Monitoring pathogenesis of natural Botrytis cinerea infections in developing grape berries

Authors: Cadle-Davidson, Lance

Submitted to: American Journal of Enology and Viticulture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/20/2008
Publication Date: 12/15/2008
Citation: Cadle Davidson, L.E. 2008. Monitoring pathogenesis of natural Botrytis cinerea infections in developing grape berries. American Journal of Enology and Viticulture. 59:387-395.

Interpretive Summary: Botrytis cinerea infects grape flowers and young grape berries soon after flowering. After colonizing the berry, fungal growth stops and the pathogen becomes quiescent. No signs or symptoms of infection, quiescence, and activation are apparent until the initiation of berry ripening, or veraison. To detect these aspects of B. cinerea development and inform vineyard management decisions, a real-time quantitative PCR (qPCR) assay was developed and tested alongside the standard assay for early detection of B. cinerea, based on tissue freezing and incubation. With the qPCR assay, quantification of B. cinerea DNA in total DNA extracted from grape berries allowed the estimation of fungal biomass, and thus, the degree to which B. cinerea had colonized the berry. In 2004 and 2005, this qPCR assay was applied with the freezing assay to detect natural infections through berry development across a diverse panel of 32 genotypes of Vitis spp. and interspecific hybrids. The qPCR and freezing assays detected infection levels of B. cinerea in both 2004 and 2005 appropriate to the actual disease severity (22.5% and 1.0% bunch rot severity, respectively). Grape genotypes varied in their resistance to infection, degree of colonization, and severity of disease. qPCR was not as effective as the freezing assay for detecting infection at early stages of development but was able to quantify fungal colonization, resulting in a new capability for monitoring B. cinerea pathogenesis. The combined ability of the two assays to detect B. cinerea early in berry development and monitor quiescence and activation provides a resource for informing disease management decisions and for identifying mechanisms of disease resistance.

Technical Abstract: Quiescent infections play key roles in Botrytis cinerea pathogenesis and in the management of Botrytis bunch rot. To detect infection, quiescence, and activation of B. cinerea, a real-time quantitative PCR (qPCR) assay was developed and tested alongside the standard assay for early detection of B. cinerea, based on tissue freezing and incubation. A SYBR Green qPCR assay quantified as little as 3.2pg of B. cinerea DNA accurately in the background of 20ng of grape DNA, but low natural levels of infection by B. cinerea could not be discriminated from non-specific amplification of grape DNA. A Taqman® qPCR assay also quantified as little as 3.2pg of B. cinerea DNA accurately, with a detection limit of 100fg, and did not amplify grape DNA. In 2004 and 2005, this qPCR assay was applied in parallel with the freezing assay to detect natural infections through berry development across a diverse panel of 32 genotypes of Vitis spp. and interspecific hybrids. The qPCR and freezing assays detected infection levels of B. cinerea in both 2004 and 2005 appropriate to the actual disease severity (22.5% and 1.0% bunch rot severity, respectively). Grape genotypes varied in their resistance to infection, degree of colonization, and severity of disease. qPCR was not as effective as the freezing assay for detecting infection at early stages of development but was able to quantify fungal colonization, resulting in a new capability for monitoring B. cinerea pathogenesis. The combined ability of the two assays to detect B. cinerea early in berry development and monitor quiescence and activation provides a resource for informing disease management decisions and for identifying mechanisms of disease resistance.