Corn Yield Response to Pyraclostrobin with Foliar Fertilizers

Strobilurin fungicides, including pyraclostrobin, protect many crops from several fungal pathogens and create opportunities to increase plant health and yields. However, corn (Zea mays L.) and many other plants’ physiological responses to pyraclostrobin include increases in processes that require nutrients. By applying foliar fertilizers, growers can adjust their nutrient-management strategy based on the plant’s reaction to pyraclostrobin. Identifying plants’ increased nutrient demands and meeting them with a foliar fertilizer at the time of fungicide application (tasselling) could increase yields. This study evaluated effects of foliar-applied pyraclostrobin at 0.11 kg ha a.i. with or without 13 commonly available foliar fertilizers on yield, tissue macroand micronutrient concentrations, severity of disease, and grain quality. Field research occurred at three University of Missouri research centers from 2008-2009. One foliar fertilizer, 0-0-30-0, caused up to 20% crop injury. Diseases affected plants in all six site-years, but overall severity was low (≤ 2%) and likely did not impact crop performance. Pyraclostrobin increased ear leaf B and Cu concentrations over all site-years seven days after treatment, and decreased N concentrations at one site-year. Grain yields increased 5% at two research sites with pyraclostrobin, and one location had increased grain moisture and grain oil at harvest. One foliar fertilizer, 30-0-0-0, increased grain yields by 10% at two sites compared to the non-treated control. However, foliar fertilizers showed no observable effects on grain quality characteristics, and none of the foliar fertilizers negatively impacted grain yield.

Several factors account for corn yield increases with pyraclostrobin.The most direct reason is the reduction in numerous foliar diseases (Bartlett et al., 2002).Worldwide, losses in corn production from fungal and bacterial pathogens alone were estimated to be about 9% from 2001-2003(Oerke, 2006)).Fungal pathogens, including grey leaf spot (Cercospora zea-maydis), caused yield losses above 44% in Iowa, 69% in Virginia and complete crop failure on occasion (Ward & Nowell, 1998;Ward et al., 1999).Fungal pathogens colonize leaf tissue and often damage the leaves themselves.Leaf necrosis can reduce plants' photosynthetic area and efficiency.Fungal infections also reduce overall physiological efficiency by redirecting plant products and photoassimilates from producing and filling grain to fungal development, growth, metabolism, reproduction, plant defense reactions, and respiration of the infected tissue (Venancio et al., 2003).The goal for most fungicide applications is to suppress or eliminate foliar infections, especially in the upper canopy, in order to maintain photosynthetic efficiency (Ward & Nowell, 1998).This is critical because yield is a direct function of photosynthesis.The eight to nine leaves in the upper canopy supply 75-90% of the photosynthate that the ear requires during grain fill (Allison & Watson, 1966).
Strobilurins, including pyraclostrobin, are preventative fungicides that work by inhibiting spore germination (Butzen et al., 2005).The active compounds in strobilurins inhibit mitochondrial respiration by binding to the quinol or ubiquinone oxidation site in the cytochrome-bc 1 complex (Bartlett et al., 2002).Binding at this site halts electron transfer in the inner membrane of the mitochondria, which prevents adenosine triphosphate (ATP) from forming and so prevents further energy transfer in the fungal spore (Bartlett et al., 2002;Taiz & Zeiger, 2006).
Certain physiological alterations in plants may increase grain yield in the absence of disease (Bartlett et al., 2002).A decrease in the progression of senescence with increasing concentrations of pyraclostrobin in spinach leaves has been observed (Grossmann & Retzlaff, 1997).Maximum retardation of senescence showed 82% higher chlorophyll levels than the control.Although a delay in senescence could result in a longer duration of grain fill, it could also prolong dry-down and result in higher grain moisture.Glaab and Kaiser (1998) showed that exposure of spinach to kresoxim-methyl (methyl (E)-methoxyimino[2-(o-tolyloxymethyl)phenyl]acetate) increased nitrate reductase activity in the treated leaves even under lighted conditions.Köhle et al. (2002) showed an increase in nitrate assimilation and subsequent yield increase in wheat (Triticum aestivum L.) plants sprayed with pyraclostrobin when compared to a non-treated control.Further grain analysis showed similar C:N ratios, and relative protein contents of treated and untreated plants offered evidence that increased nitrate assimilation in plants contributed to increased growth (Köhle et al., 2002).If protein concentrations and C:N ratios are similar with increased nitrate assimilation, then the whole plant should have increased growth to compensate for the extra nitrate.Increased activity of certain nutrient-containing processes would logically result in greater demand for specific nutrients.
Several studies have established a link between plant nutrition and severity of disease.Plant nutrients such as Ca, K, Cl, Mn, B, and P are important for reducing disease severity in some instances (Brennan, 1992;Rupe et al., 2000;Sweeney et al., 2000;Sanogo & Yang, 2001;Thomason et al., 2001;Fixen et al., 2004).When combined with a soil fertility program that builds up and maintains optimal soil test nutrient levels, foliar-applied nutrients may directly and positively manage disease.For example, potassium chloride foliar sprays reduce symptoms of several fungal foliar pathogens on several cereal grain crops (Kettlewell et al., 2000).Foliar fertilizers may work in many circumstances leading to the availability of critical nutrients for optimum plant health and productivity.
Combining a foliar fertilizer with fungicide application may reduce application costs, improve disease suppression and nutrient response, and increase flexibility in managing crop response to environmental conditions during the growing season.Although producers cannot rely solely on foliar applications for fertilizer management (Curley, 1994;Johnson et al., 2001, Fernandez et al., 2006;Nelson et al., 2010), foliar fertilizers can complement soil amendments or provide an increase in nutrient supply during plants' peak demand times or when soil conditions restrict nutrient uptake (Fernandez & Eichert, 2009).Similarly, applying foliar fertilizers to match nutrient demands from physiological responses to pyraclostrobin may result in a synergistic yield increase.Pyraclostrobin, which has a range of fungicidal activity, is labeled for a variety of crops (Bartlett et al., 2002) and received a label for plant health benefits (BASF, 2010).Strobilurin fungicides continue to grow in popularity and usefulness.Combining pyraclostrobin and foliar fertilizers has great potential, not only for its possible synergistic benefit, but also the heightened flexibility it offers producers responding to conditions during the growing season.The objective of this research was to evaluate the effect of common foliar fertilizers in the presence and absence of pyraclostrobin on the severity of disease, ear leaf nutrient concentrations, grain quality, and yield.

Site Descriptions and Experimental Design
This study was conducted during 2008 and 2009 at three University of Missouri research centers near Novelty, Portageville, and Albany, Missouri.These locations offer differences in soil and climate, and they represent three of the state's major cropping areas.The diversity of temperature, rainfall, humidity, and soils offers different challenges and issues related to high-yield corn production.19′W) near Albany.All three locations were managed for high yielding corn, including two where irrigation was added.Irrigation not only provided adequate water to eliminate yield loss from drought stress, but also provided differing environmental conditions that could affect corn diseases.Novelty had supplemental sprinkler irrigation, while Portageville was flood-irrigated.Albany was not irrigated.Irrigation events were scheduled using the Woodruff chart (Henggeler, 2008).In addition to irrigation, fertility and pest management were targeted at producing high yields.Soil type, management practices, planting and harvest dates, and environmental conditions at the time of foliar fungicide and fertilizer application are reported in Table 1.Note.† fine, smectitic, mesic Vertic Albaqualfs; ‡ fine-silty, mixed, thermic Typic Argiudolls; § fine, montmorillonitic, mesic Aquic Argiudolls.
Treatments consisted of a factorial arrangement of the 13 foliar fertilizers (Table 2) and fungicide (presence or absence of pyraclostrobin at 0.11 kg ai ha -1 ) organized in a random complete block design with three to five replications depending on site and year (Table 1).Combinations of foliar fertilizer and fungicide also included a nonionic surfactant (Induce ® , proprietary blend of alkyl aryl polyoxylkane ethers, free fatty acids, and dimothyl polysiloxane, Helena Chemical Company, Collierville, TN) at 0.25% vol./vol.All treatments simulated aerial application using a hand boom propelled by CO 2 (Bellspray, Inc.Opelousas, LA) at 28 L ha -1 .All applications were made when corn was at VT (tasseling) (Ritchie et al., 1993), which is congruent with typical preventative fungicide application (BASF, 2010).Foliar fertilizers and application rates were selected based on local availability and experience.Table 2 summarizes foliar fertilizer products and application rates.Plots were maintained weed-free as detailed in Table 3 by individual sites and years.

Measurements and Evaluations
Representative soil samples were taken from each experiment (Table 4) and evaluated to establish baseline levels for multiple essential plant macro- (2008 and 2009) and micro-nutrients (2009).Crop injury was rated on a scale of 0 (no visual injury) to 100% (complete crop death) 7 to 14 days after treatment (DAT).Severity of foliar disease was rated on a scale of 0 (no disease) to 100% (complete tissue colonization) 28 to 42 DAT.(Laverty, 1963;Keeney & Nelson, 1982) with a spectrophotometer (Genesys 10, Thermo Spectronic, Rochester, NY).Foliar P, K, Ca, Mg, Cu, Fe, Mn, Zn, and S concentrations were determined using inductively coupled plasma (ICP) spectroscopy (Mills & Jones, 1996).For Cl analysis, a 0.15 g sample was extracted with 30 mL distilled H 2 O (Brown & Jackson, 1955).The suspension was agitated on an orbital shaker (Eberbach Corporation, Ann Arbor, MI), filtered (Ahlstrom 642 filter paper), and analyzed using a chloride ion-specific electrode compared to standard solutions.Boron was determined from dry ashed samples, which were dissolved in 1 M HCl, then diluted to 0.1 M HCl, and analyzed with ICP spectroscopy.Corn grain yields were determined from the two center rows with a small-plot combine (Kincaid Equipment, Haven, KS) and adjusted to 150 g kg -1 moisture prior to statistical analysis.Grain samples from all sites except Albany in 2008 were collected for protein, oil, and starch and analyzed using near infrared spectroscopy (Foss Infratec 1241, Eden Prairie, MN).
Unless otherwise specified, data were subjected to an analysis of variance and means separated using Fisher's Protected LSD at P < 0.01.Data were subjected to an F Max test for homogeneity (Kuehl, 1994) and combined over site-years when appropriate.Main effects were presented in the absence of significant interactions (SAS, 2010).

Precipitation and Irrigation
Precipitation amounts for the six site-year growing seasons, from 1 April to 31 November, varied widely.Most were over the ten-year average for the specific site, but one site was below average.Novelty experienced greater-than-average rainfall during both growing seasons.In 2008, rainfall totaled 1182 mm, 48% greater than the 10-year average of 796 mm, and in 2009 rainfall totaled 1087 mm, 36% greater than the average.Sprinklers irrigated the Novelty site both years.In 2008, rainfall and the 149 mm of irrigation water combined for 67% more water than average on the field (Figure 1).In 2009, rainfall plus 188 mm of irrigation water made the total 60% greater than average.In 2008, Portageville experienced 601 mm of rain, which was 20% below the 10-year average of 752 mm.In 2009, 971 mm of precipitation fell, resulting in a 29% increase over the average.Both years had flood irrigation amounts totaling approximately 610 mm (Figure 2).Total water was 61% and 110% greater than the 10-year average in 2008 and 2009, respectively.Albany had higher-than-average precipitation both years.In 2008, precipitation was 861 mm, 28% greater than the 10-year average of 670 mm.The following year, 863 mm of rain fell for a 28% increase over the average.No supplemental irrigation was given at Albany (Figure 3).

Crop Injury
Crop injury, which occurred at all locations over both years, was generally persistent necrosis of leaf tissue depending on the treatment.Pyraclostrobin did not affect crop injury when compared to fertilizer-only treatments even though the treatments included surfactant (Table 5).Some fertilizer treatments caused injury.Overall and for all fertilizer treatments, crop injury was less than 10% at both Novelty and Albany for both years and less than 20% at Portageville (Table 6).The only treatment with consistently significant (P ≤ 0.05) injury (4 to 20%) for all site-years was 0-0-30-0.The form of potassium in 0-0-30-0 is K 2 CO 3 derived from potassium hydroxide (Nachurs Alpine Solutions, 2009).At high use rates, this fertilizer can cause necrosis to soybean (Nelson et al., 2012).When 0-0-30-0 was added to glyphosate, the pH of the solution was 12.0-13.0,which most likely elevated the injury levels (Nelson et al., 2012).Excluding 0-0-30-0, crop injury was inconsistent over the six site-years and was generally less than 10%.Inconsistencies most likely arose from varied environmental conditions during application, such as time of day, temperature, and relative humidity.Hotter and/or dryer conditions may have caused sprays to dry quicker on foliar surfaces, causing increased salt concentrations and leading to greater cellular damage (Neumann & Prinz, 1975;Gamble & Emino, 1987).

Disease Severity
Severity of foliar disease was less than 5% for all six site-years (Tables 7 and 8).Diseases varied by location and included: anthracnose leaf blight (Colletotrichum graminicola), bacterial stalk rot (Erwinia dissolvens), common rust (Puccinia sorghi), common smut (Ustilago zeae), gray leaf spot (Cercospora zeae-maydis), and northern corn leaf blight (Exserohilum turcicum).Neither pyraclostrobin, nor any of the foliar fertilizers had any significant effect on disease severity.For all treatments over all site-years, severity was quite low, despite elevated crop injury exhibited with some of the fertilizer treatments, optimal weather conditions for disease, and three of the six site-years having continuous corn (Table 1).The lack of disease may have resulted from using hybrids with good to excellent resistance to diseases present at all locations (Sweets & Wright, 2008;Poland et al., 2009).The disease-suppressing effects of pyraclostrobin and foliar fertilizers were not rigorously tested due to lack of disease, even in the non-treated controls.Therefore, yield response may stem from the effects of fungicide treatments or crop nutrition impacts of the fluid fertilizers.

Leaf Nutrient Concentration
All ear leaf nutrient concentrations in the non-treated control were above the sufficiency levels at the time of application (Mills & Jones, 1996), with the exception of Ca concentrations (data not presented).Calcium levels were slightly below the sufficiency range.No interactions appeared between fungicide and foliar fertilizer treatment for any nutrient, so data main effects are presented.Interactions with year are presented separately for significant interactions.A significant interaction (P = 0.05) occurred between pyraclostrobin treatment and site-year for foliar nitrogen (N) concentrations; therefore, data were presented by individual site-years for ear leaf N concentrations (Table 9).All other foliar nutrient concentrations by pyraclostrobin treatment were averaged over all six site-years (Tables 9 and 10).Only one site-year (Novelty, 2008) showed differences (P= 0.1) between the pyraclostrobin-treated and non-treated corn for ear leaf N concentration.The pyraclostrobin treatment had 6.3 g kg -1 lower ear leaf N concentration.A decrease in tissue N levels may signify an increase in the activities of certain N-containing physiological processes that can increase with strobilurin fungicides (Glaab & Kaiser, 1998;Oerke et al., 2001;Köhle et al., 2002).Table 10.The effect of pyraclostrobin on ear leaf P, K, Ca, Mg, S, Zn, Fe, Mn, and Cl concentration.Data were combined over site-years and foliar fertilizer treatments Boron concentrations in corn ear leaf samples increased with pyraclostrobin by 11.2 mg kg -1 .Strobilurin fungicides have altered various hormonal processes in plants, and B has been associated with various plant hormonal responses (Venancio et al., 2003;Marschner, 1995).In addition, B has been proposed to exhibit a role in respiration, which can be suppressed by strobilurin fungicides (Parr & Loughman, 1983;Nason et al., 2007).Strobilurin fungicides have also been shown to retard senescence in plants.Correlated with the impedance of senescence is an increase in indole-3-acetic acid (IAA) concentrations (Marschner, 1995;Grossmann & Retzlaff, 1997).Boron is closely associated with indole-3-acetic acid metabolism.Altering these biochemical processes with pyraclostrobin may explain differences in the observed B concentrations in the ear leaf.
Ear leaf Cu concentrations increased with pyraclostrobin 2.0 mg kg -1 .Köhle et al. (2003) showed that plants treated with pyraclostrobin had enhanced tolerance of oxidative stress.Copper is a component of superoxide dismutase, which along with other plant antioxidative enzymes detoxifies superoxide radicals (Taiz & Zeiger, 2006).Increased Cu in plant tissue may enhance tolerance of oxidative stress.All other nutrient concentrations showed no significant differences due to the main effect of pyraclostrobin treatment (Table 10).
Foliar fertilizer main effects are presented because limited 2-(year × fertilizer, fertilizer × fungicide) and no 3-way (year × fertilizer × fungicide) interactions were observed.A significant year × fertilizer interaction (P = 0.0009) occurred for ear leaf B concentrations; therefore, data are presented separately by site-year for B (Table 11).No consistent differences were found for ear leaf B concentrations between the non-treated control and the fertilizers that were applied.One foliar fertilizer (6-0-0-0) increased B concentrations over others in two site years.At Novelty in 2009, B concentration increased with 6-0-0-0 (P = 0.1) over 25-0-0-0 and Mn-chelate.At Albany that year, B concentration was greater than the non-treated control, 0-0-30-0, and 22-0-2-1.All other significant differences regarding foliar B concentrations were inconsistent and did not seem to show a pattern among site-years or with treatments that included B. ------------------------------------------mg kg -1 --------------------------------------- Other ear leaf nutrient concentrations were averaged over all six site-years (Table 12) because 2-and 3-way interactions were insignificant.With the exception of Cu, all foliar nutrients exhibited significant differences (P = 0.1) among treatments.Ear leaf nutrient concentration with 24-0-1-0.6,0.25% B increased over the control and/or other foliar fertilizers for several nutrient concentrations, but had lower ear leaf S concentrations compared to that of the 5-0-20-13 fertilizer treatment.The fertilizer treatment of 24-0-1-0.6,0.25% B also had a higher (59 mg kg -1 ) ear leaf Fe tissue concentration compared to the control.All other nutrient concentration increases were greater than the other foliar fertilizers for N, P, and Mg.Surprisingly, 25-0-0-0 increased ear leaf P concentration 0.19 g kg -1 compared to the control.Other differences for foliar nutrient levels were among other foliar fertilizers and were inconsistent.The fertilizer treatment of 5-0-20-13 resulted in the greatest concentrations and an increase over the control for sulfur levels with 369 mg kg -1 , a 114 mg kg -1 increase over the control.Conversely, 5-0-20-13 decreased foliar nutrient concentration of Ca and Mg compared to the control, and it decreased P and K compared to other foliar fertilizers.Ear leaf Mn concentration decreased 16 mg kg -1 with 0-0-62-0 compared to the control, while it decreased N, K, Mg, Fe, and Mn concentrations compared to several other foliar fertilizers.The micronutrient mix of Fe-Mo-Mn-B-Zn decreased Mg 0.20 g kg -1 and Zn 5.4 mg kg -1 compared to the non-treated control, while it decreased S and Fe concentrations compared to other foliar fertilizers.However, Fe-Mo-Mn-B-Zn increased Mn concentrations compared to other foliar fertilizers, which is expected with Mn in the formulation.Note.Data were combined over site-year and pyraclostrobin treatment.

Grain Yield, Moisture, Oil, and Protein Concentration
In the absence of significant interactions, main effects were presented and data were combined over site-years.Overall, grain yield data for Portageville and Novelty could be combined over all four site-years, whereas Albany data were combined over years.This could be due to differences in seeding rates, presence of irrigation, and/or hybrids.Portageville and Novelty had slightly higher yields than Albany (non-irrigated) probably due to the lack of supplemental irrigation at the Albany site.Pyraclostrobin increased grain yield 0.5 Mg ha -1 at Portageville and Novelty, but showed no effect at Albany (Table 5).Pyraclostrobin did not affect grain moisture content at Albany and Portageville.However, grain moisture at Novelty increased 5 g kg -1 with pyraclostrobin compared to the non-treated control (Figure 4).This may be due to differences in environmental conditions at the time of harvest, timing of harvest, or the difference in corn hybrids.However, applying fungicide to corn can increase grain moisture (Bradley & Ames, 2010;Munkvold et al., 2001).Such increases may result from delayed senescence, similar to reports on other crops with pyraclostrobin and other strobilurin fungicides (Grossman et al., 1999;Ruske et al., 2003;Ypema & Gold, 1999).This experiment included no measures to support delayed senescence.Similar to wheat studies, pyraclostrobin did not increase grain protein concentration compared to the non-treated control (Köhle et al., 2002).Pyraclostrobin did not increase grain oil concentration at Portageville or Albany; however it increased grain oil concentration 0.9 g kg -1 at Novelty (Table 5).Soybean studies have shown similar effects of strobilurin fungicides on grain oil concentrations (Nelson et al., 2010).
Foliar fertilizers did not affect grain protein, oil or moisture concentrations (Table 6).One fertilizer, 30-0-0-0, increased yield 0.11 Mg ha -1 over the non-treated control.Sole liquid N sources (30-0-0-0 and 25-0-0-0, 0.01% Cl) had the highest overall yields at Albany.Although no fertilizer showed a significant yield reduction compared to the non-treated control, producers should avoid applying foliar fertilizers such as 0-0-30-0 at the rates this study evaluated, which caused injury and plant necrosis.With the timing of application at tasseling, plants may not be able to recuperate from injury as well as younger plants.

Conclusions
This study and previous research show that pyraclostrobin and other strobilurin fungicides interact with plants in multiple ways.Whether there is one interaction or many is unclear, but some factors such as the presence of pyraclostrobin lead to grain yield increases in corn crops over non-treated crops without visual differences in disease severity.During this study's six site-years, no differences appeared in disease severity among any treatments or controls.This may be a result of low levels of pathogenic inoculum, but is more likely a product of corn hybrid resistance to present pathogens.Of the 13 foliar fertilizers evaluated, 0-0-30-0 consistently caused injury over all six site-years.leaf concentrations of nutrients varied widely over all macro-and micro-nutrients and site-years.Although many significant responses occurred from both main effects of pyraclostrobin and foliar fertilizers, few responses were consistent over all site-years, and no foliar fertilizer was responsible for more than one site-year's increase, except with regard to ear leaf S concentrations.Fertilizers, 5-0-20-13 and 0-0-25-17, both of which contain S, showed limited consistency in ear leaf nutrient concentrations seven DAT.
Neither pyraclostrobin nor any foliar fertilizer influenced grain protein concentrations at any of the site-years.Pyraclostrobin increased grain moisture and oil concentration at Novelty.Generally, pyraclostrobin had little effect on grain quality characteristics.Pyraclostrobin increased grain yields 0.5 Mg ha -1 at Novelty and Portageville, but showed no effect at Albany.An application of 30-0-0-0 was the only foliar fertilizer that significantly increased grain yields compared to the non-treated control.Sole N sources (30-0-0-0 and 25-0-0-0, 0.01% Cl) were the two highest yielding treatments at Albany.The combination of increases in yield from pyraclostrobin and 30-0-0-0 may provide tank mixture options for corn produced in high-yielding environments.

Figure 1 .Figure 2 .
Figure 1.Precipitation and irrigation events, as well as planting, treatment application, and harvest dates at Novelty in 2008 and 2009

Figure 4 .
Figure 4.The effect of pyraclostrobin on harvested grain moisture at Novelty, Portageville, and Albany.Data were averaged over foliar fertilizer treatment and years

Table 1 .
Field information and management practices at each location in 2008 and 2009.

Table 2 .
Foliar fertilizer characteristics, manufacturers, and application rates

Table 3 .
Crop protection chemical applications and rates for all six site-years

Table 4 .
Soil characteristics for all six site-years

Table 5 .
Pyraclostrobin main effects for crop injury 7 to 14 days after treatment, grain yield, and quality characteristics averaged over fertilizer treatments and years (Albany) or site-years (Novelty and Portageville)

Table 6 .
Foliar fertilizer main effects for crop injury, grain yield, and grain quality characteristics.Data were averaged over fungicide treatments and years unless designated otherwise

Table 7 .
Disease severity ratings from each location combined over 2008 and 2009.Data were combined over fertilizer treatments

Table 9 .
Ear leaf B, Cu, and N concentration as affected by pyraclostrobin.Boron and Cu tissue concentrations were combined over all site-years.Nitrogen foliar concentrations are presented individually by site-year.Data were combined over fertilizer treatments

Table 11 .
Ear leaf B concentrations by site-year.Data were combined over pyraclostrobin treatments

Table 12 .
Ear leaf nutrient concentrations seven days after treatment