Progress 07/01/10 to 06/30/13
Outputs
Target Audience: Michigan cherry industry; extension educators; research plant pathologists; breeders Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Undergraduate students were provided with the opportunity to engage in research on an important disease problem and learned basic techniques in plant pathology, pathogen culture and plant growth. A graduate student was engaged in thesis research on topics related to resistacne of Prunus to Armillaria. All students worked one on one with the PI as mentor. How have the results been disseminated to communities of interest? Reports to the Cherry industry; Presentation at the American Phytopathological Society; Seminar presentation on the research What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. Screening for resistance to Armillaria. Assays using colonization of cherry phloem and cambial tissues in three year old stem and branch tissues were utilized to supplement the ongoing orchard trials for resistance to Armillaria. This assay is based on the observation that Armillaria causes disease by colonization of the tissues between the periderm and secondary xylem. The colonization assay was used to assess 43 Prunus species and several breeding lines with P. maackii in their background. The assay showed that most Prunus species are susceptible to infection. Only P. maackii demonstrated significant degree of resistance to colonization of the cambium tissues. Testing P. maackii from different locations, sorces and ages gave the same result. The test was also run against 29 Prunus spp. using six A. ostoyae isolates that differed in virulence with similar results. Several rootstocks that appear to be less susceptible than the standard P. mahalab were planted for evaluation at sites in NW Michigan known to be infested with A. ostoyae and these will be assessed late as not enough time has passed for disease to express. A site with significant A. ostoyae infestation was planted with P. maackii and different scions grafted onto P. maackii. It is too soon to have results from these plots. P. maackii has also been used by the MSU breeding program to determine if resistance can be transmitted; 2. Rhizomorph and fungal infection structure biology and pathogen-produced factors: The effects Prunus periderm and cambium tissues were analyzed for effects on infection structure formation. Cambium tissue amended media from all Prunus spp. evaluated supported growth as good as or better than standard Yeast-malt-peptone glucose (YMPG) agar media. Cambium tissues from some species that was incorporated into culture media also stimulated rhizomorph formation more rapidly than unamended culture media. With the exception of P. maackii, periderm of all Prunus spp. tested supported growth of the pathogen when incorporated into culture media (both YMPG agar and water agar). Incorporating P. maackii periderm into culture media inhibitedallisolates of A. ostaoyae tested as well as isolates of A. gallica, A. geminina and A mellea. The ability to use periderm as a carbon source also suggests that A. ostaoyae may use enzymatic activity to penetrate through the periderm. Cherry fruit extracts were also found to stimulate the growth of A. ostoyae in culture and promoted rhizomorph formation. Growth was also stimulated by gallic and tannic acids. Gallic acid also promoted rhizomorph formation. Addition of both copper and manganese to the media did not stimulate rhizomorph formation but did enhance laccase activity. Cherry fruit extract also stimulated laccase activity in culture; 3. Testing of biological controls. Trichoderma was found to be inhibitory to A. ostoyae in vitro plate tests. Trichoderma was incorporated into soil which was then planted to a rootstock susceptible to A. ostoyae. The rootstocks were inoculated with A. ostoyae at that time. Trichoderma populations in the soil were monitored over the growing season and remained high. There was no effect of the different treatments on growth of the trees (as measured by diameter of the stem). Some tree death was noted, but the cause was not due to Armillaria. Due to variability of in vitro efficacy of previously isolated actinomyctes on A. ostoyae growth, no further work with these microbes was conducted; 4. A PCR technique was developed to detect Armillaria in soil. To routinely detect the pathogen, 1 kg of soil was processed by wet sieving to enrich fragments of organic matter. This fraction was used to extract DNA for PCR analysis using published primers. Much smaller soil samples were not sufficient to detect the pathogen. PCR was also used to confirm presence of A. ostoyae in several orchards in NW Michigan thus further confirming that A. ostoyae is the predominant species in this part of the state. The most significant outcomes were the identification of a potential source of resistance to Armillaria in Prunus and the ability to use PCR to detect the pathogen in soil. The former has led to incorporation of P. maackii into the breeding program and the latter technique has been shared with the MSU plant disease diagnosticians. New information of the effect of plant-derived materials on growth of the pathogen will lead to new investigations on factors that may influence both host resistance and pathogenicity.
Publications
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Progress 07/01/11 to 06/30/12
Outputs
OUTPUTS: 1. Testing of Prunus root stocks for Armillaria ostoyae resistance: Assays using colonization of cherry phloem and cambial tissues in three year old stem tissues are being utilized to supplement the ongoing orchard trials at a site adjacent to the Northwest Michigan Horticultural Research Station near Traverse City. Although there has been low disease at this site, it has proven to be a valuable resource for materials used in stem assays. New field evaluations were initiated in 2011 in other orchards that have high levels of a highly virulent isolate of Armillaria ostoyae in the soil. These sites will be examined for any evidence of infection during the next year. The use of stem segments to monitor colonization of A. ostoyae into cambial tissues has proven to be a useful tool for host evaluation. Almost all Prunus spp. and varieties tested have proven to be readily colonized except for P. maackii. Crosses have been made by the MSU cherry breeding program using P. maackii as a first step in testing the heritability of this resistance into materials suitable for root stock. We will be assaying an additional 20 Prunus species this year. The infection of stem segments has also proven to be a good method to examine fungal development in host tissue. 2. Testing of potential biological control agents: This is ongoing and in the third season of evaluation. Five different strains of Trichoderma were introduced into the soil prior to inoculation of P. mahalab rootstock with A. ostoyae. Evaluation of these trees will be carried out this fall. 3. Rhizomorph and fungal infection structure biology and pathogen-produced factors: The effects of extracts from Prunus periderm/bark and cambial tissues were analyzed for effects on infection structure formation. Samples of culture media and fungal infection structures produced in the presence of Prunus extracts and in Prunus stem tissues are being analyzed for virulence factors such as laccase. Because rhizomorphs are important for infection and growth of the pathogen through soil, the effect of various amendments to culture media on their formation and laccase production are being tested. Extracts from periderm and cambial tissues of several Prunus species have been tested for their effect on growth and rhizomorph formation. With the exception of P. maackii periderm, there were no major effects. The P. maackii periderm was highly inhibitory to growth and may be related to resistance observed in the stem assays. Addition of several different phenolic compound containing extracts to culture media increases the formation of rhizomorphs and structures that resemble fungal growth in planta. The effects of these compounds on the induction of laccase activity are underway. 4. PCR-based identification tools have been developed to examine selected orchards for the presence of Armillaria species. PARTICIPANTS: R. Hammerschmidt (PI); Linzi Kaniszewski, research staff, who conducted research on host resistance; Eric Lizotte, IPM program specialist at NW Michigan Horticulture Station who is involved in locating orchards, field screening and assisting in sampling. TARGET AUDIENCES: Michigan cherry industry, extension educators, research plant pathologists, breeders. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Screening for resistance and susceptibility to Armillaria root rot will be of use in decision making when planting into known infested areas by providing guidance on which rootstocks to use. The use of stem assays has potential of greatly speeding up the screening process. Biocontrol and agents, if proven to be effective, may lead to additional management approaches. Research on mechanisms of pathogenicity and host resistance will aid in development of future management methods and in providing information needed by cherry breeding programs.
Publications
- No publications reported this period
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Progress 07/01/10 to 06/30/11
Outputs
OUTPUTS: Resistance screening continues to utilize the site adjacent to the Northwest Michigan Horticultural Research Station near Traverse City. Disease pressure has not been high at this site, but it does serve as a resource for materials used in stem segment assays described below. New field evaluations were initiated this year in other orchards that have high levels of Armillaria ostoyae in the soil. The use of stem segments to monitor colonization of A. ostoyae into cambial tissues has proven to be a useful tool for host evaluation. Almost all Prunus spp. and varieties tested have proven to be readily colonized except for P. maackii. The infection of stem segments has also proven to be a good method to examine fungal development in host tissue. Enough fungal tissue can be collected from colonized stem segments to allow assay of enzymes such as laccase. Because rhizomorphs are important for infection and growth of the pathogen through soil, the effect of various amendments to culture media on their formation and laccase production are being tested. Extracts from periderm and cambial tissues of several Prunus species have been tested for their effect on growth and rhizomorph formation. With the exception of P. maackii periderm, there were no major effects. The P. maackii periderm was highly inhibitory to growth and may be related to resistance observed in the stem assays. Addition of several different phenolic compounds to culture media increases the formation of rhizomorphs and structures that resemble fungal growth in planta. The effects of these compounds on the induction of laccase activity is underway. A technique has been developed to detect A. ostoyae in the soil. Small soil samples (one gram or less) proved to be inadequate while much larger samples (approximately 1 kg) processed by sieving and flotation to enrich the organic fraction of the soil was successful in preparing material suitable for PCR analysis. The test has been successful in detecting the pathogen by PCR in soils from infested orchards. A test of Trichoderma species as biological controls in ongoing in microplots. These tests, which have been ongoing for two seasons, will be evaluated for Armillaria infection this fall. PARTICIPANTS: R. Hammerschmidt (PI); Linzi Kaniszewski, research staff, who conducted research on host resistance; J. Jacobs, research staff, worked on detection by PCR; Collaborator: Eric Lizotte, IPM program specialist at NW Michigan Horticulture Station who is involved in locating orchards, field screening 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 infested areas by providing guidance on which rootstocks to use. The use of stem assays has potential of greatly speeding up the screening process. Development of a protocol for detecting Armillaria in soil will aid in evaluations needed for orchard site selection and disease management. Biocontrol and agents, if proven to be effective, may lead to additional management approaches. Research on mechanisms of pathogenicity and host resistance will aid in development of future management methods
Publications
- Warnstrom, E.L., Outwater, C.A., Jacobs, J.L., and Hammerschmidt, R. (2011). Development of an in vitro bioassay to screen Prunus spp. for resistance to Armillaria ostoyae, Phytopathology, 101:S188
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