Using antagonistic soil bacteria and their cell‐free filtrates to control the black rot pathogen Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris (Xcc) is a phytopathogenic bacteria, and it is the causative agent of black rot in crucifers. Recent studies have shown that Bacillus species have strong biological control on Xanthomonas. One of the mechanisms of this control is secondary metabolites production. A collection of 257 bacteria isolated from a suppressive soil was evaluated for in vitro antagonistic activity against X. campestris, and 92 isolates (44.6%) were able to inhibit its growth. Among the 92 isolates evaluated in the double‐layer technique, 51 (55.43%) inhibited Xcc growth on the inhibition tests with cell‐free filtrates (CFF) in liquid medium. Thirteen of these isolates presented 50% or more growth inhibition, and five isolates presented 100% growth inhibition of Xcc. The CFF of the isolate TCDT‐08, which belongs to the Paenibacillus genus, was used for in vivo tests with kale crops. The artificial inoculation of kale with Xcc‐629IBSBF pretreated with CFF from the isolate TCDT‐08 demonstrated that the bacterium loses the ability of colonizing kale and of causing black rot. A Paenibacillus sp. isolate has strong inhibitory activity against X. campestris pv. campestris, and further studies can result in the use of this isolate to protect kale from Xcc infection.     

Pan Proteome of Xanthomonas campestris pv. campestris Isolates Contrasting in Virulence

Fully sequenced genomes of Xanthomonas campestris pv. campestris (Xcc) strains are reported. However, intra‐pathovar differences are still intriguing and far from clear. In this work, the contrasting virulence between two isolates of Xcc ‐ Xcc51 (more virulent) and XccY21 (less virulent) is evaluated by determining their pan proteome profiles. The bacteria are grown in NYG and XVM1 (optimal for induction of hrp regulon) broths and collected at the max‐exponential growth phase. Shotgun proteomics reveals a total of 329 proteins when Xcc isolates are grown in XVM1. A comparison of both profiles reveals 47 proteins with significant abundance fluctuations, out of which, 39 show an increased abundance in Xcc51 and are mainly involved in virulence/adaptation mechanisms, genetic information processing, and membrane receptor/iron transport systems, such as BfeA, BtuB, Cap, Clp, Dcp, FyuA, GroEs, HpaG, Tig, and OmpP6. Several differential proteins are further analyzed by qRT‐PCR, which reveals a similar expression pattern to the protein abundance. The data shed light on the complex Xcc pathogenicity mechanisms and point out a set of proteins related to the higher virulence of Xcc51. This information is essential for the development of more efficient strategies aiming at the control of black rot disease.     

Virulence analysis and genetic diversity of Xanthomonas campestris pv. campestris causing black rot of crucifers

Xanthomonas campestris pv. campestris (Xcc) is a devastating bacterium to cause black rot disease in crucifers. To study the genetic diversity and virulence analysis, 24 isolates of Xcc were collected from cole crops including cauliflower, cabbage, broccoli and knol khol from different agro-climatic regions of India ranging from temperate to subtropical climates. For virulence analysis, 24 isolates of Xcc were tested on 27 cultivars of crucifers including seven species of Brassica spp. (B. campestris, B. carinata, B. juncea, B. napus, B. nigra, B. oleracea and B. rapa), Sinapsis alba, Eruca sativa and Raphanus sativus under field conditions at IARI, New Delhi, during November 2010–March 2011. Maximum disease incidence 85.15% was found in the cultivars of crucifers caused by strains Xcc-C124, Xcc-C6, Xcc-C125, Xcc-C111 and Xcc-C131 after 15 days of inoculation and significantly increased after 30 days. Black rot severity in cultivars of crucifers varied from 0 to 6.9 and 0 to 7.9 out of 9 scale after 15 and 30 days of inoculation, respectively. But, no disease incidence was recorded on all the tested cultivars of B. juncea (Pusa Bold, Varuna, Pusa Mustard-21 and Pusa Vijay) against all the strains of Xcc after 15 days. Genetic diversity of 24 strains of Xcc was studied using REP- and BOX-PCR, indicating the existence of wide range of genetic diversity among the strains. The strains were clustered into two groups at 50% similarity coefficient and designated as Group 1 and Group 2. The majority of the strains (23 strains) were clustered under Group 1 except Xcc-C120, which formed separate group (Group 2). In the present study, genetic diversity and virulence pattern in Indian strains of Xcc were established, which will be helpful in the development of resistant genotypes against this bacterial pathogen.     

Evaluation of Paenibacillus spp. isolates for the biological control of black rot in Brassica oleracea var. capitata (cabbage)

The ability of isolates of Paenibacillus spp. to protect Brassica oleracea var. capitata (cabbage) against the black rot pathogen, Xanthomonas campestris pv. campestris (Xcc),was evaluated. Twenty-four isolates of Paenibacillus spp., isolated from New Zealand-grown brassica hosts or soil, were evaluated for in vitro antagonism towards six Xcc isolates. Seven Paenibacillus spp. isolates with different levels of in vitro suppressive activity against Xcc were screened in pot experiments for their capacity to reduce black rot symptoms on cabbage. Two Paenibacillus isolates (P10 and P16) exhibited biocontrol activity against Xcc, and four isolates (P1, P6, P9, and P24) reduced cabbage seed germination and seedling emergence. The dependence of bioactivity on inoculum rate was investigated with three Paenibacillus isolates (P6, P10, and P16) at three different concentrations (5?×?10⁸, 5?×?10⁹, and 5?×?10¹⁰?CFU?ml^?1). Negative effects on seedling emergence were detected with isolate P6 at concentrations?≥5?×?10⁹?CFU?ml^?1. All three isolates applied at the three concentrations reduced black rot symptoms on the cotyledons and true leaves. There was poor or no relationship between the inhibitory effect of Paenibacillus spp. isolates on the growth of Xcc in vitro, and their biocontrol activity in vivo. Paenibacillus isolate P16 was identified as a potential biological control of black rot in cabbage.     

Genetic Diversity and Population Structure of the Xanthomonas campestris pv. campestris Strains Affecting Cabbages in China Revealed by MLST and Rep-PCR Based Genotyping

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot for cruciferous vegetables worldwide, especially for the cole crops such as cabbage and cauliflower. Due to the lack of resistant cabbage cultivars, black rot has brought about considerable yield losses in recent years in China. Understanding of the pathogen features is a key step for disease prevention, however, the pathogen diversity, population structure, and virulence are largely unknown. In this study, we studied 50 Xcc strains including 39 Xcc isolates collected from cabbage in 20 regions across China, using multilocus sequence genotyping (MLST), repetitive DNA sequence-based PCR (rep-PCR), and pathogenicity tests. For MLST analysis, a total of 12 allelic profiles (AP) were generated, among which the largest AP was AP1 containing 32 strains. Further cluster analysis of rep-PCR divided all strains into 14 DNA groups, with the largest group DNA I comprising of 34 strains, most of which also belonged to AP1. Inoculation tests showed that the representative Xcc strains collected from diverse regions performed differential virulence against three brassica hosts compared with races 1 and 4. Interestingly, these results indicated that AP1/DNA I was not only the main pathotype in China, but also a novel group that differed from the previously reported type races in both genotype and virulence. To our knowledge, this is the first extensive genetic diversity survey for Xcc strains in China, which provides evidence for cabbage resistance breeding and opens the gate for further cabbage-Xcc interaction studies.     

Biological Control of Black Rot (Xanthomonas campestris pv. campestris) of Cabbage in Tanzania with Bacillus strains

We evaluated the biocontrol efficacy of strains of Bacillus from Tanzania against the black rot pathogen, Xanthomonas campestris pv. campestris, in cabbage and the influence of the method of application under field conditions. The incidence and severity of black rot in the foliage, stems and heads of the highly susceptible cultivar, Copenhagen Market, were significantly reduced, especially when antagonists were applied through the roots as compared to application through the seeds or foliage (cotyledons). Promising antagonists included strains of B. cereus, B. lentimorbus and B. pumilus.     

A novel 3-oxoacyl-ACP reductase (FabG3) is involved in the xanthomonadin biosynthesis of Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, produces a membrane-bound yellow pigment called xanthomonadin to protect against photobiological and peroxidative damage, and uses a quorum-sensing mechanism mediated by the diffusible signal factor (DSF) family signals to regulate virulence factors production. The Xcc gene XCC4003, annotated as Xcc fabG3, is located in the pig cluster, which may be responsible for xanthomonadin synthesis. We report that fabG3 expression restored the growth of the Escherichia coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrated that FabG3 catalyses the reduction of 3-oxoacyl-acyl carrier protein (ACP) intermediates in fatty acid synthetic reactions, although FabG3 had a lower activity than FabG1. Moreover, the fabG3 deletion did not affect growth or fatty acid composition. These results indicate that Xcc fabG3 encodes a 3-oxoacyl-ACP reductase, but is not essential for growth or fatty acid synthesis. However, the Xcc fabG3 knock-out mutant abolished xanthomonadin production, which could be only restored by wild-type fabG3, but not by other 3-oxoacyl-ACP reductase-encoding genes, indicating that Xcc FabG3 is specifically involved in xanthomonadin biosynthesis. Additionally, our study also shows that the Xcc fabG3-disrupted mutant affects Xcc virulence in host plants.     

The type III effector AvrXccB in Xanthomonas campestris pv. campestris targets putative methyltransferases and suppresses innate immunity in Arabidopsis

Xanthomonas campestris pv. campestris (Xcc) causes black rot, one of the most important diseases of brassica crops worldwide. The type III effector inventory plays important roles in the virulence and pathogenicity of the pathogen. However, little is known about the virulence function(s) of the putative type III effector AvrXccB in Xcc. Here, we investigated the immune suppression ability of AvrXccB and the possible underlying mechanisms. AvrXccB was demonstrated to be secreted in a type III secretion system‐dependent manner. AvrXccB tagged with green fluorescent protein is localized to the plasma membrane in Arabidopsis, and the putative N‐myristoylation motif is essential for its localization. Chemical‐induced expression of AvrXccB suppresses flg22‐triggered callose deposition and the oxidative burst, and promotes the in planta growth of Xcc and Pseudomonas syringae pv. tomato in transgenic Arabidopsis plants. The putative catalytic triad and plasma membrane localization of AvrXccB are required for its immunosuppressive activity. Furthermore, it was demonstrated that AvrXccB interacts with the Arabidopsis S‐adenosyl‐l ‐methionine‐dependent methyltransferases SAM‐MT1 and SAM‐MT2. Interestingly, SAM‐MT1 is not only self‐associated, but also associated with SAM‐MT2 in vivo. SAM‐MT1 and SAM‐MT2 expression is significantly induced upon stimulation of microbe‐associated molecular patterns and bacterial infection. Collectively, these findings indicate that AvrXccB targets a putative methyltransferase complex and suppresses plant immunity.     

Plant responses underlying nonhost resistance of Citrus limon against Xanthomonas campestris pv. campestris

Citrus is an economically important fruit crop that is severely afflicted by citrus canker, a disease caused by Xanthomonas citri ssp. citri (X. citri); thus, new sustainable strategies to manage this disease are needed. Although all Citrus spp. are susceptible to this pathogen, they are resistant to other Xanthomonas species, exhibiting non-host resistance (NHR), for example, to the brassica pathogen X. campestris pv. campestris (Xcc) and a gene-for-gene host defence response (HDR) to the canker-causing X. fuscans ssp. aurantifolii (Xfa) strain C. Here, we examine the plant factors associated with the NHR of C. limon to Xcc. We show that Xcc induced asymptomatic type I NHR, allowing the bacterium to survive in a stationary phase in the non-host tissue. In C. limon, this NHR shared some similarities with HDR; both defence responses interfered with biofilm formation, and were associated with callose deposition, induction of the salicylic acid (SA) signalling pathway and the repression of abscisic acid (ABA) signalling. However, greater stomatal closure was seen during NHR than during HDR, together with different patterns of accumulation of reactive oxygen species and phenolic compounds and the expression of secondary metabolites. Overall, these differences, independent of Xcc type III effector proteins, could contribute to the higher protection elicited against canker development. We propose that Xcc may have the potential to steadily activate inducible defence responses. An understanding of these plant responses (and their triggers) may allow the development of a sustained and sustainable resistance to citrus canker.     

Loss of chloroplast‐localized protein phosphatase 2Cs in Arabidopsis thaliana leads to enhancement of plant immunity and resistance to Xanthomonas campestris pv. campestris infection

Protein phosphatases (PPs) counteract kinases in reversible phosphorylation events during numerous signal transduction pathways in eukaryotes. PP2Cs, one of the four major classes of the serine/threonine‐specific PP family, are greatly expanded in plants. Thus, PP2Cs are thought to play a specific role in signal transduction pathways. Some rice PP2Cs classified in subgroup K are responsive to infection by the compatible Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight. In Arabidopsis thaliana, orthologous PP2C genes (AtPP2C62 and AtPP2C26) classified to subgroup K are also responsive to Xanthomonas campestris pv. campestris (Xcc, causal agent of black rot) infection. To elucidate the function of these subgroup K PP2Cs, atpp2c62‐ and atpp2c26‐deficient A. thaliana mutants were characterized. A double mutant plant which was inoculated with a compatible Xcc showed reduced lesion development, as well as the suppression of bacterial multiplication. AtPP2C62 and AtPP2C26 localized to the chloroplast. Furthermore, the photosynthesis‐related protein, chaperonin‐60, was indicated as the potential candidate for the dephosphorylated substrate catalysed by AtPP2C62 and AtPP2C26 using two‐dimensional isoelectric focusing sodium dodecylsulfate‐polyacrylamide gel electrophoresis (2D‐IDF‐SDS‐PAGE). Taken together, AtPP2C62 and AtPP2C26 are suggested to be involved in both photosynthesis and suppression of the plant immune system. These results imply the occurrence of crosstalk between photosynthesis and the plant defence system to control productivity under pathogen infection.     

Biochemical insights into basal and induced resistance in cabbage to black rot

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is the most devastating disease of brassica, but the mechanisms of basal or induced resistance in cabbage remain largely unknown. Here, we performed three experiments to investigate biochemical features associated with cabbage resistance to black rot. In the first experiment, biochemical changes were assessed in plants that were inoculated with a highly (UFPR 5) or a moderately (Xcc 10) aggressive Xcc isolate. In the second experiment, we examined the biochemical responses in two cultivars (Chato de Quintal [CQ] and Louco de Verão [LV], susceptible and moderately resistant to Xcc, respectively). Finally, we examined whether acibenzolar‐S‐methyl (ASM) could induce cabbage resistance to Xcc. Plants inoculated with the Xcc 10 isolate displayed higher activities of superoxide dismutase (SOD), peroxidase (POX) and ascorbate peroxidase (APX), whereas activities of chitinase (CHI), β‐1,3‐glucanase (GLU) and polyphenol oxidase (PPO) as well as the concentrations of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) were lower compared to plants inoculated with the UFPR 5 isolate. The resistance of the cultivar LV to Xcc was linked to increases in the activities of CHI, GLU, and PPO and decreases in the activities of SOD, POX and APX as well as in the concentrations of H₂O₂ and MDA relative to the cultivar CQ. In general, ASM‐sprayed plants displayed higher activities for the enzymes studied, which was associated with decreased disease symptoms and oxidative stress. Taken together, our results demonstrated that high activities of both defence and antioxidant enzymes played a major role in both basal and induced resistance of cabbage to black rot.     

Identification of genes differentially expressed in cauliflower associated with resistance to <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis>

Black rot, caused by Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc), is one of the most damaging diseases of cauliflower and other crucifers. In order to investigate the molecular resistance mechanisms and to find the genes related to black rot resistance in cauliflower, a suppression subtractive hybridization (SSH) cDNA library was constructed using resistant line C712 and its susceptible near-isogenic line C731 as tester and driver, respectively. A total of 280 clones were obtained from the library by reverse northern blotting. Sequencing analysis and homology searching showed that these clones represent 202 unique sequences. The library included many defense/disease-resistant related genes, such as plant defensin gene PDF1.2, lipid transfer protein, thioredoxin h. Gene expression profiles of 12 genes corresponding to different functional categories were monitored by real-time RT-PCR. The results showed that the expression induction of these genes in the susceptible line C712 in response to Xcc was quicker and more intense, while in C731 the reaction was delayed and limited. Our results imply that these up-regulated genes might be involved in cauliflower responses against Xcc infection. Information obtained from this study could be used to understand the molecular mechanisms of disease response in cauliflower under Xcc stress.     

Real Time Live Imaging of Phytopathogenic Bacteria Xanthomonas campestris pv. campestris MAFF106712 in ‘Plant Sweet Home’

Xanthomonas is one of the most widespread phytobacteria, causing diseases on a variety of agricultural plants. To develop novel control techniques, knowledge of bacterial behavior inside plant cells is essential. Xanthomonas campestris pv. campestris, a vascular pathogen, is the causal agent of black rot on leaves of Brassicaceae, including Arabidopsis thaliana. Among the X. campestris pv. campestris stocks in the MAFF collection, we selected XccMAFF106712 as a model compatible pathogen for the A. thaliana reference ecotype Columbia (Col-0). Using modified green fluorescent protein (AcGFP) as a reporter, we observed real time XccMAFF106712 colonization in planta with confocal microscopy. AcGFP-expressing bacteria colonized the inside of epidermal cells and the apoplast, as well as the xylem vessels of the vasculature. In the case of the type III mutant, bacteria colonization was never detected in the xylem vessel or apoplast, though they freely enter the xylem vessel through the wound. After 9 days post inoculation with XccMAFF106712, the xylem vessel became filled with bacterial aggregates. This suggests that Xcc colonization can be divided into main four steps, (1) movement in the xylem vessel, (2) movement to the next cell, (3) adhesion to the host plant cells, and (4) formation of bacterial aggregates. The type III mutant abolished at least steps (1) and (2). Better understanding of Xcc colonization is essential for development of novel control techniques for black rot.     

The role of PilZ domain proteins in the virulence of Xanthomonas campestris pv. campestris

Cyclic di‐GMP [(bis‐(3′–5′)‐cyclic di‐guanosine monophosphate)] is an almost ubiquitous second messenger in bacteria that is implicated in the regulation of a range of functions that include developmental transitions, aggregative behaviour, adhesion, biofilm formation and virulence. Comparatively little is known about the mechanism(s) by which cyclic di‐GMP exerts these various regulatory effects. PilZ has been identified as a cyclic di‐GMP binding protein domain; proteins with this domain are involved in regulation of specific cellular processes, including the virulence of animal pathogens. Here we have examined the role of PilZ domain proteins in virulence and the regulation of virulence factor synthesis in Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot of crucifers. The Xcc genome encodes four proteins (XC0965, XC2249, XC2317 and XC3221) that have a PilZ domain. Mutation of XC0965, XC2249 and XC3221 led to a significant reduction of virulence in Chinese radish. Mutation of XC2249 and XC3221 led to a reduction in motility whereas mutation of XC2249 and XC0965 affected extracellular enzyme production. All mutant strains were unaffected in biofilm formation in vitro. The reduction of virulence following mutation of XC3221 could not be wholly attributed to an effect on motility as mutation of pilA, which abolishes motility, has a lesser effect on virulence.     

Peptidoglycan hydrolysis mediated by the amidase AmiC and its LytM activator NlpD is critical for cell separation and virulence in the phytopathogen Xanthomonas campestris

The essential stages of bacterial cell separation are described as the synthesis and hydrolysis of septal peptidoglycan (PG). The amidase, AmiC, which cleaves the peptide side‐chains linked to the glycan strands, contributes critically to this process and has been studied extensively in model strains of Escherichia coli. However, insights into the contribution of this protein to other processes in the bacterial cell have been limited. Xanthomonas campestris pv. campestris (Xcc) is a phytopathogen that causes black rot disease in many economically important plants. We investigated how AmiC and LytM family regulators, NlpD and EnvC, contribute to virulence and cell separation in this organism. Biochemical analyses of purified AmiC demonstrated that it could hydrolyse PG and its activity could be potentiated by the presence of the regulator NlpD. We also established that deletion of the genes encoding amiC1 or nlpD led to a reduction in virulence as well as effects on colony‐forming units and cell morphology. Moreover, further genetic and biochemical evidence showed that AmiC1 and NlpD affect the secretion of type III effector XC3176 and hypersensitive response (HR) induction in planta. These findings indicate that, in addition to their well‐studied role(s) in cell separation, AmiC and NlpD make an important contribution to the type III secretion (T3S) and virulence regulation in this important plant pathogen.     

Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. campestris

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.