Field Ecacy of Priming Agents to Enhance Physiological Parameters of Sunower Under Stress From Charcoal Rot (M. Phaseolina)

Charcoal rot (Macrophomina phaseolina (Tassi) Goid.) can cause signicant yield losses in sunower (Helianthus annuus L.) throughout the world. Fungicide treatments are often used to manage this pathogen, but due to costs and environmental impacts, alternative methods need to be explored. Priming is an adaptive strategy which enables plant defense systems to react more effectively to pathogen attack. The priming phase in plants can be achieved by stimuli from chemicals, benecial microbes, arthropods, and abiotic stress which can induce defense systems in plants. The present study was conducted to evaluate the effect of different priming agents such as salicylic acid (SA), abscisic acid (AA), gibberellic acid (GA), and jasmonic acid (JA), as either seed or foliar treatments, on charcoal rot on the sunower. The experiments were conducted under eld conditions in two consecutive years (2017-2018). Ten seed of one susceptible (17577) and two moderately resistant (HA-259 and B-224) sunower cultivars were treated with 2, 4 and 6 mM concentrations of the individual priming agents.. The plants were articially inoculated with M. phaseolina before sowing by infesting soil, and sixty days after sowing. A number of yield parameters were quantied [head diameter (cm), 100-seed weight (g), achene per head (number), plant height (cm), stem girth (cm), shoot weight (g), root wet and dry weight (g) and chlorophyll content (mg/g)]. Among the priming agents evaluated, AA exhibited a signicant impact on all yield parameters at a higher concentration (6 mM) in both susceptible and moderately resistant cultivars, followed by SA, GA, and JA. The results also revealed that seed treatment priming was more effective than foliar priming. There were statistical differences in yield parameters between both years of the study. The AA seed priming treatment showed the most promise for managing charcoal rot of sunower. The outcome of this study will help to explore an environmentally sound and economically feasible approach for the management of charcoal rot to get sustainability in edible agents, concentrations, seasons, germplasm, and application methods while all physiological parameters were dependent variables. A test of homogeneity was performed on the data. The analysis was performed after having the P-value greater than 0.05, indicating the homogeneity of the data. Signicant differences (P ≤ 0.05) between the mean values were identied using Tukey’s honestly signicant difference (HSD) test. All statistical analyses were performed using Statistix 8.1 software (Analytical


Introduction
Plants under eld production conditions are often exposed simultaneously to several abiotic and biotic stresses (Llorens et al., 2020). The ability to perceive the stresses and activate the proper responses is crucial for plants to grow well and be productive, and sometimes even to survive (Martínez-Medina et al., 2017). The plant immune system has been thought to be far less complex than animal immune systems (David et al., 2019). To prevent damage from pests and diseases, plants can activate their defense arsenal. Sun ower (Helianthus annuus L.) is a valuable, short duration (90 to 120 days), edible oilseed crop. Sun ower oil is widely used around the globe because it is rich in healthy oils, while the seed are used as animal feed (Tewari and Arora, 2018). It has great potential to meet the domestic needs of Pakistan, yet the production of sun ower has been gradually decreasing . The production of sun ower seed is affected by several abiotic and biotic stresses (Kaur et al., 2012). Diseases, caused by several plant pathogens, have resulted in a reduction in yields and economic losses, and are a threat to food security around the world. Charcoal rot of sun ower, caused by Macrophomina phaseolina, has been a particularly challenging disease to manage, causing 60-90% yield losses in arid areas of the world (Khan, 2007).
A wide host range of M. phaseolina were recorded and more than 500 wild and cultivated plant species has been reported to be the host of charcoal rot disease, these hosts including fruits, ber crops, cereals, legumes and vegetables and also some grass species while in Pakistan 67 host species were reported (Khan, 2007).
Disease management of charcoal rot is a challenge as the disease is in uenced by a wide range of variables (Katan, 2000). Furthermore, once charcoal rot is observed in the eld, there are few if any management procedures that can be implemented (Gaige et al., 2010). Soil drenching with fungicides can be used for the management of charcoal rot but can have negative effects on bene cial organisms and the environment (Anis et al., 2010). Thus, seed treatments can sometimes be a better option (Kim et al., 2017).
Alternative environmentally friendly management methods would be bene cial for managing charcoal rot. One such approach would be priming the defense system of plants (Mauch-Mani et al., 2017). More recent studies suggest that systemically acquired resistance (SAR) can generate a prolonged "memory" of pathogen interaction, referred to as "priming", and that they pass the immunity to their offspring through epigenetic changes (Spoel and Dong, 2012). The "primed" state enables the plant to maintain a vigilant or alarmed status and enhances its ability to activate cellular defense responses quickly and effectively against pathogen attack (Fu and Dong, 2013  . The seed treated with priming agents can develop a better root system (Duman, 2006) in the plant that will help the plant to absorb more nutrients and improve water absorption. Seed treated with a priming agent has resulted in improved seed yield in Phaseolus vulgaris (Kumar et al., 2016).
The treatment of seed with priming agents has been shown to bene t the sun ower crop to mitigate stress factors such as pest damage, pathogen attack, and drought (Worrall et al., 2012). The priming agents play a vital role in protein synthesis, endosperm weakening by hydrolase activities, mobilization of storage proteins, building up of nucleic acids, cell cycle-related events, and repairing of membranes (McDonald, 2000). More production of antioxidative enzymes has been recorded in primed seed, which enables the plant to ght various biotic stresses. The positive effect of priming on seed often depends upon the duration of the priming (Hassemi-Golezani et al., 2008).
The objectives of the current study were to evaluate the e cacy of priming agents on the yield parameters of a susceptible and moderately resistant sun ower genotype under charcoal rot challenged conditions and to evaluate the effect of application methods (seed and foliar) and individual dose rates (2, 4, 6 mM) on yield parameters.

Materials And Methods
Sowing of sun ower germplasm One susceptible (17577) and two moderately resistant (HA-259 and B-224) sun ower genotypes exhibited maximum yield (Qamar et al., 2018;Qamar et al., 2019). These germplasm were selected to determine the effect of four priming agents at three different concentrations upon charcoal rot on sun ower. Three concentrations (2, 4 and 6 mM) of SA, AA, GA, and JA were prepared for the foliar and seed applications (Table 1). For seed priming, ten seeds from each selected germplasm of sun ower were dipped for 30 minutes in each concentration, washed with sterile distilled water, air-dried on double layers of sterile lter papers, and stored at 25 ± 2 º C for 24 hours in a sterile laminar ow chamber. Application of priming agents The seeds were sown in the research area with 30 cm plant to plant and 60 cm row to row distance. The eld experiment was arranged in a split-plot design based on a Randomized Complete Block Design (RCBD) with three replications independently to avoid pseudo-replication. The main plot consisted of four treatments with priming agents: SA, AA, GA and JA. Each treatment was divided into three subplots with 2, 4 and 6 mM concentrations of SA, AA, GA and JA, respectively.
To determine the effect of foliar application, a parallel eld was prepared and seeds of selected germplasm were sown with the same plant to plant and row to row distances. Each concentration (2, 4, and 6 mM) was sprayed separately on ten days old sun ower plants and 48 hours before harvesting. A negative control plot was also prepared for comparison. The arti cial inoculation of highly virulent M. phaseolina isolate (MIQ) (Qamar et al., 2018) with millet mass culture (4g/m) was applied in all experiments before sowing, and a spore suspension (100g mycelium/L in sterile distilled water) was applied 60 days after sowing (Keerio et al., 2014). All agronomic practices were conducted where applicable and the experiment was repeated twice. Impact of priming agents on physiological parameters Thirty sun ower plants from each treatment were selected randomly and harvested. Physiological parameters were recorded: 100-seed weight, achene per head, chlorophyll content, head diameter, plant height, root wet and dry weight, shoot weight, and stem girth. The effect of seed and foliar application at each concentration was recorded and compared to the control where no priming agents were applied.
Head diameter, the total number of achene per head, and 100-seed weight The diameter (cm) of each selected sun ower plant was recorded and total numbers of seeds/achene were separated and counted (number) from individual heads (Soomro et al., 2015). 100-seeds were

Root fresh and dry weight
The roots of the plants were separated carefully and washed with running tap water to remove soil particles and other debris. The roots were dried on a double layer of blotted paper towels and root fresh weight (g) was recorded. The roots were incubated at 80°C for 48 hours and their dry weight (g) was noted (Makbul et al., 2011).

Chlorophyll content
The fresh leaf samples (0.5 g) were plucked from the ten selected sun ower plants and ground in 10 mL of 80% acetone in sterile pestle and mortal. The mixture was centrifuged at 5000 rpm for 5 minutes and the supernatant was shifted to a new Eppendorf tube. The absorbance of the supernatant was computed at 663 and 645 nm and the chlorophyll content were measured (Farahmandfar et al., 2013).

Statistical analysis
Analysis of variance (ANOVA) was conducted to establish the differences among the independent and dependent variables. The independent variables were priming agents, concentrations, seasons, germplasm, and application methods while all physiological parameters were dependent variables. A test of homogeneity was performed on the data. The analysis was performed after having the P-value greater than 0.05, indicating the homogeneity of the data. Signi cant differences (P ≤ 0.05) between the mean values were identi ed using Tukey's honestly signi cant difference (HSD) test. All statistical analyses were performed using Statistix 8.1 software (Analytical Software, 2105 Miller Landing Rd, Tallahassee, FL 32312).

Head diameter (cm)
The analysis of variance revealed that all the main effects regarding head diameter were signi cant ( Table 2). The impact of priming agents on head diameter was studied during two consecutive years in the presence of M. phaseolina. Priming agents were applied to three sun ower genotypes. The maximum head diameter was recorded for HA-259 in the seed (31.46 cm) and foliar application (25.28 cm) of abscisic acid at a high concentration (6mM), followed by SA, GA, and JA. A similar trend was observed with the other two genotypes (B-224 and 17577). In susceptible genotype 17577, head diameter did not exceed 11.7cm with jasmonic acid at 2mM concentration with seed application (Fig. 1). Table 2 Analysis of variance for head diameter, seed weight, and no. of achene per head of sun ower in an experiment with two application methods, two seasons, three germplasm, four priming agents and four concentrations (total df = 575 Total number of achene per head All the main effects regarding the number of achene per head were highly signi cant ( Table 2) Test weight (100-seed weight) The analysis of variance showed that the main effects regarding test weight were signi cant (Table 2), except the seasons. The 100-seed weight tended to increase with both priming methods, with signi cantly different behaviour in the presence of charcoal rot. Abscisic acid seed treatment delivered the maximum 100-seed weight in HA-259 (9.06 g) followed by SA (8.48 g), GA (7.53 g) and JA (6.27 g) at higher concentration (Fig. 3). On the other hand, in 17577 the minimum 100-seed weight (3.55 g) was observed with JA at 2 mM concentration. A similar trend was recorded in the foliar application, with a maximum 100-seed weight (7.85 g) recorded in HA-259, followed by B-224 (6.74 g) and 17577 (6.55 g) at higher concentration.

Plant height (cm)
Signi cantly different plant heights were observed with seed and foliar application in the presence of M. phaseolina. The analysis of variance demonstrated that all the main effects regarding plant height were signi cant (Table 3). An increase in plant height was recorded with AA seed treatment in three sun ower genotypes at a higher concentration; the maximum (192.1 cm) was observed in HA-259, followed by B-224 (153.2 cm) and 17577 (112 cm). The same trend was found with foliar application of AA at a higher concentration in HA-259 (174 cm), B-224 (139 cm), and 17577 (103 cm). Plant height was slightly reduced with the application of 4 mM and 2 mM concentration of each priming agent with both application methods (seed and foliar) as compared to 6mM (Fig. 4). In SA application the height of plants was maximum at high concentration (6 mM) as compared to 4 mM, 2 mM, and control in B-224 germplasm: 146.2 cm, 118 cm, 111.2 cm, and 88 cm respectively. A similar trend was recorded in susceptible germplasm (17577), where AA exhibited the maximum height followed by SA, GA, JA, and control. Plant height did not exceed 96.9 cm with JA at 2 mM concentration with seed application. The results revealed that plant height also depends on the dose. The maximum dose increased the maximum plant height, and foliar application resulted in less plant height compared to seed treatment. Stem girth (cm) The main effects regarding stem girth (cm) were signi cant, except for the seasons (Table 3). Maximum stem girth was observed in HA-259, followed by B-224 and 17577, in both seasons. HA-259 exhibited the maximum 10.5 cm stem girth with abscisic acid seed treatment at 6 mM concentration; girth was 9.8 cm with the foliar application and 5.6 cm in control. B-224 exhibited 9.4 cm with seed treatment and 9.1 cm and 5.1 cm in foliar and control, respectively. The minimum 4.3 cm stem girth was observed in 17577 in control, while foliar application resulted in 6.5 cm (Fig. 5) and seed application resulted in 7.1 cm with abscisic acid treatment at 6 mM concentration. A similar trend was observed in SA, GA, and JA, followed by AA.

Shoot weight (g)
There was a signi cant difference in the main effects upon shoot weight, except the seasons (Table 3). Shoot weight of susceptible (17577) and moderately resistant germplasm (B-224 and HA-259) showed an increasing trend, being most evident with seed application of AA at 6mM concentration as compared to other priming agents (SA, GA, and JA) and application method (foliar application) in the presence of M. phaseolina. The maximum shoot weight was recorded in HA-259 (1216.1 g) with AA seed treatment at a higher concentration. Shoot weight also increased with SA application (1151.7 g), followed by GA (936.3 g) and JA (846.8 g) at the same concentration (Fig. 6). The minimum shoot weight was observed in 17577 (562.9 g) at 2mM concentration with foliar application of JA. Seed treatment delivered a better result than foliar application.
Root wet weight (g) The analysis of variance showed that all the main effects upon root wet weight were signi cant, except seasons (Table 4). Our ndings con rmed that the application of priming agents, be it to seed or foliar, has a positive impact on root wet weight. Seed treatment signi cantly increased root wet weight as compared to foliar application. Reduction in root wet weight (311g) was observed in 17577 with JA at 2 mM in seed priming while 103.9 g in foliar application. Among all priming agents, abscisic acid in both application methods delivered the maximum root wet weight (806.5 g) in HA-259 at maximum concentration (Fig. 7), followed by SA (759.5 g), GA (739.9g) and JA (726.2g) in seed treatments. The reduction in root wet weight of HA-259, B-224, and 17577 in control was recorded as 435.8 g and 308.1 g and 125.9 g, respectively. The analysis of variance con rmed that all the main effects of root dry weight (g) were signi cant (Table 4). Root dry weight (g) was signi cantly higher (746g) in HA-259 with AA seed treatment at higher concentration, followed by B-224 (542.9 g) and 17577 (353.3 g) in the presence of charcoal rot. Minimum dry weight (103.4g) was observed in 17577 with JA at 2 mM concentration. A signi cant difference was noted in foliar application with AA at higher concentration among germplasm HA-259 (723.9 cm), B-224 (524 cm), and 17577 (340 cm), respectively (Fig. 8). AA delivered the best results at maximum concentration, followed by SA, GA and JA.
Chlorophyll content (mg/g) The analysis of variance showed that all the main effects of chlorophyll content were signi cant (Table 4). Chlorophyll content observed in plants of susceptible (17577) and moderately resistant germplasm (B-224 and HA-259) showed an increasing trend, this being most evident with AA seed application at 6mM concentration as compared to other priming agents (SA, GA, and JA), the application method (foliar application) and control in the presence of M. phaseolina. Maximum chlorophyll content (0.74 mg/g) was recorded in HA-259 with AA seed treatment at 6mM concentration. JA also resulted in increased chlorophyll content (0.42 mg/g), but that was not signi cantly high; this was followed by of GA (0.54 mg/g) and SA (0.68 mg/g) at the same concentration (Fig. 9). Similarly, maximum chlorophyll content was observed in HA-259 (0.67 mg/g) with foliar application of AA at a high concentration (6 Mm), while JA also delivered an increased content (0.35 mg/g) but not at a signi cantly high level, followed by SA (0.62 mg/g) and GA (0.47 mg/g) at the same concentration, while in the case of nonprimed plants 0.25 mg/g chlorophyll content was recorded.

Discussion
Both seed and foliar applications of priming agents have been found to enhance germination rate, uniformity, and metabolic activities, while also triggering defense responses against biotic and abiotic stresses, with positive impacts on physiological changes in plants ( Raj  con rmed that seed priming has a signi cant impact on the yield, head diameter, achene weight and achene per head of two sun ower genotypes (Hysun-33 and FH-331) as compared to non-primed control.
A study using Pseudomonas uorescens as seed priming against abiotic stress also observed physiological parameters that increased dramatically in primed plants compared to control (Pravisya et al., 2019).
The present study found that sun ower height increased signi cantly with seed and foliar application of priming agents at three concentrations in the presence of charcoal rot as compared to unprimed plants. The present study demonstrated that seed priming with AA improves physiological parameters, including chlorophyll content of sun ower, as compared to GA and other priming agents. These ndings are supported by those of Farahmandfar et al. (2013), who compared the effects of sodium chloride, SA, GA and hydro-priming on chlorophyll content, dry weight, and length of plumule and seedling radicle of Trigonella foenum graecum. They found that seed treatment with salicylic acid enhanced the physiological parameters of fenugreek seedlings more effectively than gibberellic acid and other treatments. Our results are also in line with those of Razzaq et al. (2013), who studied the effects of seed priming with salicylic acid (50, 100, 150 ppm), abscisic acid (50, 100, 150 ppm) and ascorbic acid (10, 20, 30 ppm) on membrane stability index, chlorophyll content, relative water content (RWC) root-shoot ratio and yield in wheat cultivars, and found that salicylic acid delivered pronounced and better effects than other chemicals. Similarly, El-Hai et al. (2009) investigated the effect of exogenous and endogenous application of citric acid and salicylic acid against charcoal rot, observing various physiological parameters of sun ower (chlorophyll content, stem diameter, ower head diameter, seed plant yield, and 100-seed weight). All treatments with antioxidants reduced the incidence of charcoal rot disease, and physiological parameters were signi cantly increased in sun ower crops under eld conditions. The studies undertaken by Youssef et al. (2017) found that exogenous application on sun ower plants with SA (0.7 and 1.4mM) played a vital role in increasing yield, head diameter, plant height, total seed per head, and chlorophyll content (a,b), while also mitigating abiotic stress at higher concentration. A similar trend was found in the present study, with our results indicating that physiological parameters are enhanced with the application of priming agents at a higher concentration. Exogenous application (acibenzolar-S-methyl and exopolysaccharides) has been shown to improve physiological properties and stimulate the defense mechanism in tomato against bacterial spots (Blainski et al., 2018).
Presents results were also strengthen by the ndings of (

Conclusion
Priming technique is used not only to increase the crop yield of crops worldwide but also enhances the level of tolerance against biotic and abiotic stresses. The present study clearly showed that seed treatment with priming agents, speci cally SA,AA, GA and JA, is an environmentally sound, easy to apply, and more reliable approach to manage charcoal rot disease of sun ower as compared to foliar application. Abscisic acid has the most positive impact on plant physiological parameters (head diameter, 100-seed weight, number of achene per head, plant height, stem girth, shoot fresh and dry weight, root fresh and dry weight, and chlorophyll content), followed by salicylic acid, gibberellic acid, and jasmonic acid, in that order. Seed priming can be a valuable element of integrated disease management of charcoal rot of sun ower for its sustainable production.