E ﬀ ect of Foliar Applied Acetylsalicilic Acid on Wheat ( Triticum aestivum L.) under Field Conditions

: The exogenous application of salicylic acid prevents plant damage caused by various abiotic stresses (drought, high and low temperatures, salinity) and helps plants to build resistance to biotic stresses (pathogens). Acetylsalicylic acid, which is a synthetic salicylic acid derivative, has the same properties. In the face of climate change and a greater likelihood of extreme weather events, the use of these acids can signiﬁcantly help to ensure proper growth and development of plants, especially sensitive species, even under stress conditions. The problem, however, is choosing the right dose (concentration) and time of application, and individual species sensitivity. The aim of the study was to assess the e ﬀ ect of acetylsalicylic acid on the growth and yield parameters of spring wheat. A ﬁeld trial was carried out in 2013 and 2014 at the Institute of Plant Protection—National Research Institute in Poznan (Poland). The experiment was established on spring wheat cv. ‘Tybalt’, in four replications. Acetylsalicylic acid was tested in two concentrations: 0.3 and 0.5 mM. Each concentration was applied to the crop twice at growth stage BBCH 29 (end of tillering) and 7 days afterwards, and at growth stage BBCH 37 (ﬂag leaf just visible) and 7 days afterwards. The study evaluated 12 traits of the crop, including plant height, chlorophyll content in the ﬂag leaf, length of the ear, and quality and quantity parameters of the yield. The study showed that both the selection of the appropriate concentration and the time of acetylsalicylic acid application have a signiﬁcant impact on the growth and development of the wheat. The acetylsalicylic acid increased the amount of chlorophyll in the leaves, the number of grains in the ear, the mass of a thousand grains, and grain yield. The present study did not conﬁrm signiﬁcant di ﬀ erences between the acetylsalicylic acid concentrations. The concentration of 0.5 mM proved more e ﬀ ective only for such traits as plant height and protein content in the grain. The study showed that it is not the concentration but the time of acetylsalicylic acid application that is more important for the growth of the wheat. Although there were no signiﬁcant di ﬀ erences between the e ﬀ ects of earlier and later application time on plant height or chlorophyll content, most of the structural and qualitative parameters of the yield (number of grains in the ear, grain density, grain hardness, protein, gluten and starch content) were better after later application of acetylsalicylic acid (GS BBCH 37).


Introduction
Wheat, maize and rice make up the largest share of the world's agricultural crop production. Together, these three crops provide food for over 4.5 billion people. The area devoted to wheat production alone covers 220 million ha [1][2][3]. Recently, more and more attention has been given to of ASA in a concentration of 3 mM applied at growth stage BBCH 29 (end of tillering)-T1, and then 7 days after the first treatment-T2; treatments of ASA in a concentration of 5 mM applied at growth stage BBCH 29 (end of tillering)-T1, and then 7 days after the first treatment-T2; treatments of ASA in a concentration of 3 mM applied at growth stage BBCH 37 (flag leaf just visible, still rolled)-T3, and then 7 days after the first treatment-T4, and treatments of ASA in a concentration of 5 mM applied at growth stage BBCH 37 (flag leaf just visible, still rolled)-T3, and then 7 days after the first treatment-T4.

Spraying Parameters
Treatments were conducted using a bicycle-mounted Victoria sprayer equipped with TeeJet 110 02 VP sprayers (TeeJet Technologies, Wheaton, IL, USA) using 200 dm 3 of spray liquid per ha, with an operating pressure of 0.3 MPa. The temperature during the applications varied between 15 and 20 • C, depending on the time of application and the year of the study.

Observations
During the growing season, the height of the plants and the chlorophyll content in the flag leaf were assessed. The height was measured before the harvest, in a sample of 25 plants from each experimental plot. The chlorophyll content was measured in 30 randomly selected flag leaves from each plot using a Chlorophyll Meter SPAD-502Plus produced by Konica Minolta (Tokio, Japan). The area for a single measurement was 2 mm × 3 mm. Harvesting was performed with a Wintersteiger plot combine. Next, the following parameters were determined: mass of a thousand grains, number of grains per ear, grain density; and qualitative characteristics of the yield: protein, gluten, and starch content, sedimentation index, and grain hardness. The number of grains per ear was calculated based on a sample of 25 ears collected from each plot. The mass of a thousand grains and grain density were both measured based on a 1 kg sample from each plot. Grain density was measured using a cylinder bulk density meter, and the qualitative analysis was carried out with an Infratec TM 1241 grain analyzer produced by Foss (Hillerød, Denmark).

Weather Conditions
Weather data were obtained from the Meteorological Station in Winna Góra (52 • 12 17" N 17 • 26 48" E). Weather conditions were analyzed using the Selyaninov hydrothermal coefficient (HTC) calculated by applying the formula by G. Selyaninov: HTC= 10Σ x/Σ t; where Σ x means the monthly sum of precipitation and Σ t means the sum of temperatures in a given decade (ten days) of the month. In this paper, the sum of air temperatures > 0 • C was considered, which differs from Selyaninov's original HTC [47].
Weather conditions differed in the two study years (2013 and 2014), as presented in Table 1. The differences were recorded in the sowing period and during the initial growth of wheat. The first decades of April 2013 were very humid, whereas the same period in 2014 was marked by a considerable precipitation shortage (drought). In both years of the study, the third decade of April, and the first and second decades of May saw a shortage of precipitation. The Selyaninov coefficient (HTC) calculated for these periods indicated drought conditions. The situation changed in the third decade of May, when heavy precipitation was recorded in 2014 and HTC denoted a very humid period. In June 2014, during the ear development period, the hydrothermal conditions were highly conducive to the further development of the crop. On the other hand, throughout the entire period of intense wheat growth, ear development and grain formation in 2013 (third decade of May and entire June), the weather conditions were adverse and HTC indicated an ongoing period of drought.

Statistical Analysis
The significance of differences was assessed with Tukey's confidence half-interval at a significance level of p = 0.05. All statistical analyses for the study were carried out in R version 3.6.2 [48].

General Influence of Acetylsalicylic Acid on Wheat
The analysis of variance revealed that some of the traits studied were more sensitive to the concentration of ASA and others to the time of application (Table 2). However, the effect of ASA varied considerably for individual years and times of application. This variation is presented in Figure 1. The PCA biplot shows the trait distribution for each of the treatments studied (concentration, time of application and year). The plot clearly shows that individual areas do not overlap, which denotes a very high variability of the results obtained. The plot also demonstrates that 2014 (the second year of trials) was a year when ASA had a markedly stronger effect on wheat. Most of the traits studied are grouped in or aim towards the areas which cover this very year (areas G, H, I, J). A particular focus should be placed on area H, which denotes ASA concentration of 0.3 mM in the first application (T1/T2). This area groups such traits as ear length, mass of a thousand grains, grain density, protein content, sedimentation index, and yield. These are the traits most significantly influenced by ASA.   Figure 2, which only shows PCA for ASA concentrations (years and times of application are combined), indicates that the concentration of 0.5 mM had the greatest impact on wheat. Area ASA 5 denoting ASA concentration of 0.5 mM covers all the traits (arrows). When comparing times of application ( Figure 3), one can notice that this parameter strongly determined the impact of the experimental factors on wheat. The plot only pertains to times of application (years and concentrations have been combined). PCA shows that it was the later time of application that determined such traits as chlorophyll content, plant height, mass of a thousand grains, and grain density. From Figure 4, where cases are grouped by years, we can conclude that the results for the variables, such as plant height, ear length, thousand grains mass, number of grains per ear, grain density, sedimentation index, gluten and protein content in ears, and the grain yield are greater for the year 2014 (the second year of research) than for 2013. From this biplot, we can see the strong positive correlation between these traits (arrows). Figure 2, which only shows PCA for ASA concentrations (years and times of application are combined), indicates that the concentration of 0.5 mM had the greatest impact on wheat. Area ASA 5 denoting ASA concentration of 0.5 mM covers all the traits (arrows). When comparing times of application ( Figure 3), one can notice that this parameter strongly determined the impact of the experimental factors on wheat. The plot only pertains to times of application (years and concentrations have been combined). PCA shows that it was the later time of application that determined such traits as chlorophyll content, plant height, mass of a thousand grains, and grain density. From Figure 4, where cases are grouped by years, we can conclude that the results for the variables, such as plant height, ear length, thousand grains mass, number of grains per ear, grain density, sedimentation index, gluten and protein content in ears, and the grain yield are greater for the year 2014 (the second year of research) than for 2013. From this biplot, we can see the strong positive correlation between these traits (arrows).

Plant Height (PH) and Ear Length (EL)
When comparing the traits analyzed in the two years of the study, it was demonstrated that in 2014, both plant height and ear length were greater by 10-12% on average (Table 3). Both concentrations increased the PH in both times of application, but the concentration of 0.5 mM had a better effect. ASA increased the PH by 15%. The effect of ASA on PH did not differ between the earlier and later time of application. When comparing all experimental factors (concentration x time of application x year) in the context of PH, it was observed that there were no significant differences in wheat height between the ASA concentrations and times of application studied either in the first or second year of the study ( Figure 5A).

Plant Height (PH) and Ear Length (EL)
When comparing the traits analyzed in the two years of the study, it was demonstrated that in 2014, both plant height and ear length were greater by 10-12% on average (Table 3). Both concentrations increased the PH in both times of application, but the concentration of 0.5 mM had a better effect. ASA increased the PH by 15%. The effect of ASA on PH did not differ between the earlier and later time of application. When comparing all experimental factors (concentration x time of application x year) in the context of PH, it was observed that there were no significant differences in wheat height between the ASA concentrations and times of application studied either in the first or second year of the study ( Figure 5A).
Ear length was affected not by the ASA concentration used but by the time of application (Table 3). It was demonstrated that ASA used at stage T3/T4 significantly influenced EL, extending it by 7%. Figure 5B shows that the effect of ASA on NG in 2014 was more consistent compared to 2013, as there were no differences in EL between the treatments studied. In 2013, values similar to those obtained in 2014 were observed for both ASA concentrations applied in the later time of application (T3/T4). Ear length was affected not by the ASA concentration used but by the time of application (Table  3). It was demonstrated that ASA used at stage T3/T4 significantly influenced EL, extending it by 7%. Figure 5B shows that the effect of ASA on NG in 2014 was more consistent compared to 2013, as there were no differences in EL between the treatments studied. In 2013, values similar to those obtained in 2014 were observed for both ASA concentrations applied in the later time of application (T3/T4).

Chlorophyll in the Flag Leaf (CH)
ASA had a significant effect on the chlorophyll content in the flag leaf of wheat. Irrespective of the study year, the amount of CH increased as a result of ASA application by 18% for 0.3 mM and by 23% for 0.5 mM compared to non-treated plants (Table 3). There were also significant differences between the time of ASA application. When ASA was applied at stage T1/T2, the content of CH in the flag leaf increased by 18%, whereas at stage T3/T4, it increased by 23%. As for the years of the study, the plants had generally more CH (12%) in 2014 than in 2013. When analyzing the effect of all experimental factors (concentration, time of application, year), no differences were observed between 0.3 and 0.5 mM concentrations used at the later stage (T3/T4), and the concentration of 0.5 mM applied at the earlier stage (T1/T2) in 2013 ( Figure 5C). On the other hand, a significantly lower CH content was recorded when ASA 0.3 mM had been applied at stage T1/T2 compared to other treatments in 2013. In 2014, the effect on CH content was very consistent, and the experimental treatments studied did not differ significantly.

Number of Grains (NG) and Mass of a Thousand Grains (MTG)
The second year of the study (2014) was also more favorable to wheat in terms of the number of grains per ear and the mass of a thousand grains ( Table 3). The NG per ear that year was higher by 12% compared to 2013. As for MTG, it increased by 15% in 2014 compared to 2013. ASA concentrations of 0.5 and 0.3 mM caused an increase in NG by 13% and 9%, respectively, compared to the control. A later time of application (T3/T4) had a better effect on NG (14% increase) compared to T1/T2 (6% increase). There were no significant differences for NG between the two times of application, but MTG increased by approximately 6-7% in both these times compared to the control.
The statistical analysis of the experimental factors indicated that with regard to NG, there were no differences between ASA 0.3 and 0.5mM applied at T1/T2, and ASA 0.3 mM and ASA 0.5 mM applied at T3/T4 in the two years of the study ( Figure 5D). Time of application was not a dominating factor for NG. The highest NG was observed for the treatment that involved ASA 0.5 mM applied at T3/T4 (2014).
Varying conditions in the two years of the study strongly determined MTG. The analysis of the experimental factors demonstrated that ASA 0.3 and 0.5 mM applied at T1/T2 did not have an effect on MTG in 2013 ( Figure 6A). An increase in this value was observed for ASA applied at T3/T4, irrespective of the concentration used. In 2014, there were no significant differences in the effect of ASA between the two concentrations at T3/T4 and 0.5 mM at T1/T2. Agronomy 2020, 10, x FOR PEER REVIEW 12 of 18

Grain Density (GD) and Grain Hardness (GH)
Grain density and grain hardness were the least variable traits as regards the study years, ASA concentrations, and times of application. Although the study showed that GD and GH were significantly higher in 2014, that value was only 4%. In the case of GD, there were no significant differences between the ASA concentrations. A higher value of GD was observed only for the later time of ASA application, i.e., T3/T4 (3% increase) ( Table 3).
As for GH, a 5% increase was observed for ASA 0.5 mM. Moreover, T3/T4 proved to have a more positive impact on this trait (9% increase). The analysis of the experimental factors in the context of GD clearly shows an almost complete lack of variability in the ASA effect on this trait for individual treatments ( Figure 6B). The exception is ASA (both concentrations) applied at T1/T2 in 2013. These treatments resulted in considerably lower GD values.
The data on grain hardness ( Figure 6C) clearly show that there were no significant differences in the value of the grain hardness between the concentration of ASA 0.3 mM and 0.5 mM (the both years) and the term of application of T3/T4 and the term T1/T2 (only in 2014). In 2013, the lowest values of grain hardness were obtained for both concentrations of ASA in the application term T1/T2.

Yield (Y)
The analysis of variance revealed that more favorable weather conditions were conducive to higher yield in 2014. The yield of wheat that year was 28% higher than in 2013 (Table 3). Both ASA concentrations had a positive effect on the yield 9% and 12% yield increase was observed for ASA 0.3 mM and ASA 0.5 mM, respectively, compared to the control treatments. A similar correlation was demonstrated for the times of application. At T1/T2, the yield increased by 6%, and at T3/T4 by 15% (Table 3). Wheat yielding for individual treatment was more consistent in 2014 ( Figure 6D). In this year, ASA 0.5 mM (T1/T2) had the same effect on the yield as ASA 0.3 mM and 0.5 mM at T3/T4. In 2013, similarly higher yielding was obtained for treatments involving ASA application at T3/T4. A lack of significant differences was also observed between ASA 0.3 mM and ASA 0.5 mM at T1/T2-these were the treatments that resulted in the lowest yield.

Sedimentation Index (SI), Protein (P), Gluten (G) and Starch (S) in Grains
The analysis of variance revealed differences in the sedimentation index between the years of study. Irrespective of the other factors (ASA concentration and time of application), the index was higher by 20% in 2014 compared to the value recorded in 2013. ASA did not have a strong effect on this trait, increasing SI by 5-9% compared to the control (Table 3). T3/T4 proved to have a more positive effect on the index, which increased by 10% at that time of application. In 2013, there were no differences in SI values between the treatments studied. Such differences were observed in 2014 ( Figure 7A). The highest values were recorded for both ASA concentrations applied at T3/T4. T1/T2 was less favorable for this trait, but there were no differences between the ASA concentrations either.
In 2014, the content of protein and gluten was higher by 11% and 5%, respectively, compared to 2013, but in 2014 there was observed decrease in starch content by 8%. The P content increased by 8% after ASA 0.3 mM application, and by 14% after ASA 0.5 mM (Table 3). T3/T4 proved to have a more positive effect on this trait, as it resulted in a 14% increase. The P content increased by 8% after ASA application at T1/T2. Changes in the P content were mostly determined by the year of the study (Figure 7B). The G content increased by 5% after ASA application (for both concentrations), and significant differences were observed only for T3/T4 (8% increase). The analysis of the experimental factors demonstrated that ASA 0.3 mM applied at T3/T4 had the strongest effect on this trait in 2013 ( Figure 7C). In 2014, the highest content of G was observed after the following treatments: ASA 0.5 mM T1/T2, ASA 0.3 mM T3/T4, and ASA 0.5 T3/T4. The S content was the only trait negatively affected by ASA. Both concentrations and both times of application decreased the S content (by 2%). The analysis of interactions between the experimental factors did not show any significant effect of the study year on this trait ( Figure 7D).
( Figure 7B). The G content increased by 5% after ASA application (for both concentrations), and significant differences were observed only for T3/T4 (8% increase). The analysis of the experimental factors demonstrated that ASA 0.3 mM applied at T3/T4 had the strongest effect on this trait in 2013 ( Figure 7C). In 2014, the highest content of G was observed after the following treatments: ASA 0.5 mM T1/T2, ASA 0.3 mM T3/T4, and ASA 0.5 T3/T4. The S content was the only trait negatively affected by ASA. Both concentrations and both times of application decreased the S content (by 2%). The analysis of interactions between the experimental factors did not show any significant effect of the study year on this trait ( Figure 7D).

Discussion
The present study was conducted over two years which differed in terms of hydrothermal conditions during the critical stages of wheat growth and development. Although 2013 saw more favorable humidity conditions in the first stage of vegetative growth compared to 2014, the subsequent months were classified as very dry. This was reflected in the parameters studied, whose values were much lower in 2013 compared to 2014. However, the hydrothermal conditions in the second year of the study cannot be regarded as perfect, as the drought conditions lasted for nearly half of the wheat growing season. Yet, the difference between the study years pertained to the wheat growth stages when such adverse conditions occurred and to the duration of water shortage. In the second year of the study, drought affected the earlier stages of wheat growth and was definitely shorter compared to 2013. In the first study year, on the other hand, drought occurred in growth stages that are critical for yielding, and lasted from booting until harvest. As a result, the difference in wheat yield between the two years was over 1 t·ha −1 .
Biotic and abiotic stress accompanies every plant growing in natural conditions. It is estimated that due to abiotic stressors (drought, high temperature, salinity), crop yields can be a dozen or so times lower compared to yields from a crop affected by unfavorable biotic stressors (weeds, pathogens etc.) [49]. Spring wheat is very sensitive to water shortage, but the effects of drought depend on its intensity and duration. Given the short growing period of spring wheat, the greatest threat to this crop is a long-term drought lasting over a considerable part of its growth and development period. Long-term drought causes a large reduction in the yield, stemming from disrupted plant growth and development, and often irreversible metabolic changes in the plant. Water is essential in the initial growth stages of spring wheat (tillering, booting), but the most adverse consequences for this crop arise when drought occurs during its flowering and filling stage. A major problem faced by contemporary plant agriculture is that climate change increases the likelihood of extreme weather events, which include prolonged droughts [50,51].
The individual resistance of particular plant species or cultivars (introduction of resistant cultivars) or the use of appropriate chemical agents allow for minimizing or even preventing the effects of factors that are detrimental to plants [15,52,53]. The available research on the impact of salicylic acid on plants confirms that this compound entirely reduces the drought-induced decline of auxin levels in the plant. Thus, it has a positive effect on shoot and root mass, flower bud regeneration, flowering, and tuber formation [25][26][27]. Plants naturally contain very small amounts of salicylic acid. Studies have shown that SA is an organic acid, the amount of which differs depending on the crop species and even particular tissues. Biosynthesis, transport, and accumulation of organic acids in plants increase dramatically under stress conditions [28,29].
A study by Kaydan et al. [49] conducted on wheat under salt stress conditions showed that SA contributes to an increase in the shoot and root mass, the content of chlorophyll a and b in leaves, osmotic potential and the K+/Na+ ratio in plants. In turn, a study of sunflower plants demonstrated that the content of chlorophyll and carotenoids may decrease by as much as 64% in plants cultivated under drought conditions, and the use of SA considerably reduces this risk. It was confirmed that in sunflower, SA increases Cd, Pb and Ni accumulation in roots, Cd and Ni accumulation in shoots, shoot length, root mass, the content of chlorophyll, carotenoids and proteins in leaves, and the amount of sugar, phenols, and antioxidant enzymes [38]. Other studies showed that SA has an impact on plant growth (shoot and root mass), essential oil content in plants (basil and marjoram), oil quality, and carbohydrate and amino acid content. It also increases the content of proline, micro-and macro-elements, and polyamines (spermidine, putrescine) [35].
The present study demonstrated that the SA derivative acetylsalicylic acid (ASA) also has a positive influence on the morphological (plant height, ear length) and physiological (chlorophyll content) parameters of wheat, as well as on the structure and quality of the yield. These findings are consistent with other studies on the effect of ASA on crops [38,54]. Some authors emphasized that the effects of exogenous SA application may be more dependent on plant species and cultivar compared to those exerted by other substances. Salicylic acid occurs in every plant and its amount depends not only on the plant growth stage (as is the case with phytohormones) but also environmental conditions. In the case of endogenous salicylic acid, additional application of this substance may be toxic to the plant [32,54,55]. It has been hypothesized that a high concentration of SA may cause oxidative stress in plants, especially through hydrogen peroxide accumulation [34].
Therefore, it is of key importance to establish an effective but at the same time non-toxic concentration of SA or ASA. The present study tested two concentrations of ASA: 3 and 5 mM. However, other researchers suggested that in the case of wheat, the effective ASA concentration is 10 −2 mol/L [49]. As for salicylic acid applied on wheat seedlings, the most effective concentration is 0.05 mM. Such concentration prevented a decrease in IAA and cytokinin levels and reduced ABA accumulation under drought and salt stress conditions [39]. In the case of basil and marjoram, the optimum concentration is 0.4 mM [35]. Studies on maize showed that the SA concentration beneficial for this crop is 0.5 mM [56].
The present study did not confirm significant differences between the ASA concentrations used. The concentration of 0.5 mM proved more effective only for such traits as plant height and protein content in the grain. On the other hand, the study showed that it is not the concentration but the time of ASA application that is more important for the growth of this plant. There were no significant differences between the effects of an earlier and later ASA application time on plant height or chlorophyll content. However, most of the structural and qualitative parameters of the yield (number of grains in the ear, grain density, grain hardness, protein, gluten and starch content) were better after later application of ASA (GS BBCH 37). The present study shows that foliar ASA application may be particularly beneficial to the growth and development of wheat that was subject to drought stress in the initial stage of the growing period. Due to the scarcity of field research on salicylic acid, the present study also has a practical dimension.
The available scientific literature points to the considerable usefulness of salicylic acid and its derivative, acetylsalicylic acid, in counteracting stress consequences in plants. However, a major research problem is determining the dosing and growth stage that would allow for safe application of these substances. The use of SA and ASA in weak plants that already grow under stress conditions may prove to be an additional stressor. Another problem is the fact that due to the genotypic distinctiveness of plants, which determines their specific phytohormone composition, the concentration (dose) and optimum time of application may depend on the individual traits of a given species and cultivar. Another factor determining the effect of these substances on a plant may be its overall condition (vigor). Thus, research on these compounds should be extended to particularly include field studies which, in contrast to studies in controlled conditions, do not exclude environmental interactions.

Conclusions
Contemporary agricultural sciences need the highest possible number of studies on crop adaptation to the changing climatic conditions. Climate change and the related, increasing occurrence of abiotic stresses, including drought, will create new possibilities for the use of substances which in extreme conditions may help to preserve a constant level of yields from key crops. Wheat is one of the most important crops in the world. At the same time, it is very sensitive to water shortage in its critical growth stages. Acetylsalicylic acid is a relatively inexpensive substance which, when used in small concentrations, may significantly contribute to limiting yield losses, especially in drought conditions. In our study, we found that the acetylsalicylic acid at a concentration of 0.5 mM is much more favorable for wheat compared to the concentration 0.3 mM. It significantly increases the yield of the crop and is particularly beneficial when is used in the flag leaf growth stage. Acetylsalicylic acid has a positive influence on the morphological and physiological processes in wheat, and substantially improves the qualitative and quantitative properties of this crop.