Background: Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease of crucifers worldwide. The molecular genetic diversity and host specificity of Xcc are poorly understood. Results: We constructed a microarray based on the complete genome sequence of Xcc strain 8004 and investigated the genetic diversity and host specificity of Xcc by array-based comparative genome hybridization analyses of 18 virulent strains. The results demonstrate that a genetic core comprising 3,405 of the 4,186 coding sequences (CDSs) spotted on the array are conserved and a flexible gene pool with 730 CDSs is absent/highly divergent (AHD). The results also revealed that 258 of the 304 proved/presumed pathogenicity genes are conserved and 46 are AHD. The conserved pathogenicity genes include mainly the genes involved in type I, II and III secretion systems, the quorum sensing system, extracellular enzymes and polysaccharide production, as well as many other proved pathogenicity genes, while the AHD CDSs contain the genes encoding type IV secretion system (T4SS) and type III-effectors. A Xcc T4SS-deletion mutant displayed the same virulence as wild type. Furthermore, three avirulence genes (avrXccC, avrXccE1 and avrBs1) were identified. avrXccC and avrXccE1 conferred avirulence on the hosts mustard cultivar Guangtou and Chinese cabbage cultivar Zhongbai-83, respectively, and avrBs1 conferred hypersensitive response on the nonhost pepper ECW10R. Conclusion: About 80% of the Xcc CDSs, including 258 proved/presumed pathogenicity genes, is conserved in different strains. Xcc T4SS is not involved in pathogenicity. An efficient strategy to identify avr genes determining host specificity from the AHD genes was developed.

Dickeya zeae is the causal agent of bacterial soft rot disease, with a wide range of hosts all over the world. At present, chemical agents, especially agricultural antibiotics, are commonly used in the prevention and control of bacterial soft rot, causing the emergence of resistant pathogens and therefore increasing the difficulty of disease prevention and control. This study aims to provide a safer and more effective biocontrol method for soft rot disease caused by D. zeae. The spot-on-lawn assay was used to screen antagonistic bacteria, and three strains including SC3, SC11 and 3-10 revealed strong antagonistic effects and were identified as Pseudomonas fluorescens, P. parafulva and Bacillus velezensis, respectively, using multi-locus sequence analysis (MLSA) based on the sequences of 16S rRNA and other housekeeping genes. In vitro antimicrobial activity showed that two Pseudomonas strains SC3 and SC11 were only antagonistic to some pathogenic bacteria, while strain 3-10 had broad-spectrum antimicrobial activity on both pathogenic bacteria and fungi. Evaluation of control efficacy in greenhouse trials showed that they all restrained the occurrence and development of soft rot disease caused by D. zeae MS2 or EC1. Among them, strain SC3 had the most impressive biocontrol efficacy on alleviating the soft rot symptoms on both monocotyledonous and dicotyledonous hosts, and strain 3-10 additionally reduced the occurrence of banana wilt disease caused by Fusarium oxysporum f. sp. cubensis. This is the first report of P. fluorescens, P. parafulva and B. velezensis as potential bio-reagents on controlling soft rot disease caused by D. zeae.

Rice bacterial blight is a devastating bacterial disease threatening rice yield all over the world and Xanthomonas oryzae pv. oryzae is traditionally believed to be the pathogen. In recent years, we have received diseased rice samples with symptoms of blighted leaves from Sichuan and Guangdong provinces, China. Pathogen isolation and classification identified two different enterobacteria as the causal agents, namely Enterobacter asburiae and Pantoea ananatis. Among them, E. asburiae was isolated from samples of both provinces, and P. ananatis was only isolated from the Sichuan samples. Different from rice foot rot pathogen Dickeya zeae EC1 and rice bacterial blight pathogen X. oryzae pv. oryzae PXO99A, strains SC1, RG1, and SC7 produced rare cell wall degrading enzymes (CWDEs) but more extrapolysaccharides (EPS). E. asburiae strains SC1 and RG1 produced bacteriostatic substances while P. ananatis strain SC7 produced none. Pathogenicity tests indicated that all of them infected monocotyledonous rice and banana seedlings, but not dicotyledonous potato, radish, or cabbage. Moreover, strain RG1 was most virulent, while strains SC1 and SC7 were similarly virulent on rice leaves, even though strain SC1 propagated significantly faster in rice leaf tissues than strain SC7. This study firstly discovered E. asburiae as a new pathogen of rice bacterial blight, and in some cases, P. ananatis could be a companion pathogen. Analysis on production of virulence factors suggested that both pathogens probably employ a different mechanism to infect hosts other than using cell wall degrading enzymes to break through host cell walls.

Bayberry (Myrica rubra) is a commercial fruit in China. For the past seven years, twig blight disease has been attacking bayberry plantations in Shantou City, Guangdong Province, China, leading to destructive damage and financial loss. In this study, five fungal species associated with twig dieback and stem blight were identified based on morphological characteristics combined with multilocus sequence analysis (MLSA) on the internal transcribed spacer (ITS) region, partial sequences of β-tubulin (tub2), translation elongation factor 1-α (tef1-α), large subunit ribosomal RNA (LSU) and small subunit ribosomal RNA (SSU) genes, which are Epicoccum sorghinum, Neofusicoccum parvum, Lasiodiplodia theobromae, Nigrospora oryzae and a Pestalotiopsis new species P. myricae. P. myricae is the chief pathogen in fields, based on its high isolation rate and fast disease progression after inoculation. To our knowledge, this is the first study reporting the above five fungi as the pathogens responsible for bayberry twig blight. Indoor screening of fungicides indicates that Prochloraz (copper salt) is the most promising fungicide for field application, followed by Pyraclostrobin, 15% Difenoconazole + 15% Propiconazole, Difenoconazole and Myclobutanil. Additionally, Bacillus velezensis strain 3–10 and zeamines from Dickeya zeae strain EC1 could be used as potential ecofriendly alternatives to control the disease.

The GntR family transcription regulator HpaR1 identified from Xanthomonas campestris pv. campestris has been previously shown to positively regulate the genes responsible for hypersensitive reaction and pathogenicity and to autorepress its own expression. Here, we demonstrated that HpaR1 is a global regulator that positively regulates diverse biological processes, including xanthan polysaccharide production, extracellular enzyme activity, cell motility and tolerance to various stresses. To investigate the regulatory mechanisms of HpaR1, we began with xanthan polysaccharide production, which is governed by a cluster of gum genes. These are directed by the gumB promoter. Disruption of HpaR1 significantly reduced gumB transcription and an electrophoretic mobility shift assay demonstrated that HpaR1 interacts directly with gumB promoter. DNase I footprint analysis revealed that HpaR1 and RNA polymerase were bound to the sequences extending from −21 to +10 and −41 to +29 relative to the transcription initiation site of gumB, respectively. Furthermore, in vitro transcription assays showed that HpaR1 facilitated the binding of RNA polymerase to gumB promoter, leading to an enhancement of its transcription. These results suggest that HpaR1 regulates gumB transcription via a mechanism similar but different to what was found, until now, to only be used by some MerR family transcription activators.

Background: The virulence of the plant pathogenXanthomonas campestrispv. campestris(Xcc) involves the coordinate expression of many virulence factors, including surface appendages flagellum and type IV pili, which are required for pathogenesis and the colonization of host tissues. Despite many insights gained on the structure and functions played by flagellum and pili in motility, biofilm formation, surface attachment and interactions with bacteriophages, we know little about how these appendages are regulated inXcc.Results: Here we present evidence demonstrating the role of two single domain response regulators PilG and PilH in the antagonistic control of flagellum-dependent (swimming) and pili-dependent (swarming) motility. Using informative mutagenesis, we reveal PilG positively regulates swimming motility while and negatively regulating swarming motility. Conversely, PilH negatively regulates swimming behaviour while and positively regulating swarming motility. By transcriptome analyses (RNA-seq and RT-PCR) we confirm these observations as PilG is shown to upregulate many genes involved chemotaxis and flagellar biosynthesis but these similar genes were downregulated by PilH. Co-immunoprecipitation, bacterial two-hybrid and pull-down analyses showed that PilH and PilG were able to interact with district subsets of proteins that potentially account for their regulatory impact. Additionally, we present evidence, using mutagenesis that PilG and PilH are involved in other cellular processes, including chemotaxis and virulence.Conclusions: Taken together, we demonstrate that for the conditions tested PilG and PilH have inverse regulatory effects on flagellum-dependent and pili-dependent motility inXccand that this regulatory impact depends on these proteins influences on genes/proteins involved in flagellar biosynthesis and pilus assembly.