The research was conducted at the experimental area of the Institute of Agricultural Sciences (IAGS), University of the Punjab Lahore, located at 1° 30’ 15” N and 74° 18’ 23” E. The pot trial was carried out between August-September 2017 at temperature ranged from 27-32°C and humidity 60-70%, both conditions are favorable for EB disease in tomato.

Tomato seeds of susceptible germline (Miracle) were surface sterilized with 1% sodium hypochlorite solution and used for nursery preparation (Awan et al., 2018). The pot experiment consisted of 15 treatments [T₁-T₅ (without Zn 0.0 mg/kg), T₆-T₁₀ (with Zn 2.5 mg/kg) and T₁₁-T₁₅ (with Zn 5.0 mg/kg)] with three replicates and was laid out in a completely randomized design (Table 1). Fertilizers like the amount of zinc sulphate (Zn), urea (N), diammonium phosphate (N & P), sulphate of potash (K), boric acid (B) and magnesium sulphate (Mg) were calculated on hectare to pot basis. Two different doses of Zn (recommended: 2.5 mg/kg and double: 5.0 mg/kg) alone and in combination with recommended dose of NPK (64 kg N/ha, 20 kg P/ha and 50 kg K/ha), B (3 kg/acre) and Mg (12 kg/acre) were applied in potted soil.

Four tomato seedlings were transplanted in each sterilized potted soil (12 kg/pot). Ten days after seedling transplanting, nutrients were applied in potted soil as per requirement for the treatment. A. solani (FCBP-1401) cultural suspension (3 × 10⁴ conidia/ml) was sprayed on 15 days old-seedling in treatments (T₂-T₁₅), while negative control (T₁) received distilled water. The plants were covered with plastic bags after pathogen inoculation to maintain humidity (70-80%). The plants were regularly monitored to measure the progression of disease in terms of disease intensity (disease incidence and disease severity).

Early blight disease incidence (DI) was assessed after 15 days pathogen inoculation as the percentage of leaves with visual symptoms of the disease in relation to all the leaves of the plant (Khan et al., 2016). The disease severity was assessed in terms of percent severity index (PSI) using 0-5 scoring scale (Pandey et al., 2003) (Table 2). PSI in each pot was calculated as:

Different physiological and biochemical alteration in tomato leaves from all treatments were assessed 5 days of pathogen inoculation. Total chlorophyll contents and carotenoids were assessed in leaf ethanolic extract (0.2 g/5 ml of 80% ethanol) (Minocha et al., 2009). Total phenolics were assessed in ethanolic plant extract mixed with 20% Na₂CO₃ and 0.025 mL of Folin-Ciocalteatou’s reagent and after incubation absorbance (765 nm) was recorded (Vongsak et al., 2013).

The total protein content and enzyme activities were estimated in leaf extract prepared in chilled sodium phosphate buffer (0.1 M, pH 6.8). A modified method of Lowry (Pomory, 2008) was used for estimation of total protein content by recording absorbance of samples at 650 nm against bovine serum albumin as standard. Activities of SOD (Sugawara and Nikaido, 2014); CAT (Weydert and Cullen, 2010); POX (Sinsabaugh, 2010); PPO (Cheema and Sommerhalter, 2015) and PAL (Shi et al., 2013) were assessed and data were analyzed statistically.

Attributes related shoot and root (length, fresh and dry weight) were taken 30 days after inoculation. Dry biomass of plant material (shoot and root) was recorded by drying samples at 65°C.

The normality of the data related to disease, growth, physiological and biochemical attributes of tomato plant was tested by the Kolmogorov-Smirnov test and then the data were submitted to analysis of variance (ANOVA) and means compared by the LSD (least significant difference test) at level P ≤ 0.05 by using computer software Statistix 8.1. Two-way Factorial ANOVA was used to analyze the individual and interactive effects of zinc with other nutrients on disease intensity and physiology.

Results revealed that EB infected plants (T₂) showed the substantial increase in disease intensity (DI: 100% and PSI: 70%) with a significant reduction in growth attributes by 22-37% when compared with healthy plants (T₁). The infected plants showed typical early blight symptoms (dark brown to black spots or irregular lesions surrounded by a chlorotic halo with concentric rings leading to blighting of leaves) under pathogenic stress (Awan et al., 2018). Application of macro- and micro- nutrients (NPK, Zn, Mg & B) improved the plant resistance/tolerance against pathogen to a variable degree by reducing the disease severity (Ghorbani et al., 2008; Potarzycki 2011; Sten et al., 2017). Basal application of Zn (2.5 & 5.0 mg/kg) alone and in combination with other nutrients (NPK, Mg & B) exhibited the excellent impact on the reduction of EB disease intensity, along with improvement in health and stress markers, however, 5.0 mg/kg Zn dose showed more promising results. Zn as a micronutrient helps to induce resistance in the plant against A. solani because it is a critical nutrient for several vital biological functions in plants like photosynthesis, growth regulation, sugar formation, seed production and defense mechanisms (Khan et al., 2018). Zn may induce its toxic effect on A. solani through effecting its metabolism and enzymatic activity as reported earlier on other microorganisms by Zn nanoparticles (Chai et al., 2015; Shen et al., 2015). Collin-Hansen et al. (2005) studies indicated that Zn toxicity occurred through oxidative damages to DNA and lipids in fungus Boletus edulis. On the basis of results obtained, it could be predicted that Zn in the soil after penetrating plant cell wall, cell membrane and cortical region of root accompanied by a complex series of events to enter plant vascular bundle (xylem) and move to the stele. Through xylem, Zn finds its way to distribute and translocate in the main organs of photosynthesis and transpiration (mainly in older leaves) mainly through active transport (Arruda et al., 2015). So, Zn might alleviated EB disease stress through (i) disrupting growth, reproduction and physiology of the pathogen (ii) changing plant’s redox status, the level of signal molecules and antioxidant enzyme activities (iii) inducing synthesis of gene encoding enzymes for stress-related protein (PR proteins) and flavonoid biosynthesis pathway (Martos et al., 2016). The net outcome of Zn fertilization, therefore, might result in a faster and stronger induction of basal defense mechanisms through genetic or biochemical modifications in tomato plant upon exposure to A. solani. Likewise, Khan et al. (2018) suggested that Zn (2.5 and 5.0 mg/kg) fertilization significantly reduced charcoal rot disease and improved biological attributes in mung bean through altering physiological and biochemical attributes.

Basal fertilization with the recommended dose of NPK alone did not prove much effective in managing EB, it also increased the vegetative growth of the tomato plant. It seems that A. solani infection affected membrane permeability, impaired nutrient translocation and plant utilization, thus imbalance of NPK inside plant might affect plant tolerance to disease (Huber et al., 2012). The balance between N and K affects disease susceptibility of plants (Sten et al., 2017) due to the important role of K in photosynthesis, plant respiration, transpiration and nutrients translocation. P is involved in energy transfer compounds, cell membranes and phosphoproteins, whereas soils deficient in adequate P may also induce the severity of diseases in plants suffering from P starvation (Wissuwa, 2003). Likewise, in general application of either Mg or B proved inefficient in significantly reducing disease and improving growth attributes. Mg is an essential nutrient involved in chlorophyll formation, organic compound synthesis, protein synthesis, uptake and migration of phosphorus in plants (Aghofack-Nguemezi et al., 2014). B plays an important role in the expression of pathogenesis-related and induction of SAR in the host (Reguera et al., 2010).

The effect of Zn was uniform in combination with other nutrients including NPK, B and Mg. However, the interactive effect of NPK with both doses of Zn was the most effective in reducing disease intensity (DI: 25% and PSI: 13%) and improving growth attributes (50-168%) in pathogen-inoculated treatments over positive control that might be result of balance between required amount of nutrient for plant to cope with disease.

The positive impact of nutrients [(NPK, Mg and B) alone and in combination with Zn doses] on plant resistance to EB disease can be assessed by evaluating the alteration in health markers (photosynthetic pigments) and in the activities of various stress markers (PHE, TPC, SOD, CAT, POX, PPO, and PAL). Reduction in photosynthetic pigments (TCC and CARO) showed that toxins of A. solani damaged photosynthetic machinery by disturbing photosystems (Dehgahi et al., 2015) that might cause disruption in the food manufacturing system and also could be associated with the EB symptoms of necrosis and chlorosis (Sarkar et al., 2017). In response to infection caused by A. solani, the plant immune system possibly responds through induction of transient, low-amplitude and the first phase of ROS production suggesting a role for ROS in the establishment of the plant defenses thus resulted in 30-50% increase in activities of antioxidant enzymes (SOD, CAT, POX and PPO). Yet, due to acidification of host tissue through the production of oxalic acid by a pathogen, the calcium ion would be sequestered from the host cell walls which cause suppression of oxidative burst of the host plant (Anderson et al., 2010) (Fig. 3). Due to disruption in regulatory functions of ROS, the function of other associated plant signaling molecules like salicylic acid (SA) also disrupted. SA is essential for SAR and it synthesized from cinnamate produced by the activity of PAL (Kim and Hwang, 2014). Therefore, reduction in activity of PAL also showed failure in plant defense mechanism to induce resistance systemically through SA accumulation and plants suffered from blight disease (Chen et al., 2009).

When nutrients especially Zn was applied, activities of physiological and biochemical attributes increased significantly by 30-90%. Nevertheless, health and stress markers increased tremendously by 60-140% and reached a maximum with a greater supply of Zn in bilateral interaction with NPK. Triggering in defense response after nutrients application likely to occur sequentially through changes in Ca²⁺ influx that may results in alteration in extracellular pH, membrane potentials (Kurusu et al., 2005), along with biphasic ROS accumulation with a low-amplitude, transient first phase, followed by a sustained phase of much higher magnitude (Sharma et al., 2012; Tripathy and Oelmüller, 2012). SA would be interplay with ROS, whereas SA and ROS signals to the transcriptional control of defense genes for induction of resistance in tomato plant against disease, ultimately ROS-scavenging systems help to finally tuned ROS level (Khokon et al., 2011) (Fig. 3).

The current study revealed that synergistic crosstalk between interactive combination of Zn with NPK, Mg or B bestowed varying degree of resistance in tomato plant against EB disease through modifying activities of key players (defensives enzymes) and total phenolics which resulted in progressive effects on SA-responsive genes and defense-related proteins to elicit HR, SIR and SAR. Hence, infected plant developed sophisticated mechanisms of defense to alleviate EB stress of disease in tomato particularly through the interactive effect of Zn with NPK.