3.2.1. Relative Water Content (RWC)

In the context of RWC, all the primed plants showed a significant increase in RWC, compared to the non-primed plants; however, the greatest increases in RWC—of 30.74%, 28.09%, 17.08%, and 14.98%, for hydropriming, osmotic priming, halopriming, and hormonal priming, respectively—were observed in comparison to the control. A similar trend of increased RWC was also observed in systemic inoculation of *A. niger* in wheat plants, wherein a significantly greater increase in RWC was observed in all priming treatments than in the control (Figure 2A): the halopriming showed the highest RWC (55.85%), while

55.00%, 52.14%, and 43.52% increases were noted for osmotic priming, hydropriming, and hormonal priming, respectively.

#### 3.2.2. Shoot Length

The application of different priming techniques stimulated shoot growth. An increase in shoot length was significant in plants subjected to all priming treatments, except halopriming, as compared to the control plants (Figure 3A). In principle, the osmotic priming exerted the highest shoot length (45%) compared to the control, while the hormonal priming exhibited a 43.41% increase, the hydropriming a 41.13% increase, and the halo priming a 34.23% increase in shoot length (Figure 2B). Similarly, the shoot length was significantly increased in all primed plants, in comparison to non-primed plants, after systemic inoculation of *A. niger*, where the maximum increases in shoot length—i.e., 38.24%, 35.98%, 30.46, and 19.23%—were recorded for osmotic priming, halopriming, hydropriming, and hormonal priming, respectively.

**Figure 2.** Physiological parameters of wheat under hydropriming, osmotic priming, halopriming, and hormonal priming: (**A**) relative water content (RWC); (**B**) shoot length; (**C**) root length; (**D**) fresh root/shoot ratio; (**E**) dry root/shoot ratio. The mean values with different letter(s) indicate significant differences at *p* ≤ 0.05. Vertical bars represent standard deviation of means (*n* = 3). Sys. inoculation: Systemic inoculation.

#### 3.2.3. Root Length

All the priming treatments exhibited a pattern of increase in root length similar to that of shoot length. The highest increases—of 51.43% and 48.48%, respectively—were observed in the root length of plants subjected to osmotic priming and hormonal priming, followed by hydropriming and halopriming, with increases of 41.38% and 31.08%, respectively. Similarly, all primed plants revealed a significant increase in root length, in comparison to the control, after systemic inoculation, where the maximum root length was recorded for osmotic-primed (50.00%) and hormonal-primed plants (42.16%) (Figure 2C).

**Figure 3.** Effects of different priming methods on the growth of wheat plants. (**A**) (**I**): control; (**II**): hydropriming; (**III**): osmotic priming; (**IV**): halopriming, (**V**): hormonal priming. (**B**) Disease severity after foliar inoculation. (**I**): control vs. hydropriming; (**II**): control vs. osmotic priming; (**III**): control vs. halopriming; (**IV**): control vs. hormonal priming. (**C**) Disease severity after systemic inoculation. (**I**): control vs. hydropriming; (**II**): control vs. osmotic priming; (**III**): control vs. halopriming; (**IV**): control vs. hormonal priming.

#### 3.2.4. Fresh and Dry Root/Shoot Ratio

The fresh plant root/shoot ratio was significantly increased in all primed plants, while a non-significant increase was observed in hormonal priming, as compared to the control. With respect to the fresh root/shoot ratio, hydropriming presented the highest increase—of 58.33%—while 51.61%, 51.14%, and 40.00% increases were recorded for halo-, osmotic- , and hormonal-primed plants. The same tendency of increase in the fresh root/shoot ratio was observed after systemic inoculation, where 70.83-enhanced, 66.67%-enhanced, 61.11%-enhanced, and 53.33%-enhanced fresh root/shoot ratios were observed for osmotic priming, halopriming, hydropriming, and hormonal priming (Figure 2D).

In addition, the results exhibited a similar trend of increase in dry root/shoot ratio in all the primed plants: however, this increase was more significant in the hydro-primed plants, whose dry root/shoot ratio increased by 78.26%, while the dry root/shoot ratio of the osmotic-, halo-, and hormonal-primed plants showed 73.68%, 64.29%, and 64.29% increases, respectively, compared to the control plants. Furthermore, in the case of systemic inoculation of *A. niger*, hydropriming and osmotic priming showed the highest increase in dry root/shoot ratio (74.19 and 72.41%, respectively), followed by halopriming and hormonal priming, with enhanced dry root/shoot ratios of 57.89% and 60.00%, respectively, as compared to the control (Figure 2E).

#### *3.3. Disease Severity Analysis*

#### 3.3.1. Foliar Inoculation

Our results revealed that the foliar inoculation of *A. niger* induced a drastic disease severity in non-primed (control) plants; however, it was observed that the priming treatments significantly reduced disease severity, by alleviating the stress caused by *A. niger* inoculation. Among the priming treatments, hydropriming and osmotic priming showed the maximum decreases in disease severity, of 70.59% and 64.71%, respectively. Halo- and hormonal-primed plants also showed pronounced reductions in disease severity, of 58.82% and 47.06%, respectively, in comparison to the control plants. In general, hydropriming and osmotic priming were observed to be more effective in reducing disease severity, in comparison to halopriming and hormonal priming (Figures 3B and 4A).

**Figure 4.** Disease severity analysis of wheat plants in response to hydropriming, osmotic priming, halopriming, and hormonal priming. (**A**) Disease severity analysis after foliar inoculation of *A. niger*. (**B**) Disease severity analysis after systemic inoculation of *A. niger*. (**C**) Disease severity comparison between foliar and systemic inoculation. The mean values with different letter(s) indicate significant differences at *p* ≤ 0.05. Vertical bars represent standard deviation of means (n = 3).

#### 3.3.2. Systemic Inoculation

Similarly, the non-primed (control) wheat plants subjected to systemic inoculation showed acute disease severity, with drastically reduced growth. In the case of the primed plants, however, the hydro- and osmotic-primed plants were found to be the most resistant, significantly reducing disease severity by 75.00% and 88.33%, respectively, as compared to the control, while halopriming and hormonal priming showed comparatively less resistance than osmotic priming and hydropriming (Figures 3C and 4B). However, both halopriming and hormonal priming also induced considerable reduction in disease severity, i.e., 58.33% and 41.67%, respectively, compared to non-primed plants.

#### 3.3.3. Comparison of Foliar and Systemic Inoculation

In this study, we obtained promising results with respect to disease severity reduction for the systemic inoculation method, in comparison to the foliar spray method. Both methods were applied for the same length of time, i.e., 2 weeks, and disease symptoms appeared more rapidly in the foliar spray method than in the systemic method. The results revealed that the plants treated with systemic fungus inoculation exhibited more resistance to disease in comparison to the foliar spray technique. In particular, osmotic priming and halopriming in systemic inoculation presented significant differences in reducing disease severity—by 76.67% and 40.00%, respectively—compared to foliar-sprayed plants of the same group. In addition, halopriming and hormonal priming also revealed a considerable decrease in disease severity reduction—of 28.57% and 22.22%, respectively when compared to foliar-sprayed plants of the same treatment (Figure 4C).

#### 3.3.4. Visual Assessment of Wilting

Visual assessment of wilting also revealed the same pattern as described above for the disease severity percentage. After foliar inoculation of *A. niger*, the control plants were found to be nearly dead, while the hydro- and osmotic-primed plants were normal, but slightly wilted. The halo-primed plants showed wilting (W), while the hormonalprimed plants were wilted severely (Figures 3B and 5). Likewise, the same pattern of visual assessment of wilting was observed with systemic inoculation, where the control plants were found to be severely wilted, while the hydro- and hormonal-primed plants were wilted slightly; however, the osmotic- and halo-primed plants seemed to be normal (Figures 3C and 5).

**Figure 5.** Measurement of disease severity after foliar and systemic inoculation, by visual assessment of wilting. Different wilting conditions are described as normal (N), slightly wilted (SlW), wilted (W), severely wilted (SeW), nearly dead (ND), and dead (D).

#### *3.4. Expression Profiling of TLP, Chitinase, and β-1,3-glucanase Genes*

TLP gene expression was down-regulated in halo-primed plants compared to the control, while osmotic- and hydro-primed plants showed significantly higher expression of *TLP*. In halo-primed plants, almost no detectable expression of the *TLP* gene was seen. The expression profile of *TLP* in RT-PCR and qRT-PCR was comparable (Figures 6 and 7). Both RT-PCR and qPCR showed that *chitinase* gene expression was significantly increased in hydropriming compared to the plants treated with osmotic priming, halo priming, and hormonal priming (Figures 6 and 7). RT-PCR and qPCR results also confirmed that *β-1,3-glucanase* was highly expressed in hydro- and osmotic-primed plants compared to non-primed plants, while halo- and hormonal-primed plants also showed a considerably increased expression of *β-1,3-glucanase*; however, the change was not as significant as compared to the control (Figures 6 and 7). Overall, the analysis of the relative gene expression indicated that *β-1,3-glucanase* presented a significant role in inducing resistance to *A. niger* under each priming treatment, followed by *chitinase* and *TLP*, which played a considerable role in resistance to *A. niger* under halopriming and hormonal priming, and under hydropriming and osmotic priming, respectively.

**Figure 6.** Expression profiling of *TLP, Chitinase, and β-1, 3-glucanase* by RT-PCR.

**Figure 7.** Relative expression of *TLP, Chitinase*, and *β-1,3-glucanase*, obtained through quantitative real-time PCR analysis. The mean values with different letter(s) indicate significant differences at *p* ≤ 0.05. Vertical bars represent standard deviation of means (*n* = 3).

#### **4. Discussion**

Seed priming has been extensively used for the improvement of seed quality yield, and to lower seedling protrusion time. Different priming techniques are being used in this regard, all of which have their own advantages [56]. This study was conducted to evaluate the potential of different priming techniques—i.e., hydropriming, osmotic priming,

halopriming, and hormonal priming—to not only contribute to gain in seed growth and health, but also confer resistance against a pathogenic fungus, *A. niger*. To evaluate disease severity and resistance, we conducted disease severity analysis, as described above, and measured the expression level of the genes—namely *chitinase, TLP*, and *β-1,3-glucanase* which mainly contribute to the host resistance to pathogens.

We evaluated biochemical and physiological parameters after treatment with different priming techniques. In the present study, higher proline content was observed in all priming treatments, but this effect was more pronounced in hydropriming and osmotic priming, which enhanced proline content by 70.09% and 71.56% more than non-primed plants (control) (Figure 1A). It has been shown that under various stress conditions—e.g., high salinity, drought, and biotic stress—proline accumulates in high concentration [57–59]. Previous studies on coriander (*Coriandrum sativum*) [60] and sorghum [61] have also described the increased synthesis of proline due to priming. In the case of systemic inoculation, the hydro-, osmotic-, and halo-primed plants showed, by increased proline content, better disease resistance to fungus inoculation (47.30%, 51.26%, and 49.50%, respectively) (Figure 4). Similarly, a significant increase in proline was noted in *Brassica napus* during osmotic priming [62]. Manghwar et al. [28] also observed enhanced proline content in wheat under *Fusarium equiseti* stress. Proline is a compatible solute, usually accumulated under stress in plants, and acts in osmotic adjustment [57,63]. The results of the present study showed significantly increased protein content with all priming treatments compared to the control. Comparatively, all the primed plants inoculated with *A. niger* resulted in higher protein production than non-inoculated primed plants. The findings of [64] also showed the positive effect of priming on the protein contents of the common bean: fungal inoculation led to an overall increase in protein content and a decrease in sugar contents, which is a sign of the stimulation of osmotic material synthesis under stress conditions [65].

Moreover, an increase in sugar content after priming may be because leaves synthesize more soluble sugars after seed priming. The same beneficial effect was found in safflower (*Carthamus tinctorius*) [66], wheat [67], pepper (*Capsicum annuum* L. var Chargui) [68], and barley (*Hordeum vulgare* L.) [69]: this increase may be due to increased α-amylase activity [70]. Sugar content in our study was slightly decreased in response to fungal stress in all the pre-treated plants. Other studies have also confirmed the decrease in sugar content of primed plants after stress conditions [71]. Generally, some pathogenic infections bring changes to the photosynthetic rate and respiratory pathway, and cause fluctuation in sugar content [72–74]. The priming treatments in our study also led to increased chlorophyll content (Figure 1D). A significant increase in chlorophyll contents has been observed after osmotic priming and hydropriming. The study reported 43% and 100% increases in chlorophyll a and b contents, respectively, after priming [75]. Another study, of water, auxin, and gibberellins priming, has been reported to uplift chlorophyll content in soybean [76]. Related results after different priming methods have been observed in rice [77] and coriander [78]. An increase in the chlorophyll content of inoculated primed plants indicates the possible role of priming in disease resistance. The decrease in chlorophyll content of non-treated control plants after systemic inoculation of *A. niger* suggests the positive role of seed priming in maintaining chlorophyll content and disease resistance.

In the present study, higher RWCs were observed after seed treatments. Of all the treatments, the hydro- and osmotic-primed plants showed the highest accumulation of RWCs (Figure 2A). The same results were reported by Namdari and Baghbani [79] and by Mahboob et al. [80], who reported higher water content in *Vicia dasycarpa* and *Zea mays* with hydropriming and osmotic priming, respectively. Our findings revealed an increase in shoot and root length after priming compared to the control, which is supported by the findings of Dessalew et al. [4] and Kumar and Rajalekshmi [81]. Anwar et al. [62] observed an increase in root length in primed seeds in comparison to their control, and suggested that it could be because of embryo cell wall extensibility. In addition, it has been reported that after priming, cell division increases in the apical meristem in roots, leading to an increase in plant growth [82].

The present study showed the beneficial effects of hydropriming and osmotic priming on shoot length, root length, and fresh and dry root/shoot ratios, in response to fungal attack (Figure 2B–E). The hydro- and halo-primed China aster (*Callistephus chinensis*) plants showed significantly enhanced seed germination percentage, seedling survival, and root/shoot ratio [83]. Bourioug et al. [75] reported that hydropriming and osmotic priming in sunflower (*Helianthus annuus*) promoted overall plant growth and increased grain number and grain yield per plant by 2.5-fold and 3.3-fold, respectively. It has been suggested that seed priming enhances plant growth by decreasing the effect of oxidative reactions triggered by reactive oxygen species (ROS) in plant cells [84,85]. According to Al-Abdalall [86], laboratory treatment of both wheat and barley crops by fungi reduces root and shoot lengths and yield significantly. We also observed a decrease in all these parameters in the control (non-primed) plants after *A. niger* inoculation, in comparison to the primed plants, which could be a reason for providing resistance to the pathogen.

Zida et al. [87] reported that seed priming of sorghum plants exhibited significant increase in crop yield, of 19.6% to 51.7%. In addition, the study described threefold to fivefold decreases in the fungal species, *Curvularia* and *Epicoccum*, respectively. Similarly, Rashid et al. [88] demonstrated that, due to hydropriming, mung bean appeared to be more disease-resistant, by having fewer disease symptoms after being infected with Mung bean Yellow Mosaic Virus (MYMV). Rashid et al. [89] also reported an increase in biomass and grain weight due to priming. Likewise, our results also represent that primed plants have a considerable decrease in disease severity, by having improved biochemical (proline, protein, sugar, and chlorophyll contents) and physiological parameters (fresh root, shoot length, dry root/shoot ratio, and RWC). Foliar inoculation of *A. niger* showed a higher percentage of disease or leaf necrosis in the control (>80% leaf area) (Figure 4). The plants were found to be nearly dead, by visual assessment of wilting, as shown in Figure 5. At the same time, a considerable decrease was observed in disease severity, especially in hydroand osmotic-primed plants—70.59% and 64.71%, respectively—compared to the control, which could be effective in increasing the yield of the wheat crop. Systemic inoculation also had the same pattern of disease severity, but the capacity of disease accumulation was much less (about 60% in the control, Figure 4B,C) as compared to foliar inoculation, which gives an indication that systemic inoculation might be a vigorous method of pathogen inoculation, to show a more robust response.

Results from RT-PCR and qPCR suggest a possible role of *TLP, chitinase*, and *β-1, 3- glucanase* genes in inducing disease resistance in hydro- and osmotic-primed plants (Figure 7). Higher expression of these genes may increase resistance to *A. niger*. The higher expression of *TLP* genes in plants has been shown to provide enhanced tolerance to fungal pathogens [90,91]. Constitutive expression of *TLP*s is typically absent in healthy plants, but is induced exclusively in response to wounding or pathogenic attack [23,26]. After infecting potato plants with *Phytophthora infestan*s, the *TLP* gene was observed to be upregulated [92]. We also recorded a significant up-regulation of the *TLP* gene in both hydroand osmotic-primed plants—suggesting its positive role in disease resistance. *Chitinase* has been reported to have a prominent role in plant defense against fungi [27,28]: this gene is thought to play a dual role in fungal growth inhibition, both by cell wall digestion and by releasing pathogen-borne elicitors that induce further defense reactions in the host [93]. Plants subjected to hydropriming also have higher expression of chitinase, which could possibly be considered highly resistant to disease. It has been shown that due to pathogenic attack, the activity and expression of chitinase are elevated [94]. The best-known examples of protection conferred by transgenic expression of plant antifungal genes are represented by overexpression of *chitinases* and *β-1,3-glucanase*s [28,95]. Importantly, gene expression analysis of the current study revealed that *β-1,3-glucanases* showed the highest expression in each priming treatment, as compared to *TLP* and chitinase: their highest expression was observed in osmotic-primed plants, which resulted in the greatest disease resistance with the lowest disease severity in inoculated wheat plants. These results indicate the possible involvement of β-1,3-glucanase in disease resistance, by inducing its high expression in

both hydro- and osmotic-primed plants. In our previous study, we also observed higher expression of β-1,3-glucanase, TLP, and chitinase2, which increased the resistance of wheat plants to *F. equiseti* [28].
