No abstract

Fire blight is the most devastating disease affecting pome fruit production globally. The pathogen is native to North America and was imported to western Europe in the 1950s, progressively spreading over the continent in the ensuing decades. Previous phylogenetic studies have revealed the extreme genetic homogeneity of the pathogen outside its center of origin, which makes epidemiological studies difficult. These are generally only possible using hypervariable regions of the genome such as those represented by CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats), which are, however, not practical to sequence due to their size and variability. Here, we present a simple PCR assay targeting the duplication of a single CRISPR spacer in Erwinia amylovora that was found to be an important marker to discriminate between two main European populations of the pathogen. We implemented the assay on a total of 582 isolates to follow the spread of fire blight across the continent over several decades and, wherever possible, within single countries. The results obtained point to the occurrence of two major separate introduction events for E. amylovora in Europe that occurred approximately 20 years apart, and confirmed the existence of two principal distribution areas located in Northeastern Europe and the Eastern Mediterranean Basin from which the pathogen moved on to colonize the Eurasian continent.

No abstract

Lateral flow immunoassay (LFIA) is a convenient tool for rapid field-based control of various bacterial targets. However, for many applications, the detection limits obtained by LFIA are not sufficient. In this paper, we propose enlarging gold nanoparticles’ (GNPs) size to develop a sensitive lateral flow immunoassay to detect Ralstonia solanacearum. This bacterium is a quarantine organism that causes potato brown rot. We fabricated lateral flow test strips using gold nanoparticles (17.4 ± 1.0 nm) as a label and their conjugates with antibodies specific to R. solanacearum. We proposed a signal enhancement in the test strips’ test zone due to the tetrachloroauric (III) anion reduction on the GNP surface, and the increase in size of the gold nanoparticles on the test strips was approximately up to 100 nm, as confirmed by scanning electron microscopy. Overall, the gold enhancement approach decreased the detection limit of R. solanacearum by 33 times, to as low as 3 × 104 cells∙mL–1 in the potato tuber extract. The achieved detection limit allows the diagnosis of latent infection in potato tubers. The developed approach based on gold enhancement does not complicate analyses and requires only 3 min. The developed assay together with the sample preparation and gold enlargement requires 15 min. Thus, the developed approach is promising for the development of lateral flow test strips and their subsequent introduction into diagnostic practice.

Fire blight, a bacteriosis of apple and pear, was assayed with molecular tools to associate its origin in Russia, Slovenia and south-eastern Austria with neighboring countries. The identification of all investigated strains was confirmed by MALDI-TOF mass spectroscopy except one. Independent isolation was verified by the level of amylovoran synthesis and by the number of short sequence DNA repeats in plasmid pEA29. DNA of gently lysed E. amylovora strains from Russia, Slovenia, Austria, Hungary, Italy, Spain, Croatia, Poland, Central Europe and Iran was treated with restriction enzymes XbaI and SpeI to create typical banding patterns for PFGE analysis. The pattern Pt2 indicated that most Russian E. amylovora strains were related to strains from Turkey and Iran. Strains from Slovenia exhibited patterns Pt3 and Pt2, both present in the neighboring countries. Strains were also probed for the recently described plasmid pEI70 detected in Pt1 strains from Poland and in Pt3 strains from other countries. The distribution of pattern Pt3 suggests distribution of fire blight from Belgium and the Netherlands to Central Spain and Northern Italy and then north to Carinthia. The PFGE patterns indicate that trade of plants may have introduced fire blight into southern parts of Europe proceeded by sequential spread.