First report of giant arrowhead (Sagittaria montevidensis) with resistance to the synthetic auxin florpyrauxifen-benzyl

Abstract

Background:

Sagittaria montevidensis Cham. & Schltdl. is a weed that forms dense infestations in wet-seeded rice. The herbicide florpyrauxifen-benzyl is widely used in rice cultivation; however, there have been reports of failed S. montevidensis control, and these cases are increasing.

Objective:

This study aimed to evaluate S. montevidensis for resistance the florpyrauxifen-benzyl and assess the sensitivity of this biotype to auxin-mimicking herbicides from other chemical groups.

Methods:

The experiments were conducted in greenhouse using a completely randomized design with four replicates. Dose-response tests were performed using two biotypes of S. montevidensis, resistant and sensitive. The experiments were performed over two generations, using increasing doses of the florpyrauxifen-benzyl. The experiment with other auxin mimics was conducted with the second-generation populations, using increasing doses of the herbicides 2,4-D, dicamba, and triclopyr. In both experiments, weed control was assessed at 7, 14, and 28 days after application (DAA), and the dry mass of the aerial part was determined at 28 DAA.

Results:

Resistance of S. montevidensis to florpyrauxifen-benzyl was confirmed, with a resistance factor >700 in the two evaluated generations. The biotype was sensitive to 2,4-D, dicamba, and triclopyr achieving greater than 80% control in all treatments, and cross-resistance to synthetic auxins was ruled out.

Conclusions:

Evolution of resistance to florpyrauxifen-benzyl by S. montevidensis seriously compromises paddy rice production, highlighting the urgent need for alternative approaches.

Keywords:
Irrigated Rice; Synthetic Auxins; Chemical Control; Group 4

1. Introduction

Rice (Oryza sativa L.) is a crop of paramount economic importance in many developing nations. Its adaptability to diverse soil and climatic conditions has contributed to its widespread cultivation (Santos, 2021). In Brazil, rice cultivation is divided into two primary systems: upland and lowland or irrigated rice. The wet-seeded system, predominantly used in the southern region, involves sowing rice in previously flooded soil (Gutz et al., 2019). This method offers several advantages, including effective weed control due to the aquatic environment and reduced herbicide use (Ramos, 2017).

Sagittaria montevidensis Cham. & Schltdl. is a problematic weed for rice cultivation, infesting mainly rice-growing areas in the wet-seeded system and forming dense infestations in most crop areas (Merotto Júnior et al., 2010). It is a rooted, annual, or perennial, herbaceous, slightly lactiferous, and morphologically variable aquatic plant that spreads easily by seed and short rhizomes (Lorenzi, 2008).

In plants, the regulation of metabolism, growth, and biotic and abiotic response factors is mediated by signaling molecules that interact with specific cellular receptor proteins called phytohormones. Auxins are an important class of plant hormones (Woodward, Bartel, 2005). Due to the essentiality of this hormone for plant metabolism and growth, changes in auxin levels or its response usually lead to abnormal development and plant death. This has led to the development of herbicides that mimic the action of auxin (LeClere et al., 2018).

The herbicide florpyrauxifen-benzyl is widely used in rice cultivation and is classified as a synthetic auxin from the arylpicolinate chemical group, developed for post-emergence control of rice grass (Echinochloa crus-galli), sedges (Cyperaceae), and some eudicotyledons, including those resistant to acetolactate synthase (ALS)-inhibiting herbicides, which infest irrigated rice crops (Miller et al., 2018). The mode of action of florpyrauxifen-benzyl consists of stimulating gene transcription and ethylene biosynthesis, thus leading to the accumulation of abscisic acid and reactive oxygen species, which subsequently cause the death of sensitive species (Wang et al., 2021).

Plant resistance to herbicides is characterized by the hereditary ability of some biotypes to survive and reproduce after exposure to a herbicide dose normally lethal to the wild biotype of the same species (Heap, 2024). Reports of failure to control S. montevidensis after applying florpyrauxifen-benzyl have become frequent in recent harvests in southern Brazil's irrigated rice fields. A series of studies were conducted to determine if this was a new case of resistance to florpyrauxifen-benzyl.

The objectives of this study were as follows: a) to determine if there is resistance of S. montevidensis to the florpyrauxifen-benzyl and b) to assess the sensitivity of biotypes to auxin-mimicking herbicides from other chemical groups.

2. Materials and Methods

2.1 Plant material

Sagittaria montevidensis (R) plants that were not controlled after exposure to the recommended dose of the herbicide florpyrauxifen-benzyl were collected in an area of irrigated rice in the pre-germinated system in the region of Viamão—RS (30°01’50.6" S, 50°55’02.8" W and 6 m altitude). The collected plants were kept in pots until seeds were produced and the F1 generation (R-F1) was obtained. The second-generation seeds (R-F2) were obtained by multiplying the surviving R-F1 plants. To obtain plants from both the F1 and F2 generations, controlled artificial pollinations were conducted to purify the population. Plants from populations R and S were isolated to prevent cross-pollination.

The sensitive population (S) was collected in a floodplain area that had never been treated with the herbicide florpyrauxifen-benzyl in Turvo—SC. A preliminary experiment in the greenhouse confirmed its sensitivity to florpyrauxifen-benzyl, applying the labeled dose of florpyrauxifen-benzyl herbicide. The seeds were collected, cleaned, and stored at 5 °C. The seeds were sown in 3.6 L pots with soil saturated with water. In all experiments, the experimental unit consisted of a pot with two individual sagittaria plants. The plants were kept in a greenhouse with an average temperature of 25 °C and 55% relative humidity and manually irrigated daily throughout the experimental period to maintain a water depth of 3 cm.

2.2 Experimental design

The experiments were conducted in a completely randomized design with four replicates. The herbicides were applied using a CO₂ pressure precision sprayer with four nozzle booms, 0.5 m spacing between the nozzles, flat fan tips with air induction (model AIXR 110 015), at a working pressure of 200 kPa, speed application of 1 ms⁻¹, and an application rate of 150 Lha⁻¹. The experimental design was repeated in the F1 and F2 generations.

2.3 Florpyrauxifen-benzyl dose response

The experiment was conducted on plants with 3–4 fully expanded leaves from the F1 and F2 generations of the R and S populations. The doses of florpyrauxifen-benzyl (Loyant^®, 25 g L-1, Corteva) tested were: 0, 1/4X, 1/2X, 1X, 2X, 4X, 8X and 16X, considering that X represents the recommended dose of 20 g ha^-1 for the control of S. montevidensis.

2.4 Cross-resistance to synthetic auxin group

The experiment was conducted with plants from the F2 generation of populations R and S. The plants were treated with three synthetic auxins (group 4) from different chemical groups at increasing doses. The herbicides used were the following: 2,4-D, commercial product Aminol 806®, (0; 251.25; 502.5; 1,005; 2,010; 4,020; 8,040, and 16,080 g ae ha⁻¹), dicamba, commercial product Atectra®, (0, 80, 360, 720, 1,440, 2,880, 5,760, and 11,520 g ae ha⁻¹), and triclopyr, i.e. commercial product Triclon® (0, 240, 480, 960, 1,920, 3,840, 7,680 and 153,60 g ae ha⁻¹).

2.5 Treatment evaluation

The visual assessment of the percentage of weed control was performed at 7, 14, and 28 days after application (DAA) in all the experiments, and 0% control was deemed when no damage was observed, and 100% was the classification when the plant died (Kuva et al., 2016). Additionally, in the experiments with the F2 generation, the dry mass of the aerial part was obtained at 28 DAA by cutting the plants close to the ground and drying them in a forced-air oven for 72 hours at 65 °C until constant dry mass. For quantification, a precision scale (0.001 g) was used to obtain the variable dry mass of the aerial part.

2.6 Data analysis

The dose-response tests were subjected to analysis of variance using the F test and non-linear regression, where four parameters were adjusted:

$$y=\frac{a^{\ast }x}{(b+x))}$$

Where y is the dependent variable (dry mass or percentage of control), a is the asymptote, x is the independent variable (herbicide doses), and b is the rate that provides 50% of A (lethal dose [LD] 50 or growth reduction [GR] 50). Parameters a and b were tested using a t-test and found to be significant (p<0.05). From this equation, LD₅₀ and LD₈₀ were estimated as the doses of herbicide needed to provide 50% and 80% of control, respectively. The quotient between the LD₅₀ of the resistant biotype (R-F1 and R-F2) and the LD₅₀ of the susceptible biotype (S) was calculated to estimate the resistance factor (RF), which is characterized by the number of times the dose that provides 50% of control or reduction in dry mass production of the susceptible biotype needs to be increased for the same effect to occur on the resistant biotype (Gazziero et al., 1998). Additionally, it was necessary to estimate LD₂₀ and LD₁₀, i.e., the doses of herbicide that provide 20% and 10% of control, given that the R biotype did not achieve 50% control in the two generations, making it necessary to estimate this value. The RF₂₀ and RF₁₀ for the resistant biotype were also calculated using the LD₂₀ and LD₁₀ values.

To confirm resistance, the ten steps for reporting new cases of weed resistance to herbicides in Brazil, as proposed by the Brazilian Society of Weed Science (Sociedade Brasileira da Ciência das Plantas Daninhas, 2018), were followed. Resistance was confirmed when FR >1.0 and LD₈₀ exceeded the maximum dose recommended in the package leaflet in both generations.

3. Results and Discussion

3.1 Florpyrauxifen-benzyl dose response

For the R biotype, maximum weed control did not exceed 20% in the two evaluated generations (Figure 1), while the S biotype showed high sensitivity to the herbicide florpyrauxifen-benzyl; Florpyrauxifen-benzyl achieved satisfactory control at low doses of the herbicide, i.e., 97% of control starting at the dose of 5 g ai ha⁻¹, regardless of the generation. This dose represents only 25% of the dose recommended in the package leaflet (Figure 2). The FRs were >700 and >2,600 for the F1 and F2 generations, respectively. Additionally, the two generations of biotype R had LD₈₀ >320 g ai ha⁻¹, significantly higher than the maximum dose recommended in the package leaflet. This confirmed resistance to florpyrauxifen-benzyl.

Figure 1
Dose-response curves to florpyrauxifen-benzyl for the F1 (a) and F2 (b) generations of the R and S populations of S. montevidensis and dry mass of aerial part for the F2 generation of the R and S populations of S. montevidensis (c)

Figure 2
Dose response to florpyrauxifen-benzyl in the F2 generation of S. montevidensis

The LD₅₀ was >320 g ai ha⁻¹ for the two generations of the R biotype, while the S population had an LD₅₀ of 0.45 g ai ha⁻¹ (Table 1). Considering that the indicated field dose is 20 g ai ha⁻¹, the R biotype can be highly resistant to florpyrauxifen-benzyl. The confirmed case of resistance is the first global case of S. montevidensis resistance to the herbicide florpyrauxifen-benzyl. Cases of resistance to the same herbicide have been described for Echinochloa crus-galli (Jin et al., 2023; Hwang et al., 2021), but the RFs were lower than those reported in this study.

Table 1
Parameters obtained in the dose-response test and resistance factors in two generations of the S. montevidensis population resistant to florpyrauxifen-benzyl

Parameters	Herbicides
	Dicamba		2,4-D		Triclopyr
	Biotype S	Biotype R	Biotype S	Biotype R	Biotype S	Biotype R
Maximum control (%)	86.5	80.25	100	99.75	100	97.5
LD50 (g ai ha−1)	96.5	77.3	32	23.2	31.3	7.55
LD80 (g ai ha−1)	11520	11520	128	106.6	123.4	35.4
GR50 (g ai ha−1)	604.8	266.4	26.3	41.2	50.9	39.3
RF	-	0.8	-	0.72	-	0.24

• LD: Lethal dose: GR: Growth reduction.

Different levels of resistance may result from different mechanisms involved in resistance, different frequencies of resistance within populations, and differences in the number and dominance of alleles involved in resistance (Yang et al., 2020).

Weed resistance to auxin mimics, when related to changes in the target site of the herbicide, may be caused by a single dominant gene (Preston et al., 2009), a single recessive gene (Sabba et al., 2003), or more than one gene. Yang et al. (2020) described the relationship between the high level of resistance of Echinochloa crus-pavonis to quinclorac (RF > 570) and the change in the perception of the auxin signal by the resistant plant, assuming that resistance is controlled by a single dominant gene.

According to Preston and Malone (2014), the single dominant gene is more easily dispersed within the population and speeds up the process of resistance evolution once it has been established in a species, i.e., increases its frequency in the population.

In view of the above findings, the high levels of resistance observed in the two generations of S. montevidensis suggest that the mechanism involved in this population's resistance is related to the herbicide's target site. This hypothesis needs to be tested in future studies.

Resistance of S. montevidensis to the herbicide florpyrauxifen-benzyl is a serious problem that significantly affects irrigated rice, and measures should be considered to mitigate these problems.

3.2 Cross-resistance to synthetic auxin group

The RF of the R biotype for the herbicides dicamba, 2,4-D, and triclopyr was <1.0 (Table 1), the LD₈₀ for the R biotype using the herbicides 2,4-D and triclopyr was lower than or equal to the recommended dosage on the label. This rules out the possibility of cross-resistance to synthetic auxins and demonstrates the sensitivity of S. montevidensis biotype R to other chemical groups. The comparison of the obtained values of LDs for a 50% reduction in dry mass (GR₅₀) showed that the responses of biotypes R and S to herbicide 2,4-D are similar (Table 2). With regard to the herbicide triclopyr, the R biotype had a lower LD₅₀ than the S biotype, which shows that this biotype is more sensitive to triclopyr than the S biotype.

Table 2
Parameters obtained in the dose-response test and resistance factors for the herbicides dicamba, 2,4-D, and triclopyr in a population of S. montevidensis resistant to florpyrauxifen-benzyl

Parameters	Generation F1		Generation F2
	Biotype S	Biotype R	Biotype S	Biotype R
Maximum control (%)	100	20.6	100	13.8
LD10 (g ai ha−1)	0.0051	94	0.0135	158
LD20 (g ai ha−1)	0.0115	299.2	0.0302	>320
LD50 (g ai ha−1)	0.457	>320	0.121	>320
LD80 (g ai ha−1)	0.183	>320	0.48	>320
LD90 (g ai ha−1)	0.41	>320	1.07	>320
GR50 (g ai ha−1)	-	-	0.345	>320
RF50	-	>700.2	-	>2644
RF20	-	26017.4	-	>11703.7
RF10	-	18431.3	-	11703.7

• LD: Lethal dose; GR: Growth reduction; RF: Resistance factor

Control of the S. montevidensis species by dicamba was less effective in both the R and S biotypes, this is an intrinsic characteristic of the species and is unrelated to resistance, as it occurred in both biotypes. The low efficacy of the herbicide dicamba on monocotyledonous plants accounts for this.

These results show that although the population showed resistance to florpyrauxifen-benzyl, whose mechanism of action is that of synthetic auxins, control was effectively achieved when different herbicides with the same mechanism of action were used. Therefore, sensitivity to other chemical groups of synthetic auxins was maintained.

Differences in sensitivity to synthetic auxins were demonstrated in a study with Arabidopsis, it was found that plants with a mutation site involving auxin transport, i.e., with a change in the location of AUX1, an auxin influx carrier, are insensitive to herbicide 2,4-D but remain sensitive to dicamba. This indicates that dicamba can permeate plant cells without requiring a carrier or an alternative carrier (Gleason et al., 2011). In a study by Walsh et al. (2006), it was shown that mutations in an auxin receptor conferred resistance to picloram, but plant sensitivity to 2,4-D was maintained, indicating significant differences in the chemical perception of synthetic auxins in the upstream components of the pathway that contribute to variations in the effects of different synthetic auxins. These studies show that although synthetic auxins induce similar morphological, physiological, and molecular events in plants, the different chemical groups within them act through different mechanisms and components of perception, transduction, and signal transmission.

4. Conclusions

We concluded that the biotype of Sagittaria montevidensis from Viamão—RS is resistant to the herbicide florpyrauxifen-benzyl, and the resistance level is high. The dose recommended to achieve weed control of <21%, and resistance was maintained in two generations. The florpyrauxifen-benzyl-resistant biotype is not resistant to auxinic herbicides dicamba, 2,4-D, and triclopyr.

The resistance of S. montevidensis to florpyrauxifen-benzyl restricts available control options, necessitating the exploration of alternative methods to prevent resistance spread and development. Conversely, the herbicides 2,4-D e triclopyr demonstrated effective control of the species and offer promising alternatives.

• Funding

  This study was funded by Fundação de Amparo à Pesquisa do Estado de Santa Catarina (Fapesc) Udesc/Promop.

References

• Gazziero DLP, Brighenti AM, Maciel CDG, Christoffoleti PJ, Adegas FS, Voll E. [Resistance of the weed wild poinsettia to ALS inhibitor herbicides]. Planta daninha. 1998;16(2):117-25. Portuguese. Available from: https://doi.org/10.1590/S0100-83581998000200005
  » https://doi.org/10.1590/S0100-83581998000200005
• Gleason C, Foley RC, Singh KB. Mutant analysis in Arabidopsis provides insight into the molecular mode of action of the auxinic herbicide dicamba. PLoS ONE. 2011;6(3):1-13. Available from: https://doi.org/10.1371/journal.pone.0017245
  » https://doi.org/10.1371/journal.pone.0017245
• Gutz T, Cunha G, Olescowicz D, Bachmann G, Harthmann OEL, Guerra N et al. [Paddy rice response to phosphorus supplyand sowing density in pre-germinated system]. Rev Bras Cien Agr. 2019;14(3):1-7. Portuguese. Available from: https://doi.org/10.5039/agraria.v14i3a6631
  » https://doi.org/10.5039/agraria.v14i3a6631
• Heap I. The international herbicide-resistant weed database. Weedscience. 2024. Available from: www.weedscience.org
  » www.weedscience.org
• Hwang JI, Norsworthy JK, González-Torralva F, Priess GL, Barber LT, Butts TR. Non-target-site resistance mechanism of barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] to florpyrauxifen-benzyl. Pest Manag Sci. 2021;78(1):287-95. Available from: https://doi.org/10.1002/ps.6633
  » https://doi.org/10.1002/ps.6633
• Jin W, Sun J, Tang W, Yang Y, Zhang J, Lu Y et al. Comparative transcriptome analysis of the differential effects of florpyrauxifen-benzyl treatment on phytohormone transduction between florpyrauxifen-benzyl-resistant and -susceptible barnyard grasses (Echinochloa crus-galli (L.)) P. Beauv). Agronomy. 2023;13(3):1-16. Available from: http://dx.doi.org/10.3390/agronomy13030702
  » http://dx.doi.org/10.3390/agronomy13030702
• Kuva MA, Salgado TP, Revoredo TTO. [Herbicide efficiency and agronomic practicability experiments]. In: Monquero PA. [Experimenting with herbicides]. São Carlos: Rima; 2016. p. 75-98. Portuguese.
• LeClere S, Wu C, Westra P, Sammons RD. Cross-resistance to dicamba, 2,4-D, and fluroxypyr in Kochia scoparia is endowed by a mutation in an AUX/IAA gene. Proc Natl Acad Sci U S A. 2018;115(13):E2911-20. Available from: https://doi.org/10.1073/pnas.1712372115
  » https://doi.org/10.1073/pnas.1712372115
• Lorenzi H. [Weeds of Brazil: terrestrial, aquatic, parasitic and toxic]. 4th ed. Nova Odessa: Plantarum; 2008. Portuguese.
• Merotto Junior A, Kupas V, Nunes AL, Goulart ICGR. [Isolation of the ALS gene and investigation of the mechanism of herbicide resistance in Sagittaria montevidensis]. Cienc Rural. 2010;40(11):2381-4. Portuguese. Available from: https://doi.org/10.1590/S0103-84782010005000183
  » https://doi.org/10.1590/S0103-84782010005000183
• Miller MR, Norsworthy JK, Scott RC. Evaluation of florpyrauxifen-benzyl on herbicide-resistant and herbicide-susceptible barnyardgrass accessions. Weed Technol. 2018;32(2):126-34. Available from: https://doi.org/10.1017/wet.2017.100
  » https://doi.org/10.1017/wet.2017.100
• Moura DS, Noldin JA, Galon L, Schreiber F, Bastiani MO. [Multiple resistance of Sagittaria montevidensis biotypes to acetolactate synthase and photosystem II inhibiting herbicides]. Planta daninha. 2015;33(4):779-86. Portuguese. Available from: https://doi.org/10.1590/S0100-83582015000400016
  » https://doi.org/10.1590/S0100-83582015000400016
• Preston C, Belles DS, Westra PH, Nissen SJ, Ward SM. Inheritance of resistance to the auxinic herbicide dicamba in Kochia (Kochia scoparia). Weed Sci. 2009;57(1):43-7. Available from: https://doi.org/10.1614/WS-08-098.1
  » https://doi.org/10.1614/WS-08-098.1
• Preston C, Malone, JM. Inheritance of resistance to 2,4-D and chlorsulfuron in a multiple-resistant population of Sisymbrium orientale Pest Manag Sci. 2014;71(11):1523-8. Available from: https://doi.org/10.1002/ps.3956
  » https://doi.org/10.1002/ps.3956
• Ramos TJF. [Pre-germinated irrigated rice cultivation system: an innovative agricultural technique in the search for competitive advantages]. In: Anais do V Simpósio de Engenharia de Produção – SIMEP. Joinville, Brazil. Joinville: Univille; 2017. p. 3356-66. Portuguese.
• Sabba RP, Ray IM, Lownds N, Sterling TM. Inheritance of resistance to clopyralid and picloram in yellow starthistle (Centaurea solstitialis L.) is controlled by a single nuclear recessive gene. J Hered. 2003;94(6):523-7. Available from: https://doi.org/10.1093/jhered/esg101
  » https://doi.org/10.1093/jhered/esg101
• Santos AB. [Rice cultivation: cultivation systems]. Santo Antônio de Goiás: Embrapa Arroz e Feijão; 2021[acesse June 5, 2024]. Portuguese. Available from: https://www.embrapa.br/agencia-de-informacao-tecnologica/cultivos/arroz/producao/sistema-de-cultivo
  » https://www.embrapa.br/agencia-de-informacao-tecnologica/cultivos/arroz/producao/sistema-de-cultivo
• Sociedade Brasileira da Ciência das Plantas Daninhas – SBCPD. [10 Steps for reporting new cases of weed resistance to herbicides in Brazil]. Londrina: Comitê de Ação a Resistência aos Herbicidas; 2018. Portuguese.
• Walsh TA, Neal R, Merlo AO, Honma M, Hicks GR, Wolff K et al. Mutations in an auxin receptor homolog AFB5 and in SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis. Plant Physiol. 2006;142(2):542-52. Available from: https://doi.org/10.1104/pp.106.085969
  » https://doi.org/10.1104/pp.106.085969
• Wang H, Sun X, Yu J, Li J, Dong L. The phytotoxicity mechanism of florpyrauxifen-benzyl to Echinochloa crus-galli (L.) P. Beauv and weed control effect. Pest Biochem Physiol. 2021;179:104978. Available from: https://doi.org/10.1016/j.pestbp.2021.104978
  » https://doi.org/10.1016/j.pestbp.2021.104978
• Woodward AW, Bartel B. Auxin: regulation, action, and interaction. An Bot. 2005;95(5):707-35. Available from: https://doi.org/10.1093/aob/mci083
  » https://doi.org/10.1093/aob/mci083
• Yang X, Han H, Cao J, Li Y, Yu Q, Powles SB. Exploring quinclorac resistance mechanisms in Echinochloa crus-pavonis from China. Pest Manag Sci. 2020;77(1):194-201. Available from: https://doi.org/10.1002/ps.6007
  » https://doi.org/10.1002/ps.6007

Edited by

Editor in Chief:

Carol Ann Mallory-Smith

Associate Editor:

Javid Gherekhloo

Publication Dates

• Publication in this collection
  18 Oct 2024
• Date of issue
  2024

History

• Received
  16 May 2024
• Accepted
  12 Sept 2024