In 2020, we carried out a systematic survey about rice BLB in Guangxi (Li et al., 2021) and had isolated and identified the Xoo from the BLB frequent onset areas. The representative Xoo strains were named as GXO20-serial number; e.g., GXO20-01 in which GXO indicates the Guangxi Xanthomonas oryzae strain and 20-01 indicates chronologically the No. 1 strain isolated in 2020 (Table 1). To comparatively study Xoo strains isolated from the low land and the high mountain paddies in Guangxi, China, we have selected the representative Xoo strain GXO20-01 from Heng County, the typical low land area in Guangxi, and GXO20-06 from Napo County, the typical high mountain area in Guangxi. The distance between the two places is about 500 km, along the Tropic of Cancer. The bacterial strains used in this study are listed in Table 1. The Xoo international type strain Xoo PXO99^A was provided by the Institute of Plant Protection of Nanjing Agricultural University. The race R5 of Chinese strain YC11 was provided by the College of Life Science and Technology, Huazhong Agricultural University (Zheng et al., 2020).

The differential rice varieties used in this study were a simplified combination of near-isogenic lines (Fang et al., 1990; Liu et al., 2007) (Supplementary Table S1), IRBB2, IRBB3, IRBB5, IRBB13, IRBB14, and IR24, provided by the International Rice Research Institute (IRRI), Philippines (Ogawa et al., 1991), via the Institute of Plant Protection of Nanjing Agricultural University, China.

The Xoo strains stored at −80°C were activated by streak culture on PSG medium (10 g l-1 peptone, 10 g l-1 sucrose, 1 g l-1 glutamic acid, pH 7.0) agar plates. The batch cultures of Xoo strains were grown in PSG medium for 18 h, about to the mid-log phase and then collected, and resuspended to the optical density at 600 nm (OD₆₀₀) of 0.5.in sterile water.

The rice seeds were sown in plastic pots in a greenhouse under a 12-h light/dark cycle at 25–28°C, and the 45-day-old seedlings were clip-inoculated with a bacterial suspension of 0.5 OD₆₀₀ and maintained under conditions of 80% relative humidity. Twenty leaves of each differential were inoculated with each isolate or sterilized water (as control). The disease severity was scored based on the visual assessment of the lesion length 14 days post-inoculation. The leaves with a lesion length of <1/4 and ≥1/4 were classified as resistant (R), and susceptible (S), respectively (Chen et al., 2019). All experiments were repeated three times. The least significant difference method (LSD method) was used for data analysis and the box-plot was generated using Origin 2018. A pathotypic analysis of Xoo strains was performed according to their reactions on NIL rice (Supplementary Table S1).

For the whole genome sequencing, Xoo strains were grown in PSG medium for 36 h, about to the early stationary phase, and then harvested. The genomic DNA of each strain was isolated and purified by using QIAGEN genomic-tip 20G following the manufacturer's protocol. The DNA samples were fragmented using g-TUBE. HiFi sequencing libraries were prepared using SMRTbell™ Express Template Prep Kit 2.0. The libraries were further size selected using BluePippin Systems. The average fragment length of the sequencing library is about 15 kb for circular consensus sequencing (CCS) to achieve long and high-fidelity (HiFi) reads (Tang, 2019). The DNA sequencing was performed using PacBio Sequel II System (Pacific Biosciences, USA) at Biomarker Technologies CO., LTD (Beijing, China). The HiFi reads have been de novo assembled using the Hifiasm software (Cheng et al., 2021). The Pilon v1.22 software was used to correct the assembly with Illumina data, and Circulator v1.5.5 software was used to cyclize and adjust the starting site. The genome completeness and contamination were evaluated by CheckM v1.1.3 (Parks et al., 2015).

The preliminary genome annotation was carried out by NCBI prokaryotic genome annotation pipeline (Tatusova et al., 2016) and GeneMarkS-2 software (Lomsadze et al., 2018). The gene function and pathway analysis (Supplementary Table S2) were performed by BLASTP searches against functional databases, including Cluster of Orthologous Groups of proteins (COG) database (https://www.ncbi.nlm.nih.gov/COG/), Gene Ontology (GO) (http://geneontology.org/), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, http://www.genome.jp/kegg/).

To decipher the Xoo genome from the phytopathological perspective, the confirmed and putative pathogenicity/virulence genes in Xoo were grouped into certain categories (Supplementary Table S3) in accordance with the genome categorical system for Xanthomonas first used by da Silva et al. (2002) and further improved by Thieme et al. (2005). The TAL effectors of each genome were identified and classified by AnnoTALE (Grau et al., 2016).

For structural comparison, complete genomes were aligned using progressive Mauve (Darling et al., 2010) with default settings. For phylogenomic analysis, complete genomes were aligned using Mauve. The tree was annotated and visualized by using the CVTree3 web server (http://cvtree.online/v3/cvtree/). The phylogenetic trees were constructed by using the neighbor-joining (NJ) method. The K-tuple length is 6 (Zuo and Hao, 2015). Orthologous genes of GXO20-01 and GXO20-06 were extracted from Mauve in pairwise comparison (Darling et al., 2010).

To comparatively study the 2020-epidemic Xoo strains isolated from low land and high mountain paddies, we had chosen two Xoo strains, GXO20-01 and GXO20-02, from Heng County (the typical low land area) and two strains, GXO20-04 and GXO20-06, from Napo County (the typical high mountain area), respectively (Table 1). On the basis of molecular identification and back inoculation, the four Xoo strains were demonstrated to be quite similar in phenotypic characteristics, including EPS production, extracellular enzyme activity, motility, stress tolerance, and biofilm formation (data not shown).

The pathotypic analysis of Xoo strains was commonly performed by using a simplified combination of IRBB NILs, IRBB2, IRBB3, IRBB5, IRBB13, IRBB14, and IR24, in China (Liu et al., 2007; Chen et al., 2012). In this study, the positive control Xoo strain YC11 clearly showed the SSRRSS interaction results on the differential rice varieties (Zheng et al., 2020). The pathotyping results demonstrated that all of four representative Xoo strains from low land and high mountain paddies in Guangxi belong to race R8, indicated by the SSRSSS interaction results (Figure 1). Additionally, statistical analysis showed that there were significant differences between those Xoo strains on virulence in some certain rice varieties (Figure 1).

To quickly and effectively compare the genomic information of Xoo strains isolated from low land and high mountain paddies, GXO20-01 and GX20-06 were selected as the representative Xoo strains for low land and high mountain paddies, respectively (Table 1). Complete genome sequencing of two Xoo strains was performed using the Pacific Biosciences (PacBio) platform with high-fidelity (HiFi) long reads (see Materials and Methods). The CCS data for GXO20-01 and GX20-06 are as follows: SeqNum: 6357 and 5238 reads, SumBase: 6,106,727 and 53,252,594 bp, N50 length: 10,505 and 10,286 bp. The HiFi reads have been de novo assembled using the Hifiasm software (Cheng et al., 2021). The Pilon v1.22 software was used to correct the assembly with Illumina data, and Circulator v1.5.5 software was used to cyclize and adjust the starting site. One contig was assembled for each genome, with sizes 4,963,479 and 4,962,861 bp, respectively (Table 2). Two Xoo strains contain no plasmid. The DNA completeness for the two genomes was found to be 99.62 and 99.44%, respectively, and contamination for the two genomes was 0.37%. The basic genome features of GXO20-01 and GX20-06 and other sequenced Xoo strains were listed in Table 2. Also, the basic genome features of GXO20-01 and GX20-06 were annotated to encode 724 and 727 pseudogenes, similar to those found in PXO99^A, 759 (Salzberg et al., 2008) and in MAFF 311018, 747 (Ochiai et al., 2005), respectively (Table 2). These results also indicated that the HiFi sequencing quality is relatively high.

The genomes were annotated 4,468 and 4,462 protein encoding sequences (CDSs) in GXO20-01 and GXO20-06, respectively (Table 2; Supplementary Table S2), among which 628 and 629 in GXO20-01 and GXO20-06, respectively (Supplementary Table S3), were annotated as pathogenicity/virulence genes in accordance with the genome categorical system for Xanthomonas firstly used by da Silva et al. (2002) and further improved by Thieme et al. (2005). The Xoo genomes encode typical pathogenicity/virulence factors found in Xanthomonas spp., including extracellular hydrolases, extracellular polysaccharides, adhesins, type II secretion system, type III secretion system, and T3SEs (White et al., 2009; An et al., 2020).

In this study, the pathogenicity-related genes in Xoo were divided into 10 groups: (1) Type III secretion system (hrp/hrc genes) and its effectors, (2) other secretion systems, (3) extracellular enzymes, (4) surface polysaccharides, lipopolysaccharides, and antigens, (5) toxin and detoxification, (6) adhesion and biofilm, (7) quorum sensing and regulation of virulence factors, (8) adaptation to atypical conditions, (9) bacterial motility, and (10) other factors in bacterium–plant interaction (Supplementary Table S3). The detailed genome annotation might be helpful to elucidate the pathogenesis of this typical plant pathogenic bacterium and to pinpoint the certain genes in different infection stages (Supplementary Table S3).

To clarify the relationship between the two strains, we have conducted pairwise comparative analysis both in their genome structures and genome contents. The results showed that the chromosomes of GXO20-01 and GXO20-06 are entirely syntenous (Figure 2A). The positions of the 17 allelic tal genes show no duplications or rearrangements in one genome relative to the other (Figure 2B). The results of genome two-way alignments indicated that the two genomes are conserved both in gene contents and their allelic sites (Supplementary Table S2). Among the 4,468 and 4,462 annotated genes in GXO20-01 and GXO20-06, 4,442 genes are orthologous. Totally, there are 123 genomic variational sites between genomes, including 97 SNPs, 19 indels, and 7 complex variations. Among the SNPs, 76 were found in coding sequence genes, 33 SNPs were missense variants that resulted in 20 gene differences between two strains, as more than one SNPs in one single gene. The indels and complex variations also caused nine gene differences between the two strains (Table 3; Supplementary Tables S2, S3). Here, GXO20-01 has 6 strain-specific genes encoding a 5-amino-6-(5-phosphoribosylamino) uracil reductase (RibD), a GNAT family N-acetyltransferase, two adenylyl–sulfate kinase, outer membrane protein, and a hypothetical protein, and GXO20-06 has only one strain-specific gene, the tal5a encoding a TalDR-like TALE. Subsequently, it was found that the two TALE repertoires are nearly identical but only one TALE missing in GXO20-01. Consequently, the pairwise genomic comparison and phylogenetic analysis suggested that GXO20-01 and GXO20-06 are probably sibling Xoo strains.

Multiple alignments of the selected Xoo genomes showed that the genome structures of GXO20-01 and GXO20-06 are highly collinear with Xoo strains in lineage PX-A (Quibod et al., 2016) (Figure 2A) and the loci of the major tal genes are highly conserved (Figure 2B).

To further determine the relationships of the newly sequenced Xoo strains with other Xoo strains, we carried out a phylogenomic analysis in which representative Xoo strains were selected representing typical Xoo strains from around the world (Figure 3). The phylogenomic analysis clearly grouped GXO20-01 and GXO20-06 in the branch of PXO563 and XF89b in lineage PX-A (Quibod et al., 2016) or Xoo-A (Chien et al., 2019), but away from the Chinese mainland strains (Zheng et al., 2020).

The TALEs are the most important virulence factor used by Xoo to interact with rice, enforcing virulence, proliferation, and dissemination against rice (Erkes et al., 2017). AnnoTALE analysis indicated that GXO20-01 and GXO20-06 contain 17 and 18 tal genes, respectively. Among the 17 shared TALEs, 15 typical TALEs are assigned to classes TalAF, TalAN, TalAD, TalAB, TalAL, TalAD, TalAO, TalAE, TalAM, TalAH, TalAA, TalAR, TalAG, TalAS, TalAQ, and the iTALE/truncTALEs are assigned to class TalAI. Compared with the closely related Xoo strains, GXO20-01 and GXO20-06 contain no TalFX that is found in C9-3, no TalBK that is found in LN4, and no TalDW that is found in XF89b (Table 4).

The diversity in TALE repertoires of Xoo actually underpins the complexity of the virulence of Xoo races. A general survey of Xoo genomes in GenBank indicated that there are not two Xoo strains sharing the identical TALome. In our case, the closely related sibling Xoo strains GXO20-01 and GXO20-06 still have one TALE gene difference.

It is worth mentioning that GXO20-06 contains a tal5a gene situated in the variable tal locus that encodes a TalDR-like TALE, which is missing in GXO20-01. TalDR is a particular TALE that is found only in the PXO563 and PXO71. The tal5 locus (here referred to as GXO20-06 genome) contains a number of insertion sequences and multiple repeat sequences (Figure 4), which is considered to be the most unstable section in Xoo genomes. The genomic comparison indicated that in most Xoo genomes, tal5 locus or its homologous sections encode one or two iTALE/truncTALEs, but their CDS sequences differ largely (Table 4). In most cases, talDR/talDR-like gene totally disappeared in certain Xoo genomes. Such TALE diversities might provide a novel marker or reference for the genotyping or tracking of pathogens. Also, GXO20-06 is isolated from high mountain paddies, which still carry the talDR/talDR-like gene in the chromosome, suggesting the slow variation probably due to mild selection pressure from a single cropping rice system. As epidemic Xoo strains, it is expected GXO20-01 and GXO20-06 strains with only one TALE distinct to be the ideal reference strains in the research on the pathogenesis, virulence evolution, and pathogen tracking of rice BLB in China and even in many regions worldwide.