As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing and/or sequencing capacity, which can also introduce biases1–3. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing4,5. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We developed and deployed improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detected emerging variants of concern up to 14 days earlier in wastewater samples, and identified multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

Palmer amaranth is one of the most problematic weeds in the midsouthern United States, and the evolution of resistance to protoporphyrinogen oxidase (PPO) inhibitors in biotypes already resistant to glyphosate and acetolactate synthase (ALS) inhibitors is a major cause of concern to soybean and cotton growers in these states. A late-season weed-escape survey was conducted in the major row crop–producing counties (29 counties) to determine the severity of PPO-inhibitor resistance in Arkansas. A total of 227 Palmer amaranth accessions were sprayed with fomesafen at 395 g ha−1to identify putative resistant plants. A TaqMan qPCR assay was used to confirm the presence of the ΔG210 codon deletion or the R128G/M (homologous to R98 mutation in common ragweed) target-site resistance mechanisms in thePPX2gene. Out of the 227 accessions screened, 44 were completely controlled with fomesafen, and 16 had only one or two severely injured plants (≥98% mortality) when compared with the 1986 susceptible check (100% mortality). The remaining 167 accessions were genotypically screened, and 82 (49%) accessions were found to harbor the ΔG210 deletion in thePPX2gene. The R128G was observed in 47 (28%) out of the 167 accessions screened. The mutation R128M, on the other hand was rare, found in only three accessions. About 13% of the accessions were segregating for both the ΔG210 and R128G mutations. Sixteen percent of the tested accessions had mortality ratings <90% and did not test positive for the ΔG210 or the R128G/M resistance mechanisms, indicating that a novel target or non–target site resistance mechanism is likely. Overall, PPO inhibitor–resistant Palmer amaranth is widespread in Arkansas, and the ΔG210 resistance mechanism is especially dominant in the northeast corridor, while the R128G mutation is more prevalent in counties near Memphis, TN.

S-Metolachlor is commonly used by soybean and cotton growers, especially with POST treatments for overlapping residuals, to obtain season-long control of glyphosate- and acetolactate synthase (ALS)–resistant Palmer amaranth. In Crittenden County, AR, reports of Palmer amaranth escapes following S-metolachlor treatment were first noted at field sites near Crawfordsville and Marion in 2016. Field and greenhouse experiments were conducted to confirm S-metolachlor resistance and to test for cross-resistance to other very-long-chain fatty acid (VLCFA)–inhibiting herbicides in Palmer amaranth accessions from Crawfordsville and Marion. Palmer amaranth control in the field (soil <3% organic matter) 14 d after treatment (DAT) was ≥94% with a 1× rate of acetochlor (1,472 g ai ha–1; emulsifiable concentrate formulation) and dimethenamid-P (631 g ai ha–1). However, S-metolachlor at 1,064 g ai ha–1 provided only 76% control, which was not significantly different from the 1/2× and 1/4× rates of dimethenamid-P and acetochlor (66% to 85%). In the greenhouse, Palmer amaranth accessions from Marion and Crawfordsville were 9.8 and 8.3 times more resistant to S-metolachlor compared with two susceptible accessions based on LD50 values obtained from dose–response experiments. Two-thirds and 1.5 times S-metolachlor at 1,064 g ha–1 were the estimated rates required to obtain 90% mortality of the Crawfordsville and Marion accessions, respectively. Data collected from the field and greenhouse confirm that these accessions have evolved a low level of resistance to S-metolachlor. In an agar-based assay, the level of resistance in the Marion accession was significantly reduced in the presence of a glutathione S-transferase (GST) inhibitor, suggesting that GSTs are the probable resistance mechanism. With respect to other VLCFA-inhibiting herbicides, Marion and Crawfordsville accessions were not cross-resistant to acetochlor, dimethenamid-P, or pyroxasulfone. However, both accessions, based on LD50 values obtained from greenhouse dose–response experiments, exhibited reduced sensitivity (1.5- to 3.6-fold) to the tested VLCFA-inhibiting herbicides.

The management of glyphosate-resistant Palmer amaranth has been a challenge in southern United States cropping systems. Registration of dicamba-resistant crops will provide an alternative management option to control herbicide-resistant Palmer amaranth populations, particularly those having resistance to herbicide Groups 2, 3, 5, 9, 14, and 27. However, repeated use of sublethal doses of dicamba may lead to rapid evolution of herbicide resistance, especially in Palmer amaranth—a species with a strong tendency to evolve resistance. Therefore, selection experiments with dicamba were conducted on Palmer amaranth using sublethal doses. In the greenhouse, a known susceptible Palmer amaranth population was subjected to sublethal dicamba doses for three generations (P1–P3). Susceptibility of the individuals to dicamba was evaluated, and its susceptibility to 2,4-D was characterized. Based on the greenhouse study, following three generations of dicamba selection, the dose required to cause 50% mortality increased from 111 g ae ha−1for parental individuals (P0) to 309 g ae ha−1for the P3. Furthermore, reduced susceptibility of the P3to 2,4-D was also evident. This research presents the first evidence that recurrent use of sublethal dicamba doses can lead to reduced susceptibility of Palmer amaranth to dicamba as well as 2,4-D. Here, we show that selection from sublethal dicamba doses has an important role in rapid evolution of Palmer amaranth with reduced susceptibility to auxin-type herbicides.

It is well established that dicamba can cause severe injury to soybean that is not resistant to dicamba. Dicamba-resistant (DR) cotton became available in 2015, followed by DR soybean in 2016; in late 2016 came the release of new dicamba formulations approved for topical use in cotton and soybeans. Until this approval, use of dicamba was limited to primarily corn, small grains, range and pasture, and eco-fallow acres. Hence, studies were conducted in 2015 and 2016 to examine off-target movement of two dicamba formulations using non-DR soybean as a bio-indicator. Diglycolamine (DGA) andN,N-Bis(3-aminopropyl)methylamine (BAPMA) dicamba were applied simultaneously at 560 g ae ha–1in the center of two side-by-side 8-ha fields to vegetative glufosinate-resistant soybean. On the same day, a rate response experiment was established encompassing nine different dicamba rates of each formulation. Results from the rate response experiment indicate that soybean is equally sensitive to DGA and BAPMA dicamba. In 2015, a rain event occurring 6 to 8 h after application of the large drift trial probably limited off-target movement by incorporating some of the herbicide into the soil. As a result, secondary drift was less in 2015 than in 2016. However, minimal secondary injury (<5%) occurred 12 m farther into DGA dicamba plots in 2015. In 2016, secondary movement was decreased by 72 m when BAPMA dicamba was used compared to DGA dicamba. Appreciable secondary movement of both DGA and BAPMA dicamba is possible following in-crop applications of either formulated product to soybean in early to mid-summer. Additionally, the risk for secondary movement of BAPMA dicamba is slightly less than for DGA dicamba.