Progress 09/01/08 to 08/31/10
Outputs
OUTPUTS: A field site with well-drained sandy soils located near the Northwest Michigan Horticultural Research Station is being used for evaluating rootstock resistance. In 2009, several orchards were sampled in the cherry producing regions in NW Michigan. Samples were taken from mature trees that had died or were dying. Interestingly, we observed several fairly young trees killed by Armillaria within two years of planting. PCR analysis of samples indicated that A. ostoyae was the only species of Armillaria isolated from the sampled orchards. Potential biocontrol agents (actinomycetes) from soil were tested and found to have little activity. A Trichoderma; was isolated from Armillaria infected cherry roots and will be evaluated for potential biocontrol activity. A field test with several Trichoderma isolates was established using young Prunus trees and A. ostoyae. The fungicide propiconazole has been reported to be inhibitory to species of Armillaria such as A. mellea. This chemistry, as two commercial formulations, was further tested against three additional isolates of A. ostoyae. Some differential sensitivity among the isolates was detected. Propiconazole is mobile in woody species; therefore, use of this compound may have value in protecting against infection. Culture filtrates, fungal hyphae and rhizomorphs were assessed for laccase production by polyacrylaminde gel electrophoresis (PAGE) analysis. Several different laccases, based on electrophoretic mobility, were detected among the Armillaria species evaluated. Extraction of laccases from culture filtrates, hyphae and rhizomorphs was successful as monitored by PAGE. Culture conditions impacted the production of laccase. Soy broth stimulated the most laccase activity as compared to standard YMPG medium. PAGE analysis suggests that soy broth may have also stimulated a second isozyme with a slightly larger mass. Addition of CuSO4 decreased activity in YMPG while adding ethylenedinitrilo tetraacetic acid (EDTA) with CuSO4 as a means of chelating the CuSO4 showed a slight increase. This is of potential interest as laccase are CuSO4 containing enzymes. The isozyme patterns of laccase from A. ostoyae culture filtrates were not the same as those seen in mycelium and rhizomorphs, and this may reflect different functions of the enzymes. Higher molecular weight laccases were detected in the mycelium and rhizomorphs in addition to those around 50kD found in culture filtrates. PARTICIPANTS: R. Hammerschmidt (PI); Janette Jacobs research staff who conducted research on biocontrol agents, fungicides and Armillaria detection; Collaborator: Eric Lizotte, IPM program specialist at NW Michigan Horticulture Station who is involved in locating orchards and assisting in sampling. TARGET AUDIENCES: Michigan cherry industry, extension educators, research plant pathologists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Screening for resistance and susceptibility to Armillaria root rot will be of use in decision making when planting into known infected areas. . A survey for Armillaria in cherry producing areas was continued and verified that A. ostoyae is the predominant species. This information is important for orchard site selection and disease management Biocontrol and chemical control agents, if proven to be effective, may lead to additional management approaches. Research on mechanisms of pathogenicity and virulence will aid in development of future management methods.
Publications
- No publications reported this period
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: A variety Prunus and other plant materials at a field site located near the Northwest Michigan Horticultural Research Station are under evaluation for their response to Armillaria. Tissue culture evaluations of the Armillaria-Cherry interaction continued. Cherry cultivars Mazzard and Lapins were grown in tissue culture, and then inoculated with various isolates of Armillaria. After 1 - 3 months incubation, the lower portion of cherry stem was inspected visually for signs of Armillaria infection and DNA was extracted from the stem. Using PCR and Armillaria specific primers we were able to demonstrate that Armillaria had infected the lower stem and crown tissues. In the last year, several more orchards were sampled for Armillaria in the cherry producing regions of NW Michigan. Only A. ostoyae was detected. A new collection of potential biocontrol Actinomyctes were prepared from the cherry rhizosphere in several orchards in NW Michigan. These orchards also had a history of A. ostoyae infection and isolates of the pathogen were collected at the time the soil samples were taken for actinomyctes. Several of the actinomyctes reduced Armillaria growth by 50% or more and will be used for further testing. Propiconazole was tested against Armillaria species to determine the sensitivity of Michigan isolates to this fungicide. Using dose responses, an ED50 value propiconazole for A. ostoyae was determined to be 0.5 mg/L. There was some variability in sensitivity to the fungicide, but none of the isolates/species were resistant to propiconazole at the concentration used. Several pathogenic and saprophytic isolates were more tolerant of propiconazole than 0.5mg/L, suggesting that testing of isolate sensitivity may be needed if use of this type of fungicide is to be used to protect trees against infection. Armillaria culture filtrates were assessed for variation in laccase isozyme production as related to virulence. Several different laccases, based on electrophoretic mobility, were detected. Interestingly, the more saprophytic species, A. gallica, produced the most laccase in culture. Under the conditions used, A. ostoyae produced the least amount of laccase. A. gallica generally produces the greatest laccase activity. Among A. ostoyae isolates laccase activity is variable. Isolate SC1T1 exhibited strong laccase activity while other A. ostoyae isolates produced little to no laccase activity. SDS PAGE was used to analyze the protein profiles of culture filtrates. A. calvescens/ A. gallica isolates had a band at about 40 kD that is lacking in A. ostoyae and A. mellea isolates. This may represent the laccase produced under saprophytic growth. Armillaria isolates were grown on 3% malt extract agar, pH 7.0, containing bromophenol blue as a pH indicator to test for acid production. Thirty-four isolates representing all four species of Armillaria in our collection were tested. Media acidification only occurred where rhizomorphs were produced. This result suggests that the production of rhizomorphs may be associated with the production of organic acids that may be involved in pathogenesis or growth of the rhizomorphs through soil. PARTICIPANTS: R. Hammerschmidt (PI); Samantha Hollosy, technical research support staff who conducted research on biocontrol agents, fungicides and Armillaria detection; Collaborator: Eric Lizotte, IPM program specialist at NW Michigan Horticulture Station who is involved in locating orchards and assisting in sampling. TARGET AUDIENCES: Michigan cherry industry, extension educators, research plant pathologists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A test for infection using tissue grown seedlings was used to evaluate infection and host resistance. This test has value in screening as well as developing a tool for studying the interaction of Armillaria with cherry. A survey for Armillaria in cherry producing areas was continued and verified that A. ostoyae is the predominant species. This information is important for orchard site selection and disease management. Actinomycetes that are antagonistic to Armillaria were isolated from cherry orchards; these bacteria may be of use in future biological control of Armillaria root rot. Research on mechanisms of pathogenicity and virulence will aid in development of future management methods.
Publications
- No publications reported this period
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