Progress 05/15/05 to 05/14/08
Outputs
OUTPUTS: Results of the field trials evaluating several alternative fungicides in conjunction with varietal resistance were shared with audiences of commercial fruit growers at annual (late January) meetings of the Iowa Fruit and Vegetable Growers Association in Marshalltown, Iowa, in 2007 and 2008. Average attendance at each session was 50. A summary of the 2008 field trials will be presented at the January 2009 meeting of the IFVGA. The most meaningful data from the field trials under Objective 1 was obtained in 2008, because abundant rainfall in 2008 combined with inoculation with the pathogen, Colletotrichum acutatum, to engender a severe epidemic. In 2006 and 2007, in contrast, weather patterns during the period between bloom and harvest were too cool and/or too dry to permit development of a significant epidemic, despite repeated inoculations of plots with the pathogen and utilization of overhead irrigation to promote its splash dispersal. As a result, results from the 2006 and 2007 field trials were not judged to be of sufficient value to publicize to growers outside of Iowa. In addition to oral presentations, summaries of the annual field trials were published in the Fruit and Vegetable Trial Reports issued by Iowa State University's Muscatine Research Farm near Muscatine, Iowa and the Horticulture Research Station near Gilbert, Iowa; these booklets were distributed free of charge to the approximately 200 attendees per year at annual winter meetings and field days of the IFVGA in 2007 and 2008. Results of the effort to develop a sensitive and reliable PCR-based assay to detect C. acutatum on symptomless leaves were very promising. These results, encompassing both development of the assay on in vitro cultures of the pathogen and in vivo detection on the surface of leaves of greenhouse and field-grown strawberry plants, were shared with the research and extension communities by publication of an article in the applied research journal Plant Disease in the December 2008 issue. PARTICIPANTS: Dr. Mark L. Gleason, Project Director, Department of Plant Pathology, Iowa State University. Dr. Jean C. Batzer, Assistant Scientist, Department of Plant Pathology, Iowa State University. Dr. Laura H. Jesse, Assistant Scientist, Department of Plant Pathology, Iowa State University. Oscar Perez-Hernandez, PhD candidate, Department of Plant Pathology, Iowa State University. Dr. Myeong-Hyeon Nam, Nonsan Strawberry Experiment Station, Chungnam ARES, Nonsan, Korea. Drs. Batzer and Jesse, postdoctoral scientists in Gleason's lab, received training in both field and lab aspects of the project. Mr. Perez-Hernandez led both the field trial and development of the nested PCR assay as part of his professional training toward the PhD degree in plant pathology. Dr. Nam, a visiting scientist from Korea, gained experience in development and validation of PCR-based assays for Colleototrichum acutatum, including field validation trials of the assay. TARGET AUDIENCES: Commercial strawberry growers throughout the North Central Region. The project changes for this group that can occur as a result of the project's findings are: 1) Utilization of an early-warning assay for the anthracnose fruit rot pathogen, Colletotrichum acutatum, in transplant and production fields before the appearance of symptoms. This assay has the potential to give growers critical additional time to head off an epidemic, since it allows for a sensitive determination of presence of the pathogen on symptomless plants. In contrast, waiting for symptoms to appear is often too late to curtail an anthracnose fruit rot outbreak, and spraying fungicides prophylactically with no knowledge of pathogen presence or absence is likely to be wasteful, unnecessarily hazardous to the environment, and injurious to applicator health. 2) Confirmation that matted-row strawberry growers - the majority of growers in the North Central Region - have an effective management option in addition to fungicides: deployment of a resistant cultivar (Delmarvel). This cultivar performed well in horticultural evaluations in Iowa, and also effectively withstood a severe anthracnose fruit rot epidemic with only a very low (less than 5%) incidence of fruit damage. PROJECT MODIFICATIONS: 1) The field evaluation of alternative fungicides was conducted at only one field site in Years 2 and 3, as opposed to two sites in Year 2. The reason for this change is that the second field site, at Muscatine, Iowa, 150 miles from the ISU campus, proved too expensive to visit weekly during the growing season due to rapidly rising fuel, transportation, and labor costs that were not anticipated in the original budget. The first field site, 10 miles from the ISU campus, proved practical for implementation of this trial. 2) Validation of the nested-PCR assay for the anthracnose fruit rot pathogen in commercial fields was not attempted because a) assay development, in vitro validation, and validation in a field at the ISU Horticulture Research Station required about 1 year longer than initially envisioned, eliminating the necessary time for validation with cooperators. An additional complication was that anthracnose fruit rot outbreaks were rare in the Upper Midwest (Iowa and Minnesota) in spring and summer of 2008, so late-project field validation sites were not possible to locate in that growing season. 3) Because the PCR-based test's field validation trial was completed only in summer 2008, and the only meaningful fungicide-trial data was obtained in June 2008, outreach on project findings was pushed back to later than the project end date.
Impacts
As a result of the project's findings and outputs, a practical method was developed to check symptomless strawberry leaves for presence of the anthracnose pathogen, Colletotrichum acutatum. The value to strawberry growers is that they can now have an early warning system for this fast-spreading disease in nursery or production fields. The nested PCR assay we developed can be accomplished in a couple of days, leaving growers adequate time to apply a fungicide spray to suppress the pathogen if it is present in the symptomless phase, thereby preventing sudden epidemics once the pathogen is widely established in the field. This pre-emptive management strategy was not practical before our study because presence of the pathogen in symptomless fields could not be verified in a sufficiently sensitive, rapid, cost-effective way. We anticipate that this assay, once implemented, will provide strawberry growers with an important added margin of safety against devastating epidemics of anthracnose fruit rot. Although meaningful results were limited to one site-year, the fungicide trial results strongly suggested that cultivar resistance (e.g., Delmarvel) can effectively suppress an anthracnose fruit rot epidemic on strawberries in matted-row production in the northern U.S., and that organic alternatives such as Serenade or Milstop may provide adequate protection for resistant, but not susceptible, cultivars.
Publications
- Perez-Hernandez, O., Nam, M.H., Gleason, M.L., and Kim, H.G. 2008. Development of a nested polymerase chain reaction assay for detection of Colletotrichum acutatum on symptomless strawberry leaves. Plant Disease 92:1655-1661.
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Progress 05/15/06 to 05/14/07
Outputs
In 2006-2007, the project made substantial progress in developing a PCR-based assay for the target fungus, Colletotrichum acutatum, on symptomless strawberry leaves. A nested PCR assay was developed that used the general fungal primers ITS1F/ITS4 in the first round and specific primers CaInt2/ITS4 in the second round. Nested PCR increased the detection limit of DNA extracted from pure cultures of the fungus about 100-fold, from 10 ng using conventional PCR to 100 fg using nested PCR. Of 3 DNA extraction methods tested, NaOH lysis was equivalent in sensitivity to the other methods but required the least time to accomplish. The next step in assay development was to detect the fungus in the presence of strawberry leaves. After greenhouse-grown strawberry leaves were spray inoculated with a range of concentrations of C. acutatum conidia, leaf disks or whole leaves were assayed for the pathogen. Of four extraction methods tested, the FISW method provided to be the most
sensitive to the pathogen. The FISW method initially froze inoculated leaves for 3 hours, then incubated them for 2 days at 27 C under 100% relative humidity, then immersed the leaves in a 0.05% solution of polyoxyethylene sorbitanmonolaurate in a plastic bag, agitated the bag and contents in a sonicator. Next, the solution was concentrated by centrifugation, the pellet was resuspended, and nested PCR was performed. Extraction of DNA with the CTAB method proved to be more effective than using either PrepMan Ultra or NaOH lysis. The FISW method was able to detect as few as 1.5 conidia per ml of sprayed inoculum - the most sensitive test ever devised for detecting C. acutatum in the presence of strawberry plant tissue. A field test is currently underway at Iowa State University to validate the practical application of the FISW assay in screening strawberry fields for presence of the pathogen in periods before or after fruit are ripening. Microplots in a planting of cv. Honeoye were
spray-inoculated on August 29, 2007, with a range of inoculum levels of C. acutatum, and leaves from the inoculated plants were sampled 2 days later. PCR-based assays of the samples are ongoing. In a 2007 field trial near Gilbert, Iowa, of fungicide-based and resistance-based strategies to suppress anthracnose fruit rot, C. acutatum was spray inoculated onto all rows of a June-bearing strawberry (cvs. Honeoye and Delmarvel) twice, once before and once during bloom. Due to cool, dry weather before harvest, however, disease incidence on harvested berries was very low, and there were no significant differences among treatments with regard to anthracnose fruit rot.
Impacts
We expect that the project will help the commercial strawberry industry to prevent spread of the anthracnose pathogen, Colletotrichum acutatum, on transplants, and to give strawberry growers a sensitive technique to find out whether the pathogen is present in their fields, even when disease symptoms are absent, so they can be alerted to the possible need to deter the pathogen with fungicide sprays.
Publications
- Nam, M.H., Perez-Hernandez, O., Kim, H.G., and Gleason, M.L. 2007. Development of a nested-PCR assay for detection of Colletotrichum acutatum in vitro. Phytopathology 97:S82.
- Nam, M.H., Perez-Hernandez, O., Kim, H.G., and Gleason, M.L. 2007. A nested PCR assay for detection of Colletotrichum acutatum on symptomless strawberry leaves. Phytopathology 97:S82.
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Progress 05/15/05 to 05/14/06
Outputs
In 2006, field trials to evaluate various reduced-risk options for control of anthracnose fruit rot of strawberry were conducted at two locations (Gilbert and Muscatine, IA). The June-bearing cultivars tested included a resistant one (Delmarvel) and a susceptible one (Honeoye)that had been planted in May 2005. Experimental Design was a randomized complete block with 2 cultivars, 6 treatments, and 4 replications per treatment. Subplots consisted of 15-ft-long row segments. Guard rows (Honeoye) were alternated with treatment rows. Treatment One included Abound at 1 week pre-bloom, then Abound at 50 percent bloom, then Switch plus Captan week before start of harvest. Treatement 2 included Abound at 50% bloom, and Switch plus Captan 1 week before start of harvest Treatment 3 included Captan at 50 percent bloom and Switch plus Cptan 1 week before start of harvest. Treatment 4 involved alternating sprays of Serenade MAX weekly with Abound sprays, from 1 week pre-bloom to 1
week pre-harvest. Treatment 5 involved alternating sprays of Milstop weekly with Abound from 1 week pre-bloom to 1 week pre-harvest. Treatment 6 was an unsprayed control (no fungicide sprays). Sprays were applied with a hand-pump backpack sprayers at 30 psi to runoff. One week before bloom, we applied one million cfu/ml of C. acutatum conidia to all rows, including guard rows. At harvest, we quantified total yield, number of fruit, and incidence of anthracnose fruit rot and other diseases (e.g., Botrytis gray mold). Analysis of harvest data is ongoing. We have also begun work with use of PCR to screen for strawberry plants with symptomless infections by C. acutatum. A PhD student, Oscar Perez-Hernandez, has done preliminary experiments that have successfully used specific primers to detect the pathogen in pure culture and on strawberry leaves. Current experiments are designed to define the limit of detection of the pathogen by this assay.
Impacts
We expect that the project will help the commercial strawberry industry to prevent spread of the anthracnose pathogen, Colleototrichum acutatum, on transplants, and to give starwberry growers a sensitive technique to find out whether the pathogen is present in their fields, even when disease symptoms are absent, so they can be alerted to the possible need to deter the pathogen with fungicide sprays.
Publications
- No publications reported this period
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