Prevention of yield losses caused by glyphosate in soybeans with biostimulant

Enhanced selectivity can be achieved for some herbicide treatments through the application of chemicals that reduce or protect plants from injuries. The objective of this work was to evaluate the potential to prevent herbicide yield losses in glyphosate-resistant soybean by the use of the biostimulant Fertiactyl PÓS. Based on results from two field experiments, the association of Fertiactyl PÓS with glyphosate applied post emergence of soybeans reduced foliar injuries and prevented yield losses caused by the herbicide, either when glyphosate was applied in one single application or when it was applied in two sequential applications. However, the application of the Fertiactyl PÓS was not enough to assuage the injuries caused by the mixtures of glyphosate+lactofen and glyphosate+chlorimuron-ethyl.


INTRODUCTION
Weed control is intended to reduce or eliminate interference with crop of interest.The development of glyphosate-resistant (GR) genetically modified soybeans resulted in an alternative chemistry for farmers to deal with emerged weeds during crop cycle, mainly due to the wide spectrum of glyphosate, its systemic effect and economic viability (Ferreira et al., 2013).Despite the large adoption of GR crops and the substantial increase in glyphosate use, tolerant and resistant weeds still pose a challenge in weed management, requiring, many times, multiple sequential applications or the association of glyphosate with other herbicides (Ferreira et al., 2009).
Such herbicide treatments are usually more prone to cause injuries in soybeans due to the combined effect of herbicides, leading to decreased selectivity for the crop (Serra et al., 2011).Many farmers have observed that some GR soybean cultivars exhibit visually perceived injuries shortly after post emergence applications of glyphosate (Santos et al., 2007;Zablotowicz and Reddy, 2007).A typical symptom observed in the field after glyphosate application on GR soybeans is the yellowing of the upper leaves known as "yellow flashing".
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License a result of direct damage to chlorophyll by glyphosate (Reddy et al., 2004;Zobiole et al., 2010aZobiole et al., , b, 2012) ) or immobilization of Mg and Mn (Zobiole et al., 2010a, b), nutrients required for chlorophyll production and function.The main metabolite of glyphosate, aminomethylphosphonic acid (AMPA), may also cause injury to GRsoybeans treated with glyphosate and contribute to chlorosis (Duke et al., 2003;Reddy et al., 2004).Previous studies have also demonstrated that glyphosate or one of its metabolites can also directly affect nodulation (De Maria et al., 2006;Zobiole et al., 2010c).Reduced nodulation is associated to the direct effect on symbiotic species and/or to Ni depletion (Zobiole et al., 2010c), since Ni is both an essential element for microbial nitrogen fixation (Ureta et al., 2005).Ni deficiency symptoms in soybeans may also include chlorosis, due to lower absorption of iron.
Biostimulants are substances and/or microorganisms used to potencialize plant growth, usually enhancing the plant's ability to assimilate applied nutrients or providing benefits to plant development.The definition of plant biostimulants is still evolving, which is partly a reflection of the diversity of inputs that can be considered stimulants (Calvo et al., 2014).
Recent reviews support the potential of different types of biostimulants to improve plant biomass, crop yield and resistance to multiple types of stress (Calvo et al., 2014;Nardi et al., 2016).So far, no focus has been given to the hypothesis that biostimulants may also have an impact on preventing herbicide toxicity on cultivated species.
Herbicide application represents an abiotic stress, due to secondary effects related to metabolization in plan tissues and to the recovery from physiological injuries.The selectivity of some herbicides may, therefore, be enhanced with biostimulants.Fertiactyl Pós™ is a foliarapplied product containing a patented biostimulant complex, and its technology enables rapid and efficient nutrient absorption by the plant, ensures an active transfer of nutrients through the plant and stimulates physiological activity in the areas of stress resistance, root development and increased photosynthesis (Santos et al., 2015).
As mentioned earlier, this study aimed to evaluate the potential of a biostimulant to prevent damages in soybean plants caused by post emergence applications of herbicides.
Two simultaneous experiments were carried out, the first with a single application of the herbicide treatments (Experiment 1) and the second one with two sequential applications (Experiment 2).Treatments for both experiments are described in Table 1.
Due to the proprietary nature of the product (Fertiactyl Pós™) investigated, detailed information cannot be present on its overall composition.Its composition includes 1% of manganese complexed by heteroatom-stabilized complex (HSC) and humic and fulvic acids from seaweed extracts, but its mode of action is currently unknown.
The post emergence application of treatments in experiment 1 was performed when soybean was at V4/V5 stage (4 to 5 trifoliates leaves), and environmental conditions for the application were average (avg.)temperature of 23°C, average air moisture of 70%, wind speed of 1.0 km h -1 , moist soil and clear sky.
The first post emergence application of treatments for experiment 2 was performed when soybean was at V2/V3 stage (avg.temp.= 24°C, avg.air moisture = 69%, wind speed = 1.1 km h -1 , moist soil and few clouds) and the second sequential application was performed at V5 (avg.temp.= 24°C, avg.air moisture = 75%, wind speed =1.2 km h -1 , moist soil and cloudy sky).
For all applications, a CO2-pressurized backpack sprayer under constant pressure (2.46 kgf cm -2 -35 PSI) equipped with three XR 11002 flat-fan tips was utilized, providing an application volume of 200 L ha -1 .Both experiments were kept free of weeds by periodical hoeing.
Each experimental unit consisted of eleven 4-m long soybean rows.In the evaluations, 0.5 m of each end of the plots and the border rows were not considered, totaling an effective area of 12.1 m 2 .Climatic data (temperature and precipitation) during the field conduction of this work are as in Figure 1.
In experiment 1, soybean injuries were evaluated at 7 and 14 days after application (DAA) through the visual scale proposed by the European Weed Research Council (EWRC, 1964), where 1 means no damage and 9 means death of all plants.Plant height evaluations were also performed at 7, 14 and 42 DAA, by measuring 10 plants per plot from soil to the insertion of the last fully expanded trifoliate.In experiment 2, soybean injury evaluations were performed at 7 and 14 days after the first application (DAA-A) and at 7 and 14 days after the second sequential application (DAA-B).Plant height evaluations at 14 DAA-A and at 7, 14 and 42 DAA-B were also conducted.
To determine soybean grain yield, all plants in the effective area were manually harvested (03/04/14), threshed, and the grains were later separated from impurities and weighed.Samples for moisture determination (portable moisture analyzer model Mini GAC) were taken from each plot and grain moisture was corrected to 13%.Harvest from each plot was sampled in triplicates to perform the hundred-grains weight (HGW).
For both experiments, the experimental design was randomized blocks with seven treatments and four replications.The plots were arranged with "twofold" checks, that is, with two adjacent, nontreated checks for each treated plot.Such field arrangement has been previously used and detailed in other previous selectivity screenings (Fagliari et al., 2001;Constantin et al., 2007).The focus of all comparisons in this work is between each treatment versus non treated "twofold" checks (control) rather than comparison among herbicide treatments.All data were subjected to analysis of variance and the averages of the significant variables were compared by the F-test (p ≤ 0.10).(Tables 2 and 3), featured by the typical yellowing of the upper leaves.Symptoms were light with GLY or GLY+FP but more evident with GLY+LAC, GLY+CH, either with or without FP (Table 2).Some GR soybean cultivars are unequivocally injured by glyphosate (Zablotowicz and Reddy, 2007).However, injuries vary depending on the soybean cultivar, on the dose, on glyphosate formulation, and on environmental factors (Zobiole et al., 2011).The duration of the yellowing depends on the plants ability to restore adequate levels of these elements by root or foliar uptake (Jolley, 2004;Eker et al., 2006).

Glyphosate application resulted in soybean injuries
After one single application of herbicides, visual injuries were quickly overcome and no more symptoms were found at 14 DAA (Table 2).In experiment 2, herbicide treatments produced a similar pattern of soybean injury after first (7 DAA-A) and second (7 DAA-B and 14 DAA-B) applications (Table 3).
Application of lactofen and chlorimuron-ethyl associated with glyphosate caused more severe injuries in soybeans, with development of necrosis beyond chlorotic spots on leaves.The addition of FP to these herbicide mixtures provided no attenuation of such visual injury symptoms, both by one (Table 2) and by two sequential applications of herbicides (Table 3).
For single applications (Exp.1), the only treatment that affected soybean growth consistently in more than on evaluation was GLY+LAC (Table 2).For sequential applications (Exp.2), all treatments containing either lactofen or chlorimuron consistently affected plant height after second application (Table 4).Previous selectivity works with herbicide mixtures containing glyphosate have also reported that lactofen may cause more severe soybean injuries as compared to other herbicides (Almeida Jr. et al., 2010;Alonso et al., 2013).Addition of FP to GLY+LAC prevented the slower growth observed after one single application of herbicides (Table 2), but it was not effective for sequential applications of GLY/GLY+LAC and GLY/GLY+CHL (Table 4).
Since the second application in experiment 2 was performed in a more advanced stage of the crop development, there could have been an insufficient period of time for the crop to recover its vegetative growth before the change of sink-source balance due to the pods and grains formation, even with the use of FP.
Soybean grain yield decreased by 6 to 9% with a single application of GLY and with all mixtures containing glyphosate except GLY+FP.Use of FP alone did not provide any increase in soybean yield, but its use in tank mixture with glyphosate prevented reduction of crop yield observed in treatment GLY.No effect of treatments was found for HGW (Table 5).
A similar effect of glyphosate in soybean yield was found for sequential applications of herbicides in experiment 2. Two sequential applications of glyphosate (GLY / GLY) reduced crop yield in ≈10%, but the inclusion of FP in both applications (GLY+FP / GLY+FP) prevented yield losses.
The effect of FP is probably not directly related  lactofen and chlorimuron-ethyl found in both experiments in this report has also been described by several authors (Santos et al., 2007;Alonso et al., 2010;Zobiole et al., 2010cZobiole et al., , d, 2011)).Lower selectivity in these cases was attributed to combined negative effects on nodulation, soil microbiota, photosynthesis and efficient use of water in the soil, as well as in changes in levels of some nutrients present in the leaves and less vegetative growth.The application of FP in combination with glyphosate and the sequential applications of GLY+FP / GLY+FP were treatments that prevented yield losses caused by glyphosate.However, there was no prevention of yield loss due to the application of GLY+LAC and GLY+CHL in combination with FP in both experiments.The difference between both situations could be the type of injuries caused by the herbicide treatments, more transitory for GLY alone but more long-lasting for the necrotic lesions on leaves caused by GLY+LAC and GLY+CHL (Alonso et al., 2013).
Since the mode of action of FP is unknown, all theories to support the preventive damage effect are speculative.However, one possible explanation could be the fact that some biostimulants can enhance photosynthesis (Giannattasio et al., 2013) what could serve as a reparative effect of the already reported reduction of GR soybean photosynthesis following application of glyphosate (Zablotowicz and Reddy, 2007;Zobiole et al., 2011;2012).
Another possibility is that FP components such as humic and fulvic acids may have an effect on amelioration of the abiotic stress caused by glyphosate application.The stimulatory effects of humic and fulvic acids in some studies result in enhanced tolerance to salinity stress (Ertani et al., 2013) and drought tolerance (García et al., 2012;Anjum et al., 2011).Furthermore, humic substances have been reported to enhance some aspect of growth in several species of plants, including soybeans (Calvo et al., 2014).

Figure 1 .
Figure 1.Daily precipitation, maximum temperature and minimum temperature during the period when the experiments were carried out.Mandaguaçu (PR), 2014/2015.