Twenty-two different strains/isolates were collected for this study. Of these, the Xcc strain ATCC33913 is a type strain, isolated from Brussels sprout (Brassica oleracea var. gemmifera) in the UK in 1957 20, and the Xcc strain 8004 is a laboratory strain with spontaneous rifampicin-resistance, derived from Xcc NCPPB No.1145 isolated from cauliflower (B. oleracea var. botrytis) in the UK in 1958 14. The other 20 isolates were collected from different infected cruciferous plants in various geographic locations over a wide range of latitudes across China and named CN01 to CN20 (Table 1). These isolates were validated by morphological, virulent and molecular analyses. All the isolates formed typical X. campestris colonies of yellow mucoid texture on NYG agar medium 14 and caused typical black rot disease symptoms on the host plant radish (Raphanus sativus var. radicula; data not shown). To further confirm the isolates, their partial 16S-23S rDNA intergenic spacer (ITS) regions 41 were examined by PCR and sequencing. A PCR fragment 464 bp in length was obtained for every isolate except CN13 and CN19, for which no PCR product was obtained. Sequencing results showed that five isolates have identical ITS sequences to that of strain 8004, while the ITS sequences of the other 13 isolates differ from that of 8004 by only one or two nucleotides (Additional data files 1 and 2). The isolates CN13 and CN19 were not used for further study in this work as they were not confirmed to be Xcc by the 16S-23S rDNA ITS analysis. The phylogenetic analysis by the maximal parsimony method 42 showed that the 18 proven Xcc isolates were grouped into two clusters and each cluster contains previously identified Xcc strains (Additional data file 2). These two groups were significantly distinguished from other Xanthomonas species and X. campestris pathovars (Additional data file 2), further confirming the 18 isolates as Xcc at the molecular level. The word 'strain' will be used for the identified Xcc 'isolates' hereafter.

The in planta pathogenicity test of Xcc strains was carried out by the leaf-clipping inoculation method on eleven different cultivars (cv.) of four cruciferous species (see Materials and methods). The results showed that seven of the eleven cultivars were susceptible to all of the Xcc strains tested, whereas the other four plants manifested resistance to particular Xcc strains (Tables 1 and 2). Based upon these results, a gene-for-gene relationship governing the outcome of the interactions between the Xcc strains and the host plants could be postulated (Table 3). The key essentials are: first, the host plants that were susceptible to all of the Xcc strains possess no resistance genes against the Xcc strains; second, mustard cv. Guangtou possesses a resistance (R) gene, arbitrarily designated Rc1, for which the postulated interacting avirulence (avr) gene is designated avrRc1, present in strains 8004, ATCC33913, CN03, CN07, CN09, CN10, CN11, and CN20; third, cabbage cv. Jingfeng-1 and radish cv. Huaye possess an R gene named Rc2 that interacts with an avr gene named avrRc2, present in strains ATCC33913, CN03, CN14, CN15, and CN16; and fourth, Chinese cabbage cv. Zhongbai-83 possesses an R gene, Rc3, that interacts with the postulated avrRc3 in strains 8004, ATCC33913, CN02, CN03, CN06, CN07, CN08, CN12, CN14, CN15, CN16, CN18, as well as CN20 (Tables 2 and 3).

We also examined the hypersensitive response (HR) 43 of the Xcc strains on the nonhost pepper ECW10R, a plant commonly used to test the HR of Xcc. The results showed that eight hours after inoculation strains 8004, ATCC33913, CN01, CN03, CN09, CN10, CN11, and CN20 elicited a typical HR while the others did not (Table 2). According to the results, we postulated that strains 8004, ATCC33913, CN01, CN03, CN09, CN10, CN11, and CN20 possess an avirulence gene, designated avrRp1, that interacts with a cognate resistance gene, named Rp1, in the non-host plant pepper ECW10R (Tables 2 and 3).

To investigate genetic similarity and diversity among Xcc strains, a DNA microarray encompassing 4,186 CDSs was constructed, representing all CDSs (non-redundant) in the reference strain 8004 21. Primer design was based on the genomic sequence of 8004, which is composed of 4,273 CDSs 21. Of the 4,186 CDSs, gel electrophoresis revealed successful amplification of 4,043 CDSs, representing 96.58% of the non-redundant genome content. For the CDSs predicted to be less than 100 bp in length, for which optimized primers could not be designed, and those for which PCR amplification did not work, a 70-mer oligo probe for each CDS was designed. The word 'gene' will be used in reference to the CDS that each spot corresponds to unless otherwise indicated.

To determine the sensitivity of our aCGH analysis, self-to-self hybridization was performed using genomic DNA of the reference strain 8004. After removal of faint spots for which the intensity was lower than the average plus two standard deviations of the negative controls (blank spotting solution) on the array, it was found that more than 95% of all genes on the array could be detected and the intensity ratio of the detected genes lay between 0.6 and 1.6. aCGH analyses were then carried out using the reference strain 8004 and its derivative strain C1430nk, described previously 44. The strain C1430nk is derived from 8004 and harbors the cosmid pLAFR6 containing the open reading frames (ORFs) XC1429 and XC1430. The aCGH results revealed that only two genes, XC1429 and XC1430, had an intensity ratio of approximately 1.9-2.4 (C1430nk/8004), indicating that sole copy alteration at the genomic scale could be detected in this study (Figure 1). Based on the above results, it was presumed that the microarray can detect the 1.6-fold alteration when ignoring sequence diversity. After passing the initial tests, aCGH analyses were performed using the fully sequenced Xcc strains 8004 and ATCC33913. The results showed a good agreement with the complete genome sequences of 8004 and ATCC33913 (Figure 1). It was found that for the genes of strain ATCC33913, whose sequences are >90% identical to those of strain 8004, 99% of their spots on the array showed intensity ratios ≥0.5. Therefore, intensity ratios ≥0.5 were selected to be the threshold for genes detected as present/conserved within strain 8004. Furthermore, 98% of the genes previously reported to be specific to strain 8004 (that is, that are absent in the genome of strain ATCC33913) were detected as absent genes in the aCGH analysis of strain ATCC33913 (Figure 1). Our selected threshold for conserved genes here is similar to that described by Taboada et al. 30, who used a Log₂ ratio (sample/reference) threshold of -0.8 to detect conserved genes in aCGH analyses with an acceptable level of false positives.

The validity of the aCGH results was further tested by PCR examination of the presence or absence of 30 genes showing a range of ratios in the aCGH analysis. The PCR primers used and PCR results are presented in Additional data file 3. The results show that a ratio (sample/8004 strain) of <0.5 gives high confidence (98%) that the gene is absent/highly divergent (AHD) in the sample strain.

Using the parameters established above, the gene composition of 18 Chinese Xcc strains was analyzed by aCGH using the genome of strain 8004 as the reference. The results are shown in Tables 4 and 5, Figure 2 and Additional data file 4. Of the 4,186 CDSs spotted on the microarray slides, 3,405 are conserved in all of the strains tested (Table 5). These conserved CDSs may represent the common backbone ('core' genes) of the Xcc genome, which contains most of the genes encoding essential metabolic, biosynthetic, cellular, and regulatory functions (Table 5). The genes relevant to central intermediary metabolism, replication, transcription, translation, the TCA cycle, and nucleotide, fatty acid and phospholipid metabolism are largely conserved. Genes encoding the components involved in the type I (T1SS), type II (T2SS) and type III secretion systems (T1SS-T3SS) as well as extracellular polysaccharide production, and the rpf (regulation of pathogenicity factors) gene cluster 1112 are highly conserved among the Xcc strains investigated, although some predicted pathogenicity- and adaptation-related genes are AHD (Table 5).

The aCGH results showed that 730 CDSs are absent or highly divergent among all the Chinese strains tested (Tables 4 and 5). In addition, a total of 51 invalid hybridization spots (CDSs) were observed in all the aCGH analyses of the 18 Chinese strains. The 730 AHD genes, which account for 17.6% of all valid hybridized CDSs in the aCGH analyses, may constitute the Xcc flexible gene pool. The functional categories of all the AHD genes are given in Table 5. Half of the AHD genes have been predicted to encode proteins with unknown function.

The differences in the numbers of the AHD genes in different strains are significant (Table 4). Compared with the reference strain 8004, the most divergent Chinese Xcc strain is CN14, of which 475 CDSs are AHD; and the most closely related strain is CN07, of which only 137 CDSs are AHD. Fifty-seven Xcc 8004 CDSs, most of them encoding hypothetical proteins, are AHD in all eighteen Chinese strains. Of the 57