Some aspects of application of pesticides and fertilizers on nutritive value and other characteristics of crop plants

SUMMARY We surveyed different aspects of the application of agrochemicals (pesticides and foliar non-standard fertilizers) on the nutritive value and other non-yield characteristics of crop plants. The survey was based on results of our own trials and studies conducted by other researchers. Various parameters of plant and seedling growth, and yield, were analyzed, as well as the chemical composition, and energetic and thermodynamic parameters of plants in order to better assess the impact of these agrochemicals on crops. The application of various agrochemicals has been found to affect the germination of seeds produced by treated plants. The most significant and most diverse results have been obtained by analyzing the yield and yield components of many different crops (field crops, fruits, vegetables), as well as their chemical composition (mineral elements, different sugars, secondary metabolites, etc.) in terms of improving their nutritive quality. It was found that in maize seedlings it occurs by changing the content of various elements, as well as polyphenol profiles and thermodynamic parameters, and the effects did not only depend on the dosage of agrochemicals but also on maize genotype. We also found that agrochemicals affected the energetic and thermodynamic parameters of individual maize plants, as well as the parameters of plant growth and yield. It was noticed that these agrochemicals greatly affected the content of microelements, starch and crude proteins in maize and barley, sugar and polyphenol contents in various fruit trees and soybean. We noted that in certain agroecological situations these agrochemicals have led to spectacular magnification of yields of different crops, but there were also situations when they did not have any positive effect on crop yield, which is discussed also in the context of results of other researchers.


INTRODUCTION
Unlike conventional fertilizers, which are used as an agricultural practice to feed plants with certain elements, their treatment with non-standard nutrients aims primarily at intensifying the metabolism of plants either by applying specific metabolites (e.g. amino acids) and phytohormones  or plant extracts containing all these substances. Also, the use of plant regulators or other pesticide-active analogues of biologically active substances, mostly at sub-phytotoxic doses, also intensifies plant metabolism and thus has a beneficial effect on crop yield and quality (Dragicevic et al., 2013). This acceleration of plant metabolism occurs as a result of increased synthesis of protective substances, more intensive uptake of some essential nutrients or the presence of externally added signaling substances. This not only affects the yield of crops in quantitative terms, but more importantly their response to specific agroecological situations of abiotic, biotic and xenobiotic stress, as well as changes in some qualitative yield parameters in terms of increasing the nutritional value of consumerized plant parts. Thus, several effects are achieved which are not necessarily related only to crop mineral nutrition, but also to increased plant resistance to stressful situations, and also to possible biofortification of crops (Dragičević & Stojiljković, 2016) in a kind of organic food production. Unlike our previous article, which only applied to maize and fertilizer activity based on brassinosteroid phytohormones (Waisi et al., 2015b), here we provide a broader overview of crops and types of non-standard fertilizers. We also discuss relevant findings in the context of application of other agrochemicals, such as pesticides and plant growth and development regulators.

MATERIALS AND METHODS
Plant growth and yield parameters are described in methods given by Waisi et al. (2015b). Thermodynamic parameters were defined according to Sun (2002), and their calculation is described in more detail by Waisi (2016) and Waisi et al. (2017b). Quantification of total polyphenols, starch, sugars, crude proteins and oils contents is based on methods described by Waisi et al. (2015b), while a more detailed description of the methods for quantifying total polyphenols, proteins and polyphenolic fractions, and the antioxidant capacity of plant tissue is given by Đurović et al. (2019), and quantification of individual sugars is described by Waisi (2016). Element quantification was performed by the AAS method, as in Waisi et al. (2015b), which was described in more detail by Waisi et al. (2017b).

SURVEY OF RESULTS AND DISCUSSION
Ex-Yugoslav researchers had earlier noticed that the application of different pesticides affected nitrogen metabolism in seeds and seedlings of various cultivated plants (Štrbac, 1971;Janjić, 1975), and resultingly the germination of such seeds and seedling vigor, which has been repeatedly confirmed in later studies (e.g. Marinković et al., 1999). Also, it was noticed that some phytohormones and plant growth regulators can affect the phytotoxic process triggered by different herbicides (Nikolić et al., 2010a), indicating a possibility of their sophisticated application in terms of new formulations of so-called safeners. An overview of these aspects of the application of phytohormones as pesticides, plant growth regulators or fertilizers was explained by Nikolić et al. (2015).
In an earlier study (Nikolić & Waisi, 2012), we examined the results of micro-trials set up in two apple orchards (Obrenovac and Šid) located in northern parts of Serbia in 2011. The plots in those micro-trials were subjected to different treatments: 1) half and full doses of mancozeb and tebuconazole as two positive controls, 2) 24-EBL-based preparation combined with half the usual dose of mancozeb and tebuconazole; 3) non-standard fertilizes based on amino acids combined with half the usual dose of mancozeb and tebuconazole; and 4) plant extracts combined with half the usual dose of mancozeb and tebuconazole. We evaluated the usual parameter of fruit yield as a result of the influence of these different treatments, and determined the contents of reducing sugars in extracts of apple fruit pulp. In one trial (Obrenovac), the evaluated yield/ha of 24-EBL-treated apples was the same as in control plots, and pomological and fruit quality parameters of apples were comparable (Table 1). In another trial (Šid), the evaluated yield/ha of 24-EBLtreated apples was almost a quarter more than the apple yield from control plots (treated with half or full dose of fungicides) and the other treatment plots, also with comparable pomological and fruit quality parameters of apple fruits (Table 2).
We also assesed the efficacy of these procedures in protecting apple leaves and fruits from the notorious phytopatogenic fungus Venturia inaequalis (Stevanović et al., 2012). From the aspect of plant protection, our procedures were satisfactory with 78.71% and 77.69% efficacy of 24-EBL+half fungicide dose treatment of leaves and fruits (against 84.17% and 87.90% efficacy achieved by full fungicide dose) in Obrenovac. We got similar results in Šid, which is also satisfactory (data not shown). Our results are very similar to findings reported by other researchers (Khripach et al. 2000).
A similar trial was set up in an apple orchard near Belgrade with the application of different non-standard fertilizers (Waisi et al., 2014) and also with similar results as in the study by Nikolić and Waisi (2012). In experiments conducted near Smederevska Palanka (Nikolić et al., 2010b) we treated apple and tomato crops with several non-standard fertilizers based on phytohormones, aminoacids and plant extracts. The influence of these non-standard fertilizers on crop yield was variable, but we also detected a significant and very changeable effect of these fertilizers on seed germination following treatment of tomato plants.
We also examined the influence of non-standard fertilizers on yield and yield components of soybean and barley arable crops.
During the vegetation season 2012 we treated two soybean genotypes, one standard ("Nena"), and another with a low content of Kunitz-trypsin inhibitor protein ("Laura"), with non-standard fertilizers as a type of biofortification. This approach revealed a small impact by alterations in P phy (content of phytic phosphorus), which is an important factor in restraining the availability of mineral nutrients (Table 3). The first thing we noticed was that as the content of P phy decreased (Table 3), Zn content in soybeans grains increased (Table 5). Moreover, the influence of β-carotene was significant for the availability of mineral nutrients (Table 4), but more importantly its increase was linked with parallel Fe increase, mainly in grain of higher weight as part of a better yielding potential (Table 5). Table 3. The effects of non-standard foliar fertilizers on grain yield and contents of total phosphorus (P tot ) and phytic phosphorus (P phy ) in grain of two soybean cultivars (from: Dragičević et al., 2016b) Treatment Grain yield (t ha -1 ) P tot (g kg -1 ) P phy (  It is important to underline that ratios between P phy and β-carotene (as factors influencing the bioavabiality of minerals) and different mineral nutrients ( Mg, Fe and Zn) could be altered to some degree by applying non-standard foliar fertilizers, but these relationships also depend on soybean variety. The applied 24-EBLbased preparation and the plant extract ("Zircon") were efficient in decreasing the mentioned ratio in "Nena" grain, while some plant extracts ("Zlatno inje" and "Zircon") were efficient for "Laura" (Table 6).
Also, a correlation between 1000 grain weight (as a significant yield component) and the content of β-carotene and Zn in soybean grain was very significant (Dragičević et al., 2016b). In addition, we conducted a PCA statistical analysis of the relationships between P phy , β-carotene and mineral elements (Table 7).
In the late winters of 2013 and 2014, we sowed hull-less barley (Hordeum vulgare L. var. nudum; cv. "Apolon"), and during the coming spring of both years we treated the crop with a 24-EBL-based preparation and other non-standard fertilizers (based mainly on plant extracts and other phytohormones). After summer harvest we assessed the yield (at 14% grain moisture content; kg ha -1 ) and determined different chemical ingredients in barley grain using standard methods (Tables 8 and 9). The results (Dragičević et al., 2016a) indicated that the year affected barley grain yield and its chemical  composition, and the highest impact was found for Si under unfavourable conditions (data not shown). The applied treatments had the highest effectiveness on grain yield and increased grain quality, mainly through a reduction in P phy / β-carotene ratio (Table 9) and increase in GSH content (data not shown), thus increasing potential bioavailability of the examined mineral elements. What is more, abiotic stress caused by high precipitation amounts (in 2014) could be mitigated by applying a fertilizer, i.e. by increasing potential bioavailability of P, Mg, Ca and Fe (Table 9). Generally, the 24-EBL preparation influenced the content of P i , Zn and Fe, and the other fertilizers mainly affected potential availability of some other mineral elements and physiologically active ingredients (Ca, Mn, Si and GSH).
In previous field trials, carried out in one fruit (apple) and two field crops (soybean and barley), we showed that the preparation based on 24-EBL, considering other nonstandard fertilizers, affected not so much the yield as the quality and chemical composition of crops (Nikolić & Waisi, 2012;Dragičević et al., 2016aDragičević et al., , 2016b, acting protectively to the crop under stressful conditions (Stevanović et al., 2012).
Comparing these results with our previous studies conducted on maize treated with brassinosteroid phytohormones, i.e. reviewing the effects of brassinosteroids on germination and seedling growth (Waisi et al. 2015a(Waisi et al. , 2017a, and growth of whole plants and yield of maize in the field (Waisi et al., 2015b;Nikolić et al. 2018) by monitoring changes in chemical composition and energy and termodinamic parameters, we inferred that these are essentially related phenomena, which can be treated as specific forms of crop biofortification (Dragičević & Stojiljković, 2016), and thus as improvements in the nutritional quality of food obtained from plants so cultivated.
The mentioned concept of biofortification (Dragičević & Stojiljković, 2016) was further tested in a soybean crop to examine the impact of some non-standard fertilizers on the content of various polyphenolic fractions, as well as proteins, and their antioxidant capacity (Đurović et al ., 2019). It is known that various physico-chemical methods (heat and chemical treatment, centrifugation, exposure to electric and/or magnetic fields, etc.) are used in the processing of plant raw materials to produce food, which increases the availability of various nutrients previously existing in plant components (Đurović et al., 2018), which prompted us to combine the two approaches.
Our results (Figure 1) are in agreement with results of a previous study by Aludatt et al. (2013), in which the highest amount of phenolic acids in soybean seed was detected in free soluble form. Aludatt et al. (2013) also confirmed that ferulic acid and p-coumaric acid were the predominant phenolic compounds in full-fat soybean and in defatted soybean meal, which is similar to the results of our study (Đurović et al., 2019). Kim et al. (2016) confirmed that p-coumaric and ferulic acids were strongly bound to cell components in soybean, and released after alkaline hydrolysis. This result demonstrated that some phenolic acids were synthesized as macromolecular bound components (i.e. polyphenol, tannin, and lignin) to support structural cell wall development. Chlorogenic, caffeic and p-coumaric acids are intermediates in lignin biosynthesis, while ferulic acid is also probably linked to polysaccharides, lignin, and suberin (Boerjan et al., 2003;Dixon & Paiva, 1995). The smallest amounts of phenolic acids are present in the bound fraction, which is also consistent with the findings of Aludatt et al. (2013).
Since antioxidants may act through different mechanisms, the antioxidant activity of soybean flour extracts was determined using different assays, DPPH, FRAP and BR (Briggs-Raucher potentiometric titration). All examined plant extract-based products expressed significant alterations in total phenol content ( Figure 1) and antioxidant activity of the soybean flour extracts. An exception was treatment with "Cropmax" amino acid fertilizer, which only led to a decrease in TPC ( Figure 1) and antioxidant activity, determined by DPPH and FRAP methods ( Figure 2). All other treatments showed positive influence on the TPC, DPPH and FRAP (Figures 1 and 2), which is consistent with several other studies (Danilčenko et al., 2017;Verkleij, 2012) showing positive effects of biofertilizers (based on plant extracts) on yield, growth and antioxidant activity of different plant species.
The BR reaction method provides a "bigger antioxidant picture" and can also show synergistic effects (Milos & Makota, 2012). Therefore, the results obtained by the BR reaction method (Figure 3) demonstrate synergistic effects (phenols + proteins) on soybean treated with the "Cropmax" amino acid fertilizer, and show a much more pronounced effect than the fertilizers "Calbit-C" and "Zircon", based on plant extracts.
This indicates that it is not only the phenol content that participated in the inhibition of oscillatory regime as other species (such as proteins and some ions) also influenced the BR reaction, possibly building and/or stabilizing macromolecular structures of plant cells. In our study, the most positive influence on TPC, DPPH, FRAP data, and in the Briggs-Rauscher reaction method too, was exerted by "Calbit-C", which contains water-soluble Ca-lignosulphonate. This is consistent with the fact that Ca 2+ in the form of functional Capectate positively affects cell wall status (as well as many polyphenol compounds), which makes plants more resistant to different stressors. It also plays an important role in stabilizing the bond of phospholipids and cell membrane proteins, thus maintaining their functional stability (Murayama et al., 2016;Hepler, 2005). PCA analysis is a very useful tool to reduce a large number of variables to a small number of composite variables (main components). It explains the variability of data in a most concise manner. It reveals hidden connections and interdependence of data. The results (Figure 4) of this study indicate that the "Cropmax" amino acid fertilizer induced the h ighest variability in caffeic acid concentration, and somewhat less in the concentrations of gallic and chlorogenic acids. The "Calbit-C" and "Zircon" plant extract fertilizers were responsible mainly for variations in concentrations of trans-cinnamic, gallic and chlorogenic acids. The other treatments (particularly "Ekofus" plant extract fertilizer and control) provoked no variation in the investigated phenolic acids. It means that the application of plant extract-based products affects not only yield, but the chemical composition of grain too. Accordingly, phenolic composition and concentration can be altered in a specific way (Taie et al., 2008;Konopka et al., 2012).
Another important group of biologically active compounds in soybean seed are proteins. In this study, we have shown that using different types of natural products based on plant extracts can affect protein (and oil) contents in soybean seed, and the best effect was achieved with the "Cropmax" amino acid fertilizer ( Figure 5). This was expectable since "Cropmax" contains about 2% amino acids, which have proved to have positive effect on nitrogen metabolism and increase the content of raw proteins in plant material (Liu & Lee, 2012;Dromantiene et al. 2013). The synergistic effect of proteins and polyphenols on the antioxidant capacity of soybean meal was also observed using the Briggs-Raucher method (Figure 3).