Interaction of the Bioherbicide <i>Myrothecium verrucaria</i>with Technical-Grade Glyphosate on Glyphosate-Susceptible and -Resistant Palmer Amaranth

Previously we found that a strain of Myrothecium verrucaria (MV) exhibited bioherbicidal activity against several important weeds, and that some commercial formulations of glyphosate applied with MV resulted in synergistic interactions that improved weed control efficacy. We also found that MV had bioherbicidal activity against glyphosate-resistant Palmer amaranth. We have also reported that some commercial formulations are inhibitory to MV. Our objectives were to test the effect of unformulated glyphosate (high purity, technical-grade glyphosate) alone and in combination with MV for bioherbicidal activity on glyphosate-susceptible and -resistant Palmer amaranth biotypes under greenhouse conditions and to examine technical-grade glyphosate on the growth of this bioherbicide. High purity glyphosate (without adjuvants/surfactants) was not toxic to MV growth and sporulation at concentrations up to 2.0 mM when grown on agar supplemented with the herbicide. Both biotypes were injured by MV and MV plus glyphosate treatments as early as 19 h after application (3 h after a dew period of 16 h). These injury effects increased and were more evident through the 6-day time course, when after 120 h the MV plus glyphosate treatment had killed all glyphosate-susceptible and -resistant plants. The interaction of glyphosate plus MV was synergistic toward the control of Palmer amaranth. Data strongly suggest that the active ingredient is responsible for the synergy previously found when this bioherbicide was combined with some commercial formulations of glyphosate. Results demonstrated that MV can control both glyphosate-resistant and -susceptible Palmer amaranth seedlings and act synergistically with high-purity glyphosate to provide improved weed control.

Bioassay and Characterization of Several Palmer Amaranth (<i>Amaranthus palmeri</i>) Biotypes with Varying Tolerances to Glyphosate

The wide distribution of Palmer amaranth (Amaranthus palmeri) in the southern US became a serious weed control problem prior to the extensive use of glyphosate-resistant crops. Currently glyphosate-resistant populations of Palmer amaranth occur in many areas of this geographic region creating an even more serious threat to crop production. Investigations were undertaken using four biotypes (one glyphosate-sensitive, one resistant from Georgia and two of unknown tolerance from Mississippi) of Palmer amaranth to assess bioassay techniques for the rapid detection and level of resistance in populations of this weed. These plants were characterized with respect to chlorophyll, betalain, and protein levels and immunological responses to an antibody of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) the target site of glyphosate. Only slight differences were found in four biotypes grown under greenhouse conditions regarding extractable soluble protein and chlorophyll content, but one biotype was found to be devoid of the red pigment, betalain. Measurement of early growth (seedling shoot elongation) of seedlings was a useful detection tool to determine glyphosate resistance. A leaf disc bioassay (using visual ratings and/or chlorophyll analysis) and an assay for shikimate accumulation were effective methods for determining herbicide resistance levels. The two unknown biotypes were found to be resistant to this herbicide. Some differences were found in the protein profiles of the biotypes, and western blots demonstrated a weak labeling of antibody in the glyphosate-sensitive biotype, whereas strong labeling occurred in the resistant plants. This latter point supports research by others, that increased copy number of the EPSPS gene (and increased EPSPS protein levels) is the resistance mechanism in this species. Results indicate the utility of certain bioassays for the determination of resistance and provide useful comparative information on the levels of inherent constituents among closely related plants.

Influence of Water Quality, Formulation, Adjuvant, Rainfastness, and Nozzle Type on Efficacy of Fomesafen on Palmer Amaranth (<i>Amaranthus palmeri</i>) Control

Protoporphyrinogen oxidase (PPO) inhibitors are one of the few remaining postemergence herbicide options for controlling Palmer amaranth in soybean growing areas of Mississippi, USA. Most Palmer amaranth populations in Mississippi are resistant to both glyphosate and acetolactate synthase inhibitors. Resistance to PPO inhibiting herbicides in Palmer amaranth has very recently been reported in Arkansas, Tennessee, and isolated pockets of Mississippi. A significant proportion of reports of PPO inhibitor failures in Mississippi are not considered to be resistance-related at this time. Therefore, the objective of this research was to evaluate factors affecting the efficacy of fomesafen on Palmer amaranth including: quality of spray carrier (water), formulations, adjuvant, rainfastness, and nozzle type. All water samples and formulation combinations provided >95% control of Palmer amaranth 3 WAT. Some combinations of water samples and formulations did not result in complete control of the treated plants, with one or two individuals surviving 3 WAT. Formulation 1 provided 99% control compared to 95% from formulation 2. Irrespective of combinations of herbicide, adjuvant and height, control of Palmer amaranth was ≥91%. Formulation 1 provided 94% control compared to 88% from formulation 2. The adjuvant x height interaction was significant, owing to a 10% reduction in control of larger plants (86%) compared to smaller plants (96%) in presence of COC. COC provided better control (93%) than NIS (88%). Simulated rainfall applied ≥60 min after herbicide application did not adversely affect efficacy on Palmer amaranth when formulation 1 was applied in combination with NIS, with control ranging from 94% to 100%. Formulation 1 with COC provided ≥93% control at all rainfall application times, except 30 min after herbicide treatment, which resulted in 79% control. Formulation 2 provided better control with COC (79% to 100%) than NIS (71% to 90%), in general, across the rainfall treatments applied at various times following herbicide application. All nozzle and weed height combinations resulted in 89% or better control of Palmer amaranth. In summary, water quality, formulation, adjuvant, rainfastness, or nozzle type did not affect the activity of fomesafen under optimal application conditions in the greenhouse.

Genomic Stability of Palmer amaranth Plants Derived by Macro-Vegetative Propagation

qPCR (quantitative polymerase chain reaction) and random amplified polymorphic DNA (RAPD) were utilized to investigate genetic stability of Palmer amaranth cloned plants over 10 generations. DNA from original parent Palmer amaranth plants (grown from seeds) was re-analyzed using qPCR, and confidence levels for determining ΔΔCt (threshold crossing) values were established. ANOVA was used to determine variation (margin of error) of these ΔΔCt values. This margin of error was applied to qPCR analysis of DNA from eight individual parent plants and their descendants (10th generation) so that possible differences in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene copy number could be ascertained. This method (and the associated error) indicated a lack of agreement in ΔΔCt values of DNA from plants of these two generations. qPCR analysis showed that in five out of eight clones, EPSPS gene copy number varied more than the calculated error (P = 0.05). A second technique to monitor genetic stability, RAPD was used to determine possible changes in genomic DNA due to extended cloning of these regenerated plants. RAPD analysis showed that four out of the eight clones differed when the profiles of the two generations were compared. Results show that qPCR and RAPD analysis point to the fact that several Palmer amaranth clones experienced changes in genome structure over 10 generations. Although the glyphosate resistance trait was retained, results suggest that during cloning studies, the genetic stability of macro-vegetatively propagated lines should be monitored.

Target Site-Based Resistance to ALS Inhibitors, Glyphosate, and PPO Inhibitors in an <i>Amaranthus palmeri</i>Accession from Mississippi

Extensive acceptance of glyphosate-resistant (GR) row crops coupled with the simultaneous increase in glyphosate usage has sped the evolution of glyphosate resistance in economically important weeds. GR Amaranthus palmeri populations are widespread across the state with some exhibiting multiple resistance to acetolactate synthase (ALS) inhibiting herbicides such as pyrithiobac. A GR and ALS inhibitor-resistant accession was also resistant to the protoporphyrinogen oxidase (PPO) inhibiting herbicide fomesafen. The PPO inhibitor resistance profile and multiple herbicide resistance mechanisms in this accession were investigated. In addition to fomesafen, resistance to postemergence applications of acifluorfen, lactofen, carfentrazone, and sulfentrazone was confirmed. There was no resistance to preemergence application of fomesafen, flumioxazin, or oxyfluorfen. Molecular analysis of the ALS gene indicated the presence of point mutations leading to single nucleotide substitutions at codons 197, 377, 574, and 653, resulting in proline-to-serine, arginine-to-glutamine, tryptophan-to-leucine, and serine-to-asparagine replacements, respectively. The resistant accession contained up to 87-fold more copies of the EPSPS gene compared to a susceptible accession. A mutation leading to a deletion of glycine at codon 210 (ΔG210) of PPO2 gene was also detected. These results indicate that the mechanism of resistance in the Palmer amaranth accession is target-site based, i.e., altered target site for ALS and PPO inhibitor resistance and gene amplification for glyphosate resistance.

Effects of <i>Myrothecium verrucaria</i>on Two Glyphosate-Resistant <i>Amaranthus palmeri</i>Biotypes Differing in Betacyanin Content

Previously we found two biotypes of Amaranthus palmeri (Palmer amaranth) in a population of this economically important weed that were resistant to glyphosate but differed with respect to pigmentation. One biotype was typically red-pigmented (betacyanin) while the other was green, with no visual appearance of red hue on any plant part at any growth stage. We have also reported that a strain of Myrothecium verrucaria (MV) exhibited bioherbicidal activity against several important weeds including glyphosate-resistant Palmer amaranth. In greenhouse tests, MV was applied to these two biotypes (red and green) at two ages (3-week- and 6-week-old) and effects of this fungus monitored over a 5-day time course. Initial symptoms of MV (16 to 24 h after inoculation) were: epinastic curvature, wilting and development of lesions on leaves and stems. Generally, the younger plants tended to be more sensitive to MV than older plants. Bioherbicidal damage increased with time leading to necrosis and plant mortality and increasing disease progress. Severe loss of fresh weight occurred in both biotypes as compared to untreated plants. Results indicated that MV was effective on both biotypes, but effects on growth reduction and disease progression were more rapid and generally greater in the green biotype, suggesting that compounds responsible for red pigmentation may be more potent as defense against pathogen attack.

Morphological Characterization of <i>Amaranthus palmeri</i>x <i>A. spinosus</i>Hybrids

The growth of clones of seven Amaranthus palmeri x A. spinosus hybrids was compared to type specimens of A. palmeri and A. spinosus. The hybrids came from the field where they were originally discovered and clones of the type specimens and hybrids were established under greenhouse conditions and used to compare growth rates. A. palmeri had the highest growth rate and A. spinosus the lowest growth rate based on height, node counts, and dry weight accumulation. A. palmeri also had the greatest number of days to flowering and A. spinosus the fewest. Hybrids had intermediary growth rates and days to flowering, but differed from each other with regard to sex identity. The hybrids were either dioecious like A. palmeri or, if monoecious, had patterns unlike A. spinosus. Spine length and texture also varied in hybrids and some were without spines. Hybrid 16Ci was short compared to all others and had succulent leaves and stems, which easily separated from the plant body. These hybridizations resulted in morphologically distinct types with acquisition of physical traits intermediate to the type specimens which may drive evolution of these species.

Bioherbicidal Efficacy of a Myrothecium verrucaria-Sector on Several Plant Species

Comparative studies were conducted on mycelial preparations of the bioherbicide, Myrothecium verrucaria (MV) strain IMI 361690 and a recently discovered sector (MV-Sector BSH) of this fungus. The whitish sector was discovered, isolated, grown in pure culture on PDA and found to be a stable, non-spore producing mutant when cultured over several months under conditions that cause circadian sporulation during growth of its MV parent. Application of MV and MV-Sector BSH mycelial preparations to intact plants (hemp sesbania and sicklepod) and leaf discs (kudzu and glyphosate-resistant Palmer amaranth) showed that the sector efficacy was generally equal to, or slightly lower than MV. Bioassays of MV and this sector on seed germination and early growth of sicklepod and hemp sesbania seeds demonstrated that hemp sesbania seeds were slightly more sensitive to the fungus than sicklepod seeds and that the sector bioherbicidal activity was slightly less than that of MV. SDS-PAGE protein profiles of cellular extracts of MV and the sector and their respective culture supernatants showed several differences with respect to quantity and number of certain protein bands. Overall results showed that the isolate was a non-spore producing mutant with phytotoxicity to several weeds (including weeds tolerant or resistant to glyphosate), and that the phytotoxic effects were generally equivalent to those caused by MV treatment. Results of this first report of a non-sporulating MV mutant that suggest additional studies on protein analysis, and an extended weed host range under greenhouse and field conditions are needed in order to further evaluate its possible bioherbicidal potential.

Using Vegetation Indices as Input into Random Forest for Soybean and Weed Classification

Weed management is a major component of a soybean (Glycine max L.) production system;thus, managers need tools to help them distinguish soybean from weeds. Vegetation indices derived from light reflectance properties of plants have shown promise as tools to enhance differences among plants. The objective of this study was to evaluate normalized difference vegetation indices derived from multispectral leaf reflectance data as input into random forest machine learner to differentiate soybean and three broad leaf weeds: Palmer amaranth (Amaranthus palmeri L.), redroot pigweed (A. retroflexus L.), and velvetleaf (Abutilon theophrasti Medik). Leaf reflectance measurements were acquired from plants grown in two separate greenhouse experiments conducted in 2014. Twelve normalized difference vegetation indices were derived from the reflectance measurements, including advanced, green, greenred, green-blue, and normalized difference vegetation indices, shortwave infrared water stress indices, normalized difference pigment and red edge indices, and structure insensitive pigment index. Using the twelve vegetation indices as input variables, the conditional inference version of random forest (cforest) readily distinguished soybean and velvetleaf from the two pigweeds (Palmer amaranth and redroot pigweed) and from each other with classification accuracies ranging from 93.3% to 100%. The greatest errors were observed between the two pigweed classes, with classification accuracies ranging from 70% to 93.3%. Results suggest combining them into one class to increase classification accuracy. Vegetation indices results were equivalent to or slightly better than results obtained with sixteen multispectral bands used as input data into cforest. This research further supports using vegetation indices and machine learning algorithms such as cforest as decision support tools for weed identification.

Assessing Hyperspectral Vegetation Indices Responses of Six Pigweed Species

Pigweeds (Amaranthus species), negatively impact crop production systems throughout the world. They are distinguished from each other using manual methods that are tedious and time-consuming to complete. Hyperspectral light reflectance properties of plant leaves and canopies have shown promise for detecting and mapping weeds in crop production systems. Vegetation indices derived from hyperspectral reflectance data enhance differences between plants, leading to better detection of them from other targets. The objective was to evaluate the biomass and structural index, the biochemical index, the red edge index, the water and moisture index, the light-use efficiency index, and the lignin cellulose index for measuring differences among six pigweed species: Amaranthus albus (L), A. hybridus (L), A. palmeri (S. Wats.), A. retroflexus (L), A. spinosus (L), and A. tuberculatus [(Moq.) Sauer]. Two experiments were conducted under greenhouse conditions. Hyperspectral reflectance measurements were collected from the plant canopies on two dates for each experiment. Analysis of variance (ANOVA) and Tukey’s honest significant difference (HSD) test were used to determine if statistical differences (P ≤ 0.05) existed among the pigweed species canopies and to identify which species were statistically different for a vegetation index, respectively. The ANOVA analysis detected statistical differences among the canopy vegetation index values. Tukey’s HSD showed that the biochemical index and the red edge index detected differences between two to three pigweeds species on all dates of data collection. However, the differences were date-specific. Furthermore, statistical differences were not observed for all six species for any vegetation index. On the data collection dates, A. albus and A. tuberculatus index values were statistically different from other pigweed species for one or more of the vegetation indices. Future research should focus on using the vegetation indices in combination with each other to measure differences between the pigweed species and between them and other weeds and crops.

Efficacy of 2,4-D, Dicamba, Glufosinate and Glyphosate Combinations on Selected Broadleaf Weed Heights

Palmer amaranth, sicklepod and pitted morningglory are the three most common and troublesome weeds in soybean in South Carolina. They exhibit very aggressive growth capabilities and if left uncontrolled in fields will cause significant reductions in soybean yields. Dicamba and 2,4-D herbicides are currently having a resurgence in usage due to the recent commercialization of soybean trait technologies with tolerance to these herbicides. Dicamba and 2,4-D when tank mixed with glufosinate and glyphosate may offer additional weed control to resistant weeds through the process of herbicide synergism. Greenhouse experiments were conducted in 2013 at Edisto Research and Education Center near Blackville, SC to evaluate the efficacy of glyphosate, glufosinate, dicamba and 2,4-D treatments alone and in combination on Palmer amaranth, sicklepod, and pitted morningglory at selected heights. Results suggested that glufosinate alone provided the overall best control for all 3 weed species. Glyphosate alone provided the lowest control of all 3 species at all heights. Synergism or improved sicklepod control was observed when glufosinate was tank mixed with dicamba. However, as sicklepod increased in height, glufosinate + 2,4-D or dicamba combination offered the best control compared to glufosinate alone (90% versus 86% in 20 cm plants and 87% versus 85% in 30 cm plant). In the 5 cm Palmer amaranth, decreased control was observed when glyphosate or glufosinate was tank mixed with 2,4-D. These experiments showed that glufosinate alone and/or in combination with 2,4-D or dicamba was the overall best treatment on the three broadleaf weed species.

Weed Control and Crop Safety with Premixed <i>S</i>-Metolachlor and Sulfentrazone in Sunflower

A preliminary study conducted in the central USA near Colby and Hays, Kansas (KS) in 2010 indicated a premix of S-metolachlor & sulfentrazone codenamed F7583 (Broadaxe?) had good potential for use in sunflower (Helianthus annuus L.). Additional studies were conducted in 2011 at Colby, Hays, Manhattan, KS to refine rate and application timing of F7583 for weed control and crop safety. Four rates of F7583 (860, 1100, 1350 and 1840 g·ha-1) were compared to single rates of S-metolachlor and pendimethalin, and applied 21 days preplant versus preemergence (PRE). F7583 at ≥1100 g·ha-1 applied preplant or PRE controlled Palmer amaranth (Amaranthus palmeri S. Wats.) and kochia [Kochia scoparia (L.) Schrad.] ≥95% and 100%, respectively in neutral pH soils. In slightly acidic soils, PRE application of F7583 was more effective against Palmer amaranth and grass weeds compared to preplant application. No benefit was gained by increasing the rate of F7583 from 1100 to 1350 g·ha-1 at either application timing. Puncturevine (Tribulus terrestris L.) control was not commercially satisfactory with F7583 at any rate or time of application. Both S-metolachlor at 1070 g·ha-1 and pendimethalin at 1600 g·ha-1 applied either preplant or PRE were considerably less effective on all three broadleaf weeds compared to F7583 treatments. Individually, S-metolachlor and pendimethalin were more effective when applied PRE compared to preplant application. F7583 did not reduce sunflower plant population or visibly injure sunflower anytime during the season.

Broadleaf Weed Control in Sunflower (<i>Helianthus annuus</i>) with Preemergence-Applied Pyroxasulfone with and without Sulfentrazone

A field study was conducted at two locations in Kansas, USA in 2011 and 2012 to test weed control efficacy and crop response to preemergence-applied pyroxasulfone alone and in combination with sulfentrazone in sunflower. Treatments included three rates of pyroxasulfone (100, 200 and 400 g·ha-1) applied alone and tank-mixed with sulfentrazone at 70, 140 and 280 g·ha-1. Commercial standards sulfentrazone at 140 g·ha-1 + pendimethalin at 1390 g·ha-1 and sulfentrazone at 140 g·ha-1 + S-metolachlor at 1280 g·ha-1 were also included. Pyroxasulfone at 100 g·ha-1 controlled Palmer amaranth 87% at 3 weeks after application (WAA), but control decreased to 76% at 6 WAA. Increasing pyroxasulfone rate to ≥200 g·ha-1 or tank mixing with sulfentazone at 140 g·ha-1 provided ≥90% Palmer amaranth control for at least 6 WAA. Sulfentrazone alone at 70 g·ha-1 controlled Palmer amaranth 77% at 3 WAA, but control dropped to 69% at 6 WAA. Increasing sulfentrazone rate from 70 to 140 or 280 g·ha-1 increased control to >90% at 3 WAA, but did not maintain acceptable control at 6 WAA. Tank mixing sulfentrazone at 140 g·ha-1 with pendimethalin at 1390 g·ha-1 or S-metolachlor at 1280 g·ha-1 controlled Palmer amaranth ≥90 and 84% at 3 WAA and 6 WAA, respectively. The lowest rate of pyroxasulfone (100 g·ha-1) controlled kochia 98% and the control was complete with all other treatments. However, no treatment provided as much as 90% puncturevine control at 3 WAA and the control was commercially unacceptable (<75%) at 6 WAA. No treatment visibly injured sunflower anytime during the season or reduced sunflower plant population.