**1. Introduction**

Priming is a seed treatment in which seeds are first soaked and then dried to their original weight, during which time germination continues, but radicle protrusion does not occur [1]. Seed priming offers the following advantages: improved, uniform, and fast

**Citation:** Gul, S.; Hussain, A.; Ali, Q.; Alam, I.; Alshegaihi, R.M.; Meng, Q.; Zaman, W.; Manghwar, H.; Munis, M.F.H. Hydropriming and Osmotic Priming Induce Resistance against *Aspergillus niger* in Wheat (*Triticum aestivum* L.) by Activating *β-1, 3-glucanase, Chitinase,* and *Thaumatin-like Protein* Genes. *Life* **2022**, *12*, 2061. https://doi.org/ 10.3390/life12122061

Academic Editor: Balazs Barna

Received: 11 November 2022 Accepted: 6 December 2022 Published: 8 December 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

emergence of seedlings; healthier grain; excessive vigor; and better straw yield, tillering, allometry, and harvest index in floriculture [2], vegetables [3,4] and field crops [5–9]. Various seed treatment techniques have been established, such as osmotic priming, hydropriming, halopriming, thermopriming, and hormonal priming. In hydropriming, pre-germination starts, but actual germination does not occur. Hydro-primed plants tolerate dryness, and the negative effects of pests are decreased by the faster emergence of seedlings [10,11]. Hydropriming enhances seedling growth in rice (*Oriza sativa* L.), corn (*Zea mays* L.), chickpea (*Cicer arietinum*), and mung bean (*Vigna radiata*) seeds [12–14]. Hydropriming can be a cheap and easy seed invigoration treatment for wheat, especially in salinity and drought stresses.

In osmotic priming, the seeds are soaked in a low osmotic potential solution, having chemicals like polyethylene glycol (PEG), menthol, chemical fertilizers, sugar, glycerol, and sorbitol [15]. Osmotic priming has been known to improve seed dormancy, and to enhance vigor in soybean (*Glycine max* L.) [16] and tomato (*Solanum lycopersicum*) [17]. PEG solution enhances the emergence percentage and the homogeneity of germination, and increases water absorbance by the seeds, and the development of the shoot and radicle [15,18]. In abiotic stress conditions, cellular stability is maintained by metabolic osmo-regulators, such as glycerol, mannitol, and trehalose, which are well-known osmo-conditioners [19]. Few reports have concluded that osmotic priming agents play a key role in activating crop disease resistance [19]. In wheat, powdery mildew caused by *Blumeria graminis* is controlled by trehalose, which induces systemic acquired resistance [20].

During host–pathogen interactions, pathogenesis-related (PR) proteins are produced: these proteins are encoded by the host plant, but they are induced specifically in pathological or related situations [21]. PR proteins are of paramount importance, as they increase plant resistance to pathogens. Thaumatin-like proteins (TLPs) are important PR-proteins (PR-5), consisting of 200 amino acid residues [22–24]. TLPs are produced in plants: they protect the plants from the harmful effects of phytopathogens, stresses, and elicitors, and are also involved in a wide range of developmental signals. The antifungal property of TLPs renders them useful in genetic engineering to produce disease-resistant plants [25–28]. The role of TLPs in resistance to several basidiomycete fungi—including *Rhizoctonia solani*, *Lentinula edodes* (Berk.), and *Irpex lacteus* (Fr.)—has been reported [29]. Despite exhibiting resistance to biotic stresses, TLPs also confer resistance to abiotic stress conditions [30].

Chitinase is another PR protein (PR-3) expressed in response to a variety of stresses [31]. Chitinase has antifungal activities against plant pathogenic fungi, such as *Fusarium oxysporum*, *Botrytis cinerea*, *Rhizoctonia solani, F. udum*, *Alternaria* sp., *Bipolaris oryzae*, *Curvularia lunata*, and *Mycosphaerella arachidicola* [32–34]. The mode of action of PR-3 proteins is relatively simple, e.g., chitinases cleave the chitin polymers of the cell wall in situ, leading to a compromised cell wall that renders fungal cells osmotically sensitive [35]. Another highly complex gene family is the plant β-1,3-glucanase (β-1, 3-G); β-1,3-glucanases play a role in developmental processes and pathogen defense responses [36]. The expression of these genes is triggered by plant hormones, which also affect germination [37]. β-1,3-glucanases are well-recognized PR proteins, which belong to the PR-2 protein family. These PR proteins are strongly induced in response to wounds or infection by viral, bacterial, and fungal pathogens [38,39]. This study aimed to ascertain whether improvement in plant growth and disease resistance could be induced by using different priming techniques. For that purpose, the present study was designed to investigate the role of hydropriming, osmotic priming, halopriming, and hormonal priming in response to *A. niger* inoculation in wheat (*T. aestivum* L.). We found that, of all the priming methods, hydropriming and osmotic priming had the most significant effect on growth and development, decreasing disease severity, and increasing resistance to *A. niger* in wheat: this is most probably due to the higher expression of genes (in hydropriming and osmotic priming) involved in plant defense mechanisms, and their role in disease resistance.

#### **2. Materials and Methods**


Healthy seeds of the susceptible wheat cultivar "Sahar" were obtained from the National Seed Corporation, Fatteh Jhang, and Rawalpindi, Pakistan. The seeds were surface-sterilized, by being soaked in 70% ethanol for 3 min, washed thoroughly with sterilized distilled water many times, and then dried.

#### 2.1.2. Seed Priming

Four priming methods were used for comparative analysis. In each treatment, 20 g (g) of wheat seeds was used. The osmotic priming technique employed 30 g of polyethylene glycol (PEG 6000), which was dissolved in 100 mL of distilled water. The wheat seeds were soaked in PEG solution for 2–3 days at room temperature, dried to their original weight under shade, and used for sowing [40]. For the hydropriming, the seeds were soaked in distilled water for 24 h at room temperature: these seeds were re-dried to their original weight under a shade with continuously passing air [40]. For the hormonal priming, the wheat seeds were soaked in 200 mL of hormonal solution (100 ppm solution of Indole acetic acid (IAA)) for 12 h at room temperature; then, the seeds were re-dried to their original weight, under shade, and used for sowing. For the halopriming, the seeds were primed in 100 mL of NaCl solution (100 mM) for 12 h, and allowed to air-dry for 12 h at room temperature before sowing.

#### 2.1.3. Seed Sowing and Germination

After priming, the seeds were sown in plastic pots containing sterilized soil, and were kept under controlled conditions in a growth chamber at 20–25 ◦C day/night temperature, 60% relative humidity, and 14/10 hrs light-and-dark periods. Ten to fifteen seeds were sown in each pot. Non-treated seeds were used as the control.

#### *2.2. Fungus Inoculum Preparation*

A fresh culture of *A. niger* was obtained from the National Agricultural Research Centre (NARC), Islamabad, and observed under a microscope for confirmation. Using a sterilized spatula, the fungus was transferred to Czpeck media. The flasks were incubated in a shaker incubater (200 rpm) at 30 ◦C. After 3 days, the number of spores was calculated by hemocytometer, and adjusted to 106 spores/mL concentration. The spore suspension was filtered using a muslin cloth, and the filtrate was used for further foliar and systemic inoculations.

#### Fungus Inoculation

Two methods were used for fungus inoculation. In the foliar (surface) inoculation method, spore suspension (10<sup>6</sup> spores/mL) was sprayed on 8–10-day-old plants, with the help of a spray bottle. For one week post-inoculation, the symptoms were observed every 24 h. For systemic inoculation, sorghum seeds were used to completely disperse the fungus in the soil. The sorghum (*Sorghum bicolor*) seeds were sterilized in 70% ethanol, washed three times with distilled water, and soaked overnight in distilled water. The seeds were then dried, autoclaved, and soaked in spore suspensions for 5–7 days [41]. The inoculated sorghum seeds were isolated from the spore suspension, re-dried under shade, and 2 g of sorghum seeds was added to 1 kg of soil, which was used to grow the primed wheat seeds. In addition, non-treated sterilized sorghum seeds were used as a negative control.

#### *2.3. Disease Severity Analysis*

Disease symptoms were evaluated and defined by two different methods. In the first method, total leaf area and infected part were measured, and disease severity was calculated in percentage, using the following formula [42]:

$$\text{Disease severity} = \frac{\text{Area of plant tissue affected by disease}}{\text{Total area}} \times 100$$

In the second method, a visual assessment of wilting was performed after foliar and systemic inoculations, by following standard scaling [43–45].

#### *2.4. Determination of Biochemical Contents*

Different biochemical contents were investigated in the primed plants in response to fungal inoculation. The sugar contents of the leaves were determined by following the method of [46]. The protein, proline, and chlorophyll contents were determined by following the methods of [47–49], respectively.

#### *2.5. Analysis of Physiological Parameters*

Various physiological parameters were measured to evaluate the effectiveness of different priming techniques in response to fungal inoculation: in this respect, the lengths of freshly harvested shoots and roots were measured with measuring tape, and the root/shoot ratio was calculated. The fresh plant samples were kept in an oven at 70 ◦C for 72 h, in order to analyze the dry root/shoot ratio [50]. The relative water content of the leaves was measured after the different priming methods and induction of biotic stress by the method of [51].
