Viewing the ecological consequences of synthetic auxin herbicides from the ground up

Herbicides, although often less emphasized than other pesticides like insecticides, pose a significant risk to wild plant communities (Iriart et al., 2021). Synthetic auxin or “auxinic” herbicides such as 2,4-D and dicamba have recently surged in use due to the commercialization of transgenic crops tolerant to auxinic herbicides (Johnson et al., 2023). These chemicals act by mimicking the natural phytohormone auxin (indole-3-acetic acid [IAA]) to cause abnormal growth and death in target weeds, but are also linked to a long-standing issue known as herbicide drift, i.e., when herbicide particles (i.e., ~0.1–5% of application rate) move through the atmosphere and away from target sites, that can impair nontarget plants (Johnson et al., 2023). More insidious, however, are the potential consequences beneath the soil. For example, in the symbiotic mutualism between leguminous plants (legumes) and nitrogen-fixing soil rhizobial bacteria (hereafter rhizobia), plants exchange carbohydrates for rhizobia-fixed nitrogen (N) from the atmosphere, contributing to the N cycle and soil fertility (Ahemad and Khan, 2013). However, studies have shown that auxinic herbicides can be especially damaging to legumes (Blanchett et al., 2015; Jones et al., 2019; Iriart et al., 2022), and under certain conditions can directly reduce the growth of free-living rhizobia (Fabra et al., 1997). Moreover, because legume–rhizobia interactions are partly regulated by IAA, symbiotic interactions can be disrupted by synthetic auxins (Ahemad and Khan, 2013; Iriart et al., 2024; Iriart et al., unpublished manuscript)—yet, studies on the effects of auxinic herbicide drift on legume–rhizobia interactions or other phytohormone-regulated symbioses such as the mutualism between a plant and arbuscular mycorrhizal fungi (AMF), are limited. Here, we identify the major gaps in our knowledge of the impacts of low-dose exposures to auxinic herbicides on plant–microbe symbioses in the rhizosphere and the pathways and mechanisms that mediate them. We highlight research areas that will shed light on this issue, including some of the overlooked broader ecological consequences, potentially contributing to the footprint of disruption caused by auxinic herbicides.

Research has largely focused on the direct effects of synthetic auxin exposures on plants and some plant-associated rhizospheric microbes (i.e., Paths 1 and 2, Figure 1; Iriart et al., 2021; Ruuskanen et al., 2023), but rarely on the ecological interactions between them which could likewise be affected via indirect pathways (i.e., Path 3 and 4, Figure 1). For instance, genotypes of plants or of microbes can differ in their physiological or biochemical responses to synthetic auxins (Ahemad and Khan, 2013; Hage-Ahmed et al., 2019; Iriart et al., 2022), which could in turn affect outcomes of symbiotic interactions (i.e., Paths 5 and 6, Figure 1) via plant or microbe pathways (e.g., 1 $\to$3 $\to$5 or 2 $\to$4 $\to$5, Figure 1). However, if plant and rhizobial genotypes both vary in their responses to synthetic auxins, then genotype-by-genotype (G $\times$ G) interactions may contribute to symbiotic outcomes in unpredicted ways. To uncover which pathway(s) (i.e., pathway from Path 1 or 2 through Path 3 or 4 to 5 and 6, Figure 1) are most critical for downstream ecological consequences, more research is needed. To our knowledge, our two most recent studies (Iriart et al., 2024; Iriart et al., unpublished manuscript) represent the first attempts to address this knowledge gap using a G $\times$ G framework.

Iriart et al. (2024, unpublished manuscript) characterized plant–microbe interactions between red clover (Trifolium pratense) and its rhizobial partner (Rhizobium leguminosarum) in response to drift-level doses of dicamba. In both cases, dicamba's effects on symbiotic outcomes were primarily modulated by the rhizobial genotype (G_R) as described in Pathway 4 (Figure 1), more so than by the plant genotype (G_P) or the G_P $\times$ G_R interaction, even though herbicide was applied to different areas (plant shoot or roots) when plants were seedlings. Specifically, the impact of low-dose exposures to this synthetic auxin on the amount of fixed N provided or plant growth promoted by symbiosis ranged from strongly negative (i.e., benefits diminished) to neutral (i.e., benefits unaltered), and the identity of the rhizobial partner influenced which outcome was realized. Concordantly, this work supported a growing body of research appreciating the role of soil microbes in driving plant responses to various stressors (reviewed by Porter et al., 2020). This work also stands in contrast to the predominance of G $\times$ G effects as drivers of plant–microbe symbioses in other contexts (e.g., fluctuating nitrogen/light availability; Heath and Tiffin, 2007; Batstone et al., 2020). Thus, we amplify the argument that the indirect effects, here of synthetic auxins as mediated by microbes (Path 4, Figure 1), likely play a more important role in directing some of the ecological impacts of anthropogenic chemicals than previously thought. However, to understand whether this trend extends to other plant–microbe symbioses including plant–AMF interactions, more studies that employ a G $\times$ G approach and involve different species will be essential.

While the particular attributes of rhizobia that could underly their relevance in mediating symbiotic outcomes (Path 4, Figure 1) are still unclear, a few research questions seem fruitful to pursue. First, since it is known that during symbioses, symbiotic microbes can biosynthesize IAA and/or alter IAA levels within plants (Fu et al., 2015), could variation in microbial auxin metabolism or the rate at which they regulate it in plants interact with synthetic auxins to modulate symbiotic outcomes? For instance, could rhizobia that synthesize high amounts of IAA inadvertently facilitate the mode of action of synthetic auxins in plants? Second, it is possible that symbiotic microbes can actually degrade these herbicides and thus minimize the concentration afflicting plants. Many species of rhizospheric microbes are known to degrade herbicides (Pileggi et al., 2020); albeit evidence of this ability in rhizobia and AMF is limited (but see Fabra et al., 1997). Ultimately, investigations into rhizobial metabolism during symbiosis and with/without auxinic herbicides present will help illuminate the role of rhizobial metabolic variation in this anthropogenic context.

Alternatively, synthetic auxins might induce a change in the plant carbon balance, that is, carbon costs vs. nutritional benefits received from symbioses. Plant-rhizobia and plant-AMF symbioses have long been thought of as functioning within a “biological market,” wherein alterations in nutritional transactions between species partners can cause interactions to shift along the mutualism–parasitism continuum (Noë and Kiers, 2018). Iriart et al. (2024, unpublished manuscript) found that the rhizobial strains that had the largest declines in promoting plant growth under auxinic herbicide stress were those that nonetheless were active in providing fixed nitrogen regardless of dicamba. Thus, it may be that these negative consequence for plants are driven by increases in symbiotic costs rather than decreases in nutritional benefits received. A natural extension from this work would be to characterize carbon flux from root nodules (where rhizobia are housed in planta) to plant tissues under different synthetic auxin treatments. Additionally, it is important to note that in Iriart et al. (2024, unpublished manuscript), plants were in an N-limited environment, yet it is expected that legume investment in rhizobial symbiosis will depend on soil N availability (Heath and Tiffin, 2007). Therefore, another fruitful research direction is to assess the combined effects of N availability and auxinic herbicide presence on C and N exchange using modern isotopic analytic techniques.

The knock-on ecological consequences of herbicides have been largely understudied due to their complexity (Ruuskanen et al., 2023). Our work and that of others has shown that more often than not exposures to auxinic herbicides negatively affect plant–microbe symbioses involving rhizobia and AMF (Ahemad and Khan, 2013; García Carriquiry et al., 2024; Iriart et al., 2024, unpublished manuscript), a result which could have cascading effects for other ecosystem properties (green pathways, Figure 1). For instance, although legumes often receive a competitive advantage over other plant species by associating with rhizobia, especially when N is limited (reviewed in Traveset and Richardson, 2014), Iriart et al. (2024, unpublished manuscript) revealed that exposure to off-target levels of dicamba diminished the growth-promoting benefits acquired from rhizobia in red clover. If this result relates to wild legumes that commonly occur near agricultural fields (e.g., species of Trifolium, Medicago, Lotus; Ashman et al., unpublished data), then these chemicals may not only disrupt symbiotic N fixation processes that affect soil quality, but may also alter plant community composition by weakening the competitive ability of legumes (e.g., Paths 7a and 8a, Figure 1). Meanwhile, plants with greater auxinic herbicide tolerance may become more dominant (see Iriart et al., 2022). Furthermore, because rhizobia and AMF interactions are predicted to augment flowering (Barber and Gorden, 2015), changes in plant–microbe symbioses caused by synthetic auxins may be contributing to declines in floral resources in agroecosystems (e.g., Paths 7b and 8b, Figure 1), which in turn could be contributing to pollinator declines (Bretagnolle and Gaba, 2015). Higher-level ecological ramifications are unexplored, but if existent, would further underscore the need to start looking at the environmental impacts of synthetic auxin herbicides, and potentially other modern anthropogenic stressors, from the ground up.

V.I. and T.-L.A. conceptualized the manuscript. V.I. wrote the manuscript, and T.-L.A. revised and edited it.