Introduction

Bacterial canker of kiwifruit (Actinidia spp.) is a serious threat to the kiwifruit industry that causes substantial crop losses worldwide [1]. After the first symptoms of canker disease (such as leaf spots) appear, the disease spreads quickly to the rest of the plant, which can be destroyed within 1 year [2]. The main methods of disease control and mitigation include chemical treatment, orchard management, and breeding of resistant varietie, however, there are currently no effective methods to directly kill the causal pathogen [2]. Previous studies have reported that P. syringae pv. actinidiae is one of the main pathogenic agents of Actinidia spp. pathogenic bacterial canker disease [1,2,3,4,5,6].

P. syringae is an important phytopathogenic bacterium and one of the most relevant models for research on microbe–host interactions [1A and B) under climatic conditions consisting of low temperatures, strong winds, and heavy rainfall. During year of 2019–2021, samples of disease leaves were collected the at three different time points. From the different samples, we isolated one same bacterium forming white colonies on plates (Fig. 1C), which was identified as pseudomonads by phylogenetic analysis by 16S rDNA and SyrB gene [14] (Figure S1). By The isolated strain reproduced typical symptoms on the leaves and tender buds of HongYang and WuZhi kiwifruit plants when infiltrated into the leaves (Fig. 1D and E) or spraying onto the abaxial sides of the leaves (Fig. 1F and G). We named this pathogenic strain MHT1, which is an acronym for the Chinese pronunciation of kiwifruit (Mi Hou Tao).

Fig. 1
figure 1

Isolation and pathogenicity tests of Pseudomonas savastanoi strain MHT1. A and B. Representative leaf and stem of Actinidia chinensis plants with bacterial canker symptoms sampled from the ** County, Heyuan City, Guangdong Province, China. Morphology of Pseudomonas savastanoi MHT1 colonies. A single colony of the MH1 strain was streaked and cultivated on LB medium at 28 °C for 24 h. D and E. Symptoms on the leaves and tender buds of A. chinensis plants (HongYang kiwifruit in C; WuZhi kiwifruit in D) generated by P. savastanoi MHT1 strains 8 days post-inoculation. A. chinensis leaves were infiltrated with bacterial suspensions (108 CFU/mL in 1 mM MgCl2) and photographed 8 days later. CFU, colony-forming units. Negative control, leaves infiltrated with 10 mM MgCl2. F and G. Symptoms on the leaves of A. chinensis plants (HongYang kiwifruit) generated by P. savastanoi MHT1 strains 6 days post-infection. Abaxial side leaves of A. chinensis were sprayed with bacterial suspensions (108 CFU/mL in 1 mM MgCl2) and photographed 6 days later

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Genome sequencing, assembly, and functional annotation

To obtain more information about this pathogen and to explore the interaction mechanisms of the MHT1 strain with kiwifruit, we sequenced the MHT1 genome using the PacBio Sequel platform (Genedenovo Biotechnology Co., Ltd., Guangzhou, China). We obtained 1.12 Gb of reads representing an 187-fold coverage of the entire genome by SMRT sequencing (Genedenovo Biotechnology Co., Ltd., Guangzhou, China). After removing ambiguous and low-quality reads and adapters, we assembled the clean data into a single molecule of approximately 6.00 Mb (Fig. 2A) with the program MECAT [15]. The circular chromosome harbored 5008 predicted genes and exhibited a GC content of 58.5% (Table 1). We used several complementary strategies to predict the number of different RNAs, clustered regularly interspaced short palindromic repeats (CRISPR), genomic islands (GIs), prophage sequences, interspersed repeats, tandem repeats, and transposons along the chromosome (Table 1. The distribution statistics of the MHT1 gene complement against the Non-Redundant Protein Database (NR) (NCBI) showed that strain MHT1 shares 1949 and 1186 genes with P. syringae and P. savastanoi, respectively (Figure S2 and Table S1). According to the observed average nucleotide identity (ANI) (Figure S3), we refer to this strain as Pseudomonas savastanoi.

Fig. 2
figure 2

General genomic features of Pseudomonas savastanoi MHT1 strain. Circos plot of the MHT1 chromosome. The MHT1 chromosome contig has a full length of 5,999,881 bp and a GC content of 58.5% and harbors 5008 predicted genes. Innermost circle, GC skew curve (purple and orange indicating a GC skew lower or higher than zero, respectively); second circle out, GC content (blue and orange indicate GC content lower or higher than the average GC content across the genome, respectively); third circle, non-coding RNAs, with tRNAs in black and rRNA in red; fourth and fifth circles, annotated genes on the positive and negative strands, respectively, with colors representing the different classifications of COG functions; sixth (outermost) circle, size of the chromosome. B. Phylogenetic tree based on the whole genomes of five representative P. savastanoi strains and the MHT1 strain. C. Venn diagram showing the extent of overlap between gene families between MHT1 and the four representative P. savastanoi strains

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Table 1 General feature of the Pseudomonas savastanoi strain MHT1 genome
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Genetic relationship between MHT1 and other Pseudomonas savastanoi strains

We performed a phylogenetic analysis between MHT1 and four representative sequenced strains of P. savastanoi that have not been reported to cause bacterial canker on kiwifruit (assemblies ASM1220v1, ASM16401v3, ASM1485547v1, and ASM1714087v1)(Table S2) based on their whole genomes using the REALPHY platform [16] (Fig. 2B). We also compared these five P. savastanoi strains and investigated their strain-specific and shared genes (Fig. 2C). We determined that 230 genes are unique to MHT1. We identified the extent of synteny between MHT1 and the four representative strains using the C-Sibelia program [17] (Fig. 3). Collinear blocks in the MHT1 genome accounted for 87.8% (versus ASM1485547v1), 87.2% (versus ASM1220v1), 84.7% (versus ASM1714087v1), and 82.8% (versus ASM16401v3) of the total genome (Table 2).

Fig. 3
figure 3

Synteny map of the genomes of MHT1 and four representative P. savastanoi strains: ASM1220v1, ASM1714087v1, ASM1485547v1, and ASM16401v3

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Table 2 Comparison of collinearity between MHT1 and other strains of Pseudomonas savastanoi
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We performed Gene Ontology (GO) [18] enrichment analysis to evaluate the possible function of these MHT1-specific genes: We observed enrichment for the GO terms ‘antibiotic metabolic process’ and ‘drug metabolic process’ (Fig. 4A). Subsequently, we performed a Kyoto Encyclopedia of Genes and Genomes (KEGG) [19] pathway analysis with the same MHT1-specific genes. The top enriched KEGG pathways were associated with ‘pentose and glucuronate interconversions’, ‘metabolic pathways’, ‘amino sugar and nucleotide sugar metabolism’, ‘nonribosomal peptide structures’, ‘cysteine and methionine metabolism’, and ‘degradation of aromatic compounds’ (Fig. 4B). These results showed that although MHT1 has high similarity with these four P. savastanoi strains, it still has many unique genes that are enriched in several physiological pathways.

Fig. 4
figure 4

Top 20 enriched GO and KEGG pathways for MHT1-specific genes compared to the ASM1220v1, ASM1714087v1, ASM1485547v1, and ASM16401v3 strains. A, Enriched GO categories. B, Enriched KEGG pathways

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Prediction and analysis of conserved type III effectors in Pseudomonas savastanoi strain MHT1

Like many other gram-negative plant pathogenic bacteria, P. savastanoi may delivers many different type III effector proteins (T3Es) into host plants for its pathogenesis via a type III secretion system (T3SS) [13]. Given that different pathovars of P. savastanoi can infect several plant hosts, each strain may carry an array of conserved effector proteins that largely determines their host range [13]. To explore the characteristics of the MHT1 conserved effector protein arsenal, we searched for potential type III effectors encoded by the MHT1 genome using the T3E database and the program EffectiveT3 [20, 21]. We thus identified 26 proteins related to the T3SS and T3Es in the MHT1 genome (Table S3). We next looked for these T3Es in other P. syringae, P. amygdali, and Ralstonia solanacearum strains whose genomes have been sequenced and that can infect plant hosts [20]. We obtained eight conserved T3Es in MHT1 with strong similarity to the HopM1, AvrE1, and HopA1 effectors of P. syringae strains; the HopAH1and HopI1 effectors of P. amygdali strains; and the RipQ, RipW, and RipBG effectors of R. solanacearum strains (Table 3) [22,23,24,25,26,27]. We also predicted the subcellular localization of these potential effectors using Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) and Protein Homology/analogY Recognition Engine V 2.0 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (Table 3). These results suggest that the existence of several conserved effector proteins may be important to the pathogenicity of MHT1.

Table 3 Predicted type III effectors of MHT1
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Genomic islands and prophage elements

Genomic islands (GIs) are fragments of DNA derived from horizontal gene transfer between different bacterial genomes [28]. We screened the genome of MHT1 for GIs using Island viewer software [29]. We identified six GIs in the genome (Fig. 5) with the IslandPath-DIMOB program [29]. Over the entire chromosome, the lengths of GIs ranged from 12,298 to 64,840 bp, with an average size of 26,497 bp and covering a total length of 105,988 bp. These GIs contained 107 genes (Table S4), which mainly encoded FAD-binding oxidoreductases, short-chain dehydrogenase/reductase (SDR) family oxidoreductases, NAD(P)-dependent alcohol dehydrogenases, ATP-dependent helicase HrpB, glutathione S-transferases, transcriptional regulators, and ATP binding cassette (ABC) transporter permeases.

Fig. 5
figure 5

Circular and horizontal plots of genomic islands (GIs) identified along the MHT1 chromosome. Color bars represent the predicted GIs identified by SIGI-HMM (orange), IslandPick (green), and IslandPath-DIMOB (blue) as well as integrated results (red) from innermost to outermost circles

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Identification of prophages is important for the study of the genome of the MHT1 strain and its genetic potential [30]. In this study, we established that the MHT1 genome harbors two prophage regions (Table S5) with a total size of 55,399 bp using the program Phage_Finder [31]. The lengths of prophage regions 1 and 2 were 25,508 bp (from bp 5,334,617 to 5,360,124, with a GC content of 59.4%) and 29,891 bp (from bp 5,702,675 to 5,732,565, with a GC content of 58.1%), respectively. Sixty-five protein-coding genes were predicted in these two prophage sequences, encoding lysozymes, phage tail proteins, lipoproteins, glycoside hydrolases, and terminases (Table S6).

Carbohydrate-active enzymes, antibiotic resistance, and substitution rate (K a/K s) analysis

Carbohydrate-active enzymes (CAZymes) are proteins that break down carbohydrates into smaller molecules by forming, hydrolyzing, and modifying glycosidic bonds [S7) in the MHT1 genome, further supporting the speculation that the MHT1 strain may exhibit strong resistance to various antibiotics and other chemicals.

In addition, by KEGG pathway enrichment, we determined that the genes unique to the MHT1 strain are mainly involved in the pathways ‘pentose and glucuronate interconversions’, ‘metabolic pathways’, ‘amino sugar and nucleotide sugar metabolism’, ‘nonribosomal peptide structures’, ‘cysteine and methionine metabolism’, and ‘degradation of aromatic compounds’, which may function during infections of kiwifruit and survival in certain environments, such as low temperature and high humidity. It remains to be determined whether the MHT1 strain, like other P. savastanoi strains, has a strong ability to infect other woody hosts or whether it can only infect plants in the kiwifruit production areas where it was initially isolated. We noticed the presence of 734 genes in the MHT1 genome that were annotated as CAZyme family genes (Figure S4), among which glycoside hydrolases and glycosyl transferases were the two most abundant families. Whether this feature helps MHT1 adapt to the host plant and environment will require further research to elucidate.

In this study, we investigated conserved type III effectors in P. savastanoi MHT1 strains and found that MHT1 presents three effectors that are highly homologous to the RipQ, RipW, and RipBG effectors of R. solanacearum strains. This implies that there may be effectors with similar functions in different gram-negative plant pathogenic bacteria and that these effectors may play important roles in the infection of the host, explaining their retention over the course of evolution. The effectors may also have been obtained by horizontal gene transfer, which could provide a molecular basis for MHT1 to acquire stronger pathogenicity or the ability infect more host plants.

Conclusions

Here, we reported on the complete genome sequence of the P. savastanoi strain MHT1, isolated from infected kiwifruit plants showing bacterial canker symptoms. Comparative genomic analysis with P. savastanoi strains indicated that the MHT1 genome harbors 230 unique genes enriched in antibiotic metabolism pathways. In addition, 380 and 734 genes in the genome of the MHT1 strain were annotated as antibiotic resistance genes or as encoding carbohydrate-active enzymes, respectively. Furthermore, MHT1 has eight conserved effectors that are highly similar to other effectors from P. syringae, P. amygdali, and R. solanacearum strains. Collectively, these results provide a solid foundation for further studying the host specificity of MHT1 and for screening effective antibiotics for the prevention and control of bacterial canker caused by this pathogen.

Materials and methods

Preparation of strains

Pseudomonas savastanoi strain MHT1 was isolated from the leaves of one Actinidia chinensis plant that exhibited bacterial canker symptoms, on the ** County, Heyuan City, Guangdong Province of China. Species identification was performed by morphological and molecular analyses. Single colonies were selected after 1 day of culture in LB medium (10 g NaCl,10 g peptone,10 g agar, and 5 g yeast extract, dissolved in 1 L water, pH 7.0) at 28 °C. The selected clones were grown in LB liquid medium for 18 h at 28 °C, followed by centrifugation at 4500 r/min for 5 min to collect cells. Phylogenetic trees were constructed by using software MEGA7.0, according to (NJ) neighbor joining method, the bootstrap replications are 1000.

Pathogenicity tests

MHT1 cells were cultured in LB medium at 28 °C for 24 h, harvested by centrifugation, and adjusted to 108 CFU/mL in 10 mM MgCl2. The suspension was infiltrated into the leaves of Actinidia chinensis plants (HongYang kiwifruit and WuZhi kiwifruit) with a needleless syringe. The negative control consisted of infiltration with 1 mM MgCl2 without bacteria. For the leaf spraying infection test, the suspension was sprayed onto the abaxial sides of the leaves of Actinidia chinensis plants (HongYang kiwifruit). After infection, plants were grown under 100 μmol·m− 2·s− 1 light irradiance with a 12-h-light/12-h-dark (20 °C/18 °C) cycle in 95% relative humidity. Photographs were taken 8 days post-inoculation. The pathogenic bacteria were isolated and identified from the diseased tissues of HongYang kiwifruit and WuZhi kiwifruit.

DNA extraction, genome sequencing, and assembly

Genomic DNA from P. savastanoi strain MHT1 was extracted with the TIANamp Bacteria DNA Kit (TIANGEN BIOTECH, Bei**g, China) and quality-tested on a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). DNA concentration was measured on a Nanodrop (Thermo Fisher Scientific, Wilmington, USA).

Genome sequencing was performed on a PacBio long-read sequencer (Pacific Biosciences, Menlo Park, CA, USA). SMRTbell libraries were obtained using g-TUBE (Covaris, Woburn, Massachusetts, USA) and end repair. According to the manufacturer’s protocols of the Blue Pippin system (Pacific Biosciences), fragments with sizes larger than 10 kb were selected. The quality and average size of the fragments of the library were estimated using a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA) and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). The PacBio Sequel system (Pacific Biosciences, Menlo Park, CA, USA) was used to perform SMRT sequencing. The resulting continuous long reads were used for de novo assembly with the Falcon program (version 0.3.0) [36].

Functional genome annotation

To predict the open reading frames (ORFs), the NCBI prokaryotic genome annotation pipeline and Prokka (version 1.11) were used [37, 38]. CRISPRfinder (version 4.2.17) was used to estimate CRISPR elements [39]. Ribosomal RNAs (rRNAs), small RNAs (sRNAs), and transfer RNAs (tRNAs) were predicted using the programs rRNAmmer (version 1.2), cmscan (version 1.1.2), and tRNAscan (version 1.3.1), respectively [40,41,42]. Tandem repeat elements were predicted with the program TRF (version 4.09) [43], interspersed repeat elements were predicted with RepeatMasker (version 4.0.5) [44], and transposons were predicted with TransposonPSI (version 1.0.0) (http://transposonpsi.sourceforge.net/) [45]. Based on sequence similarity, the predicted genes in strain MHT1 were annotated by BLASTN (E-value <1e− 5), combined with analysis with Gene Ontology (GO), Cluster of Orthologous Groups of proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), Swissport, and NCBI Non-Redundant Protein (NR) databases. Annotation of protein families was conducted based on Pfam_Scan (version 1.6) and the Pfam database (version 32.0) [46]. Prophages and GIs (gene islands) were predicted using the program Phage_Finder (version 2.0) [28] and Island Viewer (version 4.0) (http://www.pathogenomics.sfu.ca/islandviewer/upload/) [31], respectively. Type III effectors in the MHT1 strain were predicted with the T3E database [20]. The phylogenetic tree was obtained by the REALPHY program (https://realphy.unibas.ch/realphy/) using the whole genome of the MHT1 and other sequenced genomes of P. savastanoi strains as input. The Comprehensive Antibiotic Resistance Database (CARD) [33] and the Carbohydrate-Active enZYmes (CAZy) [47] database were used for advanced annotations. The default parameters were used in all analyses.

Identification of orthologous genes

The alignment of the MHT1 genome and four other representative P. savastanoi strains was performed in an all-against-all comparison with the MUMmer 3 package (version 3.3.3) (http://mummer.sourceforge.net/) with default parameters [48]. Orthologous gene clusters in the genomes were estimated with default parameters and with a combination of the programs OrthoMCL (version 2.0) and DIAMOND (parameters of E-value <1e− 5, query cover > 30%) [49, 50]. The core orthologs and putative proteins in MHT1 were aligned by BLASTP. The score of significant matched proteins of each pair was assigned using a 1 × 10− 7 cut-off value [51].

Substitution rate estimation

The KaKs_Calculator Toolbox software (using the free ratio model with default parameters) (version 2.0) [52] was used to calculate the nonsynonymous mutation rate (Ka) and synonymous mutation rate (Ks). The Ka/Ks values that were higher than 0.5 [53] were considered genes under positive selection within the P. savastanoi strains.