S material Characterization
Images from the SEM-EDS, TEM, and XRD patterns are shown in Fig. S1-3. Both uncoated and coated nanoscale S showed a uniform spheroidal shape with average particle sizes of 65 and 38 nm, respectively, and average hydrodynamic sizes of 851.2 ± 28.7 nm and 982.5 ± 86.6 nm (pH = 7), respectively (Fig. S2, S4, and Table S1). The zeta potential of nS, cS, and bS (Fig. S4 and Table S1) at pH 7 was -23.6 ± 0.4, -33.5 ± 0.3, and -13.9 ± 4.3, respectively. The crystal diffraction pattern of nS, cS and bS from XRD analysis (Fig. S3) matched well with the orthorhombic structure of elemental sulfur (S8)43. The EDS analysis confirmed the elemental compositions of nS and bS as pure S, and cS as a combination of S and C (Fig. S1). The dissolution of all the three S based compounds in DI was less than 0.4% at 15 days (Fig. S5 and Table S2).
Two-photon microscopy images
Detection of S by two-photon microscopy is both size- and coating-dependent (Fig. 1, S6, and S7). In DI suspensions at 500 mg/L, much less fluorescence signal was detected from nS suspensions that were not sonicated, since particle aggregation significantly reduced signal intensity (Fig. 1e); after thorough sonication, the fluorescence signal from nS was clearly observed (Fig. 1k). At the same concentration, neither bS suspension with or without sonication, or ionic S solutions, had a fluorescence signal (Fig. 1f, g, l, m, j, and p). The finding that smaller size enables a stronger fluorescence has been reported previously for nanoscale ZnO44. Importantly, steric acid-coated nanoscale S did not fluoresce (Fig. 1h, i, n, and o), likely due to the change in surface chemical composition; an ionic SO42- solution also exhibited no fluorescence. This unique fluorescence response of uncoated nanoscale S will be important to understand the nanomaterial movement in the plant. The root uptake of S based (200 mg/L) compounds and translocation to leaf tissues as determined by two-photon microscopy is shown in Fig. 1c, d, and S6. Representative fluorescent signals from S are indicated by the arrows. These distinguishable response points are similar to the pure nanoscale S suspended in DI (Fig. 1e and k). In nS treated roots, a S fluorescent signal was evident, indicating nS accumulation; clear signals are also evident in the corresponding leaf tissue, demonstrating translocation of nanoscale S. For cS, a weak S signal was found in cS treated roots. Given the increased S content observed by ICP-OES (below) and the lack of cS response in solution, this suggests that cS was accumulated and translocated through tomato either in the surface coated form or as dissolved ions. No detectable signal S was found in bS exposed roots, indicating the absence of nanoscale S; interestingly, significant S fluorescence was shown in the corresponding leaves of bS treated plants, suggesting that active in planta transformation of bS was occurring, including likely reduction of sulfate ions to nanoscale S in leaf tissues. Spatially, S was observed near or in the vascular tissues and the intercellular spaces, indicating apoplastic route of uptake. In the leaf tissues, S resulting from nS and bS exposure was distributed similarly and primarily around the stomata, indicating a xylem-based transport pathway via the transpiration stream. Additional time resolved videos of the 3D biodistribution pattern of S within the leaves and roots and representative images taken at different depths from the surface are available (Videos 1-6 and Fig. S7) and temporally confirm the above findings.
Disease progress and plant growth
As shown in Fig. 2 and S8-9, at 4 d, there was no impact on biomass as a function of treatment or disease. However, at 8 and 16 d, disease reduced leaf biomass by 35.3% and 79.5% and root biomass by 20.1% and 73.5%, respectively. The positive impacts of S amendment were evident by day 8; cS and bS significantly increased shoot biomass by 51.2% and 43.6%, and root biomass by 26.7 and 48.2%, respectively. By day 16, the impact of bS became non-significant, but nS and cS increased shoot biomass by 264% and 378% and root biomass by 200% and 175%, respectively.
Results from the 35 d greenhouse experiment were similar (Fig. S10 and S11). Treatment with the cS and nS decreased disease progress by 54 and 56%, respectively; bS decreased disease by 32% but ionic S had no impact on disease progression. Disease decreased plant shoot mass by 87.4% but nS at 100 and 200 mg/L increased shoot mass by 1,160%-1,750% relative to the disease controls; cS increases were 819%-1,180%, respectively. Similarly, for the root mass, the increases were 263 and 412% for nS, and 219 and 221% for cS, respectively. Treatment with bS and iS had no impact on biomass under diseased conditions, regardless of concentration. Both nS and cS increased relative chlorophyll content compared to the infested control and bS in the 16 d greenhouse experiment; in the 35 d greenhouse study, cS increased linear electron flow (LEF) relative to the infested control and bS, and had thicker leaves than bS (Fig. S12). This may be related to the increment of sucrose in corresponding leaves. cS increased leaf sucrose content by 1.4-1.5-fold over controls, nS, and bS at 8 d; at 16 d, both nS and cS had higher amount of sucrose than the infested control and bS by ~50-68%. Sucrose is the end product of photosynthesis and the primary sugar transported in the phloem of most plants. It also serves as both a source of carbon skeletons and energy for plant organs unable to perform photosynthesis.
A number of nanoscale amendments have been shown to reduce disease progression and increase biomass, although these have largely been with foliar applications. Elmer and White45 showed that foliar treatment of CuO significantly suppressed Fusarium infection and increased biomass of tomato and eggplant by 37.5% and 10.6%, respectively, under field conditions. Similar fungal disease suppression capability has been reported for foliar application of different nanoscale forms of silica to watermelon46,47. Borgatta et al.16 and Ma et al.48 both demonstrated the importance of material properties, reporting that foliar application of 2 ml of 10-1000 mg/: copper phosphate nanosheets suppressed Fusarium infection and increased the biomass of watermelon and soybean at concentrations as low as 10 mg/L, whereas > 100 mg/L of spherical nanoscale CuO was needed to exert similar benefit. Less data is available on nanoscale sulfur amendments. Shang et al.49 reported that seed and foliar treatment of rice with CuS nanoparticles significantly suppressed Bakanae disease by 35.1-45.9%. Importantly, results with the nanoscale materials were more effective than with Kocide (conventional fungicide). The current findings align with a previous study from our group that focused on seed treatment or foliar application of nanoscale S in tomato50. Specifically, foliar application of 1 mg/plant signiﬁcantly decreased disease by 47.6% and increased shoot biomass by 55.6%. Importantly, the disease control eﬃcacy was 3-fold greater than hymexazol (conventional fungicide).
The mechanism of action for disease suppression clearly depends on the material used. Nanoscale Cu is known to uniquely stimulate plant defense systems and secondary metabolism48, although under sufficient concentrations, it can also directly act as an antimicrobial agent. Conversely, nanoscale silica can stimulate the deposition of cell water material in the roots, boosting the physical barrier protection against invading pathogens46,47. Nanoscale S is somewhat similar to Cu; it is known to stimulate plant defense and the glutathione system, but also directly acts as an antimicrobial agent at sufficient doses49. For example, Cao et al.24 reported that the expression of select pathogenesis and oxidative stress genes were significantly upregulated by 11−352%, although TEM confirmed the presence of nanoscale S in the plant stem, potentially yielding direct antifungal effects.
3.1 S accumulation
In the 16-day experiment, disease had no impact on root S content at day 4 and 8, but by harvest, the diseased root S content was 40% greater than the healthy controls (Fig. S13a). All S treatments increased the root S content at 16 d (range 25.2%-47.0%), although the nanoscale S treatments increased those levels more rapidly. Similar to the roots, the presence of disease significantly increased stem S content in the untreated plants by day 16 (Fig S13b). Interestingly, all S treatments increased stem S content at 4 d (10.0%-14.4%), had no impact at 8 d and significantly reduced S content at 16 d (range 11.9%-24.8%). Similar to the roots and stems, disease significantly increased leaf S content at 4 d (Fig. 2c). All S treatments further increased leaf S content (6.1%-27.0%) at 4 d; these effects were more marginal at 8 d but were again statistically significant at 16 d.
The dynamics of tissue S content as a function of time, disease and treatment are complex. It is clear that changes in tissue S content directly correlate with infection, often not evident at 4 or even 8 d but significantly greater by 16 d. Even in the absence of treatment, this is a clear indication of increased S demand as a function of biotic stress and likely activation of secondary defense pathways that utilize S. Interestingly, S amendment did impact S tissue content, although this varied significantly with time, disease status, and treatment. In treated diseased plants, the impact of particle size and coating seemed to be minimal, suggesting that S demand as a function of disease was the driving force for changes in the utilization pattern of this nutrient.
There are two known pathways for S assimilation in plants; the first involves SO42- by ion transporters SULTR, and the second with direct accumulation of elemental sulfur (S0) catalyzed by Rhodaneses (Str), which are sulfotransferases51. Sulfate is the primary form assimilated by plants; however, most sulfur in soil exists in reduced forms. Under these circumstances, sulfur may become limiting and plants may increase the expression of sulfate transporters to facilitate acquisition by the roots. ST2.1 is expressed in xylem and phloem leaf parenchyma cells, xylem parenchyma and root pericycle cells52. ST2.1 mediates the uptake of sulfate from the apoplast within the vascular tissues in roots, and promotes translocation to young tissues through shoot phloem transport. Importantly, the sulfate pathway results in the formation of SO32- and S2-, both of which are harmful to plant cells. As shown in Fig. S19, the expression of ST2.1 with both nanoscale treatments is equivalent to the controls. In addition, as the first step of primary S-assimilation in plants, ATP-sulfurylase catalyzes the activation of sulfate (SO42-) and yields adenosine-5′-phosphosulfate (APS) that is reduced to sulfide (S2-) and incorporated into cysteine (Cys)53. At 4 d, ATP sulfurylase 1 (ATPSA1) expression was significantly downregulated with bS treatment in infected plants (Fig. 3b). No significant changes in nS and cS treatments occurred at any time points, indicating no S deprivation and the S uptake through this pathway was not significantly affected54. Conversely, at 8 d the expression of thiosulfate transferase (TST), which is a gene related to the elemental sulfur accumulation pathway, is significantly upregulated by both nanoscale treatments (Fig. 3a). Importantly, TST expression is unaffected by bS treatment. These findings indicate different pathways of sulfur accumulation and assimilation as a function of particle size. In plants, Str are found in the mitochondria and chloroplasts, as well as the cytoplasm and plastids. Niu et al.51 isolated a Str gene similar to AtStr1 from wheat that was resistant to the powdery mildew fungus Erysiphe graminis55. Similarly, Walz et al.56 isolated a rhodanese-like protein displaying similarity to AtStr17 from the phloem exudates of Curcubita maxima and reported its involvement in stress and defense response by acting as a phytohormone and/or in a signaling pathway.
3.2 Tissue Nutritional Content
The effect of S treatment on the uptake of select nutrients was also assessed (Fig. S14-16, S28-29, and Table S17). Stem accumulation of Cu, Fe and Zn in the diseased plants were greater than in the healthy plants (Fig. S14). Treatment with S did not further influence stem content of these nutrients, except for Zn content with cS. Although stem Mn content was not affected by disease, exposure to the nS and cS significantly increased Mn concentration in this tissue. All S amendments, particularly cS, further increased leaf Fe content, whereas Ca content was significantly reduced with nS and cS (Fig. S15). Conversely, exposure to S did impact Cu and Zn leaf content. The translocation of Mn and Si was not affected by disease. However, under S amendment, levels of these nutrients were increased, particularly with nS and cS. In roots (Fig. S16), cS significantly increased the Mn, Mg, and Fe at 4 d and 16 d, and increased Cu, and P at 16 d compared to the corresponding infested control, while no change was evident at 8 d. No effects were noted with nS or bS at 16 d. Less alteration was observed at 4 d or 8 d compared to 16 d. Although nutrient level changes were evident with treatment, consistent trends were not evident, suggesting that the dynamics of these processes is complex and the impact on overall plant health is difficult to ascertain. The amount of bioavailable nutrient elements in soil was evaluated to examine whether the changes in root element accumulation were affected the S based exposure (Fig. S17). In the DTPA extract, higher Zn content was found in cS treated soil than nS, bS, and the controls; cS resulted in higher soluble P content than bS in the same DTPA extract. However, such increments were not observed in the extracts by DI or CaCl2 solution. However, in general, the bioavailable nutrient element content in soil was only marginally affected by treatment.
4. Gene expression
Several genes related to the S assimilation pathway were evaluated as a function of treatment and time (Fig. S19-20). In secondary SO42--assimilation, APS is phosphorylated in an adenylyl-sulfate reductase (ASR)-catalyzed reaction to produce 3′-phosphoadenosine 5′-phosphosulfate (PAPS). PAPS is involved in the production of other S-containing methionine-derived (aliphatic) or tryptophan-derived (indolic) secondary metabolites such as glucosinolates (GSs). GSs (particularly indolic type) have been reported to protect plants against several stressors and are required for plant immunity. In diseased plants, nS at 8 d and cS at 4 d and 8 d increased ASR expression by 99.2%, 718.4%, and 823.2% over the diseased control (Fig. S19). In addition, ASR expression at 4 d and 8 d in cS treated plants was 5.8- and 1.8-fold greater than bS treated plants. Glutathione (GSH) controls the redox states of many biomolecules, and functions in mediating enzymatic activity, xenobiotic detoxification, and influences plant growth, development, and stress management in response to both abiotic and biotic factors. Gamma-glutamylcysteine synthetase (γGCS) catalyzes the first rate-limiting step in the production of GSH, involving the ATP-dependent condensation of cysteine and glutamate to form the dipeptide γGC. Here, disease increased γGCS expression over time; expression at 8 and 16 d was 54.9% and 213.3% greater than levels at 4 d. Treatment with nS and cS increased γGCS expression at 4 d by 58.2-77.4% and 104.9-129.7% compared with untreated control and bS; interestingly, expression decreased at 16 d by 45.9-39.3% and 47.1-40.6%. γGCS is known to be upregulated in plants under stress, including Fusarium infection, to form GSH and can be regarded as a biomarker of plant stress status57. From 4 d to 16 d, the stress induced by Fusarium increased γGCS expression in the untreated diseased controls, but interestingly, at 16d, nS and cS reduced this response relative to bS and the controls. Similar results were evident for glutathione-S-transferases (GST), which are phase II detoxification enzymes that catalyze the conjugation of GSH to endogenous and exogenous electrophilic compounds. The presence of disease resulted in a time-dependent increase in GST expression. At 8 d, cS significantly down-regulated GST expression relative to control and bS, indicating a partial alleviation of stress. These findings align with several transcriptome-wide investigations in the literature that have shown that the expression of specific GSTs is significantly increased during the early stages of pathogen infection.
Cysteine synthase (CS) produces cysteine, which is the primary product of sulfate assimilation, and also the reactant for methionine and glutathione synthesis. Cysteine is linked to the systemic acquired resistance pathway, which in turn activates a cascade of other resistance mechanisms to promote plant immunity from diseases58. Disease resulted in upregulation of CS at 8d by 150.1% compared with the healthy control (Fig. S19). In addition, CS expression at 8 d was increased by 91.9% relative to 4 d by but by 16 d, levels had decreased again. A similar trend was evident for the other treatments in diseased plants, indicating a time-dependent activation of CS with infection. nS and cS increased CS expression at most time points. At 16d, nS and cS induced CS expression levels that were 566.5% and 603.0% greater than bS. This particle size and time-dependent CS response is particularly interesting given the different pathways of S accumulation noted above.
The ethylene pathway in plants is important in a range of developmental processes and is involved in signaling and response to stress. As shown in Fig. S20, in the first step of ethylene biosynthesis, SAMS2 catalyzes the reaction of ATP and methionine to form S-adenosylmethionine (SAM). Although disease did not cause any changes in SAMS2, cS resulted in a significant increase in SAMS2 expression (108.1%) relative to untreated controls at 4d by but not at 8 or 16 d. ER69 is related to ethylene-responsive methionine synthase. At 8 d, nS upregulated ER69 expression relative to bS and the disease control by 182.0% and 161.1%, respectively; the increase in cS treatment compared with disease control at 8 d was 85.6%. In addition, the ethylene-responsive factor (ERF) TSRF1 gene was monitored. ERFs are important in regulating pathogen resistance, abiotic stress tolerance and plant development. TSRF1 has been shown to be upregulated by ethylene, salicylic acid, and bacterial infection in tomatoes. Here, infection resulted in significantly greater expression at 8 d than at 4 or 16 d. At 8 d and 16 d, the highest level of expression was present in the control and bS, acting as a signal to fungal induced stress and clearly indicating that nanoscale treatments had alleviated the need for response17,48. The time-dependent nature by which nanoscale treatments inhibit disease progress is notable and supports previous findings suggesting a relatively narrow window of physiological opportunity where these crop protection strategies can be successful.
WRKY6 codes for a plant resistance protein that provides specific immunity by recognizing F. oxysporum f. sp. lycopersici effectors59. This recognition results in effector-triggered immunity (ETI), a rapid plant defense response that inhibits successful infection. ETI occurs only between specific plant cultivars and pathogen strains based on the presence of corresponding R and Avr proteins. As shown in Fig. 3, at 8 d, nS and cS significantly upregulated WRKY6 expression in infected tomato plants compared to control and bS by 280-365% and 212-282%, respectively. Again, expression was significantly greater at 8 d than at 4 or 16 d (Fig. 3c).
A hierarchical clustering analysis was performed to differentiate the impacts of S-based materials on the expression of S assimilation and disease-related genes in leaves harvested at 4, 8, and 16 d. The metadata heatmap (Fig. S21a) shows the overall effect of exposure time and treatment on gene expression. A two-way ANOVA (Fig. S21b and Table S4) shows the effect of the two main factors, as well as their interaction effect, on the regulation of the 13 genes. The overall correlation heatmap and correlation analysis is shown in Fig. S22-23 and Table S5-7. CS was the gene with the greatest correlation with treatment; for exposure time, TST had the highest correlation. A correlation analysis performed with WRKL6 and γGCS revealed closely related expression to SRLK4 and ERF4, respectively.
To probe the effect of particle size and surface coating, the above analysis was performed within each S material group. The correlation heatmap (Fig. S24 and Table S8-10) displays different patterns for nS, cS, and bS. A Debiased Sparse Partial Correlation (DSPC) network analysis (Fig. 4 and Table S11-12) reveals the differential effect of the S materials on the relationship between the analyzed genes. The edges represent the association measures between the two ends. Interestingly, in both nS and cS treatments, TST expression was strongly correlated with WRKL6; however, with bS, this relationship was much weaker. This highlights the importance of elemental S uptake in nanoscale treatments yielding disease suppression. In addition, TST expression was correlated with TSRF1 and SAMS2 in both nS and cS treatments, but no such relationship was found for bS. These results indicate different mechanisms by which nS, cS, and bS effect plant metabolism and impact disease. A correlation analysis of the differentially affected relationship between all genes as a function of S type is shown in Fig. S25. The PLS-DA analysis (Fig. S26 and Table S13) shows a clear separation of nS and cS, revealing a significant impact of exposure time on gene expression in these two treatments; this time-dependent effect is not evident in bS treatment.
An interesting time-dependent effect was evident after further data analysis at different time points. The correlation heatmap (Fig. S27 and Table S14-16) shows different patterns at 4 d, 8 d, and 16 d. The score plot obtained from PLS-DA (Fig. 5) shows that the separation between each group (nS, cS, bS, and control) was much clearer at 8 d than at 4 d or 16 d. Importantly, according to the VIP score analysis (Fig. 5 and Table S18), the rank of TST among all the genes increased from the 9th at 4 d to the 1st at 8 d, and then decreased to 6th at 16 d. Similarly, the VIP score of WRKY6 increased from 8th at 4 d to 3rd at 8 d, and then declined to 13th at 16 d. These data suggest a time sensitive physiological window whereby nanoscale S treatments can significantly impact disease course17. A correlation analysis and the DSPC network of gene expression within each treatment was conducted at 4 d, 8 d, and 16 d, respectively (Fig. S30 and Table S19). Interestingly, the expression of TST gene starts to strongly correlate to WRKY6 at 8d, and that remains through 16 d. This correlation does not occur at 4 d. This result is consistent with the biomass data, which again support a nanoscale-specific mechanism of disease suppression through the enhanced elemental S uptake in nS and cS treatments.
A total of 229 metabolites in tomato leaves were identified and semi-quantified. The relationship between all the metabolites and the enrichment analysis is shown in Fig. S31 and S32. The score plot obtained from the multivariate partial least-squares-discriminant analysis (PLS-DA) provides a general overview of the clustering information between groups and highlights good separation of nS and cS treatments from the bS and control groups at 16 d along the second principal component (PC2); however, bS and control are not well separated with each other (Fig. S33 and Table S20). There was no noticeable separation between the control, nS, cS, and bS groups at 8 d. A clear separation was found within each S based treatment by 16 d. This time-dependent response is in line with the gene expression and S accumulation data discussed above.
The heatmap (Fig. S35) shows the abundance of representative metabolites that affected the metabolic pathways related to disease responses. This systematic positive modulation of metabolic processes in tomato leaves upon nS and cS treatment suggests a generally beneficial impact on tomato metabolism. Specific metabolic pathways in the leaves were compared between nS and bS and also between nS and cS. At 8 d, nS enhanced 4 metabolic pathways and downregulated 3 pathways relative to bS (Fig. S36). Uridine monophosphate (UMP), L-glutamine, and sphinganine were the metabolites that were present at increased concentration with nS. The enhanced sphingolipid metabolism and aminoacyl-tRNA biosynthesis pathway was found at both 8 d and 16 d but the downregulation of anthocyanin biosynthesis, starch/sucrose metabolism, and flavone/flavonol biosynthesis was only evident at 8 d. At 16d, nS resulted in significantly different metabolism of pyrimidine, glycerophospholipid, the folate-derived single carbon pool, phenylalanine, phenylpropanoid biosynthesis, sphingolipid, lysine degradation, and aminoacyl-tRNA biosynthesis as compared to bS (Fig. S37). Specifically, nS significantly increased the level of tetrahydrofolate, sphinganine, and L-pipecolate compared to bS by 110.3%, 56.9%, and 123.6%, respectively, which likely enhanced the metabolic pathways of single carbon pool compounds (folate), aminoacyl-tRNA biosynthesis, sphingolipid, and lysine degradation. Conversely, the phenylalanine pathway was downregulated by nS compared to bS (by 119.0%). Other important metabolites associated with the above affected pathways, such as choline phosphate and ferulate, were all greater with nS treatment than bS. Among the 8 affected pathways, 4 were enhanced and 1 was inhibited by nS as compared to bS.
The phenylpropanoid pathway results in the production of compounds responsive to pathogen infection and is a complex network regulated by multiple gene families that exhibit regulatory mechanisms that are involved in the production of anti-microbial compounds and signaling molecules. Manipulation of this pathway enhanced defensive systems, including salicylic acid and antimicrobial compounds60. Separately, sphingolipids are structural components of membranes and endomembrane systems and contribute to fluidity and other cellular functions, including defense against both abiotic and biotic stressors. The folate metabolic pathway influences a salicylic acid-independent interaction with plant immunity. Taken together, a nS-specific upregulation of these important metabolites is clearly indicative of enhanced plant defensive activity across a range of pathways and aligns well with the gene expression data.
In comparing pristine and coated nanoscale sulfur treated tomato leaves at 8 d, cS enhanced 3 metabolic pathways and downregulated 3 pathways relative to nS (Fig. 38). D-glucose 6-phosphate, D-fructose 6-phosphate, pyridoxal, and 12-oxophytodienoic acid were present at greater concentrations with cS; the decreased pathways included lower levels of UMP, L-glutamine, and L-proline with cS. Interestingly, the modulated regulation of these 6 pathways was observed only at 8d; these changes were not evident at 16 d, highlighting a time and surface coating-dependent effect on cellular metabolism. At 16 d, significant differences were found in 5 metabolic pathways (Fig. S39). Treatment with cS resulted in significant enhancement of isoquinoline alkaloid biosynthesis, tyrosine metabolism, and phenylalanine metabolism relative to nS; this was mainly due to the increased levels of tyramine and phenylacetaldehyde. Conversely, nS increased sphingolipid metabolism more than cS, as evident by the increased amount of sphinganine by 56.9%.
There were additional individual metabolites in tomato leaves that were uniquely modified by disease or treatment. nS increased proline, glutamine, and indole concentrations over control, cS, and bS by 1.6-2.0-fold, 2.7-2.9-fold, and 1.4-2.2-fold, respectively. Proline not only protects plants from various stresses but also aids in recovery. Proline also plays pivotal roles in cell wall signal transduction cascades, plant development and stress tolerance61. In primary nitrogen assimilation, inorganic nitrogen is converted into glutamine and glutamate. Glutamine is a key signaling molecule and under stress conditions, participates in wound response, pathogen resistance, response and adaptation to abiotic stress, and long distance signal transduction62. Indole enhances the induction of defensive volatiles in neighboring plants in a species-specific manner63. Furthermore, the release of indole is essential for the priming of mono- and homoterpenes in plant leaves under attack. nS also increased methionine sulfoxide content by 1.6-fold relative to bS and controls. As noted above, methionine controls the level of key metabolites such as ethylene, polyamines, and biotin through SAM activity. Both biotic and abiotic stresses cause changes in methionine cycle (MTC) enzymes and these changes contribute to the ability of stress management64.
cS increased p-aminobenzoic acid (pABA) (by 19-29%) and pipecolic acid (by 48-56%) over control, nS, and bS. pABA has been identified as an antifungal metabolite against a number of plant pathogens65 and it is known to trigger a systemic acquired resistance (SAR) against bacterial and viral pathogens66,67. Pipecolic acid, a non-proteinaceous product of lysine catabolism, is an important regulator of immunity in plants and accumulates upon infection, enhances resistance, and has been associated with systemic acquired resistance68,69 cS also increased delphinidin content by 36% relative to bS, which is an anthocyanidin and an antioxidant. Fatty acids are associated in the early interactions between plants and pathogens, triggering a form of immunity that may help resist infection and colonization by pathogens70. cS increased palmitic acid by 1.4, 2.0 and 2.1-fold over the control, nS, and bS, and increased palmitic amide, a primary fatty acid amide derived from palmitic acid that has been reported to alleviate Fusarium disease in watermelon and tomato, by 1.7-fold over bS 71,72. Although jasmonic acid and methyl jasmonate were unaffected treatment, a significant increase in the precursor 12-oxo-Phytodienoic acid (12-OPDA) was induced by cS (2.9-fold) compared to controls. In addition to the link with jasmonic acid activity, 12-OPDA plays an independent role in signaling and mediating resistance to pathogens and pests.73 Accumulation of high 12-OPDA levels correlated with reduced ROS and elevated GSH74. Interestingly, no such increase was found with nS and bS.
L-tryptophan (LT), L-5-hydroxytryptophan (5-HTP; metabolite of tryptophan), and canthaxanthin, an effective antioxidant biosynthesized from β-carotene, were significantly up-regulated by cS (2.0, 2.1, and 1.7-fold, respectively) and nS (1.3, 1.7, and 2.2-fold, respectively) relative to bS. Tryptophan is an amino acid with an indole ring, and anchors membrane proteins within the cell membrane. Tryptophan is also important in glycan-protein interactions, alleviation of ROS damage, and functions as a biochemical precursor for niacin (vitamin B3), melatonin and auxins. 3′,5′-cyclic adenosine monophosphate (cAMP), a cyclic nucleotide, was increased by nS (2.3-fold), cS (2.7-fold), and bS (1.5-fold). cAMP is recognized as an important signaling molecule involved in sensing and response to biotic and abiotic environmental stresses for plant innate immunity, and potentially as a regulator of Fe and Ca homeostasis75. Tyrosine, an aromatic amino acid, was increased by nS and cS by 1.44 and 1.29-fold compared to controls. Tyrosine serves as a precursor of numerous specialized metabolites that have diverse physiological roles as electron carriers, antioxidants, attractants, and defense compounds76. Also, tyrosine-derived metabolites, such as tocopherols (vitamin E), plastoquinone, cyanogenic glycosides and suberin, have crucial roles in plant fitness. nS and cS also increased p-coumaric acid, a phenolic acid, by 1.5 and 1.2-fold over bS and the controls. p-Coumaric acid exerts beneficial effects against several diseases due to its high free radical scavenging, pathogen suppression77, and antibacterial activities78.
6. Rhizosphere microbiome analysis
There were no significant differences in the number of amplicon sequence variants (ASVs) recovered between treatments, indicating minimal influence on the rhizosphere bacterial diversity, although the mean for the disease control was lower than the healthy or sulfur treated soils (Fig. S42). Similarly, the Shannon’s diversity index returned no significant differences between the treatments, suggesting that the diversity of the community was generally resilient to the treatments. The taxonomic composition of the bacterial communities was also characterized at the phylum level (Fig. S42). Generally, the composition of the community, including the phyla Proteobacteria, Actinobacteriota, and Bacteroidota, was similar across all treatments. A PCA was performed (Fig. S42) which shows that the healthy controls clustered independently and significantly from the other groups (Table S21), suggesting the fungal pathogen exerted some effect on the rhizosphere community, independent of the form of sulfur added. Finally, a biomarker analysis was performed to identify if any bacterial taxa were enriched. Among the sulfur treatments, the enrichment of several taxa, particularly the Gammaproteobacteria and Proteobacteria, distinguished the bS treatment (Fig. S42), indicating that there were some taxa that differed in their response to the form of the sulfur amendments. Thus, taken together, these data point to small but measurable shifts in the tomato rhizosphere bacterial communities in response to the sulfur treatments, with some slight differences between the different sulfur types employed.
In conclusion, this study demonstrates that soil amended nS and cS significantly suppressed Fusarium disease in tomato plants as compared to bS. The time-dependent gene expression and metabolomic profile highlight 8d as a critical period for NP-plant-pathogen interaction and disease suppression. Specifically, the expression of many defense and stress-related genes was significantly enhanced at 8d, whereas many of these alterations were reduced at 16d. Similarly, unique metabolomic profiles were evident 8d and 16d but not earlier. The potential mechanisms of nS and cS mediated disease suppression was investigated at the molecular level and was shown to be closely correlated with the enhanced expression of S bioassimilation and disease defense-related genes, increased disease resistance and plant immune system related metabolites, and more importantly, the unique S assimilation pathway in which elemental S was directly transferred into plant tissue. This unique accumulation pathway allowed more efficient utilization of S0 NPs by the plants and avoided the excessive formation of SO32- and S2- which could be harmful to plant cells. This finding is supported by the upregulation of gene expression related to the element S assimilation pathway in nS/cS treatments, the increased S content in plant tissues and the unique S translocation ratio in nS/cS treated samples relative to bS, as well as by two-photon microscopic images and videos. The findings demonstrate that soil application of nS and cS at an appropriate time offers great potential as a novel crop defense management strategy for disease suppression and significantly advances efforts to develop sustainable nanoscale treatments to achieve food security.