Abstract
Fire blight is a destructive plant disease caused by Erwinia amylovora affecting pome fruit trees, and responsible for large yield declines, long phytosanitary confinements, and high economic losses. In Portugal, the first major fire blight outbreaks occurred in 2010 and 2011, and although later considered eradicated, the emergence of other outbreaks in recent years stressed the need to characterize the E. amylovora populations associated with these outbreaks. In this regard, CRISPR genotyping, assessment of three virulence markers, and semi-quantitative virulence bioassays, were carried out to determine the genotype, and assess the virulence of thirty-six E. amylovora isolates associated with outbreaks occurring between 2010 and 2017 and affecting apple and pear orchards located in the country central-west, known as the main producing region of pome fruits in Portugal. The data gathered reveal that 35 E. amylovora isolates belong to one of the widely-distributed CRISPR genotypes (5-24-38 / D-a-α) regardless the host species, year and region. Ea 680 was the single isolate revealing a new CRISPR genotype due to a novel CR2 spacer located closer to the leader sequence and therefore thought to be recently acquired. Regarding pathogenicity, although dot-blot hybridization assays showed the presence of key virulence factors, namely hrpL (T3SS), hrpN (T3E) and amsG from the amylovoran biosynthesis operon in all E. amylovora isolates studied, pathogenicity bioassays on immature pear slices allowed to distinguish four virulence levels, with most of the isolates revealing an intermediate to severe virulence phenotype. Regardless the clonal population structure of the E. amylovora associated to the outbreaks occurring in Portugal between 2010 and 2017, the different virulence phenotypes, suggests that E. amylovora may have been introduced at different instances into the country. This is the first study regarding E. amylovora in Portugal, and it discloses a novel CRISPR genotype for this bacterium.
Highlights
Erwinia amylovora, an Erwiniaceae bacterium species [1], is the etiological agent of fire blight, a destructive plant disease that affects the productive potential of trees, and the entire pome fruit trees production sector, of enormous value in Portugal, belonging to the Amygdaloideae subfamily, previously named Spiraeoideae [2] of the Rosaceae family [3, 4]
The 36 bacterial isolates were identified as E. amylovora, using four species-specific DNA markers recommended by EPPO for diagnosis [59]
For the 36 bacterial isolates tested, all four markers were amplified with product sizes corresponding to the expected length of the markers (187, 391, 458, and 1296 bp, for G1-F+G2-R, PEANT1+PEANT2, FER1-F+rgER2R, and FER1-F+FER1-R, respectively), except for the isolate Ea 630, for which no amplification was obtained with primers PEANT1+PEANT2, targeting the plasmid pEA29 specific marker (Fig 1)
Summary
An Erwiniaceae bacterium species [1], is the etiological agent of fire blight, a destructive plant disease that affects the productive potential of trees, and the entire pome fruit trees production sector, of enormous value in Portugal, belonging to the Amygdaloideae subfamily, previously named Spiraeoideae [2] of the Rosaceae family [3, 4]. E. amylovora enters through natural openings and wounds in host plants, colonizing the intercellular spaces, migrating into the vascular system, and producing biofilms that block xylem vessels inhibiting water transport. This excessive biofilm production can lead to originate bacterial exudates [9,10,11,12]. These exudates are acknowledged as major sources of E. amylovora dissemination to other nearby hosts through wind, water, insects and by cultural practices, namely pruning using contaminated tools [11, 13]
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