RESUMO
P. brasiliense is an important bacterial pathogen causing blackleg (BL) in potatoes. Nevertheless, P. brasiliense is often detected in seed lots that do not develop any of the typical blackleg symptoms in the potato crop when planted. Field bioassays identified that P. brasiliense strains can be categorized into two distinct classes, some able to cause blackleg symptoms and some unable to do it. A comparative pangenomic approach was performed on 116 P. brasiliense strains, of which 15 were characterized as BL-causing strains and 25 as non-causative. In a genetically homogeneous clade comprising all BL-causing P. brasiliense strains, two genes only present in the BL-causing strains were identified, one encoding a predicted lysozyme inhibitor Lprl (LZI) and one encoding a putative Toll/interleukin-1 receptor (TIR) domain-containing protein. TaqMan assays for the specific detection of BL-causing P. brasiliense were developed and integrated with the previously developed generic P. brasiliense assay into a triplex TaqMan assay. This simultaneous detection makes the scoring more efficient as only a single tube is needed, and it is more robust as BL-causing strains of P. brasiliense should be positive for all three assays. Individual P. brasiliense strains were found to be either positive for all three assays or only for the P. brasiliense assay. In potato samples, the mixed presence of BL-causing and not BL-causing P. brasiliense strains was observed as shown by the difference in Ct value of the TaqMan assays. However, upon extension of the number of strains, it became clear that in recent years additional BL-causing lineages of P. brasiliense were detected for which additional assays must be developed.
RESUMO
BACKGROUND: Bacterial plant pathogens of the Pectobacterium genus are responsible for a wide spectrum of diseases in plants, including important crops such as potato, tomato, lettuce, and banana. Investigation of the genetic diversity underlying virulence and host specificity can be performed at genome level by using a comprehensive comparative approach called pangenomics. A pangenomic approach, using newly developed functionalities in PanTools, was applied to analyze the complex phylogeny of the Pectobacterium genus. We specifically used the pangenome to investigate genetic differences between virulent and avirulent strains of P. brasiliense, a potato blackleg causing species dominantly present in Western Europe. RESULTS: Here we generated a multilevel pangenome for Pectobacterium, comprising 197 strains across 19 species, including type strains, with a focus on P. brasiliense. The extensive phylogenetic analysis of the Pectobacterium genus showed robust distinct clades, with most detail provided by 452,388 parsimony-informative single-nucleotide polymorphisms identified in single-copy orthologs. The average Pectobacterium genome consists of 47% core genes, 1% unique genes, and 52% accessory genes. Using the pangenome, we zoomed in on differences between virulent and avirulent P. brasiliense strains and identified 86 genes associated to virulent strains. We found that the organization of genes is highly structured and linked with gene conservation, function, and transcriptional orientation. CONCLUSION: The pangenome analysis demonstrates that evolution in Pectobacteria is a highly dynamic process, including gene acquisitions partly in clusters, genome rearrangements, and loss of genes. Pectobacterium species are typically not characterized by a set of species-specific genes, but instead present themselves using new gene combinations from the shared gene pool. A multilevel pangenomic approach, fusing DNA, protein, biological function, taxonomic group, and phenotypes, facilitates studies in a flexible taxonomic context.
Assuntos
Pectobacterium , Solanum tuberosum , Europa (Continente) , Pool Gênico , Pectobacterium/genética , Filogenia , Doenças das Plantas , Solanum tuberosum/genéticaRESUMO
Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.