Characterization of Pseudomonas syringae Isolated from Systemic Infection of Zucchini in Australia

Abstract

Zucchini plants, with symptoms including twisted petioles, necrotic leaves, crown-rot and internal fruit-rot, were found in Bundaberg, Australia at a commercial field for the first time during late autumn 2016, resulting in direct yield losses of 70 to 80%. Three Pseudomonas syringae strains that isolated from symptomatic leaf (KL004-k1), crown (77-4C) and fruit (KFR003-1) were characterised and their pathogenicity evaluated on pumpkin, rockmelon, squash and zucchini. Biochemical assays showed typical results for P. syringae. The three isolates differed, however, in that two produced fluorescent pigment (KFR003-1 and 77-4C) whilst the third, KL004-k1, was non-florescent. Multi-locus sequence analysis classified the isolates to phylogroup 2b. The SNP analysis of core genome from the Australian and closely related international isolates of P. syringae showed two separate clusters. The Australian isolates were clustered based on fluorescent phenotype. Pathogenicity tests demonstrated all three isolates moved systemically within the inoculated plants and induced necrotic leaf symptoms in zucchini plants. Their identities were confirmed with specific PCR assays for P. syringae and phylogroup 2. Pathogenicity experiments also showed the Eva variety of zucchini was more susceptible than Rosa for all three isolates. Isolate KL004-k1 was more virulent than 77-4C on pumpkin, rockmelon, squash and zucchini. This study expands the knowledge of P. syringae isolates that infect cucurbits and provides useful information for growers about the relative susceptibility of a range of cucurbit species.

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

“…In 2016, unusual symptoms were observed in zucchini fields in Bundaberg including twisted petioles, necrotic leaves, crown-rots and internal fruit-rots. Eleven P. syringae isolates that obtained from the outbreak were in clade 2b [ 2 ] and closely related to other P. syringae in clade 2b-a [ 3 ]. The recent detection of this unusual disease and identification of P. syringae isolates genetically distinct from previously detected isolates affecting cucurbit in Australia [ 2 ] suggest a recent introduction of these isolates.…”

“…Eleven P. syringae isolates that obtained from the outbreak were in clade 2b [ 2 ] and closely related to other P. syringae in clade 2b-a [ 3 ]. The recent detection of this unusual disease and identification of P. syringae isolates genetically distinct from previously detected isolates affecting cucurbit in Australia [ 2 ] suggest a recent introduction of these isolates. This was most likely through imported seed for production.…”

“…Comparative genomics of virulence factors in the P. syringae complex have shown that strains from phylogroup 2 harbor fewer type III effectors (T3Es) but can produce more phytotoxins, compared to strains from the other primary phylogroups [ 6 – 8 ]. Over the last few years, many P. syringae strains in phylogroup 2 associated with Cucurbitaceae have been isolated and their genetic diversity, pathogenicity and host-association have been studied in detail [ 2 , 3 , 9 – 11 ]. Pan-genome association analysis of P. syringae phylogroup 2 cucurbit-infecting strains identified seven genes that potentially contribute to niche adaptation to cucurbit hosts including T3Es hopZ5 , hopA1 and its chaperone shcA , type VI secreted effector vgrG , and three uncharacterised proteins [ 3 ].…”

“…Clade 2b-a is a branch group within clade 2b [ 3 ] and consists of P. syringae isolated from Cucurbitaceae including pumpkin ( Cucurbita moschata ), melon ( Cucumis melo ), squash ( Cucurbita pepo ), watermelon ( Citrullus lanatus ) and zucchini ( Cucurbita pepo ) [ 2 , 3 , 9 – 11 ]. This clade emerged from genome-wide homologous recombination between clade 2a and 2b [ 3 ].…”

“…In this study, eleven P. syringae isolates obtained from a zucchini disease outbreak in Australia [ 2 ] were compared to the publicly available genomes of P. syringae phylogroup 2. As these Australian isolates were determined to be P. syringae clade 2b, their genetic diversity and effector profile in relation to previous reports [ 3 , 11 ] will further inform the genetic basis of their observed phenotypes.…”