Faculty of Sciences of University of Porto, Porto, Portugal.
LAQV/REQUIMTE, Faculty of Sciences of University of Porto, Porto, Portugal.
PLoS One. 2021 Apr 16;16(4):e0250280. doi: 10.1371/journal.pone.0250280. eCollection 2021.
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.
火疫病是一种由韧皮部杆菌引起的破坏性植物疾病，影响苹果和梨等果实树木，导致产量大幅下降、植物检疫长期受限和经济损失巨大。在葡萄牙，2010 年和 2011 年首次发生大规模火疫病疫情，尽管后来认为已被根除，但近年来其他疫情的爆发强调了需要对与这些疫情相关的韧皮部杆菌种群进行特征描述。在这方面，进行了 CRISPR 基因分型、三个毒力标记物的评估和半定量毒力生物测定，以确定基因型，并评估与 2010 年至 2017 年期间发生并影响该国中西部（称为葡萄牙主要的果实产区）的苹果和梨园有关的 36 个韧皮部杆菌分离株的毒力。收集的数据表明，35 个韧皮部杆菌分离株属于广泛分布的 CRISPR 基因型（5-24-38 / D-a-α）之一，无论宿主物种、年份和地区如何。Ea 680 是唯一一个由于新的 CR2 间隔子位于靠近前导序列而揭示新 CRISPR 基因型的分离株，因此被认为是最近获得的。关于致病性，尽管斑点杂交分析显示所有研究的韧皮部杆菌分离株都存在关键毒力因子，即 hrpL（T3SS）、hrpN（T3E）和来自淀粉聚糖生物合成操纵子的 amsG，但在不成熟的梨片上进行的致病性生物测定可以区分出四种毒力水平，大多数分离株显示出中度至重度的毒力表型。尽管 2010 年至 2017 年期间在葡萄牙发生的疫情中与韧皮部杆菌相关的克隆种群结构不同，但不同的毒力表型表明，韧皮部杆菌可能在不同时间被引入该国。这是葡萄牙首次关于韧皮部杆菌的研究，揭示了该细菌的一个新的 CRISPR 基因型。
