Application of Biostimulants and Herbicides as a Promising Co-Implementation: The Incorporation of a New Cultivation Practice

: Over the last decades, biostimulants have been the center of discussion as a sustainable cultivation practice to promote plant growth and protect crops from abiotic stress. Agrochemical products are abundantly used for this purpose, which has resulted in raised international concern. Biostimulants, when used in combination with herbicides in some cases, could act as safeners, reducing the harmful and stressful effects of herbicides, and as a result, this combination can be considered a relatively new agricultural technique. However, they can also have adverse or non-signiﬁcant effects, something that is strongly affected by the operating mechanisms of their components. In practice, there is a need to identify plant species-biostimulant-herbicide working systems from all the stakeholders of agricultural production. While research is still in its early stages, several studies have been conducted to evaluate various biostimulant and herbicide combinations to contribute to this goal. In this review, studies of their combination in sequential or mixed tank applications have been gathered to see how and if each speciﬁc combination can have a potential use in agricultural practice. The results indicated that there are various effects on crops, some of which were positive and others negative or non-signiﬁcant. The fact that there is a countless number of possible plant species-biostimulant-herbicide combinations to be evaluated is a challenging task. Nevertheless, this review could serve as a foundation for the upcoming research. The aim of this review is to summarize the knowledge of some successful working examples of these three factors that could facilitate the incorporation of biostimulant and herbicide application, either sequentially or in a tank mixture, as a part of the agricultural practice for ﬁeld crops.


Introduction
The use of biostimulants is increasingly incorporated as a part of the cultivation practice for many field crops.Thus, the fundamental principles of their application should be defined by all the stakeholders of agricultural production [1].It is well accepted that biostimulants have a diverse nature (from a single compound to a mixture of microorganisms), and their actions on plant processes vary (from tolerance to abiotic stress to increased N efficiency) [2,3].Given these specificities, there is an effort to identify the most efficient period to apply these products [1].A focus on the published research regarding the application period of biostimulants on field crops verifies that this period is very often close to the period of herbicide application, and a single spray application is sufficient [4].This could be explained as reasoning to assist the cultivation at the specific stage of growth, because of the research for the more effective period of application [5], or likely because of the convenience of making this application close (sequentially) or even together (tank mixture) with the basic cultivation practice of chemical weed control [6,7].However, it should be Agronomy 2023, 13, 2634 2 of 19 noted that the timing of the application is a major issue that affects the performance of the biostimulants, and it is also affected by the type of the biostimulant that is applied [8].
Especially when the biostimulants are microbial, the combined use with herbicides should examine the effect of the different chemical substances of the herbicides on the living microorganisms.It is known that many herbicide active ingredients disturb the balance of the soil microbial community, while others show no effect [9].For example, relevant research revealed that atrazine reduced the diversity of bacterial populations in soils cultivated with maize [10].Moreover, sulfonylurea reduced the abundance of bacteria in the rhizosphere of winter wheat [11], while mesotrione reduced the abundance of soil microbe [12].
Regarding the group of non-microbial biostimulants, which are predominantly organic compounds, there is also an interaction with herbicides; however, this effect is highly affected by the chemical composition of the biostimulant [13].For example, proteins of low molecular weight could increase soil microorganisms that degrade herbicides, while low molecular weight proteins have an inferior effect [14].Furthermore, the results of research indicated that the toxic effect of a mix of 2,4-D, MCPA, and dicamba herbicide on the enzyme activity and soil ergosterol was significantly reduced when biostimulants like hydrolyzed poultry feathers and wheat condensed distillers soluble were applied [15].
The use of herbicides could cause injuries to the crops, depending on the active ingredient of the herbicide, the application rate and time, and the method of the application [16].For this reason, it is very common for agricultural practice to use some compounds called herbicide safeners in order to minimize the injury that herbicides cause to the crops [16] without affecting the efficacy of the herbicides [17].Many biostimulants have the potential to induce plant tolerance to different types of abiotic stresses [18].Therefore, some biostimulants could be used as potential safeners [17].
It is worth mentioning that because of the fact that the use of biostimulants is relatively new in the agricultural sector and because of the lack of extended research, many researchers choose to control the weeds mechanically in their experimental trials in order to avoid unpredictable interactions of biostimulants and herbicides [19].
The research results of many biostimulant products have gained worldwide interest from the scientific community, as well as the directly involved crop producers, as they promise to enhance plant growth and productivity.Actually, this is reflected globally, as the biostimulants industry is estimated to reach US$4.14 billion by 2025 [20].Various definitions of biostimulants have been proposed and reviewed over the last few years [2].It is still unclear which would be the categorization of biostimulants in the future, and two possible justifications for this uncertainty are the diverse nature of biostimulants, as well as the potential of a multiple claim of function for some of them.Biostimulants contain organic or inorganic compounds and are mainly of natural origin.However, synthetic compounds, such as inorganic salts and phenolic compounds (e.g., nitrophenolates) are also used in the preparation of biostimulants as they have a beneficial effect on plants and enhance their growth [2,21].
Biostimulants are a promising strategy for improving productivity and increasing plant resistance to abiotic stress, such as low and high temperatures, drought, salinity, and nitrogen deficiency [3,22,23].Research has also shown that biostimulants can reduce the amount of mineral fertilizers in the soil and contribute to the improvement in plant growth and quality traits [24,25].Different methods of application have been recorded.For example, biostimulants containing seaweed are usually applied foliarly, while humic substances and nitrogen compounds are often used in the form of a ground application [26].The use of biostimulants is one more input for agriculture; however, this agricultural practice has no adverse effects on soil conditions and could ensure sustainability.
The potential plant species-biostimulant-herbicide combinations are practically impossible to count.The knowledge of some working systems of these three factors could contribute to the incorporation of this cultivation practice (application of biostimulants and herbicides sequentially or in a tank mixture) for the field crops (Figure 1).contribute to the incorporation of this cultivation practice (application of biostimulants and herbicides sequentially or in a tank mixture) for the field crops (Figure 1).

Biostimulants and Herbicides
One of the factors that can have a significant effect on the combined application of herbicides and biostimulants is the way their application is conducted.Separate applications (Table 1) and mixed tank applications (Table 2) of herbicides and biostimulants can induce different interactions among them.It has been noted that herbicides affect soil microbes and can have various interactions with them.A mixture of terbuthylazine, mesotrione, and S-metolachlor at high dosages had a negative impact on the soil microbiome of maize cultivation [27].The use of atrazine and diuron affected the composition of microbiota in the soil intensely compared to the 2,4-D, which was milder [28].On the other hand, many microorganisms have the ability to degrade or transform herbicides, like the bacterium Serratia marcescens that degrades nicosulfuron and other sulfonylurea herbicides [29] and some strains of Bacillus sp. that biodegrade trifluralin [30].Genera like Pseudomonas can degrade 2,4-D, fungal strains like Beauveria bassiana are able to transform diuron, while Pseudomonas sp. and Arthrobacter atrocyaneus degrade glyphosate [31].This, from a point of view, is a very useful feature of the microorganisms that could contribute to control herbicide pollution [31]; however, at the same time, it could reduce the efficiency of the herbicides and reduce the weed control.

Biostimulants and Herbicides
One of the factors that can have a significant effect on the combined application of herbicides and biostimulants is the way their application is conducted.Separate applications (Table 1) and mixed tank applications (Table 2) of herbicides and biostimulants can induce different interactions among them.It has been noted that herbicides affect soil microbes and can have various interactions with them.A mixture of terbuthylazine, mesotrione, and S-metolachlor at high dosages had a negative impact on the soil microbiome of maize cultivation [27].The use of atrazine and diuron affected the composition of microbiota in the soil intensely compared to the 2,4-D, which was milder [28].On the other hand, many microorganisms have the ability to degrade or transform herbicides, like the bacterium Serratia marcescens that degrades nicosulfuron and other sulfonylurea herbicides [29] and some strains of Bacillus sp. that biodegrade trifluralin [30].Genera like Pseudomonas can degrade 2,4-D, fungal strains like Beauveria bassiana are able to transform diuron, while Pseudomonas sp. and Arthrobacter atrocyaneus degrade glyphosate [31].This, from a point of view, is a very useful feature of the microorganisms that could contribute to control herbicide pollution [31]; however, at the same time, it could reduce the efficiency of the herbicides and reduce the weed control.

Interaction Effects on Biostimulant and Herbicidal Efficacy
In the last decade, researchers tested the application of biostimulants in a different time interval from herbicide application.Matysiak et al. [32] carried out a two-year experiment assessing the interaction of auxin mimic and acetolactate synthase inhibitors (ALS) herbicides such as MCPA + dicamba, dicamba + triasulfuron, and florasulam + 2,4-D with seaweed extracts and nitrophenols in spring wheat with an interval of three days between the applications.Their results showed that auxin mimic and ALS inhibitor herbicides, combined with seaweed extracts and nitrophenols, during the separate application, controlled the weeds and did not affect biostimulant action in most of the cases, showing great values in the yield and its parameters.
Seaweed extract and nitrophenol biostimulants have also been tested in combination with other herbicides, such as linuron (inhibitor of Photosystem II) + clomazone (inhibitor of deoxy-d-xylulose phosphate synthase) and metribuzin (inhibitor of Photosystem II) in potato [33].Herbicide application was performed 7-10 days after the planting of the tubers, while the biostimulants were implemented in two applications, the first at the emergence of the potato plants and the second 14-28 days after.The results showed that overall, the application of seaweed extract and nitrophenols was not affected negatively by the herbicides and helped the potato plants to produce a slightly higher yield.However, it is not clear yet how this interaction is explained.Zarzecka et al. [34] took this research a step further, where they evaluated also how this interaction affected protein content of potato tubers.Linuron + clomazone, with the addition of the seaweed extract, did not have significant differences from treatments, where only herbicides were used.That means that even though linuron + clomazone did not affect the biostimulant effect of seaweed extracts and nitrophenols (helping them achieve better yields), it did not had significant effects on the quality measurement of protein content.Gugala et al. [35] also found that the combinations of linuron + clomazone and metribuzin with seaweed extracts and nitrophenol biostimulants can increase the glycoalkaloids (TGA) in potatoes, when applied in separate applications.Seaweed extracts and nitrophenol biostimulants have been compared in a study that evaluates how they influence potato growth, while the weed control for this experiment was conducted using metribuzin + flufenacet (inhibitor of very long-chain fatty acids synthesis) [36].Their results varied over the years and the treatments; one possible explanation is that the herbicide substances that were used, interacted with the tested biostimulants.
Some other biostimulants and herbicide combinations have been tested on the potato.Ginter et al. [37] and Zarcecka et al. [38] evaluated the action of three biostimulants (combinations of amino acids with organic materials and N-containing substance with lactic acid bacteria and actinomycetes) with a very common herbicide in potato crop, clomazone + metribuzin.The application of herbicide was performed at BBCH 00-08, while the biostimulant's first application was at BBCH 13-19 and the second at BBCH 31-35.The combination of the inhibitor of deoxy-d-xylulose phosphate synthase and the inhibitor of Photosystem II showed great effectiveness in reducing the number and fresh weight of weeds.Especially with the amino acid-based product, which positively affected the potato's growth and its competitiveness with weeds.The total yield increased by 30%, and the share of marketable yield in the total yield increased by almost 10%.It is possible that the N that was provided via the biostimulants to the potato plants resulted in an increase in the yield, while the herbicide mechanism of action did not disrupt the biostimulant's action.This combination of biostimulants and herbicides was also found to enhance polyphenol content in potato tubers [39] but interestingly decreased glycoalkaloid concentration [40].In an experiment that utilized the application of the three aforementioned biostimulants to evaluate the possibility of a profitable implementation in potatoes, it has been found that the addition of biostimulants at BBCH 13-19 (first application) and BBCH 31-35 (second application) raised yield significantly, compared to just mechanical (16-26%) and chemical (clomazone + metribuzin) weeding (up to 13%) [41].They also noted that there is a significant profit to be made with the use of biostimulants in potato cultivation in combination with herbicides.Nevertheless, this raises the question of whether these biostimulants could have assisted the cultivation even more if the control of the weeds had been conducted in a non-chemical way and also been more cost-effective and environmentally friendly.In another experiment that was focused on the combination of sulfosulfuron (ALS), biostimu-lants, and fungicides in spring wheat, [42] it was found that the biostimulant with humic and fulvic acids combined with sulfosulfuron produced a higher yield than the control in every year.It seems that humic and fulvic acids had a positive effect regardless of the chemical application.

Effect of Biostimulants on Mitigating Stress from Herbicidal Application on Plants and Soil
It is common for herbicides to cause injuries to crops depending on the active ingredient and the dosage.Biostimulants can be used as herbicide safeners to mitigate injury effects that herbicides cause to the crops without affecting their efficacy.For example, the use of commercial biostimulant Fertiactyl Pós reduced the injuries of glyphosate on the leaves of soybeans and mitigated the negative effect of the herbicide on the yield [43].Similarly, the use of commercial biostimulant Megafol reduced the injuries caused by metolachlor herbicide on maize plants [44].In the case of imidazolinone-resistant sunflower hybrid, an amino acid bioostimulant, reduced the negative effects of imazamox application [18].Moreover, melatonin, an important animal hormone that is considered a biostimulant, could be used as a natural safener to reduce the damage from herbicides [45].
Biostimulants, for example, have been tested as sequential applications after total herbicides.In soil-focused research, Means et al. [46] evaluated the effect of beneficial chemical compounds and urea on the microorganisms in the rhizosphere of glyphosateresistant soybean after the application of glyphosate, a broad-spectrum systemic herbicide that belongs to the inhibitors of enolpyruvyl shikimate phosphate synthase (EPSP) or a combination of clethodim inhibitor of acetyl-CoA carboxylase (ACC) + fomesafen inhibitors of protoporphyrinogen oxidase (Protox).They observed that urea and biostimulant's sequential application only enhanced dehydrogenase activity in the rhizosphere, whereas it did not have a significant effect on the other enzyme activities.
The combination of plant growth promoting bacteria (PGPB) with herbicides is not a very common implementation, as it is unnatural to combine living organisms with chemical herbicidal substances.In an experiment in alfalfa, native bacteria of the rhizosphere were isolated and used to evaluate their effect on the herbicidal abiotic stress [47].In particular, bacteria Serratia rubidaea, Pseudomonas putida, Serratia sp., and Synorhizobium meliloti and their combinations were used to mitigate the stress from imazethapyr (ALS herbicide).The combinations of S. rubidaea and P. putida, Serratia sp., and S. meliloti, as well as the combination of the above three bacteria, were able to increase plant biomass, antioxidant activities, and the general microbial population.These attributes, on the contrary, were decreased when other combinations were implemented (i.e., P. putida and Serratia sp.).Chemical substances are natural to affect other organisms, even though some of the bacteria strains were able to tolerate imazethapyr and help alfalfa overcome the herbicide stress.However, many of the combinations tested produced negative results, as expected.These results prove that there is a prospect of using the right PGPB combinations to overcome herbicidal stress in alfalfa; however, much research should be conducted to find the optimal practices.
Another interesting use for biostimulants is the damage control from the herbicidal unwanted effects on the crops.Neshev et al.
[48] used a biostimulant with amino acids, chemical macro-and micro-elements, and organic substances as a medicative application to herbicide-damaged sunflower.The ALS herbicides tribenuron-methyl and imazamox were applied at the 5-6 leaf stage, while the biostimulant application followed four days later.The biostimulant application that followed the application of the tribenuron-methyl herbicide did not have significant differences in chlorophylls (a and b) and carotenoids compared to the application with no biostimulant.However, it was able to increase significantly net photosynthesis rate, sunflower head diameter, plant height, and yield.Furthermore, it increased absolute seed mass and the seed oil content probably by increasing the N-uptake of the plants.It should be noted, though, that it never fully overcame the damage from the herbicide.On the contrary, the biostimulant effect exceeded expectations after the imazamox application.In some parameters, such as seed yield, total leaf area, head diameter, and absolute seed mass, it managed to reach the levels of the untreated control.The ameliorative effect of biostimulants after imazamox herbicide application was also noticed in a field experiment in pumpkins, where plants were able to recover quickly and overcome the harmful effects of the herbicide [6].Similar results were obtained in a maize experiment where a simulation of imazamox drift effect was performed to see if the ameliorative effect of protein hydrolysates could help the cultivation overcome the herbicide stress [49].Their results show that the application of protein hydrolysates can increase yield compared to the sole application of herbicide and even reach, in some cases, the control values.Imazamox stress effect seems to be relieved when combined with various biostimulants, helping crops overcome the unwanted effects.In another experiment, three different biostimulants (nitrophenolates, seaweed extract, and amino acid derivatives) were evaluated in buckwheat cultivation to explore their influence on the negative effects of herbicides (metazachlor (inhibitor of very long-chain fatty acids synthesis)/clomazone and linuron) [50].However, none of the biostimulants demonstrated any improvement in buckwheat affected by the herbicides.It seems that the disruption of specific paths in enzyme and photosynthesis cannot be easily dealt even with the addition of biostimulants.It is natural to assume that the use of specific biostimulants as an ameliorative application in crops, after the use of different herbicides, has a huge potential and more research needs to be conducted.
The use of biostimulants as safeners to prevent excess herbicidal stress has been evaluated in wheat.Gaafar et al. [51] soaked wheat seeds with beneficial cyanobacterial extracts (Arthrospira platensis and Nostoc muscorum) and amino acid (tryptophan) to improve the tolerance/protection of wheat against the selective broadleaf herbicide bromoxynil of the Nitriles family, that was applied 55 days after sowing.Interestingly enough, the activity of the antioxidant enzymes on wheat leaves, plant height, and fresh and dry weight were increased at the application with the biostimulant-soaked seeds compared to untreated seeds in which bromoxynil was used.However, in a pot experiment, a botanical extract biostimulant was evaluated as seed treatment and postemergence leaf application in wither barley, under different concentrations to overcome the herbicidal stress of cinmethylin (inhibitor of fatty acid thioesterase) [52].Their results showed that this specific plant-based biostimulant was able to prevent excess herbicidal stress to barley's dry biomass 21 days after the seed treatment, with the application rates being insignificant.Melatonin has also been used to prevent herbicidal stress induced by paraquat application on maize, which interacts with the electron transfer components associated with Photosystem I [53].Seed priming with melatonin proved that it could demonstrate tolerance to paraquat in sweet corn by increasing the activity of the antioxidant enzymes.The use of biostimulants on seeds (coating, priming, soaking, etc.) has good potential to overcome the difficulties of herbicide stress in field crops.
In a multifactorial experiment in wheat, Iwaniuk et al. [54] evaluated, among others, the combination of herbicide sulfosulfuron with humic + fulvic acids biostimulants.Three applications of biostimulants were conducted (BBCH 31, 47, and 71), while the herbicide was applied during the wheat stem elongation stage when the second node is at least two cm above the first (BBCH 32).They found that the biostimulants, as protection from abiotic stress induced by herbicide and fungicide application in wheat, can contribute to the absorption of assimilable phosphorous and thus improve crop nutrition.In another multifactorial four-year experiment, Iwaniuk et al. [55] evaluated the effect of different combinations of chemical and biostimulant regimes on the concertation of 20 amino acids and yield characteristics of wheat.Wheat yield was increased when herbicide sulfusulfuron was applied along with the combination with the humic acid (liquid form).Specifically, in the year 2018, it reached an increase of around 17% compared to the control and sole herbicide application.In general, most of the yield traits were increased from sulfonelurea application, with or without the biostimulator.However, where only biostimulants were applied, the yield traits were not increased at all.In addition, it should be noted that the amino acid concentrations were higher on sole sulfonelurea application, while the lowest was observed during the sole liquid humic acid application.These results raise the question of whether humic acid biostimulants can only provide assistance or protection from the unwanted effects of chemical protection applications.This question is reinforced by the fact that humic acids have also been used in beans to protect the cultivation by the auxin mimic herbicide 2,4-D [56].They were applied when beans were seedlings, while the herbicide application was performed at the 3-4 leaf stage.This study's results showed that the application of humic acids was able to protect, to some extent, the adverse effects of 2,4-D genotoxic and DNA methylation.So, can biostimulants be considered and used as safeners to prevent the stress from herbicides?This needs to be thoroughly investigated in various combinations of biostimulants and herbicides in order to reach a trustworthy conclusion.
The implementation of biostimulants and herbicide applications in agriculture practices is a novel and very complicated goal.That being said, the separate application of these two can overcome some of the difficulties of their coexistence and become a valuable tool for agriculture.The seeds were soaked in solutions with biostimulant, and herbicide was applied at 5-6th leaf stage [53] n.d.= non defined.

Biostimulant-Herbicides Mixed Tank Applications 2.2.1. Interaction Effects on Biostimulant and Herbicidal Efficacy
Biostimulants and herbicides have been mostly used in sequential applications to avoid possible unfavored effects of their direct interaction.However, notable research has also been conducted when these categories were implemented as a mixed tank application.In particular, Matysiak et al. [32], besides the assessment of the interaction between auxin mimic and acetolactate synthase inhibitors (ALS) herbicides MCPA + dicamba, dicamba + triasulfuron and florasulam + 2,4-D with seaweed extracts and nitrophenols, with three days intervals between the applications in spring wheat, also evaluated their combination in a tank mixture at BBCH 30.In the case of weed control, mixed tank applications did not present statistically significant differences against the weeds that were tested.The only exceptions were the use of dicamba + triasulfuron with the addition of a seaweed extract, against Veronica agrestis and florasulam + 2,4-D in tank mixture with the seaweed extract, or the nitrophenol compound against Veronica agrestis and Viola arvensis, that were able to provide an average control of these weed species.The mixed tank application of herbicides and biostimulants did not present noteworthy results that can lead to further research.These combinations, when used in separate applications, were able to control all the weeds that were tested.
Seaweed extracts were also tested in combination with total herbicides in Roundup Ready soybean since phytotoxicity symptoms can happen even in the resistant variety [57,58].In a field experiment with RR soybean, de Andrade et al. [57] noticed that seaweed extract formulations MC Extra and Megafol, when applied as a tank mixture, can increase the yield of soybean significantly compared to sole application of glyphosate.However, the formulation MC Cream did not improve yield significantly. Usually, broadspectrum herbicides tend to present unintended stress symptoms due to their total herbicidal action.However, it seems very promising to implement biostimulants such as seaweed extracts to reinforce the glyphosate-resistant varieties.In another experiment, de Andrade et al. [58] used seaweed extract MC Extra in a tank mixture with different glyphosate formulations.Their results showed that the seaweed formulation, when applied in a higher dose, presented a higher yield than most of the glyphosate formulations that were tested; still, they were glyphosate formulations that did not affect yield significantly.Since there is an availability of different commercial formulations of biostimulants and herbicides, it is understandable that different effects may occur per combination.Giving farmers the opportunity to have different options of formulations to select in order to include them in their cultivation practice is crucial for improving their crop's productivity.

Effect of Biostimulants on Mitigating Stress from Herbicidal Application on Plants and Soil
Some biostimulants have been tested in a mixed tank with glyphosate in field crops.In a large-scale experiment that contained 37 fields of winter wheat, corn, and oats, Soltani et al. [59] evaluated the addition of two beneficial chemical compounds mixed with different herbicide applications (glyphosate, glyphosate + topramezone + atrazine, glyphosate + thiencarbazone/tembotrione, bromoxynil/MCPA) on crop injury and herbicidal efficacy.They found that the addition of the biostimulants did not affect crop injury from herbicides and only, in a few cases, negatively affected weed control.This suggests that combining herbicides with biostimulants in a tank mixture may not yield the anticipated results, which can occur when these products are used individually.How biostimulants can improve crop stress tolerance from herbicides has been tested by many researchers.Balavanova et al. [18] and Navarro-León et al. [17] evaluated the effect of an amino acid compound in a tank mixture with imazamox on sunflower tolerance to the particular herbicide.Balabanova et al. [18] found that this combination provided an increase in fresh weight, plant height, and leaf area of the sunflowers after 14 days from the application compared to sunflowers that only imazamox was applied, proving that biostimulants potentially can be used as an ameliorative application.However, they also noted that leaf gas exchange parameters decreased.Navarro-León et al. [17] found that the amino acid compound can be used to increase stress tolerance from imazamox on sunflowers.ALS herbicides such as imazamox can ameliorate stress in both separate and mixed tank applications with amino acid compounds; this might be explained by the provision of N-based compounds from the biostimulants that can provide plants with the necessary N to overcome the herbicidal stress effects.Panfili et al. [44] used a seaweed extract in a mixed tank with metolachlor to see if it can increase maize tolerance.They confirmed that maize, even with high doses of metolachlor, has benefited from the addition of the seaweed extract to the mixture.
When herbicide-induced stress is under the scope of research, it is quite crucial to evaluate the productivity of crops.Having that in mind, Bezuglova et al. [11] evaluated different dosage combinations of a humic preparation with the sulfonelurea tribenuron-methyl in a tank mixture on winter wheat production.They found that the tribenuron-methyl toxic effect on wheat was alleviated by the addition of the humic preparation in the mixture.Wheat's yield was increased in all applications, where the humic preparation was added along with tribenuron-methyl compared to the sole application of the sulfonelurea.In the second year of the experiment, humic preparation at a rate of 4 L/ha and tribenuron-methyl at a rate of 15 g/ha was the combination that performed the best among the treatments.Researchers pointed out that a possible explanation for this decrease in phytotoxicity is the enhanced phosphorus mobilization by plants via root microbiota.
There are cases, though, where biostimulants could not ameliorate the toxicity symptoms of herbicides.In a field experiment of white beans, Soltani et al. [60] used various herbicides in a mixed tank with two beneficial chemical compounds, but no significant differences were noticed in visual injury, dry weight, plant height, and yield in most of these combinations.

How the Regulatory Framework Affects Biostimulants and Herbicides Use
The excessive use of agrochemicals has adverse effects on agricultural biodiversity and is strongly associated with environmental pollution, as herbicide residues remain in the soil or end up in surface and groundwater [61,62].In that context, several international organizations and governing authorities have focused on the decrease in the use of herbicides and on a related increase in biostimulants in order to preserve agricultural sustainability.This strategy is in line with the objectives of the Sustainable Development Goals (SGDs) adopted by the United Nations (UN) in 2015 [63].The main objectives of the SDGs are to transform conventional farming and current ecosystems into climate-resilient and sustainable agricultural systems and to secure food and healthy nutrition worldwide by 2030.It should be noted that the population is projected to reach 9.8 billion by 2050 [64], and thus, there is an urgent issue of improving plant production while protecting the natural ecosystem.Although pesticide application often achieves crop protection, excessive use has a negative impact on non-target species and causes environmental problems, posing a threat to the achievement of specific SDGs such as human health, conservation of agricultural biodiversity, and clean water resources.Achieving the SDGs will contribute to the adoption of agroecological farming practices to mitigate the environmental footprint of conventional farming approaches.Hence, the reduction in herbicides and the increase in biostimulants might be of high importance.
In line with the SDGs, several governing authorities have focused on setting a specific regulatory framework to promote this strategy.The high chemical input has raised concerns globally, and thus, the European Commission presented a set of proposals in the European Green Deal (EGD), which aims at the transition to sustainable and climate-neutral agriculture [65].Sustainable Use of Pesticides (SUD) regulation is part of the EGD and highlights the soil pollution due to the reckless use of chemicals [66].Therefore, it proposes to reduce the chemical pesticide input by 50%, as well as to reduce the use of the most harmful pesticides by 2030.The European Commission supports the sustainable use of chemicals and sets high ambitions to adopt alternative ways for plant protection to maintain and improve soil health and fertility.The aforementioned changes in the use of herbicides and biostimulants have a central role in this framework.
A similar-scope policy is followed worldwide by countries such as the United States, Brazil, etc.The Food and Agricultural Organization (FAO) proposed the 10 elements of agroecology, presented between 2015 and 2019, to achieve sufficient food production and a green transition of agricultural and food systems [67].Agroecology is a holistic approach that supports the transition to sustainable agri-food systems and provides solutions to modern agricultural problems.This strategy has set long-term goals for environmentally friendly agriculture and focuses on 10 agroecological principles (efficiency, resilience and diversity of ecosystems, synergies, recycling agricultural products, improvement in rural livelihoods, food security and human health, improvement in national and global governance mechanisms and circular economy).FAO aims to raise awareness of the risk of harmful conventional agricultural practices, in order to develop sustainable and profitable farming systems, with less environmental and economic impact.Preserving ecosystems, ensuring food security, and improving efficiency in agriculture are a priority and a major challenge worldwide.These strategies aim to guide farmers and policymakers and support international cooperation to adopt greener approaches and use chemicals in a sustainable and safe way to maintain environmental security.The reduction in herbicides and the related increase in biostimulants has a central role in this effort as well.
The categorization of biostimulants is usually based on their composition; however, some other aspects of categorization have been raised during the last few years.The European Biostimulant Industry Consortium (EBIC) [68] has defined biostimulants as "containing substance(s) and/or microorganisms, whose function when applied to plants or the rhizosphere, is to stimulate natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality".Furthermore, there are some other current definitions for plant biostimulants.The new EU Regulation (2019) [69] separates the biostimulants into two basic types, the microbial and the non-microbial, and then defines five categories according to their ability to claim that they can improve (a) nutrient use efficiency; (b) tolerance to abiotic stress; (c) quality traits; or (d) availability of confined nutrients in the soil or rhizosphere".On the other hand, du Jardin [2] defined as a biostimulant, any substance or microorganism that could be used for plant growth and to improve yield and quality characteristics, regardless of its content in nutrients.The same author classified biostimulants according to their content and proposed seven categories: humic and fulvic acids, free amino acids and N-containing substances, seaweed extracts and botanicals, chitosan other biopolymers, beneficial chemical compounds, beneficial fungi and beneficial bacteria.
In the era when biostimulants were making their first steps in the newly developing market of such products and the regulatory framework was weak, in countries of the EU, including Greece, the easiest solution for the producing companies of these products was to obtain trading licenses as fertilizer products.A significant step forward has been taken in the EU with the Fertilizing Products Regulation (2019/1009), which is a framework that recognizes biostimulants.This Regulation was approved by the European Parliament and the Council of the European Union on 5 June 2019 and entered into force on 16 July 2022.In order to ensure the effectiveness of biostimulants, the Regulation specifies that every biostimulant product should clearly mention on its label what is the effect (claim) that it has on the plants or the soil.The biostimulant manufacturers should have available a conformity assessment conducted by an independent body in order to be able to use the CE-Mark on their plant biostimulant product and be able to launch it on the EU Market.After determining the framework of the certification of biostimulants, the EU moved one step further by adding the use of biostimulants as a potential "eco-scheme" that could support the future common agricultural policy (CAP).The "eco-schemes" are a new tool of the EU Commission designed to "reward farmers that choose to go one step further in terms of environmental care and climate action".Since the summer of 2023, the use of biostimulants has been a part of the new eco-schemes' actions of the CAP 2023-2027 of Greece.
The Fertilizing Products Regulation (2019/1009) apparently represents a significant step toward progress; however, the challenges are still in front of us.The scientific community has a major role in this challenge, and the results of the ongoing research should contribute and possibly give a direction to the future of the use of biostimulants.For example, there are rising concerns about how the use of microbial and non-microbial biostimulants affects and disturbs the sensitive ecosystem of the soil community.Moreover, it should be investigated and then regulated the import and use of microorganisms of exotic places and climates, as they could possibly compete with the local microorganism populations and act as invasion species.The use of indigenous species and strains of microorganisms could be a solution.Other aspects of the use of biostimulant products are their stability, the release rate of their substances, the survival rate of the microorganisms, and maybe, when we talk about microbial biostimulants, their expiration date.An also important topic is the use of biostimulants combined with other plant protection products such as herbicides, which is the topic of this review article.In the future, after a lot of research, it is possible to have information on the labels of the products regarding their ability or not to be combined in a tank mix or separate use with other products and the possibility of this use to affect their effectiveness.All of these issues are already of concern to the scientific community, and the results are updating constantly the data related to this issue.

Challenges and Perspectives
Published works that investigate the effects of biostimulants are constantly increasing, while at the same time, the results of the research are applied by many emerging small or large companies who want to be established in this new evolving market.Knowledge about biostimulants and herbicides interaction is still limited, and the situation is getting more complicated when this interaction is getting more complex by adding the plant species.
In such complicated systems, it is very common to identify synergistic and antagonistic relationships.Especially when we talk about microbial biostimulants, the use of herbicides needs to be thoroughly investigated, as herbicides, although applied in low quantities, are well-accepted that they have deleterious effects on the microbial activity of the soil [70].However, the selection of the appropriate combination of plants and bacteria could be an effective way for the remediation of herbicide-contaminated soils [71].
For a relatively new cultivation technique, such as the use of biostimulants, the main challenge is to warrant the effectiveness of the application and further increase their reliability and usage among producers [72].Field crop producers steadily and gradually increase the use of biostimulants in their cultivations, trying to adopt more environmentally friendly techniques that contribute to plant growth in a more sustainable way.A major issue for the farmers that have already decided to use a biostimulant for their cultivation is whether to make this application close (sequentially) or together (tank mixture) with the chemical weed control.However, as mentioned before, it seems practically impossible to assess and evaluate all the combinations of biostimulants and herbicides that exist.There are many research teams that try to give as many as possible answers to this question.The number of combinations is indeed one part of this challenge, but it is not the only one.Dosages, crop type, and varieties, time of application, method of application as well as the soil-climatic conditions of each region increase the variability of this challenge.Even biostimulants are usually used as combinations of substances, where synergistic or not relations occur [73].For example, soil moisture is probably an important regulator that affects PGPB-plant interactions [74].The implementation of the biostimulants at the cultivation technique assumes to address the concerns of the farmers regarding the cost of the cultivation and the same time to be more precise regarding the functions and potential usage of them [26].For this reason, it would be very useful to emerging research results with neutral or even negative findings regarding the effect of biostimulants, which could contribute to a better understanding of their functions and be more precise regarding their optimum frame of operation.
The combination of biostimulant substances and herbicides can be an important agricultural practice to overcome the difficulties that herbicides as active substances are causing to non-target crops and the environment.Implementing biostimulants that improve productivity and abiotic stress resistance while protecting crops from the unwanted effects of herbicides is a major goal for agriculture.A promising tool that could contribute to reducing the negative effects of biostimulants and herbicide coexistence is the encapsulation of biostimulants.This technique could be considered a practical and low-cost solution to protect biostimulant substances, microbial or not, and increase their stability under various field conditions [75].

Conclusions
Overall, the combination of biostimulants and herbicides of the research results so far demonstrated various effects on crops, some of which were positive and others negative or non-significant.It appears that seaweed extracts and nitrophenols do not have a negative interaction with auxin mimic and ALS herbicides, and their action makes them an interesting subject for further investigation.Additionally, these biostimulants were not affected by the use of inhibitors of Photosystem II and inhibitors of deoxy-d-xylulose phosphate synthase, making them an interesting tool as they can combined with a broader spectrum of different herbicides.The use of total herbicides with urea and other beneficial chemical compounts did not have significant effect in the enzyme activity in rhizosphere, however it did enhance dehydrogenase activity (DHA), which is a very sensitive indicator of soil fertility.Notably, the ameliorative effect of various biostimulants was very interesting, relieving the ALS herbicide imazamox stress effect.This approach of combining ALS herbicides with biostimulants that increase N-uptake has the potential for further investigation.On the other hand, the disruption of specific paths, such us the inhibition of deoxy-d-xylulose phosphate synthase, inhibition of Photosystem II, and the inhibition of very long-chain fatty acids synthesis, is more difficult to overcome and usually presents no significant effects.In general, it seems that biostimulants and herbicides are more promising to be combined as a practice in separate applications.The vast number of different implementations of these combined substances can be considered a difficulty or a challenging opportunity for researchers to explore which of these combinations can be utilized in agriculture.New biostimulant products are introduced every day, while the availability of herbicides constantly changes.It is up to researchers to find the best-suited combination and implementation way to promote crop growth, quality, and quantity characteristics and to benefit the efficacy of herbicides while also mitigating their adverse effect on non-target plants by either protecting the crop or assisting them in overcoming the damage.The implementation of such practices has the potential to stop the excessive use of agrochemicals (fertilizers, herbicides, pesticides, etc.) and readapt the agriculture practices to a more sustainable path, respecting the environment and contributing to the Green Deal goals.This manuscript constitutes a novelty for researchers and farmers, as it collects and compares some successful working examples of plant species-biostimulant-herbicide interactions that could be the foundations for further research.

Figure 1 .
Figure 1.Determination of the principal factors that play a key role in the incorporation of biostimulants in the cultivation practice of field crops.

Figure 1 .
Figure 1.Determination of the principal factors that play a key role in the incorporation of biostimulants in the cultivation practice of field crops.

Table 1 .
Examples of application of biostimulants and herbicides sequentially.

Table 2 .
Examples of application of biostimulants and herbicides in tank mixture.