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2013. Plant Management Network. This article is in the public domain.
Accepted for publication 25 June 2013. Published 17 September 2013.


Evaluating Headline Fungicide on Alfalfa Production and Sensitivity of Pathogens to Pyraclostrobin


Deborah A. Samac, USDA-ARS, Plant Science Research Unit, Saint Paul, MN 55108; Bill Halfman, UWEX Monroe County, University of Wisconsin, Sparta, WI 54656; Bryan Jensen, Department of Entomology, University of Wisconsin, Madison, WI 53706; Fritz Brietenbach and Lisa Behnken, UM Extension Regional Office, University of Minnesota, Rochester, MN 55904; Jaime Willbur, USDA-ARS, Plant Science Research Unit, Saint Paul, MN 55108; Daniel Undersander, Department of Agronomy, University of Wisconsin, Madison, WI 53706; Greg Blonde, UWEX Waupaca County, University of Wisconsin, Waupaca, WI 54981; and JoAnn F. S. Lamb, USDA-ARS, Plant Science Research Unit, Saint Paul, MN 55108


Corresponding author: Deborah A. Samac. debby.samac@ars.usda.gov


Samac, D. A., Halfman, B., Jensen, B., Brietenbach, F., Behnken, L., Willbur, J., Undersander, D., Blonde, G., and Lamb. J. F. S. 2013. Evaluating Headline fungicide on alfalfa production and sensitivity of pathogens to pyraclostrobin. Online. Plant Health Progress doi:10.1094/PHP-2013-0917-01-RS.


Abstract

Headline fungicide was recently registered for management of foliar diseases on alfalfa. The effect of disease control on yield, forage quality, and potential return on investment for fungicide application was determined for field experiments conducted at five locations in 2012, three in Wisconsin and two in Minnesota. Headline reduced defoliation in 12 out of 14 harvests and increased forage yield and return on investment in 5 out 12 harvests compared to the untreated control. Headline plus the insecticide Warrior II reduced defoliation in 10 out of 14 harvests and increased yield and return on investment in four harvests compared to Warrior II alone. Two common foliar pathogens were highly sensitive to pyraclostrobin, the active ingredient in Headline. For Phoma medicaginis, the in vitro EC50 was 2.3 ng pyraclostrobin/ml and for Stemphylium globuliferum the EC50 was 52 ng pyraclostrobin/ml. The results indicate that fungicide application can increase yields under higher disease pressure and increase crude protein when the crop is harvested at later developmental stages.


Introduction

Alfalfa hay prices have reached historic highs across the United States due to reduced acreage and recent drought conditions, leading to a re-evaluation of crop management strategies that maximize yield and provide a net positive return on investment. Previous studies demonstrated that fungicides reduce leaf spot diseases, increase forage yield (1,2,3,6,7,12), and may increase return on investment (1). The higher the potential yields, the greater the benefits from fungicides (6). Until recently, growers have been limited to cultural methods for managing foliar diseases because cupric hydroxide, the only fungicide labeled for alfalfa, had poor disease control (12). The fungicide Headline (pyraclostrobin, BASF, Research Triangle Park, NC) was registered for use on alfalfa in 2011. However, information is needed on the impact of fungicide application on foliar disease control, yield, and forage nutritive quality.

Leaf spotting leads to premature defoliation starting at the lower nodes and progressing upwards (5) and loss of primary leaves at a node reduces formation of secondary branches (12). Studies conducted in the 1970s and 1980s found leaf spot diseases caused a reduction in dry matter yields of 5 to 44% (3). The amount of disease and corresponding effect on yield was highly influenced by weather conditions, with less disease associated with drier conditions (1,3,5,8,13). A more recent study in several states found an average annual yield reduction of 9.6 to 16.5% depending on year and location (12), demonstrating that leaf spot diseases continue to impact alfalfa production. Few studies have investigated the effect of leaf spot diseases on forage quality. Alfalfa leaves contain higher amounts of crude protein than stems and are more digestible by ruminant animals. Thus, hay from diseased plants with a smaller leaf fraction is expected to have lower crude protein and higher acid detergent fiber than hay from healthy plants. Interestingly, disease severity was found to have little effect on total digestible nutrients and acid detergent fiber, but could reduce crude protein (3,8,10).

In preliminary trials conducted in 2011, foliar diseases were observed in Headline-treated plots, raising the question of the sensitivity of alfalfa foliar pathogens to pyraclostrobin. The strobilurins, also known as the quinone outside inhibitor (QoI) class of fungicides, inhibit mitochondrial respiration of fungi by binding to the cytochrome bc1 enzyme complex (11). Soon after QoIs were introduced to the market, resistant isolates of important plant pathogens were reported. Several mutations were identified that confer resistance and have low fitness costs (11). Recurrent use of QoI fungicides has been shown to result in dominance of resistant isolates in pathogen populations. Additionally, information is needed on the potential interactions of the fungicide in a tank-mix with an insecticide, because most growers would prefer to apply both chemicals in a single application. The objectives of this study were to conduct field research trials in Wisconsin and Minnesota to: (i) examine the benefit of using a foliar fungicide, alone or in combination with the foliar insecticide Warrior II, on alfalfa yield and forage quality; (ii) measure the effect of fungicide and insecticide application on disease severity; (iii) identify the diseases present over the course of the season; and (iv) determine the sensitivity of major fungal foliar pathogens to pyraclostrobin.


Experimental Field Plots

Trials were conducted at three locations in Wisconsin (Arlington, Tomah, and Waupaca), and two locations in Minnesota (Rosemount and Waseca). The trials at the Arlington, Waseca, and Rosemount locations were conducted on university experiment stations, while the trials at the Tomah and Waupaca locations were conducted on farms in commercial production fields. At each location, a randomized complete block experimental design was used with four replicates. Treatments were: Headline (9 fl oz/acre); Headline (9 fl oz/acre) + Warrior II (1.2 fl oz/acre); Warrior II (1.2 fl oz/acre); and an untreated check. All plots measured 20 ft wide and were a minimum of 30 ft long. Total application volumes ranged from 23.7 to 24.7 gal/acre depending on the equipment used at the location. Application timing was between 6 and 9 inches of growth. Trials were conducted on the first, second, and fourth harvests, except at Tomah, which did not have a last cutting due to drought conditions.

Plots in Wisconsin were harvested on a cutting schedule to maximize alfalfa quality for use in dairy forage, at about 10% bloom. The Minnesota plots were harvested on a schedule for good quality heifer and beef cattle forage at about 25-50% bloom. Subsamples for quality analysis were harvested separately by hand from three 1-ft² areas at a 2.5-inch stubble height, immediately weighed to obtain fresh weight, and dried at 120°F in a forced-air oven for 3 days. Samples were ground using a Wiley mill to pass a 1-mm screen. Subsamples were evaluated by near infrared (NIR) spectroscopy to determine percent crude protein, acid detergent fiber (ADF), neutral detergent fiber (NDF), NDF digestibility, relative feed value (RFV), and relative feed quality (RFQ). Numerical data were subjected to analysis of variance to examine the effects of the different treatments. The level of significance was set to 10%.


Disease Assessment, Pathogen Isolation, and Fungicide Sensitivity

From each plot, ten stems were harvested randomly by hand at a 2.5-inch stubble height and transported to the laboratory for disease rating. Percent defoliation (D) was determined by counting the number of nodes on the primary stem missing a leaf, dividing by the total number of nodes on the primary stem, and multiplying by 100. The mean percent diseased leaf area for each stem (X) was estimated visually by comparing diseased leaves to an alfalfa foliar disease assessment key (9). Disease severity (S) of each stem was calculated using the equation:

S = [ (100−D)(X/100) ] + D.

Diseases were diagnosed by symptoms and spore morphology of isolated pathogens.

A collection of pathogen isolates was made from leaves exhibiting symptoms of Stemphylium leaf spot (Stemphylium spp.) and spring black stem and leaf spot (Phoma medicaginis). Diseased leaves were placed in moist chambers to stimulate spore production for 2 to 7 days. Conidia were removed using a sterile glass rod or needle and germinated on water agar. Single hyphae were excised and grown on either V-8 agar (Stemphylium spp.) or potato dextrose agar (P. medicaginis) for spore production.

Pyraclostrobin sensitivity of Stemphylium spp. was determined by a spore germination assay on 2% water agar with 0.1 mg/ml salicylhydroxamic acid (SHAM) and 1.0, 10, or 100 ng/ml pyraclostrobin (15). The number of germinated spores was counted 24 h after plating at 100× from at least three fields and a minimum of 100 spores. The concentration of pyraclostrobin inhibiting 50% spore germination (ED50) was calculated by linear regression of spore germination and pyraclostrobin concentration. Conidia of P. medicaginis were germinated in 4% (v/v) potato dextrose broth with 0.1 mg/ml SHAM and 1, 5, or 25 ng/ml pyraclostrobin. Spore germination was determined at 24 h after inoculation using an inverted microscope at 200× by counting three fields and a minimum of 200 spores. For both fungi, a germinated spore had a germ tube at least twice the length of the spore. The ribosomal DNA internal transcribed spacer (rDNA ITS) was amplified from representative cultures, sequenced, and compared to rDNA ITS sequences in GenBank to confirm identity of isolated strains as described previously (4).


Calculating Return on Investment in Fungicide and Insecticide Treatments

Yield and forage quality values were used to calculate the total value of the forage harvested from plots receiving each treatment using the spreadsheet tools Milk 2006 for Alfalfa and Grass and FeedVal-2012 (www.uwex.edu/ces/crops/uwforage/dec_soft.htm). Values were then adjusted for the cost of the treatment and applications fees obtained from a survey of agronomy dealers. Corn grain, soybean meal, good quality alfalfa hay, poor quality alfalfa hay, and corn silage were used as benchmark feeds for pricing using average feed prices from January 2012 through November 2012. Alfalfa hay prices were obtained from records of actual sales of known quality tested hay (Ken Barnett, UW Extension Center for Dairy Profitability, personal communication).


Effect of Treatments on First Forage Harvest

Symptoms of spring black stem and leaf spot and Stemphylium leaf spot were observed at all locations. Percent defoliation and disease severity were highly correlated (R² = 0.95 to 0.99) for all treatments across all locations. Scoring percent defoliation is simpler, more rapid, and is less prone to rater error and rater-to-rater variation. Percent defoliation was previously found to be the best visual predictor of yield loss (7). Thus, percent defoliation was used for measuring effects of fungicide treatments even though not all leaf loss is due to disease. Headline treatment significantly reduced percent defoliation compared to the untreated control (Fig. 1A). Headline + Warrior II significantly reduced defoliation at three locations, but at Waseca and Waupaca the addition of Warrior II appeared to interfere with fungicide activity (Fig. 1B). At two locations, Headline treatment increased average dry matter yield compared with the control, and was numerically higher than the control at two additional locations, but was not significant due to high variation among replicates (Fig. 1C). The 0.17 tons/acre increase at Waupaca after Headline treatment corresponds to a 8.4% dry matter yield increase compared to the untreated control and the 0.22 tons/acre increase at Arlington after Headline corresponds to a 10.6% increase compared to the control. Similarly, at the locations where Headline + Warrior significantly reduced defoliation, there was an average increase in yield compared to the treatment with Warrior alone, but differences were not significant (Fig. 1D). Headline treatment resulted in an increase in percent crude protein and small increase in net energy for lactation at the two locations where the crop was harvested at a later stage of development, Waseca and Rosemount, and Headline + Warrior II increased percent crude protein and net energy for lactation at Rosemount (Table 1). At the other locations where the crop was harvested at an earlier stage, Headline or Headline + Warrior did not increase percent crude protein even though percent defoliation was significantly reduced by these treatments.


 

Fig. 1. First harvest percent defoliation and dry matter yield (tons/acre). Numbers above bars are the difference in percent defoliation and dry matter yield between treatments. ns = not significantly different. Harvest dates by location: Arlington, 9 May 2012; Waseca, 21 May 2012; Waupaca, 14 May 2012; Tomah, 15 May 2012; Rosemount, 1 June 2012. (A) Percent defoliation in untreated control and Headline treatments. (B) Percent defoliation in Warrior II and Warrior II + Headline treatments. (C) Dry matter yield in untreated control and Headline treatments. (D) Dry matter yield in Warrior II and Warrior II + Headline treatments.

 

Table 1. First harvest forage quality. % CP is percent crude protein. NEL is Net Energy for Lactation, presented as MCal per pound of dry matter. nsd is no significant difference.

    Arlington Waseca Waupaca Tomah Rosemount
%CP NEL %CP NEL %CP NEL %CP NEL %CP NEL
Control 26.5 0.73 19.3 0.65 24.7 0.72 22.8 0.70 17.8 0.66
Headline 26.1 0.72 19.5 0.66 24.6 0.72 22.6 0.70 19.2 0.67
Difference -0.4 -0.01 0.2 0.01 nsd nsd nsd nsd 1.4 0.01
Warrior 26.0 0.71 20.9 0.67 24.8 0.72 22.7 0.74 17.5 0.67
Warrior II +
Headline
25.9 0.71 18.2 0.65 24.5 0.72 22.6 0.74 18.6 0.70
Difference nsd nsd -2.7 -0.02 nsd nsd nsd nsd 1.1 0.03

Effect of Treatments on Second Harvest

The diseases observed at the second harvest were spring black stem and leaf spot and Leptosphaerulina leaf spot. Headline treatment significantly reduced defoliation at four locations and Headline + Warrior II reduced defoliation at the same four locations (Fig. 2A, B). Headline treatment increased yields at three locations from 0.2 to 0.28 tons/acre and Headline + Warrior II increased yield at three locations compared to Warrior II alone from 0.13 to 0.43 tons/acre (Fig. 2C, D). The effect of Headline and Headline + Warrior II on defoliation was greatest at Rosemount and Tomah and there was a corresponding increase in yield. The 0.23 tons/acre increase at Tomah corresponds to a 15% increase and the 0.28 tons/acre increase at Rosemount corresponds to a 14.5% yield increase. There was an increase in crude protein at Rosemount but not at Tomah (Table 2).


 

Fig. 2. Second harvest percent defoliation and dry matter yield (tons/acre). Numbers above bars are the difference in percent defoliation and dry matter yield between treatments. ns = not significantly different. Harvest dates by location: Arlington, 11 June 2012; Waseca, 25 June 2012; Waupaca, 12 June 2012; Tomah, 15 June 2012; Rosemount, 5 July 2012. (A) Percent defoliation in untreated control and Headline treatments. (B) Percent defoliation in Warrior II and Warrior II + Headline treatments. (C) Dry matter yield in untreated control and Headline treatments. (D) Dry matter yield in Warrior II and Warrior II + Headline treatments.

 

Table 2. Second harvest forage quality. % CP is percent crude protein. NEL is Net Energy for Lactation, presented as MCal per pound of dry matter. nsd is no significant difference.

     Arlington Waseca Waupaca Tomah Rosemount
%CP NEL %CP NEL %CP NEL %CP NEL %CP NEL
Control 27.3 0.67 22.1 0.65 24.1 0.66 25.0 0.70 23.9 0.68
Headline 27.4 0.67 21.6 0.66 24.7 0.66 24.5 0.70 24.7 0.67
Difference nsd nsd -0.5 0.01 nsd nsd nsd nsd 0.8 -0.01
Warrior 27.0 0.71 21.9 0.65 24.2 0.66 24.7 0.70 25.1 0.66
Warrior II +
Headline
26.8 0.71 22.5 0.66 24.3 0.66 24.9 0.70 25.6 0.67
Difference nsd nsd 0.6 nsd nsd nsd nsd nsd 0.5 0.01

Effect of Treatments on Fourth Harvest

The diseases observed at the fourth harvest were Leptosphaerulina leaf spot, summer black stem and leaf spot, a low amount of spring black stem and leaf spot, and occasional observations of rust. The Tomah location was not harvested as drought conditions resulted in uneven growth across the field. Headline and Headline + Warrior II decreased defoliation in three of the four locations (Fig. 3A, B). There was no significant effect of Headline on yield and Headline + Warrior II increased yield significantly only at Arlington (Fig. 3C, D), where the 0.2 ton/acre increase corresponded to an 18% increase in yield. Treatments had little to no effect on forage quality (Table 3).


 

Fig. 3. Fourth harvest percent defoliation and dry matter yield (tons/acre). Numbers above bars are the difference in percent defoliation and dry matter yield between treatments. ns = not significantly different. Harvest dates by location: Arlington, 15 August 2012; Waseca, 6 September 2012; Waupaca, 6 August 2012; Rosemount, 24 September 2012. (A) Percent defoliation in untreated control and Headline treatments. (B) Percent defoliation in Warrior II and Warrior II + Headline treatments. (C) Dry matter yield in untreated control and Headline treatments. (D) Dry matter yield in Warrior II and Warrior II + Headline treatments.

 

Table 3. Fourth harvest forage quality. % CP is percent crude protein. NEL is Net Energy for Lactation, presented as MCal per pound of dry matter. nsd is no significant difference.

     Arlington Waseca Waupaca Tomah Rosemount
%CP NEL %CP NEL %CP NEL %CP NEL %CP NEL
Control 26.8 0.68 24.2 0.72 27.7 0.69       23.5 0.73
Headline 27.8 0.68 23.3 0.71 27.9 0.69       23.7 0.71
Difference nsd nsd -0.9 -0.01   nsd nsd       nsd -0.02  
Warrior 27.4 0.71 23.0 0.70 27.9 0.69       24.9 0.72
Warrior II +
Headline
27.0 0.71 22.9 0.70 27.6 0.69       24.3 0.74
Difference nsd nsd nsd nsd nsd nsd       -0.6 0.02

Total annual yields were numerically highest at each location for the Headline + Warrior II treatment followed by the Headline treatment, and then either the Warrior II or control treatment. However, differences were not significant due to variation among harvests and replications.


Return on Investment

For the treatments in which a significant yield increase was observed, there was a net positive return on investment. The highest net return was for the second harvest. At Waseca, Tomah, and Rosemount there was a net increase for the Headline vs. control treatments and at Arlington, Tomah, and Rosemount there was a net increase for the Warrior vs. Warrior + Headline treatment. Overall, the return on investment ranged from −$104 per acre to $93.91 per acre (Fig. 4).


 

Fig. 4. Return on investment for Headline and Headline + Warrior II treatments. (A) First harvest. (B) Second harvest. (C) Fourth harvest.

 


Sensitivity of Pathogens to Pyraclostrobin

A total of 30 Stemphylium strains were isolated in pure culture. In contrast to previous reports in which S. botryosum was found causing the disease in the Midwest (1,13), the species identified in 2012 in Wisconsin and Minnesota was S. globuliferum, based on spore morphology and cultural characteristics (14). All of the S. globuliferum strains were very sensitive to pyraclostrobin with an average EC50 = 52 ng/ml. There was no difference in sensitivity between strains isolated from Headline-treated plots and control plots (Fig. 5).


 

Fig. 5. EC50 values for individual Stemphylium isolates from control and Headline treated plots.

 

Fifty Phoma medicaginis strains were isolated in pure culture. Isolates were confirmed to be P. medicaginis from rDNA sequence and spore morphology. All isolates were very sensitive to pyraclostrobin with an average EC50 = 2.3 ng/ml. There was no difference in sensitivity between strains isolated from Headline-treated plots and control plots (Fig. 6).


Fig. 6. EC50 values for individual Phoma medicaginis isolates from control and Headline treated plots.


Fourteen comparisons of treatments were possible across locations and harvests. While significant reductions in foliar disease were observed in response to the application of Headline fungicide either alone or in combination with Warrior II, the effect on yield was inconsistent. In general, larger differences in percent defoliation measured between control and fungicide treated plots were associated with greater yield increases and a positive net return on investment. The percent crude protein increased with fungicide treatment only when forage was harvested at 25 to 50% bloom. Alfalfa foliar diseases are strongly affected by weather, particularly leaf moisture. Drought conditions prevailed over Wisconsin throughout the season. Rainfall was near normal at Rosemount but was below normal at Waseca from June-September. The dry conditions likely contributed to the low amount of foliar disease observed. More foliar disease and a larger response to fungicide application is to be expected with greater rainfall or in fields using overhead irrigation. Also, a greater response may be observed in alfalfa grown to flowering stage compared to alfalfa harvested at early bud.

All strains of two major foliar pathogens isolated from experimental plots, S. globuliferum and P. medicaginis, were very sensitive to the active ingredient in Headline. The EC50 values obtained in this study will be valuable for monitoring fungicide sensitivity in these fungi over time. Because P. medicaginis was ubiquitous in the plots throughout the season, the potential exists for selection of resistant strains by repeated applications of a fungicide with a single mode of action. Reducing spring black stem and leaf spot symptoms may have benefits to stand longevity because P. medicaginis also attacks the crown and roots of alfalfa plants. The long-term impact of Headline application on fungicide resistance and stand persistence warrants further investigation.

Fungicides have the potential to increase alfalfa yields during periods of high disease pressure when used with management practices and fertility programs that maximize yields. Headline is a protectant and must be applied before disease symptoms develop, reducing the predictability of a yield response and positive net return on investment.


Acknowledgments and Disclaimer

We thank Dr. Victor Cabrera, UW Extension Dairy Management Specialist and Dr. Randy Shaver, UW Extension Dairy Nutrition Specialist, for developing procedures for determining return on investment. We thank Melinda Dornbusch for excellent technical help on the project. This research was partially supported by funds from the Midwest Forage Association and USDA-ARS-MWA.

Mention of any trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.


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