Glyphosate reduced seed and leaf concentrations of calcium, manganese, magnesium, and iron in non-glyphosate resistant soybean

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Abstract

Greenhouse experiments were conducted to study the effects of glyphosate drift on plant growth and concentrations of mineral nutrients in leaves and seeds of non-glyphosate resistant soybean plants (Glycine max, L.). Glyphosate was sprayed on plant shoots at increasing rates between 0.06 and 1.2% of the recommended application rate for weed control. In an experiment with 3-week-old plants, increasing application of glyphosate on shoots significantly reduced chlorophyll concentration of the young leaves and shoots dry weight, particularly the young parts of plants. Concentration of shikimate due to increasing glyphosate rates was nearly 2-fold for older leaves and 16-fold for younger leaves compared to the control plants without glyphosate spray. Among the mineral nutrients analyzed, the leaf concentrations of potassium (K), phosphorus (P), copper (Cu) and zinc (Zn) were not affected, or even increased significantly in case of P and Cu in young leaves by glyphosate, while the concentrations of calcium (Ca), manganese (Mn) and magnesium (Mg) were reduced, particularly in young leaves. In the case of Fe, leaf concentrations showed a tendency to be reduced by glyphosate. In the second experiment harvested at the grain maturation, glyphosate application did not reduce the seed concentrations of nitrogen (N), K, P, Zn and Cu. Even, at the highest application rate of glyphosate, seed concentrations of N, K, Zn and Cu were increased by glyphosate. By contrast, the seed concentrations of Ca, Mg, Fe and Mn were significantly reduced by glyphosate. These results suggested that glyphosate may interfere with uptake and retranslocation of Ca, Mg, Fe and Mn, most probably by binding and thus immobilizing them. The decreases in seed concentration of Fe, Mn, Ca and Mg by glyphosate are very specific, and may affect seed quality.

Introduction

Glyphosate (N-[phosphonomethyl] glycine) is the most commonly applied herbicide in cropping systems (Duke and Powles, 2008). The major herbicidal action of glyphosate is based on the inhibition of the enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) that results in reduced biosynthesis of aromatic amino acids and alterations in protein metabolism. Glyphosate also causes adverse effects on photosynthetic carbon metabolism and sucrose translocation within plants (Geiger et al., 1999, Riberio et al., 2008). Impairments in nitrate assimilation and nitrogen fixation are reported as further detrimental effects of glyphosate in plants (King et al., 2001, De Maria et al., 2006, Bellaloui et al., 2006), especially under water stress conditions (Zablotowicz and Reddy, 2007). Even at relatively low application doses (e.g., 1.25 mM), glyphosate caused significant decreases in nitrogenase activity in nodules of lupine plants 24 h after its spray (De Maria et al., 2006).

Plant organs with high metabolic activity and growth rates such as nodules, root tips and shoot apex represent a high sink activity for glyphosate. Experimental evidence is available showing substantial accumulation of foliar-applied glyphosate in such sink tissues (Schulz et al., 1990, Hetherington et al., 1999, Feng et al., 2003). According to Feng et al. (2003) up to 80% of the glyphosate absorbed after foliar applications is translocated into shoot apex and root tips. Even at low foliar application rates, the sink tissues accumulate glyphosate at very high concentrations. Previously, it has been reported that only a single foliar application of glyphosate at a rate of 0.5 kg ha−1 was effective to cause an accumulation of glyphosate up to 0.3 mM in the sink organs, and this concentration could be much higher when glyphosate is applied at greater rates or repeatedly (McWhorter et al., 1980, Honegger et al., 1986, cited in King et al., 2001). In tomato and spinach plants treated with foliar glyphosate applications, it is estimated that glyphosate may account for up to 16% of the dry weight of sink tissues (Schulz et al., 1990).

Accumulation of glyphosate in shoot apex or root tips at high amounts may induce impairments in cellular utilization of cationic mineral nutrients via reducing the free activity of these nutrients by chelation. Glyphosate has high ability to complex several divalent cationic nutrients such as calcium (Ca), magnesium (Mg), manganese (Mn) and iron (Fe) (Lundager-Madsen et al., 1978, Motekaitis and Martell, 1985, Barja et al., 2001). These cationic nutrients easily bind to the glyphosate molecule via the carboxyl and phosphonate groups forming poorly soluble or very stable complexes. Formation of such insoluble glyphosate complexes with cations in spray solution reduces effectiveness of glyphosate to kill weed plants. In velvetleaf plants, Bernards et al. (2005) showed that the presence of Mn in spray solution caused formation of stable Mn–glyphosate complexes which in turn resulted in reduced penetration and translocation of glyphosate from the treated leaves. Similar antagonistic reactions with glyphosate have been also shown for Ca and Mg (Nalewaja and Matysiak, 1991, Thelen et al., 1995). The presence of monovalent cations in glyphosate spray solutions did not cause any change in the glyphosate efficacy (Stahlman and Phillips, 1979). Calcium has been shown to precipitate glyphosate by forming a 1:1 complex in aqueous solutions (Gauvrit et al., 2001, Schoenherr and Schreiber, 2004). Therefore, it is often recommended by the manufacturer companies not to use hard water in preparing glyphosate spray solutions.

These results and observations indicate that divalent cations may reduce the efficacy of glyphosate in plant and soil systems. When complexed by glyphosate, the activity of divalent cations at the physiological level might also be reduced. This can be considered as a significant side-effect for non-target plants exposed to glyphosate spray drift. Glyphosate spray drift is currently an increasing concern in cropping systems where glyphosate is being repeatedly applied (Burke et al., 2005, Bellaloui et al., 2006, Buehring et al., 2007, Rolder et al., 2007). Up to 10% of the foliarly applied glyphosate may move to non-target plants (Al-Khatib and Peterson, 1999, Snipes et al., 1991) and this spray drift may be as high as 37% of the applied glyphosate rate depending on the speed of wind and accuracy of the glyphosate application method (Nordby and Skuterud, 1975).

There are, however, limited data on the effects of glyphosate drift on mineral nutritional status of the non-target plants. Applying glyphosate up to 12.5% of the recommended rate adversely affected nitrate assimilation and nitrogen fixation of soybean plants (Bellaloui et al., 2006). In both glyphosate resistant and glyphosate non-resistant crops, it has been shown that shoot concentrations of micronutrients, especially Mn and Fe, show a decrease upon glyphosate applications (Eker et al., 2006, Bott et al., 2008). The results of Bott et al. (2008) clearly indicate higher demand for Mn of the glyphosate-tolerant crops when treated with glyphosate. Because of high affinity of glyphosate to chelate and immobilize divalent cations glyphosate reduces shoot Mn concentration irrespective of whether crops are glyphosate tolerant or not. Interestingly, Gordon (2007) showed that leaf concentrations of Mn in glyphosate-tolerant soybean are lower than the glyphosate-sensitive soybean plants and therefore glyphosate-tolerant soybean cultivars require adequate Mn application to achieve highest yield. The fact that glyphosate is a very strong chelator for divalent cations indicates that glyphosate can physiologically immobilize these nutrients in the tissues. The ‘yellow flashing’ commonly observed in RR crops after glyphosate applications is attributable to immobilization of divalent cations especially Fe and Mn (Franzen et al., 2003, Hansen et al., 2004, Jolley et al., 2004, Eker et al., 2006). Most probably, the length of this ‘flashing’ is dependent on the ability of the plants to recover by adequate root uptake of the concerned elements which are immobilized by the glyphosate in plant tissues, assuming no foliar fertilization of these nutrients have been made.

In the present study, we tested the effects of foliar application of glyphosate on mineral nutrient concentrations of non-glyphosate resistant soybean plants at both early growth stage and at the grain maturation. To our knowledge, the effects of increasing glyphosate rates on concentration of mineral nutrients in soybean grain have not been described before. In the case of the experiment conducted until grain maturation, glyphosate has been sprayed at V4, V6 and R1 stages. These growth stages are widely considered application times for glyphosate, and suggested to be critical stages for an effective weed control (Halford et al., 2001, Krausz and Young, 2001, Bellaloui et al., 2008, Miller et al., 2008).

Section snippets

Growth conditions

Non-glyphosate resistant soybean plants (Glycine max [L.] Merr. Cultivar: Nova) were grown under greenhouse conditions equipped with an evaporative cooling system (24–28; 21–24 °C day/night) under natural daylight during the summer season. Soybean seeds were sown in a 3.3-l plastic pots containing 2.8 kg loamy clay soil with pH 7.6, organic matter 1.5%, CaCO3 17.6%. The soil used in the experiments was transported from the Eskisehir region in Central Anatolia in Turkey where soil Zn deficiency

Results

The effect of foliar-applied glyphosate on the shoot growth and leaf concentrations of mineral nutrients was first studied in 21-day-old plants by analyzing young and old leaves separately. The first reaction of plants to increasing glyphosate application up to 0.6% of the recommended dose was the development of chlorosis on the youngest leaves. As expected, chlorophyll concentrations (SPAD values) showed a clear decrease by glyphosate (Table 1). Increasing application of glyphosate

Discussion

Reproductive organs are known to be more sensitive to glyphosate than the vegetative tissues. As shown in cotton, tobacco and soybean plants (Pline et al., 2003, Walker et al., 2006, Yasuor et al., 2007), glyphosate application results in greater damage to generative organs than the vegetative parts of plants. The reason for higher sensitivity of reproductive tissue to glyphosate might be related to higher accumulation of glyphosate in the reproductive tissues. Actively growing parts of plants

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