Strategies to Reduce Damage from Aphanomyces Root Rot on Alfalfa

Transcription

1 Strategies to Reduce Damage from Aphanomyces Root Rot on Alfalfa PI: Deborah Samac, 1991 Upper Buford Circle, 495 Borlaug Hall, St. Paul, MN Phone: (612) ; Co-PI: Dan Undersander, 353 Moore Hall-Plant Sciences, 1575 Linden Dr., Madison, WI Phone: (608) , Co-PI: Jeff Coulter, 1991 Upper Buford Circle, 411 Borlaug Hall, St. Paul, MN Phone: (612) ; Co-PI: Craig Sheaffer, 1991 Upper Buford Circle, 411 Borlaug Hall, St. Paul, MN Phone: (612) ; Co-PI: Scotty Wells, 1991 Upper Buford Circle, 411 Borlaug Hall, St. Paul, MN Phone: (612) ; Co-PI: Doug Holen, UM Extension Regional Office-Morris, State Hwy 329, Morris, MN Phone: ; Abstract Diseases are a major source of yield loss and stand decline in alfalfa in the Midwest. Surveys were conducted to determine the distribution of root rot and seed rot pathogens in Minnesota. A total of 44 soil samples were surveyed in for the presence of Aphanomyces root rot (ARR). In 72% of soils there was a high risk of ARR. In these soils, race 2 of the pathogen was the most common race. In 16% of the soil samples, there was low survival of the race 1+2 resistant check cultivar indicating that there are ARR pathotypes in those soils that can overcome the resistance in current cultivars. In 7% of the soil samples, damping off organisms were present at very high population densities, preventing identification of ARR races. In 21% of the soil samples, there was a low risk of ARR. A novel seed treatment (zeolite mineral) was tested in four field trials for control of soil borne seedling diseases. There were no significant differences in final stand density or forage yield among the seed treatments. This result demonstrated that the source of the zeolite material is critical to pathogen control. In vitro tests with a Stamina fungicide seed treatment found that some Pythium strains are resistant to the fungicide. The Stamina seed treatment was effective in protecting plants from ARR. A highly effective zeolite seed treatment was found to lyze zoospores of Aphanomyces euteiches, the causal agent of ARR, indicating that control of the pathogen occurs prior to pathogen germination. Introduction Aphanomyces root rot (ARR), caused by Aphanomyces euteiches, is one of the most important diseases of alfalfa in the U.S. The disease causes stunting and death of seedlings,

2 which can result in complete stand loss under conducive environmental conditions. Non-lethal damage reduces forage yields and reduces winter survival. Two races of the pathogen infecting alfalfa are currently recognized. Most alfalfa cultivars have resistance to race 1 but fewer have resistance to both race 1 and 2. Although surveys for ARR have been conducted in Wisconsin, the risk of ARR in Minnesota is largely unknown. This information will help alfalfa producers select appropriate cultivars to maximize production and will guide seed producers in developing new cultivars to meet grower needs. The common seed treatment with Apron XL does not protect alfalfa from ARR. Moreover, Apron-treated seed cannot be used in organic production systems. Recently, a natural mineral seed treatment (zeolite) was shown to provide excellent control of ARR and other seedling diseases in controlled environment tests (1). The seed treatment needs to be tested under field conditions with a range of disease pressures to determine if it will be a useful alternative in conventional and organic production systems. The mechanism of action of the mineral treatment is unknown. Experiments to investigate whether the seed treatment inhibits pathogen spore germination are needed to guide development of treatments with broader activity against alfalfa pathogens. The objectives of this study were to: (i) survey alfalfa fields throughout Minnesota to determine the risk of ARR and Phytophthora root rot (PRR) and identify soils with race 1 and/or 2; (ii) compare seed treatments for control of seedling diseases of alfalfa in replicated field trials; and (iii) determine if the mineral seed treatment inhibits germination of pathogen spores. Materials and Methods Soil collection and assays to identify pathogens. Soil was collected from transects across alfalfa fields or fields recently terminated from alfalfa, mixed thoroughly, and a 1-gallon bag of soil retained for testing. Soils were sieved to remove rocks and plant debris and then placed in plastic flat inserts. Seeds of ARR susceptible cultivars (Saranac, Agate), race 1 resistant (WAPH-1), and race 1+2 resistant (WAPH-5) cultivars were planted and grown under controlled environmental conditions. After 7 days, the soils were flooded for 5 days to promote disease. The amount of disease was rated at 21 days after planting. Greater than 65% of WAPH-1 plants rated as susceptible indicates race 2 present while greater than 65% of WAPH-5 plants rated as susceptible indicates a new pathotype is present. Total DNA was extracted from three replicate 100 mg samples from 10 field soils using the Powersoil DNA kit (MoBio). The DNA recovered was used in PCR-based assays for the ARR pathogen using purified pathogen DNA to generate a standard curve for quantifying the amount of pathogen present (2). Field trials of alfalfa seed treatments. Field experiments were established in Lancaster, WI, and Rosemount, St. Paul, and Becker, MN to compare seed treatments for control of seedling diseases. The Lancaster site has documented evidence for pathotypes overcoming ARR race 1+2 resistance in current cultivars. The root rot potential at the Minnesota sites was unknown at planting. Soil from each location was used in the assays described above to identify pathogens and races present. There were six replications at the Lancaster site and four replications of each treatment at the Minnesota locations. Seed treatments included Apron XL, Stamina (BASF, a QoI fungicide labeled for alfalfa seed treatment), natural zeolite mineral treatment, and a control without a treatment. The cultivar PGI557 (Producer s Choice) was used with resistance to PRR and ARR race 1. Seed was treated and provided by Summit Seed

3 Coatings, Caldwell, ID. At days after planting, the number of emerged seedlings was counted in 1-meter segments of the center three rows in each plot. Plots were harvested two times during the season to determine fresh weight and dry weight. Treated seed was used in controlled environment trials to compare the level of control against ARR and Pythium seed rot. Effect of zeolite on pathogens. Seeds treated with different amounts of zeolite (100%, 75%, 50%, 25%, 10%) were tested in the standard plant bioassays (1) to determine if there was a response to the amount of zeolite used. Briefly, treated seed were planted into medium grade vermiculite and grown for 7 days in a controlled environment. Plants were inoculated and flooded to promote disease then rated at 21 days after planting. Seeds treated with different amounts of zeolite were used in an in vitro assay with Pythium paroecandrum to determine the response to zeolite concentration. Finally, zeolite was added to A. euteiches zoospore solutions to measure the effect on spore germination and pathogen growth over time. Results and Discussion Root rot pathogens in Minnesota. Aphanomyces root rot is difficult to diagnose in the field. The disease is associated with wet and poorly drained soils. Plants infected with Aphanomyces usually become stunted and chlorotic (yellow) before they wilt and die. Cotyledons usually turn yellow first, and seedlings often develop a purple tint before they die. Roots turn light to dark brown. Symptoms typically appear on slopes or low areas of fields. The similar pathogens Phytophthora and Pythium tend to kill seedlings quickly before plants become severely chlorotic or discolored. The plant bioassay of soils from and Minnesota found that race 2 was the most common race (Figure 1.) This is based on a total of 44 soil samples, Figure 1. Result of survey for races of Aphanomyces. 14 from 2013 and 30 from Surveys in New York, Illinois, and Wisconsin have also found that race 2 is the most common race in those states. Races that can overcome race 1 and race 2 resistant cultivars have been reported in several states. Planting resistant varieties is the most effective way to manage Aphanomyces root rot in commercial production. An increasing number of commercial varieties have resistance to both race 1 and race 2. Total DNA was extracted from eight soil samples positive for the presence of Aphanomyces and two control soil samples with low or undectable levels of Aphanomyces by the plant bioassay. The DNA from two soil samples had very low amounts of A. euteiches DNA. Although more rapid than the plant bioassay, the PCR assay is not sensitive enough to detect damaging levels of pathogen DNA in soil. Field trials of alfalfa seed treatments. Treated seeds were planted in four locations in the spring of There were no significant differences among the seed treatments and the control treatment with no fungicide for stand counts (Figure 2) or forage yield (data not shown). Treated seeds were used in in vitro assays for ARR and Pythium seed rot. These tests confirmed that the Stamina and zeolite seed treatments from the field tested seeds were ineffective against the two pathogens. The zeolite was from a different source than that used in previous tests, indicating that the source of the mineral is critical for activity.

4 Figure 2. Final stand counts at four field locations. Effect of zeolite on pathogens. Seeds treated with different amounts of zeolite (100%, 75%, 50%, 25%, 10%) were tested in the ARR and Pythium seed rot bioassays to determine if there was a response to the amount of zeolite used. The zeolite was from a third source and different from that used in the field study. Assays included the original zeolite treatment, ApronXL and Stamina treatments. In the Pythium seed rot assay, the original zeolite and Apron XL treatments provided protection from the pathogen (Figure 3A). The strain used, P. paroecandrum L3 appears to be resistant to Stamina. In the ARR assay, the Stamina and original zeolite treatments provided protection (Figure 3B). In both assays the newest zeolite treatment provided little protection from the pathogens. Further examination of the composition of the original zeolite is needed to determine the component that results in disease control. A suspension of zeolite was made using the original effective zeolite treated seeds. Dilutions of the suspension were made and mixed with freshly prepared A. euteiches zoospores so that treatments consisted of 0.75, 1.5, 3, or 6 mg zeolite/ml in a peptone-glucose growth medium. The control contained no zeolite. Zoospores were incubated at 24 C for 4 h and then the percent germination determined. At all concentrations, the zeolite treatment resulted in zoospore lysis. An average of 10% zoospores germinated in the zeolite treatments compared to 95% germination in the control treatment. This suggests that the zeolite treatment controls the pathogen prior to spore germination, which would reduce the number of spores that could infect susceptible plants.

5 Figure 3. Effect of zeolite concentration. A, Pythium seed rot bioassay. B, Aphanomyces root rot bioassay. Acknowledgements Funding for this study was provided by the Midwest Forage Research Program of the Midwest Forage Association and USDA-Agricultural Research Service. References 1. Samac, D. A., Schraber, S., and Barclay, S A mineral seed treatment for control of seedling diseases of alfalfa suitable for organic production systems. Plant Dis Vandemark, G.J., Barker, B.M., and Gritsenko Quantifying Aphanomyces euteiches in alfalfa with a fluorescent polymerase chain reaction assay. Phytopathology 92: Keywords: alfalfa, Aphanomyces root rot, Phytophthora root rot, zeolite