First Report of Bacterial Canker of Pecan Caused by Pseudomonas syringae pv. syringae in China.

Pecan (Carya illinoinensis) is a world-famous nut tree that is widely cultivated in China, especially in Jiangsu Province (Zhang et al. 2015). In April 2022, cankers on trunks were recorded in pecan (cv. Pawnee) fields located in Taizhou (32°27'58″ N, 120°0'49″ E), Jiangsu. Cankers on the trunks resulted in wilt of the plants. Usually, the color of infected bark on the trunk became darker than the healty bark. When the outer bark was peeled away, the inner tissues were water-soaked, often with reddish streaks. In the surveyed orchards, disease incidence ranged from 10 to 20% among young saplings (about 200 three-year-old trees). While no fungal mycelium or spores were found in the diseased areas by microscope, bacterial colonies were isolated by surface-sterilizing small fragments (25 mm2) of symptomatic tissue in 0.5% NaOCl, rinsing the sections twice in sterilized water, and then streaking them on Luria-Bertani (LB) plates. More than 20 bacterial isolates were obtained and all isolates induced a hypersensitive response on Nicotiana tabacum. All isolates were fluorescent on King's medium B, and were gram-negative based on lysis by KOH. Isolates were positive for levan formation, negative for oxidase and arginine dihydrolase, and did not cause soft rot on potato slices. Based on above information, the isolates thus belonged to Lelliot's LOPAT group 1, P. syringae (Lelliott and Stead 1988). The 16S rRNA sequences of five representative isolates (accession numbers OP175939-OP175943) were amplified by PCR, sequenced, and compared with the NCBI GenBank database (Weisburg et al. 1991; Sarkar and Guttman 2004), finding a 99.92% genetic similarity with a previously reported 16S rRNA sequence of a Pseudomonas syringae pv. syringae (Pss) isolate (accession numbers NW389777). Additional housekeeping genes gap1(accession numbers OP186937-OP186941), rpoD (accession numbers OP186952-OP186956), gyrB (accession numbers OP186947-OP186951), and gltA (accession numbers OP186942-OP186946) were PCR-amplified and sequenced as reported by Hwang et al. (2005), followed by multilocus sequence typing analysis (MLSA). Molecular phylogenetic trees (MEGA vesion 6.0, maximum likelihood with Jukes-Cantor model, 1,000 bootstraps) were generated based on each of these five DNA regions and revealed that all five isolates were clustered together with the strains in P. syringae genomospecies 2, and grouped these isolates with Pss in the PAMDB database (Hwang et al. 2005). As a result, these isolates were identified as Pss. Pathogenicity on pecan (cv. Pawnee) was confirmed by cutting the trunks of two-year-old pecan trees with sterilized blades dipped in cell suspensions containing 107 CFU/ml of each isolate. Plants inoculated in a similar manner with sterile water served as negative controls. The inoculated plants were incubated in a greenhouse maintained at 25°C and 80% relative humidity. After 7 to 8 days, all inoculated plants showed the symptoms of necrosis previously described for the original field plants, while the control plants did not show symptoms. The bacteria reisolated from the inoculated plants were identified as Pss using the LOPAT tests. These results and the sequence analysis of the 16S rRNA and four housekeeping genes described above, fulfilled Koch's postulates. No target bacteria were isolated from the control plants. To our knowledge, this is the first report of Pseudomonas syringae pv. syringaecausing bacterial canker of pecan worldwide. The identification of this pathogen will allow the study of strategies for managing the disease. References: Hwang, M. S., et al. 2005. Applied and Environmental Microbiology, 71:5182-5191. Lelliott, R. A., and Stead, D. E. 1988. Blackwell Scientific, Sussex, UK. Sarkar, S. F., and Guttman, D. S. 2004. Applied and Environmental Microbiology, 70:1999. Weisburg, W. G., et al. 1991. Journal of Bacteriology, 173: 697. Zhang, R., et al. 2015. Scientia Horticulturae, 197: 719-727. The author(s) declare no conflict of interest. Keywords: Carya illinoinensis, Pseudomonas syringae, Canker, Identification †Indicates the corresponding author.Y. Q. Zhao; zhaoyuqiang123@126.com.

yggS Encoding Pyridoxal 5'-Phosphate Binding Protein Is Required for Acidovorax citrulli Virulence.

Bacterial fruit blotch, caused by seed-borne pathogen Acidovorax citrulli, poses a serious threat to the production of cucurbits globally. Although the disease can cause substantial economic losses, limited information is available about the molecular mechanisms of virulence. This study identified that, a random transposon insertion mutant impaired in the ability to elicit a hypersensitive response on tobacco. The disrupted gene in this mutant was determined to be Aave_0638, which is predicted to encode a YggS family pyridoxal phosphate-dependent enzyme. YggS is a highly conserved protein among multiple organisms, and is responsible for maintaining the homeostasis of pyridoxal 5'-phosphate and amino acids in cells. yggS deletion mutant of A. citrulli strain XjL12 displayed attenuated virulence, delayed hypersensitive response, less tolerance to H2O2 and pyridoxine, increased sensitivity to antibiotic ß-chloro-D-alanine, and reduced swimming. In addition, RNA-Seq analysis demonstrated that yggS was involved in regulating the expression of certain pathogenicity-associated genes related to secretion, motility, quorum sensing and oxidative stress response. Importantly, YggS significantly affected type III secretion system and its effectors in vitro. Collectively, our results suggest that YggS is indispensable for A.citrulli virulence and expands the role of YggS in the biological processes.

Hfq, a RNA Chaperone, Contributes to Virulence by Regulating Plant Cell Wall-Degrading Enzyme Production, Type VI Secretion System Expression, Bacterial Competition, and Suppressing Host Defense Response in Pectobacterium carotovorum.

Hfq is a RNA chaperone and participates in a wide range of cellular processes and pathways. In this study, mutation of hfq gene from Pectobacterium carotovorum subsp. carotovorum PccS1 led to significantly reduced virulence and plant cell wall-degrading enzyme (PCWDE) activities. In addition, the mutant exhibited decreased biofilm formation and motility and greatly attenuated carbapenem production as well as secretion of hemolysin coregulated protein (Hcp) as compared with wild-type strain PccS1. Moreover, a higher level of callose deposition was induced in Nicotiana benthamiana leaves when infiltrated with the mutant. A total of 26 small (s)RNA deletion mutants were obtained among a predicted 27 sRNAs, and three mutants exhibited reduced virulence in the host plant. These results suggest that hfq plays a key role in Pectobacterium virulence by positively impacting PCWDE production, secretion of the type VI secretion system, bacterial competition, and suppression of host plant responses.

Loss-of-Function Mutations in the Dpp and Opp Permeases Render Erwinia amylovora Resistant to Kasugamycin and Blasticidin S.

Extensive use of the antibiotic streptomycin to control fire blight disease of apples and pears, caused by the enterobacterial plant pathogen Erwinia amylovora, leads to the development of streptomycin-resistant strains in the United States and elsewhere. Kasugamycin (Ksg) has been permitted to be used as an alternative or replacement to control this serious bacterial disease. In this study, we investigated the role of two major peptide ATP-binding cassette transporter systems in E. amylovora, the dipeptide permease (Dpp) and oligopeptide permease (Opp), in conferring sensitivity to Ksg and blasticidin S (BcS). Minimum inhibitory concentration and spot dilution assays showed that the dpp deletion mutants exhibited slightly enhanced resistance to Ksg in rich medium, whereas the opp mutant exhibited slightly enhanced resistance to Ksg in minimal medium and BcS in rich medium. Deletion of both dpp and opp conferred a higher level of resistance to Ksg in both rich and minimal media, whereas deletion of opp alone was sufficient to confer high level of resistance to BcS in minimal medium. In addition, bioinformatic analysis combined with reverse transcription-quantitative polymerase chain reaction showed that the Rcs phosphorelay system negatively regulates opp expression and the rcsB mutant was more sensitive to both Ksg and BcS in minimal medium as compared with the wild type. An electrophoresis motility shift assay further confirmed the direct binding of the RcsA/RcsB proteins to the promoter region of the opp operon. However, neither the Dpp nor the Opp permeases contributed to disease progress on immature pears, hypersensitive response on tobacco leaves, or exopolysaccharide amylovoran production. These results suggested that Ksg and BcS employ the Dpp and Opp permeases to enter E. amylovora cells and the Dpp and Opp permeases act synergistically for illicit transport of antibiotics.

AsnB, regulated by diffusible signal factor and global regulator Clp, is involved in aspartate metabolism, resistance to oxidative stress and virulence in Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak in rice, which is a destructive disease worldwide. Xoc virulence factors are regulated by diffusible signal factor (DSF) and the global regulator Clp. In this study, we have demonstrated that asnB (XOC_3054), encoding an asparagine synthetase, is a novel virulence-related gene regulated by both DSF and Clp in Xoc. A sequence analysis revealed that AsnB is highly conserved in Xanthomonas. An asnB mutation in Xoc dramatically impaired pathogen virulence and growth rate in host rice, but did not affect the ability to trigger the hypersensitive response in nonhost (plant) tobacco. Compared with the wild-type strain, the asnB deletion mutant was unable to grow in basic MMX (-) medium (a minimal medium without ammonium sulphate as the nitrogen source) with or without 10 tested nitrogen sources, except asparagine. The disruption of asnB impaired pathogen resistance to oxidative stress and reduced the transcriptional expression of oxyR, katA and katG, which encode three important proteins responsible for hydrogen peroxide (H(2)O(2)) sensing and detoxification in Xanthomonas in the presence of H(2)O(2), and nine important known Xoc virulence-related genes in plant cell-mimicking medium. Furthermore, the asnB mutation did not affect extracellular protease activity, extracellular polysaccharide production, motility or chemotaxis. Taken together, our results demonstrate the role of asnB in Xanthomonas for the first time.

epv, Encoding a hypothetical protein, is regulated by DSF-mediating quorum sensing as well as global regulator Clp and is required for optimal virulence in Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola causes bacterial leaf streak in rice, a destructive disease worldwide. In this study, six putative hypothetical secreted proteins, which were absent in X. oryzae pv. oryzae, were detected from X. oryzae pv. oryzicola strain BLS256. Disruption-based mutagenesis study revealed that one of them, Xoc_15235, named as extracellular polysaccharide and virulence-related gene (epv), was required for the optimal virulence in host rice but not for the induction of a hypersensitive reaction in nonhost tobacco. Sequence analysis revealed that epv was highly conserved in Xanthomonas spp. (except X. oryzae pv. oryzae). In-frame deletion of epv in X. oryzae pv. oryzicola dramatically impaired pathogen virulence and extracellular polysaccharide (EPS) production, one of the important known virulence-associated functions in Xanthomonas spp. Quantitative real-time reverse-transcription polymerase chain reaction showed that expression of both gumB (a gene encoding exopolysaccharide xanthan biosynthesis export protein) and a known virulence-related gene, pgk (encoding phosphoglycerate kinase), were obviously reduced in the epv-deletion mutant compared with the wild-type strain Rs105. In addition, we observed that epv was positively regulated by both diffusible signal factor and global regulator Clp in X. oryzae pv. oryzicola. Taken together, the novel roles and genetics of epv of X. oryzae pv. oryzicola in the EPS production and virulence were investigated for the first time.

[Relationship of histidine auxotrophy of Acidovorax citrulli with pathogenicity].

UNLABELLED: Acidovorax citrulli (Ac) is an important bacterium that occurs in watermelon, melon and other cucurbits. It mainly damages watermelon and melon, and can cause leaf blight, fruit rot, and even mortality. OBJECTIVE: To verify the relationship between defects in the synthesis of histidine and the pathogenicity of Ac. METHODS: We generated a transposon (Tn5) mutant library on the background of strain xjl12 of Ac. Then we used subclone technology to identify the gene. RESULTS: The mutant could not elicit the hypersensitive response (HR) in nonhost tobacco, and its virulence was reduced. It is impaired in hisC, which encodes the protein histidinolphosphate aminotransferase. The other three genes (hisA, hisB and hisD) involved in the process of histidine synthesis were also studied. These mutants could not elicit the hypersensitive response (HR) in nonhost tobacco; their virulence was reduced significantly and disease symptoms caused by mutants were delayed for 48 hours when compared to the wild type strain. By adding exogenous histidine, pathogenicity of the mutants was restored. CONCLUSION: The change of the characteristics of the mutants was directly related to the synthesis of histidine.

[Analysis of the flgD and figE genes regulated by diffusible signal factor in Xanthomonas oryzae pv. oryzicola].

OBJECTIVE: To investigate functions of flgDxoc and flgExoc genes regulated by diffusible signal factor (DSF) in Xanthomonas oryzae pv. oryzicola(Xoc)Rs105. METHODS: TheflgDxoc and flgExoc genes were amplified by PCR. We constructed deltaflgDxoc and deltaflgExoc, the deletion mutants from Rs105 by using double crossover method, and determined cell morphology, motility, pathogenicity in host rice and hypersensitive response (HR) in nonhost tobacco. We tested the differential expression of flgDxoc and flgExoc gene by reverse transcriptional polymerase chain reaction (RT-PCR) between the wide type and deltarpfFxoc (the deletion mutant of rpfFxoc gene, which could not produce DSF). RESULTS: We cloned flgDxoc and flgExoc from genomic DNA of Rs105. PCR and Southern blot analysis demonstrated that the flgDxoc and flgExoc genes were knocked out successfully. Both mutants were non-flagellated and significantly attenuated motility on the 0.3% semi-solid medium. The pathogenicity on rice were obviously attenuated in deltaflgDxoc and deltaflgExoc compared to the wild type. All the changes in mutant could be restored through the complementation. However, there was no significant difference in bacterial growth in MMX medium and induction of HR between mutant (deltaflgDxoc or deltaflgExoc) and the wild type. In addition, the results of RT-PCR demonstrated that the transcription level of flgDxoc and flgExoc were downregulated in deltarpfFxoc. CONCLUSION: This study showed that expressions of flgDxoc and flgExoc were positively regulated by DSF, and necessary for flagellar hook assembly and flagellar structure in Xoc. Meanwhile, FlgD and FlgE contributed to pathogen's virulence, motility and chemotaxis, but no differences at growth rate in MMX medium and HR in nonhost. In addition, our results provided molecular evidences that the contribution of DSF-type quorum sensing to pathogen's virulence might be, at least partially, dependent on bacterial flagellar in Xoc.

Identification and molecular characterization of twin-arginine translocation system (Tat) in Xanthomonas oryzae pv. oryzae strain PXO99.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases in rice. This study identified and characterized the contribution of the twin-arginine translocation (Tat) pathway to motility, chemotaxis, extracellular polysaccharide (EPS) production and virulence in X. oryzae pv. oryzae strain PXO99. The tatC disruption mutant (strain TCM) of strain PXO99 were generated, and confirmed both by PCR and Southern blotting. Strain PXO99 cells were highly motile in NYGB 0.3% soft agar plate. In contrast, the tatC mutation impaired motility. Furthermore, strain TCM cells lacked detectable flagella and exhibited almost no chemotaxis toward glucose under aerobic conditions, indicating that the Tat secretion pathway contributed to flagellar biogenesis and chemotactic responses. It was also observed that strain TCM exhibited a reductive production of extracellular polysaccharide (EPS) and a significant reduction of virulence on rice plants when compared with the wild type PXO99. However, the tatC mutation in strain PXO99 did not affect growth rate and the ability to induce hypersensitive response (HR) in nonhost tobacco (Nicotiana tabacum L. cv. Samsun). Our findings indicated that the Tat system of X. oryzae pv. oryzae played an important role in the pathogen's virulence.

The luxS gene is involved in AI-2 production, pathogenicity, and some phenotypes in Erwinia amylovora.

Erwinia amylovora causes fire blight of apple, pear, and other members of the Rosaceae family. The enzyme LuxS catalyzes the last step in the production of autoinducer-2 (AI-2), a molecule implicated with quorum sensing in many bacterial species. It is now well recognized that LuxS also plays a central role in sulfur metabolism and in the activated methyl cycle, which is responsible for the generation of S-adenosyl-L-methionine. A research paper has reported that luxS is not involved with quorum sensing in Er. amylovora, but in our study, Er. amylovora strain NCPPB1665 (Ea1665) produced luxS-dependent extracellular AI-2 activity. Additionally, the maximal AI-2 activity occurred during late-exponential and early-stationary growth phases and diminished during the stationary phase. The luxS mutant of Ea1665 was constructed, and the phenotypes of a defined luxS mutant have been characterized. Inactivation of luxS in Ea1665 impaired motility, extracellular polysaccharide (EPS) production, and tolerance for hydrogen peroxide, and reduced virulence on pear leaves.

Characterization of monoclonal antibodies for lead-chelate complexes: applications in antibody-based assays.

Monoclonal antibodies against lead were generated by immunizing BALB/c mice with lead conjugated to keyhole limpet hemocyanin (KLH) via a bifunctional chelator, S-2-(4-aminobenzyl)diethylenetriamine pentaacetic acid (DTPA). Stable hybridoma cell lines were produced by fusion of murine splenocytes and SP2/0 myeloma cells. One of the hybridomas generated from this fusion (4/7) synthesized and secreted an antibody that bound tightly to Pb2+-DTPA complexes but not to metal-free DTPA. The performance for a competitive inhibition enzyme-linked immunosorbent assay (ELISA) incorporating this antibody was assessed for its sensitivity to changes in pH, ionic strength, and blocking reagents. The cross-reactivities in this ELISA were less than 3% for Fe3+, Cd2+, Hg2+, and Cu2+ and less than 0.3% for Cr3+, Mn2+, Mg2+, In3+, Ag1+, Ni2+, Co2+, Zn2+, Ca2+, Cu1+, and Hg1+. The IC50 value achieved for lead was 2.72 +/- 0.034 microM, showing the detection range of 0.092-87.2 microM and the lowest detection limit of 0.056 +/- 0.005 microM. Recoveries from the analyte-fortified tap water and ultrapure water were in the range of 80-114% . These results indicate that the ELISA could be a convenient analytical tool for monitoring lead residues in drinking water.

Zinc uptake regulator (zur) gene involved in zinc homeostasis and virulence of Xanthomonas oryzae pv. oryzae in rice.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases in rice. In order to understand the gene of zinc uptake regulator (zur) involved in virulence of the pathogen in rice, we generated a mutant OSZRM by homologous suicide plasmid integration. The mutant failed to grow in NYGB medium supplemented with Zn(2+) or Fe(3+) at a concentration of 500 muM or 6 mM, whereas the wild-type strain grew well at the same conditions. The zur mutant was hypersensitive to hydrogen peroxide and exhibited reduction catalase activity and the production of extracellular polysaccharide (EPS). Interestingly, the mutant showed a reduction in virulence on rice but still kept triggering hypersensitive response (HR) in tobacco. When the mutant was complemented with the zur gene, the response was recovered to wild-type. These results suggested that zur gene is a functional member of the Zur regulator family that controls zinc and iron homeostasis, oxidative stress, and EPS production, which is necessary for virulence in X. oryzae pv. oryzae.