Mitigation of glyphosate side effects on non-target plants : use of different agrochemicals as protectants in common bean plants

Glyphosate is an herbicide widely used in agriculture. However, it may causes phytotoxic effects on non-target plants (crops), generating productivity losses. Currently there are evidences about agrochemicals as potential mitigators of glyphosate side effects. Therefore, the objective of this study was to evaluate the effects of distinct doses of fulvic acid 25% (k-tionic® 1000 and 2000 mL ha-1), amino acids (Codamin BR® 500 and 1000 mL ha-1), phosphite (Nutex 0-3020®, 2000 and 4000 mL ha-1) and biostimulant (Byozime TF® 250 and 500 mL ha-1) in reversion of fitotoxicity caused by glyphosate on ‘Carioca’ common bean plants. For comparison, plants treated only with glyphosate, and untreated plants (control) were also evaluated. Glyphosate spray (Roundup WG 0.06 kg ha-1) was performed at 31 days after planting (DAP), and agrochemicals were applied at 36 DAP. Phytotoxicity symptoms, as well as leaf area, dry mass of leaves and stems, and physiological indexes were evaluated at 54 DAP. Application of amino acid-based product (500 mL ha-1) significantly reduced (29.90%) phytotoxicity symptoms. Both doses of amino acid-based product increased dry mass of leaves; therefore, they were able to maintain the leaf weight ratio similar to the control plants. However, there was a decrease in leaf area, which was not reverted by any of the agrochemicals. It is concluded that application of amino acid-based product 1 Dra.; Engenheira Agrônoma; Funcionária da Isagro Brasil Comercio de Produtos Agroquímicos Ltda; Endereço: Rua Joaquim Floriano, 466 / Cj 1205, Itaim Bibi, CEP: 04534-002, São Paulo, São Paulo, Brasil; E-mail: chryz.serciloto@fmc.com 2 MSc.; Bióloga; Doutoranda em Genética e Melhoramento de Plantas na Escola Superior de Agricultura “Luiz de Queiroz” ESALQ/USP; Endereço: Avenida Pádua Dias, 11, São Judas, CEP: 13418-900, Piracicaba, São Paulo, Brasil; E-mail: marcia198811@usp.br 3 Dr.; Engenheiro Agrônomo; Professor Titular da Universidade de São Paulo, USP; Endereço: Avenida Pádua Dias, 11, Agronomia, Caixa Postal: 09, CEP: 13418-900, Piracicaba, São Paulo, Brasil; E-mail: prcastro@usp.br (*) Autor para correspondência.


Introduction
Tillage system is used in approximately 25 million hectares, representing 50% of the area with annual crops in Brazil.The adoption of this practice was possible due to development of herbicides, among which glyphosate is the most used (YAMADA et al., 2009).This herbicide affects shikimic acid pathway by inhibiting the synthesis of 5-enolpyruvoylshikimate-3-phosphate synthase, thus preventing the synthesis of three essential amino acids -tryptophan, phenylalanine and tyrosine (RODRIGUES et al., 1982).
The fast translocation of glyphosate from treated leaves to roots, rhizomes and apical meristems, is one of its most important features.The in vivo stability of glyphosate also contributes to its irreversible phytotoxic effects (GRUYS; SIKORSKI, 1999).In addition of drift and residual glyphosate in soil, this herbicide may be transferred from target (weed) to non-target plants (crop), through contact among roots (RODRIGUES et al., 1982).Leaf chlorosis followed by necrosis are the common symptoms due to glyphosate application.Other symptoms in leaves are wrinkling or malformations (especially in growth areas) and necrosis of meristem, rhizomes and stolons of perennial plants (GRUYS;SIKORSKI, 1999).
H owe ve r, s e ve r a l c om p o u n d s are potential mitigators of glyphosate phytotoxic effects on non-target plants.The application of sucrose 2% on coffee plants, a week after glyphosate pulverization, reversed phytotoxicity caused by this herbicide (MARTIN, 2003).The use of amino acid-based products as seed treatment and/or foliar sprays also reduced symptoms of phytotoxicity in glyphosateresistant soybean (ZOBIOLE et al., 2010).Therefore, the objective of this study was to evaluate the effects of some agrochemicals as mitigators of glyphosate phytotoxicity in 'Carioca' common bean plants.

Materials and Methods
The experiment was carried out in a greenhouse without humidity and temperature control, at Piracicaba in southeastern of Brazil (latitude 22º 42' S, longitude 47º 38' W ), from September to November.Common bean (Phaseolus vulgaris cv.Carioca) seeds were sown in 8 dm 3 plastic pots filled with clay, sand and manure in the ratio of 2:2:1 (v:v:v), respectively; and three plants remained in each pot.
Phytotoxicity was evaluated by three reviewers, who gave scores to symptomatology caused by glyphosate at 36, 40, 46 and 54 DAP.These scores were according to the scale of visual assessment of herbicides phytotoxicity proposed by European Weed Research Council (EWRC, 1964), ranging from 1 (no symptoms) to 9 (plant death).At 54 DAP, leaf area was determined through Area Meter Li-Cor 2000 equipment.Subsequently, leaves and stems were placed in an oven at 65 ± 2 °C for 4 days, to obtain the dry mass of leaves (DML), stems (DMS), and shoots (DMS = DML+DMS).The leaf weight ratio (LWR = DML/ DMS), leaf area ratio (LAR = LA/DMS) and specific leaf area (SLA = AF/DML) were also calculated.
The experiment was carried out in a completely randomized design with 10 treatments and 6 replications with three plants each.Data were subjected to analysis of variance (ANOVA) at 5% of significance level.Subsequently, Duncan's test (α≤0.05)was used to compare means among treatments.All procedures were performed through SAS ® statistical program (SAS, 2011).
All plants treated with glyphosate showed phytotoxicity symptoms, but the use of agrochemicals partially mitigated them.This reversion tended to be less pronounced with time, which indicates that other applications may be a suitable strategy to alleviate these symptoms (Figure 1).
Amino acid-based product (both doses) was the more efficient mitigator because it provided a high reduction of phytotoxicity symptoms, and also maintained the reversion during a time more long than the other agrochemicals.Probably these results were due to amino acids replaced by product, since glyphosate's negative effects are mainly due to interruption of aromatic amino acids synthesis (RODRIGUES et al., 1982) The dry mass of stems was not affected by herbicide (figure 2), but there was a decrease in dr y mass of leaves (Figure 2).Only amino acidbased product was able to mitigated this effect, indicating again that exogenous application of amino acids can reverts the side effects of glyphosate on nontarget plants.The decrease of leaf biomass was responsible by reduction of shoot dry mass and leaf weight ratio, as shown in figure 3.These effects probably were due to deregulation of shikimic acid pathway, which results in loss of carbon available (approximately 20%) for other reactions, since tyrosine, phenylalanine and tryptophan are precursors of the most of aromatic compounds in plant (FERREIRA et al., 2005).Carioca) treated with distinct agrochemicals (doses ranging from 250 to 4000 mL ha -1 ) after glyphosate application Source: Authors (2012).
The loss of biomass also can be related to increase of stomatal resistance, reduction of chlorophyll content and decrease of photosynthetic activity in leaves, which impairs the production of photosynthates (BRECKE;DUKE, 1980;TAIZ;ZEIGER, 2010;ZOBIOLE et al., 2010;CARVALHO et al., 2013).This herbicide also restricts nutrient absorption (CAKMAK et al., 2009), so there are a probability that mitigation of side effects can be due, even partially, to replacement of nutrients found in amino acid-based product, since some nutrients take part in stomatal aperture, chlorophyll molecules, and photosynthetic mechanisms (TAIZ; ZEIGER, 2010).However, all glyphosatetreated plants showed a decrease in leaf area, which was not reverted by any of the used agrochemicals (Figure 4).In fact, inhibition of leaf expansion of bean plants can start after 25 hours of glyphosate application, as demonstrated by Brecke and Duke (1980).Leaf area of coffee plants was also reduced (CARVALHO et al., 2013), and flag leaves of rice plants showed slow and stunted growth, emerging as a shortened organ (DAVIS et al., 2011).Taken together, these results indicate that reduction of leaf area is a fast and common effect caused by glyphosate.
However, leaf area ratio and specific leaf area did not varied among treatments (figure 4), this results was due to a proportional reduction in leaf area and dry mass of leaves, which provide a similar value when theses physiological indexes were calculated.

Conclusion
It can be conclude that the amino acidbased product used in this study (Codamin BR 500 mL ha -1 ) acts as protectant against the side effects of glyphosate in 'Carioca' common bean plants, because it alleviated the phytotoxic symptoms and maintained the regular biomass partition of plants treated with this herbicide.However, this agrochemical is not able to revert the leaf area reduction caused by glyphosate, therefore it can be state that the side effects of glyphosate in 'Carioca' common bean plants can be partially mitigate by Codamin BR 500 mL ha -1 .Furthermore, the doses of the other agrochemicals tested did not are efficient in the reversion of negative effects of glyphosate on 'Carioca' bean plants.

Figure 2 -
Figure 2 -Dry mass of stems and leaves of bean plants (Phaseolus vulgaris cv.Carioca) treated with distinct agrochemicals (doses ranging from 250 to 4000 mL ha-1) after glyphosate application

Figure 3 -
Figure 3 -Dry mass of shoots and leaf weight ratio of bean plants (Phaseolus vulgaris cv.Carioca) treated with distinct agrochemicals (doses ranging from 250 to 4000 mL ha -1 ) after glyphosate application

Figure 4 -
Figure 4 -Leaf area, leaf area ratio and specific leaf area of bean plants (Phaseolus vulgaris cv.Carioca) treated with distinct agrochemicals (doses ranging from 250 to 4000 mL ha -1 ) after glyphosate application