New Pseudomonas spp. Are Pathogenic to Citrus

Abstract

Five putative novel Pseudomonas species shown to be pathogenic to citrus have been characterized in a screening of 126 Pseudomonas strains isolated from diseased citrus leaves and stems in northern Iran. The 126 strains were studied using a polyphasic approach that included phenotypic characterizations and phylogenetic multilocus sequence analysis. The pathogenicity of these strains against 3 cultivars of citrus is demonstrated in greenhouse and field studies. The strains were initially grouped phenotypically and by their partial rpoD gene sequences into 11 coherent groups in the Pseudomonas fluorescens phylogenetic lineage. Fifty-three strains that are representatives of the 11 groups were selected and analyzed by partial sequencing of their 16S rRNA and gyrB genes. The individual and concatenated partial sequences of the three genes were used to construct the corresponding phylogenetic trees. The majority of the strains were identified at the species level: P. lurida (5 strains), P. monteilii (2 strains), P. moraviensis (1 strain), P. orientalis (16 strains), P. simiae (7 strains), P. syringae (46 strains, distributed phylogenetically in at least 5 pathovars), and P. viridiflava (2 strains). This is the first report of pathogenicity on citrus of P. orientalis, P. simiae, P. lurida, P. moraviensis and P. monteilii strains. The remaining 47 strains that could not be identified at the species level are considered representatives of at least 5 putative novel Pseudomonas species that are not yet described.

Fig 1. Pathogenicity tests and disease severity comparison for all Pseudomonas strains analyzed.

A. Pathogenicity test in Sour Orange citrus leaves for each group of strains. B. Disease severity comparison of Pseudomonas strains of each group tested in three plant genotypes, measured by the lesion area expressed in mm² two weeks after inoculation. The number of strains and the number of non-pathogenic strains for each group tested are indicated below each graph. The black horizontal line represents the median and the red horizontal line represents the mean values (if only 2 values are considered, mean and median values are identical); the boundary of the box closest to zero indicates the 25^th percentile and the boundary farthest from zero indicates 75^th percentile. Whiskers (error bars) above and below the box indicate the 90^th and 10^th percentiles. The black dots indicate outlier values.

Fig 2. Phenotypic clustering of the studied strains.

A similarity matrix based on the phenotypic traits was generated using a simple matching coefficient and the dendrogram constructed by UPGMA.

Fig 3. Phylogenetic tree of the Pseudomonas strains based on the nucleotide sequences of the rpoD gene (717 nt).

The scale bar represents the number of substitutions per site. The number shown next to each node indicates the percentage bootstrap values of 1000 replicates. Cellvibrio japonicus was used as the outgroup.

Fig 4. Phylogenetic tree based on the rpoD gene sequence (455 nt) of the strains assigned to the P. syringae species complex including the pathovar reference strains and strains of the 13 phylogroups (PG) defined by Berge et al. [26].

Fig 5. Phylogenetic consensus tree of the Pseudomonas strains based on the nucleotide sequences of the 16S rRNA, rpoD, and gyrB genes of 53 selected strains.

The scale bar represents the number of substitutions per site. The number shown next to each node indicates the percentage bootstrap values of 1000 replicates. Cellvibrio japonicus was used as the outgroup.