Comparative genomics and genomic diversity of Pseudomonas syringae clade 2b-a in Australia

Abstract

Background A zucchini disease outbreak with unusual symptoms associated with Pseudomonas syringae clade 2b was identified in Bundaberg, Australia during autumn 2016. To investigate the genetic diversity of the 11 Australian isolates obtained from the outbreak, the genomes were compared to the publicly available P. syringae strains in phylogroup 2. Results Average nucleotide identity refined the P. syringae clade 2b-a into four clusters (Cluster A, B, C1 and C2), an expansion from the previously identified A, B and C. Australian isolates were in Cluster A, C1 and C2. Genomic analyses highlighted several key factors that may contribute to the virulence of these isolates. Six orthologous groups, including three virulence factors, were associated with P. syringae phylogroup 2 cucurbit-infecting strains. A region of genome plasticity analysis identified a type VI secretion system pathway in clade 2b-a strains which could also contribute to virulence. Pathogenicity assays on isolates KL004-k1, KFR003-1 and 77-4C, as representative isolates of Cluster A, C1 and C2, respectively, determined that all three isolates can infect pumpkin, squash, watermelon and zucchini var. Eva with different levels of disease severity. Subsequently, type III effectors were investigated and four type III effectors (avrRpt2, hopZ5, hopC1 and hopH1) were associated with host range. The hopZ effector family was also predicted to be associated with disease severity. Conclusions This study refined the taxonomy of the P. syringae clade 2b-a, supported the association between effector profile and pathogenicity in cucurbits established in a previous study and provides new insight into important genomic features of these strains. This study also provided a detailed and comprehensive resource for future genomic and functional studies of these strains.

Development of tools to detect and identify strains belonging to thePseudomonas syringaespecies complex responsible for vein clearing of zucchini

“…The test based on sylC was multiplexed with qPCR tests targeting avrRpt2 and hopZ5. The gene encoding the T3E AvrRpt2 is present in all VCZ strains that have a narrow host range on cucurbits (Lacault et al 2020;Djitro et al 2022a) but also in the pathovar tomato (Innes et al 1993) and was found in some strains of pathovars maculicola, spinaceae and tagetis that were not known to have it. Indeed, avrRpt2 was not included in the large analysis of the distribution of virulence encoding genes including 53 T3Es in Pseudomonas syringae made by Sarkar et al (2006).…”

“…Individually the three PCR tests targeting genes encoding the pathogenicity factors SylC, AvrRpt2 and HopZ5 were not specific for VCZ strains (Supplementary Table 2). However, VCZ strains from main clusters are characterized by the simultaneous presence of sylC and either avrRpt2 or hopZ5 (Figure 2 and Table 1), as avrRpt2 characterizes 2ba strains that have a narrow host range on cucurbits whereas hopZ5 is present in strains with a wider host range (Lacault et al 2020;Djitro et al 2022a). Clade-2b* and -2d strains were also isolated from zucchini seeds and gave a positive signal with 4Ba test (targeting sylC) justifying the need to include their corresponding qPCR tests in the multiplex.…”

“…Two closely related clusters, clusters 2ba-C and 2a-F, group strains that have been sampled from watermelon, cantaloupe and squashes in the USA (Newberry et al 2019). Recently, clusters 2ba-A and 2ba-C strains causing unusual symptoms on zucchini, such as twisted petioles, necrotic leaves, crown-rots and internal fruit rots have been reported in Australia (Djitro et al 2022a, b). An extended MultiLocus Sequence Analysis (MLSA) scheme has been proposed to allocate strains in these clusters and facilitate the analysis of large strain collections (Lacault et al 2020).…”