Xanthomonas Campestris Pv Research Articles

The FabA-FabB Pathway Is Not Essential for Unsaturated Fatty Acid Synthesis but Modulates Diffusible Signal Factor Synthesis in Xanthomonas campestris pv. campestris.

Most bacteria use type II fatty acid synthesis (FAS) systems for synthesizing fatty acids, of which the conserved FabA-FabB pathway is considered to be crucial for unsaturated fatty acid (UFA) synthesis in gram-negative bacteria. Xanthomonas campestris pv. campestris, the phytopathogen of black rot disease in crucifers, produces higher quantities of UFAs under low-temperature conditions for increasing membrane fluidity. The fabA and fabB genes were identified in the X. campestris pv. campestris genome by BLAST analysis; however, the growth of the X. campestris pv. campestris fabA and fabB deletion mutants was comparable to that of the wild-type strain in nutrient and minimal media. The X. campestris pv. campestris ΔfabA and ΔfabB strains produced large quantities of UFAs and, altogether, these results indicated that the FabA-FabB pathway is not essential for growth or UFA synthesis in X. campestris pv. campestris. We also observed that the expression of X. campestris pv. campestris fabA and fabB restored the growth of the temperature-sensitive Escherichia coli fabA and fabB mutants CL104 and CY242, respectively, under non-permissive conditions. The in-vitro assays demonstrated that the FabA and FabB proteins of X. campestris pv. campestris catalyzed FAS. Our study also demonstrated that the production of diffusible signal factor family signals that mediate quorum sensing was higher in the X. campestris pv. campestris ΔfabA and ΔfabB strains and greatly reduced in the complementary strains, which exhibited reduced swimming motility and attenuated host-plant pathogenicity. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Antibacterial Activity of Different Strains of the Genus Trichoderma

The main pathogens causing plant diseases are bacteria, viruses, and fungi. A number of strategies are usually used for plant protection and control of pathogenic microorganisms. The main interest of researchers is focused on the development of alternative synthetic chemicals to control bacterial diseases of plants. Among such approaches, biological control of bacterial diseases using agents such as antagonistic fungi and some other microorganisms is considered to be one of the most effective strategies. Species of the genus Trichoderma are known for their antagonistic activity against plant pathogenic fungi and bacteria and can be an effective safety strategy to control them. An important peculiarity of fungi of this genus is their ability to inhibit target pathogenic organisms without harming non-target (beneficial) microorganisms. The study of the antagonistic activity of fungi of the genus Trichoderma was conducted mainly against pathogenic fungi of agricultural plants. At the same time, the study of the antibacterial activity of fungi of this genus has attracted much less attention. Therefore, the aim of our work was to determine the antibacterial activity of microscopic fungi of the genus Trichoderma against test cultures of bacteria causing pathogenesis of agricultural plants. Methods. The objects of research were 100 fungal strains of the genus Trichoderma and six economically important plant pathogenic bacteria such as Pseudomonas syringae UCM В-1027Т, Pseudomonas fluorescens 8573, Pectobacterium carotovorum UCM В-1095T, Xanthomonas campestris pv. campestris UCM В-1049, Clavibacter michiganensis subsp. michiganensis 102, and Agrobacterium tumefaciens UCM В-1000. Cultures of the studied fungi were grown on potato-dextrose agar. The antagonistic activity of fungi of the genus Trichoderma against plant pathogenic bacteria was studied using the conventional method of diffusion in agar and method of dual culture. The antibacterial activity of culture filtrates of Trichoderma strains was evaluated via the zone of growth inhibition of plant pathogenic bacteria. The percentages of growth inhibition of plant pathogenic bacteria were calculated, and the antagonistic activity of strains was concluded on the basis of the obtained values. Results. In general, the studied Trichoderma strains had the antagonistic activity against plant pathogenic bacteria. Using method of diffusion in agar, it was shown that among the 100 studied Trichoderma strains, 12 had the effect of growth inhibition (bacteriostatic effect) of all six studied species of pathogenic bacteria; 20 strains inhibited the growth of five ones, 36 — four, 12 — three, and 7 — of two strains. The strains with a wide spectrum of antibacterial activity were studied by the double culture method. This made it possible to demonstrate the high selectivity of the antagonistic effect of Trichoderma strains on individual test cultures of phytopathogenic bacteria. For example, strain No7A inhibited the growth of C. michiganensis subsp. michiganensis 102 by 47% and the growth of P. syringae UCM B-1027T by 30%, while the zones of growth inhibition of these test cultures, determined by the method of diffusion in agar, were 5 and 6 mm, respectively. Conclusions. The obtained results indicated the potential and overall ability of Trichoderma strains to biologically control bacterial pathogens. The most promising for the use of plant pathogenic bacteria as agents for biocontrol were strains F-60, 1515, and 320, which were active against all studied bacteria. Such strains may have the potential as a preventive biocontrol agent of plant pathogens with a wide range of action. On the other hand, Trichoderma strains with high activity against certain pathogens may have the potential to be used as a control agent against a specific target pathogen.

Antimicrobial Activity of Bee Queen Larvae and Royal Jelly

Royal jelly has unique healing properties due to the presence of a number of biologically active compounds and yet unidentified components that are used for the prevention and treatment of many diseases. Purpose. To investigate the antimicrobial activity of homogenate of bee queen larvae and royal jelly against four phytopathogenic bacteria. Methods. Method of diffusion of biologically active compounds into agar medium sown with test cultures of phytopathogenic bacteria. Homogenates of bee queen larvae and royal jelly were diluted with sterile distilled water 1:5, centrifuged at 10,000 rpm to precipitate insoluble components, and 100 μL were added to the holes of the medium in Petri dishes seeded with phytopathogenic bacteria. After incubation of the dishes at 28ºC, the bactericidal effect of royal jelly was detected in the form of non-growth zones of phytobathogenic bacteria. Results. Royal jelly inhibited the growth of Clavibacter michiganensis subsp. michiganensis 102, Pectobacterium carotovorum 8982, Xanthomonas campestris pv. campestris 8003b, and Pseudomonas syringae pv. syringae 8511 around the holes in the medium. The largest zone of no bacterial growth (45.0 mm) was observed around the wells with royal jelly on the lawn of P. carotovorum 8982, which indicates the high sensitivity of these bacteria to an unidentified compound in royal jelly. Homogenate of bee queen larvae delayed the growth of only X. campestris pv. campestris 8003b. Thin layer chromatography revealed one similar and three different compounds in bee queen larvae and royal jelly. Conclusions. For the first time, the antibacterial activity of royal jelly against four phytopathogenic bacteria С. michiganensis subsp. michiganensis 102, P. carotovorum 8982, X. campestris pv. campestris 8003b, and P. syringae pv. syringae 8511 was established, which expands the spectrum of its antagonistic activity and can be used in the future for practical purposes.

Xanthomonas campestris VemR enhances the transcription of the T3SS key regulator HrpX via physical interaction with HrpG.

VemR is a response regulator of the two-component signalling systems (TCSs). It consists solely of a receiver domain. Previous studies have shown that VemR plays an important role in influencing the production of exopolysaccharides and exoenzymes, cell motility, and virulence of Xanthomonas campestris pv. campestris (Xcc). However, whether VemR is involved in the essential pathogenicity determinant type III secretion system (T3SS) is unclear. In this work, we found by transcriptome analysis that VemR modulates about 10% of Xcc genes, which are involved in various cellular processes including the T3SS. Further experiments revealed that VemR physically interacts with numerous proteins, including the TCS sensor kinases HpaS and RavA, and the TCS response regulator HrpG, which directly activates the transcription of HrpX, a key regulator controlling T3SS expression. It has been demonstrated previously that HpaS composes a TCS with HrpG or VemR to control the expression of T3SS or swimming motility, while RavA and VemR form a TCS to control the expression of flagellar genes. Mutation analysis and in vitro transcription assay revealed that phosphorylation might be essential for the function of VemR and phosphorylated VemR could significantly enhance the activation of hrpX transcription by HrpG. We infer that the binding of VemR to HrpG can modulate the activity of HrpG to the hrpX promoter, thereby enhancing hrpX transcription. Although further studies are required to validate this inference and explore the detailed functional mechanism of VemR, our findings provide some insights into the complex regulatory cascade of the HpaS/RavA-VemR/HrpG-HrpX signal transduction system in the control of T3SS.

Smart farming: modeling distribution of Xanthomonas campestris pv. oryzae as a leaf blight-causing bacteria in rice plants

Regarding plant protection in agriculture, it has been known that the cause of leaf blight is the bacteria named Xanthomonas campestris pv. oryzae. This paper aims to conduct a species distribution model of leaf blight-causing bacteria in rice plants and elaborates on its habitat suitability throughout Indonesia within the climate change context. The occurrences data was extracted from the Global Biodiversity Information Facility (GBIF), whereas the climate data was obtained from the Worldclim data set. The Species Distribution Model used the Maximum entropy method available on the Ecocommons website. The result shows that the bacteria occurrences positively correlate with several climatic variables and spread throughout the archipelago presented into five classes. Main islands such as Java, Bali, and Sumatra share areas with the highest suitability values. While Kalimantan and Sulawesi only share small areas with high suitability area. Papua has a less suitable location for the bacteria to spread. Rice cultivation is inseparable from the threat of pests and diseases. It can cause losses in the form of decreased production to crop failure. Therefore, The Species Distribution Model needed to identify areas where the vector is likely to occur. This way, mitigation or even prevention efforts could be made effectively.

Bacterial outer membrane vesicles induce a transcriptional shift in arabidopsis towards immune system activation leading to suppression of pathogen growth in planta.

Gram-negative bacteria form spherical blebs on their cell periphery, which later dissociate from the bacterial cell wall to form extracellular vesicles. These nano scale structures, known as outer membrane vesicles (OMVs), have been shown to promote infection and disease and can induce typical immune outputs in both mammal and plant hosts. To better understand the broad transcriptional change plants undergo following exposure to OMVs, we treated Arabidopsis thaliana (Arabidopsis) seedlings with OMVs purified from the Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. campestris and performed RNA-seq analysis on OMV- and mock-treated plants at 2, 6 and 24h post challenge. The most pronounced transcriptional shift occurred at the first two time points tested, as reflected by the number of differentially expressed genes and the average fold change. OMVs induce a major transcriptional shift towards immune system activation, upregulating a multitude of immune-related pathways including a variety of immune receptors. Comparing the response of Arabidopsis to OMVs and to purified elicitors, revealed that OMVs induce a similar suite of genes and pathways as single elicitors, however, pathways activated by OMVs and not by other elicitors were detected. Pretreating Arabidopsis plants with OMVs and subsequently infecting with a bacterial pathogen led to a significant reduction in pathogen growth. Mutations in the plant elongation factor receptor (EFR), flagellin receptor (FLS2), or the brassinosteroid-insensitive 1-associated kinase (BAK1) co-receptor, did not significantly affect the immune priming effect of OMVs. All together these results show that OMVs induce a broad transcriptional shift in Arabidopsis leading to upregulation of multiple immune pathways, and that this transcriptional change may facilitate resistance to bacterial infection.

Evaluation of the biological effectiveness of the biofungicide Serenade ASO, SC in the fight against bacterial black spot of tomatoes (Xanthomonas campestris pv. vesicatoria (Doidge) on indoor tomatoes

The article presents the results of two-year studies on the biological effectiveness of biofungicide Serenade, ASO, SC (Bacillus amyloliquefaciens, strain QST-713, titer not less than 1x10 9 CFU/ml) in the fight against bacterial black spot on the leaves and fruits of indoor tomatoes in the conditions of the II soil-climatic zone of the Crimea. It was found that a five-time treatment with biofungicide Serenade, ASO, SC, the rate of application of 5.0, 6.5 and 8.0 l/ha, contributed to a decrease in the intensity of development of Xanthomonas campestris pv. vesicatoria on leaves by 2.3-2.9 times, on fruits by 2.2-2.7 times. On the 10th day after the fifth spraying, the biological effectiveness of biofungicide Serenade, ASO, SC in the application rates of 5.0, 6.5 and 8.0 l/ha, with an average development of bacterial black spot on the control variant (3.3-5.2% on leaves, 2.9-3.3% on fruits), on leaves was 76.5-85.1%, on fruits – 71.6-75.0%. On the day of the last harvest, on the 29th day after the last treatment, the biological efficiency on leaves was 57.3-65.4%, on fruits – 54.5-62.6%. The difference of five-time treatment with biofungicide Serenade, ASO, SC in the norm of application of 8.0 l/ha, with the standard is insignificant, within the limits of experimental error. The positive effect of biofungicide Serenade, ASO, SC on yield was noted. The difference with the control (77.9 c/ha) is 34.9, 36.9 and 41.0 c/ha.

Biological effects of Thymol loaded chitosan nanoparticles (TCNPs) on bacterial plant pathogen Xanthomonas campestris pv. campestris.

Engineered nanomaterials can provide eco-friendly alternatives for crop disease management. Chitosan based nanoparticles has shown beneficial applications in sustainable agricultural practices and effective healthcare. Previously we demonstrated that Thymol loaded chitosan nanoparticles (TCNPs) showed bactericidal activity against Xanthomonas campestris pv campestris (Xcc), a bacterium that causes black rot disease in brassica crops. Despite the progress in assessing the antibacterial action of TCNPs, the knowledge about the molecular response of Xcc when exposed to TCNPs is yet to be explored. In the present study, we combined physiological, spectroscopic and untargeted metabolomics studies to investigate the response mechanisms in Xcc induced by TCNPs. Cell proliferation and membrane potential assays of Xcc cells exposed to sub-lethal concentration of TCNPs showed that TCNPs affects the cell proliferation rate and damages the cell membrane altering the membrane potential. FTIR spectroscopy in conjunction with untargeted metabolite profiling using mass spectrometry of TCNPs treated Xcc cells revealed alterations in amino acids, lipids, nucleotides, fatty acids and antioxidant metabolites. Mass spectroscopy analysis revealed a 10-25% increase in nucleic acid, fatty acids and antioxidant metabolites and a 20% increase in lipid metabolites while a decrease of 10-20% in amino acids and carbohydrates was seen in in TCNP treated Xcc cells. Overall, our results demonstrate that the major metabolic perturbations induced by TCNPs in Xcc are associated with membrane damage and oxidative stress, thus providing information on the mechanism of TCNPs mediated cytotoxicity. This will aid towards the development of nano- based agrochemicals as an alternative to chemical pesticides in future.

Isolation, characterization and genome analysis of an orphan phage FoX4 of the new Foxquatrovirus genus

The growing interest in the therapeutic application of bacteriophages leads to a drastic increase in the number of sequenced genomes. Luckily, recent insights in phage taxonomy facilitate the classification of phages in a comprehensive and data-driven manner as recently proposed by the International Committee on Taxonomy of Viruses. In this research, we present the taxonomical classification of a novel, narrow host range Xanthomonas phage FoX4, isolated from a Brussels sprouts field in Belgium infested with Xanthomonas campestris pv. campestris. The phage has a limited ability to lyse a bacterial culture, yet adsorbs efficiently to its host. Based on its genome sequence and low similarity to previously described phages, the phage comprises the novel phage genus Foxquatrovirus.

Metabolic Promiscuity of an Orphan Small Alarmone Hydrolase Facilitates Bacterial Environmental Adaptation.

Small alarmone hydrolases (SAHs) are alarmone metabolizing enzymes found in both metazoans and bacteria. In metazoans, the SAH homolog Mesh1 is reported to function in cofactor metabolism by hydrolyzing NADPH to NADH. In bacteria, SAHs are often identified in genomes with toxic alarmone synthetases for self-resistance. Here, we characterized a bacterial orphan SAH, i.e., without a toxic alarmone synthetase, in the phytopathogen Xanthomonas campestris pv. campestris (XccSAH) and found that it metabolizes both cellular alarmones and cofactors. In vitro, XccSAH displays abilities to hydrolyze multiple nucleotides, including pppGpp, ppGpp, pGpp, pppApp, and NADPH. In vivo, X. campestris pv. campestris cells lacking sah accumulated higher levels of cellular (pp)pGpp and NADPH compared to wild-type cells upon amino acid starvation. In addition, X. campestris pv. campestris mutants lacking sah were more sensitive to killing by Pseudomonas during interbacterial competition. Interestingly, loss of sah also resulted in reduced growth in amino acid-replete medium, a condition that did not induce (pp)pGpp or pppApp accumulation. Further metabolomic characterization revealed strong depletion of NADH levels in the X. campestris pv. campestris mutant lacking sah, suggesting that NADPH/NADH regulation is an evolutionarily conserved function of both bacterial and metazoan SAHs and Mesh1. Overall, our work demonstrates a regulatory role of bacterial SAHs as tuners of stress responses and metabolism, beyond functioning as antitoxins. IMPORTANCE Small alarmone hydrolases (SAHs) comprise a widespread family of alarmone metabolizing enzymes. In metazoans, SAHs have been reported to control multiple aspects of physiology and stress resistance through alarmone and NADPH metabolisms, but their physiological functions in bacteria is mostly uncharacterized except for a few reports as antitoxins. Here, we identified an SAH functioning independently of toxins in the phytopathogen Xanthomonas campestris pv. campestris. We found that XccSAH hydrolyzed multiple alarmones and NADPH in vitro, and X. campestris pv. campestris mutants lacking sah displayed increased alarmone levels during starvation, loss of interspecies competitive fitness, growth defects, and strong reduction in NADH. Our findings reveal the importance of NADPH hydrolysis by a bacterial SAH. Our work is also the first report of significant physiological roles of bacterial SAHs beyond functioning as antitoxins and suggests that SAHs have far broader physiological roles and share similar functions across domains of life.

A novel bacterial strain Burkholderia sp. F25 capable of degrading diffusible signal factor signal shows strong biocontrol potential.

Vast quantities of synthetic pesticides have been widely applied in various fields to kill plant pathogens, resulting in increased pathogen resistance and decreased effectiveness of such chemicals. In addition, the increased presence of pesticide residues affects living organisms and the environment largely on a global scale. To mitigate the impact of crop diseases more sustainably on plant health and productivity, there is a need for more safe and more eco-friendly strategies as compared to chemical prevention. Quorum sensing (QS) is an intercellular communication mechanism in a bacterial population, through which bacteria adjust their population density and behavior upon sensing the levels of signaling molecules in the environment. As an alternative, quorum quenching (QQ) is a promising new strategy for disease control, which interferes with QS by blocking intercellular communication between pathogenic bacteria to suppress the expression of disease-causing genes. Black rot caused by Xanthomonas campestris pv. campestris (Xcc) is associated with the diffusible signal factor (DSF). As detailed in this study, a new QQ strain F25, identified as Burkholderia sp., displayed a superior ability to completely degrade 2 mM of DSF within 72h. The main intermediate product in the biodegradation of DSF was identified as n-decanoic acid, based on gas chromatography-mass spectrometry (GC-MS). A metabolic pathway for DSF by strain F25 is proposed, based on the chemical structure of DSF and its intermediates, demonstrating the possible degradation of DSF via oxidation-reduction. The application of strain F25 and its crude enzyme as biocontrol agents significantly attenuated black rot caused by Xcc, and inhibited tissue maceration in the host plant Raphanus sativus L., without affecting the host plant. This suggests that agents produced from strain F25 and its crude enzyme have promising applications in controlling infectious diseases caused by DSF-dependent bacterial pathogens. These findings are expected to provide a new therapeutic strategy for controlling QS-mediated plant diseases.

Physical, chemical, and biological control of black rot of brassicaceae vegetables: A review.

As one of the important sources of human nutrition, Brassicaceae vegetables are widely grown worldwide. Black rot caused by Xanthomonas campestris pv. campestris (Xcc) seriously affects the quality and yield of Brassicaceae vegetables. Therefore, it is important to study control methods of Xcc for Brassicaceae vegetable production. This paper reviews the physical, chemical, and biological control methods of Xcc in Brassicaceae vegetables developed in recent years, and the underlying mechanisms of control methods are also discussed. Based on our current knowledge, future research directions for Xcc control are also proposed. This review also provides a reference basis for the control of Xcc in the field cultivation of Brassicaceae vegetables.

Genome-Wide Characterization, Identification and Expression Profile of MYB Transcription Factor Gene Family during Abiotic and Biotic Stresses in Mango (Mangifera indica).

Mango (Mangifera indica) is an economically important fruit tree, and is cultivated in tropical, subtropical, and dry-hot valley areas around the world. Mango fruits have high nutritional value, and are mainly consumed fresh and used for commercial purposes. Mango is affected by various environmental factors during its growth and development. The MYB transcription factors participates in various physiological activities of plants, such as phytohormone signal transduction and disease resistance. In this study, 54 MiMYB transcription factors were identified in the mango genome (371.6 Mb). A phylogenetic tree was drawn based on the amino acid sequences of 222 MYB proteins of mango and Arabidopsis. The phylogenetic tree showed that the members of the mango MYB gene family were divided into 7 group, including Groups 1, -3, -4, -5, -6, -8, and -9. Ka/Ks ratios generally indicated that the MiMYBs of mango were affected by negative or positive selection. Quantitative real-time PCR showed that the transcription levels of MiMYBs were different under abiotic and biotic stresses, including salicylic acid, methyl jasmonate, and H2O2 treatments, and Colletotrichum gloeosporioides and Xanthomonas campestris pv. mangiferaeindicae infection, respectively. The transcript levels of MiMYB5, -35, -36, and -54 simultaneously responded positively to early treatments with salicylic acid, methyl jasmonate, and H2O2. The transcript level of MiMYB54 was activated by pathogenic fungal and bacterial infection. These results are beneficial for future interested researchers aiming to understand the biological functions and molecular mechanisms of MiMYB genes.

A plant growth-promoting bacteria Priestia megaterium JR48 induces plant resistance to the crucifer black rot via a salicylic acid-dependent signaling pathway.

Xanthomonas campestris pv. campestris (Xcc)-induced black rot is one of the most serious diseases in cruciferous plants. Using beneficial microbes to control this disease is promising. In our preliminary work, we isolated a bacterial strain (JR48) from a vegetable field. Here, we confirmed the plant-growth-promoting (PGP) effects of JR48 in planta, and identified JR48 as a Priestia megaterium strain. We found that JR48 was able to induce plant resistance to Xcc and prime plant defense responses including hydrogen peroxide (H2O2) accumulation and callose deposition with elevated expression of defense-related genes. Further, JR48 promoted lignin biosynthesis and raised accumulation of frees salicylic acid (SA) as well as expression of pathogenesis-related (PR) genes. Finally, we confirmed that JR48-induced plant resistance and defense responses requires SA signaling pathway. Together, our results revealed that JR48 promotes plant growth and induces plant resistance to the crucifer black rot probably through reinforcing SA accumulation and response, highlighting its potential as a novel biocontrol agent in the future.

Integrated metabolome and transcriptome analysis reveals salicylic acid and flavonoid pathways' key roles in cabbage's defense responses to Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc) is a vascular bacteria pathogen causing black rot in cabbage. Here, the resistance mechanisms of cabbage against Xcc infection were explored by integrated metabolome and transcriptome analysis. Pathogen perception, hormone metabolisms, sugar metabolisms, and phenylpropanoid metabolisms in cabbage were systemically re-programmed at both transcriptional and metabolic levels after Xcc infection. Notably, the salicylic acid (SA) metabolism pathway was highly enriched in resistant lines following Xcc infection, indicating that the SA metabolism pathway may positively regulate the resistance of Xcc. Moreover, we also validated our hypothesis by showing that the flavonoid pathway metabolites chlorogenic acid and caffeic acid could effectively inhibit the growth of Xcc. These findings provide valuable insights and resource datasets for further exploring Xcc-cabbage interactions and help uncover molecular breeding targets for black rot-resistant varieties in cabbage.

CHARACTERIZATION AND IDENTIFICATION OF Xanthomonas spp. ISOLATED FROM INFECTED Brassicaceae AND SELECTION OF POTENTIAL XANTHAN GUM PRODUCER

Xanthomonas spp. synthesize a complex exopolysaccharide called xanthan gum, which has significant commercial value. Xanthomonas-infected vegetables exhibiting typical spot symptoms were used in this study. Isolation was done by streaking the sample suspension on the Yeast Malt agar. A colony that resembled Xanthomonas spp. was purified before a simple phenotypic test and identified using BIOLOG and 16 rRNA sequencing. The screening was done based on their performance by fermentation in a shake flask, under controlled conditions. The yield and viscosity of xanthan gum produced from each bacterium were compared to Xanthomonas campestris PV. campestris strain ATCC33913. There were 411 wild types of Xanthomonas spp. successfully isolated and ten strains were selected for xanthan gum evaluation. The results showed, the xanthan production (g/L) varied from (1.57 - 8.24) with the yield of xanthan from 0.64 to 4.71 g/g biomass. Strain C206 produced the highest xanthan gum concentration (8.24±0.20 g/L) compared to others and the control strain, ATCC 33913 (2.27±0.10g/L). The highest yield of xanthan 4.71±0.18 (g/g biomass) was produced by strain C298 followed by strain C279 with 3.88±0.04 (g/g biomass). From our investigation, the production and yield of xanthan gum and the viscosity of the polymers were significantly dependent on the bacterial strain. Based on the stable viscosity and yield of xanthan produced, X. campestris C279 was selected for further studies on product optimization.

Combined effect of thyme and clove phenolic compounds on Xanthomonas campestris pv. campestris and biocontrol of black rot disease on cabbage seeds.

The seed-borne bacterium Xanthomonas campestris pv. campestris (Xcc) as a causal organism of black rot disease remains the most serious bacterial problem of agricultural production of cruciferous plants worldwide. The eradication of a primary inoculum originating in seeds is available, but no treatment is totally effective. With the threat of developing chemical resistance and increasing pressure for sustainable disease management, biocontrol methods represent one of the main strategies currently applied in agriculture. Natural antimicrobials, including essential oils, are promising tools in disease management with low risks of environmental pollution and impact on human health. Thyme and clove essential oils were demonstrated to be highly effective in Xanthomonas studies in vitro; therefore, their application in black rot control was evaluated in this study. From five phenolic substances originating from thyme and clove essential oils (carvacrol, eugenol, linalool, p-cymene and thymol), the most promising in vitro results were observed with carvacrol, for which 0.0195% led to the death of all Xcc cells in 30 min. Moreover, a synergistic antibacterial effect of carvacrol and thymol solutions decreased the minimal inhibition concentration to 0.0049% and 0.0195% for carvacrol and thymol, respectively. Using the quadruple bactericidal values, the complete elimination of Xcc from the surface of infested cabbage seeds was obtained for both carvacrol and thymol solutions and their combined mixture at 2 MIC value. The elimination of bacterial infection from germinated cabbage plants was observed for both plate counting and quantitative real-time PCR methods. We also evaluated the effect of the application of phenolic treatment on the seed germination and germinated plants. Our results suggest a high potential of the application of carvacrol and thymol in vegetable seed production, specifically for cabbage, thus representing a suitable alternative to cupric derivatives.

STUDY OF XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS ISOLATES ORIGINATING FROM SOIL AND BRASSICA SPP. SEEDS

Xanthomonas campestris pv. campestris (Xcc) is a harmful pathogen of the Brassicaceae agricultural plants in Serbian agroecological conditions. The bacterium survives in soil, plant residues, seeds, and wild and weedy plants. This study aimed to characterize Xcc isolates that persist in field soil and roots where they represent a risk to causing disease of Brassicaceae plants. Collection of soil samples was carried out in the period 2015 - 2017 in four locations regions Vojvodina with cabbage cultivation in monoculture or crop rotations. The cabbage seed samples were collected in the same period in Futog and Despotovo locations (agricultural farms) and local farmer markets. All isolations were done on SX and YDC agar plates with the addition of cycloheximide at 250 mg/l. Based on the pathogenicity tests and morphological and biochemical characteristics, 28 identified Xcc isolates were selected, 12 from soil and 16 from seeds. Molecular confirmation of the Xcc isolates was done using multiplex PCR (m-PCR) with Xcc-specific primer sets DLH 120/125 and Zup 2309/2310. In all selected isolates and the reference Xcc strain NCPPB1144, the expected 370 and 619 bp PCR fragments were amplified. Characterizing isolates by rep-PCR fingerprinting using BOX A1R and (GTG)5 primers resulted in informative patterns. Cluster analysis grouped the strain Xcc NCPPB 1144 with all Xcc isolates, while other tested Xanthomonads were clustered separately. Based on the similarity level of 90%, we obtained three out of 12 representative isolates from soil and four out of 16 representative seed isolates. The characterization of Xcc isolates from soil and seeds will enable their comparison with those pathogenic isolated from leaves of Brassicaceae plants.

A serralysin-like protein of Candidatus Liberibacter asiaticus modulates components of the bacterial extracellular matrix.

Huanglongbing (HLB), the current major threat for Citrus species, is caused by intracellular alphaproteobacteria of the genus Candidatus Liberibacter (CaL), with CaL asiaticus (CLas) being the most prevalent species. This bacterium inhabits phloem cells and is transmitted by the psyllid Diaphorina citri. A gene encoding a putative serralysin-like metalloprotease (CLIBASIA_01345) was identified in the CLas genome. The expression levels of this gene were found to be higher in citrus leaves than in psyllids, suggesting a function for this protease in adaptation to the plant environment. Here, we study the putative role of CLas-serralysin (Las1345) as virulence factor. We first assayed whether Las1345 could be secreted by two different surrogate bacteria, Rhizobium leguminosarum bv. viciae A34 (A34) and Serratia marcescens. The protein was detected only in the cellular fraction of A34 and S. marcescens expressing Las1345, and increased protease activity of those bacteria by 2.55 and 4.25-fold, respectively. In contrast, Las1345 expressed in Nicotiana benthamiana leaves did not show protease activity nor alterations in the cell membrane, suggesting that Las1345 do not function as a protease in the plant cell. Las1345 expression negatively regulated cell motility, exopolysaccharide production, and biofilm formation in Xanthomonas campestris pv. campestris (Xcc). This bacterial phenotype was correlated with reduced growth and survival on leaf surfaces as well as reduced disease symptoms in N. benthamiana and Arabidopsis. These results support a model where Las1345 could modify extracellular components to adapt bacterial shape and appendages to the phloem environment, thus contributing to virulence.

HpaP divergently regulates the expression of hrp genes in Xanthomonas oryzae pathovars oryzae and oryzicola.

The bacterial pathogens Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc) cause leaf blight and leaf streak diseases on rice, respectively. Pathogenesis is largely defined by the virulence genes harboured in the pathogen genome. Recently, we demonstrated that the protein HpaP of the crucifer pathogen Xanthomonas campestris pv. campestris is an enzyme with both ATPase and phosphatase activities, and is involved in regulating the synthesis of virulence factors and the induction of the hypersensitive response (HR). In this study, we investigated the role of HpaP homologues in Xoo and Xoc. We showed that HpaP is required for full virulence of Xoo and Xoc. Deletion of hpaP in Xoo and Xoc led to a reduction in virulence and alteration in the production of virulence factors, including extracellular polysaccharide and cell motility. Comparative transcriptomics and reverse transcription-quantitative PCR assays revealed that in XVM2 medium, a mimic medium of the plant environment, the expression levels of hrp genes (for HR and pathogenicity) were enhanced in the Xoo hpaP deletion mutant compared to the wild type. By contrast, in the same growth conditions, hrp gene expression was decreased in the Xoc hpaP deletion mutant compared to the wild type. However, an opposite expression pattern was observed when the pathogens grew in planta, where the expression of hrp genes was reduced in the Xoo hpaP mutant but increased in the Xoc hpaP mutant. These findings indicate that HpaP plays a divergent role in Xoo and Xoc, which may lead to the different infection strategies employed by these two pathogens.