Tracking the dissemination of Erwinia amylovora in the Eurasian continent using a PCR targeted on the duplication of a single CRISPR spacer

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.

Variability within a clonal population of Erwinia amylovora disclosed by phenotypic analysis

“…First fire blight outbreaks appeared in 2010 in Portugal, being persistent and with periodic outbreaks, which cause dramatic economic impacts mostly to apple and pear producers ( EPPO, 2018 ; Mendes et al, 2021a ; Mendes et al, 2021b ). A study from Kurz et al (2021) that includes six strains from E. amylovora isolates from 2010 and 2011 has shown low diversity regarding those strains, with only two CRISPR profiles disclosed, similar to our previous findings ( Mendes et al, 2021a ), namely a CRISPR profiling of Portuguese representative temporal/local populations of E. amylovora associated with outbreaks occurring between 2010 and 2017, which displayed two CRISPR profiles. Nevertheless, it was also revealed that despite the high clonality of the studied E. amylovora isolates, distinct levels of virulence were observed ( Mendes et al, 2021a ).…”

Function and Application of the CRISPR-Cas System in the Plant PathogenErwinia amylovora

“…Since the late 18th century, the WP clade has spread from New York State to nearly all apple producing countries and currently accounts for all sequenced E. amylovora isolates found outside North America ( 3 , 7 ). These non-North American isolates are found exclusively within group Ia.…”

“…Recently, CRISPR spacer analysis was used to track the dissemination of E. amylovora across Eurasia, Portugal, and New York State. PCRs were designed to amplify either the entire CRISPR array ( 4 , 5 ) or a spacer, designated 1029, which had duplicated within the CRR1 array ( 3 ). Both of these methodologies are capable of a high level of differentiation within the WP clade.…”

“…There are three major Amygdaloideae -infecting clades of E. amylovora , all of which originate from North America. The Widely Prevalent (WP) clade, the most commonly isolated group of E. amylovora , was disseminated globally through the movement of infected material in the early to mid 1900s ( 3 , 6 ). The Eastern North American (ENA) and Western North American (WNA) clades are found strictly in the continental USA and Canada in the eastern and western pome fruit growing regions, respectively ( 7 , 8 ).…”

“…The presence and order of CRISPR spacers creates a heritable record of significant iMGE pressures within the bacterium’s environment and creates a pattern by which phylogeny can be elucidated ( 1 , 2 ). This technique has been used extensively to describe the WP clade across Eurasia, Portugal, and the northeastern USA ( 3 – 5 , 13 ). PCR-based techniques can effectively describe the WP clade and can be used to create the observed CRISPR profiles because of the small size and high level of conservation found within those CRISPR arrays.…”