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United States Department of Agriculture

Agricultural Research Service

Research Project: SUSTAINABLE VINEYARD PRODUCTION SYSTEMS

Location: Crops Pathology and Genetics Research

Title: A RAPID INFECTION ASSAY FOR ARMILLARIA AND REAL-TIME PCR QUANTITATION OF THE FUNGAL BIOMASS IN PLANTA

Authors
item BAUMGARTNER, KENDRA
item Bhat, Ravindra -
item FUJIYOSHI, PHILLIP

Submitted to: Fungal Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 1, 2009
Publication Date: December 10, 2009
Citation: Baumgartner, K., Bhat, R., Fujiyoshi, P.T. 2010. A RAPID INFECTION ASSAY FOR ARMILLARIA AND REAL-TIME PCR QUANTITATION OF THE FUNGAL BIOMASS IN PLANTA. Fungal Biology. 114:107-119.

Interpretive Summary: The lack of a procedure for reliably infecting plants with the root fungus Armillaria mellea in the greenhouse has been a barrier to research on Armillaria root disease. The existing infection assay takes 7-18 months for detectable infection, during which time the inoculum often dies, resulting in some plants never having the chance to become infected. Also, plants do not show symptoms of infection, nor do they die, and this makes it difficult to conduct studies on how the pathogen colonizes roots, when symptoms appear after colonization, and which plant species are better able to resist colonization. We tested a new infection assay, inoculating plants grown in an agar-based gel and comparing grape rootstocks previously characterized from the existing infection assay as tolerant (Freedom) or susceptible (3309C). Culture medium of 25 plants per rootstock was inoculated and 5 plants per rootstock were harvested 0, 2, 4, 6, and 8 weeks post-inoculation; the experiment was completed twice. A microscopy-based method (confocal microscopy) and a DNA-based method (quantitative PCR; Q-PCR) were used to measure the degree of infection. Roots were treated with WGA-AlexaFluor488, hyphae and roots were scanned on green and red channels on a confocal microscope, and percent root colonization was quantified. A fungal gene (EF1') was determined to have a single copy in A. mellea, and both EF1' and a single-copy grape gene (UFGT) were amplified by Q-PCR; fungal DNA: plant DNA served as a measure of fungal biomass. Armillaria was detected by culture, microscopy, and Q-PCR starting 2 weeks post-inoculation from all inoculated plants, demonstrating that the new infection assay is rapid and plants do not escape infection. Our findings of higher percent root colonization (as measured by microscopy) of 3309C than Freedom at all harvests (P<0.0001), consistently higher fungal biomass (as measured by Q-PCR) of 3309 than Freedom, and a significant positive correlation between percent root colonization and fungal biomass (P=0.01) suggests that the quantitative methods of our new assay give similar results to the qualitative method of the existing infection assay.

Technical Abstract: Slow and unreliable infection in the greenhouse has been a barrier to research on Armillaria root disease. The existing infection assay takes 7-18 months for detectable infection, during which time the inoculum often dies, resulting in unequal challenge among plants. As symptom expression and mortality are rare, presence/ absence of infection is the only piece of data from the existing infection assay, which prevents both routine comparisons of strain virulence and complex investigations of pathogenesis, neither of which have been done for Armillaria mellea. We tested a new infection assay, inoculating plants in tissue culture medium and comparing grape rootstocks previously characterized from the existing infection assay as tolerant (Freedom) or susceptible (3309C). Culture medium of 25 plants per rootstock was inoculated and 5 plants per rootstock were harvested 0, 2, 4, 6, and 8 weeks post-inoculation; the experiment was completed twice. Confocal microscopy and quantitative PCR (Q-PCR) were used to quantify infection. Roots were treated with WGA-AlexaFluor488, hyphae and roots were scanned on green and red channels on a confocal microscope, and percent root colonization was quantified. A fungal gene (EF1') was determined to have a single copy in A. mellea, and both EF1' and a single-copy grape gene (UFGT) were amplified by Q-PCR; fungal DNA: plant DNA served as a measure of fungal biomass. Armillaria was detected by culture, microscopy, and Q-PCR starting 2 weeks post-inoculation from all inoculated plants, demonstrating that the new infection assay is rapid and plants do not escape infection. Our findings of higher percent root colonization (as measured by microscopy) of 3309C than Freedom at all harvests (P<0.0001), consistently higher fungal biomass (as measured by Q-PCR) of 3309 than Freedom, and a significant positive correlation between percent root colonization and fungal biomass (P=0.01) suggests that the quantitative methods of our new assay give similar results to the qualitative method of the existing infection assay.

Last Modified: 8/27/2014
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