Non-Target-Site Based Tolerance to Herbicides in Amaranthus palmeri

Palmer amaranth, one of the most aggressive and damaging broadleaf weeds in the USA, has evolved resistance to multiple herbicide modes of action. The overall objective of this research was to elucidate the mechanisms by which Palmer amaranth adapt to herbicide selection stress. This research aimed (1) to evaluate the efficacy of fomesafen, glufosinate, glyphosate and trifloxysulfuron to Amaranthus populations; (2) identify candidate genes for endowing tolerance to glufosinate; (3) investigate the involvement of non-target-site resistance (NTSR) mechanism in an ALS-resistant population; and (4) to examine the molecular basis of resistance to PPO inhibitors in Palmer amaranth populations from Arkansas. For objective 1, a total of 124 populations were collected in Arkansas between 2008 and 2015. Overall, 33%, 81%, and 100% of the populations were resistant to fomesafen, glyphosate, and trifloxysulfuron, respectively. Thirty percent of the populations were multiple resistant to fomesafen, glyphosate, and trifloxysulfuron. All populations were controlled \u3e88% by glufosinate. For objective 2, the transcriptomes of glufosinate-tolerant and –sensitive biotypes were assembled using RNA-Seq. Thirteen candidate non-target genes were highly expressed in glufosinate-tolerant biotypes, including glutathione S-transferase (GST), two cytochrome P450, and nine additional genes related to stress signaling and detoxification. Validation of differential gene expression by quantitative real-time PCR revealed increased expression CYP72A219 and GST in glufosinate-treated tolerant biotypes, indicating their involvement in glufosinate tolerance. For objective 3, a population with cross resistance to multiple ALS-inhibiting herbicides was investigated. Two of the nine resistant plants harbored Ser653Asn mutation in the ALS gene. Resistant plants that lacked ALS mutations had elevated levels of CYP81B and GSTF10 genes. This Palmer amaranth population from Arkansas exhibit both target-site (TS) and NTSR to ALS inhibitors. For objective 4, resistance to PPO inhibitors was first detected in a population collected in 2011 with resistance attributed to PPO Gly210 deletion. Several PPO-resistant populations were confirmed in 2014 and 2015; the majority (55%) of the resistant biotypes carried the same mutation. An alternative target-site mutation Arg128Gly was also identified in at least one population. Overall, this research showed that Palmer amaranth has multiple genetic adaptation traits to counteract the lethal effects of herbicides

High [CO2] and Temperature Increase Resistance to Cyhalofop-Butyl in Multiple-Resistant Echinochloa colona

Changes in the environment, specifically rising temperature and increasing atmospheric carbon dioxide concentration [CO2], can alter the growth and physiology of weedy plants. These changes could alter herbicide efficacy, crop-weed interaction, and weed management. The objectives of this research were to quantify the effects of increased atmospheric [CO2] and temperature on absorption, translocation and efficacy of cyhalofop-butyl on multiple-resistant (MR) and susceptible (S) Echinochloa colona genotypes. E. colona, or junglerice, is a troublesome weed in rice and in agronomic and horticultural crops worldwide. Cyhalofop-butyl is a grass herbicide that selectively controls Echinochloa spp. in rice. Maximum 14C-cyhalofop-butyl absorption occurred at 120 h after herbicide treatment (HAT) with >97% of cyhalofop-butyl retained in the treated leaf regardless of [CO2], temperature, or genotype. Neither temperature nor [CO2] affected herbicide absorption into the leaf. The translocation of herbicide was slightly reduced in the MR plants vs. S plants either under elevated [CO2] or high temperature. Although plants grown under high [CO2] or high temperature were taller than those in ambient conditions, neither high [CO2] nor high temperature reduced the herbicide efficacy on susceptible plants. However, herbicide efficacy was reduced on MR plants grown under high [CO2] or high temperature about 50% compared to MR plants at ambient conditions. High [CO2] and high temperature increased the resistance level of MR E. colona to cyhalofop-butyl. To mitigate rapid resistance evolution under a changing climate, weed management practitioners must implement measures to reduce the herbicide selection pressure. These measures include reduction of weed population size through reduction of the soil seedbank, ensuring complete control of current infestations with multiple herbicide modes of action in mixture and in sequence, augmenting herbicides with mechanical control where possible, rotation with weed-competitive crops, use of weed-competitive cultivars, use of weed-suppressive cover crops, and other practices recommended for integrated weed management

A Novel Single-Site Mutation in the Catalytic Domain of Protoporphyrinogen Oxidase IX (PPO) Confers Resistance to PPO-Inhibiting Herbicides

Protoporphyrinogen oxidase (PPO)-inhibiting herbicides are used to control weeds in a variety of crops. These herbicides inhibit heme and photosynthesis in plants. PPO-inhibiting herbicides are used to control Amaranthus palmeri (Palmer amaranth) especially those with resistance to glyphosate and acetolactate synthase (ALS) inhibiting herbicides. While investigating the basis of high fomesafen-resistance in A. palmeri, we identified a new amino acid substitution of glycine to alanine in the catalytic domain of PPO2 at position 399 (G399A) (numbered according to the protein sequence of A. palmeri). G399 is highly conserved in the PPO protein family across eukaryotic species. Through combined molecular, computational, and biochemical approaches, we established that PPO2 with G399A mutation has reduced affinity for several PPO-inhibiting herbicides, possibly due to steric hindrance induced by the mutation. This is the first report of a PPO2 amino acid substitution at G399 position in a field-selected weed population of A. palmeri. The mutant A. palmeri PPO2 showed high-level in vitro resistance to different PPO inhibitors relative to the wild type. The G399A mutation is very likely to confer resistance to other weed species under selection imposed by the extensive agricultural use of PPO-inhibiting herbicides

Assessment of Efficacy and Mechanism of Resistance to Soil-Applied PPO Inhibitors in <i>Amaranthus palmeri</i>

Resistance to protoporphyrinogen oxidase (PPO) inhibitors in Palmer amaranth is a major concern, given the high selection pressure and increasing number of populations with reduced sensitivity to PPO herbicides in the US. We evaluated the effect of five soil-applied herbicides on Palmer amaranth (Amaranthus palmeri S. Wats.) populations collected in 2014 and 2015 in Arkansas, USA. Soil-applied saflufenacil, sulfentrazone, and flumioxazin reduced the seedling emergence 91–100%; however, fomesafen and oxyfluorfen showed reduced (63–90%) efficacy on some populations. Target-site mutation (TSM) is the major mechanism of resistance to PPO herbicides; therefore, six populations showing resistance to soil-applied fomesafen were selected for molecular investigations. A total of 81 survivors were genotyped for all known resistance-conferring mutations. A total of 64% and 36% survivors had single and double TSMs, respectively, with 69% of plants carrying TSM in both alleles of PPO2. Three survivors from two populations showed an additional copy of PPO2, whereas all other survivors had one copy. Expression analysis showed 3- to 6-fold upregulation of PPO2 in all plants from resistant populations tested. Transgenic overexpression of WT-ApPPO2 and dG210-Apppo2 in A. thaliana confirmed the reduced sensitivity to soil-applied fomesafen compared to the wild type. Collectively, PPO inhibitors applied pre-emergence are still effective in controlling populations resistant to foliar-applied PPO herbicides. Mechanically, elevated expression of resistant PPO2, alongside functional TSM, contribute to reduced sensitivity to soil-applied fomesafen

RNA-Seq transcriptome analysis of Amaranthus palmeri with differential tolerance to glufosinate herbicide.

Amaranthus palmeri (Amaranthaceae) is a noxious weed in several agroecosystems and in some cases seriously threatens the sustainability of crop production in North America. Glyphosate-resistant Amaranthus species are widespread, prompting the use of alternatives to glyphosate such as glufosinate, in conjunction with glufosinate-resistant crop cultivars, to help control glyphosate-resistant weeds. An experiment was conducted to analyze the transcriptome of A. palmeri plants that survived exposure to 0.55 kg ha-1 glufosinate. Since there was no record of glufosinate use at the collection site, survival of plants within the population are likely due to genetic expression that pre-dates selection; in the formal parlance of weed science this is described as natural tolerance. Leaf tissues from glufosinate-treated and non-treated seedlings were harvested 24 h after treatment (HAT) for RNA-Seq analysis. Global gene expression was measured using Illumina DNA sequence reads from non-treated and treated surviving (presumably tolerant, T) and susceptible (S) plants. The same plants were used to determine the mechanisms conferring differential tolerance to glufosinate. The S plants accumulated twice as much ammonia as did the T plants, 24 HAT. The relative copy number of the glufosinate target gene GS2 did not differ between T and S plants, with 1 to 3 GS2 copies in both biotypes. A reference cDNA transcriptome consisting of 72,780 contigs was assembled, with 65,282 sequences putatively annotated. Sequences of GS2 from the transcriptome assembly did not have polymorphisms unique to the tolerant plants. Five hundred sixty-seven genes were differentially expressed between treated T and S plants. Of the upregulated genes in treated T plants, 210 were more highly induced than were the upregulated genes in the treated S plants. Glufosinate-tolerant plants had greater induction of ABC transporter, glutathione S-transferase (GST), NAC transcription factor, nitronate monooxygenase (NMO), chitin elicitor receptor kinase (CERK1), heat shock protein 83, ethylene transcription factor, heat stress transcription factor, NADH-ubiquinone oxidoreductase, ABA 8'-hydroxylase, and cytochrome P450 genes (CYP72A, CYP94A1). Seven candidate genes were selected for validation using quantitative real time-PCR. While GST was upregulated in treated tolerant plants in at least one population, CYP72A219 was consistently highly expressed in all treated tolerant biotypes. These genes are candidates for contributing tolerance to glufosinate. Taken together, these results show that differential induction of stress-protection genes in a population can enable some individuals to survive herbicide application. Elevated expression of detoxification-related genes can get fixed in a population with sustained selection pressure, leading to evolution of resistance. Alternatively, sustained selection pressure could select for mutation(s) in the GS2 gene with the same consequence

Summary of statistics for transcriptome assembly.

<p>Summary of statistics for transcriptome assembly.</p

Volcano plots depicting differential gene expression between treatments.

<p>A) Treated susceptible (S) relative to non-treated S plants (SWT vs SWO), B) Treated tolerant (T) relative to treated S plants (TWT vs SWT), C) treated T relative to non-treated T plants (TWT vs TWO), and D) treated T plants relative to non-treated S plants (TWO vs SWO). The x-axis shows the log fold change or relative abundance. The <i>P</i> value (-log base 10) for differential gene expression is plotted on the y axis. Dots in black represent genes that did not achieve significant changes in expression; colored dots on the left indicate genes with significantly downregulated expression and colored dots on the right indicates genes with significantly upregulated expression.</p

Heat map analysis of genes that are putatively related to abiotic stress response in <i>A</i>. <i>palmeri</i>.

<p>TWO (non-treated tolerant), SWO (non-treated susceptible), TWT (glufosinate-treated tolerant), SWT (glufosinate-treated susceptible).</p

The number of differentially expressed genes common or specific to treated and non-treated T and S plants.

<p>A 4-way Venn diagram depicting the distribution of differentially expressed genes across all pairwise comparisons. The number within each shaded area is the number of differentially expressed genes common in each compared treatments.</p