Nano-selenium enhances melon resistance to Podosphaera xanthii by enhancing the antioxidant capacity and promoting alterations in the polyamine, phenylpropanoid and hormone signaling pathways | Journal of Nanobiotechnology

October 17, 2023

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Characterization of the synthesized Nano-Se

The synthesis of Nano-Se in this study utilized ascorbic acid and chitosan to prevent the aggregation of nanoparticles and improve the bioavailability of Nano-Se [30]. The synthetic process was taken as complete after solution became red (Additional file 1: Fig. S1A). In order to study the average particle size and shape of the synthesized Nano-Se, SEM and TEM observations were conducted. Dynamic Light Scattering (DLS) analysis was also used to define the particle size distribution and polydispersity index (PDI) [31]. The surface characteristics and morphology of the synthesized Nano-Se were studied by SEM and TEM. Sodium selenite was dispersed by chitosan and ascorbic acid, and the surface of Nano-Se was uniform and clear. SEM (Additional file 1: Fig. S1B) and TEM (Additional file 1: Fig. S1G, J) images monodisperse spherical Nano-Se with a size between 50 and 200 nm. The morphology was further validated by AFM (Additional file 1: Fig. S1K). DLS technology defined the typical particle size distribution, which was estimated to be 72.4 nm (Additional file 1: Fig. S1M). The polydispersity index (PDI) calculated from DLS data, where PDI values < 0.05 are fit best to the monodisperse model, whereas PDI values > 0.7 are considered to indicate a polydispersity diffusion of particles [32]. In our study, the synthesized Nano-Se has a PDI value of 0.085, indicative of a medium dispersity. Energy dispersive X-ray (EDX) research is a method used for elemental analysis [33]. The basic structure and purity of the synthesized Nano-Se were determined by EDX, as shown in Additional file 1: Fig. S1D–J. The specific absorption peak of Nano-Se (1.74 keV) corresponds to Selenium, with no other element peak detected in the spectrum (Additional file 1: Fig. S1E). EDX mapping confirmed the composition of the Nano-Se, indicating the presence of C, O and Se (Additional file 1: Fig. S1H–J). The X-ray diffraction (XRD) spectra were consistent with the Se phase (Additional file 1: Fig. S1N). Additional file 1: Fig. S1O depicts the FTIR spectrum of the synthesized Nano-Se, which shows an expanded band width of 3266 cm⁻¹ (≡C–H stretching band) indicating the existence of the alkyne. The medium-strong spike evident at 1637 cm⁻¹ is caused by carbonyl C = O, confirming the presence of an amide group.

Effects of Nano-Se on the incidence of powdery mildew disease in melon leaves

The effects of the Nano-Se treatment on the incidence of powdery mildew in melon cultivars of differing resistances is shown in Fig. 1. At 4 dpi, the Nano-Se treatment significantly reduced the incidence of powdery mildew in JSG, ZZX and JX plants by 18, 20 and 28%, respectively. At 9 dpi, the incidence rate of powdery mildew in JSG, ZZX, HMC and JX plants were significantly reduced by 12, 8, 18 and 16%, respectively (Fig. 1A). Relative to their controls, the Nano-Se treatment significantly reduced the number of disease spots in JSG and HMC plants by 27 and 51%, respectively and at 9 dpi by 28 and 24% in JSG and ZZX plants, respectively (Fig. 1B). No significant effect of the Nano-Se treatment on the index of powdery mildew disease could be detected at 4 dpi, whereas at 9 dpi, the index was significantly reduced in all four cultivars by 21–45% (Fig. 1C). Nano-Se was reported to an effective and economic alternative to control fungal plant pathogens of faba bean [34].

Nano-Se treatments of soil or plants can enhance plant resistance to fungal pathogens [35,36,37]. However, how Nano-Se achieves this protective effect remains unclear. In this study, Nano-Se was shown to reduce the disease index in melon infected with a main agent of powdery mildew, P. xanthii by 21–45% (Fig. 1) and that this occurred with increases in active oxygen species scavenging, polyamine synthesis, phenylpropanoid metabolism and changes in plant hormone levels. The effects of the foliar application of Nano-Se on the melon stem weights in the four melon cultivars are shown in Additional file 2: Table S3. Compared with the control group, the Nano-Se treatment significantly increased the fresh weight of stems in JSG, ZZX and JX plants at 0 dpi by 62, 76 and 24%, respectively, and increased dry weight of stems in JSG, ZZX and HMC plants by 79, 23 and 93%, respectively.

Effect of the Nano-Se treatment on structural alterations in melon leaves infected with P. xanthii

The alterations in melon leaf anatomical organization after powdery infection are reduced in Nano-Se treated plants

Leaf structure is affected by and can adapt to changing environments [38, 39]. As shown in Fig. 2, the anatomical structure of melon leaves largely consists of the (top to bottom) adaxial epidermis (Ade), palisade tissue (Pt), spongy tissues (St) and the abaxial epidermis (Abe). The Ade and Abe are both cellular monolayers. The Ade cells were flat and nearly rectangular, whereas those of the Abe were small, irregular in shape and tightly packed. The tissue compactness after the Nano-Se treatment was higher than that of the control group (Additional file 2: Table S4), and a large amount of mycelium was attached to the adaxial epidermis of the control leaves of the susceptible cultivar at 10 dpi. The tissue sections of inoculated melon leaves (Fig. 2b1, d1, h1) clearly showed a large number of hyphae (Hp) attached to the upper surface of leaf epidermal cells at 10 dpi, while no invading hyphae were observed in the Pt or St. At 1 dpi, the tissue structure of melon leaves (Fig. 2a1–h1) showed an orderly arrangement to the Pt cells and a relatively compact St. However, at 10 dpi (Fig. 2a2–h2), the Pt and St were loosely arranged.

The anatomical characteristics of infected melon leaves at 1 dpi were shown in Additional file 2: Table S4. Leaf thickness, stomatal density and spongy tissue thickness significantly affected the resistance of loquat leaf spot disease [40]. The leaf thickness of all four cultivars were significantly increased in the Nano-Se treated plants by 11–34%. The thickness of the Ade of HMC and JX leaves was also significantly increased after Nano-Se treatment by 33 and 31%, respectively. The palisade tissue thickness of ZZX and JX Nano-Se treated leaves significantly increased by 17 and 21%, respectively. The anatomical characteristics at 10 dpi melon leaves are shown in Additional file 2: Table S4. Relative to their controls, the Nano-Se treatment resulted in significantly increased leaf thickness in JSG, HMC and JX cultivars by 12, 17 and 42%, respectively. The Ade thickness of JSG after Nano-Se treatment significantly increased by 41%. In the Nano-Se group, the thickness of the Abe and Pt layers in HMC and JX leaves showed significant increases of 53, 17, 37 and 73%, respectively. In ZZX, HMC and JX, the Nano-Se treatment significantly increased the Pt to St ratio by 63, 33 and 20%, and the tissue compactness by 50, 17 and 24%, respectively.

Effects of Nano-Se foliar treatment on fungal development and melon leaf cell ultrastructural changes after infection with P. xanthii

Scanning electron microscopy (SEM) observations of melon leaves at different stages of powdery mildew infection are shown in Additional file 1: Fig. S2. To highlight the ultrastructural effects of P. xanthii and the amelioration of such by Nano-Se, only the susceptible cultivars were studied. As the infection progresses the mycelial network becomes denser, with greater hyphal leaf penetration and the production of many conidiophores. When the mycelium invades into the leaves, it produced haustoria, which may have an impact on leaves. Compared with the control plants, the mycelial density on the leaf surface of Nano-Se treated plants was reduced. In the present research, Nano-Se was shown to help maintain the structure of the palisade and spongy tissue layers and to reduce the number of mycelia detected by SEM in these plants (Additional file 1: Fig. S2a2–f2).

The cell ultrastructural characteristics of melon leaf cells were studied with transmission electron microscope (TEM) at different stages of powdery mildew infection (Fig. 3). As the infection progressed, the palisade chloroplasts swelled, became more spherical and clumped together. With the increase in chloroplast deformation, the starch granules changed from long strips to oval or triangular, the osmiophilic granules in cells increased, and the grana lamellar structure changed significantly (panels b1, c1–2, f1). The cell wall serves as a primary defense against fungal invasion and is rapidly and locally modified in response to fungal infection, including the formation of encapsulating papillae [41]. In the early stage of fungal contact and cell penetration, plants cells form a papilla between the cell wall and the plasma membrane to impede fungal penetration. An example of this is given in panel b₂. However, no papillae were found in JSG leaves of the control group, which was consistent with the SEM results. The intrusion plugs of powdery mildew penetrate the papillae to form haustoria. The epidermal cells of JSG leaves treated by Nano-Se were filled by multiple haustoria at 11 dpi. The papilla structure was observed in Nano-Se treated plants by TEM (Fig. 3b2), which indicated that the Nano-Se pretreatment enhanced the resistance of melon plants.

Effects of Nano-Se on melon leaf chitinase (CHT) and β-1, 3-glucanase (GLU) after infection with P. xanthii

Pathogenesis-related (PR) proteins are important components of plant defense [42]. Members of the GLU and CHI PR families catalyze the degradation of fungal cell walls [43]. The influence of Nano-Se on melon leaf CHT and GLU at the levels of enzyme and transcriptional activity is shown in Fig. 4. Prior to infection (0 dpi), Nano-Se treatment resulted in significantly higher GLU activities in all four cultivars (10–36%). At 10 dpi, Nano-Se treatment also significantly increased GLU activity (7–15%) in four cultivars. Before infection (0 dpi), the levels of GLU mRNA were largely unaffected by Nano-Se treatment in all four cultivars. At 10 dpi, GLU mRNA levels in ZZX and HMC remained largely unaffected, while GLU mRNA levels in JSG and JX were significantly increased by 15–36%. GLU activity was shown to induce the systemic response in plants by releasing polymers from the fungal cell wall [44].

Chitin is one of the main components of fungal cell walls consisting of a homopolymer of N-acetyl-D-glucosamine linked through β (1–4) bonds and can induce the plant defensive response [45]. Nano-Se treatment significantly increased CHT activities at 0 dpi (12–95%) and at 10 dpi, (19–42%) in the four cultivars. At 10 dpi, Nano-Se significantly upregulated the expressions of CHT1 in JSG and CHT2 in ZZX by 70% (D) and 20% (E), respectively. Conversely, Nano-Se treatment group significantly increased CHT2 expression in JSG and ZZX at 0 dpi by 259 and 38%, respectively. In this study, Nano-Se was shown to increase the activities and mRNA levels of GLU and CHI (Fig. 4), indicating they could play a role in the Nano-Se mediated enhanced resistance to P. xanthii. Sugar signaling is closely related with cell proliferation, plant growth and development and interacts with many other signal pathways [46].

Effects of Nano-Se treatment on LOX and plant hormones in melon leaves infected with P. xanthii

The effects of Nano-Se treatment groups on LOX activity and mRNA levels of melon leaves infected with powdery mildew were not significant (Additional file 1: Fig. S3). The effects of Nano-Se on plant hormone levels in melon leaves inoculated with powdery mildew pathogens are shown in Additional file 1: Fig. S4. Infection was seen to decrease the IAA levels in control plants without the Nano-Se treatment. Conversely, Nano-Se treatment significantly increased the IAA content in the four cultivars at both 0 dpi (28–124%) and 10 dpi cultivars (43–172%). IAA treatment increased the resistance of kiwifruit to Botrytis cinerea, with the promotion of high activities of pathogen resistance-related defense enzymes [47].

The JA and SA signaling pathways have been reported to play key and integrated roles in the poplar response to fungal infection [48]. The leaf SA content was also increased in response to infection in the control group without the Nano-Se treatment. However, relative to the control levels, the SA levels in Nano-Se treated plants were significantly increased the SA content in the four cultivars at both 0 dpi (61–122%) and 10 dpi (24–73%). Chitosan enhanced the resistance of apple to Glomerella leaf spot through the SA signal pathways [49], which was consistent with this study (Additional file 1: Fig. S4). The synthesis and signal transduction of JA pathway were inhibited during the infection of Botrytis cinerea [50]. In contrast, although infection with P. xanthii induced higher JA levels, the Nano-Se treatment had no further significant effect. In this study, there was no significant difference in gene expression involved in JA synthesis, including LOX1, LOX2, LOX8, LOX9 and LOX10 (Additional file 1: Fig. S3). The upregulation of gene expression in plant hormone signal pathways, including those of SA and JA, has been reported to be involved in fruit defense against molds [51]. SA was shown to activate catechin and proanthocyanidins biosynthesis in poplar trees against rust infection [52].

Effects of Nano-Se on antioxidant capacity of melon leaves infected with P. xanthii

The effects of Nano-Se on the leaf antioxidant enzyme activities before (0 dpi) and after (10 dpi) P. xanthii infection are shown in Fig. 5. Nano-Se treatment resulted in a significant increase in SOD activity in all four cultivars. Both prior to (41–215%) and following infection (31–120%; A). Before infection (0 dpi), SOD mRNA levels were largely unaffected by Nano-Se in ZZX, HMC, and JX, but significantly increased in JSG by 44%. In contrast, at 10 dpi, Nano-Se treatment significantly increased SOD mRNA levels in ZZX, HMC and JX (37–72%; B). Quiterio-Gutierrez et al. [53] showed that the combined application of Nano-Se and Nano-Cu reduced the incidence of Alternaria solani in tomato plants, with the induction of higher leaf activities of SOD, APX, glutathione peroxidase (GPX) and PAL. Nano-Se treatment increased CAT activity both before (38–126%) and after infection (42–65%; C) in the four cultivars. Before infection (0 dpi), the CAT mRNA levels were largely unaffected by Nano-Se in ZZX, HMC and JX, but significantly increased by 30% in JSG. At 10 dpi, the Nano-Se treatment resulted in significant increases (58–96%) in CAT mRNA levels in JSG and ZZX, but not in HMC or JX (D). Nano-Se treatment increased APX activity (45–279%) in the four cultivars both before (45–279%) and after infection (24–94%; E).

Before infection (0 dpi), Nano-Se treatment resulted in increased APX mRNA levels were increased by Nano-Se before infection in JSG and ZZX (8–23%). In contrast, at 10 dpi, Nano-Se treatment significantly increased APX mRNA levels only in HMC (32%; F). POD activity was increased by Nano-Se in all four cultivars prior to (19–41%) and following infection (21–58%; G). Increases in enzymes involved in ROS scavenging, including POD, has been linked to the improved disease resistance of loquat fruit to C. gloeosporioides [54]. However, before infection (0 dpi), Nano-Se treatment resulted in increased POD mRNA levels were only increased prior to infection in HMC and JX (34–43%) and after infection in JSG, HMC and JX (28–51%; H). Following infection, the antioxidant capacity of disease-resistant cultivars was stronger than in disease-susceptible cultivars in the control group without Nano-Se application. The antioxidant capacity of melon seedlings treated with Nano-Se increased both before and after infection with P. xanthii powdery mildew, but to different degrees.

The immune response of plants to pathogens can involve callose deposition, the accumulation of ROS and cell death [55]. The effect of Nano-Se on ROS accumulation in leaves at different ages of powdery mildew infection are shown in Fig. 6. Tissue observations of H₂O₂ and O₂⁻ at 2, 4, 8 and 10 dpi (Fig. 6A) showed that both ROS were induced to higher levels after infection, indicating a higher level of oxidative stress, but that increases in O₂⁻ were detectable after 2 dpi, whereas increases in H₂O₂ were seen only after 8 dpi. Reactive oxygen species (ROS) rapidly accumulate in plants infected by microbial pathogens, and are important components of the plant defense response [56]. Notably, the accumulation of both ROS was reduced by the Nano-Se treatment in all cultivars. To further examine the effect of Nano-Se on the antioxidant capacity, radical scavenging and lipid peroxidation activities in melon leaves during powdery mildew infection, the levels of H₂O₂, MDA, GSH, proline and free radical scavenging/antioxidant activity (using DPPH) were determined (Fig. 6B–F). Fungal ROS metabolism is central to the infection process, and crucial in plant colonization [57]. Nano-Se was seen to increase leaf activities of SOD, CAT, APX and POD as well as their gene expression (Fig. 5), which occurred with a reduction in H₂O₂, O₂⁻ and MDA and an increase in GSH, proline and radical scavenging capacity (Fig. 6).

The regulation of ROS levels in pathogen-challenged plants plays a decisive role between susceptible and resistant interactions [58]. In confirmation of histological observation (Fig. 6A), Nano-Se treatment significantly reduced H₂O₂ accumulation in leaves at 0 and 10 dpi in all cultivars by 12–26% and 9–25%, respectively (B). Bai et al. [59] showed that H₂O₂ accumulation increased significantly with the decrease of total antioxidant capacity. Nano-Se significantly increased the levels of GSH at both 0 and 10 dpi in the all four cultivars by 18–63% and 19–34%, respectively (D). Nano-Se also promoted a significant increase in proline levels at both 0 and 10 dpi in all cultivars by 39–46% and 16–61%, respectively (E). Based on the DPPH assay, Nano-Se significantly increased the antioxidant activity at both 0 and 10 dpi in all cultivars by 3–10% and 5–27%, respectively (F). Nano-Se reduced the leaf MDA content by 21–36% at 0 dpi, and by 30 and 48% at 10 dpi in cultivars HMC and JX, respectively (C). Wang et al. [60] reported that Na₂SeO₃ reduced H₂O₂ and MDA levels in alfalfa and Cunha et al. [61] demonstrated that Na₂SeO₄ enhanced enzymatic and non-enzymatic antioxidant metabolism in peanut plants.

Effects of Nano-Se on the primary metabolism of P. xanthii infected melon leaves

Sugar content

Higher sugar contents have been reported to induce higher pathogen resistance in plants by promoting cell wall lignification, flavonoids synthesis and the levels of PR proteins [62]. Prior to the onset of infection (0 dpi), Nano-Se treated plants of all cultivars showed significant increases in leaf soluble sugar, reducing sugar and sucrose contents by 16–36%, and 17–40% and 22–65%, respectively (Additional file 1: Fig. S5A, B, C). The increased levels of sucrose were accompanied by increases in both sucrose phosphate synthase (52–206%) and sucrose synthase (24–91%) activities (D, E). At 10 dpi, the contents of soluble sugar, reducing sugar, sucrose and sucrose synthase activity were all decreased, but not significantly different between the control treatment without Nano-Se application and the Nano-Se treatment. In our study, at the early stage of powdery mildew infection, Nano-Se was seen to promote increases in soluble and reducing sugars, sucrose and related enzyme activities (Additional file 1: Fig. S5).

Effects of Nano-Se on amino acid metabolism in melon leaves infected with P. xanthii

Amino acid homeostasis is interconnected with plant immune signaling pathways [63]. Relative to their controls, Nano-Se treatment had no discernable effect on total free amino acid contents of the four cultivars either prior to or following infection with powdery mildew (Additional file 1: Fig. S6A). However, Nano-Se treated plants of all cultivars showed a significantly higher content of glutamate of at 0 dpi (52–107%; B) and those of ZZX, HMC and JX also showed significant increases of 44–56% at 10 dpi. Nano-Se also promoted increases in glutamine synthase activities in all cultivars prior to infection (16–24%) and in JSG and JX at 10 dpi by 32% and 17%, respectively (C). Significant increases in leaf GABA content were observed in all cultivars tested prior to infection (21–88%) and at 10 dpi (50–199%) (D). The application of GABA was reported to increase plant tolerance to stress by regulating the expression of genes involved in plant signal transduction, hormone, ROS and polyamine metabolism [64]. Hydroxyproline levels were also significantly increased by Nano-Se in all four cultivars by 17–86% at 0 dpi and by 8–37% at 10 dpi (E). In the present study, the observed increase in amino acid content of melon leaf after Nano-Se treatment occurred with increases in the levels of glutamate, GABA and GS activity (Additional file 1: Fig. S6).

Effects of Nano-Se on secondary metabolism

Polyamine metabolism

A possible role for free polyamines and the regulation of polyamine catabolism in plant resistance has been proposed [65]. The PAO activity in all cultivars was enhanced by the Nano-Se treatment at 0 and 10 dpi by 48–182% and 26–92%, respectively (Fig. 7A). These Nano-Se induced changes were accompanied by relative increases in PAO expression in JSG (150%) and HMC (34%) prior to inoculation and in all infected cultivars at 10 dpi by 12–86% (B). At 0 dpi, the Nano-Se treatment significantly increased SPD contents in JSG, ZZX and HMC by 78, 24 and 32%, respectively. However, at 10 dpi, the changes in SPD content of four cultivars were not significant (C). The Nano-Se treatment significantly increased SPD synthase mRNA levels in JSG, ZZX at 0 dpi by 36 and 27%, respectively (D).

Compared with the control group, Nano-Se treated plants showed significantly increased SPM levels in the four cultivars at 0 and 10 dpi by 30–139% and 36–118%, respectively (E). At 0 dpi, the Nano-Se treatment significantly increased SPM synthase mRNA levels in JSG and JX by 122 and 58%, respectively and at 10 dpi, by 200, 78 and 37% in the JSG, ZZX and HMC cultivars, respectively (F). The Nano-Se treatment increased PUT in the four cultivars at 0 and 10 dpi by 56–165% and 43–112%, respectively (G). Polyamines were also suggested to play an important role in environmental stress responses [66] and the metabolism of mainly putrescine, spermine and spermidine can affect the ability of fungi to cope with environmental challenges [67].

S-adenosylmethionine decarboxylase mRNA was increased in all four cultivars at 0 dpi by 57–115%, whereas at 10 dpi, only JSG showed an increase of 92% by Nano-Se (H). Except for ZZX, Nano-Se treatment increased arginine decarboxylase expression in three cultivars at 0 and 10 dpi by 34–140% and 34–76%, respectively (I). Conversely, no significant effects of Nano-Se treatment on the expression of ornithine decarboxylase and N-carbamyl putrescine hydrolase were observed (J, K). Conversely, the Nano-Se pretreatment potentiated PAO activity, the levels of SPD, SPM and PUT, as well as the mRNA levels of PAO, SPMS, S-adenosylmethionine decarboxylase and arginine decarboxylase (Fig. 7).

Effects of Nano-Se on melon leaf lignin metabolism

Additional file 1: Fig. S7 shows that Nano-Se promoted increases in lignin content in all cultivars at both 0 and 10 dpi (17–49%; A). The accumulation of phenylpropanoid-derived secondary metabolites, such as phenolic acids, flavonoids, and lignin have been associated with citric plant defense against fungi [68]. In our study, the improvement of melon resistance to powdery mildew disease by Nano-Se was also shown to enhance lignin synthesis (Additional file 1: Fig. S7). In agreement, the accumulation of lignin was shown to be linked to the improvement of antifungal activity and the defensive response in citrus fruits [68]. Conversely, Nano-Se had no significant effect on leaf CAD activity or CAD and CCR mRNA levels in the four cultivars at 0 or 10 dpi (B, C, D).

Alterations in phenylpropanoid metabolism in response to P. xanthii infection and Nano-Se treatment

The influence of Nano-Se treatment groups on phenylpropanoid metabolism in melon leaves at different of P. xanthii infection is depicted in Fig. 8. The Nano-Se treatment was seen to significantly increase the total phenols in ZZX and JX cultivars at 0 dpi by 12–22%, respectively, whereas at 10 dpi, Nano-Se treatment groups significantly increased the total phenol content in all four cultivars by 17–27%, respectively (A). The flavonoid contents in all Nano-Se plants were significantly increased at 0 and 10 dpi by 13–27% and 5–15%, respectively (B). The Nano-Se treatment increased PAL activity in the four cultivars by 40–80% at 0 dpi and by 22–38% at 10 dpi (C). This was accompanied by significant increases in PAL mRNA levels of in all four cultivars at 0 and 10 dpi by 37–76% and 18–64%, respectively (D). The flavonoids and phenolic acids were shown linked to the improvement of antifungal activity and the defensive response in citrus fruits [68]. Increases in PAL activity, flavonoid and total phenol contents contribute to plant disease resistance [69].

Nano-Se treatment also increased C4H activity in the four cultivars at 0 and 10 dpi by 61–140% and 24–126%, respectively (E), which were similarly accompanied by respective increases at the mRNA level of 40–141% and 12–80% (F). Before infection (0 dpi), Nano-Se treatment increased the 4CL activity (50–84%) in four cultivars. At 10 dpi, Nano-Se treatment significantly increased the 4CL activity of the four cultivars by 19–64% (G). Nano-Se treatment groups significantly increased 4CL mRNA levels of all four cultivars at 0, 10 dpi by 18–67% and 14–65%, respectively (H). The Nano-Se treatment also increased the CHS mRNA levels in the four cultivars by 69–135% at 0 dpi and by 52–150% at 10 dpi (I). The Nano-Se treatment had no significant effect on the mRNA levels of FLS or LDOX in any cultivar (J, K). In our study, the improvement of melon resistance to powdery mildew disease by Nano-Se was shown to enhance phenylpropanoid metabolism (Fig. 8).

RNA-Seq analysis of Nano-Se effects on melon leaf gene expression in uninfected and P. xanthii infected seedlings

A PCA analysis of leaf RNA-Seq data was conducted with the samples of the susceptible and resistant cultivars at 0 and 10 dpi, with and without Nano-Se pretreatment. The PCA readily discriminated the 0 dpi and 10 dpi treatments, with PC1, PC2 and PC3 explaining 21.9%, 15.2% and 10.6% of the total variance, respectively. The melon leaf samples at 0 dpi and 10 dpi, as well as the control and Nano-Se treated plants were clearly distinguished (Fig. 9A). Based on the correlation heat map analysis of FPKM values, it was found that there was a good correlation between the three biological replicates (Fig. 9B). A total of 1,110,261,430 clean reads and 166.56 G clean bases were obtained in all samples (Additional file 2: Table S5). As shown in the visualization of the heat map, obvious hierarchical clustering of different types of samples could be seen (Fig. 9C). Prior to infection (0 dpi), the numbers of DEGs detected in response to the Nano-Se treatment in the susceptible (S; JSG) and resistant (R; HMC) cultivars were 539 (338 up- and 201 down-regulated, Additional file 1: Fig S8A) and 1621 (902 up- and 719 down-regulated, Additional file 1: Fig S8B), respectively. In response to P. xanthii infection (10 dpi), a larger number of DEGs were detected in seedlings untreated with Nano-Se, where 2285 (1191 up- and 1094 down-regulated) were detected in the susceptible cultivar JSG (Additional file 1: Fig. S8C), and 2629 (1392 up- and 1237 down-regulated) were detected in the resistant cultivar, HMC (Additional file 1: Fig. S8D). Conversely, much fewer DEGs were detected in infected seedlings that had been subjected to the Nano-Se pretreatment, where 188 (102 up- and 86 down-regulated), and 23 (10 up- and 13 down-regulated) were detected in the susceptible and resistant cultivars, respectively. These results are summarized in a bar chart in Fig. 9D.

The functions of the DEGs identified were annotated from matched identifiers from 7 databases (KEGG, KOG, NR, Pfam, Swissprot, Tremble, and GO) (Additional file 2: Table S6). The biological functions of DEGs were subjected to GO enrichment analysis. In the comparison of control and Nano-Se treatment groups at 0 dpi, the main enriched terms indicated alterations in catalytic activity, DNA polymerase activity, monooxygenase activity, nucleotidyl transferase activity, RNA-directed DNA polymerase activity, transferase activity, chloroplast thylakoid, plastid thylakoid, coenzyme and tetrapyrrole binding. Between the conditions of 0 and 10 dpi, both R and S cultivars mainly showed enrichment in the terms chloroplast/thylakoid, photosynthetic membrane, plastid thylakoid, and thylakoid part (Additional file 1: Fig S9). The main differentially enriched terms between the control and Nano-Se treatment group at 10 dpi were bacterium and drug, secondary metabolic and biosynthetic process and tetrapyrrole binding (Additional file 1: Fig S10).

KEGG enrichment analysis was also performed. The DEGs obtained from the comparison groups of 0 S ck vs. 0 S Nano-Se, 0 R ck vs. 0 R Nano-Se, 0 S ck vs. 10 S ck, 0 R ck vs. 10 R ck, 10 S ck vs. 10 S Nano-Se, and 10 R ck vs. 10 R Nano-Se were annotated on 78, 115, 123, 125, 64, and 126 KEGG pathways, respectively (Additional file 2: Tables S7–S12), of which the 20 most significantly enriched pathways in each comparison are shown in Fig. 10. In particular, the biosynthesis of secondary metabolites (ko01110) was significantly enriched in four comparisons (0 S ck vs. 0 S Nano-Se, 0 R ck vs. 0 R Nano-Se, 0 S ck vs. 10 S ck, and 0 R ck vs. 10 R ck). The flavonoid biosynthesis (ko00941; 0 R ck vs. 0 R Nano-Se), isoflavonoid biosynthesis (ko00943; 0 S ck vs. 10 S ck) and flavone and flavonol biosynthesis (ko00944; 0 R ck vs. 10 R ck) were significantly enriched in two cultivars. KEGG pathway analysis of tea plants treated with Na₂SeO₃ and Na₂SeO₄ revealed that many genes involved in amino acid and glutathione metabolism were up-regulated, and genes and proteins associated with glutathione metabolism and biosynthesis of ubiquinone and terpenoid were highly expressed [70]. The DEGs associated with the biosynthesis pathway of catechins, caffeine and theanine in tea were affected by Nano-Se [71].

Therefore, the biosynthesis of secondary metabolites, including flavonoids and isoflavonoids might play an important role in the enhanced resistance to powdery mildew infection displayed by Nano-Se treated plants. In addition, flavone and flavonol biosynthesis (ko00944) were also enriched in the process of Nano-Se inducing resistance to P. xanthii infection. After Na₂SeO₃ treatment, genes related to phenylpropanoid, flavonoid and anthocyanin biosynthesis were significantly up-regulated, resulting in the accumulation of anthocyanin metabolites in grain wheat [72], which is similar to the results of this study. The beneficial effects of Nano-Se against melon powdery mildew therefore appears to be achieved through its effects on multiple pathways. A list of the 18 most affected DEGs, including genes for two antioxidant enzyme genes, nine phenylpropanoid pathway genes, two flavonoid synthesis pathway genes and five glycosyl transferase genes, is provided in Additional file 2: Table S13 as genes worthy of further investigation. The effects of Nano-Se appear to be most evident prior to mildew infection, indicating that Nano-Se mostly affects melon basal resistance, rather than reinforcing the plant response to infection.

The concentration of Nano-Se in this study was 5.0 mg·L⁻¹. We verified that the prepared Nano-Se were not toxic to plants. Melon seedlings at different stages of treatment with Nano-Se are shown in Additional file 1: Fig S11. Low doses of selenium improved plant stress resistance through photosynthesis, while high doses of selenium interfered with nitrogen assimilation, resulting in decreased nitrogen compound synthesis ability [73]. In this study, the levels of glutamate, GS and GABA associated with the nitrogen cycle increased after Nano-Se treatment, which further indicated that 5 mg·L⁻¹ Nano-Se is not toxic to melon. Low concentrations (20 μM) of selenates were also shown to be beneficial for plant growth, which was related to the antioxidant effect of selenium [74].