Published online 4 April 2007
Published in Agron J 99:645-649 (2007)
DOI: 10.2134/agronj2006.0067
© 2007 American Society of Agronomy
677 S. Segoe Rd., Madison, WI 53711 USA
Soybean
Effect of Lactofen Application Timing on Yield and Isoflavone Concentration in Soybean Seed
Kelly A. Nelsona,*,
George E. Rottinghausb and
Teak E. Nelsonc
a Div. of Plant Sciences, Univ. of Missouri, Novelty, MO 63460
b Veterinary Medicine Diagnostic Lab., Univ. of Missouri, Columbia, MO 65211
c Dep. of Nursing, Truman State Univ., Kirksville, MO 63501
* Corresponding author (nelsonke{at}missouri.edu)
Received for publication March 3, 2006.
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ABSTRACT
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Postemergence application of the herbicide lactofen is known to increase phenolic compounds such as isoflavones in soybean [Glycine max (L.) Merr.] plants. We hypothesized that lactofen increases isoflavone levels in seed when applied at the R1 and R5 stages of development. Field research evaluated the effect of lactofen application timing and cultivar (‘Garst 3712’, glyphosate-resistant; ‘Big Bubba’, high-protein; and ‘Envy’, edamame) on crop response and seed isoflavone concentration. Lactofen at 70 g a.i. ha–1 injured soybean 7 to 15% when compared with the untreated control 7 d after treatment. Lactofen applied to Big Bubba at the R5 stage of development increased concentration of genistein 78 µg g–1 (11%), daidzein 82 µg g–1 (9%), and total isoflavones 169 µg g–1 (10%) compared with the nontreated control. Lactofen applied to Garst 3712 at the R5 stage of development increased daidzein concentration 52 µg g–1 (7%). There was no effect of lactofen on isoflavone concentration in Envy seed. Seed yield averaged across the three cultivars was reduced by 290 kg ha–1 when lactofen was applied at the R1 stage in 2002 and by 330 kg ha–1 when lactofen was applied at the R5 stage in 2004. Results suggest a late application of lactofen may increase isoflavone concentration in seed of high-protein soybean cultivars, but this effect may be accompanied by a reduction in seed yield.
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INTRODUCTION
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ISOFLAVONES HAVE BEEN IMPLICATED in many health-related effects. Genistein and daidzein are heterocyclic phenols with a structure similar to estrogen (Wu et al., 1996), likely explaining their correlation with breast cancer risk reduction (Ingram et al., 1997; Wu et al., 1996) and use in management of numerous perimenopausal symptoms (Atkinson et al., 2004; Messina and Hughes, 2003). Diets high in these and other isoflavones have been associated with subsequent improvement in total, low-density lipoprotein, and high-density lipoprotein cholesterol levels (Sagara et al., 2003; Wang et al., 2004); blood pressure (Sagara et al., 2003); and platelet aggregation/antithrombolytic effects (Nakashima et al., 1991; Tham et al., 1998). Consumption of isoflavone-rich foods may decrease the risk of prostate cancer development (Adlercreutz and Mazur, 1997) and slow its rate of progression due to their antioxidant properties (Wei et al., 1995).
Lactofen {(±)-2-ethoxy-1-methyl-2-oxoethyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate} is a postemergence herbicide labeled for broadleaf weed control in soybean up to 45 d before harvest or R6 stage of development (Fehr and Caviness, 1985), and for Sclerotinia stem rot [Sclerotina sclerotiorum (Lib.) de Bary] suppression when applied before the R1 stage of development (Valent, 2005). Several studies have reported increased levels of phenolic compounds in soybean plants that were in the same pathway of the phytoestrogenic isoflavones when protoporphyrinogen oxidase inhibitor herbicides such as lactofen or acifluorfen were applied postemergence (Dann et al., 1999; Kömives and Casida, 1983; Levene et al., 1998; Nelson et al., 2002a). Bioassay research indicated that lactofen increased phenolic compound levels in treated leaves up to 28 d after treatment in the field, reduced the incidence of Sclerotinia stem rot in soybean (Nelson et al., 2002a), and the effects were systemic (Nelson et al., 2002b). Landini et al. (2002) reported that lactofen, acifluorfen, and fomesafen were strong isoflavone inducers which resulted in the accumulation of daidzein, a precursor of glyceollin, and the formation of genistein in distal cells of cotyledons which was considered partially systemic (Landini et al., 2002). An increase in isoflavone levels in cotyledons treated with lactofen was the result of pathogenesis-related protein gene expression (Graham, 2005).
Isoflavone concentration in soybean seed may range from 300 to 3300 µg g–1 depending on climatic conditions (Hoeck et al., 2000; Tsukamoto et al., 1995), cultivar (Chiari et al., 2004; Eldrige and Kwolek, 1983; Hoeck et al., 2000; Lee et al., 2004; Sequin et al., 2004; Wang and Murphy, 1994; Wu et al., 2004), planting date (Tsukamoto et al., 1995), K fertilization (Vyn et al., 2002; Yin and Vyn, 2004), herbicide treatment (Duke et al., 2003), location, year (Eldrige and Kwolek, 1983; Hoeck et al., 2000; Sequin et al., 2004; Wang and Murphy, 1994), and yield level (Yin and Vyn, 2005). Cultivar isoflavone variability was affected by crop management decisions (Vyn et al., 2002) and planting dates (Tsukamoto et al., 1995), and discussion has been related to temperatures during seed fill or unexplainable climatic differences (Hoeck et al., 2000; Tsukamoto et al., 1995; Vyn et al., 2002). No research has evaluated the effect of lactofen on isoflavone concentration in soybean seed and the interaction with cultivar selection or herbicide application timing. Since there may be a systemic effect of lactofen on phenolic compound production in the plant, we hypothesized that a postemergence application of lactofen would increase isoflavone concentration in soybean seed at a timing that may reduce the incidence of disease (R1) or during early seed fill (R5) when a late herbicide application may be needed for additional weed control. The objective of this research was to determine the impact of lactofen application timing on yield and isoflavone concentration of three different cultivars.
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MATERIALS AND METHODS
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Field research was conducted at the University of Missouri Greenley Research Center near Novelty (40°01' N, 92°11' W) on a Putnam silt loam (fine, smectitic, mesic Vertic Albaqualfs) in 2002 and 2004. The site in 2003 was abandoned due to a poor stand. The soil had 29 and 26 g kg–1 organic matter and 6.0 and 6.3 pH measured in 0.01 M CaCl2 solution in 2002 and 2004, respectively (Nathan et al., 2006). Preplant soil K (NH4OAc extraction), which is known to affect isoflavone concentration (Vyn et al., 2002), was high at 450 kg ha–1 in 2002 and 370 kg ha–1 in 2004. The soil contained 71 kg ha–1 P (Bray 1 extraction), 5070 kg ha–1 Ca, and 420 kg ha–1 Mg in 2002, and 50 kg ha–1 P, 4330 kg ha–1 Ca, and 380 kg ha–1 Mg in 2004, which were in the high range (Buchholz, 1992). The field was spring disk-harrowed, cultivated two times, and mulched before planting each year.
The experiment was a randomized complete block design with a split-plot arrangement of treatments and four replications. Cultivar was the main plot and herbicide treatment timing was the subplot. Subplots were 3 by 9.1 m. Envy (edamame), Big Bubba (high-protein), and Garst 3712 (glyphosate-resistant) were planted 6 June 2002 and 24 May 2004 in 0.76-m rows at 346 000 seed ha–1. Treatments included a nontreated control and lactofen application of 70 g a.i. ha–1 plus 0.25% v/v nonionic surfactant (NIS) Agri-Dex (Helena Chemical Co., Memphis, TN) plus 2.3 L ha–1 28% urea ammonium nitrate (UAN) applied at the R1 and R5 stages of development (Fehr and Caviness, 1985). Agri-Dex is a proprietary blend of heavy range paraffin base petroleum oil polyol fatty acid esters polyethoxylated derivatives. Additive controls such as UAN or NIS were not included since these treatments had no effect on phytoallexin production in the leaves of soybean plants (Dann et al., 1999; Nelson et al., 2002a, 2002b). Plots with or without herbicide applications were maintained weed-free by cultivation and manual weeding throughout the season. Plant height and environmental conditions at the time of application are reported in Table 1. Herbicide treatments were applied with a CO2 propelled hand-boom calibrated to deliver 187 L ha–1 at 130 kPa, traveling 4.7 km h–1. The hand-boom was equipped with 8003 flat-fan nozzles (Spraying Systems Co., Wheaton, IL) spaced 0.51 m apart and 0.48 m above the canopy.
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Table 1. Application date, air temperature, relative humidity, and soybean height at the time of lactofen application in 2002 and 2004.
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Plant injury due to herbicide was evaluated 7 d after treatment based on combined visual score (0 = no visual crop injury and 100 = complete plant death) of herbicide-induced leaf necrosis, chlorosis, and stunting. Plant samples of Envy, a determinate cultivar, were taken at the R6 stage of development (Fehr and Caviness, 1985) from a 1-m section of nonharvest rows on 21 Aug. 2002 and 20 Aug. 2004. Pod number and fresh weight were recorded and seed brix determined using a portable refractometer (Sper Scientific, Scottsdale, AZ). No plant samples were taken from the other two cultivars, as these cultivars are usually not harvested for fresh pods. Seed yield of all three cultivars was determined by machine-harvesting the two center rows 8 m long at R8 (Fehr and Caviness, 1985) with a Massey 10 small plot harvester (Kincaid Equipment Manufacturing, Haven, KS). Yield was adjusted to 130 g kg–1 moisture. Envy seed were machine harvested 13 Sept. 2002 and 4 Sept. 2004, while Big Bubba and Garst 3712 seed were harvested 19 Oct. 2002 and 10 Nov. 2004.
Mature seed from each plot was collected and analyzed for genistin, daidzin, and glycitin content and converted to genistein, daidzein, and glycitein as the primary isoflavones in soybean (Duke et al., 2003; Sequin et al., 2004; Vyn et al., 2002; Wu et al., 2004). Primary standards (500 ppm) and working standards (10 ppm) of genistin, daidzin, and glycitin (LC Laboratories, Woburn, MA) were prepared in 80% methanol and stored at –20°C. Seed was ground in a Stein Mill (Steinlite, Inc., Atchison, KS), washed two times in hexane, dried, and reground. A 0.5-g ground seed sample and a standard soybean seed sample with known isoflavone concentration as a control was weighed in 50-mL polypropylene tubes with 20 mL of 80% methanol in water. Each tube was weighed and recorded to correct for loss due to evaporation during extraction. Samples were placed in a water bath for 3 d at 55°C with occasional shaking. The malonyl isoflavone derivatives were reduced to the genistin, daidzin, and glycitin moieties by the 3-d heat treatment period. Following incubation, samples were weighed and evaporative loss was corrected with 80% methanol, centrifuged at 2050 g for 5 min, and an aliquot transferred to an autosample vial for high pressure liquid chromatography (HPLC) analysis. The HPLC system consisted of a Hitachi L-7100 pump with isocratic flow at 1.0 mL min–1, Hitachi L-7200 autosampler (20-µL injection volume), and Hitachi L-7400 UV detector (detection wavelength = 260 nm) with a Hypersil 5 µm, C18 (BDS) 250 by 4.6 mm reversed-phase analytical column (Phenomenex) fitted with a SecurityGuard C18 (ODS) 4.0- by 3.0-mm guard column (Phenomenex). The mobile phase was water:0.1% acetic acid (solvent A) and acetonitrile:0.1% acetic acid (solvent B) with a gradient from 93% A:7% B to 80% A and 20% B over 28 min, 50% A and 50% B over 2 min, and reequilibrated to 93% A and 7% B. Data were recorded and processed by a Hitachi D-7000 data acquisition package with ConcertChrom software on a microcomputer. All values for genistin, daidzin, and glycitin were converted to genistein, daidzein, and glycitein equivalents, respectively. For example, the conversion of genistin to genistein was calculated as: concentration of genistin x (molecular weight of genistein/molecular weight of genistin). The conversion factors for genistin, daidzin, and glycitin to genistein, daidzein, and glycitein were 0.625, 0.611, and 0.637, respectively. Total isoflavone concentration was the sum of the genistein, daidzein, and glycitein concentrations.
Data were subjected to an ANOVA using the general linear model procedure (PROC GLM) (SAS Institute, 2005). Percentage data for necrosis ratings were transformed to the arcsine before the analysis. All data were combined across years and main effects of cultivar or herbicide treatment are presented where interactions were not observed. Means were separated using Fisher's Protected LSD at P = 0.01 unless otherwise specified.
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RESULTS AND DISCUSSION
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Soybean injury 7 d after lactofen application was 10 to 14% following the R1 application and 7 to 15% following the R5 application (Table 2). Injury due to lactofen was similar to injury reported in other studies evaluating soybean tolerance, weed control, disease management, and canopy development (Nelson et al., 2002a; Wichert and Talbert, 1993). Lactofen injury to Envy was evident up to 28 d after the R1 application and up to harvest following the R5 application, while Garst 3712 and Big Bubba cultivars recovered by 28 d after treatment (data not presented). It is likely that crop injury was more evident with Envy because it was a determinate cultivar with limited vegetative growth following flowering.
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Table 2. Soybean plant injury (0 = no visual crop injury and 100 = complete plant death) 7 d after application of lactofen at 70 g ha–1 plus nonionic surfactant at 0.25% v/v plus 28% urea ammonium nitrate at 2.3 L ha–1 in 2002 and 2004.
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Envy, an edamame type cultivar, is commonly harvested at the R6 stage of development and consumed as fresh or frozen (Wu et al., 2004). Lactofen applied to this cultivar during flowering (R1) reduced the number of two-seed pods by 60 m–2, total pods by 77 m–2, and total pod yield by 1100 kg ha–1 when compared with the nontreated control (Table 3). Lactofen applied during early seed development (R5) increased yield of one-seed pods by 13 m–2, but had no effect on total pod yield or pods with two or three seeds. None of the treatments affected brix levels in the seed of Envy.
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Table 3. Pod yield and brix levels of ‘Envy’ soybean treated with lactofen at 70 g ha–1 plus nonionic surfactant at 0.25% v/v plus 28% urea ammonium nitrate at 2.3 L ha–1 at the R1 and R5 development stages in 2002 and 2004. Pods were hand harvested at the R6 stage of development.
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Seed yields were greater in 2004 than 2002 (Table 4) due to more favorable rainfall amounts and distribution (data not presented). Envy seed yield was low compared with the yield of the other cultivars probably due to its determinate growth habit and early relative maturity. Garst 3712 seed yield was 410 kg ha–1 greater than Big Bubba in 2002, while there was no significant seed yield difference between these two cultivars in 2004. Lactofen applied at the R1 stage in 2002 and at the R5 stage in 2004 reduced seed yield, averaged across cultivars, by 290 and 330 kg ha–1, respectively, when compared with the nontreated control. Late applications of lactofen at rates greater, similar, or less than this research had variable effects on grain yield depending on the presence or absence of weeds or disease (Dann et al., 1999; Nelson et al., 2002a; Wichert and Talbert 1993).
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Table 4. Seed yield of cultivars in 2002 and 2004, and soybean treated with lactofen at 70 g ha–1 plus nonionic surfactant at 0.25% v/v plus 28% urea ammonium nitrate at 2.3 L ha–1 applied at the R1 and R5 development stages.
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Cultivar ranking for genistein and daidzein concentration was Big Bubba > Garst 3712 > Envy, while ranking for glycitein concentration was Garst 3712 > Big Bubba > Envy for nontreated seed samples (Table 5). Techniques for analyzing isoflavone concentration in the immature seed were recently developed and validated for edamame (Wu et al., 2004), which is consumed in the vegetable form. The focus of our study was isoflavone concentration in the mature seed, which is known to contain a higher total concentration than immature seed (Wu et al., 2004). Therefore, all plots in our research were mechanically harvested for mature seed and analyzed for isoflavone concentration due to the hypothesized increase in isoflavone concentration in the seed.
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Table 5. Seed concentration of genistein, daidzein, glycitein, and total isoflavone of soybean cultivars treated with lactofen at 70 g ha–1 plus nonionic surfactant at 0.25% v/v plus 28% urea ammonium nitrate at 2.3 L ha–1 at the R1 and R5 stages of development in 2002 and 2004. Seed isoflavone concentration was measured for each cultivar and lactofen application stage mechanically harvested at R8.
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Lactofen at 70 g ha–1 applied to Big Bubba in the R5 stage of development increased the concentration of genistein 78 µg g–1, daidzein 82 µg g–1, and total isoflavones 169 µg g–1 compared with the nontreated control (Table 5). Daidzein concentration increased 52 µg g–1 in Garst 3712 when lactofen was applied at the R5 stage of development, but there was no effect of lactofen on genistein or total isoflavone concentrations. Knowledge of specific isoflavone concentrations in soybean cultivars and the effect of management systems on these levels would allow consumers to select foods according to the specific health benefits desired, such as the cardiovascular benefits attributed to genistein and daidzein (Nakashima et al., 1991; Wang et al., 2004).
Lactofen applied at the R1 development stage (6–7 wk after planting) did not affect isoflavone concentration in any of the three cultivars (Table 5). This finding is similar to other research evaluating an early application of acifluorfen plus bentazon and clethodim 6 wk after planting (Duke et al., 2003). Lactofen significantly increased total isoflavone levels 10% in Big Bubba when applied at the R5 stage of development. No increase in total isoflavone concentration was observed for Envy or Garst 3712 due to lactofen applied at the R5 stage. The lack of response by Garst 3712 or Envy to the R5 application timing may be due to the protein content of the seed since isoflavone concentration has been negatively correlated with seed protein content (Chiari et al., 2004). Other research has indicated that high temperature stress on the plant during seed fill resulted in lower isoflavone content (Tsukamoto et al., 1995). However, plant stress alone during flowering or seed fill does not explain the increase in isoflavone concentration in this research, or there would have been an increase in isoflavone concentration due to the lactofen treatment for all of the cultivars. A late lactofen application to control weeds such as common waterhemp (Amaranthus rudis Sauer), common ragweed (Ambrosia artemisiifolia L.), or giant ragweed (Ambrosia trifida L.) in soybean may increase soybean isoflavone concentration, but the effect depends on the cultivar. The value of an increase in isoflavone concentration in Big Bubba, a high-protein cultivar commonly processed for tofu, with lactofen applied at the R5 stage of development would need to balance a reduction in grain yield, or an application timing may need to be identified so that isoflavone content can be increased without negatively affecting seed yield.
Treatments that increase isoflavone concentration may hold potential for farmers seeking value-added opportunities and consumers seeking health promotion and disease prevention benefits of soy products. Additional research should evaluate additional high-protein cultivars and the impact of lactofen and other protoporphyrinogen oxidase inhibiting herbicides such as fomesafen, acifluorfen, flumiclorac, and carfentrazone on isoflavone concentrations and pathogenesis-related protein gene expression (Graham, 2005) in the seed. Since elevated isoflavone concentration was shown to be negatively correlated with protein content (Chiari et al., 2004), protein levels should also be quantified.
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ACKNOWLEDGMENTS
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The authors would like to thank Dana Harder, Matthew Jones, Randall Smoot, Adam Jones, Martin Schmidt, Heather Collier, and Sandra Devlin for their technical assistance with this research.
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ABSTRACT
INTRODUCTION
