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Use of Uptake Spraying Oil to Improve Fungicidal Activity of the Triazole Fungicide Fenbuconazole on Puccinia triticina and Puccinia striiformis Rusts of Wheat
Gerrit J. deBoer, Dow AgroSciences Discovery Research, 9330 Zionsville Road, Indianapolis, IN 46268 USA; Peter Nott, Dow AgroSciences Australia Ltd., 20 Rodborough Road, Frenchs Forest, New South Wales 2086 Australia; and Gregory M. Kemmitt, Dow AgroSciences, European Development Centre, 3 Milton Park, Abingdon, Oxfordshire, OX144RN, UK
deBoer, G. J., Nott, P., and Kemmitt, G. M. 2013. Use of Uptake Spraying Oil to improve fungicidal activity of the triazole fungicide fenbuconazole on Puccinia triticina and Puccinia striiformis rusts of wheat. Online. Plant Health Progress doi:10.1094/PHP-2013-0528-01-RS.
Fenbuconazole, a broad spectrum triazole fungicide with restricted uptake and consequent redistribution within wheat plants, has been perceived to possess limited curative properties when formulated as a suspension concentrate (SC). Field and greenhouse studies demonstrated that curative and to a lesser extent protectant activity of fenbuconazole on wheat leaf rust (Puccinia triticina) and stripe rust (Puccinia striiformis f.sp. tritici) was improved by the addition of the adjuvant Uptake Spraying Oil (Uptake is a trademark of Dow AgroSciences). The purpose of this study was to determine if the addition of adjuvant increased penetration of fenbuconazole into wheat tissue and if this was correlated with enhanced redistribution within the plant and the observed increase in activity. These data confirm that the addition of adjuvant improved penetration of fenbuconazole into wheat leaves and was responsible for the observed increase in biological activity of the fenbuconazole suspension concentrate formulation. In addition, adjuvant decreased the amount of material washed off with water a result suggesting the potential for improved rain-fastness in the field. Fenbuconazoles fungicidal activity against cereal rusts could be limited by poor penetration into plant tissue unless a suitable adjuvant is incorporated into the spray solution.
Fenbuconazole is a broad spectrum triazole fungicide developed by Rohm and Haas in the late 1980s (1,2). It was first introduced into Europe in 1992 for management of Septoria species and rusts on cereals. Today fenbuconazole is mainly used in the pome fruit, stone fruit, grapevine, and citrus markets for management of a range of key diseases. When formulated as a suspension concentrate (SC) fenbuconazole shows limited local penetration and consequently relatively poor redistribution and movement within the plant apoplast (3,4), which is unusual for a triazole. Restricted uptake and movement within the plant has resulted in a perception that fenbuconazole lacks curative properties. In fact, fenbuconazole (like other triazole fungicides) is highly active against fungal mycelium (unpublished data) and this suggests the lack of curative efficacy maybe a function of poor penetration and movement within the host plant and/or poor penetration into the target fungus (1).
Uptake Spraying Oil is sold by Dow AgroSciences (DAS) in Australia as an adjuvant for use with cereal and grass herbicides (5). The product is a blend of paraffin oil, alkoxylated alcohol, and non-ionic surfactants and is used to improve spreading and wetting on leaf surfaces as well as an aid to leaf penetration (6). When applying an active ingredient to a plant to achieve biological control, Wirth et al. (7) suggested five important factors that are affected by an adjuvant. These consisted of formulation, atomization, trajectory, retention, and finally penetration. The final factor, penetration, which occurs directly on the leaf surface, is exceedingly complex involving spray retention, spreading, and coverage. Even the type of deposit that is formed can influence penetration of the pesticide (8,9,10). In addition to the physical effects that allow for more active ingredient to be in contact with the leaf surface, there are the adjuvant effects of surfactants that enhance transcuticular diffusion as well as permeability of the plasma membrane and oils that actually increase active ingredient penetration (11,12,13,14). Most initial research on adjuvants was directed to herbicidal uptake due to the development of phloem translocated herbicides that need improved penetration to increase efficacy (15). However, with the advent of systemic fungicides the need for increased penetration spurred fungicidal adjuvant research (16,17).
Previous exploratory studies indicated that curative, and to a lesser extent protectant, activity of fenbuconazole on wheat brown rust (Puccinia triticina) and stripe rust (Puccinia striiformis f.sp. tritici) could be improved by the addition of Uptake Spraying Oil (unpublished). The purpose of this study was to determine if the addition of Uptake as a tank-mixture in foliar applications of fenbuconazole to wheat (Triticum aestivum) plants increased penetration of fenbuconazole into wheat tissue and if this was correlated with enhanced redistribution within the plant and subsequent observation of improved rust control.
We also examined the metabolic stability of fenbuconazole in wheat. Activity could be limited by a rapid rate of metabolism which has previously been observed to occur in animals, plants and soil (18,19). Although more fenbuconazole might be moved into the plant tissue with the aid of an adjuvant, rapid metabolism could prevent accumulation of higher leaf titers of the fungicide.
14C Uptake into Wheat Leaves
A 0.5 ml 14C-fenbuconazole (Fig. 1) suspension concentrate formulation (SC) (10% active ingredient) was formulated from 450 mg of blank formulation (without active ingredient), 48 mg of non-radioactive fenbuconazole, and 2 mg of radioactive fenbuconazole. The 14C-fenbuconazole SC formulation was diluted with water to 830 mg/ml (62.5 g a.i./ha in a typical spray volume of 75 liters/ha). A 2-µl drop line of the suspension was applied horizontally 6 cm from the tip of the first leaf blade of wheat plants at the two true leaf stage. Applications with the added adjuvant involved mixing 0.5% vol/vol (v/v) of adjuvant (Uptake Spraying Oil) in water with the SC formulation. At 0, 1, 3, and 6 days after application, the treated leaf was excised 2 cm below the application point and washed with three solvents for 10 seconds each. Leaves were washed first with water to mimic rain, second with 20% ethanol to remove surface fungicide, and third with chloroform to remove leaf cuticle. Washed leaves were oxidized to determine the concentration of 14C fenbuconazole that had entered the leaf. Four plants were treated at each time point: three for washing and oxidizing and one plant without washing for phosphor imaging.
The effect of adjuvant on disease control by fenbuconazole (Indar™, Table 1) was associated with improved fungicide penetration into the leaf (Table 2). From the phosphor images comparing 0 hour drop line application with (Fig. 2A) and without (Fig. 2E) adjuvant, it was noted that the application zone showed much greater spreading on the wheat leaf surface with adjuvant than without adjuvant. There was no further increase in spread after the initial application (Fig. 2C). The improved uptake observed in the presence of adjuvant could reflect increased surface contact and/or improved penetration. The improved absorption with adjuvant also allowed for significantly greater transport to the tip of the wheat leaf (Fig. 2C) as compared to no movement in the application without adjuvant (Fig. 2G). The penetration distribution profile of fenbuconazole in wheat leaves (Table 2) indicates that addition of an adjuvant decreased the amount of material washed off with water at all times, a result suggesting improved rain-fastness in the field. After 24 h in treatments without adjuvant, > 50% of the surface material was washed off by water as compared to < 20% with adjuvant.
Addition of adjuvant increased the quantity of material penetrating the leaf over time (Table 2). Once in the wheat leaf, xylem movement was observed, as fenbuconazole moved the entire length of the wheat leaf to the tip by 6 days (Fig. 2C), similar to triadimefon and fluoxastrobin in barley (20,21). No movement was observed below the treated section as would be expected if phloem mobility was occurring.
Table 1. Mean percent visual leaf surface free of P. triticina in mobility bioassay looking at the effect of adjuvant on efficacy of fenbuconazole (Indar) and epoxiconazole (Opus).
x Uptake at 0.5% vol/vol.
y Curative treatment applied 2 days after P. triticina application.
z Preventive treatment applied 1 days before P. triticina application.
Table 2. % penetration distribution results in wheat for 14C-fenbuconazole without and with Uptake adjuvant added.
y ± standard deviation from three replicates, means in same column followed by same letter do not significantly differ (P = 0.05) Tukey HSD.
14C Metabolism in Wheat Plants
Metabolism was determined by injecting hydroponically grown wheat at the junction of roots and stem with 0.1 µCi of technical 14C-fenbuconazole in a volume of 1 µl of acetonitrile. Plants were harvested 0, 6, 26, 72, and 144 h after application. The entire wheat plant was frozen in liquid N2 and ground to a powder with 100% methanol then extracted a second time with 80% methanol. The extraction pellet was oxidized to measure non-extractable radioactivity. Four replicated plants were taken at each harvest time so that three could be extracted for metabolic evaluation and one plant for phosphor imaging to illustrate movement and uniform coverage. The plant extracts were concentrated then resuspended in 10% acetonitrile for HPLC analysis.
No metabolism was observed in the HPLC evaluation of the wheat plant extracts; by 144 h only the parent material was observed and less than 8% was unextractable (data not presented). Fenbuconazole was very stable in wheat plants as evidenced by no metabolites observed by 6 days consistent with results discussed in the registration of fenbuconazole in Australia (22).
Greenhouse Mobility Bioassay In Wheat with a Puccinia triticina Test System
Winter wheat seeds (cv. Yuma) were sown in plastic pots in a soilless compost medium Metro Mix 360 (SunGro Horticulture, Bellevue, WA, USA) and thinned to 3 plants per pot following seedling emergence. Seedlings were held at 22°C in greenhouse until they reached the stage of the first true leaf fully extended (approximately 7 days post-seeding).
An isolate of P. triticina originally isolated from a wheat field in Indiana was maintained in the greenhouse by routine culturing on wheat seedlings (cv. Yuma) in order to provide a continuous supply of fresh urediniospores for the bioassay inoculations. Planting density was 10-12 seedlings per 7.6 × 7.6 cm pot. Plants were inoculated at the 2 true leaf stage by foliar application of a spore suspension (1 × 106 urediniospores/ml in deionized H2O) with a compressed air-assisted hand held DeVilbiss spray gun. 300 ml of spore suspension was used to evenly cover 80 pots of wheat plants. Immediately after inoculation the plants were moved into a darkened dew chamber (24°C, 95% RH) for 24 h in order to facilitate infection. The plants were then moved into the greenhouse and held at 22°C with disease symptoms typically expressing 7-8 days after inoculation.
Compounds tested included (Table 1) Indar 2F (fenbuconazole 240 g a.i./liter SC) and Uptake spray oil adjuvant which are trademarks of Dow AgroSciences, Indianapolis, Indiana, while Opus™ (epoxiconazole 250 g a.i./liter SC) is a trademark of BASF Limited, Ludwigshafen, Germany. Opus was included as a commercial standard known to have excellent redistribution properties in wheat (3). The concentrations tested were based on final spray concentration of a proposed commercial application rate of Indar 2F under Australian conditions (i.e., a rate of 62.5 g a.i./ha in a typical spray volume of 75 liter/ha and is equivalent to 830 ppm of fenbuconazole in the spray tank). All test material solutions were in deionized H2O. Uptake Spraying Oil adjuvant was added to give a final concentration of 0.5% v/v. Test materials were applied 5 cm below the leaf tip by placing a 2-µl drop of formulation on the adaxial surface of the first true leaf of the test wheat plants. Three replicate seedlings were used per treatment. Two separate tests were conducted: a curative study in which P. triticina was inoculated onto the plants 48 h before the compounds were applied; and a protectant test in which the plants were inoculated 24 h after application of the test materials to the plant tissue. After inoculation with P. triticina urediniospores, the plants were placed in a dew room for 24 h and then moved to a greenhouse with a temperature of 22°C. The disease expressed at 7-8 days after inoculation.
Plants were evaluated 8-9 days after inoculation by visually estimating the percent surface area of disease-free leaf lamina (adaxial surface) in the area extending from the site of compound application to the leaf tip.
The bioassay data reported in Table 1 illustrated that addition of Uptake spray oil to the fenbuconazole treatments had a markedly positive effect on the level of curative efficacy. In the absence of an adjuvant, fenbuconazole did not curatively control P. triticina. Protectant efficacy of fenbuconazole was also improved following addition of Uptake spray oil, although the trend was not as great as that seen in the curative study. The epoxiconazole standard gave excellent control in both protectant and curative studies irrespective of whether or not it was combined with Uptake spray oil.
Due to the design of this bioassay, in order for the test compounds to control disease they had to be redistributed away from the initial application point on the leaf. The redistribution pattern for triazoles such as fenbuconazole and epoxiconazole, which are known to move in the plants apoplast, would typically be expected to be in an acropetal direction due to redistribution via the xylem (23). As noted for the 14C transport results, the adjuvant improved penetration of material into the wheat leaf and in so doing increased the amount of fenbuconazole movement to the leaf tips.
Both fenbuconazole formulations performed well and were (P = 0.05) more active when applied with 0.5% v/v Uptake Spraying Oil, with the same treatments demonstrating brown rust control very closely aligned to that achieved by epoxiconazole.
A field trial was conducted in 2004 near Hamilton, New Zealand in order to evaluate the effect of addition of Uptake adjuvant into the spray solution on efficacy of a commercial fenbuconazole formulation (240 g a.i./liters SC) to control stripe rust, in wheat (cv. Tiritea). All fungicide treatments were applied at 125 g a.i./ha once, just prior to flag leaf emergence - GS37, and before any disease was present. Where included uptake was tested at a rate of 500 ml/100 liters. Four replicate (3 × 5 m) plots per treatment were sprayed using a compressed air-propelled, backpack-mounted, precision plot sprayer fitted with a 2-m boom, through XR110003 flat fan jet nozzles at 220 kPa, and delivering a water volume of 300 liters/ha. Opus was used as a commercial reference standard in the trial. Rust severity was assessed visually on a whole plot/plant basis at various intervals after application depending on the epidemic development.
Two more field trials were conducted near New Plymouth in New Zealand. Methods were similar to those described above with the exception that the target disease was leaf rust and fungicide treatments were applied on two occasions: the first at second node (GS 32) and the second 13 days later at approximately full flag leaf emergence (GS 39). Low levels of leaf rust (approximately 1% severity/plot) were present in trials at the first application. In addition, a suspo-emulsion (SE) formulation of fenbuconazole (GF-1582) was also included alongside the SC formulation.
In the stripe rust trial (Table 3), the epidemic was initially slow to build, with 21% severity present in the untreated area on a whole plant/plot basis 27 DAA (days after application). However, a week later this had increased to almost 70% severity and by the final assessment, 53 DAA there was > 90% severity in the untreated controls.
Table 3. Severity of stripe rust in wheat
x Means followed by same letter do not significantly differ (P = 0.05) Duncans New MRT.
x DAA = days after application.
All fungicide treatments provided very good control of stripe rust. However, at the final assessment, with the untreated at 92% severity, there were (P = 0.05) treatment differences, Opus at 32% severity was superior (P = 0.05) to the other fungicide treatments. Fenbuconazole applied with Uptake Spraying Oil (42% severity) was superior (P = 0.05) to fenbuconazole applied without Uptake Spraying Oil.
In the second trial on leaf rust (Table 4), epidemic development was rapid, with 8% severity on a whole plant basis in the untreated control, 11 days after the first application, rising to 33% severity at the second assessment, 20 days after first application (or 7 days after second application) (Table 4). Eight days later, the epidemic had reached 80% severity and at the final assessment, 36 days after the first application (or 23 days after second application), the untreated controls were essentially destroyed at 90% severity. The final column in Table 4 shows the AUDPC or area under the disease curve, which was essentially "% severity per day" over the duration of the epidemic. This allows a precise comparison between treatments over the course of the epidemic (24).
Table 4. Percent severity of leaf rust on wheat 23 days after second application.
x Means followed by same letter do not significantly differ (P = 0.05) Duncans New MRT.
y AUDPC = area under the disease progress curve.
These data confirm that penetration of fenbuconazole into wheat leaves was enhanced by addition of Uptake Spraying Oil adjuvant and correlated with an observed increase in biological activity of the SC fenbuconazole formulation. The fenbuconazole SC formulations fungicidal activity against cereal rusts is likely to be limited by poor penetration into plant tissue when a suitable adjuvant is not incorporated into the spray solution. The effect on rust efficacy was seen in both the glasshouse and the field environment, an important step when considering the possible effects of differences in cuticle morphology between greenhouse versus field-grown wheat plants, a phenomenon which did not correlate for the parameter affecting formulation drop retention (12). In the literature, adjuvants have been shown to increase biological activity of fungicides due to improved spray deposition and uptake (25,26,27,28,29). These results are consistent with studies with prothioconazole applied as an EC with and without spiroxamine, in which improved uptake into wheat leaves was observed in the presence of spiroxamine and attributed to an adjuvant effect (30). From the wheat plant phosphor images it was also apparent that improved penetration allowed more of the fenbuconazole to be transported in the leaf. The penetration and distribution data indicated that as penetration improved, the amount of fenbuconazole not washed off with water (rain fast) also increased proportionally.
Fenbuconazole was stable in wheat plants such that after 6 days 93% of the applied fenbuconazole was still present as parent material, with the only loss apparently due to incorporation of metabolites into plant constituents (31).
The authors would like to thank Todd Mathieson for technical assistance and Carla Klittich for comments to the manuscript.
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