Development of a real-time PCR method for the specific detection of the novel pear pathogen Erwinia uzenensis

Abstract

Erwinia uzenensis is a plant-pathogenic bacterium, recently described in Japan, which infects pear trees, causing the ‘bacterial black shoot disease of European pear’ (BBSDP). Like other Erwinia pear pathogens, E . uzenensis causes damp, black lesions on young shoots resembling those of E . amylovora , but not blossom blight, fruitlet blight or wilting of the shoot tip. The distribution of E . uzenensis seems restricted to the country where it was reported up to now, but it may spread to other countries and affect new hosts, as is the current situation with E . piriflorinigrans and E . pyrifoliae . Fast and accurate detection systems for this new pathogen are needed to study its biology and to identify it on pear or other hosts. We report here the development of a specific and sensitive detection protocol based on a real-time PCR with a TaqMan probe for E . uzenensis , and its evaluation. In sensitivity assays, the detection threshold of this protocol was 10 1 cfu ml -1 on pure bacterial cultures and 10 2 –10 3 cfu ml -1 on spiked plant material. The specificity of the protocol was evaluated against E . uzenensis and 46 strains of pear-associated Erwinia species different to E . uzenensis . No cross-reaction with the non-target bacterial species or the loss of sensitivity were observed. This specific and sensitive diagnostic tool may reveal a wider distribution and host range of E . uzenensis initially considered restricted to a region and will expand our knowledge of the life cycle and environmental preferences of this pathogen.

Development of a multiplex real-time PCR method for the detection of Pseudomonas savastanoi pv. glycinea and Curtobacterium flaccumfaciens pv. flaccumfaciens in soybean seeds

“…Bacterial suspension was spread using a sterile spatula and incubated at 28 °С for 96 h. Bacterial colonies were counted and used to calculate concentrations in each dilution. Each suspension was used for qPCR analysis as described by Holeva et al (2019). For this, 2 µl of bacterial suspension was added to 23 µl of the reaction mixture for PCR.…”

Pre-Symptomatic Detection of Viral Infection in Tobacco Leaves Using PAM Fluorometry

“…In current agricultural practice, the presence of diseases in plants is most often assessed visually; however, this approach cannot be considered sufficiently reliable and objective. Along with traditional visual diagnostics, ELISA, PCR, sequencing, and fluorescence in situ hybridization (FISH) methods are used to accurately determine plant diseases [14][15][16][17][18]. These methods make it possible to precisely detect the pathogen and determine its taxonomic affiliation.…”