MATERIALS AND METHODS

Transcription

1 Investigating the Effects of Foliar Fungicide Applications on Alfalfa Production and Fungicide Resistance in 2012 and 2013 PI: Deborah Samac, USDA-ARS and UM Department of Plant Pathology, 1991 Upper Buford Circle, 495 Borlaug Hall, St. Paul, MN 55108, Phone: (612) , Co-PI: Bill Halfman, Associate Professor, UWEX Monroe County, County Highway B, Room 1, Sparta, WI , Phone: (608) , Co-PI: Bryan Jensen, UW Department of Entomology, 1630 Linden Dr., Madison, WI 53706, Phone: (608) , Co-PI: Fritz Breitenbach, UM Extension Regional Office, th Ave SE, Rochester, MN 55904, Phone: (507) , Co-PI: Lisa Behnken, UM Extension Regional Office, th Ave SE, Rochester, MN 55904, Phone: (507) , Co-PI: Dan Undersander, UW Department of Plant Sciences, 1575 Linden Dr., Madison, WI 53706, Phone: (608) , INTRODUCTION Foliar diseases are a serious problem for alfalfa management in all areas where alfalfa is grown. Defoliation due to foliar diseases has been measured from 3-71% depending on time of year, environmental condition, and locale. In addition to yield loss, foliar diseases can reduce forage quality. Until recently, growers have been limited to cultural methods for managing foliar diseases. In 2011 the fungicide Headline (pyraclostrobin, BASF, Research Triangle Park, NC) was registered for used on alfalfa. However, information on the benefits of fungicide application on disease severity, forage yield, and forage quality is needed. In preliminary trials conducted in 2011, foliar diseases were observed in Headline treated plots, raising the question of the sensitivity of foliar pathogens to pyraclostrobin. Additionally, information is needed on the potential interactions of the fungicide in a tank-mix with an insecticide because many growers would be interested in applying 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 a foliar insecticide 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 the major pathogens to pyraclostrobin. MATERIALS AND METHODS 2102 Trials were conducted at three locations in Wisconsin (Arlington, Tomah, and Waupaca) and two locations in Minnesota (Waseca and Rosemount). 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 in commercial production fields. At each location, a randomized complete block experimental design was used with four replicates. Treatments were: Headline (9 fl oz/a), Headline (9 fl oz/a) + Warrior II (1.2 fl oz/a), Warrior II (1.2 fl oz/a), and an untreated check. All plots measured 20 feet wide x a minimum of 30 feet long. Total application volumes ranged from 23.7 to 24.7gallons per acre depending on the equipment used at the location. Application timing was between 6 and 9 inches of growth. Trials were conducted on first, second, and the last cutting before September 1, except at Tomah, which did not have a last cutting due to drought conditions Trials were conducted at two locations in Wisconsin (Cashton and Waupaca) and one location in Minnesota (Rochester). The trials at the Cashton and Waupaca locations were conducted in commercial production fields, while the trial at the Rochester location was conducted on a University Experiment Station. At each location, a randomized complete block experimental design was used with six replicates. Two treatments were used, Headline (9 fl oz/a) + Warrior II (1.2 fl oz/a) and Warrior II (1.2 fl oz/a). All plots measured 20 feet wide x a minimum of 30 feet long. Total application volumes ranged from 23.7 to 24.7gallons per acre depending on the equipment used at the location. Application timing was between 6 and 9 inches of growth. Trials were conducted as a four-cut system before

2 September 1 at Cashton and Waupaca. At Rochester, two cutting management systems were implemented; a four-cut dairy and a three-cut heifer-beef management system with all harvests finished before September and 2013 The following data were collected from each site: dry matter yield (tons/acre), forage quality, insect sweep counts, stem height, percent defoliation, and percent diseased leaf area. Yields were taken using small plot harvesters. Subsamples for quality analysis were harvested separately by hand from a 3 square foot area 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). From each plot 10 stems were harvested randomly by hand and transported to the laboratory for disease rating. Percent defoliation was determined by counting the number of leaves missing at each primary node, dividing by the number of nodes, and multiplying by 100. The percent diseased leaf area was estimated visually by comparing diseased leaves to an alfalfa foliar disease assessment key. Diseases were diagnosed by symptoms and spore morphology. Numerical data was subjected to analysis of variance to examine the effects of the different treatments. The level of significance was set to 10%. A collection of pathogen isolates was made from leaves exhibiting symptoms of Stemphylium leaf spot (caused by several Stemphylium species) and spring black stem and leaf spot (caused by Phoma medicaginis). Diseased leaves were placed in moist chambers to stimulate spore production. Conidia of 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 0.001, 0.01 or 0.1 µg/ml pyraclostrobin. The numbers of germinated spores were counted 24 h after plating at 100X from at least three fields and a minimum of 100 spores. The concentration of pyraclostrobin inhibiting 50% spore germination (ED 50 ) was calculated by linear regression of spore germination and pyraclostrobin concentration. Conidia of P. medicaginis were germinated in 4% potato dextrose broth with 0.1 mg/ml SHAM and 2, 10 or 100 ng/ml pyraclostrobin. Spore germination was determined at 24 h after inoculation using an inverted microscope at 200X by counting 3 fields and a minimum of 200 spores. 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. Milk 2006 and the FeedVal 2012 spreadsheet tools were used to determine dollar values of the alfalfa harvested from the plots when feed value differences (α=0.10) were measured between treatments at locations. The FeedVal 2012 spreadsheet uses benchmark feeds of known quality and prices to make economic comparisons with feeds of known nutritional values. Milk 2006 was used to calculate net energy of lactation values (nel) for the alfalfa samples. FeedVal 2012 was then used for calculating economic values of the alfalfa samples using crude protein and nel. Annual average prices for corn grain, soybean meal, good quality alfalfa hay, poor quality alfalfa hay and corn silage were used as benchmark prices. Alfalfa hay prices were obtained from records of actual sales of known quality tested hay from Ken Barnett, UW Extension Center for Dairy Profitability.

3 2012 RESULTS First harvest. Symptoms of spring black stem and leaf spot and Stemphylium leaf spot were observed at all locations. 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 addition of Warrior II appeared to interfere with fungicide activity (Fig. 1B). Headline significantly increased yield in two locations (Fig. 2A) but Headline + Warrior II did not increase yield compared to Warrior II alone (Fig. 2B). The 0.17 ton/acre increase at Waupaca after Headline treatment corresponds to an 8.4% increase and the 0.22 ton/acre increase at Arlington after Headline corresponds to a 10.6% increase. Headline treatment resulted in an increase in crude protein at two locations and Headline + Warrior II increased crude protein at one location (Table 1). The relationship between defoliation, yield, and forage quality was inconsistent. Figure 1. First harvest percent defoliation. Numbers above bars are the difference in percent defoliation between treatments. ns= not significantly different. (A) Untreated control and Headline treatment. (B) Warrior II and Warrior II + Headline treatment. Figure 2. First harvest dry matter yield (tons/acre). Numbers above bars are the difference between treatments. ns= not significantly different. (A) Untreated control and Headline treatment. (B) Warrior II and Warrior II + Headline treatment.

4 Table 1. First harvest forage quality. %CP is percent crude protein. NE L is Net Energy for Lactation, presented as MCal per pound of dry matter, UW Milk nsd = no significant difference 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. Response to Headline and Headline + Warrior II was similar (Fig. 3). 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 (Fig. 4). 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 ton/acre increase at Tomah corresponds to a 15% increase and the 0.28 ton/acre increase at Rosemount corresponds to a 14.5% yield increase. There was an increase in crude protein at Rosemount but not at Tomah. A B Figure 3. Second harvest percent defoliation. Numbers above bars are the difference in percent defoliation between treatments. ns=not significantly different. (A) Untreated control and Headline treatment. (B) Warrior II and Warrior II + Headline treatment.

5 Figure 4. Second harvest dry matter yield (tons/acre). Numbers above bars are the difference between treatments. ns= not significantly different. (A) Untreated control and Headline treatment. (B) Warrior II and Warrior II + Headline treatment Table 2. Second harvest forage quality. %CP is percent crude protein. NE L is Net Energy for Lactation, presented as MCal per pound of dry matter, UW Milk nsd = no significant difference Fourth harvest. The diseases observed at the fourth harvest were Leptosphaerulina leaf spot, summer black stem and leaf spot, some 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. 5). Response to Headline and Headline + Warrior II was similar. There was no significant effect of Headline on yield and Headline + Warrior II increased yield significantly at only one location (Fig. 6). Treatments had little to no effect on forage quality (Table 3).

6 A B Figure 5. Fourth harvest percent defoliation. Numbers above bars are the difference in percent defoliation between treatments. ns=not significantly different. (A) Untreated control and Headline treatment. (B) Warrior II and Warrior II + Headline treatment. A B Figure 6. Fourth harvest dry matter yield (tons/acre). Numbers above bars are the difference between treatments. ns=not significantly different. (A) Untreated control and Headline treatment. (B) Warrior II and Warrior II + Headline treatment.

7 Table 3. Fourth harvest forage quality. %CP is percent crude protein. NE L is Net Energy for Lactation, presented as MCal per pound of dry matter, UW Milk nsd = no significant difference. Figure 7. Return on investment for Headline and Headline + Warrior II treatments. (A) First harvest. (B) Second harvest. (C) Fourth harvest. Return on investment was calculated for all treatment observations, using average feed prices from January 2012 through November 2012 for the benchmark feeds. Treatment costs were obtained from a survey of agronomy dealers requesting the costs of Headline (9 fl. oz/a) and applications fees. A treatment cost of $35/A was assigned to the Headline treatment and included the application fee ($8/A). It reflects the average cost of applying only the fungicide. A treatment cost of $27 was assigned to the Headline + Warrior treatment. It excludes the application fee and the cost of Warrior. This figure reflects the cost of adding Headline to an already planned application of Warrior. For all treatment observations (positive or negative) the economic gain or loss was determined from using the Milk 2006 and FeedVal 2012 spreadsheets. In cases where there were statistically significant yield or quality responses the return on investment ranged from -$104 per acre to $93.91 per acre (Fig. 7) A total of 30 Stemphylium strains were isolated in pure culture, 14 from control plots and 16 from Headline treated plots. In contrast to older reports in which S. botryosum was found causing the disease in the Midwest, the species identified in 2012 in Wisconsin and Minnesota was S. globuliferum, previously found only in Australia on Medicago spp. This is the first time this pathogen has been identified in the U.S. and the first report of the pathogen on alfalfa. All of the Stemphylium strains were very sensitive to pyraclostrobin with an average EC 50 =52 ng/ml. There was no difference in sensitivity between strains isolated from Headline treated plots and control plots (Fig. 8).

8 Figure 8. EC 50 values for individual Stemphylium isolates from control and Headline treated plots. Fifty Phoma medicaginis strains were isolated in pure culture, 26 from control plots and 24 from Headline treated plots. Isolates were confirmed to be P. medicaginis from rdna sequence and spore morphology. All isolates were very sensitive to pyraclostrobin with an average EC 50 =2.3 ng/ ml. There was no difference in sensitivity between strains isolated from Headline treated plots and control plots (Fig. 9). Figure 9. EC 50 values for individual Phoma medicaginis isolates from control and Headline treated plots.

9 2013 Harvest: Harvest dates for 2013 are listed in Table 4. Symptoms of spring black stem and leaf spot were observed in the first harvest of alfalfa. During midseason, spring black stem and leaf spot, Leptosphaerulina leaf spot and summer black stem and leaf spot were observed. Headline significantly reduced defoliation at Rochester for all four dairy management harvests and for the second and third harvests of the heifer-beef management system. (Fig.10). The most consistent response to Headline fungicide was in the three-cut heifer-beef management system, with a yield increase in the first and second cuttings and for the total season yield. However, yield response was inconsistent for all other locations and systems (Fig ) as was quality for all systems (Tables 5-8.) Table 4. Figure 10. Figure 11. First harvest dry matter yield (tons/acre). Figure 12. Second harvest dry matter yield (tons/acre). Numbers above bars are the difference between treatments. ns = not significantly different

10 Figure 13. Third harvest dry matter yield (tons/acre). Figure 14. Fourth harvest dry matter yield (tons/acre). Numbers above bars are the difference between treatments. ns = not significantly different Figure 15. Season total dry matter yield (tons/acre). Numbers above bars are the difference between treatments. ns = not significantly different

11 Table 5. Table 6. Table 7. Table 8. %CP is percent crude protein. NE L is Net Energy for Lactation, presented as MCal per pound of dry matter, UW Milk nsd = not significantly different

12 DISCUSSION In 2012, fourteen unique comparisons of treatments were possible across locations and cuttings. While significant reductions in foliar disease was observed in response to the application of Headline fungicide either alone or in combination with Warrior II, the response was inconsistent for both yield and forage quality. 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. Years with greater rainfall or fields in which overhead irrigation is used would expect to see a greater amount of foliar disease and a larger response to fungicide application. Also, a greater response may be observed in alfalfa grown for lower quality hay that is harvested at later stages of development. Additional studies are needed before conclusive recommendations on the economic benefits of Headline application can be made. Identification of a new pathogen of alfalfa in the U.S. was unexpected. Symptoms observed are distinct from those of S. botryosum, the Stemphylium leaf spot pathogen previously identified in the Midwest. The pathogen identified in this study, S. globuliferum, was believed to be a pathogen of alfalfa and red clover based on herbarium specimens. Most likely, S. globuliferum has been present in the area for some time, but has been misidentified. Two major foliar pathogens, S. globuliferum and P. medicaginis, are very sensitive to the active ingredient in Headline. The EC 50 values obtained in this study will be valuable for monitoring fungicide sensitivity in these fungi over time in locations where strobilurin fungicides are applied. 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. Reducing the foliar phase of the disease should reduce the amount of inoculum that can attack crowns and roots. The long-term impacts of Headline application on stand persistence and nitrogen credits warrants investigation. DISCUSSION 2013 In 2013, thirteen comparisons of treatments were possible across locations and cuttings. Once again, significant reductions in foliar disease were observed in response to the application of the fungicide. However, the relationship between defoliation, yield, and forage quality has been inconsistent. Five of thirteen cuttings resulted in a significant yield increase with Headline fungicide. Of these five harvests, the significant dry matter yield increases ranged from 0.07 to 0.28 tons dry matter /acre. There was only one significant increase in quality (measured as Net Energy for Lactation (NEL) when Headline was applied on the fourth cutting of Rochester Dairy System. No other increase in quality was attained with the treatments. Results are still highly variable due to environmental conditions. Economic return on investment has also been difficult to achieve. Yield responses, where significant, have been too low to provide a return on investment in these trials. SUMMARY Headline fungicide does control many foliar many diseases on alfalfa, significantly reducing alfalfa defoliation in both 2012 and However, yield response has been variable and inconsistent, and quality response has been difficult to achieve. Defoliation is not necessarily correlated with yield reduction. Fungicides may influence quality, but not to a point to make it pay. With today s alfalfa market prices, and the inconsistent yield responses, it will be difficult to achieve a return on investment of a fungicide application. Alfalfa genetics are much better compared to 30 years ago which may allow for better disease tolerance.