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Effect of relative humidity on the uptake, translocation, and efficacy of glufosinate ammonium in wild oat (Avena fatua)
Pesticide Biochemistry and Physiology 73 (2002) 1–8 www.academicpress.com
Effect of relative humidity on the uptake, translocation, and efficacy of glufosinate ammonium in wild oat (Avena fatua) R.J.L. Ramsey, G.R. Stephenson, and J.C. Hall* Department of Environmental Biology, University of Guelph, Guelph, Ont., Canada N1G 2W1 Received 18 October 2001; accepted 18 April 2002
Abstract Relative humidity (RH) can greatly affect the uptake and efficacy of glufosinate ammonium (D ,L -homoalanine-4yl-(methyl)-phosphinate) in wild oat (Avena fatua L.). Exposure to high (>95%) RH as opposed to low (40%) RH increased glufosinate ammonium efficacy and it was high RH within 12 h of spraying that was most crucial in affecting the efficacy. Subsequent dose–response experiments at 40% RH indicated that exposure of wild oat to high RH for as little as 30 min before and after spraying significantly increased the efficacy when compared to plants grown continuously at 40% RH. Furthermore, when [14 C]glufosinate ammonium was applied as a spray to wild oat plants grown at 40% RH, there was a significant increase in uptake into wild oat plants exposed to high RH for 30 min before and after treatment compared to those left continuously at low RH. Conversely, when the same experiment was conducted with [14 C]glufosinate ammonium applied as ten 1-ll droplets, exposure to high RH for 30 min before and after treatment did not increase the herbicide uptake. Based on these results, we hypothesize that droplet size and hence drying time is the major factor in effecting changes in glufosinate ammonium uptake in response to exposure to high versus low RH. Applying [14 C]glufosinate ammonium as large droplets resulted in an overestimation of uptake and masked the inhibitory effect of low RH on glufosinate ammonium as determined by dose–response experiments. Conversely, spray application of [14 C]glufosinate ammonium produced data that correlated with those from dose–response experiments. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction Wild oat (Avena fatua L.) is a weed that poses a serious problem to cereal and canola crops in western Canada [1]. Among the herbicides registered for control of wild oat is glufosinate
*
Corresponding author. Tel.: +519-824-4120x8598. E-mail address: [email protected] (J.C. Hall).
ammonium (D ,L -homoalanine-4yl-(methyl)-phosphinate), sold under the trade names of Liberty and Ignite. Glufosinate ammonium is a post-emergent herbicide with a broad spectrum of activity. An inhibitor of glutamine synthetase, glufosinate ammonium competes with glutamate for the active site and once bound becomes phosphorylated, irreversibly inhibiting the enzyme [2,3]. Inhibition of glutamine synthetase results in a rapid accumulation of ammonia and inhibition of photosynthesis through depression of photorespiration [2,4,5].
0048-3575/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 4 8 - 3 5 7 5 ( 0 2 ) 0 0 0 1 7 - 2
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Despite the susceptibility of wild oat to glufosinate ammonium, poor control can occur when relative humidity (RH) is low at the time of spraying. Low RH has been correlated with reduced efficacy of many herbicides [6–10], including glufosinate ammonium [11–14]. The mechanism through which RH influences the efficacy of foliarapplied herbicides is not completely understood, but some researchers have suggested that it could involve interactions between the herbicide droplet, leaf cuticle, and availability of water in or around droplets [15,16]. It has also been reported that the activity of polar, water-soluble herbicides is particularly sensitive to changes in RH [17]. In western Canada, RH can change by 40–50% within a few hours (i.e., high RH at night, low RH during the day). These conditions provide several possible scenarios to explain the poor efficacy of glufosinate ammonium. Early hypotheses developed in our laboratory involved the premise that optimal herbicide uptake would result in rapid, localized tissue damage, reducing loading into the phloem and subsequent translocation of glufosinate ammonium, causing poor efficacy. While self-limitation of herbicide translocation has been reported for glyphosate and chlorsulfuron [18], similar findings for glufosinate have not been reported. Glufosinate ammonium has some phloem and xylem mobility [19]; however, it does not tend to translocate to a major degree out of the leaves of treated weeds [12,20,21]. Furthermore, the RH in western Canada tends to be low, offering few instances where optimal herbicide uptake would be expected. In these studies, the effects of high and low RH on the uptake, translocation, and efficacy of glufosinate ammonium in wild oat were examined through dose–response experiments and uptake and translocation studies using [14 C]glufosinate ammonium conducted at various RH regimes. Through the course of our investigations, the following hypotheses were developed with regard to glufosinate ammonium uptake, translocation, and efficacy in wild oat: 1. Short exposure to high RH within hours of spraying will increase uptake and efficacy as much as exposure to high RH for longer periods (days). 2. Exposure to high RH after spraying is more important to increase efficacy than is high RH before spraying The objectives of our research were twofold: to further elucidate the interaction between RH and glufosinate ammonium efficacy in wild oat in terms of uptake, translocation, and efficacy and to
determine why preliminary 14 C uptake data as affected by RH did not correlate with dose– response data.
2. Materials and methods 2.1. Growth of plants Wild oat seeds were germinated in Promix (Plant Products, Brampton, Ont.) and transplanted to 8-cm square pots filled with crushed particulate expanded baked clay (‘‘Turface,’’ Applied Industrial Materials, Deerfield, IL). Plants were watered every second day, alternating between tap water and half-strength Hoagland’s solution [22]. The growing conditions were 16-h photoperiod with a light intensity of 450 lEm2 s1 and 25/15 °C day/night temperatures. Prior to exposure to different RH regimes, plants were grown in a controlled environment growth room (70% RH). High humidity treatments (>95% RH) consisted of placing plants inside Plexiglas chambers (111 46 51 cm), each of which contained an ultrasonic mist humidifier (Sunbeam Household Products, Boca Raton, FL, USA). Low humidity treatments were provided by placing plants in a growth cabinet set for 40% RH. Relative humidity and temperature were continuously monitored with a solid state RH probe (Cole-Parmer, Vernon Hills IL, USA). 2.2. Dose–response experiments Herbicide treatments were applied with an automatic hood sprayer (RC-5000-100EP Mandel Scientific, Guelph, Ont., Canada) using a flat fan nozzle (SS80015E Spraying Systems, Wheaton, IL, USA). The spray volume used was equivalent to 110 l/ha of spray solution delivered at 200 kPa. Wild oat plants were at the 3-leaf stage of development at the time of herbicide application. At harvest, shoots were removed and dried at 60 °C for 3 days and weighed. All experiments were repeated at least once. Preliminary results suggested that the RH 12 h before and after spraying had a greater impact on efficacy than the RH 6 days before and after spraying. It was also determined that high RH was always associated with increased efficacy and that low RH was always associated with poor efficacy. With this in mind, subsequent dose–response experiments focused on the effect short (12 h or less)
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exposure to 99% RH, before and after spraying, had on the efficacy of glufosinate ammonium applied to wild oat plants grown at 40% RH. In Experiment 1, plants were exposed to: (1) 40% RH for 6 days, prior to exposure to 99% RH for 3, 6 or 12 h before and after treatment or (2) left at 40% RH until treatment. Once all plants that completed their respective treatment regimes were treated with glufosinate ammonium, they were returned to 40% RH and left at this RH for 3 days after which they were returned to 70% RH until harvest (Table 1). Experiment 1 was repeated several times using shorter exposures to high RH before and after herbicide treatment (i.e., 180, 120, 60, 40, 30, and 20 min). The exact treatment regimes are outlined in Table 1. Experiment 2 was designed to determine if exposure to high RH before or after herbicide treatment was responsible for the increase in efficacy associated with high RH as was observed in Experiment 1. As in Experiment 1, wild oat plants
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were grown at 40% RH for 6 days and separated into groups that were exposed to 40 min of high RH: (1) before and after, (2) only before or (3) only after herbicide application. Plants exposed to high RH only after spraying remained at low RH until herbicide application while those plants exposed to high RH only before spraying were returned to 40% RH immediately after spraying. As with Experiment 1, plants were returned from 40% RH to 70% RH 3 days after herbicide treatment. The exact treatment regimes are outlined in Table 1. 2.3. Uptake and translocation experiments Radiolabeled glufosinate ammonium was produced using methods similar to those of Mersey et al. [12]. D ,L -Homoalanine-4-yl-(methyl)-phosphinate [3,4] (14 C labeled) was incubated in excess NH4 OH for 30 min to yield [14 C]glufosinate ammonium (873.65 MBq/g). Water and NH4 OH were
Table 1 Relative humidity regimes for dose–response and uptake/translocation experiments Experiment
#1
Time course RH pre-treatment RH before (6 days) spraying
Spray treatments
RH after spraying
RH post-treatment until harvest
Harvest times
40%
Ignite 0, 100, 200, 400 g ai/ha
40%, 99%, 99%, 99%, 40%, 99%, 99%, 99%, 40%, 99%, 99%, 99%, 40%, 99%,
40%, 3 days then 70% until harvest
14 DAT
40%, 3 days then 70% until harvest
14 DAT
40%, 3 days then 70% until harvest
14 DAT
40%, 3 days then 70% until harvest
14 DAT
40%
40%
40%
40%, 99%, 99%, 99%, 40%, 99%, 99%, 99%, 40%, 99%, 99%, 99%, 40%, 99%,
3h 6h 12 h 1h 2h 3h 20 min 40 min 60 min 30 min
Ignite 0, 100,200, 400 g ai/ha Ignite 0, 100, 200, 400 g ai/ha Ignite 0, 100, 200, 400 g ai/ha
3h 6h 12 h 1h 2h 3h 20 min 40 min 60 min 30 min
#2
40%
40%, 40%, 99%, 40 min 99%, 40 min
Ignite 0, 100, 200, 400 g ai/ha
40%, 99%, 40 min 40%, 99%, 40 min
40%, 3 days then 70% until harvest
14 DAT
#3
40% 40% 40% 40%
40% 99%, 1 h 40% 99%, 12 h
[14 C]Ignite (400 g ai/ha) [14 C]Ignite (400 g ai/ha)
40% 99%, 1 h 40% 99%, 12 h
40% 40% 40% 40%
1, 2, 4, 8 h 1, 2, 4, 8 h 1, 2, 4, 72 h l, 2, 4, 72 h
#4
Six days prior to treatment, all plants were placed at 40% RH (i.e., 6 days RH pre-treatment) until several hours before application of glufosinate ammonium. * DAT, days after treatment.
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evaporated under a stream of nitrogen gas and [14 C]glufosinate ammonium re-dissolved in ‘‘Ignite’’ formulation blank. Plants were treated at the 3-leaf stage with approximately 300,000 dpm in 10 ll (0.00289 mol/L) of [14 C]glufosinate ammonium. The 14 C-labeled herbicide solution was applied using 10 ll Wiretrol micropipettes to place ten 1-ll droplets midway up the second leaf. To achieve a herbicide concentration in the dosing solution that would simulate a field dose of 400 g ai/ha in 110 l/ha water (0.0183 mol/L), a calculated amount of unlabelled glufosinate ammonium was added from an aqueous, unformulated 600 g ai/L stock provided by Aventis CropScience. There were six replicates per treatment and all experiments were repeated at least once. Experiment 3 was designed to examine the effect of short exposure to high RH on the uptake of [14 C]glufosinate ammonium into leaves of wild oat plants. Plants were exposed to low RH for 6 days before treatment with [14 C]glufosinate ammonium with half the plants exposed to high RH for 1 h before and after treatment (Table 1). [14 C]Glufosinate ammonium was applied as ten 1-ll droplets. Plants were harvested 1, 2, 4, and 8 h after treatment. The treated leaf was removed and rinsed with an aqueous 20% ethanol, 0.5% Tween 20 (a polyoxyethylene based nonionic detergent) solution. Remaining plant parts were not used because preliminary experiments indicated that little or no herbicide translocates out of the treated leaf 8 h after application of [14 C]glufosinate ammonium. The results of preliminary experiments and Experiment 3 indicated that the method used to apply [14 C]glufosinate ammonium may affect the uptake of the herbicide. This hypothesis was tested by applying [14 C]glufosinate ammonium to the leaf as a fine spray, as opposed to several large 1ll droplets as is commonly done to evaluate 14 Clabeled herbicide uptake. In Experiment 4, [14 C]glufosinate ammonium was applied as a 10 ll spray, completely covering the adaxial side of the second leaf. This was accomplished through the use of a hand-held sprayer calibrated to deliver a low volume (10 ll) of radiolabelled herbicide solution to the leaf. Briefly, a 10 ll Wiretrol micropipette was inserted into a device so that the end of the micropipette was directly in front of an air stream. Pushing the plunger forced the treatment solution to enter the air stream, forming a fine spray. Construction of a similar device has been previously described [23]. As with the device constructed by Bucholtz and Hess [23], our
sprayer produced droplets that were not significantly different in size than those produced by the 80015 nozzle used in dose–response experiments. While 1-ll droplets have a diameter of about 1500 lm, the droplets produced by our sprayer and an 80015 nozzle were in the range of 400– 500 lm in diameter. Plants were exposed to low RH for 6 days before treatment with half being exposed to 30 min of high RH before and after 14 C-labeled herbicide application (Table 1). As in Experiment 3, the treated leaves were removed at 1, 2, 4, and 72 h after treatment and rinsed as described above. In Experiments 3 and 4, all harvested plant parts were dried at 60 °C for 3 days. Dried plant parts were oxidized in a biological oxidizer (OX 300, R.J. Harvey Instrument, Hillsdale, NJ), in which liberated 14 CO2 was trapped in a scintillation cocktail (R.J. Harvey Instrument) and quantified by liquid scintillation spectroscopy (LSS). The efficiency of combustion and recovery was greater than 95%. Leaf rinses were collected and 14 C content was quantified by LSS. Uptake data are expressed as a percentage of recovered 14 C. Data for all experiments were analyzed using SigmaStat 2.0 (Jandel Scientific, San Rafael, CA) using one-way ANOVA followed by Tukey’s multiple comparison test if significant differences were detected. Comparisons were restricted to data within a harvest interval or herbicide dose.
3. Results and discussion Preliminary experiments established that high RH was associated with increased uptake and efficacy of glufosinate ammonium, while low RH was associated with poor efficacy and uptake. These findings are similar to those reported by other researchers examining the uptake of glufosinate ammonium or other herbicides at high and low RH [7,10–12,24,25]. In the first part of Experiment 1, wild oat plants grown at 40% RH were exposed to high RH (>95%) for 0, 3, 6 or 12 h before and after spraying (Table 1). Dry weight analysis indicated that all three exposures to high RH produced a significant increase in glufosinate ammonium efficacy compared to a continuous regime of 40% RH (Fig. 1). Furthermore, the efficacy of glufosinate ammonium, when plants were exposed to short durations of high RH, was the same as when plants were grown continuously at high RH (>95%). Further modifications of Experiment 1 established that at the
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Fig. 1. Influence of RH regimes on dry weight of wild oat treated with glufosinate ammonium. RH regimes were: continuous 40% RH (d) or continuous 40% RH except for 99% RH 3 ( ), 6 (.), or 12 (r) h before and after application of glufosinate ammonium. Letters denote statistically significant differences between treatments within harvest times as determined by ANOVATukey’s test (a ¼ 0:05).
Fig. 2. Influence of RH regimes on dry weight of wild oat treated with glufosinate ammonium. RH regimes were: continuous exposure to 40% RH (d) or continuous 40% RH except for exposure to 99% RH for 40 min before (.), after (r), or before and after ( ) spraying with glufosinate ammonium. Letters denote statistically significant differences between treatments within harvest times as determined by ANOVA-Tukey’s test (a ¼ 0:05).
doses tested, even 30 min of exposure to high RH before and after treatment produced an increase in efficacy similar to that associated with 12 h of high RH exposure before and after treatment. This is the first instance where such short exposures to high RH have been reported to significantly increase herbicide efficacy. In Experiment 2, it was found that high RH after, but not before spraying, was required to improve glufosinate ammonium efficacy. In fact, 40 min of exposure to high RH after spraying resulted in increased efficacy equal to that produced by exposing the plants to high RH for 40 min both before and after treatment (Fig. 2). These results suggest that the increase in efficacy observed is a result of events that occur after herbicide droplets come into contact with the leaf surface. We speculate that exposure to high RH after spraying may increase the droplet drying time thereby allowing a longer time or ‘‘window’’ for the herbicide to penetrate the cuticle. In Experiment 1, it was found that exposure to 99% RH for 30 min before and after treatment was sufficient to increase glufosinate ammonium efficacy on wild oat. To examine the effect of similar conditions on the uptake of [14 C]glufosinate ammonium, Experiment 3 was performed, exposing wild oat plants grown at 40% RH to either 99% RH for 1 h before and after treatment or a continuous 40% RH regime. [14 C]Glufosinate ammonium was applied as ten 1-ll droplets and short harvest times were used to examine the effect
of high RH on the uptake of glufosinate ammonium. It was observed that there was no difference in uptake of [14 C]glufosinate ammonium between plants exposed to 99% RH 1 h before and after application versus those left at 40% RH (Fig. 3), even though exposure to 1 h of 99% RH before and after spraying caused an increase in efficacy as measured by dry weights (i.e., Experiment 1; dose–response experiment). In a study to determine the effect of RH on the uptake and efficacy of glufosinate ammonium in several amaranth species, Coetzer et al. [14] observed a similar disagreement between the results from [14 C]glufosinate ammonium uptake experiments and dose– response experiments. They found that continuous exposure to 90% RH resulted in a significant increase in glufosinate ammonium efficacy as measured by dose–response, but there were no differences in glufosinate ammonium uptake. They proposed that increased translocation of glufosinate ammonium in plants exposed to continuous 90% RH may be the mechanism through which increased efficacy was achieved [14]. While [14 C]glufosinate ammonium translocation patterns may be altered by long exposures to high RH, it seems unlikely that the short duration exposures to high RH used in Experiment 3 would change translocation sufficiently to increase herbicide efficacy. Furthermore, preliminary experiments suggested that little or no glufosinate ammonium translocated out of the treated leaf (data not shown). Other researchers have also
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Fig. 4. Percentage of recovered 14 C found in the treated leaf of wild oat plants treated with [14 C]glufosinate ammonium applied as a spray. Plants were grown continuously at 40% RH ( ) except for half the plants which were exposed to 99% RH for 30 min before and after herbicide application (d). Letters denote statistically significant differences between treatments at a harvest time as determined by ANOVA-Tukey’s test (a ¼ 0:05).
shown that while there are some species-dependent differences, glufosinate ammonium tends not to translocate to a great extent out of treated leaves [20,21,26,27]. Although no prior reports of increased herbicide efficacy in response to short (1 h or less) exposure to high RH exist, an RHdependent increase in herbicide uptake is the most logical explanation for increased efficacy of glufosinate ammonium on wild oat observed in dose–response experiments. This lack of agreement in data from the dose–response (Experiment 1) and 14 C uptake (Experiment 3) experiments and several studies in the literature indicates a serious flaw in methodology that may subsequently result in the overestimation of herbicide uptake. This problem led to the hypothesis that applying [14 C]glufosinate ammonium as 1-ll droplets may mask differences in uptake observed following short exposures to high RH. When [14 C]glufosinate ammonium was applied as a spray (Experiment 4), there were significant differences in herbicide uptake at high versus low RH conditions (Fig. 4), in contrast to the results obtained with application of [14 C]glufosinate ammonium as droplets (Fig. 3). Uptake of [14 C]glufosinate ammonium was fourfold higher in plants exposed to 99% RH for 30 min before and after treatment than those exposed continuously to 40% RH. These results agree with our dose–response data obtained using the same RH regimes and with previously published studies by other investigators (using longer exposures to high RH)
that reported increased herbicide uptake in response to high RH [7,10–12,24,25]. While the average recovery of [14 C]glufosinate in these experiments was low, it was consistent (36% 0:71). When herbicide recovery in all low RH treatments was compared with the corresponding recoveries for high RH treatments at each harvest time, there were no significant differences observed indicating that while recovery was low, it was uniform across treatments. The difference in herbicide uptake observed between experiments using 1-ll droplets or spray application of [14 C]glufosinate ammonium is likely related to differences in drying time between large and small droplets. For every tenfold decrease in droplet volume, the surface area of an equivalent volume (composed of 10 smaller droplets) increases by 2.15 times. As the area of a liquid exposed to air increases, so too will the rate of evaporation, with a few large droplets taking significantly longer to dry than many small ones. Thus, at any RH, small herbicide droplets from spray application will dry faster than 1-ll droplets. Therefore, increases in herbicide absorption that occur under high RH regimes may be a result of slower droplet drying [16]. In addition, herbicide uptake from dried/crystalline deposits has been reported to be limited [15], suggesting that the longer the herbicide droplet remains aqueous the greater herbicide absorption will be. This may be particularly true in the case of glufosinate ammonium, which was observed to have very
Fig. 3. Percentage of recovered 14 C found in the treated leaf of wild oat plants treated with [14 C]glufosinate ammonium applied as ten 1-ll droplets. Plants were grown continuously at 40% RH ( ) except for half the plants which were exposed to 99% RH 1 h before and after herbicide application (d). There were no significant differences between RH regimes within harvest times as determined by ANOVA (a ¼ 0:05).
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rapid initial uptake into leaves of Xanthium strumarium, Ipomoea purpurea, and Commelina difusa followed by little or no absorption 1 h after application, when the herbicide droplets had presumably dried [28]. It has been reported that the activity of polar, water-soluble herbicides is more sensitive to RH than lipophilic herbicides and that an extended droplet drying time (caused by high RH) may have a greater effect on the uptake of herbicides that do not quickly partition into the lipophilic constituents of the cuticle [17]. The reduced uptake of [14 C]glufosinate ammonium into the leaves of wild oat at low RH observed in this study is likely a result of rapid drying of droplets to solid herbicide deposits which cannot penetrate into the cuticle. At high RH, the droplets will remain aqueous for a longer period, allowing significantly more [14 C]glufosinate ammonium to enter the plant. The fact that these differences in uptake were not observed when [14 C]glufosinate ammonium was applied as 1-ll droplets may be related to the drying time for such a large droplet, which was long enough to allow optimal herbicide uptake at either low or high RH. At present, even with good coverage of glufosinate ammonium on wild oat plants treated at the 3–4 leaf stage using a flat fan nozzle, efficacy may be poor if the RH is low (40% RH). Most likely, the uptake of glufosinate into leaves occurs very rapidly when the droplets are aqueous and little or no uptake occurs once the droplets have dried, a process that will be hastened during conditions of low RH. This suggests that some instances of poor wild oat control using glufosinate ammonium in western Canada may well be due to application during conditions of low RH. Using experiments with [14 C]glufosinate ammonium applied as a spray, differences in uptake under high and low RH were observed. These differences were not seen in 14 C uptake experiments using conventional application of the herbicide solution with a micropipette, indicating that application [14 C]glufosinate ammonium as large droplets can result in an overestimation of glufosinate ammonium uptake. In the future, we will examine the effect of droplet drying time on glufosinate ammonium uptake in wild oat using smaller scale studies with isolated cuticles.
Acknowledgments We thank the Natural Sciences and Engineering Research Council (NSERC) of Canada for
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providing an NSERC PGS A scholarship to Ryan J.L. Ramsey as well as NSERC and Aventis CropScience Company for providing funding to J. Christopher Hall for this research. We thank Aventis for the gift of formulated glufosinate ammonium, formulation blank, and radiolabeled glufosinate.
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[21] J.R. Maschhoff, S.E. Hart, J.L. Baldwin, Effect of ammonium sulfate on the efficacy, absorption, and translocation of glufosinate, Weed Science 48 (2000) 2–6. [22] D.R. Hoagland, D.I. Arnon, The water culture method for growing plants without soil, California Agriculture Experiment Station Circular 346 (1950). [23] D.L. Bucholtz, F.D. Hess, An atomizer for application of very low volumes of herbicide solutions, Weed Science 36 (1988) 406–409. [24] R.G. Lym, Absorption and translocation of foliarapplied sulfometuron in leafy spurge (Euphorbia esula), Weed Science 40 (1992) 477–481. [25] T.M. Sterling, N.K. Lownds, Picloram absorption by broom snakeweed (Gutierrezia sarothrae) leaf tissue, Weed Science 40 (1992) 390–394. [26] J.N. Beriault, G.P. Horsman, M.D. Devine, Phloem transport of D ,L -glufosinate and acetyl-L -glufosinate in glufosinate-resistant and -susceptible Brassica napus, Plant Physiology 121 (1999) 619– 627. [27] R.E. Hoagland, in: S.J.C. Horace, G. Cutler (Eds.), Biochemical Interactions of the Microbial Phytotoxin Phopsphinothricin and Analogs with Plants and Microbes, CRC Press, Boca Raton, FL, 1999, p. 299. [28] F. Skora Neto, H.D. Coble, F.T. Corbin, Absorption, translocation and metabolism of [14 C]glufosinate in Xanthium strumarium, Commelina difusa and Ipomoea purpurea, Weed Science 48 (2000) 171–175.
Effect of relative humidity on the uptake, translocation, and efficacy of glufosinate ammonium in wild oat (Avena fatua) R.J.L. Ramsey, G.R. Stephenson, and J.C. Hall* Department of Environmental Biology, University of Guelph, Guelph, Ont., Canada N1G 2W1 Received 18 October 2001; accepted 18 April 2002
Abstract Relative humidity (RH) can greatly affect the uptake and efficacy of glufosinate ammonium (D ,L -homoalanine-4yl-(methyl)-phosphinate) in wild oat (Avena fatua L.). Exposure to high (>95%) RH as opposed to low (40%) RH increased glufosinate ammonium efficacy and it was high RH within 12 h of spraying that was most crucial in affecting the efficacy. Subsequent dose–response experiments at 40% RH indicated that exposure of wild oat to high RH for as little as 30 min before and after spraying significantly increased the efficacy when compared to plants grown continuously at 40% RH. Furthermore, when [14 C]glufosinate ammonium was applied as a spray to wild oat plants grown at 40% RH, there was a significant increase in uptake into wild oat plants exposed to high RH for 30 min before and after treatment compared to those left continuously at low RH. Conversely, when the same experiment was conducted with [14 C]glufosinate ammonium applied as ten 1-ll droplets, exposure to high RH for 30 min before and after treatment did not increase the herbicide uptake. Based on these results, we hypothesize that droplet size and hence drying time is the major factor in effecting changes in glufosinate ammonium uptake in response to exposure to high versus low RH. Applying [14 C]glufosinate ammonium as large droplets resulted in an overestimation of uptake and masked the inhibitory effect of low RH on glufosinate ammonium as determined by dose–response experiments. Conversely, spray application of [14 C]glufosinate ammonium produced data that correlated with those from dose–response experiments. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction Wild oat (Avena fatua L.) is a weed that poses a serious problem to cereal and canola crops in western Canada [1]. Among the herbicides registered for control of wild oat is glufosinate
*
Corresponding author. Tel.: +519-824-4120x8598. E-mail address: [email protected] (J.C. Hall).
ammonium (D ,L -homoalanine-4yl-(methyl)-phosphinate), sold under the trade names of Liberty and Ignite. Glufosinate ammonium is a post-emergent herbicide with a broad spectrum of activity. An inhibitor of glutamine synthetase, glufosinate ammonium competes with glutamate for the active site and once bound becomes phosphorylated, irreversibly inhibiting the enzyme [2,3]. Inhibition of glutamine synthetase results in a rapid accumulation of ammonia and inhibition of photosynthesis through depression of photorespiration [2,4,5].
0048-3575/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 4 8 - 3 5 7 5 ( 0 2 ) 0 0 0 1 7 - 2
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Despite the susceptibility of wild oat to glufosinate ammonium, poor control can occur when relative humidity (RH) is low at the time of spraying. Low RH has been correlated with reduced efficacy of many herbicides [6–10], including glufosinate ammonium [11–14]. The mechanism through which RH influences the efficacy of foliarapplied herbicides is not completely understood, but some researchers have suggested that it could involve interactions between the herbicide droplet, leaf cuticle, and availability of water in or around droplets [15,16]. It has also been reported that the activity of polar, water-soluble herbicides is particularly sensitive to changes in RH [17]. In western Canada, RH can change by 40–50% within a few hours (i.e., high RH at night, low RH during the day). These conditions provide several possible scenarios to explain the poor efficacy of glufosinate ammonium. Early hypotheses developed in our laboratory involved the premise that optimal herbicide uptake would result in rapid, localized tissue damage, reducing loading into the phloem and subsequent translocation of glufosinate ammonium, causing poor efficacy. While self-limitation of herbicide translocation has been reported for glyphosate and chlorsulfuron [18], similar findings for glufosinate have not been reported. Glufosinate ammonium has some phloem and xylem mobility [19]; however, it does not tend to translocate to a major degree out of the leaves of treated weeds [12,20,21]. Furthermore, the RH in western Canada tends to be low, offering few instances where optimal herbicide uptake would be expected. In these studies, the effects of high and low RH on the uptake, translocation, and efficacy of glufosinate ammonium in wild oat were examined through dose–response experiments and uptake and translocation studies using [14 C]glufosinate ammonium conducted at various RH regimes. Through the course of our investigations, the following hypotheses were developed with regard to glufosinate ammonium uptake, translocation, and efficacy in wild oat: 1. Short exposure to high RH within hours of spraying will increase uptake and efficacy as much as exposure to high RH for longer periods (days). 2. Exposure to high RH after spraying is more important to increase efficacy than is high RH before spraying The objectives of our research were twofold: to further elucidate the interaction between RH and glufosinate ammonium efficacy in wild oat in terms of uptake, translocation, and efficacy and to
determine why preliminary 14 C uptake data as affected by RH did not correlate with dose– response data.
2. Materials and methods 2.1. Growth of plants Wild oat seeds were germinated in Promix (Plant Products, Brampton, Ont.) and transplanted to 8-cm square pots filled with crushed particulate expanded baked clay (‘‘Turface,’’ Applied Industrial Materials, Deerfield, IL). Plants were watered every second day, alternating between tap water and half-strength Hoagland’s solution [22]. The growing conditions were 16-h photoperiod with a light intensity of 450 lEm2 s1 and 25/15 °C day/night temperatures. Prior to exposure to different RH regimes, plants were grown in a controlled environment growth room (70% RH). High humidity treatments (>95% RH) consisted of placing plants inside Plexiglas chambers (111 46 51 cm), each of which contained an ultrasonic mist humidifier (Sunbeam Household Products, Boca Raton, FL, USA). Low humidity treatments were provided by placing plants in a growth cabinet set for 40% RH. Relative humidity and temperature were continuously monitored with a solid state RH probe (Cole-Parmer, Vernon Hills IL, USA). 2.2. Dose–response experiments Herbicide treatments were applied with an automatic hood sprayer (RC-5000-100EP Mandel Scientific, Guelph, Ont., Canada) using a flat fan nozzle (SS80015E Spraying Systems, Wheaton, IL, USA). The spray volume used was equivalent to 110 l/ha of spray solution delivered at 200 kPa. Wild oat plants were at the 3-leaf stage of development at the time of herbicide application. At harvest, shoots were removed and dried at 60 °C for 3 days and weighed. All experiments were repeated at least once. Preliminary results suggested that the RH 12 h before and after spraying had a greater impact on efficacy than the RH 6 days before and after spraying. It was also determined that high RH was always associated with increased efficacy and that low RH was always associated with poor efficacy. With this in mind, subsequent dose–response experiments focused on the effect short (12 h or less)
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exposure to 99% RH, before and after spraying, had on the efficacy of glufosinate ammonium applied to wild oat plants grown at 40% RH. In Experiment 1, plants were exposed to: (1) 40% RH for 6 days, prior to exposure to 99% RH for 3, 6 or 12 h before and after treatment or (2) left at 40% RH until treatment. Once all plants that completed their respective treatment regimes were treated with glufosinate ammonium, they were returned to 40% RH and left at this RH for 3 days after which they were returned to 70% RH until harvest (Table 1). Experiment 1 was repeated several times using shorter exposures to high RH before and after herbicide treatment (i.e., 180, 120, 60, 40, 30, and 20 min). The exact treatment regimes are outlined in Table 1. Experiment 2 was designed to determine if exposure to high RH before or after herbicide treatment was responsible for the increase in efficacy associated with high RH as was observed in Experiment 1. As in Experiment 1, wild oat plants
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were grown at 40% RH for 6 days and separated into groups that were exposed to 40 min of high RH: (1) before and after, (2) only before or (3) only after herbicide application. Plants exposed to high RH only after spraying remained at low RH until herbicide application while those plants exposed to high RH only before spraying were returned to 40% RH immediately after spraying. As with Experiment 1, plants were returned from 40% RH to 70% RH 3 days after herbicide treatment. The exact treatment regimes are outlined in Table 1. 2.3. Uptake and translocation experiments Radiolabeled glufosinate ammonium was produced using methods similar to those of Mersey et al. [12]. D ,L -Homoalanine-4-yl-(methyl)-phosphinate [3,4] (14 C labeled) was incubated in excess NH4 OH for 30 min to yield [14 C]glufosinate ammonium (873.65 MBq/g). Water and NH4 OH were
Table 1 Relative humidity regimes for dose–response and uptake/translocation experiments Experiment
#1
Time course RH pre-treatment RH before (6 days) spraying
Spray treatments
RH after spraying
RH post-treatment until harvest
Harvest times
40%
Ignite 0, 100, 200, 400 g ai/ha
40%, 99%, 99%, 99%, 40%, 99%, 99%, 99%, 40%, 99%, 99%, 99%, 40%, 99%,
40%, 3 days then 70% until harvest
14 DAT
40%, 3 days then 70% until harvest
14 DAT
40%, 3 days then 70% until harvest
14 DAT
40%, 3 days then 70% until harvest
14 DAT
40%
40%
40%
40%, 99%, 99%, 99%, 40%, 99%, 99%, 99%, 40%, 99%, 99%, 99%, 40%, 99%,
3h 6h 12 h 1h 2h 3h 20 min 40 min 60 min 30 min
Ignite 0, 100,200, 400 g ai/ha Ignite 0, 100, 200, 400 g ai/ha Ignite 0, 100, 200, 400 g ai/ha
3h 6h 12 h 1h 2h 3h 20 min 40 min 60 min 30 min
#2
40%
40%, 40%, 99%, 40 min 99%, 40 min
Ignite 0, 100, 200, 400 g ai/ha
40%, 99%, 40 min 40%, 99%, 40 min
40%, 3 days then 70% until harvest
14 DAT
#3
40% 40% 40% 40%
40% 99%, 1 h 40% 99%, 12 h
[14 C]Ignite (400 g ai/ha) [14 C]Ignite (400 g ai/ha)
40% 99%, 1 h 40% 99%, 12 h
40% 40% 40% 40%
1, 2, 4, 8 h 1, 2, 4, 8 h 1, 2, 4, 72 h l, 2, 4, 72 h
#4
Six days prior to treatment, all plants were placed at 40% RH (i.e., 6 days RH pre-treatment) until several hours before application of glufosinate ammonium. * DAT, days after treatment.
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evaporated under a stream of nitrogen gas and [14 C]glufosinate ammonium re-dissolved in ‘‘Ignite’’ formulation blank. Plants were treated at the 3-leaf stage with approximately 300,000 dpm in 10 ll (0.00289 mol/L) of [14 C]glufosinate ammonium. The 14 C-labeled herbicide solution was applied using 10 ll Wiretrol micropipettes to place ten 1-ll droplets midway up the second leaf. To achieve a herbicide concentration in the dosing solution that would simulate a field dose of 400 g ai/ha in 110 l/ha water (0.0183 mol/L), a calculated amount of unlabelled glufosinate ammonium was added from an aqueous, unformulated 600 g ai/L stock provided by Aventis CropScience. There were six replicates per treatment and all experiments were repeated at least once. Experiment 3 was designed to examine the effect of short exposure to high RH on the uptake of [14 C]glufosinate ammonium into leaves of wild oat plants. Plants were exposed to low RH for 6 days before treatment with [14 C]glufosinate ammonium with half the plants exposed to high RH for 1 h before and after treatment (Table 1). [14 C]Glufosinate ammonium was applied as ten 1-ll droplets. Plants were harvested 1, 2, 4, and 8 h after treatment. The treated leaf was removed and rinsed with an aqueous 20% ethanol, 0.5% Tween 20 (a polyoxyethylene based nonionic detergent) solution. Remaining plant parts were not used because preliminary experiments indicated that little or no herbicide translocates out of the treated leaf 8 h after application of [14 C]glufosinate ammonium. The results of preliminary experiments and Experiment 3 indicated that the method used to apply [14 C]glufosinate ammonium may affect the uptake of the herbicide. This hypothesis was tested by applying [14 C]glufosinate ammonium to the leaf as a fine spray, as opposed to several large 1ll droplets as is commonly done to evaluate 14 Clabeled herbicide uptake. In Experiment 4, [14 C]glufosinate ammonium was applied as a 10 ll spray, completely covering the adaxial side of the second leaf. This was accomplished through the use of a hand-held sprayer calibrated to deliver a low volume (10 ll) of radiolabelled herbicide solution to the leaf. Briefly, a 10 ll Wiretrol micropipette was inserted into a device so that the end of the micropipette was directly in front of an air stream. Pushing the plunger forced the treatment solution to enter the air stream, forming a fine spray. Construction of a similar device has been previously described [23]. As with the device constructed by Bucholtz and Hess [23], our
sprayer produced droplets that were not significantly different in size than those produced by the 80015 nozzle used in dose–response experiments. While 1-ll droplets have a diameter of about 1500 lm, the droplets produced by our sprayer and an 80015 nozzle were in the range of 400– 500 lm in diameter. Plants were exposed to low RH for 6 days before treatment with half being exposed to 30 min of high RH before and after 14 C-labeled herbicide application (Table 1). As in Experiment 3, the treated leaves were removed at 1, 2, 4, and 72 h after treatment and rinsed as described above. In Experiments 3 and 4, all harvested plant parts were dried at 60 °C for 3 days. Dried plant parts were oxidized in a biological oxidizer (OX 300, R.J. Harvey Instrument, Hillsdale, NJ), in which liberated 14 CO2 was trapped in a scintillation cocktail (R.J. Harvey Instrument) and quantified by liquid scintillation spectroscopy (LSS). The efficiency of combustion and recovery was greater than 95%. Leaf rinses were collected and 14 C content was quantified by LSS. Uptake data are expressed as a percentage of recovered 14 C. Data for all experiments were analyzed using SigmaStat 2.0 (Jandel Scientific, San Rafael, CA) using one-way ANOVA followed by Tukey’s multiple comparison test if significant differences were detected. Comparisons were restricted to data within a harvest interval or herbicide dose.
3. Results and discussion Preliminary experiments established that high RH was associated with increased uptake and efficacy of glufosinate ammonium, while low RH was associated with poor efficacy and uptake. These findings are similar to those reported by other researchers examining the uptake of glufosinate ammonium or other herbicides at high and low RH [7,10–12,24,25]. In the first part of Experiment 1, wild oat plants grown at 40% RH were exposed to high RH (>95%) for 0, 3, 6 or 12 h before and after spraying (Table 1). Dry weight analysis indicated that all three exposures to high RH produced a significant increase in glufosinate ammonium efficacy compared to a continuous regime of 40% RH (Fig. 1). Furthermore, the efficacy of glufosinate ammonium, when plants were exposed to short durations of high RH, was the same as when plants were grown continuously at high RH (>95%). Further modifications of Experiment 1 established that at the
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Fig. 1. Influence of RH regimes on dry weight of wild oat treated with glufosinate ammonium. RH regimes were: continuous 40% RH (d) or continuous 40% RH except for 99% RH 3 ( ), 6 (.), or 12 (r) h before and after application of glufosinate ammonium. Letters denote statistically significant differences between treatments within harvest times as determined by ANOVATukey’s test (a ¼ 0:05).
Fig. 2. Influence of RH regimes on dry weight of wild oat treated with glufosinate ammonium. RH regimes were: continuous exposure to 40% RH (d) or continuous 40% RH except for exposure to 99% RH for 40 min before (.), after (r), or before and after ( ) spraying with glufosinate ammonium. Letters denote statistically significant differences between treatments within harvest times as determined by ANOVA-Tukey’s test (a ¼ 0:05).
doses tested, even 30 min of exposure to high RH before and after treatment produced an increase in efficacy similar to that associated with 12 h of high RH exposure before and after treatment. This is the first instance where such short exposures to high RH have been reported to significantly increase herbicide efficacy. In Experiment 2, it was found that high RH after, but not before spraying, was required to improve glufosinate ammonium efficacy. In fact, 40 min of exposure to high RH after spraying resulted in increased efficacy equal to that produced by exposing the plants to high RH for 40 min both before and after treatment (Fig. 2). These results suggest that the increase in efficacy observed is a result of events that occur after herbicide droplets come into contact with the leaf surface. We speculate that exposure to high RH after spraying may increase the droplet drying time thereby allowing a longer time or ‘‘window’’ for the herbicide to penetrate the cuticle. In Experiment 1, it was found that exposure to 99% RH for 30 min before and after treatment was sufficient to increase glufosinate ammonium efficacy on wild oat. To examine the effect of similar conditions on the uptake of [14 C]glufosinate ammonium, Experiment 3 was performed, exposing wild oat plants grown at 40% RH to either 99% RH for 1 h before and after treatment or a continuous 40% RH regime. [14 C]Glufosinate ammonium was applied as ten 1-ll droplets and short harvest times were used to examine the effect
of high RH on the uptake of glufosinate ammonium. It was observed that there was no difference in uptake of [14 C]glufosinate ammonium between plants exposed to 99% RH 1 h before and after application versus those left at 40% RH (Fig. 3), even though exposure to 1 h of 99% RH before and after spraying caused an increase in efficacy as measured by dry weights (i.e., Experiment 1; dose–response experiment). In a study to determine the effect of RH on the uptake and efficacy of glufosinate ammonium in several amaranth species, Coetzer et al. [14] observed a similar disagreement between the results from [14 C]glufosinate ammonium uptake experiments and dose– response experiments. They found that continuous exposure to 90% RH resulted in a significant increase in glufosinate ammonium efficacy as measured by dose–response, but there were no differences in glufosinate ammonium uptake. They proposed that increased translocation of glufosinate ammonium in plants exposed to continuous 90% RH may be the mechanism through which increased efficacy was achieved [14]. While [14 C]glufosinate ammonium translocation patterns may be altered by long exposures to high RH, it seems unlikely that the short duration exposures to high RH used in Experiment 3 would change translocation sufficiently to increase herbicide efficacy. Furthermore, preliminary experiments suggested that little or no glufosinate ammonium translocated out of the treated leaf (data not shown). Other researchers have also
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Fig. 4. Percentage of recovered 14 C found in the treated leaf of wild oat plants treated with [14 C]glufosinate ammonium applied as a spray. Plants were grown continuously at 40% RH ( ) except for half the plants which were exposed to 99% RH for 30 min before and after herbicide application (d). Letters denote statistically significant differences between treatments at a harvest time as determined by ANOVA-Tukey’s test (a ¼ 0:05).
shown that while there are some species-dependent differences, glufosinate ammonium tends not to translocate to a great extent out of treated leaves [20,21,26,27]. Although no prior reports of increased herbicide efficacy in response to short (1 h or less) exposure to high RH exist, an RHdependent increase in herbicide uptake is the most logical explanation for increased efficacy of glufosinate ammonium on wild oat observed in dose–response experiments. This lack of agreement in data from the dose–response (Experiment 1) and 14 C uptake (Experiment 3) experiments and several studies in the literature indicates a serious flaw in methodology that may subsequently result in the overestimation of herbicide uptake. This problem led to the hypothesis that applying [14 C]glufosinate ammonium as 1-ll droplets may mask differences in uptake observed following short exposures to high RH. When [14 C]glufosinate ammonium was applied as a spray (Experiment 4), there were significant differences in herbicide uptake at high versus low RH conditions (Fig. 4), in contrast to the results obtained with application of [14 C]glufosinate ammonium as droplets (Fig. 3). Uptake of [14 C]glufosinate ammonium was fourfold higher in plants exposed to 99% RH for 30 min before and after treatment than those exposed continuously to 40% RH. These results agree with our dose–response data obtained using the same RH regimes and with previously published studies by other investigators (using longer exposures to high RH)
that reported increased herbicide uptake in response to high RH [7,10–12,24,25]. While the average recovery of [14 C]glufosinate in these experiments was low, it was consistent (36% 0:71). When herbicide recovery in all low RH treatments was compared with the corresponding recoveries for high RH treatments at each harvest time, there were no significant differences observed indicating that while recovery was low, it was uniform across treatments. The difference in herbicide uptake observed between experiments using 1-ll droplets or spray application of [14 C]glufosinate ammonium is likely related to differences in drying time between large and small droplets. For every tenfold decrease in droplet volume, the surface area of an equivalent volume (composed of 10 smaller droplets) increases by 2.15 times. As the area of a liquid exposed to air increases, so too will the rate of evaporation, with a few large droplets taking significantly longer to dry than many small ones. Thus, at any RH, small herbicide droplets from spray application will dry faster than 1-ll droplets. Therefore, increases in herbicide absorption that occur under high RH regimes may be a result of slower droplet drying [16]. In addition, herbicide uptake from dried/crystalline deposits has been reported to be limited [15], suggesting that the longer the herbicide droplet remains aqueous the greater herbicide absorption will be. This may be particularly true in the case of glufosinate ammonium, which was observed to have very
Fig. 3. Percentage of recovered 14 C found in the treated leaf of wild oat plants treated with [14 C]glufosinate ammonium applied as ten 1-ll droplets. Plants were grown continuously at 40% RH ( ) except for half the plants which were exposed to 99% RH 1 h before and after herbicide application (d). There were no significant differences between RH regimes within harvest times as determined by ANOVA (a ¼ 0:05).
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rapid initial uptake into leaves of Xanthium strumarium, Ipomoea purpurea, and Commelina difusa followed by little or no absorption 1 h after application, when the herbicide droplets had presumably dried [28]. It has been reported that the activity of polar, water-soluble herbicides is more sensitive to RH than lipophilic herbicides and that an extended droplet drying time (caused by high RH) may have a greater effect on the uptake of herbicides that do not quickly partition into the lipophilic constituents of the cuticle [17]. The reduced uptake of [14 C]glufosinate ammonium into the leaves of wild oat at low RH observed in this study is likely a result of rapid drying of droplets to solid herbicide deposits which cannot penetrate into the cuticle. At high RH, the droplets will remain aqueous for a longer period, allowing significantly more [14 C]glufosinate ammonium to enter the plant. The fact that these differences in uptake were not observed when [14 C]glufosinate ammonium was applied as 1-ll droplets may be related to the drying time for such a large droplet, which was long enough to allow optimal herbicide uptake at either low or high RH. At present, even with good coverage of glufosinate ammonium on wild oat plants treated at the 3–4 leaf stage using a flat fan nozzle, efficacy may be poor if the RH is low (40% RH). Most likely, the uptake of glufosinate into leaves occurs very rapidly when the droplets are aqueous and little or no uptake occurs once the droplets have dried, a process that will be hastened during conditions of low RH. This suggests that some instances of poor wild oat control using glufosinate ammonium in western Canada may well be due to application during conditions of low RH. Using experiments with [14 C]glufosinate ammonium applied as a spray, differences in uptake under high and low RH were observed. These differences were not seen in 14 C uptake experiments using conventional application of the herbicide solution with a micropipette, indicating that application [14 C]glufosinate ammonium as large droplets can result in an overestimation of glufosinate ammonium uptake. In the future, we will examine the effect of droplet drying time on glufosinate ammonium uptake in wild oat using smaller scale studies with isolated cuticles.
Acknowledgments We thank the Natural Sciences and Engineering Research Council (NSERC) of Canada for
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providing an NSERC PGS A scholarship to Ryan J.L. Ramsey as well as NSERC and Aventis CropScience Company for providing funding to J. Christopher Hall for this research. We thank Aventis for the gift of formulated glufosinate ammonium, formulation blank, and radiolabeled glufosinate.
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