Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Row crop growers (specifically cotton, soybean, corn), vegetable growers, Extension Agents, Chemical Company representatives, University researchers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The graduate student working on this project was trained on molecular biology protocols, specifically DNA/RNA extraction, primer design, gene amplification by PCR, cDNA synthesis, RT-QPCR gene expression analysis, and PCR-based assay of SNPs. The graduate student also received training on statistical analysis softwares, oral presentation, and writing scientific papers How have the results been disseminated to communities of interest?Through presentations of research at Professional Weed Science meetings. Publication of research in refereed journals. Publication of Extension Bulletins. What do you plan to do during the next reporting period to accomplish the goals? Conduct RNA-seq analysis experiment in response to fomesafen, dicamba, S-metolachlor, and glufosinate. Perform dose-response studies with populations selected for the RNA-seq experiments. Generate the full resistance profile of this line to determine other resistance traits driven by NTSR mechanisms. Parallel to the above, test the effect of Cyt P450 inhibitors and GST inhibitors on the activity of fomesafen. Produce F2 of purified PPO and dicamba NTSR lines (about 10 months) to cross with SS line to produce segregating population for QTL analysis.
Impacts
What was accomplished under these goals?
General screening for herbicide resistance, overview. Resistance was testedwith 1X dose of each herbicide: fomesafen 264 g ha-1, glufosinate-ammonium 655 g ha-1, dicamba 560 g ae ha-1, and S-metolachlor 1120 g ai ha-1. Standard protocol was followed. At least 100 plants per accession were tested for each herbicide. A susceptible standard was included in each assay. The assay was repeated. Seedlings were sprayed when 3- to 4 inches tall on average, in a spray chamber. Herbicide effect was assessed visually 21 days after treatment (DAT) using a 0 to 100% scale, where 0 represents absence of symptoms and 100% is dead. For S-metolachlor, 100 seeds were planted in pots filled with the same amount of field soil, sprayed with herbicide, and irrigated with a fine mist to wet the soil and activate the herbicide. Treated pots were replicated three times and the test was repeated. Non-treated checks were planted as reference. Putative resistant populations were subjected to dose response assays to determine the level of resistance. A population from an organic farm was used as the susceptible standard. Resistance to fomesafen In the mid-south (AR, MO, MS, TN), 15% of accessions tested were susceptible, with mortality between 90 and 100% in general and the few survivors incurring generally >90% injury.The majority of Arkansas populations had high mortality, but the survivors were highlyresistant, with about 40% average injury. About 80% of Mississippi populations had low mortality (about 45% average), but high injury of survivors (about 70% average). Thus, many plants survive the field dose, the survivors were highly injured, but will most likely recover without follow-up intervention. The most problematic populations were from Missouri where mortality was generally low and the survivors incurred only about 50% injury.To verify the possibility of and G399 mutations occurring in the same allele of the PPO2 gene, crosses between specific genotypes were made. We are also studying NTSR resistance. Cross resistance to PPO inhibitors is being conducted in five Palmer populations that do not carry any mutations based on previous study. This will allow us to choose which population to use for a transcriptome study, to identify genes driving NTSR to PPO herbicides. Survey of PPO mutations among fomesafen survivors A total of 167 Palmer amaranth populations were sprayed with a labeled rate of Flexstar (264 g ai ha-1). The PPO2 gene of survivors wasassayed for mutations. The cross-resistance profile of selected populations was characterized using a representative of chemical families in this group, including the new chemistries: saflufenacil (Sharpen) and trifludimoxazin (Tirexor). Resistance to PPO inhibitors in Palmer amaranth is widespread across the mid-southern States. Only 12% of the populations were completely controlled by fomesafen. Some populations showed extremely high resistance, with survival rate higher than 90%. The deletion of G210 codon in the ppo2 gene is the most prevalent mutation; occasionally occurring with other mutations within a population, or in the same plant. High resistance level is generally associated with a high frequency of individuals carrying single mutations or populations containing multiple mutations. Saflufenacil and trifludimoxazin, which belong to different chemical families compared to fomesafen, could control one-half of the resistant populations tested >90%. Thus, the new herbicides are more effective than the old chemistries, but pre-selection rendered the new PPO herbicides highly at risk. This information reinforces the need for growers to use the new tools judiciously and inform the integration of these new tools into existing weed management programs. Characterization of Palmer amaranth ?G210 mutation Transgenic rice overexpressing the Palmer amaranth ppo2 ΔG210 gene was generated via particle bombardment. Transgenic seedlings were sprayed postemergence with 0.78 kg ha-1 fomesafen (2x) and the plants containing the construct showed low injury from herbicide in contrast to the non-transformed plants. In a soil-based assay, fomesafen caused 27% reduction in germination of T2 seeds demonstrating that the insertion of Palmer amaranth ppo2 containing ΔG210 mutation conferred resistance to fomesafen in rice. All survivors carried the ppo2 transgene and showed 155- to 1144-fold increase in expression of the Palmer ppo2 gene compared to the non-transformed rice line. T3 progenies of survivors from soil-applied fomesafen carried the Palmer amaranth ppo2 transgene. Resistance to S-metolachlor Six opulations were significantly less controlled by the labeled rate in comparison to the susceptible standard. At least one accession required 3 times more S-metolachlor to be controlled 90%. Early detection of S-metolachlor-resistant Palmer amaranth accessions in Arkansas will help raise awareness on the loss of important herbicides for controlling troublesome weeds.Dose-response assays were conducted on selected populations. The constitutive induction of candidate NTSR genes associated with metolachlor, and the induction of these same genes in response to S-metolachlor application has been evaluated in six resistant populations from AR. The data indicate that GST genes are responsible for resistance. Resistant plants have high expression of certain GST genes even without herbicide treatment, and further induction of such genes in response to herbicide treatment. Resistance to dicamba We have generated F1 populations from survivors of dicamba application. Phenotyping of those is on-going. The data thus far show a shift in resistance level/survival rates in some cases. The resistance testing of F1 lines was repeated. F1 seeds harvested in September are being tested. A dose-response study of selected populations with dicamba is being conducted and the one with the highest resistance has been selected for transcriptome study to identify genes that confer resistance to dicamba or other auxinic herbicides. We observed that the regrowth pattern of survivors from a dicamba application is variable. For example, some plants lose their apical dominance in response to dicamba, but others do not. Also, sometimes the newly developed leaves are injury-free, sometimes the new leaves exhibit auxinic herbicide symptoms. Because of that, the survivors were grouped according to their recovery phenotype, disregarding their population source. Resistance to glufosinate (Liberty) A set of eight F1 populations derived from glufosinate survivors was provided by Dr. Taghi Bararpour. The amount of seed and germination percentage was highly variable, which limited the number of plants that could be tested. Glufosinate was able to control the majority of the F1 plants tested. However, there were some survivors, up to 11 plants in one F1 population. The F1 glufosinate survivors are being grown to produce F2 seed.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Noguera MM, Rangani, G, Heiser J, Bararpour T, Steckel LE, Betz M, Porri A, Lerchl J, Zimmermann S, Nichols RL and Roma?Burgos N. (2020), Functional PPO2 mutations: co?occurrence in one plant or the same ppo2 allele of herbicide?resistant Amaranthus palmeri in the US mid?south. Pest Manag Sci. https://doi.org/10.1002/ps.6111
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Roma-Burgos N, MM Noguera, L Steckel, JW Heiser, T Bararpour, RL Nichols (2020). How is Dicamba Doing on Palmer Amaranth (Amaranthus palmeri) in the US Mid-South? Proc Annual Meeting, Weed Sci Soc Amer, Maui, HI (403)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Rangani G, L Benedetti, M Noguera, N Roma-Burgos (2020). Understanding the mechanism of resistance to S-metolachlor in Palmer amaranth. Proc 73rd Annual Meeting Southern Weed Sci Soc, Biloxi, MS. p 96 (Poster)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Carvalho de Lima P, G Rangani, S Zhao, AC Langaro, V Srivastava, N Roma-Burgos (2020). Characterization of Amaranthus palmeri ppo2 (?G210) mutation in rice. Proc 73rd Annual Meeting Southern Weed Sci Soc, Biloxi, MS. p 96 (Oral) p 130
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Noguera MM, Rangani G, N Roma-Burgos (2020). Patterns of PPO resistance evolution in Palmer amaranth from mid-southern US. Proc 73rd Annual Meeting Southern Weed Sci Soc, Biloxi, MS. p 96 (Oral) p 157
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Row crop growers (specifically cotton, soybean, corn), vegetable growers, Extension Agents, Chemical Company representatives, University researchers. Changes/Problems:Work in 2019 focused more heavily on resistance to PPO and screening for resistance to dicamba. Research on glufosinate was set aside momentarily as resources dictated. What opportunities for training and professional development has the project provided?The graduate student working on this project was trained on molecular biology protocols, specifically DNA/RNA extraction, primer design, gene amplification by PCR, cDNA synthesis, RT-QPCR gene expression analysis, and PCR-based assay of SNPs. The graduate student also received training on statistical analysis softwares, oral presentation, and writing scientific papers The post-doctoral research associate (molecular biologist) working on this project had training on resistance screening, phenotyping, plant culture, research presentations, and bioinformatics. How have the results been disseminated to communities of interest? Through presentations of research at Professional Weed Science meetings.Publication of research in refereed journals. What do you plan to do during the next reporting period to accomplish the goals?Resistance to PPO inhibitors Publish the PPO resistance and mutation survey data from the US mid-south. The manuscript has now undergone one round of peer review. (Resubmit to journal March 9) Grow individuals from a population known to have NTSR resistance to PPO inhibitors for RNAseq experiment to identify the metabolic genes or other genes involved in NTSR mechanisms to PPO herbicides. Generate the full resistance profile of this line to determine other resistance traits driven by NTSR mechanisms. Parallel to the above, test the effect of Cyt P450 inhibitors and GST inhibitors on the activity of fomesafen. Produce F2 of purified PPO NTSR lines (about 10 months) to cross with SS line to produce segregating population for QTL analysis in the following year. Verification of putative resistance to dicamba Screening for resistance to dicamba of all Tennessee, 2017 and 2019 Mississippi, and 2018 Arkansas samples Conduct dicamba dose response assays, comparing field populations and the offspring of survivors. Submit a short report about Palmer amaranth response to dicamba across the US mid-south for publication. Collect new samples from AR and recover seeds from these Prepare plant materials for RNAseq experiment using the most dicamba-tolerant plants. Produce F2 of purified dicamba NTSR lines Resistance to S-metolachlor Screen populations from AR, MO, MS, TN Determine level of resistance in resistant populations Validate the induction of candidate NTSR genes in resistant populations Differential tolerance to glufosinate (The least advanced component of the project) Screen AR18, MO18, and TN18 (around 90 accessions) with glufosinate. Analyze strong glufosinate survivors for possible mutation in the GS gene; if mutations are not detected, these plants will be studied for NTSR mechanism(s). Study F1 glufosinate survivors from Mississippi, from Dr. Bararpour's screen of recent samples. Conduct dose response assays on most tolerant populations. Prepare plant materials for RNAseq experiment using the most tolerant lines, to generate a new transcriptome data set in response to glufosinate.
Impacts
What was accomplished under these goals?
General screening for herbicide resistance, overview. The screening for resistance was done with 1X dose of each herbicide: fomesafen 264 g ha-1, glufosinate-ammonium 655 g ha-1, dicamba 560 g ae ha-1, and S-metolachlor 1120 g ai ha-1. For the foliar herbicide, aach accession was sown in a 50-cell tray filled with Sunshine® potting medium (Sunshine premix No. 1®; Sun Gro Horticulture, Bellevue, WA). Five to ten seeds were planted per cell; seedlings were thinned to one per cell. One 50-cell plastic tray was considered a replication, and each herbicide had two replications. Therefore, 100 plants per accession were tested for each herbicide. A set of 10 plants per accession was grown separately as non-treated checks. A susceptible standard was included in each assay. The assay was repeated. Seedlings were sprayed when 3- to 4 inches tall on average, in a spray chamber. Herbicide effect was assessed visually 21 days after treatment (DAT) using a 0 to 100% scale, where 0 represents absence of symptoms and 100% is dead. For S-metolachlor, 100 seeds were planted in pots filled with the same amount of field soil, sprayed with herbicide, and irrigated with a fine mist to wet the soil and activate the herbicide. Treated pots were replicated three times and the test was repeated. Non-treated checks were planted as reference. Putative resistant populations were subjected to dose response assays to determine the level of resistance. A population from an organic farm was used as the susceptible standard. Response to fomesafen (Flexstar) In the mid-south (AR, MO, MS, TN), 15% of accessions tested were susceptible, with mortality between 90 and 100% in general and the few survivors incurring generally >90% injury. The majority of susceptible populations were from Arkansas. The majority of Arkansas populations had high mortality, but the survivors were most resistant, with about 40% average injury. About 80% of Mississippi populations had low mortality (about 45% average), but high injury of survivors (about 70% average). Thus, many plants survive the field dose, the survivors were highly injured, but will most likely recover without follow-up intervention. The most problematic populations were from Missouri where mortality was generally low and the survivors incurred only about 50% injury. Survey of PPO mutations among fomesafen survivors The presence of target site mutations was verified using TaqMan® qPCR assays for the known mutations ?G210 and R128G/M. The G399A mutation will be genotyped using the classical PCR/electrophoresis approach. The PPO mutation assay was done on the survivors from 120 field populations. The majority (80%) of populations with survivors carried at least one of the four resistance-conferring mutations. About ¼ of populations with survivors did not have any known PPO mutations. Roughly 1/3 of populations with PPO mutations, had survivors that carry only 1 of 4 known mutations; 25% had 2 of 4 mutations. Three field populations carried all four mutations among its resistant plants. In past studies, the mutations usually occur in different resistant plants, rather than all in one plant. The ?G210 mutation was detected in 80% of accessions with mutations, followed by G399A (51%) and R128G (43%). Out of 122 individuals genotyped, 37 carried two PPO mutations. The R128M mutation was rare. The PPO gene of survivors that do not carry any known mutations will be sequenced. Survivors with <70% injury, but do not harbor any PPO mutation will be studied for nontarget-site resistance mechanisms. Response to glufosinate (Liberty) Seeds from survivors of Liberty and Dicamba, from the earlier batch of screening, were harvested and processed (18 F1 populations from dicamba survivors and 6 F1 populations from glufosinate survivors). Seeds of these were planted and are being evaluated for the inheritance of tolerance or resistance to either herbicide. The healthiest survivors of full dose glufosinate application were grown for seed production and F1 lines were evaluated for their response to glufosinate. F1 seeds from survivors or previous screenings from Arkansas are also being evaluated. Thus far, no field population can be classified as resistant to glufosinate. Response to dicamba Two field samples from Missouri showed the highest tolerance to dicamba. F1 lines were produced. The tolerance level to dicamba is being evaluated with respect to the field population in dose response assays. The samples from MO were tested with dicamba and survivors are growing for seed production. Response to S-metolachlor Palmer amaranth accessions from Arkansas are not equally controlled by the labeled rate of S-metolachlor. Three accessions were significantly less controlled by the labeled rate in comparison to the susceptible standard. At least one accession required 3 times more S-metolachlor to be controlled 90%. Early detection of S-metolachlor-resistant Palmer amaranth accessions in Arkansas will help raise awareness on the loss of important herbicides for controlling troublesome weeds.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Rangani G, RA Salas-Perez, RA Aponte, M Knapp, IR Craig, T Mietzner, AC Langaro, MM Noguera, A Porri, N Roma-Burgos� (2019) A novel single-site mutation in the catalytic domain of protoporphyrinogen oxidase IX (PPO) confers resistance to PPO-Inhibiting Herbicides. Front Plant Sci 10:568. doi: 10.3389/fpls.2019.00568
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