CRISPR genotyping as complementary tool for epidemiological surveillance of Erwinia amylovora outbreaks

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.

Fig 1. Identification of Erwinia amylovora Portuguese isolates by PCR, using specific primer pairs, according to EPPO protocol [59].

Bacterial isolates are identified by strain numbers on the top. Primers used are listed on the right, and DNA product sizes in bp are indicated on the left. Positive control (C+): Type Strain LMG 2024. Negative control (C-): H₂O.

Fig 2. Dot blot results.

Dot blot using three probes (hrpL^M, hrpN^M and amsG^M) and genomic DNA from 36 Erwinia amylovora isolates. The top grid represents the position of the DNA from each E. amylovora isolate in the nylon membrane. Positive control (C+): Type Strain LMG 2024. Negative control (C-): TE Buffer.

Fig 3. CRISPR arrays patterns of the 36 Portuguese Erwinia amylovora isolates and type strain LMG 2024.

CR1/2 and 3 array patterns for the strains ATCC 49946, were retrieved from McGhee and Sundin [40] and Rezzonico et al. [39]. Isolates identification are listed on the left of each color pattern. Each spacer is represented by a single color for each CRISPR array. Spacers were considered unique if they contained at least 5 nucleotide differences compared to other spacers. Each spacer is identified by a color and a number in the upper side of each CR. Spacers that are equal are aligned in column for the different isolates in study. The blank intervals represent a spacer that it is not present in that CR pattern. Patterns/genotypes are identified by a number and a letter respectively in the right side of the color pattern. Nomenclature of each spacer, and pattern/genotype were based on the classification of McGhee and Sundin [40], and Rezzonico et al. [39], respectively.

Fig 4. Virulence assay results.

Symptoms observed in immature pear slices 7 days after infection with Erwinia amylovora isolates, were categorized according a damage scale adapted from Schwarczinger et al. [69]. Positive control (C+): Type Strain LMG 2024. Negative control (C-): PBS Buffer.