Interaction between saflufenacil and ammonium glufosinate to control ryegrass

Interação entre saflufenacil e glufosinato de amônio no controle do azevém

Abstract

Annual ryegrass (Lolium multiflorum Lam.) is one of the main weeds in subtropical cropping systems of Europe, Oceania and South America. Therefore, the hypothesis of this work is that the interaction between ammonium glufosinate and saflufenacil can be synergistic for ryegrass control. Our main goal was to evaluate the effects of the mixture of saflufenacil with ammonium glufosinate in the control of ryegrass by graphical analysis of isobologram and a surface response graphic in addition to the Colby’s test. Two experiments were carried out, in greenhouse (isobolograma experiment) and field conditions (Surface response and Colby). The behavior of the mixture between saflufenacil and ammonium glufosinate was a synergistic interaction for the control of ryegrass by the isobologram test on the dry matter variable . By the Colby test, it was confirmed that at the dose of 400 g ha^-1 of ammonium glufosinate and at the doses of 10.5, 21.0 and 31.5 of saflufenacil the effect was synergistic for control of ryegrass for as control variables (%).The mixture between these two herbicides has synergistic potential to increase ryegrass control, mainly with a high proportion of ammonium glufosinato over saflufenacil.

Keywords:
Lollium multiflorum; isobologram; Colby’s test

Resumo

O azevém (Lolium multiflorum Lam.) é uma das principais plantas daninhas em sistemas de cultivo subtropical e temperado. Diante disso a hipótese desse trabalho é que a interação entre saflufenacil e glufosinato de amônio pode ser sinérgica para controle de azevém promovendo controle mais eficiente. O objetivo do trabalho foi avaliar os efeitos da mistura de saflufenacil com glufosinato de amônio no controle de azevém por análise gráfica de isobolograma e superfície resposta além do teste de Colby. Foram conduzidos dois experimentos, em casa de vegetação e campo, avaliando as misturas de glufosinato de amônio e saflufenacil. Através do método de elaboração de isobolograma em estufa e do método de colby em conjunto com uma superfície resposta a campo. O comportamento da mistura entre saflufenacil e glufosinato de amônio foi de interação sinérgica para o controle de azevém pelo teste de isobolograma na variável de matéria seca. Já pelo teste de Colby, verificou-se que na dose de 400 g i.a ha^-1 de glufosinato de amônio e nas doses de 10,5; 21,0; e 31,5 de saflufenacil o efeito foi de sinergismo para controle de azevém para as variáveis de controle (%). Ou seja, a mistura entre esses dois herbicidas tem potencial sinérgico para aumentar o controle de azevém, principalmente com uma maior proporção de glufosinato de amônio sobre saflufenacil.

Palavras-chave:
Lollium multiflorum; isobolograma; teste de Colby

1. Introduction

Ryegrass (Lollium multiflorum Lam.) is one of the main weeds in subtropical cropping systems. It is a problematic species in winter and summer crops (Oliveira et al., 2021). It constitutes the main weed in wheat (Triticum aestivum L.), promoting yield unitary losses between 0.19 and 0.58% (Galon et al., 2019). Another characteristic of the species that implies in the difficulty of control in Brazil are the multiple cases of herbicide resistance, in mechanisms such as acetolactate synthase (ALS), 5-enolpyruvylshikimate-3-phosphate (EPSPs) and acetil co-a carboxylase (ACCase) inhibitors are known (Heap, 2022).

One of the main alternatives for managing herbicide-resistant weed biotypes is the use of different mechanisms of action (Norsworthy et al., 2012). To control the ryegrass biotypes with resistance mentioned above, non-systemic herbicides have been used to manage in the pre-sowing burndown of winter or summer crops, one of the main ones being paraquat (Schneider et al., 2015). However, from 2021 to beyond, paraquat cannot be used in Brazil (Albrecht et al., 2022), and producers must use alternatives to the herbicide. Among the herbicides used as alternatives to paraquat, glufosinate ammonium and saflufenacil stand out, depending on the target species.

Ammonium glufosinate is a mechanism of action herbicide of glutamine synthetase (GS) inhibitors (group 10 HRAC), classified as a non-selective herbicide, except in transgenic crops (Takano and Dayan, 2020). Saflufenacil, on the other hand, is a protoporphyrinogen oxidase (PPO) (group 14 HRAC) inhibitor herbicide, controlling mainly eudicotyledonous weeds (Grossmann et al., 2010). There are reports of low control efficiency of these herbicides for the control of monocotyledonous weeds, mainly from the Poaceae family such as ryegrass (Kumaratilake et al., 2002; Soltani et al., 2020). In this sense, these herbicides need mixtures to broaden the spectrum or promote an increase in the control of certain weed species.

In addition to increasing efficiency and control spectrum, mixing herbicides can be a practice for weed resistance management (Evans et al., 2016). However, knowing the effects of mixtures of different herbicides is essential for successful management. When two compounds are mixed there can be three types of interactions: synergism, antagonism and additivity.

Synergism occurs when the mixture provides greater control than expected by the action of the products applied in isolation, whereas antagonism is characterized by the decrease of the expected effect due to the interaction between the products. Meanwhile additivity occurs when there is no influence of the binary mixture on the alone result (Hernández et al., 2017). One of the main ways of testing and quantifying these interactions can be done by comparing their mixed and isolated effects (Colby, 1967), which is one of the simplest and easiest methods to reproduce. Another way to evaluate mixtures is the graphical analysis of isobolograms, where binary mixtures of compounds are evaluated in order to observe the interaction behavior in relation to the isobole line (Ritz and Streibig, 2014).

Therefore, the hypothesis of this work is that the interaction between glufosinate ammonium and saflufenacil can be synergistic for ryegrass control, promoting a more efficient control. The objective of this work was to evaluate the effects of the mixture of saflufenacil with glufosinate of ammonium in the control of annual ryegrass by graphical analysis of isobologram and surface response in addition to the Colby’s test.

2. Materials and Methods

Two experiments were carried out, the first being conducted under greenhouse conditions in the city of Santa Maria (experiment 1), Rio Grande do Sul and the second under field conditions in the year 2020 in the city of Cruz Alta (experiment 2), Rio Grande do Sul.

2.1. Experiment 1

The experiment was carried out in a completely randomized design with four replications. The experimental units were pots with a volumetric capacity of 0.2 L, filled with soil classified as greyish brown argisol (Santos et al., 2018) containing one ryegrass plant per pot.

The experiment consisted of 36 treatments, consisting of five proportions (0:100, 25:75, 50:50, 75:25, 100:0) of seven doses of mixtures between saflufenacil herbicides (Heat^®, Basf, Germany) and ammonium glufosinate (Finale^®, Basf, Germany) (Table 1). The treatments were applied in an automatic application sprayer (Model III, DeVries, United States), with XR 110015 flat-fan nozzles (TeeJet, USA), calibrated for an application with a carrier water volume of 150 L ha^-1, working pressure of 30 Kpa and displacement speed of 3.6 km h^-1. The application was carried out when the ryegrass plants were at the stage of three expanded leaves.

Table 1
Proportions of doses for ammonium glufosinato and saflufenacil used in graphical analysis of isobolograma.

Proportion (S:G1)	ammonium glufosinate (g i.a. ha-1)
	d/16	d/4	d	2d	4d	8d	32d
0:100	37.5	150	600	1200	2400	4800	38400
25:75	28.125	112.5	450	900	1800	3600	28800
50:50	18.75	75	300	600	1200	2400	19200
75:25	9.375	37.5	150	300	600	1200	9600
100:0	0	0	0	0	0	0	0
	saflufenacil (g i.a.2 ha-1)
0:100	0	0	0	0	0	0	0
25:75	0.5469	2.1875	8.75	17.5	35	70	560
50:50	1.0938	4.375	17.5	35	70	140	1120
75:25	1.6406	6.5625	26.25	52.5	105	210	1680
100:0	2.1875	8.75	35	70	140	280	2240

• ¹
  S = saflufenacil, G = ammonium glufosinate;
• ²
  Active ingredients.

The variables evaluated in the experiment were injury (%) at 10 and 20 days after treatment (DAT) and shoot dry weight at 20 DAT. The control was evaluated on a percentage scale in which zero indicates absence of lesions and 100% means the death of the plants. The dry weight was performed by cutting the aerial part of the ryegrass plants close to the ground, the material being placed in an oven for 72 hours at 60 °C and then weighed on a precision scale.

For statistical analysis and for making the graphs, the R software (R Core Team, 2020) and the drc package (Ritz et al., 2015) were used. First, the ratios were observed in the form of dose-response curves using the four-parameter logistic model (Equation 1):

$$y=a-c/{\left[1+\left(x/E{D}_{50}\right)\right]}^b$$ (1)

where: y = is the response variable (percentage of control or Dry weight); x = doses of herbicides; a is the maximum point of the curve; c represents the minimum point; ED₅₀ is the dose that provides a 50% reduction in the y-variable response; and b is the slope of the curve. For a better fit of the data, parameter c was limited to 0.

All models of proportions in each variable were analyzed using the lack-of-fit test (p>0.05). After rejecting the model, the variables were submitted to graphical analysis of the isobolograms. The assessments of the different proportions were calculated based on the ED₅₀ values. In the previous statistical model, it was assumed that the “zero” dose is the same for all associations, therefore the parameter c is independent in associations. The isobologram analysis was performed by adjusting the additive concentration model.

2.2. Experiment 2

An experiment was carried out under field conditions, in the site of Cruz Alta, in a randomized complete block design with four replications. The experimental units consisted of 3 x 3 m plots, totaling 9 m². The ryegrass plants occurring in the area came from natural reseeding, and the soil in the area was classified as Dystrophic Red Latosol (Santos et al., 2018).

The treatments were arranged in a factorial scheme, with factor A doses of the herbicide glufosinate ammonium: 0, 200, 400 and 600 g i.a. ha^-1. And factor D doses of saflufenacil herbicide: 0, 10.5, 21.0 and 31.5 g i.a. ha^-1. In all mixtures mineral oil (0.5 v/v) was added.

The treatments were applied with a backpack sprayer pressurized by CO₂, calibrated for a carrier water volume equivalent to 120 L ha^-1 through 6 ST 015 application nozzles. The treatments were applied when the ryegrass plants were between 3 and 4 tillers. The analyzed variable was herbicide efficiency at 2, 7, 14 and 25 DAT, following the methodology previously described.

For the statistical analyzes and for making the graphs, the R software (R Core Team, 2020) and the SigmaPlot (Systat, UK) were used. The mixtures were evaluated using the Colby (1967) test to verify the interaction between the herbicides glufosinate ammonium and saflufenacil (Equation 2).

$$E=100-\left\{\left[\left(100-X\right)*\left(100-Y\right)\right]/100\right\}$$ (2)

where E: Expected value of the herbicide mixtures in each combination. X and Y: The visual control variable (%). When the effect of the observed value (O) resulting from the application of the mixture of herbicides X+Y is greater, lesser or significantly equal to the expected value (E), synergism, antagonism, or additivity occur, respectively. Observed and expected values ​​were compared by Student's t test (p≤0.05). A response surface graphical analysis was performed with the arrangement of the A and D factors of the experiment.

3. Results

All the variables analyzed in experiment 1 had the null hypothesis accepted by the lack-of-fit test (Table 2), showing adjustment of the non-linear regressions in each variable. However, for the fit of the isobole line in the additive concentration model, only the dry weight variable fit the model. Experiment 2 had all variables significant by the analysis of variance and found a synergistic interaction at doses of 400 g ha^-1 of ammonium glufosinate mixed with saflufenacil. The response surface graph did not obtain any inflection point, demonstrating behavior similar to that found in the Colby test.

Table 2
ED₅₀ parameter and its Standard error (SE) and p value. Lack-of-fit test p value of the non-linear regressions in the proportions of 100. 75. 50 and 25 and 0 of the herbicide ammonium glufosinate. considered principal in the mixture with saflufenacil and in the isobologram analysis.

Proportion	ED50	Std Error	p	Lack-of-fit
	10 DAT
100	142.62	9.63	<0.001	0.61
75	19.54	1.8	<0.001
50	11.64	1.29	<0.001
25	3.99	0.45	<0.001
0	386.42	36.81	<0.001
	20 DAT
100	316.44	41.88	<0.001	0.06
75	59.05	59.05	0.98
50	19.83	19.83	<0.001
25	6.75	6.75	0.8
0	343.22	343.2	0.47
	DW
100	134.96	41.25	0.001	0.41
75	6.41	3.38	0.06
50	2.23	1.71	0.19
25	1.26	0.61	0.03
0	225.38	33035	<0.001

3.1. Experiment 1

For the dose-response curves obtained in each proportion (Figure 1), ED₅₀ values ​​of 142.62, 316.44 and 134.96 were observed (Table 2) in the injury variables (%) at 10, 20 DAT and dry matter at 20 DAT, respectively for the herbicide ammonium glufosinate (proportion 100). As for the herbicide saflufenacil (proportion 0), ED₅₀ values ​​of 386.42, 343.22 and 225.38 were observed in the same variables. These results demonstrate better control of ryegrass through the application of ammonium glufosinate alone than saflufenacil in the same condition.

Figure 1
Dose response curve in the proportions (100. 75. 50. 25 and 0) in the 10 (A) and 20 (B) DAT (days after treatment) and dry weight (g) (C) in 20 DAT.

The lowest ED₅₀ values ​​were found in the proportion of 25% of ammonium glufosinate, standing at 3.99, 6.75, and 1.25 in the variables of 10, 20 DAT and DW at 20 DAT (Table 2) . At the same time, a trend towards a reduction in ED₅₀ was observed according to the lower proportion of ammonium glufosinate in mixture, compared to the 100% proportion. This result indicates that the greater the proportion of saflufenacil in the mixture, the lower the ED₅₀ values ​​observed in all evaluations. This suggests a predominant effect of saflufenacil that may indicate the synergism of this mixture.

For the graphic analysis of the isobologram, a synergistic effect was observed in all proportions in the DW variable at 21 DAT, visualized due to the points of each proportion being below the additive line of the isobole (Figure 2). This fact confirms the existence of synergism between these herbicides for ryegrass. Therefore, this observation supports the results found from the dose-response curves, in which the values ​​of the isolated proportions of ammonium glufosinate and saflufenacil were higher when isolated than in the mixture condition (Table 2, Figure 1). Overall, isobologram studies are an efficient way to understand the interaction of different compounds.

Figure 2
Isobologram graphic analysis of ammonium glufosinate (g ha^-1) and saflufenacil (g ha-1) for the dry weight variable at 21 days after treatment (DAT). If the points were below the continuous line (isobole line) the observed behavior is one of synergism.

3.2. Experiment 2

For experiment 2, in the response surface graphical analysis (Figure 3). In the first evaluation at 2 DAT (Figure 3A) we did not observe differences in the control, except for the low efficiency for the herbicide saflufenacil when isolated and mixed at a dose of 100 g i.a. of ammonium glufosinate. However, we observed that the control peak was in the herbicide efficiency evaluation at 10 DAT (Figure 3B), always at doses of 400 and 600 g of ammonium glufosinate a.i., mainly in mixtures with 10.5 and 21 g of saflufenacil. In the evaluation after 15 DAT (Figure 3C) we also observed control similar to those treatments, but with a reduced control effect, similarly to that found in the evaluation at 25 DAT (Figure 3D).

Figure 3
Graphic analysis of surface response for the mixture of different doses of saflufenacil and glufosinate ammonium. at 7 (A). 14 (B). 21 (C) and 25 (D) days after treatment (DAT) for herbicide efficiency (%) in ryegrass control.

For the evaluation by the Colby test, an additive effect was observed in all glufosinate ammonium mixtures at doses of 200 and 600 g a.i. in all evaluations (Table 3). However, for mixtures with 400 g of ai, all saflufenacil doses showed synergistic effects, promoting greater control than when these herbicides were applied alone.

Table 3
Observed injury (obs) and expected(exp) estimated by Colby’s test in 2. 7. 14 and 25 days after treatment (DAT).

Rate ammonium glufosinate	Rate saflufenacil	2 DAT (obs)	2 DAT (exp)	7 DAT (obs)	7 DAT (exp)
0	10.5	8.00	-	3.75	-
0	21	8.00	-	3.75	-
0	31.5	10.00	-	5.00	-
200	0	27.50	-	42.50	-
200	10.5	28.00	33.31	38.25	44.62
200	21	32.00	33.64	50.00	44.51
200	31.5	34.75	34.75	50.00	45.37
400	0	34.50	-	58.75	-
400	10.5	36.25*	19.04	66.25*	32.62
400	21	33.75*	19.41	57.50*	33.12
400	31.5	43.75*	20.8	63.25*	33.51
600	0	41.25	-	65.00	-
600	10.5	47.50	44.80	62.50	61.50
600	21	48.75	44.85	70.00	61.57
600	31.5	46.25	46.00	68.75	62.00
		14 DAT (obs)	14 DAT (exp)	25 DAT (obs)	25 DAT (exp)
0	10.5	3.50	-	0.00	-
0	21	2.75	-	2.25	-
0	31.5	4.75	-	0.00	-
200	0	38.90	-	22.00	-
200	10.5	38.75	40.96	18.00	23.77
200	21	43.75	40.23	15.00	33.22
200	31.5	40.00	41.85	20.25	24.82
400	0	56.25	-		-
400	10.5	55.00*	27.62	31.25*	5.18
400	21	56.25*	27.06	27.50*	18.27
400	31.5	48.75*	28.56	32.50*	6.39
600	0	58.75	-		-
600	10.5	43.75	45.81	32.50	34.26
600	21	52.50	53.83	37.50	47.88
600	31.5	52.50	54.97	36.25	38.56

• ^*
  Significance by Student’s T test (p<0.05).

It is worth mentioning two important points between the two experiments that caused differences between the visual control evaluations: The development stage for experiment 1 was 3-4 leaves, while in experiment 2 the annual ryegrass was in full tillering; the other difference is that the isobologram graphic analysis in experiment 1 uses higher doses, always aiming to promote the additivity of the mixtures, while in the Colby test conducted, doses around the registration doses of the products in Brazil were used.

4. Discussion

4.1. Experiment 1

In a similar work, synergism was also observed for the mixture of glufosinate ammonium with saflufenacil in the control of Amaranthus tuberculatus (Moq.) J.D.Sauer by isobologram graphic analysis (Takano et al., 2020). Although the species studied is within the spectrum of action of these herbicides, it is visible that their mixture has a synergistic relationship. Also, the joint action of a GS inhibitor and a PPO can cause an increase in protoporphyrin and reactive oxygen species (ROS) production (Takano et al., 2020), explaining the mechanism of improved weed control. On the other hand, when an isobologram of the mixture of metribuzin, a non-systemic herbicide according to our object of study, with halosulfuron (systemic) and flumioxazin (non-systemic) was evaluated, the analysis of isobolograms showed antagonism of these mixtures to control common lambsquarters (Chenopodium album L.) and redroot pigweed (Amaranthus retroflexus L.) (Kalkhoran et al., 2021).

On the other hand, mixtures of the herbicides desmedipham + phenmedipham + ethofumesate with clopyralid or ethofumesate showed different behaviors for the tested species: being desmedipham + phenmedipham + ethofumesate and clopyralid synergistic for the control of Portulaca oleracea, Solanum nigrum, and A. retroflexus, while desmedipham + phenmedipham + ethofumesate with ethofumesate tended to be antagonistic for C. album and P.oleracea (Chitband et al., 2021). Meanwhile in a work with the mixture between atrazine with doses of 140 nM of clomazone may have a synergistic interaction for the control of sunflower (Helianthus annuus) (Kruse et al., 2006). And in a similar trial, the mixture of atrazine with mesotrione also demonstrated the ability to be synergistic to control Palmer amaranth (Amaranthus palmeri S.Wats.) in any mathematical model of isobologram interaction (Abendroth et al., 2011).

Herein, the use of isobolograms is an efficient way to understand the dynamics of the mixture between two compounds, whether it is synergistic, antagonistic or additive, with similar or different mechanisms of action.Also, mainly in relation to the dose, in order for the study to seek the widest range of possible doses. However, due to some limitations in the use of isobolograms, a simpler way may be to study interactions via the Colby (1967) test, in order to know the interaction between certain doses.

4.2. Experiment 2

The use of ammonium glufosinate mixtures is of great importance to increase the herbicide's spectrum of action and increase efficiency. The mixture of ammonium glufosinate with saflufenacil as previously mentioned is quite efficient for weed control. In addition, it can also increase the efficiency of flumioxazin, pyraflufen, lactofen and fomesafen, PPO inhibitors for controlling Kochia (Bassia scoparia (L.) A.J.Scott) (Takano et al., 2020). However, in general, the interactions found in glufosinate ammonium mixtures are antagonism. When mixed with glyphosate and clethodim to control grasses such as Urochloa spp. and Echinochloa spp (Meyer et al., 2021). In fact, unlike what was found in this work, the mixture of ammonium glufosinate with atrazine was antagonistic for the control of ryegrass (Ulguim et al., 2019). In addition, the mixture of glyphosate and ammonium glufosinate also proved to be antagonistic to control Cirsium arvense, C. album, Setaria faberi and Abutilon theophrasti, but no changes were found in photosystem II electron transport and fluorescence (Bethke et al., 2013). However, it was found that when mixed with ammonium glufosinate, dicamba degradation was reduced in Palmer amaranth and E. crus-galli (Meyer et al., 2020). That is, the use of ammonium glufosinate can cause a reduction in the metabolism of systemic herbicides, consequently reducing the efficiency of the herbicide.

Saflufenacil manages to be more versatile in relation to mixing with other herbicides. One of the best known interactions is the synergism in relation to the mixture with glyphosate to control broadleaf (Knezevic et al., 2009). This mixture stands out for the control of Conyza spp. resistant to glyphosate (Dalazen et al., 2015). Also, when mixed with paraquat and metribuzin, a synergistic interaction can also be achieved to control Alternantera tenella (Trezzi et al., 2016). Furthermore, the mixture of imazethapyr with saflufenacil can increase the control of weeds such as Echinochloa spp., Sesbania virgata, Ipomoea spp. and Palmer amaranth (Montgomery et al., 2015). Similarly, this mixture did not interfere with the control of weedy rice by imazethapyr (Camargo et al., 2012). Evidencing additive activity for some weeds. This was similar to what was found when mixing saflufenacil with clomazone, imazapir + imazapic and cyhalofop to control Echinochloa spp. (Pigatto et al., 2020).

5. Conclusions

Ammonium glufosinate was able to control ryegrass at a dose of 600 g ha^-1, but this represents an increase of 50% of the recommended application rate of this herbicide in production fields in Brazil (Brasil, 2022). However, we observed a synergistic action for the dose of 400 g ha^-1 with all saflufenacil doses used in the experiment. This fact may suggest a brief corroboration with the theory of additive concentration which, among other concepts, states that the mixture of two compounds can cause a similar toxicological response (Berenbaum, 1989). Since it is observed that the dose of 600 g ha^-1 of ammonium glufosinate obtains the same control response with or without the saflufenacil mixture.

According to the results we found, the behavior of the mixture between saflufenacil and glufosinate ammonium is a synergistic interaction for the control of ryegrass by the isobologram test. Using the Colby test, it was verified that at a dose of 400 g i.a ha^-1 of ammonium glufosinate and at doses of 10.5, 21 and 31.5 of saflufenacil the interaction found is of synergism to control ryegrass. That is, this mixture has synergistic potential to control ryegrass.

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Publication Dates

• Publication in this collection
  10 Jan 2025
• Date of issue
  2024

History

• Received
  10 May 2024
• Accepted
  09 Oct 2024