Evaluating Soil Micro-nutrient Bioavailability of Manganese in Glyphosate-resistant Soybeans

1. Abstract: Laboratory experiments, greenhouse studies and field research were performed in the spring and summer of 2011 to determine the root cause of possible Manganese (Mn) deficiencies in glyphosate-resistant crops. The goal was to examine whether the herbicide caused chelation of Mn, thus rendering it non-bioavailable for plant root uptake, or if soil matrix conditions (i.e., soil pH, electrical conductivity, cation exchange capacity, bioavailable micronutrients, and/or sorption capabilities) were involved in the response of Mn in the soil-plant environment. The greenhouse experiment involved growing three varieties of glyphosate-resistant soybeans, amending the soil with five rates of Mn prior to planting, and utilizing two rates of Roundup® Weathermax herbicide plus untreated blanks of all factors. After a 12-week growth period, determination of biomass production, chlorophyll production, and total Mn concentrations in the soil and plants were evaluated. During the growth phase, several laboratory experiments were conducted to determine soil solution sorption kinetics of Mn, glyphosate, and glyphosate-Mn interactions. In addition, experimentation of water-soluble Mn, bioavailable Mn fractions, and total Mn concentrations were determined. Field experiments were then conducted to investigate similar Mn and herbicide conditions; however, an additional glyphosate-resistant soybean variety was used, and the high Mn amendment in the greenhouse study was traded for a lower concentration. The soil used for this study was an eastern Wyoming Mollisol (fine-​​loamy, mixed, mesicFluventicHaplustoll), which was collected from the Sustainable Agriculture and Research Extension Center (SAREC) in Lingle, Wyoming. Relevance to Soil and Plant Science: An evolution in farming practices from conventional weed control practices to the use of glyphosate-resistant crops has generated interest in both disciplines as to the effects of the herbicide, Roundup®, its active ingredient, glyphosate, and the metabolite, aminomethylphosphonic acid (AMPA) (Figure 1). Research in the field of glyphosate-resistant crops and reciprocal interactions in soil fertility is of upcoming importance in relation to bioavailability (Table 2, Figure 3) and crop uptake of cationic, secondary micronutrients such as Manganese (Mn) in glyphosate-resistant crops as a function of glyphosate application. Manganese as Mn2+ is important as a co-factor in enzymatic processes in new leaf tissue (Heenan and Campbell, 1980). Although most soils contain sufficient Mn concentrations as Mn2+, Mn3+, and Mn4+(Kabata-Pendias, 2010), published research suggests that glyphosate and AMPA bind these cationic ions, and render them non-bioavailable for plant uptake (Zobiole et al., 2010). • EXPERIMENTAL OBJECTIVES: • Construct soil solution isotherms for the sorption of: • - Glyphosate • - Manganese • Use isotherms to explain Mn bioavailability in glyphosate-resistant soybeans in comparison to tissue concentrations and bioavailable Mn concentrations . RESULTS AND DISCUSSION: Results of our studies are important to sorption chemistry as it relates to glyphosate-resistant soybean’s ability to uptake Mn (Figures 5 and 6). Our soil has a slight basic pH and low organic matter that allow for the cation to be sorbed at a greater capacity (Table 1). When the soils were treated with 1200 mg Mn2+ kg-1 soil, the labile fraction increased exponentially as the cation was no longer able to be held as tightly on the soil surface (Table 2 and Figure 3) and subsequent Mn toxicity occurred in the soybeans (Figure 2), which is shown by plant chlorosis and necrosis throughout the high rate amendments. The exchangeable fraction experiment showed that if a glyphosate interaction should occur, a significant portion of the excess fraction could have been chelated by glyphosate, which would support Mn crop deficiency due to high zwitterionization potential of glyphosate and AMPA (Figure 1). The sorption isotherm results offered reasonable support to the greenhouse growth study in that glyphosate may not be the cause of Mn deficiency in glyphosate resistant crops and as was reflected by the tissue analysis of the glyphosate-resistant soybeans (Figure 4). In both of the isotherms, the compound and the cation showed higher affinity for binding to soil surfaces, which would lower the affinity for a chelation effect in the soil solution system. (a) (b) • MATERIALS: • Standard soil and solutions: • McCook Series; fine-loamy, mixed, mesicFluventicHaplustoll (NRCS, 2010) from Lingle, WY • Prepare 6 levels of Mn standards from MnCl2·4H2O • Prepare 5 levels of glyphosate from a 10 g L-1 standard solution. • Analytical equipment: • Inductively Coupled Plasma-Mass Spectrometer (ICP- MS) with ionic suppressor • Spectrophotometer with capability of 830 nm • Scales with both 2 and 4-place sensitivity • Other laboratory supplies: • - Sample shaker, 40 mL vials, centrifuge tubes, and centrifuge • - Water bathes, and temperature- controlled hot plate • - Spectrophotometer cuvettes Alix C. Hakala1, George F. Vance1, Andrew R. Kniss2, and Jay B. Norton1 Departments of Ecosystem Sciences and Management1 and Plant Science2, University of Wyoming Figure 1 (above). The compound of interest, glyphosate (a) and its metabolite, AMPA (b). Figure 2 (below). Not all treatments are created equal! Results of 5 levels of Mn (L to R in mg kg-1: 0, 200, 400, 800, 1200) are shown on the soybeans one-week post herbicide application. • METHODS: • General soil properties: • Determine pH, electrical conductivity, cation exchange capacity (CEC), % organic matter, and water soluble Mn by saturated paste extract • Find values for the exchangeable fraction of Mn via NH4OAc method (Methods of Soil Analysis, 2001) • Digest Mn amendments for total concentration by Solid and Waste method D8260 (EPA, 2001) • Soil solution chemistry: • Examine Mn sorption to the target soil • Formulate and execute an appropriate method based on Glass (1981) and Moser (2009) for sorption of glyphosate • Other methods: • Compare bioavailable Mn with plant tissue concentrations of Mn for correlation. Evaluating Soil Micro-nutrient Bioavailability of Manganese in Glyphosate-resistant Soybeans Figure 5 (above): Sorption of glyphosate by the McCook soil. Sorption affinity increased with glyphosate concentrations. A Freundlich K value calculated from the isotherm was 8.08 L kg-1. Figure 6 (left): Sorption of Mn by McCook soil. Langmuir Isotherm fit was better than Freundlich. Langmuir values calculated from the isotherm were kl = 0.016 L kg-1 and b = 2,500 mg kg-1 Post soil amendment test for exchangeable fraction of Manganese: Table 2: Post-amendment solution Mn Experiment Conclusions: In conclusion, this series of experiments showed that glyphosate has little effect on uptake of the micronutrient, Mn. Throughout the laboratory experiments and the greenhouse studies, little effect of glyphosate chelation on the cation was demonstrated, while the plant was able to continually uptake Mn for physiological processes until toxicity occurred. Sorption and bioavailable fraction experiments involving Mn demonstrated that Mn saturation on the binding sites of soil surfaces can overcome effects of pH and organic matter of the system and still cause toxicity. Also, glyphosate sorption is preferential towards the same soil surfaces, which has a stronger affinity than chelation in the soil solution system. Both conclusions are supported by the tissue analysis of Mn in the above and belowground biomass portions of the plant, where plant uptake of Mn follows a non-linear relationship with respect to the bioavailable fraction of Mn in the soil solution system. References: EPA (2001). Analytical Methods of Solid and Wastes Method D8260. Internet citation found at www.epa.org. Glass, R.L. Colorimetric determination of glyphosate in water after oxidation to orthophosphate. Anal Chem., 1981. 53 (6): 921-923. Heenan, D.P. and L.C. Campbell (1980). Transport and distribution of manganese in two cultivars of soybean (Glycine max (L.) Merr.). Australian Journal of Agricultural Research 31 (5) 943 - 949 Kabata-Pendias, A. Trace Elements in Soils and Plants (3rd Ed.). CRC Press, Boca Raton, FL (2001) pp. 80-87; 286-295; 299-304. Moser, S.C. New extraction and solid phase purification techniques for the analysis of glyphosate in vegetation, soil and water. EPA Region 6 QA meeting presentation in Powerpoint (Oct 20, 2009). Nollet, M.L., and H.S. Rathore (2010). Handbook of Pesticides (1st Ed.). CRC Press, Boca Raton, FL. pp.: 28; 343-348; 401-420. Methods of Soil Analysis: Part 3- Chemical Analysis. 2001. Soil Science Society of America, Madison, WI. Third printing pp. 581-586. Soil Survey Staff. 2001. Soil taxonomy: A basic system of soil classification for making and interpreting soil surveys. 2nd edition. Natural Resources Conservation Service. U.S.D.A. Handbook 436. Zobiole, L.H., R.S. de Oliveira, D.M. Huber, J. Constantin, C. de Castro, F.A. de Oliveira, and A. de Oliveira. Glyphosate reduces shoot concentrations of mineral nutrients in glyphosate-resistant soybeans. Plant Soil (2010) 328:57-69. Acknowledgements: The lead author would like to thank Drs. Kniss, Vance, and Norton, and the Wyoming Analytical Laboratories in Laramie, WY for the endless support of this project, especially Mr. Monte Ellis, Ms. Connie Boese, Mrs. Debra Augustin, and Mr. Frank Piraino. Also, thank you to both the staff at the Laramie Research and Extension Center (LREC) and the James E. Hageman SAREC research station.