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Distribution of [14C]glyphosate in mature glyphosate-resistant cotton from application to a single leaf or over-the-top spray
PESTICIDE Biochemistry & Physiology
Pesticide Biochemistry and Physiology 82 (2005) 36–45 www.elsevier.com/locate/ypest
Distribution of [14C]glyphosate in mature glyphosate-resistant cotton from application to a single leaf or over-the-top spray Paul C.C. Feng*, Tommy Chiu Monsanto Co., GG4D 700 Chesterfield Parkway West, Chesterfield, MO 63017-1732, USA Received 29 March 2004; accepted 28 July 2004 Available online 2 February 2005
Abstract Distribution of [14C]glyphosate was examined in mature glyphosate-resistant cotton plants at the 13-node stage in the absence of phytotoxicity. Initial experiments employed manual application of glyphosate to individual leaves within a relatively immature (Node 9) or mature (Node 5) sympodium (i.e., fruiting node) in the plant. We measured glyphosate export out of the treated leaf to the fruiting structures and foliage in the sympodium as well as out of the sympodium into the plant. Application to the Stem leaf, Leaf 1 or Leaf 2 in Node 9 showed 30–37% glyphosate export by 14 days after treatment. While Stem leaf exported mainly to the plant, Leaves 1 and 2 exported equally between the plant and the sympodium. Within the sympodium, glyphosate was distributed almost entirely to the fruiting structures with Boll 1 containing the highest level irrespective of the treated leaf. In Node 5, application to the Stem leaf or Leaves 1–3 showed overall lower glyphosate export (20–27%) with Stem leaf and Leaf 3 exporting mainly to the plant, and Leaves 1 and 2 exporting mainly to the sympodium. Within Node 5, the subtending boll of the treated leaf was not the main distribution target, but instead the boll at the next higher sympodial position relative to the treated leaf. Subsequent studies were conducted using over-the-top spray application of [14C]glyphosate at field use-rates in mature plants at the 13node stage. Analysis of open bolls at full maturity showed high residues in seeds and fiber of Bolls 1 and 2 in the mature sympodia. Our results suggest that glyphosate distribution in mature plants was affected by sympodial age, position of the treated leaf, and position and sink strength of the bolls. 2004 Elsevier Inc. All rights reserved. Keywords: Glyphosate; Glyphosate-resistant cotton; Roundup Ultra; Roundup Ready
*
Corresponding author. Fax: +1 636 737 6060. E-mail address: [email protected] (P.C.C. Feng).
0048-3575/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2004.07.010
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1. Introduction The development of glyphosate resistance has revolutionized agricultural practices in cotton. The current commercial product, Roundup Ready cotton, was engineered through expression of a glyphosate insensitive CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene [1]. Roundup Ready cotton was commercialized in 1997 and permits over-the-top (OT) spray application of glyphosate through the four-leaf stage. Applications beyond that growth stage can lead to plant injuries, which have been the subject of numerous investigations. Uptake and translocation studies showed that [14C]glyphosate was readily distributed to bolls [2]. Other studies [3,4] demonstrated lower expression of CP4-EPSPS in male reproductive tissues that contributed to reduced pollen viability, male sterility, and boll drop [5,6]. Translocation studies thus far have focused on young plants due to potential injurious effects of glyphosate in mature cotton. The next generation of glyphosate-resistant cotton (Roundup Ready Flex) is currently under development and demonstrates enhanced resistance to glyphosate [7]. The new glyphosate-resistant cotton permits an expanded window of application between 3 and 14 leaf stages, and offers growers greater flexibility in weed control. Boll retention and development have a direct impact on fiber and seed yield. Boll drop is a physiological response to environmental stresses such as drought or cold temperature [8,9], and is determined by an intricate balance of assimilate supply, demand, and distribution [10]. Pinkhasov [11] demonstrated that assimilates in cotton are photosynthesized mainly by the stem (33%) and lateral branch leaves (55%). Sink strength plays a major role in assimilate distribution. Bolls in the first three positions proximal to the main-stem are dominant sinks and receive the majority of assimilates [12]. Assimilate distribution is also affected by proximity with the subtending leaf being the most important source for the associated boll [13]. The objectives of our research were to examine the distribution of [14C]glyphosate in mature cotton plants and to model photoassimilate distribution using [14C]glyphosate as a marker.
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Glyphosate is a highly systemic herbicide that is readily translocated in the phloem following a pattern of source to sink tissues. Several reports [14,15] suggest that glyphosate is translocated with photoassimilates; however, glyphosate translocation can be affected by its own toxicity, which occurs within hours after application [16,17]. Our studies employed the new Roundup Ready Flex plants to exclude potential interference from glyphosate toxicity. We examined glyphosate translocation in mature plants with application to individual lateral branch leaves in a relatively mature or immature sympodium. We also examined distribution of [14C]glyphosate from OT spray to simulate field application.
2. Materials and methods 2.1. Plant rearing Glyphosate-resistant plants were raised in a greenhouse (29/25 C, day/night temperatures, 16-h photoperiod with supplemental lighting) and were treated with Pix Plus plant regulator (mepiquat chloride, 4.2% and Bacillus cereus, 0.0058%, BASF) with OT sprays (3 ml/L) at the four- and seven-leaf stages to control stature. Plants were acclimated in the growth chamber (29/25 C day/night temperatures, 16-h photoperiod, 700 lE/s/m2 light intensity, and 75% relative humidity) for at least 7 days prior to experimentation. Plants were visually selected to match in size, growth stage, and the maturity of fruiting structures. 2.2. Application of [14C]glyphosate dosing solutions Both drop and spray applications employed a glyphosate formulation comprised of an equal mixture of Roundup Ultra and Accord at a rate of 1.68 kg ae/ha and diluted to a spray volume of 187 L/ha. The Accord formulation contains no surfactant and served to reduce foliar surfactant injury. [14C]Phosphonomethyl labeled glyphosate (457 MBq/mmol specific activity, 98.2% purity, New England Nuclear) was fortified into the glyphosate formulation. Manual drop
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application to a leaf employed 50 ll of glyphosate formulation containing [14C]glyphosate (50 kBq) and was applied as 1 ll droplets to the adaxial leaf surface. OT spray application employed 35 ml of glyphosate formulation containing [14C]glyphosate (2.5 MBq). The formulation was sprayed in an enclosed track sprayer using a commercial flat fan nozzle (XR TeeJet 9501, Spraying Systems) calibrated to deliver the desired dose at a height of 46 cm and a volume of 187 L/ha. 2.3. Treatment design The drop application consisted of two studies utilizing plants at the 13-node stage. The first study involved application of glyphosate to individual branch leaves in the immature Node 9. The three treatments (three plants each) consisted of applications to the Stem leaf, Leaf 1 or Leaf 2. At the time of application, Node 9 contained a small boll, a square and a tiny square at positions 1, 2, and 3, respectively. The second study consisted of four treatments with application of glyphosate to individual branch leaves (Stem leaf, Leaf 1, Leaf 2 or Leaf 3) in the mature Node 5. At the time of application, Node 5 contained large to small bolls in positions 1–4. The OT spray study employed plants at the 13or 15-node stages (three plants per treatment). At the time of spray, fruiting structures were present at positions 1–4 in Nodes 5–12 with no open bolls. 2.4. Plant harvest Plants from drop studies were harvested at 14 days after treatment (DAT). The sympodium containing the treated leaf was excised and separated into individual fruiting structures, leaves, and stems. The treated leaf (Stem leaf or Leaves 1–3) was washed with water (40 ml) and dried with methanol (10 ml). The combined washes were analyzed by liquid scintillation counting for unabsorbed, surface radioactivity. Tissues from the harvested sympodium were packed into multiple cones and combusted in a biological oxidizer (Packard 387 oxidizer with Oximate 80 robotics, Perkin–Elmer Life Sci.). The mature bolls were further separated into seed, fiber, and carpels.
Fiber was subjected to repeated washing with water; the washes and the fiber were then analyzed for radioactivity. Radioactivity was summed for individual fruiting structures, subtending leaves, and the foliage (stems and side branches). Since the same glyphosate dose was applied in each treatment, results were calculated as percentage of applied dose. Average and standard error of means (SEM) were calculated based on three replicates. Following OT spray, plants were maintained in the growth chamber until full maturity and allowed to desiccate prior to harvest (40 DAT). All open bolls were individually harvested from each plant, and separated into seed and fiber. Tissue dry weights were measured prior to combustion analysis. As glyphosate undergoes little to no metabolism in cotton [18], tissue radioactivity was converted to glyphosate mass and expressed as ppm dry weights. Average and standard error of means were calculated based on three replicates. It is estimated that the combined drop and spray studies generated approximately 4000 combustion samples.
3. Results and discussion Our studies employed Roundup Ready Flex plants to exclude interference from glyphosate toxicity. As glyphosate is translocated with photoassimilates [14,15], we also wished to examine assimilate translocation using glyphosate as a marker. Assimilate distribution in plants is typically studied with radiolabeled substrates (e.g., sucrose and CO2) [15]; however, analysis can be complicated due to metabolism. Since glyphosate undergoes little to no metabolism in cotton [18], we hoped to generate a simpler picture of assimilate distribution. 3.1. Plant growth and development A cotton plant is comprised of a main-stem with nodes that give rise to a Stem leaf and side branch. The nodes are numbered from bottom up with the first fruiting branch (i.e., sympodium) usually appearing at Node 5. Within the fruiting branch are also nodes that give rise to a leaf and
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a fruiting structure, and these are numbered sequentially away from the main-stem (e.g., Boll 1 and Leaf 1 are located proximate to the main stem). The fruiting structure begins with a square that develops to a flower, which after pollination forms a boll. We monitored the growth of bolls and leaves during the 14-day study for the purpose of gauging the source or sink strengths of individual tissues. At the time of application, Node 5 contained bolls in positions 1–4. Position 1 was closest to the main-stem and most mature. Bolls 1 and 2 were at their maximum size and expanded no further during the 14-day study. Bolls 3 and 4 both increased in size; by 14 DAT, Boll 3 was at its maximum size while Boll 4 was about half its maximum size. Stem leaf and Leaves 1–3 were fully expanded and showed no further increase in diameter during the study. Only Leaf 4 showed a slight increase in diameter. There is a direct relationship between tissue growth, sink strength, and assimilate demand. A good example is a boll, which peaks in size at about 25 days post-anthesis (DPA) with assimilate import peaking at around 30 DPA [10]. Based on size and growth stage, we estimated the relative sink strength of the bolls in Node 5 as Boll 1 P Boll 2 > Boll 3 > Boll 4. At the time of application, Node 9 contained a small boll, a square, and a tiny square in positions 1, 2, and 3, respectively. By 14 DAT, positions 1 and 2 contained expanding bolls with a flower and a square in positions 3 and 4, respectively. The Stem leaf, Leaves 1 and 2 remained unchanged in size, while Leaves 3 and 4 expanded greatly during the 14-day study. Based on size and growth stage, the relative sink strengths of the fruiting structures in Node 9 were estimated as Boll 1 > Boll 2 > Flower 3 > Square 4. 3.2. Glyphosate export out of the treated leaf [14C]Glyphosate was fortified into a formulation (1.68 kg/ha at 187 L/ha volume) and was manually applied (50 · 1 ll) to the adaxial surface of the leaf. To examine the effect of leaf position, application was made individually to either the Stem leaf or Leaves 1–3 in Node 5; Leaf 3 in Node 9 was not treated due to its small size.
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Our previous studies showed that the surfactant system in Roundup Ultra causes rapid absorption and translocation of glyphosate that peaks at approximately 1 DAT [19–21]. In the current studies, the 14 DAT harvest is expected to represent end points of glyphosate uptake and translocation. At harvest, the treated sympodium was excised and separated into individual bolls, leaves, and stem. Mature bolls (Bolls 1 and 2) in Node 5 were further dissected into fiber, seed, and carpels. The treated leaf was washed to recover surface glyphosate followed by combustion analysis to determine tissue glyphosate. Distribution was calculated as percentages of applied dose. Table 1 summarizes glyphosate that was recovered in the leaf wash, localized in or exported from the treated leaf. Application of glyphosate to the Stem leaf, Leaf 1 or Leaf 2 in Node 9 recovered 37–48% of applied dose in the leaf wash. In comparison, applications to leaves in Node 5 recovered 46–66%. These results indicated higher average uptake in leaves of Node 9 than Node 5, and suggest that cuticle of mature leaves posed a greater barrier to glyphosate absorption. Studies have shown that foliar absorption of glyphosate is also affected by numerous physical variables including glyphosate concentration [22], surfactant type [23,24], and droplet size [25,26]. Table 1 also shows glyphosate that remained localized in the treated leaf at 14 DAT. This localization is believed to result from surfactant injury associated with manual application of large size droplets [26,27]. Localization averaged 25 and 20% among treated leaves in Node 9 and Node 5, respectively. Our main interest was monitoring glyphosate exported from the treated leaf, and this was calculated by the difference between applied and recovered radioactivities in the treated leaf and the wash. Export among treated leaves averaged 33% in Node 9 and 23% in Node 5, and indicated that glyphosate is more efficiently absorbed and exported from leaves in immature (Node 9) than mature (Node 5) sympodium. 3.3. Glyphosate distribution in immature Node 9 Glyphosate that was exported from the treated leaf can either remain in the sympodium or trans-
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Table 1 The distribution of [14C]glyphosate (percentage of applied dose) in mature glyphosate-resistant cotton plants (13-node stage) from manual application to individual branch leaves (Stem leaf, Leaf 1, Leaf 2 or Leaf 3) in an immature (Node 9) or mature (Node 5) sympodium at 14 DAT Treatment node
Appl. leafa (N = 3)
% appl.
SEM
% appl.
SEM
% appl.
SEM
9
Stem leaf Leaf 1 Leaf 2
41.8 36.6 48.0
3.1 0.5 8.3
27.8 26.0 20.4
2.5 0.4 6.3
30.4 37.4 31.6
4.1 0.5 2.3
Average
42.1
4.0
24.7
3.1
33.1
2.3
Stem leaf Leaf 1 Leaf 2 Leaf 3
56.6 46.0 65.7 61.9
3.7 1.8 1.2 4.5
22.7 27.3 14.2 13.7
3.8 1.4 1.0 2.9
20.7 26.7 20.1 24.4
1.9 0.6 1.3 1.6
Average
57.6
2.8
19.5
2.3
23.0
1.4
5
a
Leaf washb
Localizedc
Exportedd
14
Radiolabeled [ C]glyphosate was fortified into formulated glyphosate (1.68 kg/ha) and applied as 50 · 1 ll droplets to the adaxial leaf surface of Stem leaf, Leaf 1, Leaf 2 or Leaf 3 in Node 9 or 5. b Treated leaf was washed at 14 DAT with water followed by methanol; combined washes were analyzed by liquid scintillation counting and expressed as percentage of applied dose. Average and standard error of means (SEM) were calculated from three replicate plants. c After washing, glyphosate localized in the treated leaf was determined by combustion analysis and expressed as percentage of applied dose. d Glyphosate exported from the treated leaf was calculated by the difference between applied and recovered dose (wash and localized).
ported out of the sympodium into the plant. Within the sympodium, distribution could be to fruiting structures, leaves or stems. These data are summarized in Fig. 1. When applied to the Stem leaf, gly-
Fig. 1. The distribution of [14C]glyphosate (percentage of applied dose) from manual application to individual branch leaves (Stem leaf, Leaf 1 or Leaf 2) in the immature Node 9 of glyphosate-resistant cotton plants at 14 DAT.
phosate export was mainly to the plant (22% of applied dose) with much less (8%) to the sympodium. In comparison, application to Leaf 1 or Leaf 2 resulted in comparable distribution between the sympodium and the plant. Assuming that glyphosate is a suitable marker for photoassimilates, our results suggest that all leaves, and especially the Stem leaf, in immature sympodia are important sources for the plant. Fig. 2 shows the distribution of glyphosate as percentage of applied dose between the fruiting structures and the foliage within Node 9. The results showed that irrespective of the application leaf, glyphosate was principally distributed to the fruiting structures (bolls, flowers, and squares) which represented the dominant sinks in the immature sympodium. Fig. 3 further breaks down the distribution to individual fruiting structures in Node 9 as a function of application leaf. At 14 DAT, positions 1–4 contained a medium boll, a small boll, a flower, and a square, respectively, with an estimated relative sink strengths of Boll 1 > Boll 2 > Flower
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3.4. Glyphosate distribution in mature Node 5
Fig. 2. The distribution of [14C]glyphosate (percentage of applied dose) between sympodial foliage and fruiting structures from manual application to individual branch leaves (Stem leaf, Leaf 1 or Leaf 2) in the immature Node 9 of glyphosateresistant cotton plants at 14 DAT.
Fig. 4 summarizes glyphosate export as percentage of applied dose from application to leaves in Node 5. When applied to the Stem leaf, glyphosate was once again distributed mostly to the plant (15%) and not to the sympodium (6%); this distribution pattern was similar to the corresponding Stem leaf in Node 9. Application to Leaf 1 produced a different pattern with distribution to sympodium (18%) far exceeding that of the plant (8%). Application to Leaf 2 produced a similar pattern as Leaf 1 with the sympodium receiving greater distribution than the plant (12 vs 8%). However, with application to Leaf 3, the pattern reverted to that of the Stem leaf with plant distribution exceeding that of the sympodium (15 vs 9%). The conclusion is that Stem leaf and Leaf 3 source mainly the plant while Leaves 1 and 2 source mainly the sympodium. Our results demonstrated a difference in glyphosate distribution between Node 9 and Node 5. In the immature sympodium, plant export was the major role for all the leaves, whereas in the mature sympodium, some leaves (Leaves 1 and 2) sourced the sympodium while others (Stem leaf and Leaf 3) the plant. Fig. 5 shows the distribution as percentage of applied dose between the foliage and the bolls within Node 5. The results showed that irrespective of the application leaf, glyphosate was almost
Fig. 3. The distribution of [14C]glyphosate (percentage of applied dose) among sympodial fruiting structures from manual application to individual branch leaves (Stem leaf, Leaf 1 or Leaf 2) in the immature Node 9 of glyphosate-resistant cotton plants at 14 DAT.
3 > Square 4. The distribution results showed that Boll 1 contained the majority of glyphosate irrespective of the application leaf. Boll 1 was the strongest sink and apparently out-competed other sinks accessing glyphosate not only from its subtending Leaf 1 but also from distant leaves (i.e., Stem leaf and Leaf 2). Our results suggest that in an immature sympodium, boll sink strength was the principal determinant for glyphosate distribution.
Fig. 4. The distribution of [14C]glyphosate (percentage of applied dose) from manual application to individual branch leaves (Stem leaf, Leaf 1, Leaf 2 or Leaf 3) in the mature Node 5 of glyphosate-resistant cotton plants at 14 DAT.
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Fig. 5. The distribution of [14C]glyphosate (percentage of applied dose) between sympodial foliage and bolls from manual application to individual branch leaves (Stem leaf, Leaf 1 or Leaf 2) in the mature Node 5 of glyphosate-resistant cotton plants at 14 DAT.
entirely distributed to the bolls. These results were similar to that of Node 9 and suggest that in both immature and mature sympodia, the fruiting structures are the dominant sinks. Fig. 6 further breaks down the distribution of glyphosate to bolls as a function of application leaf in Node 5. Application to the Stem leaf produced most distribution to Boll 1, which was indicative of its high sink strength. Much to our surprise, application to Leaf 1 showed most distribution to Boll 2. Boll 1 received much less glyphosate in spite of
its sink strength and subtending position to Leaf 1. The same pattern was observed with application to Leaf 2, which showed most distribution to Boll 3 and not Boll 2. These results demonstrated that distribution is primarily to the boll in the next higher position from the treated leaf. Application to Leaf 3 did show the highest distribution to Boll 3. Our results differed from the common understanding that the subtending leaf is the primary source for the associated boll [13]. As these plants were fully resistant to glyphosate, we were confident that glyphosate distribution was not affected by its own toxicity. The mature Bolls 1 and 2 in Node 5 were further separated into seed, fiber, and carpel. Table 2 shows tissue levels of glyphosate expressed as percentages of boll-contained radioactivity. The results showed that irrespective of the application leaf, the majority of glyphosate in Bolls 1 and 2 were in the seeds (46–52%) with the remaining split between the fiber (25–27%) and carpel (23–27%). These results showed that distribution of glyphosate within the boll is very consistent with seeds as the dominant sink. Our results also showed that fibers contained significant glyphosate residues, which were mostly (78%) removed by repeated washing with water (data not shown). Limited analysis was also conducted to determine the destiny of glyphosate that was exported out of the treated sympodium (Node 5) and into the plant. Acropetal or basipetal movement was assessed by analysis of the apical meristem and Boll 1 above or below the treated sympodium. Results showed the presence of glyphosate in Boll 1 below the treated sympodium, but not in the apical meristem or Boll 1 above the treated sympodium (data not shown). These results suggest that glyphosate exported out of the sympodium is mainly translocated basipetally to source the roots and proximate bolls along the main stem. 3.5. Modeling assimilate distribution in mature sympodia
Fig. 6. The distribution of [14C]glyphosate (percentage of applied dose) among sympodial bolls from manual application to individual branch leaves (Stem leaf, Leaf 1 or Leaf 2) in the mature Node 5 of glyphosate-resistant cotton plants at 14 DAT.
The distribution of glyphosate from leaves in Node 5 was very unexpected. Based on glyphosate distribution, we developed a model for assimilate distribution in mature sympodia (Fig. 7). The
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Table 2 The distribution of [14C]glyphosate (percentage boll-contained activity) in Bolls 1 and 2 from manual application to individual branch leaves (Stem leaf, Leaf 1, Leaf 2 or Leaf 3) in Node 5 of glyphosate-resistant cotton plants at 14 DAT Node 5 Appl. leafa
Boll 1b % distribution
Boll 2b % distribution
(N = 3)
Seed
Fiber
Carpel
Seed
Fiber
Carpel
Stem leaf Leaf 1 Leaf 2 Leaf 3
61.3 51.3 41.6 54.6
18.5 22.5 38.4 21.3
20.2 26.2 19.9 24.1
49.0 37.6 49.3 48.5
28.9 31.4 24.4 24.6
22.1 31.0 26.4 27.0
Average SEM
52.2 4.1
25.2 4.5
22.6 1.5
46.1 2.8
27.3 1.7
26.6 1.8
a Radiolabeled glyphosate was fortified into formulated glyphosate (1.68 kg/ha) and applied as 50 · 1 ll droplets to the adaxial leaf surface of Stem leaf, Leaf 1, Leaf 2 or Leaf 3 in Node 5. b Boll 1 or 2 from individual treatments were dissected into seed, fiber, and carpel. Tissue radioactivity was determined by combustion analysis and expressed as a percentage of total radioactivity in the boll. Average and standard error of means (SEM) were calculated from three replicate plants.
Fig. 7. A proposed model of photoassimilate distribution in a mature sympodium showing directional phloem flow that reduces assimilate distribution to upstream bolls to insure assimilate export to the plant.
model suggests that assimilate transport in the sympodial phloem is directional flowing up from the main-stem and back down in separate streams. Bolls and leaves are connected only to the upward stream. Since the movement of the phloem is towards sympodial terminal, only bolls downstream from the leaf can access the assimilates. As shown in the model, application of glyphosate to Leaf 2 targets distribution mainly to Boll 3, and precluded upstream Bolls 1 and 2 from much distribution. Excess assimilates are exported to the plant through the downward stream that is not accessible by the bolls. The model also explains the result from application to Leaf 3. As immature Boll 4 was not yet a strong sink, more glyphosate was exported to the plant.
Our studies suggest a distinction in assimilate translocation between mature and immature sympodia. The immature sympodium appears to contain only one phloem where assimilate distribution is mainly based on boll sink strength regardless of distance from the source leaf. The leaves produce ample assimilates, but since demand is low from the immature fruiting structures most is exported to the plant. In contrast, the first three position bolls in a mature sympodium are at their peak assimilate demand, and probably could consume all the assimilates from the sympodial leaves. The proposed dual phloem model insures that some assimilates in mature sympodia are exported to the plant. This is likely an adaptation to favor the survival of the plant over the bolls under less than ideal growing conditions. Future studies are required to further support this model. 3.6. Glyphosate distribution from OT spray application The above studies using single-leaf applications were useful in defining the role of individual leaves on glyphosate distribution. The results showed that glyphosate distribution varied depending on leaf age and position, as well as sympodium age. In field spray applications, plant coverage is rarely uniform and can be affected by biological variables such as canopy density and leaf morphology that affect droplet interception and rebound [28,29].
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Plant coverage can also be affected by physical variables such as spray volume [30], droplet size [25], and formulation concentration [22,31]. As a consequence, glyphosate distribution in a spray application is very complex and dependent on which leaves actually intercept and retain the spray. Glyphosate distribution from spray application is not likely to be modeled by application to a single leaf. We examined [14C]glyphosate distribution with OT spray in mature cotton plants to simulate field application. Plants (13-node stage) were sprayed at a field use-rate of 1.68 kg/ha in 187 L/ha volume and maintained in the growth chamber until full maturity (40 DAT). After desiccation, open bolls were individually harvested and separated into seeds and fiber. There were no open bolls at the time of spray; therefore, residues in the harvested bolls represented translocated glyphosate. Fig. 8 reports the seed concentration of glyphosate (ppm dry weight) from the open bolls. High concentrations were detected in Boll 1 (3.5– 5.2 ppm) and Boll 2 (1.0–4.9 ppm) between Nodes 5 and 10. Seed residues from Boll 2 diminished by Node 9 while that from Boll 1 diminished by Node 11. This was consistent with our earlier results showing Boll 1 as the dominant sink in immature sympodia. Boll 3 or Boll 4, if present, showed very low residues (<1.0 ppm) in all sympodia. Glyphosate distribution was also examined from OT spray application to older plants at the 15-node stage. Analysis showed that peak seed residues were
shifted to higher nodes (8–11) and higher position bolls (2 and 3) which was coincident with maturation of Boll 1 in lower sympodia. Foliar analysis showed that the majority of the spray-dose were intercepted by leaves in the upper and outer canopy (data not shown) that efficiently exported glyphosate out of the sympodium to the plant. Studies from drop application in Node 5 showed that glyphosate can be delivered to higher position bolls (e.g., Boll 3), if applied to the appropriate leaves (i.e., Leaves 2 and 3). Our results suggest that during OT spray, the plant coverage pattern leads to extensive sympodial export and basipetal translocation that sourced the strong sink bolls (positions 1 and 2) proximal to the main stem. Analysis of fiber showed residue concentrations (ppm dry wt) that averaged approximately 1.5 times higher than that of seeds (data not shown). Boll retention, fiber, and seed yield are of primary importance to cotton growers. Glyphosate distribution in mature cotton plants was examined in the absence of toxicity from applications to a single leaf or OT spray. Our results showed that distribution of glyphosate was dependent on leaf age and position as well as sympodium age, and likely to be affected by the pattern of foliar coverage during spray application. As glyphosate moves freely in the phloem [14,15], our results are also indicative of photoassimilate distribution in mature cotton plants. A complex pattern of assimilate distribution was evident in mature sympodia to balance the needs of maturing bolls with that of the plant.
Acknowledgments We thank Amy Martens and Jesse Hart for assistance in the greenhouse work. We also thank Minhtien Tran and Doug Sammons for discussions, and Claire CaJacob and Mark Oppenhuizen for supporting this research.
References Fig. 8. Seed concentration of glyphosate (ppm dry weight) from open bolls at full plant maturity from over-the-top spray application of [14C]glyphosate (1.68 kg/ha) to 13-node glyphosate-resistant cotton plants.
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tolerant cotton: genetic characterization and protein expression, J. Agric. Food Chem. 44 (1996) 1960–1966. W.A. Pline, J.W. Wilcut, K.L. Edmisten, R. Wells, Absorption and translocation of glyphosate in glyphosate-resistant cotton as influenced by application method and growth stage, Weed Sci. 49 (2001) 460–467. Y.S. Chen, C. Hubmeier, Histochemical analysis of male reproductive development in glyphosate-tolerant cotton (Gossypium hirsutum), in: Proceedings of the 18th AsianPacific Weed Science Society Conference, Beijing, China, 2001. W.A. Pline, R. Viator, J.W. Wilcut, K.L. Edmisten, J. Thomas, R. Wells, Reproductive abnormalities in glyphosate-resistant cotton caused by lower CP4-EPSPS levels in the male reproductive tissue, Weed Sci. 50 (2002) 438–447. W.A. Pline, K.L. Edmisten, J.W. Wilcut, R. Wells, J. Thomas, Glyphosate-induced reductions in pollen viability and seed set in glyphosate resistant cotton and attempted remediation by giberellic acid (GA3), Weed Sci. 51 (2003) 19–27. M.A. Jones, C.E. Snipes, Tolerance of transgenic cotton to topical applications of glyphosate, J. Cotton Sci. 3 (1999) 19–26. O.L. May, A.S. Culpepper, R.E. Cerny, C.B. Coots, C.B. Corkern, J.T. Cothren, K.A. Croon, K.L. Ferreira, J.L. Hart, R.M. Hayes, S.A. Huber, A.B. Martens, W.B. McCloskey, M.E. Oppenhuizen, M.G. Patterson, D.B. Reynolds, Z.W. Shappley, J. Subramani, T.K. Witten, A.C. York, B.G. Mullinix Jr., Transgenic cotton with improved resistance to glyphosate herbicide, Crop Sci. 44 (2004) 234–240. G. Guinn, J.R. Mauney, Fruiting of cotton. II. Effects of plant moisture status and active boll load on boll retention, Agron. J. 76 (1984) 94–98. G. Guinn, Causes of square and boll shedding, in: Proceedings of the Beltwide Cotton Research Conference, San Diego, CA, 1998, pp. 1355–1364. A.M. Schubert, C.R. Benedict, R.J. Kohel, Carbohydrate distribution in bolls, in: J.R. Mauney, J.M. Stewart (Eds.), Cotton Physiology, The Cotton Foundation, Memphis, TN, 1986, pp. 311–324. Y.I. Pinkhasov, Photosynthesis, transport of photosynthetic products, and development of methods of regulating transport in cotton, Biol. Bull. Acad. Sci. USSR 8 (1981) 231–241. J.M. Munro, An analysis of earliness in cotton, Cotton Growth Rev. 48 (1971) 28–41. D.A. Ashley, 14C-labeled photosynthate translocation and utilization in cotton plants, Crop. Sci. 12 (1972) 69–74. J.A. Gougler, D.R. Geiger, Carbon partitioning and herbicide translocation in glyphosate treated sugar beet (Beta vulgaris), Weed Sci. 32 (1984) 546–551. R.S. McAllister, L.C. Haderlie, Translocation of 14Cglyphosate and 14CO2-labeled photoassimilates in Canada thistle (Cirsium arvense), Weed Sci. 33 (1985) 153–159.
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[16] D.R. Geiger, H.D. Bestman, Self-limitation of herbicide mobility by phytotoxic action, Weed Sci. 38 (1990) 324– 329. [17] D.R. Geiger, W.J. Shieh, M.A. Fuchs, Causes of selflimited translocation of glyphosate in Beta vulgaris plants, Pest. Biochem. Physiol. 64 (1999) 124–133. [18] M.S. Bleeke, The metabolism of glyphosate in glyphosate tolerant crops, in: Proceedings of the Third Pan Pacific Conference on Pesticide Science, Hawaii, 2003. [19] P.C.C. Feng, J.S. Ryerse, R.D. Sammons, Correlation of leaf damage with uptake and translocation of glyphosate in velvetleaf (Abutilon theophrasti), Weed Technol. 12 (1998) 300–307. [20] P.C.C. Feng, J.S. Ryerse, C.R. Jones, R.D. Sammons, Analysis of surfactant leaf damage using microscopy and its relation to glyphosate or deuterium oxide uptake in velvetleaf (Abutilon theophrasti), Pestic. Sci. 55 (1999) 385–386. [21] P.C.C. Feng, J.J. Sandbrink, R.D. Sammons, Retention, uptake and translocation of 14C-glyphosate from trackspray applications and correlations to rainfastness in velvetleaf (Abutilon theophrasti), Weed Technol. 14 (2000) 127–132. [22] S.H. Liu, R.A. Campbell, J.A. Studens, R.G. Wagner, Absorption and translocation of glyphosate in Aspen (Populus tremuloides Michx.) as influenced by droplet size, droplet number, and herbicide concentration, Weed Sci. 44 (1996) 482–488. [23] H. De Ruiter, A.J.M. Uffing, E. Meinen, Influence of surfactants and ammonium sulfate on glyphosate phytotoxicity to quackgrass (Elytigia repens), Weed Technol. 10 (1996) 803–808. [24] M.H. Denis, S. Delrot, Effects of salt and surfactants on foliar uptake and long distance transport of glyphosate, Plant Physiol. Biochem. 35 (1997) 291–301. [25] P.C.C. Feng, T. Chiu, R.D. Sammons, J.S. Ryerse, Droplet size affects glyphosate retention, absorption and translocation in corn, Weed Sci. 51 (2003) 443–448. [26] J.S. Ryerse, R.A. Downer, R.D. Sammons, P.C.C. Feng, Effect of glyphosate spray droplets on leaf cytoarchitecture in velvetleaf (Abutilon theophrasti), Weed Sci. 52 (2004) 302–309. [27] J.S. Ryerse, P.C.C. Feng, R.D. Sammons, Endogenous fluorescence identifies dead cells in plants, Microsc. Today 1 (2001) 22–24. [28] J.C. Casely, D. Coupland, Environmental and plant factors affecting glyphosate uptake, movement and activity, in: E. Grossbard, D. Atkinson (Eds.), The Herbicide Glyphosate, Butterworth, London, 1985, pp. 92–123. [29] D.D. Buhler, O.C. Burnside, Effects of application variables on glyphosate phytotoxicity, Weed Technol. 1 (1987) 14–17. [30] C.N. Duncan Yerkes, S.C. Weller, Diluent volume influences susceptibility of field bindweed (Convolvulus arvensis) biotypes to glyphosate, Weed Technol. 10 (1996) 565–569. [31] J.R. Cranmer, D.L. Linscott, Droplet makeup and the effect on phytotoxicity of glyphosate in velvetleaf (Abutilon theophrasti), Weed Sci. 38 (1990) 406–410.
Pesticide Biochemistry and Physiology 82 (2005) 36–45 www.elsevier.com/locate/ypest
Distribution of [14C]glyphosate in mature glyphosate-resistant cotton from application to a single leaf or over-the-top spray Paul C.C. Feng*, Tommy Chiu Monsanto Co., GG4D 700 Chesterfield Parkway West, Chesterfield, MO 63017-1732, USA Received 29 March 2004; accepted 28 July 2004 Available online 2 February 2005
Abstract Distribution of [14C]glyphosate was examined in mature glyphosate-resistant cotton plants at the 13-node stage in the absence of phytotoxicity. Initial experiments employed manual application of glyphosate to individual leaves within a relatively immature (Node 9) or mature (Node 5) sympodium (i.e., fruiting node) in the plant. We measured glyphosate export out of the treated leaf to the fruiting structures and foliage in the sympodium as well as out of the sympodium into the plant. Application to the Stem leaf, Leaf 1 or Leaf 2 in Node 9 showed 30–37% glyphosate export by 14 days after treatment. While Stem leaf exported mainly to the plant, Leaves 1 and 2 exported equally between the plant and the sympodium. Within the sympodium, glyphosate was distributed almost entirely to the fruiting structures with Boll 1 containing the highest level irrespective of the treated leaf. In Node 5, application to the Stem leaf or Leaves 1–3 showed overall lower glyphosate export (20–27%) with Stem leaf and Leaf 3 exporting mainly to the plant, and Leaves 1 and 2 exporting mainly to the sympodium. Within Node 5, the subtending boll of the treated leaf was not the main distribution target, but instead the boll at the next higher sympodial position relative to the treated leaf. Subsequent studies were conducted using over-the-top spray application of [14C]glyphosate at field use-rates in mature plants at the 13node stage. Analysis of open bolls at full maturity showed high residues in seeds and fiber of Bolls 1 and 2 in the mature sympodia. Our results suggest that glyphosate distribution in mature plants was affected by sympodial age, position of the treated leaf, and position and sink strength of the bolls. 2004 Elsevier Inc. All rights reserved. Keywords: Glyphosate; Glyphosate-resistant cotton; Roundup Ultra; Roundup Ready
*
Corresponding author. Fax: +1 636 737 6060. E-mail address: [email protected] (P.C.C. Feng).
0048-3575/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2004.07.010
P.C.C. Feng, T. Chiu / Pesticide Biochemistry and Physiology 82 (2005) 36–45
1. Introduction The development of glyphosate resistance has revolutionized agricultural practices in cotton. The current commercial product, Roundup Ready cotton, was engineered through expression of a glyphosate insensitive CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene [1]. Roundup Ready cotton was commercialized in 1997 and permits over-the-top (OT) spray application of glyphosate through the four-leaf stage. Applications beyond that growth stage can lead to plant injuries, which have been the subject of numerous investigations. Uptake and translocation studies showed that [14C]glyphosate was readily distributed to bolls [2]. Other studies [3,4] demonstrated lower expression of CP4-EPSPS in male reproductive tissues that contributed to reduced pollen viability, male sterility, and boll drop [5,6]. Translocation studies thus far have focused on young plants due to potential injurious effects of glyphosate in mature cotton. The next generation of glyphosate-resistant cotton (Roundup Ready Flex) is currently under development and demonstrates enhanced resistance to glyphosate [7]. The new glyphosate-resistant cotton permits an expanded window of application between 3 and 14 leaf stages, and offers growers greater flexibility in weed control. Boll retention and development have a direct impact on fiber and seed yield. Boll drop is a physiological response to environmental stresses such as drought or cold temperature [8,9], and is determined by an intricate balance of assimilate supply, demand, and distribution [10]. Pinkhasov [11] demonstrated that assimilates in cotton are photosynthesized mainly by the stem (33%) and lateral branch leaves (55%). Sink strength plays a major role in assimilate distribution. Bolls in the first three positions proximal to the main-stem are dominant sinks and receive the majority of assimilates [12]. Assimilate distribution is also affected by proximity with the subtending leaf being the most important source for the associated boll [13]. The objectives of our research were to examine the distribution of [14C]glyphosate in mature cotton plants and to model photoassimilate distribution using [14C]glyphosate as a marker.
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Glyphosate is a highly systemic herbicide that is readily translocated in the phloem following a pattern of source to sink tissues. Several reports [14,15] suggest that glyphosate is translocated with photoassimilates; however, glyphosate translocation can be affected by its own toxicity, which occurs within hours after application [16,17]. Our studies employed the new Roundup Ready Flex plants to exclude potential interference from glyphosate toxicity. We examined glyphosate translocation in mature plants with application to individual lateral branch leaves in a relatively mature or immature sympodium. We also examined distribution of [14C]glyphosate from OT spray to simulate field application.
2. Materials and methods 2.1. Plant rearing Glyphosate-resistant plants were raised in a greenhouse (29/25 C, day/night temperatures, 16-h photoperiod with supplemental lighting) and were treated with Pix Plus plant regulator (mepiquat chloride, 4.2% and Bacillus cereus, 0.0058%, BASF) with OT sprays (3 ml/L) at the four- and seven-leaf stages to control stature. Plants were acclimated in the growth chamber (29/25 C day/night temperatures, 16-h photoperiod, 700 lE/s/m2 light intensity, and 75% relative humidity) for at least 7 days prior to experimentation. Plants were visually selected to match in size, growth stage, and the maturity of fruiting structures. 2.2. Application of [14C]glyphosate dosing solutions Both drop and spray applications employed a glyphosate formulation comprised of an equal mixture of Roundup Ultra and Accord at a rate of 1.68 kg ae/ha and diluted to a spray volume of 187 L/ha. The Accord formulation contains no surfactant and served to reduce foliar surfactant injury. [14C]Phosphonomethyl labeled glyphosate (457 MBq/mmol specific activity, 98.2% purity, New England Nuclear) was fortified into the glyphosate formulation. Manual drop
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application to a leaf employed 50 ll of glyphosate formulation containing [14C]glyphosate (50 kBq) and was applied as 1 ll droplets to the adaxial leaf surface. OT spray application employed 35 ml of glyphosate formulation containing [14C]glyphosate (2.5 MBq). The formulation was sprayed in an enclosed track sprayer using a commercial flat fan nozzle (XR TeeJet 9501, Spraying Systems) calibrated to deliver the desired dose at a height of 46 cm and a volume of 187 L/ha. 2.3. Treatment design The drop application consisted of two studies utilizing plants at the 13-node stage. The first study involved application of glyphosate to individual branch leaves in the immature Node 9. The three treatments (three plants each) consisted of applications to the Stem leaf, Leaf 1 or Leaf 2. At the time of application, Node 9 contained a small boll, a square and a tiny square at positions 1, 2, and 3, respectively. The second study consisted of four treatments with application of glyphosate to individual branch leaves (Stem leaf, Leaf 1, Leaf 2 or Leaf 3) in the mature Node 5. At the time of application, Node 5 contained large to small bolls in positions 1–4. The OT spray study employed plants at the 13or 15-node stages (three plants per treatment). At the time of spray, fruiting structures were present at positions 1–4 in Nodes 5–12 with no open bolls. 2.4. Plant harvest Plants from drop studies were harvested at 14 days after treatment (DAT). The sympodium containing the treated leaf was excised and separated into individual fruiting structures, leaves, and stems. The treated leaf (Stem leaf or Leaves 1–3) was washed with water (40 ml) and dried with methanol (10 ml). The combined washes were analyzed by liquid scintillation counting for unabsorbed, surface radioactivity. Tissues from the harvested sympodium were packed into multiple cones and combusted in a biological oxidizer (Packard 387 oxidizer with Oximate 80 robotics, Perkin–Elmer Life Sci.). The mature bolls were further separated into seed, fiber, and carpels.
Fiber was subjected to repeated washing with water; the washes and the fiber were then analyzed for radioactivity. Radioactivity was summed for individual fruiting structures, subtending leaves, and the foliage (stems and side branches). Since the same glyphosate dose was applied in each treatment, results were calculated as percentage of applied dose. Average and standard error of means (SEM) were calculated based on three replicates. Following OT spray, plants were maintained in the growth chamber until full maturity and allowed to desiccate prior to harvest (40 DAT). All open bolls were individually harvested from each plant, and separated into seed and fiber. Tissue dry weights were measured prior to combustion analysis. As glyphosate undergoes little to no metabolism in cotton [18], tissue radioactivity was converted to glyphosate mass and expressed as ppm dry weights. Average and standard error of means were calculated based on three replicates. It is estimated that the combined drop and spray studies generated approximately 4000 combustion samples.
3. Results and discussion Our studies employed Roundup Ready Flex plants to exclude interference from glyphosate toxicity. As glyphosate is translocated with photoassimilates [14,15], we also wished to examine assimilate translocation using glyphosate as a marker. Assimilate distribution in plants is typically studied with radiolabeled substrates (e.g., sucrose and CO2) [15]; however, analysis can be complicated due to metabolism. Since glyphosate undergoes little to no metabolism in cotton [18], we hoped to generate a simpler picture of assimilate distribution. 3.1. Plant growth and development A cotton plant is comprised of a main-stem with nodes that give rise to a Stem leaf and side branch. The nodes are numbered from bottom up with the first fruiting branch (i.e., sympodium) usually appearing at Node 5. Within the fruiting branch are also nodes that give rise to a leaf and
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a fruiting structure, and these are numbered sequentially away from the main-stem (e.g., Boll 1 and Leaf 1 are located proximate to the main stem). The fruiting structure begins with a square that develops to a flower, which after pollination forms a boll. We monitored the growth of bolls and leaves during the 14-day study for the purpose of gauging the source or sink strengths of individual tissues. At the time of application, Node 5 contained bolls in positions 1–4. Position 1 was closest to the main-stem and most mature. Bolls 1 and 2 were at their maximum size and expanded no further during the 14-day study. Bolls 3 and 4 both increased in size; by 14 DAT, Boll 3 was at its maximum size while Boll 4 was about half its maximum size. Stem leaf and Leaves 1–3 were fully expanded and showed no further increase in diameter during the study. Only Leaf 4 showed a slight increase in diameter. There is a direct relationship between tissue growth, sink strength, and assimilate demand. A good example is a boll, which peaks in size at about 25 days post-anthesis (DPA) with assimilate import peaking at around 30 DPA [10]. Based on size and growth stage, we estimated the relative sink strength of the bolls in Node 5 as Boll 1 P Boll 2 > Boll 3 > Boll 4. At the time of application, Node 9 contained a small boll, a square, and a tiny square in positions 1, 2, and 3, respectively. By 14 DAT, positions 1 and 2 contained expanding bolls with a flower and a square in positions 3 and 4, respectively. The Stem leaf, Leaves 1 and 2 remained unchanged in size, while Leaves 3 and 4 expanded greatly during the 14-day study. Based on size and growth stage, the relative sink strengths of the fruiting structures in Node 9 were estimated as Boll 1 > Boll 2 > Flower 3 > Square 4. 3.2. Glyphosate export out of the treated leaf [14C]Glyphosate was fortified into a formulation (1.68 kg/ha at 187 L/ha volume) and was manually applied (50 · 1 ll) to the adaxial surface of the leaf. To examine the effect of leaf position, application was made individually to either the Stem leaf or Leaves 1–3 in Node 5; Leaf 3 in Node 9 was not treated due to its small size.
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Our previous studies showed that the surfactant system in Roundup Ultra causes rapid absorption and translocation of glyphosate that peaks at approximately 1 DAT [19–21]. In the current studies, the 14 DAT harvest is expected to represent end points of glyphosate uptake and translocation. At harvest, the treated sympodium was excised and separated into individual bolls, leaves, and stem. Mature bolls (Bolls 1 and 2) in Node 5 were further dissected into fiber, seed, and carpels. The treated leaf was washed to recover surface glyphosate followed by combustion analysis to determine tissue glyphosate. Distribution was calculated as percentages of applied dose. Table 1 summarizes glyphosate that was recovered in the leaf wash, localized in or exported from the treated leaf. Application of glyphosate to the Stem leaf, Leaf 1 or Leaf 2 in Node 9 recovered 37–48% of applied dose in the leaf wash. In comparison, applications to leaves in Node 5 recovered 46–66%. These results indicated higher average uptake in leaves of Node 9 than Node 5, and suggest that cuticle of mature leaves posed a greater barrier to glyphosate absorption. Studies have shown that foliar absorption of glyphosate is also affected by numerous physical variables including glyphosate concentration [22], surfactant type [23,24], and droplet size [25,26]. Table 1 also shows glyphosate that remained localized in the treated leaf at 14 DAT. This localization is believed to result from surfactant injury associated with manual application of large size droplets [26,27]. Localization averaged 25 and 20% among treated leaves in Node 9 and Node 5, respectively. Our main interest was monitoring glyphosate exported from the treated leaf, and this was calculated by the difference between applied and recovered radioactivities in the treated leaf and the wash. Export among treated leaves averaged 33% in Node 9 and 23% in Node 5, and indicated that glyphosate is more efficiently absorbed and exported from leaves in immature (Node 9) than mature (Node 5) sympodium. 3.3. Glyphosate distribution in immature Node 9 Glyphosate that was exported from the treated leaf can either remain in the sympodium or trans-
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Table 1 The distribution of [14C]glyphosate (percentage of applied dose) in mature glyphosate-resistant cotton plants (13-node stage) from manual application to individual branch leaves (Stem leaf, Leaf 1, Leaf 2 or Leaf 3) in an immature (Node 9) or mature (Node 5) sympodium at 14 DAT Treatment node
Appl. leafa (N = 3)
% appl.
SEM
% appl.
SEM
% appl.
SEM
9
Stem leaf Leaf 1 Leaf 2
41.8 36.6 48.0
3.1 0.5 8.3
27.8 26.0 20.4
2.5 0.4 6.3
30.4 37.4 31.6
4.1 0.5 2.3
Average
42.1
4.0
24.7
3.1
33.1
2.3
Stem leaf Leaf 1 Leaf 2 Leaf 3
56.6 46.0 65.7 61.9
3.7 1.8 1.2 4.5
22.7 27.3 14.2 13.7
3.8 1.4 1.0 2.9
20.7 26.7 20.1 24.4
1.9 0.6 1.3 1.6
Average
57.6
2.8
19.5
2.3
23.0
1.4
5
a
Leaf washb
Localizedc
Exportedd
14
Radiolabeled [ C]glyphosate was fortified into formulated glyphosate (1.68 kg/ha) and applied as 50 · 1 ll droplets to the adaxial leaf surface of Stem leaf, Leaf 1, Leaf 2 or Leaf 3 in Node 9 or 5. b Treated leaf was washed at 14 DAT with water followed by methanol; combined washes were analyzed by liquid scintillation counting and expressed as percentage of applied dose. Average and standard error of means (SEM) were calculated from three replicate plants. c After washing, glyphosate localized in the treated leaf was determined by combustion analysis and expressed as percentage of applied dose. d Glyphosate exported from the treated leaf was calculated by the difference between applied and recovered dose (wash and localized).
ported out of the sympodium into the plant. Within the sympodium, distribution could be to fruiting structures, leaves or stems. These data are summarized in Fig. 1. When applied to the Stem leaf, gly-
Fig. 1. The distribution of [14C]glyphosate (percentage of applied dose) from manual application to individual branch leaves (Stem leaf, Leaf 1 or Leaf 2) in the immature Node 9 of glyphosate-resistant cotton plants at 14 DAT.
phosate export was mainly to the plant (22% of applied dose) with much less (8%) to the sympodium. In comparison, application to Leaf 1 or Leaf 2 resulted in comparable distribution between the sympodium and the plant. Assuming that glyphosate is a suitable marker for photoassimilates, our results suggest that all leaves, and especially the Stem leaf, in immature sympodia are important sources for the plant. Fig. 2 shows the distribution of glyphosate as percentage of applied dose between the fruiting structures and the foliage within Node 9. The results showed that irrespective of the application leaf, glyphosate was principally distributed to the fruiting structures (bolls, flowers, and squares) which represented the dominant sinks in the immature sympodium. Fig. 3 further breaks down the distribution to individual fruiting structures in Node 9 as a function of application leaf. At 14 DAT, positions 1–4 contained a medium boll, a small boll, a flower, and a square, respectively, with an estimated relative sink strengths of Boll 1 > Boll 2 > Flower
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3.4. Glyphosate distribution in mature Node 5
Fig. 2. The distribution of [14C]glyphosate (percentage of applied dose) between sympodial foliage and fruiting structures from manual application to individual branch leaves (Stem leaf, Leaf 1 or Leaf 2) in the immature Node 9 of glyphosateresistant cotton plants at 14 DAT.
Fig. 4 summarizes glyphosate export as percentage of applied dose from application to leaves in Node 5. When applied to the Stem leaf, glyphosate was once again distributed mostly to the plant (15%) and not to the sympodium (6%); this distribution pattern was similar to the corresponding Stem leaf in Node 9. Application to Leaf 1 produced a different pattern with distribution to sympodium (18%) far exceeding that of the plant (8%). Application to Leaf 2 produced a similar pattern as Leaf 1 with the sympodium receiving greater distribution than the plant (12 vs 8%). However, with application to Leaf 3, the pattern reverted to that of the Stem leaf with plant distribution exceeding that of the sympodium (15 vs 9%). The conclusion is that Stem leaf and Leaf 3 source mainly the plant while Leaves 1 and 2 source mainly the sympodium. Our results demonstrated a difference in glyphosate distribution between Node 9 and Node 5. In the immature sympodium, plant export was the major role for all the leaves, whereas in the mature sympodium, some leaves (Leaves 1 and 2) sourced the sympodium while others (Stem leaf and Leaf 3) the plant. Fig. 5 shows the distribution as percentage of applied dose between the foliage and the bolls within Node 5. The results showed that irrespective of the application leaf, glyphosate was almost
Fig. 3. The distribution of [14C]glyphosate (percentage of applied dose) among sympodial fruiting structures from manual application to individual branch leaves (Stem leaf, Leaf 1 or Leaf 2) in the immature Node 9 of glyphosate-resistant cotton plants at 14 DAT.
3 > Square 4. The distribution results showed that Boll 1 contained the majority of glyphosate irrespective of the application leaf. Boll 1 was the strongest sink and apparently out-competed other sinks accessing glyphosate not only from its subtending Leaf 1 but also from distant leaves (i.e., Stem leaf and Leaf 2). Our results suggest that in an immature sympodium, boll sink strength was the principal determinant for glyphosate distribution.
Fig. 4. The distribution of [14C]glyphosate (percentage of applied dose) from manual application to individual branch leaves (Stem leaf, Leaf 1, Leaf 2 or Leaf 3) in the mature Node 5 of glyphosate-resistant cotton plants at 14 DAT.
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P.C.C. Feng, T. Chiu / Pesticide Biochemistry and Physiology 82 (2005) 36–45
Fig. 5. The distribution of [14C]glyphosate (percentage of applied dose) between sympodial foliage and bolls from manual application to individual branch leaves (Stem leaf, Leaf 1 or Leaf 2) in the mature Node 5 of glyphosate-resistant cotton plants at 14 DAT.
entirely distributed to the bolls. These results were similar to that of Node 9 and suggest that in both immature and mature sympodia, the fruiting structures are the dominant sinks. Fig. 6 further breaks down the distribution of glyphosate to bolls as a function of application leaf in Node 5. Application to the Stem leaf produced most distribution to Boll 1, which was indicative of its high sink strength. Much to our surprise, application to Leaf 1 showed most distribution to Boll 2. Boll 1 received much less glyphosate in spite of
its sink strength and subtending position to Leaf 1. The same pattern was observed with application to Leaf 2, which showed most distribution to Boll 3 and not Boll 2. These results demonstrated that distribution is primarily to the boll in the next higher position from the treated leaf. Application to Leaf 3 did show the highest distribution to Boll 3. Our results differed from the common understanding that the subtending leaf is the primary source for the associated boll [13]. As these plants were fully resistant to glyphosate, we were confident that glyphosate distribution was not affected by its own toxicity. The mature Bolls 1 and 2 in Node 5 were further separated into seed, fiber, and carpel. Table 2 shows tissue levels of glyphosate expressed as percentages of boll-contained radioactivity. The results showed that irrespective of the application leaf, the majority of glyphosate in Bolls 1 and 2 were in the seeds (46–52%) with the remaining split between the fiber (25–27%) and carpel (23–27%). These results showed that distribution of glyphosate within the boll is very consistent with seeds as the dominant sink. Our results also showed that fibers contained significant glyphosate residues, which were mostly (78%) removed by repeated washing with water (data not shown). Limited analysis was also conducted to determine the destiny of glyphosate that was exported out of the treated sympodium (Node 5) and into the plant. Acropetal or basipetal movement was assessed by analysis of the apical meristem and Boll 1 above or below the treated sympodium. Results showed the presence of glyphosate in Boll 1 below the treated sympodium, but not in the apical meristem or Boll 1 above the treated sympodium (data not shown). These results suggest that glyphosate exported out of the sympodium is mainly translocated basipetally to source the roots and proximate bolls along the main stem. 3.5. Modeling assimilate distribution in mature sympodia
Fig. 6. The distribution of [14C]glyphosate (percentage of applied dose) among sympodial bolls from manual application to individual branch leaves (Stem leaf, Leaf 1 or Leaf 2) in the mature Node 5 of glyphosate-resistant cotton plants at 14 DAT.
The distribution of glyphosate from leaves in Node 5 was very unexpected. Based on glyphosate distribution, we developed a model for assimilate distribution in mature sympodia (Fig. 7). The
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Table 2 The distribution of [14C]glyphosate (percentage boll-contained activity) in Bolls 1 and 2 from manual application to individual branch leaves (Stem leaf, Leaf 1, Leaf 2 or Leaf 3) in Node 5 of glyphosate-resistant cotton plants at 14 DAT Node 5 Appl. leafa
Boll 1b % distribution
Boll 2b % distribution
(N = 3)
Seed
Fiber
Carpel
Seed
Fiber
Carpel
Stem leaf Leaf 1 Leaf 2 Leaf 3
61.3 51.3 41.6 54.6
18.5 22.5 38.4 21.3
20.2 26.2 19.9 24.1
49.0 37.6 49.3 48.5
28.9 31.4 24.4 24.6
22.1 31.0 26.4 27.0
Average SEM
52.2 4.1
25.2 4.5
22.6 1.5
46.1 2.8
27.3 1.7
26.6 1.8
a Radiolabeled glyphosate was fortified into formulated glyphosate (1.68 kg/ha) and applied as 50 · 1 ll droplets to the adaxial leaf surface of Stem leaf, Leaf 1, Leaf 2 or Leaf 3 in Node 5. b Boll 1 or 2 from individual treatments were dissected into seed, fiber, and carpel. Tissue radioactivity was determined by combustion analysis and expressed as a percentage of total radioactivity in the boll. Average and standard error of means (SEM) were calculated from three replicate plants.
Fig. 7. A proposed model of photoassimilate distribution in a mature sympodium showing directional phloem flow that reduces assimilate distribution to upstream bolls to insure assimilate export to the plant.
model suggests that assimilate transport in the sympodial phloem is directional flowing up from the main-stem and back down in separate streams. Bolls and leaves are connected only to the upward stream. Since the movement of the phloem is towards sympodial terminal, only bolls downstream from the leaf can access the assimilates. As shown in the model, application of glyphosate to Leaf 2 targets distribution mainly to Boll 3, and precluded upstream Bolls 1 and 2 from much distribution. Excess assimilates are exported to the plant through the downward stream that is not accessible by the bolls. The model also explains the result from application to Leaf 3. As immature Boll 4 was not yet a strong sink, more glyphosate was exported to the plant.
Our studies suggest a distinction in assimilate translocation between mature and immature sympodia. The immature sympodium appears to contain only one phloem where assimilate distribution is mainly based on boll sink strength regardless of distance from the source leaf. The leaves produce ample assimilates, but since demand is low from the immature fruiting structures most is exported to the plant. In contrast, the first three position bolls in a mature sympodium are at their peak assimilate demand, and probably could consume all the assimilates from the sympodial leaves. The proposed dual phloem model insures that some assimilates in mature sympodia are exported to the plant. This is likely an adaptation to favor the survival of the plant over the bolls under less than ideal growing conditions. Future studies are required to further support this model. 3.6. Glyphosate distribution from OT spray application The above studies using single-leaf applications were useful in defining the role of individual leaves on glyphosate distribution. The results showed that glyphosate distribution varied depending on leaf age and position, as well as sympodium age. In field spray applications, plant coverage is rarely uniform and can be affected by biological variables such as canopy density and leaf morphology that affect droplet interception and rebound [28,29].
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Plant coverage can also be affected by physical variables such as spray volume [30], droplet size [25], and formulation concentration [22,31]. As a consequence, glyphosate distribution in a spray application is very complex and dependent on which leaves actually intercept and retain the spray. Glyphosate distribution from spray application is not likely to be modeled by application to a single leaf. We examined [14C]glyphosate distribution with OT spray in mature cotton plants to simulate field application. Plants (13-node stage) were sprayed at a field use-rate of 1.68 kg/ha in 187 L/ha volume and maintained in the growth chamber until full maturity (40 DAT). After desiccation, open bolls were individually harvested and separated into seeds and fiber. There were no open bolls at the time of spray; therefore, residues in the harvested bolls represented translocated glyphosate. Fig. 8 reports the seed concentration of glyphosate (ppm dry weight) from the open bolls. High concentrations were detected in Boll 1 (3.5– 5.2 ppm) and Boll 2 (1.0–4.9 ppm) between Nodes 5 and 10. Seed residues from Boll 2 diminished by Node 9 while that from Boll 1 diminished by Node 11. This was consistent with our earlier results showing Boll 1 as the dominant sink in immature sympodia. Boll 3 or Boll 4, if present, showed very low residues (<1.0 ppm) in all sympodia. Glyphosate distribution was also examined from OT spray application to older plants at the 15-node stage. Analysis showed that peak seed residues were
shifted to higher nodes (8–11) and higher position bolls (2 and 3) which was coincident with maturation of Boll 1 in lower sympodia. Foliar analysis showed that the majority of the spray-dose were intercepted by leaves in the upper and outer canopy (data not shown) that efficiently exported glyphosate out of the sympodium to the plant. Studies from drop application in Node 5 showed that glyphosate can be delivered to higher position bolls (e.g., Boll 3), if applied to the appropriate leaves (i.e., Leaves 2 and 3). Our results suggest that during OT spray, the plant coverage pattern leads to extensive sympodial export and basipetal translocation that sourced the strong sink bolls (positions 1 and 2) proximal to the main stem. Analysis of fiber showed residue concentrations (ppm dry wt) that averaged approximately 1.5 times higher than that of seeds (data not shown). Boll retention, fiber, and seed yield are of primary importance to cotton growers. Glyphosate distribution in mature cotton plants was examined in the absence of toxicity from applications to a single leaf or OT spray. Our results showed that distribution of glyphosate was dependent on leaf age and position as well as sympodium age, and likely to be affected by the pattern of foliar coverage during spray application. As glyphosate moves freely in the phloem [14,15], our results are also indicative of photoassimilate distribution in mature cotton plants. A complex pattern of assimilate distribution was evident in mature sympodia to balance the needs of maturing bolls with that of the plant.
Acknowledgments We thank Amy Martens and Jesse Hart for assistance in the greenhouse work. We also thank Minhtien Tran and Doug Sammons for discussions, and Claire CaJacob and Mark Oppenhuizen for supporting this research.
References Fig. 8. Seed concentration of glyphosate (ppm dry weight) from open bolls at full plant maturity from over-the-top spray application of [14C]glyphosate (1.68 kg/ha) to 13-node glyphosate-resistant cotton plants.
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