Retrospective survey of Dickeya fangzhongdai using a novel validated real-time PCR assay

Abstract

Dickeya fangzhongdai, an aggressive plant pathogen, causes symptoms on a variety of crops and ornamental plants including bleeding canker of Asian pear trees. Historical findings stress the need for a specific detection tool for D. fangzhongdai to prevent overlooking the pathogen or assigning it to general Dickeya spp. Therefore, a qualitative real-time PCR for specific detection of D. fangzhongdai has been developed and validated. The developed assay shows selectivity of 100%, diagnostic sensitivity of 76% and limit of detection with 95% confidence interval in plant matrices ranging from 311 to 2,275 cells/mL of plant extracts. The assay was successfully used in a retrospective survey of selected host plants of relevance to Europe and environmental niches relevant to D. fangzhongdai. Samples of potato tubers and plants, plants from the Malinae subtribe (apple, pear, quince, and Asian pear tree) and fresh surface water from Slovenia were analyzed. D. fangzhongdai was not detected in any plant samples, however, 12% of surface water samples were found to be positive.

Keywords: Dickeya; diagnostics; molecular testing; plant pathogen; real-time PCR; survey; water.

FIGURE 1

Logarithmic amplification curves of D. fangzhongdai DNA extracted from spiked plant matrices and spiked surface water for the novel real-time PCR assay Df_tr (A) and assay described by Tian et al. (2020) (B). The curves show bacterial standard curves prepared in potato matrix (green curves), in apple matrix (orange curves), in orchid matrix (blue curves) and in surface water (gray curves). The threshold line for the real-time PCR assay Df_tr is shown in orange and for Tian et al. (2020) in red.

FIGURE 2

Non-linear modeling of probability of detection on spiked plant matrixes (A) and spiked surface water (B) for Df_tr real-time PCR assay (shown in blue and gray) and real-time PCR assay described by Tian et al. (2020) (shown in green). The concentrations shown are expressed as log(cells/mL of plant extract), and in the brackets as cells/mL of plant extracts. The model used for both assays on spiked plant matrixes (A) is two-parameter log-logistic function (LL.2), and models used on spiked surface water (B) is log-logistic function (LL.2) for Df_tr real-time PCR assay and two-parameter Weibull function (W2.2) for real-time PCR assay described by Tian et al. (2020). The dotted line denotes 95% probability of detection.

FIGURE 3

Logarithmic amplification curves of positive surface water samples of real-time PCR Df_tr. Threshold line is shown in magenta. Each sample was analyzed in duplicates, and it was considered positive if at least one reaction was positive.