Real‐time PCR detection of Erwinia amylovora on blossoms correlates with subsequent fire blight incidence

Abstract

Fire blight is the most devastating bacterial disease of rosaceous plants. Forecasting fire blight infections is important to allow for countermeasures that reduce economic damage in pome fruit production. Current computerized forecasting models are solely based on physical factors such as temperature and moisture, but not on the actual presence of the pathogen Erwinia amylovora. Although the inoculum concentration is considered to be crucial for infection and disease outbreak, most current approaches used for identification of fire blight inoculum including morphological, biochemical, serological, and DNA-based methods are nonquantitative. Based on a real-time PCR approach previously published, an improved protocol to be used directly on whole bacteria in the field is described. The method allows for early detection and quantification of the pathogen prior to the occurrence of first symptoms. There is a clear correlation between bacterial abundance and subsequent disease development. However, in some cases, no disease symptoms could be observed despite a pathogen load of up to 3Á4 9 10 6 cells per blossom. Integration of the amount of pathogen detected into refined prediction algorithms may allow for the improvement of applied forecasting models, finally permitting a better abatement of fire blight.

Alternative inoculum sources for fire blight: the potential role of fruit mummies and non‐host plants

“…Real‐time PCR analyses were performed using a modified protocol derived from Salm & Geider () using primers p29TF and p29TR as previously described in Voegele et al . () (for details see Hinze et al ., ). Samples consisted of extraction or washing fluids containing intact bacteria.…”

“…The quantification was individually adapted to the different PCR cycler systems and reaction kits used as described in Hinze et al . (). Each sample was measured twice and the resulting cell counts were averaged.…”

“…To correlate E. amylovora detection with subsequent infestation in an orchard the severity of each observed fire blight incidence was graded (Hinze et al ., ) as follows: grade 0, no symptoms visible; grade 1, single blossom clusters with symptoms in an orchard on <10% of trees; grade 2, ≥10% of trees infested of all trees in an orchard showing symptoms; grade 3, symptoms appearing on every tree in an orchard; grade 4, multiple blossom clusters with symptoms on every tree.…”

“…These plants might also be exposed to insect vectors, wind and rain splash, which are well‐known mechanisms for the dispersal of bacteria (McManus & Jones, ; Thomson, ; Sabatini et al ., ). Their blossoms might offer similar conditions for epiphytic survival of the bacteria as those of host plants (Hinze et al ., ). Only a few studies have dealt with the occurrence of E. amylovora on non‐host plants, especially outside the family Rosaceae.…”

“…In this study, an improved real‐time PCR approach (Hinze et al ., ) is used to investigate the possible role of fruit mummies and buds in overwintering and as alternative inoculum sources for dissemination of E. amylovora in the spring. Furthermore, the role of symptomless non‐host plants in epiphytic survival and multiplication of the bacteria, inconspicuously contributing to the epidemiology of fire blight, is investigated by analysis of field samples and in vitro experiments.…”