Phylogeny and virulence of naturally occurring type III secretion system-deficient Pectobacterium strains

Abstract

Pectobacterium species are enterobacterial plant-pathogenic bacteria that cause soft rot disease in diverse plant species. Previous epidemiological studies of Pectobacterium species have suffered from an inability to identify most isolates to the species or subspecies level. We used three previously described DNA-based methods, 16S-23S intergenic transcribed spacer PCR-restriction fragment length polymorphism analysis, multilocus sequence analysis (MLSA), and pulsed-field gel electrophoresis, to examine isolates from diseased stems and tubers and found that MLSA provided the most reliable classification of isolates. We found that strains belonging to at least two Pectobacterium clades were present in each field examined, although representatives of only three of five Pectobacterium clades were isolated. Hypersensitive response and DNA hybridization assays revealed that strains of both Pectobacterium carotovorum and Pectobacterium wasabiae lack a type III secretion system (T3SS). Two of the T3SS-deficient strains assayed lack genes adjacent to the T3SS gene cluster, suggesting that multiple deletions occurred in Pectobacterium strains in this locus, and all strains appear to have only six rRNA operons instead of the seven operons typically found in Pectobacterium strains. The virulence of most of the T3SS-deficient strains was similar to that of T3SS-encoding strains in stems and tubers.

FIG. 1.

Sixteen of 45 Pectobacterium isolates obtained in 2004 and P. wasabiae SCRI488 were unable to elicit an HR on N. tabacum cv. Xanthi. Bacterial isolates were infiltrated into the leaves of 6- to 7-week-old N. tabacum cv. Xanthi, and results were recorded the following day. Leaf panels 1 through 8 were infiltrated with Pectobacterium sp. strains WPP14, WPP17, WPP161, WPP163, WPP165, and WPP172, P. wasabiae SCRI488, and a water control, respectively.

FIG. 2.

Pectobacterium isolates unable to elicit an HR did not contain the genes for the Pectobacterium T3SS. (A) The P. carotovorum WPP14 T3SS is indicated by open arrows, and the border genes, hecB, and ECA2075 are indicated by filled arrows. (B) Genomic DNA from isolates unable to elicit an HR did not hybridize to DNA amplified from the P. carotovorum WPP14 T3SS gene cluster, including hrpL, hrpN, hrpW, dspE, dspF, hrpD, hrpE, hrpF, hrpG, hrcC, hrpT, hrpV, hrpB, and hrcJ.

FIG. 3.

Genomic DNA from WPP17 and WPP172 were unable to hybridize to two genes flanking the T3SS region, ECA2075 and hecB, respectively. Genomic DNA was digested with SacII and EcoRV and then hybridized with hecB or ECA2075 probes. The presence of T3SS and the flanking regions is indicated on the right. Lane 1, WPP14; lane 2, WPP17; lane 3, WPP161; lane 4, WPP163; lane 5, WPP165; lane 6, WPP168; lane 7, WPP172; lane 8, WPP178.

FIG. 4.

Presence of the Pectobacterium T3SS was not correlated with the ability to macerate potato tubers (A) or cause disease in stems (B). For the inoculated tubers, the bars indicate the amounts of tissue macerated, and the error bars indicate the standard errors for 30 to 36 replicates. Bars with the same letter are not significantly different according to a least significant difference test at a P value of 0.05. Pc, Pw, and Pb indicate that isolates were identified as P. carotovorum subsp. carotovorum, P. wasabiae, and P. carotovorum subsp. brasiliensis, respectively. For the inoculated stems, the bars indicate lesion lengths 7 days postinoculation, and the error bars indicate the standard errors for 14 to 20 replicates. Bars with the same letter were not significantly different according to a least significant difference test at a P value of 0.05. dH2O, distilled water; NC, control.

FIG. 5.

Character and substitution weight parsimony phylogeny generated by using concatenated sequences of six housekeeping genes of 51 isolates, including 4 Brenneria sp. isolates, 10 Dickeya sp. isolates, 4 Yersinia sp. isolates, and 33 Pectobacterium sp. isolates. The numbers for Pectobacterium sp. indicate the clades, as follows; 1, P. carotovorum subsp. brasiliensis; 2, P. carotovorum subsp. carotovorum; 3, P. atrosepticum; 4, P. betavasculorum; and 5, P. wasabiae. The strains whose designations begin with ATCC are the type strains of species. Primer sets were designed to amplify fragments of six conserved housekeeping genes: acnA, gapA, icdA, mdh, pgi, and proA. Bootstrap values (expressed as percentages for 1,000 replications) are indicated only for branches also retrieved by MP and NJ analyses. Yersinia sp. strains were used as an outgroup for these analyses. Details for strains used in a previous analysis were described by Ma et al. (20), and the new strains are described in Table 1.

FIG. 6.

16S-23S ITS-PCR-RFLP patterns of Pectobacterium strains. The 16S-23S ITS region was amplified with primers L1 and G1, and the amplified DNA was digested with RsaI and then analyzed by gel electrophoresis. The strains used are indicated above the lanes, and strains are grouped into the clades defined by the MLSA. dH₂O, distilled water.

FIG. 7.

Four representative I-CeuI patterns for the 42 Pectobacterium isolates obtained from diseased potato tubers in Wisconsin in 2004. WPP14 was isolated in 2001 from a diseased potato plant with aerial stem rot. Fragments more than 1,000 kb long are not shown. The fields from which the strains originated and the numbers of strains with each pattern are indicated at the bottom. Lane M contained markers.