than non-AM plants in all varieties due to the lower or same concentration of Zn_g and Fe_g in combination with greater phytate content. In the Low P treatment, AM fungal inoculation reduced the estimated Zn and Fe bioavailability in rice grains by up to 50% (PA:Zn_g and PA:Fe_g ratio mean values ranged from 13.90 and 16.62 in non-AM plants to 25.72 and 31.80 in AM plants, respectively). In the High P treatment, the inoculated Langi and Reiziq plants had substantially higher mean PA:Zn_g ratio, with 42.82 and 38.90, respectively (lower Zn bioavailability) compared to the other varieties and treatments (Figure S3a). This trend was also found for Fe bioavailability (Figure S3b) in Langi and Reiziq AM plants, but there was no AM fungal inoculation effect on the Zn and Fe bioavailability in other varieties at high P treatment.

Transcriptome profiling of rice grain

Mean gene expression as counts per million for each treatment are reported in Table S1. Differentially expressed (DE) genes were detected in the different comparisons as follows: 29 genes in the Low P, non-AM vs AM fungal inoculated samples (AMF_LP; Table S2), 1,072 genes in the AM-inoculated, non P-treated vs P-treated samples (P_AM; Table S3), 40 genes in the non-inoculated, non P-treated vs P-treated samples (P_NM; Table S4) and 8 genes in the interaction between soil P treatment and AM fungal inoculation (P_AMFinter; Table S5). Gene expression changes for the induced DE genes are shown in Figure 6. In addition, 22 DE genes were present as DE genes in both AMF_LP and P_AM, but displayed inverse regulation direction (i.e. AM fungal inoculation induced the gene expression, and soil P treatment repressed expression and vice versa) (Figure 6). Six of those DE genes were also present in the P_AMFinter comparison. This suggested that AM fungal inoculation and soil P treatment could have some interaction by regulating the same set of genes in grain. The genes with the strongest response to the treatments were Os02g0757100 “similar to phosphate induced-1 protein” and Os04g0635100 “similar to H0315F07.12 protein” which were up-regulated by AM colonisation and down-regulated by soil P addition, and Os03g0231150 “similar to Hox19 (Fragment)” and Os03g0229500 “similar to MATE efflux family protein, expressed” which showed the opposite expression pattern (ie., down-regulated by AM colonisation).

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DISCUSSION

Soil P fertiliser increased rice grain yield but decreased grain micronutrient bioavailability and AM colonisation

In agricultural management, it is common for P fertiliser to be applied to soils to overcome plant P limitation, thereby enhancing crop yields when there are no additional limitations to plant growth (e.g. water, N, light). Indeed, in this study, P addition increased rice yields by approximately 30% across both experiments. This is in line with previous field studies where P fertilisers increased the yield of aerobic rice by around 32% (Jana et al., 2020; Jinger et al., 2022). In the present study, the increase in soil P availability, and/or P uptake by AM fungi, led directly to greater grain phytate accumulation and reduced grain Zn and Fe concentrations. Due to the co-existence of higher phytate accumulation and less Zn and Fe concentration in the High P treatment grains, the PA:Zn and PA:Fe ratios in whole grain doubled with soil P addition, suggesting substantially reduced Zn and Fe bioavailability, and a less nutritious rice product for human or animal consumers. The results from this work suggest there is a trade-off between maximising stored P reserves in the grain and producing grain with greater bioavailable micronutrient concentrations. Here, the rice plants prioritised utilisation of luxury P to increase grain yield and/or store more phytate to store P and micronutrients reserve for the developing plant embryo, rather than increased uptake of Zn and Fe in whole grain. This is similar to results with 10 diverse durum wheat genotypes, which demonstrated that greater soil P supply always resulted in lower grain Zn and Fe bioavailability, regardless of AM inoculation treatment (Tran et al., 2021).

The availability of soil P in this study affected the association between R. irregularis and rice plants. In the low P treatment, AM colonisation was greater in three of the five varieties (Sherpa, Topaz and Calrose) than at High P. Plants tend to form associations with AM fungi as a strategy to enhance P acquisition. At Low P, the mean proportion of root colonisation nearly reached 55%, which also lead to greater shoot and grain biomass. At High soil P availability, AM colonisation was reduced to as low as around 40% in these three varieties confirming previous studies in other species (Balzergue et al., 2013; Higo et al., 2020; Nguyen et al., 2019; Watts-Williams et al., 2022). This might be due to reduced need for nutrient acquisition from fungal partners, altered root exudates and/or physiological changes induced in plants that are unfavourable for fungal colonisation (Zeilinger et al., 2016).

AM fungi increased phytate accumulation and reduced micronutrient bioavailability in whole grain

In rice, approximately 70% of the total grain P concentration is stored as PA (Perera et al., 2018). Therefore, it is anticipated that changes in the total P levels of the seeds would relate to phytate concentrations within the seeds (Jeong et al., 2017; Rose et al., 2016). It was also observed that high soil P availability typically results in high seed P concentrations and PA content in rice grains (Rose et al., 2016). Here, our work also demonstrated in five rice varieties that AM fungi indirectly increased rice phytate content through the uptake of P via the mycorrhizal pathway. The activity of the mycorrhizal P uptake pathway in soil with low P availability likely indirectly induced the expression of OsPHI-1 which is a phosphate-induced gene that encodes a protein with a putative ATP-binding domain, involved in the cell expansion and organ size (Aya et al., 2014; Sano et al., 1999). Inoculation with AM fungi led to greater phytate content in rice plants, which may be due to increased P uptake from the combination of two P uptake pathways, in the inoculated plants.

Because AM colonisation did not increase whole grain Zn and Fe concentrations in line with P, the overall outcome was that AM colonised rice had ratios of PA to Zn and Fe (whole grain basis) that were more than double that of the non-AM control plants, and lead to an overall reduction in whole grain Zn and Fe bioavailability. These results do not concur with other findings in winter wheat plants by Ma et al. (2019) and Jing et al. (2023). The authors found that AM fungal inoculation enhanced P nutrition uptake and increased grain Zn concentration from 1.13 to 2.76 times in Ma et al. (2019) or 0.64 to 0.81 times in Jing et al. (2023) with a net higher Zn bioavailability in grain. Furthermore, Watts-Williams et al. (2022) demonstrated that AM fungi colonisation increased sorghum Zn and Fe bioavailability compared to non-colonised plants. On the other hand, the elemental localisation of Zn in the sectioned grain as revealed by XFM mapping showed that AM fungal inoculation led to increased Zn concentration in the aleurone layer. This resulted in a lower ratio of PA:Zn_a in the aleurone layer in some treatments. While Zn loading is under the control of the plant, if the overall Zn budget of the plant is greater due to AM colonisation, then the plant may allocate more to the aleurone layer than a non-AM plant. Consequently, brown rice, which retains the aleurone layer, may be a preferred choice for Zn intake in the human diet. However, this also means that greater amounts of phytate are also being consumed.

AM fungi have been reported to improve aerobic rice grain yield, nutrition accumulation and plant resilience to biotic and abiotic stressors, and also require lower P inputs (Gao et al., 2007; Narwal et al., 2018; Sangothari et al., 2023). However, the nature of the AM-rice association is also dependent on many factors such as plant species and variety, soil characteristics (nutrient availability and microbiome) and AM fungal species, diversity and abundance (Pickles et al., 2020). Our current data indicate that under low soil available P conditions, AM fungi enhance P uptake and phytate accumulation in rice grain, without a concurrent increase in Zn and Fe in the whole grain. This suggests that for rice, AM fungi may be best managed in conjunction with micronutrient fertiliser application for optimal outcomes in terms of both yield and micronutrient bioavailability.

Rice variety-based differences in response to AM colonisation

Rice plants exhibit a greater propensity to form a functional association with AM fungi under aerobic soil conditions (i.e., upland, rainfed) compared to flooded conditions (i.e., lowland), where the anoxic environment is thought to impede the colonisation process (Watanarojanaporn et al., 2013; Xavier Martins & Rodrigues, 2020). Cultivation of rice under alternative irrigation methods or rainfed conditions would not only reduce water usage but also facilitate improved nutrient and water uptake function of AM fungi (Mitra et al., 2023). Under controlled environment aerobic soil conditions, we found that the five rice varieties grown in this study were well-colonised by R. irregularis; the effects of AM fungal inoculation on grain yield and other physiological parameters, however, differed for each variety, in line with previous work using these varieties (Watts-Williams et al., 2025).

Greater AM colonisation did not necessarily lead to an increased mycorrhizal response in shoot or grain biomass. For example, Topaz had the greatest percentage root length colonised at Low P, but no increase in shoot or grain dry weight relative to the non-AM control plants.. This highlights that the association of AM fungi with rice can either enhance or have a neutral effect on grain yield, depending on the plant genotype. A previous study reported that 12 cultivated rice varieties (japonica subspecies) displayed root colonisation by either Funnelformis mosseae or R. irregularis but the relationship between plant growth response measures and AM colonisation were either positive, negligible, or negative between different rice varieties (Campo et al., 2020). A similar observation was also reported by Guigard et al. (2023), who found that AM inoculation of two subspecies rice (indica: IR64 & Phka Rumduol; and japonica: Nipponbare, Kitaake, Azucena & Zhonghua 11) and 3 AM fungal genotypes (F. mosseae, R. irregularis, & R. intraradices) resulted in biomass response ranging from −21% to 227%.

CONCLUSIONS AND FURTHER WORK

Our study sheds light from both physiological and molecular standpoints on the relationship between AM fungi and nutrient uptake in rice, particularly regarding phytate accumulation and the bioavailability of micronutrients. We have demonstrated variety-based variability in grain micronutrient content and phytate accumulation in rice as influenced by soil P availability. We found that AM fungal colonisation exerts little direct influence on the transcriptome of developing rice grain, but may influence gene expression through increased P acquired by the plant via the mycorrhizal pathway of uptake.

While our findings highlight the positive impact of AM fungi on phosphorus uptake in all varieties, and grain yield in certain rice varieties, their contribution to micronutrient bioavailability appears to be positive in the aleurone layer but negative in whole grain. Moving forward, it is crucial to consider optimal strategies for concurrently managing AM fungi, rice variety selection and micronutrient addition.

AUTHOR CONTRIBUTIONS

TDN was involved in the design of the experiment, performed the research and data analysis and contributed to data interpretation and writing the manuscript. NS was involved in the design of the experiment, contributed to data interpretation and wrote the manuscript. AATJ was involved in data interpretation and wrote the manuscript. EL, ES and CD were involved in the design of the experiment, performed the research, data analysis and wrote the manuscript. TRC contributed to data interpretation and wrote the manuscript. SJWW was involved in the design of the experiment, contributed to data analysis and interpretation, and wrote manuscript as it appears in its final form.

ACKNOWLEDGEMENTS

SJWW acknowledges the Australian Research Council Discovery Early Career Researcher Award (DE210100908) and TDN the University of Adelaide Research Scholarship and AgriFutures Australia PhD top-up scholarship (PRO-017382), for support. The authors thank A/Prof Ehsan Tavakkoli and Ms Bogumila Tomczak for access to the ICP-OES, members of the lab, and Dr Alison Gill for technical assistance, Dr Lishi Cai for assistance with R statistical analyses. This research was undertaken on the XFM beamline at the Australian Synchrotron, part of ANSTO (AS242/XFM/21663)

CONFLICTS OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The sequencing data that support the findings of this study are openly available in NCBI at 415 , reference number PRJNA1176553. All other data are available upon reasonable request to the corresponding author.

References

Aya, K., Hobo, T., Sato‐Izawa, K., Ueguchi‐Tanaka, M., Kitano, H., & Matsuoka, M. (2014). A novel AP2‐type transcription factor, SMALL ORGAN SIZE1, controls organ size downstream of an auxin signaling pathway. Plant and Cell Physiology, 55(5), 897–912. https://doi.org/10.1093/pcp/pcu023

Balzergue, C., Chabaud, M., Barker, D. G., Bécard, G., & Rochange, S. F. (2013). High phosphate reduces host ability to develop arbuscular mycorrhizal symbiosis without affecting root calcium spiking responses to the fungus. Frontiers in Plant Science, 4, 426. https://doi.org/10.3389/fpls.2013.00426

Beasley, J. T., Bonneau, J. P., Moreno‐Moyano, L. T., Callahan, D. L., Howell, K. S., Tako, E., Taylor, J., Glahn, R. P., Appels, R., & Johnson, A. A. (2022). Multi‐year field evaluation of nicotianamine biofortified bread wheat. The Plant Journal, 109(5), 1168–1182. https://doi.org/10.1111/tpj.15623

Bhattacharya, P. T., Misra, S. R., & Hussain, M. (2016). Nutritional aspects of essential trace elements in oral health and disease: An extensive review. Scientifica, 2016(1), 5464373. https://doi.org/10.1155/2016/5464373

Bouis, H. E., & Welch, R. M. (2010). Biofortification—A sustainable agricultural strategy for reducing micronutrient malnutrition in the global south. Crop Science, 50, S‐20‐S‐32. https://doi.org/10.2135/cropsci2009.09.0531

Bouis, H. E., Hotz, C., McClafferty, B., Meenakshi, J., & Pfeiffer, W. H. (2011). Biofortification: A new tool to reduce micronutrient malnutrition. Food and Nutrition Bulletin, 32(1_suppl1), S31–S40. https://doi.org/10.1177/15648265110321S105

Cakmak, I. (2008). Enrichment of cereal grains with zinc: Agronomic or genetic biofortification? Plant and Soil, 302(1), 1–17. https://doi.org/10.1007/s11104-007-9466-3

Cakmak, I., McLaughlin, M., & White, P. (2017). Zinc for better crop production and human health. Plant and Soil, 411(1), 1–4. https://doi.org/10.1007/s11104-016-3166-9

Campo, S., Martín‐Cardoso, H., Olivé, M., Pla, E., Catala‐Forner, M., Martínez‐Eixarch, M., & San Segundo, B. (2020). Effect of root colonization by arbuscular mycorrhizal fungi on growth, productivity and blast resistance in rice. Rice, 13(1), 42. https://doi.org/10.1186/s12284-020-00402-7

Cavagnaro, T. R., Dickson, S., & Smith, F. A. (2010). Arbuscular mycorrhizas modify plant responses to soil zinc addition. Plant and Soil, 329, 307–313. https://doi.org/10.1007/s11104-009-0158-z

Colombo, F., Paolo, D., Cominelli, E., Sparvoli, F., Nielsen, E., & Pilu, R. (2020). MRP transporters and low phytic acid mutants in major crops: Main pleiotropic effects and future perspectives. Frontiers in Plant Science, 11, 576091. https://doi.org/10.3389/fpls.2020.01301

Dhaliwal, S. S., Sharma, V., Shukla, A. K., Verma, V., Kaur, M., Shivay, Y. S., Nisar, S., Gaber, A., Brestic, M., & Barek, V. (2022). Biofortification—A frontier novel approach to enrich micronutrients in field crops to encounter the nutritional security. Molecules, 27(4), 1340. https://doi.org/10.3390/molecules27041340

Dhiman, K., Sharma, D., Kumari, R., & Tomar, P. (2023). Biofortification of crops using microbes—A promising sustainable agriculture strategy. Journal of Plant Nutrition, 46(12), 2912–2935. https://doi.org/10.1080/01904167.2022.2160755

Ercoli, L., Schüßler, A., Arduini, I., & Pellegrino, E. (2017). Strong increase of durum wheat iron and zinc content by field‐inoculation with arbuscular mycorrhizal fungi at different soil nitrogen availabilities. Plant and Soil, 419, 153–167. https://doi.org/10.1007/s11104-017-3319-5

Gao, X., Kuyper, T. W., Zou, C., Zhang, F., & Hoffland, E. (2007). Mycorrhizal responsiveness of aerobic rice genotypes is negatively correlated with their zinc uptake when nonmycorrhizal. Plant and Soil, 290, 283–291. https://doi.org/10.1007/s11104-006-9160-x

Giovannetti, M., & Mosse, B. (1980). An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytologist, 84(3), 489–500. https://doi.org/10.1111/j.1469-8137.1980.tb04556.x

Godswill, A. G., Somtochukwu, I. V., Ikechukwu, A. O., & Kate, E. C. (2020). Health benefits of micronutrients (vitamins and minerals) and their associated deficiency diseases: A systematic review. International Journal of Food Science, 3(1), 1–32. https://doi.org/10.47604/ijf.1024

Goudia, B. D., & Hash, C. T. (2015). Breeding for high grain Fe and Zn levels in cereals. International Journal of Innovation and Applied Studies, 12(2), 342–354.

Guigard, L., Jobert, L., Busset, N., Moulin, L., & Czernic, P. (2023). Symbiotic compatibility between rice cultivars and arbuscular mycorrhizal fungi genotypes affects rice growth and mycorrhiza‐induced resistance. Frontiers in Plant Science, 14, 1278990. https://doi.org/10.3389/fpls.2023.1278990

Gupta, P., Balyan, H., Sharma, S., & Kumar, R. (2021). Biofortification and bioavailability of Zn, Fe and Se in wheat: Present status and future prospects. Theoretical and Applied Genetics, 134, 1–35. https://doi.org/10.1007/s00122-020-03709-7

Gupta, R. K., Gangoliya, S. S., & Singh, N. K. (2015). Reduction of phytic acid and enhancement of bioavailable micronutrients in food grains. Journal of Food Science and Technology, 52, 676–684. https://doi.org/10.1007/s13197-013-0978-y

Hambidge, M. (2000). Human zinc deficiency. The Journal of Nutrition, 130(5), 1344S–1349S. https://doi.org/10.1093/jn/130.5.1344S

Higo, M., Azuma, M., Kamiyoshihara, Y., Kanda, A., Tatewaki, Y., & Isobe, K. (2020). Impact of phosphorus fertilization on tomato growth and arbuscular mycorrhizal fungal communities. Microorganisms, 8(2), 178. https://doi.org/10.3390/microorganisms8020178

Holm, P. B., Kristiansen, K. N., & Pedersen, H. B. (2002). Transgenic approaches in commonly consumed cereals to improve iron and zinc content and bioavailability. The Journal of Nutrition, 132(3), 514S–516S. https://doi.org/10.1093/jn/132.3.514S

Hu, Y., Pandey, A. K., Wu, X., Fang, P., & Xu, P. (2022). The role of arbuscular mycorrhiza fungi in drought tolerance in legume crops: A review. Legume Research‐An International Journal, 45(1), 1–9. https://doi.org/10.18805/LRF-660

Hurrell, R., & Egli, I. (2010). Iron bioavailability and dietary reference values. The American Journal of Clinical Nutrition, 91(5), 1461S–1467S. https://doi.org/10.3945/ajcn.2010.28674F

Jana, K., Mondal, R., & Mallick, G. (2020). Growth, productivity and nutrient uptake of aerobic rice (Oryza sativa L.) as influenced by different nutrient management practices. Oryza‐An International Journal on Rice, 57(1), 49–56. https://doi.org/10.35709/ory.2020.57.1.6

Jeong, K., Julia, C. C., Waters, D. L., Pantoja, O., Wissuwa, M., Heuer, S., Liu, L., & Rose, T. J. (2017). Remobilisation of phosphorus fractions in rice flag leaves during grain filling: Implications for photosynthesis and grain yields. PLoS ONE, 12(11), e0187521. https://doi.org/10.1371/journal.pone.0187521

Jing, Y., Zhang, C., Yifan, L., Yuanzhe, M., Xiangyao, W., Jun, C., & Fuyong, W. (2023). Effects of arbuscular mycorrhizal fungi on uptake, translocation and accumulation of Zn in winter wheat during whole plant growth stages. Pedosphere, 34(2), 374–384. https://doi.org/10.1016/j.pedsph.2023.07.021

Jinger, D., Dhar, S., Dass, A., Sharma, V., Paramesh, V., Parihar, M., Joshi, E., Singhal, V., Gupta, G., & Prasad, D. (2022). Co‐fertilization of silicon and phosphorus influences the dry matter accumulation, grain yield, nutrient uptake, and nutrient‐use efficiencies of aerobic rice. Silicon, 14, 4683–4697. https://doi.org/10.1007/s12633-021-01239-5

Kumar, A., Lal, M. K., Kar, S. S., Nayak, L., Ngangkham, U., Samantaray, S., & Sharma, S. G. (2017). Bioavailability of iron and zinc as affected by phytic acid content in rice grain. Journal of Food Biochemistry, 41(6), e12413. https://doi.org/10.1111/jfbc.12413

Lehmann, A., Veresoglou, S. D., Leifheit, E. F., & Rillig, M. C. (2014). Arbuscular mycorrhizal influence on zinc nutrition in crop plants—A meta‐analysis. Soil Biology and Biochemistry, 69, 123–131. https://doi.org/10.1016/j.soilbio.2013.11.001

Lucca, P., Hurrell, R., & Potrykus, I. (2002). Fighting iron deficiency anemia with iron‐rich rice. Journal of the American College of Nutrition, 21(sup3), 184S–190S. https://doi.org/10.1080/07315724.2002.10719264

Ma, X., Luo, W., Li, J., & Wu, F. (2019). Arbuscular mycorrhizal fungi increase both concentrations and bioavilability of Zn in wheat (Triticum aestivum L) grain on Zn‐spiked soils. Applied Soil Ecology, 135, 91–97. https://doi.org/10.1016/j.apsoil.2018.11.007

Meng, F., Wei, Y., & Yang, X. (2005). Iron content and bioavailability in rice. Journal of Trace Elements in Medicine and Biology, 18(4), 333–338. https://doi.org/10.1016/j.jtemb.2005.02.008

Mitra, D., Panneerselvam, P., Senapati, A., Chidambaranathan, P., Nayak, A. K., & Mohapatra, P. K. D. (2023). Arbuscular mycorrhizal fungi response on soil phosphorus utilization and enzymes activities in aerobic rice under phosphorus‐deficient conditions. Life, 13(5), 1118. https://doi.org/10.3390/life13051118

Narwal, E., Annapurna, K., Choudhary, J., & Sangwan, S. (2018). Effect of arbuscular mycorrhizal fungal colonization on nutrient uptake in rice aerobic conditions. International Journal of Current Microbiology and Applied Sciences, 7(4), 1072–1093. https://doi.org/10.20546/ijcmas.2018.704.118

Nguyen, T. D., Cavagnaro, T. R., & Watts‐Williams, S. J. (2019). The effects of soil phosphorus and zinc availability on plant responses to mycorrhizal fungi: A physiological and molecular assessment. Scientific Reports, 9(1), 14880. https://doi.org/10.1038/s41598-019-51369-5

Oatway, L., Vasanthan, T., & Helm, J. H. (2001). Phytic acid. Food Reviews International, 17(4), 419–431. https://doi.org/10.1081/FRI-100108531

Perera, I., Seneweera, S., & Hirotsu, N. (2018). Manipulating the phytic acid content of rice grain toward improving micronutrient bioavailability. Rice, 11, 1–13. https://doi.org/10.1186/s12284-018-0200-y

Pickles, B. J., Truong, C., Watts‐Williams, S. J., & Bueno, C. G. (2020). Mycorrhizas for a sustainable world. The New Phytologist, 225(3), 1065–1069. https://doi.org/10.1111/nph.16307

Reddy, N. R. (2001). Occurrence, distribution, content, and dietary intake of phytate. In Food phytates (pp. 41–68). CRC Press.

Rose, T. J., Kretzschmar, T., Liu, L., Lancaster, G., & Wissuwa, M. (2016). Phosphorus deficiency alters nutrient accumulation patterns and grain nutritional quality in rice. Agronomy, 6(4), 52. https://doi.org/10.3390/agronomy6040052

Ryan, M. H., McInerney, J. K., Record, I. R., & Angus, J. F. (2008). Zinc bioavailability in wheat grain in relation to phosphorus fertiliser, crop sequence and mycorrhizal fungi. Journal of the Science of Food and Agriculture, 88(7), 1208–1216. https://doi.org/10.1002/jsfa.3200

Samtiya, M., Aluko, R. E., & Dhewa, T. (2020). Plant food anti‐nutritional factors and their reduction strategies: An overview. Food Production, Processing and Nutrition, 2(1), 1–14. https://doi.org/10.1186/s43014-020-0020-5

Sangothari, A., Radhamani, S., Janaki, P., & Ravichandran, V. (2023). Effect of arbuscular mycorrhizal fungi and different combination of foliar application of nutrients on nutrient uptake and yield of aerobic rice. Agricultural Science Digest, 44(2), 265–296. https://doi.org/10.18805/ag.D-5814

Sano, T., Kuraya, Y., Amino, S.‐I., & Nagata, T. (1999). Phosphate as a limiting factor for the cell division of tobacco by‐2 cells. Plant and Cell Physiology, 40(1), 1–16. https://doi.org/10.1093/oxfordjournals.pcp.a029464

Sharma, D., Ghimire, P., Bhattarai, S., Adhikari, U., Khanal, S., & Poudel, P. B. (2020). Biofortification of wheat: Genetic and agronomic approaches and strategies to combat iron and zinc deficiency. International Journal of Environment, Agriculture and Biotechnology, 5(4), 1077–1088. https://doi.org/10.22161/ijeab.54.29

Sperotto, R. A., Boff, T., Duarte, G. L., Santos, L. S., Grusak, M. A., & Fett, J. P. (2010). Identification of putative target genes to manipulate Fe and Zn concentrations in rice grains. Journal of Plant Physiology, 167(17), 1500–1506. https://doi.org/10.1016/j.jplph.2010.05.003

Statista. (2024). Worldwide production of grain in 2023/24, by type (in million metric tons)*. M. Shahbandeh. https://www.statista.com/statistics/263977/world-grain-production-by-type/

Watts‐Williams S. J, Gill, Nguyen T. D, Tavakkoli E, Jewell N, Brien C. (2025). Arbuscular mycorrhizal fungi can improve the water use and phosphorus acquisition efficiencies of aerobically grown rice. Journal of Sustainable Agriculture and Environment, in press. https://doi.org/10.1002/sae2.70040

Subramanian, K., Balakrishnan, N., & Senthil, N. (2013). Mycorrhizal symbiosis to increase the grain micronutrient content in maize. Australian Journal of Crop Science, 7(7), 900–910.

Sun, J., Miller, J. B., Granqvist, E., Wiley‐Kalil, A., Gobbato, E., Maillet, F., Cottaz, S., Samain, E., Venkateshwaran, M., & Fort, S. (2015). Activation of symbiosis signaling by arbuscular mycorrhizal fungi in legumes and rice. The Plant Cell, 27(3), 823–838. https://doi.org/10.1105/tpc.114.131326

Team, R. C. (2020). R: A language and environment for statistical computing. R Foundation for Statistical Computing.

Tran, B. T., Cavagnaro, T. R., Able, J. A., & Watts‐Williams, S. J. (2021). Bioavailability of zinc and iron in durum wheat: A trade‐off between grain weight and nutrition? Plants, People, Planet, 3(5), 627–639. https://doi.org/10.1002/ppp3.10151

Vierheilig, H., Coughlan, A. P., Wyss, U., & Piche, Y. (1998). Ink and vinegar, a simple staining technique for arbuscular‐mycorrhizal fungi. Applied and Environmental Microbiology, 64(12), 5004–5007. https://doi.org/10.1128/AEM.64.12.5004-5007.1998

Watanarojanaporn, N., Boonkerd, N., Tittabutr, P., Longtonglang, A., Young, J. P. W., & Teaumroong, N. (2013). Effect of rice cultivation systems on indigenous arbuscular mycorrhizal fungal community structure. Microbes and Environments, 28(3), 316–324. https://doi.org/10.1264/jsme2.ME13011

Watts‐Williams, S. J., & Cavagnaro, T. R. (2018). Arbuscular mycorrhizal fungi increase grain zinc concentration and modify the expression of root ZIP transporter genes in a modern barley (Hordeum vulgare) cultivar. Plant Science, 274, 163–170. https://doi.org/10.1016/j.plantsci.2018.05.015

Watts‐Williams, S. J., Gill, A. R., Jewell, N., Brien, C. J., Berger, B., Tran, B. T., Mace, E., Cruickshank, A. W., Jordan, D. R., & Garnett, T. (2022). Enhancement of sorghum grain yield and nutrition: A role for arbuscular mycorrhizal fungi regardless of soil phosphorus availability. Plants, People, Planet, 4(2), 143–156. https://doi.org/10.1002/ppp3.10224

Wei, Y., Shohag, M., & Yang, X. (2012). Biofortification and bioavailability of rice grain zinc as affected by different forms of foliar zinc fertilization. PLoS ONE, 7(9), e45428. https://doi.org/10.1371/journal.pone.0045428

Welch, R. M., & Graham, R. D. (2004). Breeding for micronutrients in staple food crops from a human nutrition perspective. Journal of Experimental Botany, 55(396), 353–364. https://doi.org/10.1093/jxb/erh064

WHO. (1996). Trace elements in human nutrition and health. World Health Organization.

Xavier Martins, W. F., & Rodrigues, B. F. (2020). Identification of dominant arbuscular mycorrhizal fungi in different rice ecosystems. Agricultural Research, 9, 46–55. https://doi.org/10.1007/s40003-019-00404-y

Zarcinas, B., Cartwright, B., & Spouncer, L. (1987). Nitric acid digestion and multi‐element analysis of plant material by inductively coupled plasma spectrometry. Communications in Soil Science and Plant Analysis, 18(1), 131–146. https://doi.org/10.1080/00103628709367806

Zeilinger, S., Gupta, V. K., Dahms, T. E., Silva, R. N., Singh, H. B., Upadhyay, R. S., Gomes, E. V., Tsui, C. K.‐M., & Nayak, S.,. C. (2016). Friends or foes? Emerging insights from fungal interactions with plants. FEMS Microbiology Reviews, 40(2), 182–207. https://doi.org/10.1093/femsre/fuv045