Preharvest UV-C Hormesis Induces Key Genes Associated With Homeostasis, Growth and Defense in Lettuce Inoculated With Xanthomonas campestris pv. vitians

Preharvest application of hormetic doses of ultraviolet-C (UV-C) generates beneficial effects in plants. In this study, within 1 week, four UV-C treatments of 0.4 kJ/m2 were applied to 3-week-old lettuce seedlings. The leaves were inoculated with a virulent strain of Xanthomonas campestris pv. vitians (Xcv) 48 h after the last UV-C application. The extent of the disease was tracked over time and a transcriptomic analysis was performed on lettuce leaf samples. Samples of lettuce leaves, from both control and treated groups, were taken at two different times corresponding to T2, 48 h after the last UV-C treatment and T3, 24 h after inoculation (i.e., 72 h after the last UV-C treatment). A significant decrease in disease severity between the UV-C treated lettuce and the control was observed on days 4, 8, and 14 after pathogen inoculation. Data from the transcriptomic study revealed, that in response to the effect of UV-C alone and/or UV-C + Xcv, a total of 3828 genes were differentially regulated with fold change (|log2-FC|) > 1.5 and false discovery rate (FDR) < 0.05. Among these, of the 2270 genes of known function 1556 were upregulated and 714 were downregulated. A total of 10 candidate genes were verified by qPCR and were generally consistent with the transcriptomic results. The differentially expressed genes observed in lettuce under the conditions of the present study were associated with 14 different biological processes in the plant. These genes are involved in a series of metabolic pathways associated with the ability of lettuce treated with hormetic doses of UV-C to resume normal growth and to defend themselves against potential stressors. The results indicate that the hormetic dose of UV-C applied preharvest on lettuce in this study, can be considered as an eustress that does not interfere with the ability of the treated plants to carry on a set of key physiological processes namely: homeostasis, growth and defense.

Evaluating the Antibacterial Activity and Mode of Action of Thymol-Loaded Chitosan Nanoparticles Against Plant Bacterial Pathogen Xanthomonas campestris pv. campestris

The bacterium Xanthomonas campestris pv. campestris (Xcc) causes black rot disease in cruciferous crops, resulting in severe yield loss worldwide. The excessive use of chemical pesticides in agriculture to control diseases has raised significant concern about the impact on the environment and human health. Nanoparticles have recently gained significant attention in agriculture owing to their promising application in plant disease control, increasing soil fertility and nutrient availability. In the current study, we synthesized thymol-loaded chitosan nanoparticles (TCNPs) and assessed their antibacterial activity against Xcc. The synthesis of TCNPs was confirmed by using ultraviolet–visible spectroscopy. Fourier-transform infrared spectroscopy, transmission electron microscopy, and scanning electron microscopy analysis revealed the functional groups, size, and shape of TCNPs, with sizes ranging from 54 to 250 nm, respectively. The antibacterial activity of TCNPs against Xcc was investigated in vitro by liquid broth, cell viability, and live dead staining assay, and all of them demonstrated the antibacterial activity of TCNPs. Furthermore, TCNPs were found to directly inhibit the growth of Xcc by suppressing the growth of biofilm formation and the production of exopolysaccharides and xanthomonadin. The ultrastructure studies revealed membrane damage in TCNP-treated Xcc cells, causing a release of intracellular contents. Headspace/gas chromatography (GC)–mass spectrometry (MS) analysis showed changes in the volatile profile of Xcc cells treated with TCNPs. Increased amounts of carbonyl components (mainly ketones) and production of new volatile metabolites were observed in Xcc cells incubated with TCNPs. Overall, this study reveals TCNPs as a promising antibacterial candidate against Xcc.

Induction of Isochromanones by Co-Cultivation of the Marine Fungus Cosmospora sp. and the Phytopathogen Magnaporthe oryzae

Microbial co-cultivation is a promising approach for the activation of biosynthetic gene clusters (BGCs) that remain transcriptionally silent under artificial culture conditions. As part of our project aiming at the discovery of marine-derived fungal agrochemicals, we previously used four phytopathogens as model competitors in the co-cultivation of 21 marine fungal strains. Based on comparative untargeted metabolomics analyses and anti-phytopathogenic activities of the co-cultures, we selected the co-culture of marine Cosmospora sp. with the phytopathogen Magnaporthe oryzae for in-depth chemical studies. UPLC-MS/MS-based molecular networking (MN) of the co-culture extract revealed an enhanced diversity of compounds in several molecular families, including isochromanones, specifically induced in the co-culture. Large scale co-cultivation of Cosmospora sp. and M. oryzae resulted in the isolation of five isochromanones from the whole co-culture extract, namely the known soudanones A, E, D (1-3) and their two new derivatives, soudanones H-I (4-5), the known isochromans, pseudoanguillosporins A and B (6, 7), naphtho-γ-pyrones, cephalochromin and ustilaginoidin G (8, 9), and ergosterol (10). Their structures were established by NMR, HR-ESIMS, FT-IR, electronic circular dichroism (ECD) spectroscopy, polarimetry ([α]D), and Mosher’s ester reaction. Bioactivity assays revealed antimicrobial activity of compounds 2 and 3 against the phytopathogens M. oryzae and Phytophthora infestans, while pseudoanguillosporin A (6) showed the broadest and strongest anti-phytopathogenic activity against Pseudomonas syringae, Xanthomonas campestris, M. oryzae and P. infestans. This is the first study assessing the anti-phytopathogenic activities of soudanones.

Interactive Regulation of Hormone and Resistance Gene in Proline Metabolism Is Involved in Effector-Triggered Immunity or Disease Susceptibility in the Xanthomonas campestris pv. campestris–Brassica napus Pathosystem

To characterize cultivar variations in hormonal regulation of the transition between pattern-triggered immunity (PTI) and effector-triggered immunity or susceptibility (ETI or ETS), the responses of resistance (R-) genes, hydrogen peroxide, and proline metabolism in two Brassica napus cultivars to contrasting disease susceptibility (resistant cv. Capitol vs. susceptible cv. Mosa) were interpreted as being linked to those of endogenous hormonal levels and signaling genes based on a time course of disease symptom development. Disease symptoms caused by the Xanthomonas campestris pv. campestris (Xcc) infections were much more developed in cv. Mosa than in cv. Capitol, as shown by an earlier appearance (at 3 days postinoculation [3 DPI]) and larger V-shaped necrosis lesions (at 9–15 DPI) in cv. Mosa. The cultivar variations in the R-genes, hormone status, and proline metabolism were found in two different phases (early [0–3 DPI] and later [9–15 DPI]). In the early phase, Xcc significantly upregulated PTI-related cytoplasmic kinase (Botrytis-induced kinase-1 [BIK1]) expression (+6.3-fold) with salicylic acid (SA) accumulation in cv. Capitol, while relatively less (+2.6-fold) with highly increased jasmonic acid (JA) level in cv. Mosa. The Xcc-responsive proline accumulation in both cultivars was similar to upregulated expression of proline synthesis-related genes (P5CS2 and P5CR). During the later phase in cv. Capitol, Xcc-responsive upregulation of ZAR1 (a coiled-coil-nucleotide binding site-leucine-rich repeat [CC-NB-LRR-type R-gene]) was concomitant with a gradual increase in JA levels without additional proline accumulation. However, in cv. Mosa, upregulation of TAO1 (a toll/interleukin-1 receptor-nucleotide binding site-leucine-rich repeat [TIR-NB-LRR-type R-gene]) was consistent with an increase in SA and abscisic acid (ABA) levels and resulted in an antagonistic depression of JA, which led to a proline accumulation. These results indicate that Xcc-induced BIK1- and ZAR1-mediated JA signaling interactions provide resistance and confirm ETI, whereas BIK1- and TAO1-enhanced SA- and/or ABA-mediated proline accumulation is associated with disease susceptibility (ETS).

Molecular characterization of xanthan gum producing Xanthomonas Campestris isolated from dark rot spotted leaves in Keffi, Nasarawa State, Nigeria

Background: Despite the wide application of Xanthan gum, its commercial production remains a global challenge. In recent years, considerable research has been carried out using agro-industrial wastes, which are renewable and abundantly available to produce value-added products. The present study was set out for molecular identification of Xanthomonas campestris from leaves of four different plants with indications of dark rot spots and evaluation of their xanthan gum production capacity. Methods: Twenty-five (25) samples of leaves from four different plants with indications of dark rot spots were collected from the study area and isolated for Xanthomonas campestris following standard microbiological methods. Cultural, morphological and biochemical tests were conducted to confirm the organism. Results: The results revealed that of the total 100 samples taken, 6 leaves (24%) were infected with Xanthomonas species in mint, 3(12%) were infected in mango, 1(4%) were infected in rice and 2(8%) were infected in pepper. Further molecular identification of the isolates was carried out to reveal Xanthomonas campestris pv. vesicatoria strain 85-10 and Xanthomonas perforans strain 91-118. These were further used for the production of xanthan gum using sugar cane molasses substrates extracted from sugar cane, which was used as fermentation medium for the production. Isolates from plants varying ability in Xanthan gum production, with the mint plant having the highest Xanthan gum production (0.10 ± 0.02 to 0.9 ± 0.00 g/l). Conclusion: The present study confirmed the high xanthan gum production capacity of Xanthomonas campestris from dark rot spots containing mint leaves and should be considered during local and industrial production of the xanthan gum

The lolB gene in Xanthomonas campestris pv. campestris is required for bacterial attachment, stress tolerance, and virulence

Background Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium that can cause black rot disease in crucifers. The lipoprotein outer membrane localization (Lol) system is involved in the lipoprotein sorting to the outer membrane. Although Xcc has a set of annotated lol genes, there is still little known about the physiological role in this phytopathogen. In this study, we aimed to characterize the role of LolB of Xcc in bacterial attachment, stress tolerance, and virulence. Results To characterize the role of LolB, lolB mutant was constructed and phenotypic evaluation was performed. The lolB mutant revealed reductions in bacterial attachment, extracellular enzyme production, and virulence. Mutation of lolB also resulted in reduced tolerance to a myriad of stresses, including heat and a range of membrane-perturbing agents. Trans-complementation of lolB mutant with intact lolB gene reverted these altered phenotypes to the wild-type levels. From subsequent reporter assay and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, the expression of genes that encode the major extracellular enzymes and the stress-related proteins was reduced after lolB mutation. Conclusions The results in this work contribute to the functional understanding of lolB in Xanthomonas for the first time, and provide new insights into the function of lolB in bacteria.

Xanthomonas campestris pv. musacearum bacterial infection induces organ-specific callose and hydrogen peroxide production in banana

Xanthomonas campestris pv. musacearum (Xcm) bacteria cause banana Xanthomonas wilt (BXW), the most destructive disease of bananas in East and Central Africa. During early stages of infection in susceptible banana cultivars, incomplete systemic movement of Xcm limits bacterial colonization in the upper organs. Mechanistic basis of this delayed movement is unknown. We hypothesized that Xcm infection triggers basal pattern triggered immune (PTI) responses whose spatial and temporal variability along banana’s anatomical structure accounts for initially limiting Xcm in upper organs. Hence, we examined PTI responses such as callose deposition and hydrogen peroxide (H2O2) production in different organs in response to Xcm infection in BXW susceptible Kayinja and Mbwazirume banana cultivars and wild resistant progenitor Musa balbisiana. Xcm-induced callose increased and peaked at 14 days post inoculation (dpi) and 28dpi as assessed by fluorescence microscopy and enzyme-linked immunosorbent assays, respectively. The levels of Xcm-induced H2O2 and callose were highest in the pseudostems and corms, respectively, and were independent of host susceptibility or resistance to BXW. H2O2 production showed a biphasic transient pattern with an initial increase at 1-hour post Xcm-inoculation (hpi), followed by a decline 3-6hpi and then a second increase by 12hpi. Our findings point to organ-specific responses to Xcm infection in bananas. The corm which doubles as a subterranean parenating organ and interface between mother plants and lateral shoots, was the most responsive organ in callose production while the pseudostem was the most responsive organ in H2O2 production, suggesting the significance of these organs in banana response to BXW.

The cis -2-dodecenoic acid (BDSF) quorum sensing system in Burkholderia cenocepacia

It has been demonstrated that quorum sensing (QS) is widely employed by bacterial cells to coordinately regulate various group behaviors. Diffusible signal factor (DSF)-type signals have emerged as a growing family of conserved cell-cell communication signals. In addition to the DSF signal initially identified in Xanthomonas campestris pv. campestris, B urkholderia d iffusible s ignal f actor (BDSF, cis -2-dodecenoic acid) has been recognized as a conserved DSF-type signal with specific characteristics in both signal perception and transduction from DSF signals. Here, we review the history and current progress of the research of this type of signal, especially focusing on its biosynthesis, signaling pathways, and biological functions. We also discuss and explore the huge potential of targeting this kind of QS system as a new therapeutic strategy to control bacterial infections and diseases.

BDSF is a degradation-prone quorum-sensing signal detected by the histidine kinase RpfC of Xanthomonas campestris pv. campestris

Diffusible signal factors (DSFs) are medium-chain fatty acids that induce bacterial quorum sensing. Among these compounds, BDSF is a structural analog of DSF that is commonly detected in bacterial species (e.g., Xanthomonas, Pseudomonas, and Burkholderia). Additionally, BDSF contributes to the interkingdom communication regulating fungal life stage transitions. How BDSF is sensed in Xanthomonas spp. and the functional diversity between BDSF and DSF remain unclear. In this study, we generated genetic and biochemical evidence that BDSF is a low-active regulator of X. campestris pv. campestris quorum sensing, whereas trans-BDSF seems not a signaling compound. BDSF is detected by the sensor histidine kinase RpfC. Although BDSF has relatively low physiological activities, it binds to the RpfC sensor with a high affinity and activates RpfC autophosphorylation to a level that is similar to that induced by DSF in vitro. The inconsistency in the physiological and biochemical activities of BDSF is not due to RpfC–RpfG phosphorylation or RpfG hydrolase. Neither BDSF nor DSF controls the phosphotransferase and phosphatase activities of RpfC or the ability of RpfG hydrolase to degrade the bacterial second messenger cyclic di-GMP. We demonstrated that BDSF is prone to degradation by RpfB, a critical fatty acyl-CoA ligase involved in the turnover of DSF-family signals. rpfB mutations lead to substantial increases in BDSF-induced quorum sensing. Although DSF and BDSF are similarly detected by RpfC, our data suggest that their differential degradation in cells is the major factor responsible for the diversity in their physiological effects.

Comparative genomics of the black rot pathogen Xanthomonas campestris pv. campestris and non-pathogenic co-inhabitant Xanthomonas melonis from Trinidad reveal unique pathogenicity determinants and secretion system profiles

Black-rot disease caused by the phytopathogen Xanthomonas campestris pv. campestris (Xcc) continues to have considerable impacts on the productivity of cruciferous crops in Trinidad and Tobago and the wider Caribbean region. While the widespread occurrence of resistance of Xcc against bactericidal agrochemicals can contribute to the high disease burdens, the role of virulence and pathogenicity features of local strains on disease prevalence and severity has not been investigated yet. In the present study, a comparative genomic analysis was performed on 6 pathogenic Xcc and 4 co-isolated non-pathogenic Xanthomonas melonis (Xmel) strains from diseased crucifer plants grown in fields with heavy chemical use in Trinidad. Native isolates were grouped into two known and four newly assigned ribosomal sequence types (rST). Mobile genetic elements were identified which belonged to the IS3, IS5 family, Tn3 transposon, resolvases, and tra T4SS gene clusters. Additionally, exogenous plasmid derived sequences with origins from other bacterial species were characterised. Although several instances of genomic rearrangements were observed, native Xcc and Xmel isolates shared a significant level of structural homology with reference genomes, Xcc ATCC 33913 and Xmel CFBP4644, respectively. Complete T1SS hlyDB, T2SS, T4SS vir and T5SS xadA, yapH and estA gene clusters were identified in both species. Only Xmel strains contained a complete T6SS but no T3SS. Both species contained a complex repertoire of extracellular cell wall degrading enzymes. Native Xcc strains contained 37 T3SS and effector genes but a variable and unique profile of 8 avr, 4 xop and 1 hpa genes. Interestingly, Xmel strains contained several T3SS effectors with low similarity to references including avrXccA1 (~89%), hrpG (~73%), hrpX (~90%) and xopAZ (~87%). Furthermore, only Xmel genomes contained a CRISPR-Cas I-F array, but no lipopolysaccharide wxc gene cluster. Xmel strains were confirmed to be non-pathogenic by pathogenicity assays. The results of this study will be useful to guide future research into virulence mechanisms, agrochemical resistance, pathogenomics and the potential role of the co-isolated non-pathogenic Xanthomonas strains on Xcc infections.