Silicate Fertilization Improves Microbial Functional Potential For Stress Tolerance in Arsenic-Enriched Rice Cropping Systems

doi:10.21203/rs.3.rs-147549/v1

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Silicate Fertilization Improves Microbial Functional Potential For Stress Tolerance in Arsenic-Enriched Rice Cropping Systems

https://doi.org/10.21203/rs.3.rs-147549/v1

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published 31 Aug, 2021

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Background: The host plant and its rhizosphere microbiome are similarly exposed to abiotic stresses under arsenic (As)-enriched cropping systems. Since silicon (Si) fertilization is effective in alleviating As-induced stresses in plants, and plant-microbe interactions are tightly coupled, we hypothesized that Si-fertilization would improve soil microbial functional potential to environmental stress tolerance, which was surprisingly not yet studied. With the help of high throughput metagenome, microarray and analyzing plant impacts on soil microbiome and the environment, we tested the hypothesis in two geographically different rice (i.e., Japonica and Indica) grown on As- enriched soils.

Results: Silicate fertilization in rice grown on As-enriched soils altered rhizosphere bacterial communities and increased several commensal microorganisms and their genetic potential to tolerate oxidative stress, osmotic stress, oxygen limitation, nitrogen and phosphate limitation, heat and cold shock, and radiation stress. The stress resistant microbial communities shifted with the changes in rhizosphere nutrient flows and cumulative plant impacts on the soil environment.

Conclusions: The study highlights a thus-far unexplored behavior of Si-fertilization to improve microbial stress resilience under As-laden cropping systems and open up promising avenue to further study how commonalities in plant-microbe signaling in response to Si-fertilization alleviates As-induced stresses in agro-systems.

General Microbiology

Silicon fertilization

Stress resistant gene

Rhizosphere microbiome

Plant traits

Geogenic arsenic (As) contamination in groundwater is widespread throughout the globe. The use of As-contaminated groundwater for drinking and domestic use is well recognized as a serious health hazard, affecting tens of millions of people worldwide [1]. Although, several novel technologies have been developed to remove As from drinking water [2], the As-contaminated groundwater still has been extensively used for irrigating a vast crop land, especially in South and Southeast Asian countries [1]. Long-term use of As-contaminated groundwater for irrigation of crop lands accumulates As in the soil and then the edible parts of the plant. Human exposure to As through consumption of As-contaminated rice is a great concern [3]. Arsenic contamination in the food chain is a pressing issue not only for human health safety, but also for food security [4]. Plant grown on As-enriched arable soils experiences stunted growth and yield decline due to As-induced stresses (e.g., oxidative stress, nutritional disorders, photosynthetic inhibition, metabolic and genetic alternations) in plants [5, 6]. Silicon fertilization has been documented to alleviate an impressive range of abiotic stresses, including heavy metal toxicity, oxidative stress, osmotic stress, nutrient imbalance, radiation stress, and temperature (heat and cold) stress in plants [7, 8]. Silicon mediated molecular regulation of genes with potential roles in stress tolerance of plants has been well documented [9]. In addition, being a chemical analog of arsinite, Si shares same transport system for root uptake and competitively inhibits arsenite uptake by rice [10] and can potentially mitigate As-induced stresses in the plant [11].

Microbes, the most natural inhabitants of the ecosystem exhibit vast metabolic potentials to abate abiotic stresses [12]. Since microbial interactions with plants are a vital part of the dynamic ecosystem, they are considered as the natural partners that modulate local and systematic mechanisms in plants to fight against abiotic and biotic stresses [13]. Plant under stress seek urgent help from microbes (‘cry for help’ strategy) to alleviate stresses [14]. Plants are closely associated with microbes in the rhizosphere, the dynamic region of plant-microbe interactions, where plant root exudates sustain high microbial activities under stressed conditions. Several mechanisms related to plant-microbes interactions that help in alleviating stresses have been postulated, (i) the stress factors can directly enrich particular microorganisms that developed mechanisms to respond to and minimize the negative effects of the stressor, (ii) abiotic stresses bring forth a cascade of changes in plant, which potentially attract beneficial microorganisms towards the rhizosphere and assist the plant to adapt and alleviate the stresses, (iii) synergistic and cooperative interactions in microbial communities benefit for the survival of both plant and microbes under the stresses [14].

Although, Si-fertilization effects to alleviate As-induced stresses in plants has been well documented, its effects on microbes-mediated stress responses in As-enriched agricultural soils are unknown. In recent years, Si-fertilization effects in improving soil microbial community structure and functions in agro-systems have been greatly accented [15–17], its effects on microbial functional potential to respond environmental stresses are, however, surprisingly not yet reported. Since Si-fertilization is effective in improving As-induced stress tolerance in plants and plant-microbe interactions are tightly coupled, the hypothesis of our study posits that Si-fertilization would improve soil microbial functional potential for environmental stress tolerance under As-laden cropping systems. To test the hypothesis, we used a functional gene microarray (i.e., GeoChip 5) to study microbial functional potential to tolerate stresses such as oxidative stresses, osmotic stress, oxygen limitation, nitrogen limitation, phosphorus limitation, glucose limitation, protein stress, radiation stress, heat and cold shock in As-enriched rice cropping systems. In addition, bacterial community structure (16 s rRNA), soil enzyme activities involved in nutrient cycling, pore-water chemistry, plant nutrient uptake, and photosynthetic parameters were measured. Multivariate analyses were performed to better understand the key factors shaping potential soil microbial functional communities responding to environmental stresses. The study provides new insights into our understanding of Si-fertilization impacts on the soil microbial functional potential to respond abiotic stresses in As-enriched cropping systems.

Greenhouse experiments

The pot experiments were conducted in controlled greenhouse conditions. The soil was collected from 0-20 cm depth, air-dried, ground and passed through a 2 mm sieve. The initial soil properties were presented in Table S2. The sieved soils was spiked with 40 mg kg^-1 of Na₂HAsO₄.7H₂O, packed into Wagner pots (24 cm diameter and 30 cm height) with a bulk density of 1.1 g cm^-3 and left for one month for ageing. After ageing, the soil total As content was 43.6 mg kg^-1 (n=10), which was relatively higher than the provisional limit of As in arable soils (20 mg kg^-1) and was within the range of the total As content of As-contaminated paddy soils of South and Southeast Asian countries [34].

The silicon fertilizer used in this study was a granular calcium silicate fertilizer made up of blast furnace slag, manufactured and marketed by POSCO, South Korea. The chemical composition of the silicon fertilizer was given in Table S3. The fertilizer has been found to be effective in increasing rice yield by 10-15% without bringing any heavy metal contamination in the soil and crop in South Korea [35]. The silicate fertilizer at a rate of 2 Mg ha^-1(recommended rate for rice cultivation in Si poor soil in South Korea) was applied to the potted soil three days prior to transplanting. In control, no silicate fertilizer was added to the rice planted pot. NPK fertilizer (110 kg N ha^-1, 45 kg P₂O₅ ha^-1, and 58 kg K₂O ha^-1) as urea, fused superphosphate, and potassium chloride, respectively were applied to all the pots following the standard Korean rice cultivation guideline. Twenty eight days old Indica (cv. Rc158) and Japonica (cv. Dongjinbyeo) rice seedlings were transplanted with two seedlings per pot. The pots were remained flooded throughout the experiment.

Sampling and analysis

Soil pore-water was collected in acid-washed and N₂-flushed crimp-sealed vials using a rhizo-sampler (EcoTech^®, Germany). The pore water was filtered with 0.45 µm syringe filter and 2% nitric acid was added to it. The concentrations of total As and As species (AsIII and AsV) in the pore-water was measured by an inductively coupled plasma-mass spectrometry (ICP-MS) and HPLC-ICP-MS (Agilent 7500ce series, Agilent Technologies, USA), respectively. The total Fe and Si concentrations were measured by an inductively coupled plasma-optical emission spectrometry (ICP-OES), (Vista-MPX, Varian, Australia). To measure As, Si, and nutrient (N, P, and K) uptake by the plant, their concentration in plant parts and the plant biomass were recorded. Total As concentration in rice straw and grain was estimated as reported previously [36]. The concentrations of Si and major plant nutrient (N, P, and K) in rice straw were estimated by the standard methods as described by Das et al. [15]. Photosynthetic parameters such as net photosynthetic rate (P_n), stomatal conductance (g_s), and transpiration rate (T_r) in fully expended flag leaves were measured between 9:00 a.m. to 10:00 a.m. at different growth stages of rice using a portable photosynthesis system (CIRAS-2, PP-Systems, UK). For microbiological study, soils from the rhizosphere were collected at the harvesting stage of the plant.

Microbial 16S rRNA and functional gene microarray analyses

Total genomic DNA from freshly collected rhizosphere soils (500 mg) was extracted in triplicate using a Fast-DNA spin kit for soil (MPBio, USA) according to the manufacturer’s instructions and quantified using PicoGreen (Molecular Probes, Eugene, OR, USA). Oligomers containing the Illumina overhang adapter sequence as well as the following 16S rDNA region specific sequence, V3–4 region for bacteria [37] was used as amplicon primers. Sample DNAs were PCR-amplified with the annealing temperature set to 58°C. The purified amplicons were PCR-indexed using the Illumina Nextera XT index kit and the library was constructed. The library was paired-end (600 cycles v3) sequenced using the MiSeq platform (Illumina Inc., San Diego, CA, USA). The paired reads were first merged and then processed to remove short or low-quality sequences and potential chimeras. Operational taxonomic units (OTUs) were defined at 97% sequence-identity cutoff using the VSEARCH algorithm [38]. Taxonomic assignment was conducted using the on-line RDP classifier (https://rdp.cme.msu.edu/classifier/).

Functional gene microarray (i.e., GeoChip) has been popularly used to study functional potentials of microbial communities important to biogeochemistry, ecology, and environmental sciences. The latest functional gene microarray (i.e., GeoChip 5) covers 1517 gene family and ~57,000 probes, including 86 gene family and 25,732 probes targeting stress response genes [39]. In this study, the soil microbial functional potential involved in stress response was evaluated using GeoChip 5, according to the method previously described by Das et al. [15]. GeoChip 5 analysis was conducted at Institute for Environmental Genomics, University of Oklahoma, USA. DNA (2 µg) was labeled with Cy5 fluorescent dye (Thermo Fisher Scientific, CA, USA) by a random priming method, purified with a QIAquick purification kit (Qiagen, CA, USA), and dried in a SpeedVac (ThermoSavant, NY, USA) at 45 °C for 45 min. GeoChip hybridization was carried out at 42 °C for 16 h on a HS4800 Pro Hybridization Station (Tecan US, Durham, USA). After purification, microarrays were scanned by a NimbleGen MS200 Microarray Scanner (Roche NimbleGen, Madison, WI, USA) at 633 nm, using a laser power and photomultiplier tube gain of 100% and 75%, respectively. The data were processed and analyzed using Agilent Feature Extraction 11.5 software and Microarray Data Manager (http://ieg.ou.edu/microarray/).

APIZYM assay

APIZYM, a semi-quantitative enzyme assay system (Biomerieus, USA) has been used considerably to study a broad range of enzyme activities in soil [40]. The kit is supplied with 20 microcupules (19 microcupules for different hydrolytic enzyme and one for control) containing different dehydrated chromogenic substrates (Table S3). Relative soil enzyme activities were estimated using the APIZYM system as described by Das et al. [41]. In brief, freshly collected rhizosphere soils were mixed with sterile nano-pure water (1: 1.25 w-v), homogenized in a horizontal shaker for 10 min and centrifuged at 2000g for 15 min. The supernatant (70 μl) was dispensed into each microcupules and incubated at 30°C for 24 h. This was followed by the addition of 30 μl of each ZYM A and ZYM B reagent (supplied with the kit) to the microcupule, left for 5 minutes, and the intensity of soil enzyme activities was measured.

Shift in rhizosphere bacterial community structure by silicate fertilization under As stress

Irrespective of the treatment, Proteobacteria was the most dominant phylum comprising 33.4 to 46.7% relative abundance. Besides, Firmicutes (13.4–22.5%), Actinobacteria (4.6–16.6%), Bacteroidetes (2.9–8.8%), and Acidobacteria (1.0–12.5%) were the other major phyla detected in the As-enriched paddy soils. Although, α-diversity of bacterial community did not change significantly, among the phyla, Acidobacteria and Fusobacteria were significantly decreased while Gemmatimonadetes was significantly increased in response to silicate fertilization in both Japonica and Indica rice (Fig. 1). Chloroflexi, Cyanobacteria, Caldithrix, and Verrucomicrobia were significantly increased in Japonica rice, whereas Actinobacteria was significantly increased in Indica rice. Likewise, Nitrospirae in Japonica rice and Cyanobacteria and Verrucomicrobia in Indica rice were significantly decreased by silicate fertilization. Among the dominant genera, Bacillus, Ramlibacter, Geobacter, and Azospirillum were significantly increased while Acidobacterium was significantly decreased by silicate fertilization, irrespective of the rice cultivar. Although, there was no report of Si-fertilization impact on rhizosphere microbial community of rice grown on As-rich soils, some of the recent study reported that (a) silicate fertilization in rice grown on flooded soil (not contaminated) markedly altered rhizosphere bacterial community structure and increased Alphaproteobacteria, Betaproteobacteria, Cyanobacteria, and Verrucomicrobia but decreased Actinobacteria [15], (b) a slag-based Si-fertilizer application to Pakchoi (Brassica chinensis L.) grown on soil contaminated with multiple heavy metals reduced the selective pressure of heavy metals on the bacterial communities and increased the richness of soil bacteria [17], (c) foliar application SiO₂ nanoparticles enriched Rhodobacteraceae and Paenibacillus albeit bacterial community structure was unchanged in rhizosphere of Pakchoi grown on contaminated mine soil [16].

Functional genes involved in stress response are increased by silicate fertilization under As stress

Functional genes of a particular stress category responded differently to silicate fertilization, irrespective of rice cultivar. Since there are multiple gene probes for each stress category on the GeoChip microarray, the relative abundance of all gene probes for the individual category were summed. Among the stress gene categories, the relative normalized signal intensities of genes related to oxidative stress (ahpC, ahpE, katA, katE), osmotic stress (opuE, proV), oxygen limitation (arcA, narH), nitrogen limitation (glnA, glnR), phosphate limitation (phoA, phoB, pstA, pstB, pstC), heat shock (dnaK, groEL, hrcA), cold shock (cspB), and radiation stress (obgE) were significantly increased by 37.7 and 24.4%, 40.2 and 24.0%, 18.8 and 15.6%, 51.9 and 66.4%, 34.4 and 43.7%, 27.0 and 23.7%, 24.0 and 15.3%, and 34.4 and 32.5% in Japonica and Indica rice, respectively, in response to silicate fertilizer application in As-enriched paddy soils (Fig. 2). The oxidative stress gene (oxyR), osmotic stress gene (proX), oxygen limitation stress gene (narJ), heat shock stress gene (grpE), and cold shock stress gene (cspA) were, however, increased in either Japonica or Indiac cultivar. The cultivar variation significantly altered the normalized signal intensity of genes involved in osmotic stress (opuE, proX), oxygen limitation (arcA) and nitrogen limitation (glnA) (Supplementary Table S1).

The texa-function relationship revealed that variations in oxidative stress tolerance in response to silicate fertilization were largely attributed to the genera Oribacterium, Botryotinia, Corynebacterium, Haloarcula, Lyngbya, Atopobium, Lactobacillus, Bacteroides, and Staphylococcus (Supplementary Fig. S1). Differences in osmotic stress tolerance in silicate amended and no-amended soils were mostly attributed to the genera Halothermothrix, Actinomyces, Bacillus, Ruegeria, Desulfatibacillum, Alkalilimnicola, Brachybacterium, and Thermobifida. Differences in oxygen limitation were mostly related to the genera Shewanella, Staphylococcus, Geobacillus, and Mycobacterium, nitrogen limitation were mostly related to the genera Synechococcus, Sodalis, Octadecabacter, Sulfolobus, Leptospira, Thermoplasma, Mesorhizobium, Shewanella, Granulicatella, Enterococcus, Gemella, and uncultured cyanobacterium, and phosphate limitation were mostly attributed to the genera Methylococcus, Methanosarcina, Roseovarius, Cellvibrio, Marinobacter, Rhizobium, Shewanella, Grimontia, Bradyrhizobium, Dictyoglomus, Providencia, Vibrio, and Methylophaga. Variations in gene indicative pathways related to heat shock stress tolerance in silicate amended and no-amended soils were largely attributed to the genera Geobacillus, Bacteroides, Thauera, Cyanothece, Mycobacterium, Ajellomyces, Schizosaccharomyces, Mobiluncus, and Enterococcus, and cold shock stress tolerance were mostly attributed to the genera Lysinibacillus, Bradyrhizobium, Streptococcus, Gramella, Corynebacterium, and Proteus. Differences in radiation stress tolerance were largely attributed to the genera Borrelia, Alkaliphilus, Roseiflexus, Anaplasma, Acidobacterium, Clostridium, Thiomonas, Lactobacillus, Candidatus, and Bacillus (Fig. S1).

Silicate fertilization increases soil enzyme activities involved in nutrient cycling under As stressed conditions

Soil enzyme activities involved in C, N, and P cycling altered in response to the silicate fertilizer amendment in rice grown on As-enriched soils. The labile C degrading enzymes, e.g., α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase, α-mannosidase, and α-fucosidase that respectively degrade maltose, cellobiose, melibiose, lactose, mannose, and fructose, distinctly increased in silicate fertilizer amended soils compared to that of unamended soils, irrespective of the cultivar (Fig. 3). In contrast, recalcitrant C degrading enzymes such as esterase, lipase, and N-acetyl-β-glucosaminidase that degrade hemicelluloses and polysaccharide, respectively, exhibited either no increase or little increase in response to the silicate fertilization. Soil enzyme activities associated with N cycling (i.e., leucine-aminopeptidase and trypsin) and P cycling (i.e., alkaline phosphomonoesterase and phosphohydrolase) remarkably increased in silicate fertilizer amended soils compared to that of unamended soils, irrespective of the cultivar. Overall, total soil enzyme activities remarkably increased, indicating an improvement of nutrient cycling in response to silicate fertilizer application in rice paddy grown on As-enriched soils. Silicon-fertilization to improve soil enzyme activities in arable and forest soil has been proved effective [18, 19].

Photosynthetic parameters are affected by silicate fertilization under As stressed conditions

Silicate fertilization significantly (P < 0.001) increased plant photosynthetic rate, transpiration efficiency, and stomatal conductance, while significantly (P < 0.001) decreased the transpiration rate, irrespective of the cultivar (Fig. 4). On an average, the photosynthetic rate, transpiration efficiency, and stomatal conductance were increased by 18.1 and 19.6%, 39.1 and 37.0%, and 12.5 and 10.4%, while the transpiration rate was decreased by 15.0 and 13.1% in Japonica and Indica cultivar, respectively. All the studied photosynthetic parameters were also significantly influenced by the cultivar variation, however, the cultivar effects were relatively smaller than the silicate fertilization effects. The increase in photosynthetic parameters in response to silicate fertilization in As-enriched rice paddies could be indicative of the alleviation of As toxicity effects by the silicate fertilization.

Silicate fertilization alters pore-water chemistry and increases nutrient uptake and the crop yield

The pore-water As concentration significantly (P < 0.001) decreased, whereas pore-water Si and Fe concentration significantly (P < 0.001) increased as a result of the silicate fertilization in both Japonica and Indica rice. The dissolved organic carbon (DOC) was also significantly (P < 0.001) increased by the silicate fertilization, irrespective of the cultivar. No noticeable effect of cultivar on pore-water As and Fe concentrations were detected, whereas a significant cultivar effect on pore-water Si concentration was observed (Fig. S2).

Regardless of treatment, there was no significant changes in grain N, P, and K concentrations, however, the grain Si concentration significantly (P < 0.001) increased by 85.0 and 87.5% and grain As concentration significantly (P < 0.001) decreased by 29.8 and 26.1% in Japonica and Indica rice, respectively, in response to the silicate fertilization. On the contrary, N, P, and Si concentrations in rice straw markedly increased by 24.3 and 27.7%, 16.4 and 17.2%, and 87.7 and 83.5%, and As concentrations in straw significantly (P < 0.001) decreased by 44.4 and 51.4% in Japonica and Indica rice, respectively. The increase in grain yield (27.8% in Japonica and 22.7% in Indica) and straw yield (35.2% in Japonica and 36.5% in Indica) as a result of the silicate fertilization was significant. Converting the value of yield and element concentration into uptake, we found that, silicate fertilization significantly induced an increase in grain N uptake by 33.1 and 27.3%, grain P uptake by 38.6 and 30.3%, grain K uptake by 37.4 and 32.9%, grain Si uptake by 136 and 130%, while decreasing grain As uptake by 10.3 and 9.4% in Japonica and Indica rice, respectively. Likewise, it increased straw N uptake by 68.1 and 74.3%, straw P uptake by 57.4 and 60.0%, straw K uptake by 46.3 and 49.0%, straw Si uptake by 154 and 151%, while decreasing straw As uptake by 24.8 and 33.6 % in Japonica and Indica rice, respectively. Taking grain and straw uptake together, plant N, P, K, and Si, uptake were increased by 59.9 and 65.5%, 53.4 and 55.0%, 45.7 and 48.2%, and 152 and 149%, while plant As uptake was decreased by 24.5 and 33.2% in Japonica and Indica rice, respectively (Table 1). The increase in plant N, P, and K uptake was attributed to the increase in yield rather than changes in N, P, and K concentrations in grain. The decrease in As uptake and increase in plant N, P, K, and Si uptake, and the crop yield suggested the ameliorative effect of silicate fertilization against As toxicity in the rice agro-system.

Table 1

Silicate fertilization (SF) impacts on yield and nutrient uptake of rice grown on As-enriched soils
Parameters	Japonica		Indica		Statistical analysis
Parameters	Control	SF	Control	SF	SF	Cultivar	SF × Cultivar
Grain yield (g pot^− 1)	4.20	5.37	3.97	4.87	8.96^**	1.12^ns	0.149 ^ns
Straw yield (g pot^− 1)	25.3	34.2	32.4	44.2	10.8^**	7.46^*	0.217 ^ns
Grain N conc. (mg g^− 1)	8.80	9.17	9.40	9.76	0.311 ^ns	0.842 ^ns	0.000 ^ns
Grain P conc. (mg g^− 1)	2.21	2.40	2.30	2.44	1.25 ^ns	0.194 ^ns	0.021 ^ns
Grain K conc. (mg g^− 1)	5.77	6.20	6.03	6.53	1.45	0.6 ^ns	0.007 ^ns
Grain Si conc. (mg g^− 1)	17.8	32.9	18.5	34.6	153^***	0.875 ^ns	0.167 ^ns
Grain As conc. (mg g^− 1)	1.36	0.96	1.30	0.95	19.3^**	0.158 ^ns	0.162 ^ns
Straw N conc. (mg g^− 1)	4.80	5.97	4.93	6.30	88.8^***	3.01 ^ns	0.553 ^ns
Straw P conc. (mg g^− 1)	1.36	1.58	1.40	1.64	29.7^***	1.11 ^ns	0.038 ^ns
Straw K conc. (mg g^− 1)	13.5	14.6	13.9	15.1	2.65 ^ns	0.331 ^ns	0.012 ^ns
Straw Si conc. (mg g^− 1)	34.2	64.1	35.3	64.8	73.9^***	0.072 ^ns	0.004 ^ns
Straw As conc. (mg g^− 1)	8.60	4.78	10.4	5.04	38.9^***	1.91 ^ns	1.05 ^ns
Grain N uptake (mg pot^− 1)	37.0	49.2	37.3	47.5	14.4^**	0.055 ^ns	0.119 ^ns
Grain P uptake (mg pot^− 1)	9.28	12.9	9.11	11.9	20.9^***	0.699 ^ns	0.346 ^ns
Grain K uptake (mg pot^− 1)	24.2	33.3	23.9	31.8	27.1^***	0.295 ^ns	0.134 ^ns
Grain Si uptake (mg pot^− 1)	74.8	177	73.3	169	254^***	0.613 ^ns	0.292 ^ns
Grain As uptake (mg pot^− 1)	5.73	5.13	5.14	4.66	7.84	1.82 ^ns	0.019 ^ns
Straw N uptake (mg pot^− 1)	121	204	160	279	507^***	160^***	16.4^**
Straw P uptake (mg pot^− 1)	34.4	54.1	45.3	72.4	199^***	77.2^***	4.97^*
Straw K uptake (mg pot^− 1)	342	500	444	669	52.8^***	28.3^***	1.41 ^ns
Straw Si uptake (mg pot^− 1)	863	2191	1145	2868	130^***	12.8^**	2.18 ^ns
Straw As uptake (mg pot^− 1)	217	163	336	223	13.0^**	14.8^**	1.61 ^ns
Plant N uptake (mg pot^− 1)	158	253	197	326	312^***	78.2^***	2.98 ^ns
Plant P uptake (mg pot^− 1)	43.6	67.0	54.4	84.3	181^***	50.2^***	2.78 ^ns
Plant K uptake (mg pot^− 1)	366	533	473	701	56.0^***	27.1^***	1.32 ^ns
Plant Si uptake (mg pot^− 1)	938	2368	1218	3036	152^***	13.2^**	2.18 ^ns
Plant As uptake (mg pot^− 1)	223	168	341	228	13.0^**	14.5^**	1.59 ^ns
Values are the mean of three replicate observations. In ‘Statistical analysis’ F value, followed by significant label (P) are provided. ^*, ^, ^*, and ns indicates significant at P < 0.001, at P < 0.01, at P < 0.05, and non significance, respectively.

In this study, we found that silicate fertilizer application to rice grown on As-enriched soils remarkably increased several commensal microorganisms and their genetic potential to tolerate oxidative stress, osmotic stress, oxygen limitation, nitrogen and phosphate limitation, heat and cold shock, and radiation stress. Emerging evidence reveals that the shift in microorganisms following stresses generally benefits plant to adapt and survive under stresses and can have a legacy effect to increase progeny fitness [14].

Arsenic induced increase in reactive oxygen species (ROS) can trigger oxidative stress. The excessive production of ROS can cause oxidative damage and ultimately cell death. Under exposure to potentially toxic elements, glutathione and sulfhydryl groups in proteins are depleted, which leads to production of ROS such as superoxide iron, H₂O₂, as well as hydroxyl radicals [20]. Microorganisms protect themselves to the oxidative stresses by producing antioxidant enzymes such as superoxide dismutase (regulated by sodA gene) and catalase (regulated by katA,E), induction of alkyl hydroperoxide reductase (regulated by ahpCF), DNA repair following damage by ROS, and induction of the regulon oxyR that increases ROS scavenging activities and limits H₂O₂ generation [21]. The increase in signal intensity of most of these genes involved in oxidative stress tolerance with silicate fertilization in As-enriched rice paddies suggested its ameliorative role against oxidative stress induced by As. Microbial As detoxification metabolism cross-protected them from As-induced osmotic stress [21]. Microbes living under osmotic stress experience multiple tolerances due to wider gene expression under the stress. To protect themselves from the osmotic stress, microbes alter cell osmoprotectant concentrations by using transport systems such as opuE, a sodium/proline symporter or the ProU transport system consisting of proV, proW, and proX [22]. The signal intensity of these genes involved in osmotic stress tolerance was remarkably increased with the silicate fertilization in As-enriched rice cropping system, indicating its beneficial role against osmotic stress induced by As. Likewise, the ameliorative effect of silicate fertilization on As-induced oxygen limitation in microbes was observed. In an oxygen-limiting environment, cytochrome genes of microbes (cydA and cydB) are activated via regulatory genes such as arcA and arcB [23]. Moreover, certain microbes contain genes that allow them to use other electron acceptors, such as nitrate reductase genes (narG, narH, narJ, and narI) in oxygen-limiting conditions [24]. In this study, particularly, we observed a significant increase of arcA, narH, and narJ signal intensity with the silicate fertilization. Arsenic contamination in arable soils commonly reduces microbial metabolic activities for nutrient acquisition [25]. The common nutrients that are often limited are C, N, and P. We observed an increase in gene signal intensities involved in N and P, but not C acquisition with the silicate fertilization. Considerable research evidenced that Si amendment improves plant photosynthetic activities by enhancing mesophyll conductance in rice [26], which in turn increases root exudation and thereby the carbon content in the soil not become limited. On the contrary, higher plant growth under silicate fertilization increases plant N and P uptake from the soil, thereby making soil N and P limited. The N limitation is sensed by glutamine in bacteria and glutamine synthase (glnA) genes are activated via regulatory genes tnrA and glnR [27]. Likewise, P-limitation activate the response regulator phoB that induces expression of the Pst inorganic phosphate (Pi) uptake system comprised of subunits pstS, pstA, pstB, and pstC of the transporter and activation of alkaline phosphatase (phoA) releases Pi [28]. Significant increase in signal intensity of genes glnA and glnR, and phoA, phoB, pstA, pstB, and pstC with silicate fertilization indicates its ameliorative effects to counter As-induced N and P limitation in soil under As-contaminated rice agro-systems. Microbes are usually exposed to temperature variations in the environment and often experience heat and cold shock. They alleviate heat shock by activating heat shock proteins such as dnaK, grpE, groEL and groES, molecular chaperones that prevent denaturation, and the regulatory gene hrcA [29]. Similarly, microbes alleviate cold shock by inducing cold shock proteins (cspA and cspB), and increasing the ratio of unsaturated to saturated fatty acids in membrane lipids, which is accomplished via the desaturase gene (des), comprised of two-component system genes, desK-desR [30]. The silicate fertilization remarkably increases the signal intensity of genes dnaK, grpE, groEL, and hrcA and cspA, cspB, and desk, suggesting the importance of silicate fertilization to enhance microbial adaptation to heat and cold shock in the rice agro-system.

Upon exposure to As, microorganisms develop different mechanisms for As-detoxification (e.g., As oxidation and reduction, extrusion of arsenite out of cells, As methylation etc.) either acquiring resistance against the toxic substance or evolving under high selection pressure to withstand the metalloid stress. The resistance mechanisms can help microbes to not only prevent As-toxicity, but also provide cross-protection against As-induced abiotic stresses [21]. The acute effect of As stress, however, resulted in an inhibition of As-sensitive microbes and the loss of biochemical activities essential for soil functions [31]. Silicon-rich amendments have been demonstrated to reduce As bioavailability, alter soil biochemical properties, increase root exudation and nutrient uptake by plants, which in turn can influence microbial communities, activities and functions [15–17]. A majority of functional stress response gene categories showed significant (P ≤ 0.05) or marginally significant (P ≤ 0.10) correlations with the measured variables of plant photosynthetic parameters and nutrient uptake, pore-water chemistry, and soil enzyme activities involved in C, N, and P cycle (Fig. S3). Canonical correspondence analysis (CCA) showed that 54.4% of the variation in potential stress response microbial communities was explained by the first two canonical axes of the CCA plot (Fig. 5). A partial CCA-based variation partitioning analysis (VPA) revealed that the three categories of variables, i.e., plant variables (photosynthetic parameters and plant nutrient uptake), pore-water chemistry (pore water As, Si, and Fe), and soil enzyme activities (Soil enzyme activities involved in C, N, and P cycling) explained 33.6, 6.3, and 7.7% of the total variance, respectively (Fig. 5), suggesting rhizosphere microbial communities involved in stress response could shift with changes in rhizosphere nutrient flows and cumulative plant impacts on the soil environment. Emerging evidence demonstrated that rhizosphere carbon and nutrient flows and plant nutrient uptake markedly shape rhizosphere microbial community structure and activities in agricultural soils [32]. Changes in soil environment often modulate plant metabolites that shape rhizosphere microbiome [14]. For example, under N-limiting conditions, leguminous plants release more flavonoids to attract N-fixing bacteria [33]. It is assumed that the shift in stress resilience microorganisms with silicate fertilization could be mediated by systematic root metabolite exudation and commonalities in plant-microbe signaling to enhance the capacity to combat stress, which needs further investigation. In fact, despite past efforts to raise awareness of the importance of Si fertilization to improve crop growth and yield, even under stressed conditions [7–9], coupled with some recent findings on its ameliorative effects on rhizosphere microbial communities necessary to boost crop productivity [15–17], many soil scientists and microbiologists remain indifferent to or unaware of the potential roles of this quasi-essential element. A multidisciplinary experimentation is the current need for a holistic understanding of the underlying role of Si in improving plant-microbe interactions necessary to combat stresses, and therefore its ability to improve crop productivity.

Since the host plant and its natural partner rhizosphere microbiome are similarly exposed to abiotic stresses under As-laden agro-systems and emerging evidence demonstrated the beneficial role of Si to alleviate As-induced stresses in plant, it makes sense that microbial resilience to the stresses would be improved by silicate fertilization. We found that silicate fertilization in rice grown on As-enriched soils altered rhizosphere microbial community structure and improved the genetic potential of microbes to tolerate oxidative stress, osmotic stress, oxygen limitation, nitrogen and phosphate limitation, heat and cold shock, and radiation stress. The stress resistant microbial communities shifted with the changes in rhizosphere nutrient flows and cumulative plant impacts on the soil environment. It is beyond the scope of the current study to elucidate Si induced microbe mediated stress resistance in plant, however, our findings pave the way for future system based study approach integrating metabolomics, transcriptomics, and metagenomics to better understand so far unexplored plant-microbes signaling phenomenon induced by Si under multiple stresses. A vigilant amalgamation of these system-based approaches enable developing bio-based solutions for improving agricultural productivity and environmental sustainability.

Supporting information

Additional information as noted in the text.

Acknowledgements

Due appreciation is given to Dr. Joy D. Van Nostrand, Institute for Environmental Genomics, University of Oklahoma, USA and Dr. Ganesh M. Nawkar, Division of Applied Life Sciences and Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South Korea for helping with the analyses of GeoChip and plant photosynthetic parameters, respectively.

Authors’ contributions

SD and PJK conceived the original idea and designed the work. SD, GWK, and PPV performed sampling, lab procedure, and statistical analysis. SD performed microbial analyses and Bioinformatics. SD wrote the manuscript with support from PJK and MSIB. All authors approved the final manuscript.

Funding

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1A2B2002239).

Availability of data and materials

Sequences supporting the study are publicly available at the National Center for Biotechnology Information (NCBI) under BioProject accession number PRJNA687064.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Institute of Agriculture and Life Sciences, Gyeongsang National University, Jinju, 660-701, South Korea. ²Division of Applied Life Science, Gyeongsang National University, Jinju, 660-701, South Korea.

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Silicate Fertilization Improves Microbial Functional Potential For Stress Tolerance in Arsenic-Enriched Rice Cropping Systems

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Shift in rhizosphere bacterial community structure by silicate fertilization under As stress

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