Foliar-applied glyphosate substantially reduced uptake and transport of iron and manganese in sunflower (Helianthus annuus L.) plants

Evidence clearly shows that cationic micronutrients in spray solutions reduce the herbicidal effectiveness of glyphosate for weed control due to the formation of metal-glyphosate complexes. The formation of these glyphosate-metal complexes in plant tissue may also impair micronutrient nutrition of nontarget plants when exposed to glyphosate drift or glyphosate residues in soil. In the present study, the effects of simulated glyphosate drift on plant growth and uptake, translocation, and accumulation (tissue concentration) of iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu) were investigated in sunflower (Helianthus annuus L.) plants grown in nutrient solution under controlled environmental conditions. Glyphosate was sprayed on plant shoots at different rates between 1.25 and 6.0% of the recommended dosage (i.e., 0.39 and 1.89 mM glyphosate isopropylamine salt). Glyphosate applications significantly decreased root and shoot dry matter production and chlorophyll concentrations of young leaves and shoot tips. The basal parts of the youngest leaves and shoot tips were severely chlorotic. These effects became apparent within 48 h after the glyphosate spray. Glyphosate also caused substantial decreases in leaf concentration of Fe and Mn while the concentration of Zn and Cu was less affected. In short-term uptake experiments with radiolabeled Fe (59Fe), Mn (54Mn), and Zn (65Zn), root uptake of 59Fe and 54Mn was significantly reduced in 12 and 24 h after application of 6% of the recommended dosage of glyphosate, respectively. Glyphosate resulted in almost complete inhibition of root-to-shoot translocation of 59Fe within 12 h and 54Mn within 24 h after application. These results suggest that glyphosate residues or drift may result in severe impairments in Fe and Mn nutrition of nontarget plants, possibly due to the formation of poorly soluble glyphosate-metal complexes in plant tissues and/or rhizosphere interactions.     

Changes in Soluble Manganese and Iron Concentrations of Tropical Wetland Soils as Influenced by Glyphosate Dosage

Glyphosate is largely used to control weeds in wetland soils of Brazil. We investigated changes in the chemistry of soluble manganese (Mn) and iron (Fe) in these soils as affected by glyphosate dosage. Triplicate samples of the A horizon of wetland soils with different organic-matter contents were incubated with deionized water (1:2) for 1, 3, and 30 days under flooding. Three different glyphosate doses (0, 0.048, and 0.096 g L^?1 m^?2) were spiked on the flooded water at the beginning of the incubation periods. After incubation, pH was measured and samples of the supernatant were collected for determination of Mn/Fe concentrations by atomic absorption. Glyphosate application impacted Mn but had no effect on pH and Fe. Soluble Mn concentrations decreased as glyphosate dosage increased for the high organic-matter soil after 3 days of incubation. It indicated that glyphosate application can change the chemistry of soil metals. The intensity of these changes depends on the glyphosate dosage, evolved metal, incubation time, and soil properties.     

Influence of glyphosate on amino acid composition of Egyptian broomrape

The parasitic plant broomrape is entirely dependent on its host for reduced carbon and nitrogen and is also susceptible to inhibition by glyphosate that is translocated to the parasite through a host. Studies were conducted to examine the effect of broomrape parasitism on amino acid concentrations of two hosts: common vetch that is tolerant of low levels of glyphosate and oilseed rape that has been genetically engineered for glyphosate resistance. The influence of glyphosate on the amino acid content of broomrape and the two hosts was also examined. Amino acid concentrations in leaves and roots of parasitized common vetch plants were generally similar to those of the corresponding tissues of nonparasitized plants. Amino acid concentrations in broomrape were lower than those of the parasitized common vetch root. For common vetch, glyphosate applied at rates that selectively inhibited broomrape growth did not alter individual amino acid concentrations in the leaves, but generally increased amino acid levels at 0.18 kg ha-1. Glyphosate application also increased the amino acid concentrations, with the exception of arginine, of broomrape growing on common vetch and did not generally influence concentrations in leaves or roots of common vetch. In oilseed rape, parasitization by broomrape generally led to higher amino acid concentrations in leaves but lower concentrations in roots of parasitized plants. Broomrape had higher amino acid concentrations than roots of the parasitized oilseed rape. Glyphosate applied at 0.25 and 0.5 kg ha-1 generally increased the amino acid concentrations in oilseed rape leaves, but the 0.75 kg ha-1 application caused the amino acid concentrations to decrease compared to those of untreated plants. In oilseed rape root the general trend was an increase in the concentration of amino acids at the two highest rates of glyphosate. Individual amino acid concentrations in broomrape attachments growing on oilseed rape were generally increased following glyphosate application of 0.25 kg ha-1. These results indicate that low rates of glyphosate alter amino acid profiles in both host and broomrape and raise questions about the regulation of amino acid metabolism in the parasite.     

Response of Mycorrhizal Infection to Glyphosate Applications and P Fertilization in Glyphosate-Tolerant Soybean,Maize, and Cotton

Glyphosate and phosphorus (P) fertilizer may alter arbuscular mycorrhizal (AM) fungal infection rates of glyphosate-tolerant cotton, maize, and soybean in low-P soil. Microbial biomass, water soluble P, Mehlich-3 P, and acid and alkaline phosphatase activities were not significantly impacted by glyphosate or P in the greenhouse. Phosphorus fertilization decreased mycorrhizal infection rates in cotton and maize and increased shoot biomass and shoot P in soybean in 2005, and decreased mycorrhizal infection in soybean and increased shoot biomass in cotton and maize and shoot P in all three crops in 2006. In pasteurized soil, glyphosate decreased percent mycorrhizal infection in maize, increased infection in cotton, and did not significantly affect infection in soybean. When soil was not pasteurized, glyphosate did not significantly alter mycorrhizal infection in any crop. The potential for glyphosate to alter AM fungal infection in glyphosate-tolerant plants may depend on whether soil microbial communities are compromised by other factors.     

Effect of Glyphosate and Zinc Application on Yield,Soil Fertility,Yield Components,and Nutritional Status of Soybean

Glyphosate is a widely used nonselective herbicide for the control of agricultural weeds. It is being increasingly used in glyphosate resistant genetically modified plants. However, there are few studies on its effects on the nutritional status of soybean, particularly on the uptake of zinc (Zn). Two experiments were conducted under field conditions in a Typic Quartzipsamment and an Orthic Ferralsol to investigate the effect of glyphosate application × Zn interaction on soil fertility, yield components, seed yield (SY), shoot dry weight (SDW) yield, and nutritional status of soybean. The five Zn rates 0, 3, 6, 9, and 12 kg ha^?1 were used in two soybean varieties [BRS 133 (conventional—NGM) and its essentially derived transgenic line BRS 245RR (GM), which was divided into: with (+Gly) and without (–Gly) glyphosate application. Only the P (phosphorus) and Zn available concentrations in the soil were impacted by Zn rates. However, the available P concentration only decreased in the soil planted with GM soybean. Mehlich 1 and diethylenetriaminepenta acetic acid–triethanolamine (DTPA–TEA), 7.3 extractants were effective to determine the available Zn. In the two crop sites, the number of pods per plant (NPP) and the SDW yield were affected by the interaction varieties × Zn. SY was influenced by the application of the herbicide, reducing a potential phytotoxic effect with the use of high rates. Regarding the nutrients, only the foliar calcium (Ca), boron (B), iron (Fe), and manganese (Mn) concentrations were negatively affected by glyphosate, and in the case of Zn, the difference occurred only between the varieties BRS 133 and BRS 245RR.     

Accumulation of amino acids,proline, and carbohydrates in response to aluminum and manganese stress in maize

Synthesis of amino acids, proline, and carbohydrates was studied in roots and shoots of 5 maize accessions, differing in aluminum (Al) and manganese (Mn) tolerance, in response to Al and Mn stress at the seedling stage in solution culture. The concentrations of these metabolites increased in roots and shoots of the seedlings in the nutrient solution with added Al (0.22 mM), and Mn (2.0 mM). Both Al and/or Mn tolerant and non‐tolerant accessions accumulated more metabolites under stress than control. Generally, the tolerant accessions accumulated more solutes than the non‐tolerant maize accessions examined.     

Microbial activity,community structure and potassium dynamics in rhizosphere soil of soybean plants treated with glyphosate

With the advent of glyphosate [N-(phosphonomethyl)glycine] tolerant crops, soils have now been receiving repeated applications of the herbicide for over 10 years in the Midwestern USA. There is evidence that long-term use of glyphosate can cause micronutrient deficiency but little is known about plant potassium (K) uptake interactions with glyphosate. The repeated use of glyphosate may create a selection pressure in soil microbial communities that could affect soil K dynamics and ultimately K availability for crops. Therefore, the objectives of this study were to characterize the effect of foliar glyphosate applied to GR (glyphosate resistant) soybeans on: (1) rhizosphere microbial community profiles using ester linked fatty acid methyl ester (EL-FAME) biomarkers, (2) exchangeable, non-exchangeable, and microbial K in the rhizosphere soil, and (3) concentrations of soybean leaf K. A greenhouse study was conducted in a 2 × 2 × 3 factorial design with two soil treatments (with or without long-term field applications of glyphosate), two plant treatments (presence and absence of soybean plants), and three rates of glyphosate treatments (0×, 1× at 0.87, and 2× at 1.74 kg ae ha^?1, the recommended field rate). After each glyphosate application, rhizosphere soils were sampled and analyzed for microbial community structure using ester linked fatty acid methyl ester biomarkers (EL-FAME), and exchangeable, plant tissue and microbial biomass K. Glyphosate application caused a significant decrease in the total microbial biomass in soybean rhizosphere soil that had no previous exposure to glyphosate, at 7 days after glyphosate application. However, no significant changes were observed in the overall microbial community structure. In conclusion, the glyphosate application lowered the total microbial biomass in the GR soybean rhizosphere soil that had no previous exposure to glyphosate, at 7 days after glyphosate application; caused no changes in the microbial community structure; and did not reduce the plant available K (soil exchangeable or plant tissue K).     

Behavior of glyphosate and aminomethylphosphonic acid (AMPA) in soils and water of reservoir Radeburg II catchment (Saxony/Germany)

The behavior of the herbicide glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) was investigated in soils and water in a well-defined catchment of the reservoir Radeburg II near Dresden (Saxony/Germany). The half-life of glyphosate in soil ranged from 11 to 17 days. Glyphosate and AMPA completely disappeared from soil after about 5 months following application of the products ”︁Roundup Ultra” and ”︁Touchdown”. The aquatic system in the test areas (surface water, soil solution, and groundwater) was not significantly affected by direct application of the compound. In general, there was a clear indication of strong sorption of the two substances by soil particles. Settlement areas were recognized as possible sources of glyphosate and AMPA intake in aquatic systems.     

MANGANESE TOXICITY IN THREE SPECIES OF ANNUAL MEDICS

The selection of Medicago spp. cultivars tolerant to high levels of manganese (Mn) is needed if they are to be established in acidic soils. We compared the growth of three species of annual medics (M. polymorpha, M. murex and M. tornata) in an acid soil with a high level of extractable Mn, and in nutrient solutions with 100 and 500 μM Mn. M. polymorpha accumulated the greatest biomass in the field, in pots, and in nutrient solution, but M. murex was the most tolerant of a high level of Mn in nutrient solution and in the unlimed acid soil in pots. M. tornata produced a similar biomass to M. murex in limed soil and in a nutrient solution with a low level of Mn. However, it was very sensitive to a high level of Mn, both in solution and in unlimed soil. The relative growth between the three species in the field, in pots, and in nutrient solution was comparable, suggesting that selection in nutrient solution can be used to identify ecotypes tolerant to Mn.     

Shikimate accumulates in both glyphosate-sensitive and glyphosate-resistant horseweed (Conyza canadensis L. Cronq.)

Horseweed (Conyza canadensis) is a cosmopolitan weed that commonly grows throughout North America. Horseweed that is not completely controlled by normal applications of glyphosate has been reported in western Tennessee. This research had three objectives: (1) to develop and validate an analytical procedure for the quantitative determination of shikimate, an important indicator of glyphosate activity in plants; (2) to confirm resistance to glyphosate in a horseweed population; and (3) to examine the accumulation of shikimate in both glyphosate-resistant and glyphosate-susceptible horseweed plants. The analytical procedure to determine shikimate used extraction with 1 M HCl for 24 h, followed by liquid chromatography using photodiode array detection, and shikimate recoveries were >or=82%. Glyphosate applications of both 0.84 kg ae/ha (the standard application rate) and 3.8 kg ae/ha to susceptible plants caused complete plant death. The same glyphosate applications to putative resistant populations caused less than 15% growth reduction as determined by visual evaluations, and fresh weights of these resistant plants 17 days after glyphosate treatment (DAT) were reduced an average of 45% in one population and were not affected in a different population. This direct comparison conclusively confirms that horseweed plants collected in western Tennessee in 2002 are resistant to 4 times the normal application dosage of glyphosate. The glyphosate-resistant horseweed biotypes still exhibited some herbicidal effects from the glyphosate, such as yellowing in the most actively growing, apical shoot meristems. The yellowing in the shoot apexes was transitory, and the plants recovered from this damage. Shikimate concentrations in all untreated horseweed plants were less than 100 microg/g, which was significantly less than that in all plants which had been treated with 0.84 kg ae/ha of glyphosate. Unexpectedly, shikimate accumulated (>1000 microg/g) in both resistant populations and the susceptible population. However, there were differences in shikimate accumulation patterns between resistant and susceptible horseweed biotypes. Shikimate concentrations in resistant populations declined about 40% from 2 to 4 DAT, while shikimate concentrations in the susceptible horseweed plants increased about 35% from 2 to 4 DAT. The confirmed resistance of a widespread weed implies that alternative control strategies for glyphosate-resistant horseweed will be needed in those no-tillage production systems where it commonly occurs.     

Tolerance and accumulation of shikimic acid in response to glyphosate applications in glyphosate-resistant and nonglyphosate-resistant cotton (Gossypium hirsutum L.).

Measurement of shikimic acid accumulation in response to glyphosate inhibition of 5-enolpyruvylshikimate-3-phosphate synthase is a rapid and accurate assay to quantify glyphosate-induced damage in sensitive plants. Two methods of assaying shikimic acid, a spectrophotometric and a high-performance liquid chromatography (HPLC) method, were compared for their accuracy of recovering known amounts of shikimic acid spiked into plant samples. The HPLC method recovered essentially 100% of shikimic acid as compared with only 73% using the spectrophotometric method. Relative sensitivity to glyphosate was measured in glyphosate-resistant (GR) and non-GR cotton leaves, fruiting branches, and squares (floral buds) by assaying shikimic acid. Accumulation of shikimic acid was not observed in any tissue, either GR or non-GR, at rates of 5 mM glyphosate or less applied to leaves. All tissues of non-GR plants accumulated shikimic acid in response to glyphosate treatment; however, only fruiting branches and squares of GR plants accumulated a slight amount of shikimic acid. In non-GR cotton, fruiting branches and squares accumulated 18 and 11 times, respectively, more shikimic acid per micromolar of translocated glyphosate than leaf tissue, suggesting increased sensitivity to glyphosate of reproductive tissue over vegetative tissue. GR cotton leaves treated with 80 mM of glyphosate accumulated 57 times less shikimic acid per micromolar of translocated glyphosate than non-GR cotton but only 12.4- and 4-fold less in fruiting branches and squares, respectively. The increased sensitivity of reproductive structures to glyphosate inhibition may be due to a higher demand for shikimate pathway products and may provide an explanation for reports of fruit abortion from glyphosate-treated GR cotton.     

CHARACTERIZATION OF MANGANESE TOXICITY IN TWO SPECIES OF ANNUAL MEDICS

There is interest in the development of cultivars of Medicago spp. tolerant to soil acidity, where manganese (Mn) can be a major constraint to plant growth. We evaluated the response of several ecotypes of M. polymorpha and M. murex to a high level of Mn (50 mg Mn kg^?1 soil), and its impact on biological N₂ fixation and mineral composition. Plant growth in pots was decreased by Mn toxicity, whether plants were dependent on N₂ fixation or provided with fertilizer nitrogen (N). Plant growth was impaired by high levels of Mn to a greater extent than was N₂ fixation. Tolerance to high levels of Mn differed between the ecotypes, with accession 66 of M. polymorpha and 194 of M. murex being the most tolerant. A 500 μM concentration of Mn in nutrient solution led to decreased concentration of zinc (Zn) in the whole plant, and to modifications in the partition of iron (Fe), potassium (K), and calcium (Ca) between roots and shoots. Tolerance to Mn was negatively correlated with the concentrations of Mn and Zn in the roots.     

Electron paramagnetic resonance studies of manganese toxicity,tolerance, and amelioration with silicon in snapbean

Plant genotypes within species differ widely in tolerance to excess manganese (Mn) that may occur in acid soils, or in neutral or alkaline soils having poor aeration caused by imperfect drainage or compaction. However, Mn tolerance mechanisms in plants are largely unknown. Silicon (Si) is reported to detoxify Mn within plants, presumably by preventing localized accumulations of Mn associated with lesions on leaves. Because Mn is paramagnetic, electron paramagnetic resonance (EPR) spectroscopy, shows promise as a tool for characterizing toxic and non‐toxic forms of Mn in tolerant and sensitive plants. The objective of our study was to use EPR to: i) determine the chemical/ physical state of Mn in Mn‐tolerant and ‐sensitive snapbean cultivars; and ii) characterize the protective effects of Si against Mn toxicity. Manganese‐sensitive Wonder Crop 2 (WC) and Mn‐tolerant Green Lord (GL) cultivars of snapbean were grown at pH 5.0, in a greenhouse, in a modified Steinberg solution containing: Mn=0.05mg.L^‐1 (optimal); Mn=1.0mgL^‐1 (toxic); Mn=1.0 mg L^‐1 plus Si=4 mg L^‐1; and Mn=0.05 mg L^‐1 plus 4 mg Si L^‐1. All trifoliate leaf samples exhibited a 6‐line EPR signal that is characteristic of hexaaquo Mn²⁺. In both cultivars, a higher EPR Mn²⁺ signal‐intensity generally correlated with lower total leaf mass, higher total Mn concentrations and more pronounced symptoms of toxicity. Tolerance to excess Mn coincided with lower Mn²⁺ signal intensity. Silicon treatments ameliorated Mn toxicity symptoms in both genotypes, decreased total leaf Mn concentrations, and decreased EPR Mn²⁺ signal intensity. Results suggest that Mn toxicity is associated with reduced electron transport and accumulation of oxidation products in leaves. Amelioration of Mn toxicity by Si is regarded as connected with a reduction in this Mn‐induced process. Results indicated that EPR spectroscopy can be useful in investigating the biochemical basis for differential Mn tolerance in plants. The EPR observations might also help plant breeders in developing Mn‐tolerant cultivars.     

The composition of grain and forage from glyphosate tolerant wheat MON 71800 is equivalent to that of conventional wheat (Triticum aestivum L.)

Glyphosate tolerant wheat MON 71800, simply referred to as MON 71800, contains a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein from Agrobacterium sp. strain CP4 (CP4 EPSPS) that has a reduced affinity for glyphosate as compared to the endogenous plant EPSPS enzyme. The purpose of this work was to evaluate the compositional equivalence of MON 71800 to its nontransgenic parent as well as to conventional wheat varieties. The compositional assessment evaluated the levels of proximates, amino acids, fatty acids, minerals, vitamins, secondary metabolites, and antinutrients in wheat forage and grain grown during two field seasons across a total of eight sites in the United States and Canada. These data demonstrated that with respect to these important nutritional components, the forage and grain from MON 71800 were equivalent to those of its nontransgenic parent and commercial wheat varieties. These data, together with the previously established safety of the CP4 EPSPS protein, support the conclusion that glyphosate tolerant wheat MON 71800 is as safe and nutritious as commercial wheat varieties.     

Glyphosate Formulations on Nutritional Condition and Productivity of Roundup Ready Soybean

We evaluate the effect of different commercial formulations of glyphosate on the nutritional status of Roundup Ready soybeans (TMG 125). The experiment was conducted under field conditions for two agricultural years. The experimental design used was randomized blocks with four repetitions. The treatments were the implementation of Roundup Original, Trop, Roundup Ultra, Roundup WG, Roundup Transorb R and Zapp QI formulations, all at a dose of 720 g e.a. ha^?1 of glyphosate and two controls (weeding and no weeding). There was no difference in the accumulation of macronutrients. In the second year of evaluation (2011/2012), manganese (Mn) accumulation was affected by the Trop formulations, Roundup Transorb, Zapp QI, Roundup Ultra, Roundup WG. To the accumulation of zinc (Zn), there was a reduction in the application of Trop formulations and Zapp QI. However, this reduction observed did not influence the response of plants in relation to the productive yield.     

Tolerance of seven tropical pasture grasses to excess manganese

Abstract

Setaria and paspalum were found to be very tolerant of excess Mn. Green panic and sorghum were somewhat less tolerant with foliar symptoms due to excess Mn being exhibited in plants containing 1000 ppm Mn and yield reductions occurring in plants containing Mn concentrations of the order of 2000 ppm. Excess Mn did not effect the early seedling growth of sabi grass but regrowth was severely depressed. Rhodes grass and buffel grass were severely effected by excess manganese. Regrowth of these two species was more adversely effected than initial seedling growth indicating that these species probably would not survive to maintain a stable pasture in Mn toxic situations.

Accumulation of excess Mn was accompanied by a linear decline in Ca concentrations in all species.     

Glyphosate effects on phenolic metabolism of nodulated soybean (Glycine max L. merr.).

Glyphosate is a herbicide that blocks the shikimic acid pathway. Three Bradyrhizobium japonicum strains with different sensitivities to glyphosate were used to test the effect of this herbicide on the phenolic metabolism of nodulated soybeans and on the bacteroid nitrogenase activity. Glyphosate caused an inhibition in the bacteroid nitrogenase activity that was related with the sensitivity of the nodule-forming strains. Both leaves and nodules accumulated huge amounts of shikimate and phenolic acids (mainly protocatechuic acid), indicating that the herbicide was translocated to the nodule and disturbed phenolic metabolism. However, this accumulation was not clearly related to the sensitivity of the different strains. Bacteroids from control plants were incubated with the same concentration of shikimate, and phenolic acid accumulated in glyphosate-treated plants. Despite the high levels found in nodules, they were not responsible for the decrease of the nitrogenase activity. Glyphosate by itself caused a small inhibition of the bacteroid nitrogenase activity.     

草甘膦对烟草叶片超微结构的影响

草甘膦是农业生产中广泛使用的广谱非选择性除草剂,它抑制5-烯醇丙酮酸莽草酸-3-磷酸合成酶。该酶是莽草酸途径中的关键酶,广泛存在于微生物与植物中。用30mmol／L草甘膦处理烟草叶片2、6、12、24、48h,结果显示,处理12h内烟草叶片无明显变化,24、48h后叶片黄化,并出现坏死。对各处理时期烟草叶片的超微结构研究显示,草甘膦处理导致烟草叶片细胞的叶绿体基粒片层结构被破坏,线粒体电子密度下降。这些结果提示,草甘膦通过阻碍植物的光命作用和呼晖作用面导致叶片坏死。     

Foliar Application of Manganese Increases Seed Yield and Improves Seed Quality of Cotton Grown on Calcareous Soils

Cotton (Gossypium hirsutum L.) is often grown on calcareous soils where manganese (Mn) deficiency is quite common. Despite the fact that Mn deficiency can limit cotton productivity the effect of foliar application of Mn was not determined. A field study was conducted to determine if foliar Mn applications during anthesis affects the growth and yield of cotton. Manganese increased the chlorophyll content by up to 17% compared with the control treatment with no difference between the two rates of Mn. Moreover, Mn increased the seed yield by an average of 30%, the number of bolls per plant by 31%, the number of bolls per square meter by 31% and boll retention by 37% compared with the control, but it did not affect the mean boll weight and the quality of the fibers. Foliar application of Mn increased seed germination by an average of 14% and increased seed vigor (AA) by an average of 28% compared with the control treatment. The results obtained here suggest that foliar Mn application can improve the lint and seed yield and also the seed quality of cotton grown for seed production on calcareous soils.     

Evaluating quantitative screening methods for manganese toxicity in cotton genotypes 1

Identification of cotton genotypes more tolerant of toxic concentrations of soil solution manganese (Mn²⁺) would integrate well with soil ameliorations of that problem. Several quantitative and semi‐quantitative methods to determine the amount of Mn toxicity were evaluated on three genotypes of Gossypium hirsutum (LaDSIS 12513, LaDASS 5175, and Coker gl 79–501) and one genotype of Gossypium barbadense (Pitnas S‐5). Specific leaf weight (SLW) and the semi‐quantitative, ‘percentage of leaves that were damaged’ (PLD) correlated the least with other methods of Mn toxicity determination. Neither SLW or PLD provided more separation between genotypes than area/leaf (AL), peroxidase (POD) activity, and indole‐3‐acetic acid oxidase (IAAO) activity. Similar genotype separations occurred for AL, POD, and IAAO at 10 mg/L Mn in solution, but POD and IAAO produced more genotype separations than AL at 5 mg/L of Mn. There were differences in enzyme activity between genotypes at control (0.25 mg/L) Mn solution concentration, making assessment difficult, especially between species. Barring this caveat, the relatively fast POD activity assay was considered to be the best method since it paralleled activity of IAAO, the functional enzyme of Mn toxicity, which had a relatively slow assay method.