Design, Synthesis, and Bioactivity Evaluation of New Thiochromanone Derivatives Containing a Carboxamide Moiety.

In this study, using the botanical active component thiochromanone as the lead compound, a total of 32 new thiochromanone derivatives containing a carboxamide moiety were designed and synthesized and their in vitro antibacterial activities against Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicolaby (Xoc), and Xanthomonas axonopodis pv. citri (Xac) were determined, as well as their in vitro antifungal activities against Botryosphaeria dothidea (B. dothidea), Phomopsis sp., and Botrytis cinerea (B. cinerea). Bioassay results demonstrated that some of the target compounds exhibited moderate to good in vitro antibacterial and antifungal activities. In particular, compound 4e revealed excellent in vitro antibacterial activity against Xoo, Xoc, and Xac, and its EC50 values of 15, 19, and 23 µg/mL, respectively, were superior to those of Bismerthiazol and Thiodiazole copper. Meanwhile, compound 3b revealed moderate in vitro antifungal activity against B. dothidea at 50 µg/mL, and the inhibition rate reached 88%, which was even better than that of Pyrimethanil, however, lower than that of Carbendazim. To the best of our knowledge, this is the first report on the antibacterial and antifungal activities of this series of novel thiochromanone derivatives containing a carboxamide moiety.

Bactericidal In-Vitro Effect of Zinc Ferrite Nanoparticles and the Orange Wax Extracts on Three Phytopathogen Microorganisms.

Phytopathogenic bacteria affect a wide variety of crops, causing significant economic losses. Natural biocides are the alternative to chemical methods of phytopathogens control. The goal of the present study is the evaluation of the biocidal activity of the following: 1) the extract of orange wax (EOW); 2) zinc ferrite nanoparticles (ZF-NPs); 3) the EOW adsorbed on the ZF-NPs; and 4) the EOW/ZF-NPs washed with 40% ethanol. For the biocidal activity, three phytopathogenic bacteria were used, namely, Xanthomonas axonopodis pv. Vesicatoria (Xav) Erwinia amylovora (Ew), and Pseudomonas syringae pv. Phaseolicola (Psph). For the ZF-NPs, an inhibitory effect higher than 50% ( ) was observed for Xav respect to the antibiotic used as positive control. On the other hand, the ZF-NPs did not show inhibitory effects on both Ew and Psph. In addition, the EOW in dimethyl sulfoxide (DMSO) at 100% caused growth inhibition on Xav, bacteriostatic activity on Ew, and had not biological activity on Psph. To the best of our knowledge, the control of Xav by zinc ferrites and orange wax, and the bacteriostatic effect produced by orange wax extract on Ew have not been reported elsewhere. Orange wax and zinc ferrite nanoparticles show potential in control of phytopathogenic bacteria. However, the bactericidal effect depends on the bacterium, the concentration of treatments, and the method of preparation.

Biological and transcriptomic studies reveal hfq is required for swimming, biofilm formation and stress response in Xanthomonas axonpodis pv. citri.

BACKGROUND: Hfq is a widely conserved bacterial RNA-binding protein which generally mediates the global regulatory activities involv ed in physiological process and virulence. The goal of this study was to characterize the biological function of hfq gene in Xanthomonas axonpodis pv. citri (Xac), the causal agent of citrus canker disease. RESULTS: An hfq mutant in Xac was generated by plasmid integration. The loss of hfq resulted in attenuation of bacterial growth, motility and biofilm formation. In addition, the hfq mutation impaired Xac resistance to H2O2 and both high and low pH environments, but did not affect the virulence to citrus. RNA-Seq analyses indicated that Hfq played roles in regulating the expression of 746 genes. In hfq mutant, gene expression related to chemotaxis, secretion system, two-component system, quorum sensing and flagellar assembly were repressed, whereas expression of ribosomal genes were significantly up-regulated. The down-regulated expression of three bacterial chemotaxis related genes and seven flagella genes, which involved in cell growth and biofilm formation, were further validated by RT-qPCR. CONCLUSIONS: The study demonstrated that hfq was involved in multiple biological processes in Xac. The results could serve as initiate points for identifying regulatory sRNAs and genes controlled by Hfq-sRNA interactions in Xac.

Phytohormone priming elevates the accumulation of defense-related gene transcripts and enhances bacterial blight disease resistance in cassava.

Cassava bacterial blight (CBB) disease caused by Xanthomonas axonopodis pv. manihotis (Xam) is a severe disease in cassava worldwide. In addition to causing significant cassava yield loss, CBB disease has not been extensively studied, especially in terms of CBB resistance genes. The present research demonstrated the molecular mechanisms underlining the defense response during Xam infection in two cassava cultivars exhibiting different degrees of disease resistance, Huay Bong60 (HB60) and Hanatee (HN). Based on gene expression analysis, ten of twelve putative defense-related genes including, leucine-rich repeat receptor-like kinases (LRR-RLKs), resistance (R), WRKY and pathogenesis-related (PR) genes, were differentially expressed between these two cassava cultivars during Xam infection. The up-regulation of defense-related genes observed in HB60 may be the mechanism required for the reduction of disease severity in the resistant cultivar. Interestingly, priming with salicylic acid (SA) or methyl jasmonate (MeJA) for 24 h before Xam inoculation could enhance the defense response in both cassava cultivars. The disease severity was decreased 10% in the resistant cultivar (HB60) and was remarkably reduced 21% in the susceptible cultivar (HN) by SA/MeJA priming. Priming with Xam inoculation modulated cassava4.1_013417, cassava4.1_030866 and cassava4.1_020555 (highest similarity to MeWRKY59, MePR1 and AtPDF2.2, respectively) expression and led to enhanced resistance of the susceptible cultivar in the second infection. The putative cis-regulatory elements were predicted in an upstream region of these three defense-related genes. The different gene expression levels in these genes between the two cultivars were due to the differences in cis-regulatory elements in their promoter regions. Taken together, our study strongly suggested that the induction of defense-related genes correlated with defense resistance against Xam infection, and exogenous application of SA or MeJA could elevate the defense response in both cultivars of cassava. This finding should pave the way for management to reduce yield loss from disease and genetic improvement in cassava.

Antimicrobial textiles: Biogenic silver nanoparticles against Candida and Xanthomonas.

This paper introduces cotton fibers impregnated with biogenic silver nanoparticles (AgNPs), synthesized from a Fusarium oxysporum fungal filtrate (FF) solution, and open up the possibility for their use in medical environment and agriculture clothing as means to avoid microbial spreading. After thorough AgNPs characterization, regarding their physical, chemical and biochemical properties, Minimum Inhibitory Concentrations (MIC) against some human and orange tree pathogens were determined. We report the strong AgNPs activity against Candida parapsilosis and Xanthomonas axonopodis pv. citri (Xac) that was morphologically characterized, pointing to strong AgNPs effects on microorganisms' membranes. Cotton fibers were then impregnated with AgNPs suspension and these maintained strong antimicrobial activity even after repeated mechanical washing cycles (up to 10). Reported data might point to an application for biogenic AgNPs as potent agrochemicals, as well as, to their application in textiles for antiseptic clothing for medical and agronomic applications.

Foliar application of the leaf-colonizing yeast Pseudozyma churashimaensis elicits systemic defense of pepper against bacterial and viral pathogens.

Yeast associates with many plant parts including the phyllosphere, where it is subject to harsh environmental conditions. Few studies have reported on biological control of foliar pathogens by yeast. Here, we newly isolated leaf-colonizing yeasts from leaves of field-grown pepper plants in a major pepper production area of South Korea. The yeast was isolated using semi-selective medium supplemented with rifampicin to inhibit bacterial growth and its disease control capacity against Xanthomonas axonopodis infection of pepper plants in the greenhouse was evaluated. Of 838 isolated yeasts, foliar spray of Pseudozyma churashimaensis strain RGJ1 at 108 cfu/mL conferred significant protection against X. axonopodis and unexpectedly against Cucumber mosaic virus, Pepper mottle virus, Pepper mild mottle virus, and Broad bean wilt virus under field conditions. Direct antagonism between strain RGJ1 and X. axonopodis was not detected from co-culture assays, suggesting that disease is suppressed via induced resistance. Additional molecular analysis of the induced resistance marker genes Capsicum annuum Pathogenesis-Related (CaPR) 4 and CaPR5 indicated that strain RGJ1 elicited plant defense priming. To our knowledge, this study is the first report of plant protection against bacterial and viral pathogens mediated by a leaf-colonizing yeast and has potential for effective disease management in the field.

Effect of cellulose and lignin on disintegration, antimicrobial and antioxidant properties of PLA active films.

This study reports the effects on antimicrobial, antioxidant, migration and disintegrability activities of ternary nanocomposite films based on poly(lactic acid) incorporating two biobased nanofillers, (cellulose nanocrystals (CNC) and lignin nanoparticles (LNP)), in two different amounts (1 and 3% wt.). Results from antimicrobial tests revealed a capacity to inhibit the Gram negative bacterial growth of Xanthomonas axonopodis pv. vesicatoria and Xanthomonas arboricola pv. pruni along the time, offering innovative opportunities against dangerous bacterial plant pathogens. LNP proved to be highly efficient in antioxidation activity, based on the disappearance of the absorption band at 517nm of the free radical, 2,2-diphenyl-1-picrylhydrazyl (DPPH) upon reduction by an antiradical compound; moreover the combination of LNP and CNC generates a synergistic positive effect in the antioxidation response of PLA ternary films. Furthermore, all the studied formulations showed a disintegrability value up to 90% after 15days of incubation in composting conditions. Migration results showed that the films can be considered suitable for application in food packaging field.

Comparison of gene activation by two TAL effectors from Xanthomonas axonopodis pv. manihotis reveals candidate host susceptibility genes in cassava.

Xanthomonas axonopodis pv. manihotis (Xam) employs transcription activator-like (TAL) effectors to promote bacterial growth and symptom formation during infection of cassava. TAL effectors are secreted via the bacterial type III secretion system into plant cells, where they are directed to the nucleus, bind DNA in plant promoters and activate the expression of downstream genes. The DNA-binding activity of TAL effectors is carried out by a central domain which contains a series of repeat variable diresidues (RVDs) that dictate the sequence of bound nucleotides. TAL14Xam668 promotes virulence in Xam strain Xam668 and has been shown to activate multiple cassava genes. In this study, we used RNA sequencing to identify the full target repertoire of TAL14Xam668 in cassava, which includes over 50 genes. A subset of highly up-regulated genes was tested for activation by TAL14CIO151 from Xam strain CIO151. Although TAL14CIO151 and TAL14Xam668 differ by only a single RVD, they display differential activation of gene targets. TAL14CIO151 complements the TAL14Xam668 mutant defect, implying that shared target genes are important for TAL14Xam668 -mediated disease susceptibility. Complementation with closely related TAL effectors is a novel approach to the narrowing down of biologically relevant susceptibility genes of TAL effectors with multiple targets. This study provides an example of how TAL effector target activation by two strains within a single species of Xanthomonas can be dramatically affected by a small change in RVD-nucleotide affinity at a single site, and reflects the parameters of RVD-nucleotide interaction determined using designer TAL effectors in transient systems.

Piper betle-mediated synthesis, characterization, antibacterial and rat splenocyte cytotoxic effects of copper oxide nanoparticles.

The study reports a simple, inexpensive, and eco-friendly synthesis of copper oxide nanoparticles (CuONPs) using Piper betle leaf extract. Formation of CuONPs was confirmed by UV-visible spectroscopy at 280 nm. Transmission electron microscopy (TEM) images showed that the CuONPs were spherical, with an average size of 50-100 nm. The scanning electron microscopy (SEM)-energy dispersive spectroscopy (EDS) peak was observed approximately at 1 and 8 keV. The X-ray diffraction (XRD) studies indicated that the particles were crystalline in nature. CuONPs effectively inhibited the growth of phytopathogens Ralstonia solanacearum and Xanthomonas axonopodis. The cytotoxic effect of the synthesized CuONPs was analyzed using rat splenocytes. The cell viability was decreased to 94% at 300 µg/mL.

RNAseq analysis of cassava reveals similar plant responses upon infection with pathogenic and non-pathogenic strains of Xanthomonas axonopodis pv. manihotis.

KEY MESSAGE: An RNAseq-based analysis of the cassava plants inoculated with Xam allowed the identification of transcriptional upregulation of genes involved in jasmonate metabolism, phenylpropanoid biosynthesis and putative targets for a TALE. Cassava bacterial blight, a disease caused by the gram-negative bacterium Xanthomonas axonopodis pv. manihotis (Xam), is a major limitation to cassava production worldwide and especially in developing countries. The molecular mechanisms underlying cassava susceptibility to Xam are currently unknown. To identify host genes and pathways leading to plant susceptibility, we analyzed the transcriptomic responses occurring in cassava plants challenged with either the non-pathogenic Xam strain ORST4, or strain ORST4(TALE1 Xam ) which is pathogenic due to the major virulence transcription activator like effector TALE1 Xam . Both strains triggered similar responses, i.e., induction of genes related to photosynthesis and phenylpropanoid biosynthesis, and repression of genes related to jasmonic acid signaling. Finally, to search for TALE1 Xam virulence targets, we scanned the list of cassava genes induced upon inoculation of ORST4(TALE1 Xam ) for candidates harboring a predicted TALE1 Xam effector binding element in their promoter. Among the six genes identified as potential candidate targets of TALE1 Xam a gene coding for a heat shock transcription factor stands out as the best candidate based on their induction in presence of TALE1 Xam and contain a sequence putatively recognized by TALE1 Xam .

Glutamate transport and xanthan gum production in the plant pathogen Xanthomonas axonopodis pv. citri.

L-glutamate plays a central role in nitrogen metabolism in all living organisms. In the genus Xanthomonas, the nitrogen nutrition is an important factor involved in the xanthan gum production, an important exopolysaccharide with various industrial and biotechnological applications. In this report, we demonstrate that the use of L-glutamate by the phytopathogen Xanthomonas axonopodis pv. citri as a nitrogen source in defined medium significantly increases the production of xanthan gum. This increase is dependent on the L-glutamate concentration. In addition, we have also characterized a glutamate transport system that is dependent on a proton gradient and on ATP and is modulated by amino acids that are structurally related to glutamate. This is the first biochemical characterization of an energy substrate transport system observed in a bacterial phytopathogen with a broad economic and industrial impact due to xanthan gum production.

A LOV protein modulates the physiological attributes of Xanthomonas axonopodis pv. citri relevant for host plant colonization.

Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease.

The Xanthomonas axonopodis pv. citri flagellum is required for mature biofilm and canker development.

Xanthomonas axonopodis pv. citri (Xac) is the causative agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. To evaluate the participation of the single flagellum of Xac in biofilm formation, mutants in the fliC (flagellin) and the flgE (hook) genes were generated. Swimming motility, assessed on 0.25 % agar plates, was markedly reduced in fliC and flgE mutants. However, the fliC and flgE mutants exhibited a flagellar-independent surface translocation on 0.5 % agar plates. Mutation of either the rpfF or the rpfC gene, which both encode proteins involved in cell-cell signalling mediated by diffusible signal factor (DSF), led to a reduction in both flagellar-dependent and flagellar-independent surface translocation, indicating a regulatory role for DSF in both types of motility. Confocal laser scanning microscopy of biofilms produced in static culture demonstrated that the flagellum is also involved in the formation of mushroom-shaped structures and water channels, and in the dispersion of biofilms. The presence of the flagellum was required for mature biofilm development on lemon leaf surfaces. The absence of flagellin produced a slight reduction in Xac pathogenicity and this reduction was more severe when the complete flagellum structure was absent.

The monofunctional catalase KatE of Xanthomonas axonopodis pv. citri is required for full virulence in citrus plants.

BACKGROUND: Xanthomonas axonopodis pv. citri (Xac) is an obligate aerobic phytopathogen constantly exposed to hydrogen peroxide produced by normal aerobic respiration and by the plant defense response during plant-pathogen interactions. Four putative catalase genes have been identified in silico in the Xac genome, designated as katE, catB, srpA (monofunctional catalases) and katG (bifunctional catalase). METHODOLOGY/PRINCIPAL FINDINGS: Xac catalase activity was analyzed using native gel electrophoresis and semi-quantitative RT-PCR. We demonstrated that the catalase activity pattern was regulated in different growth stages displaying the highest levels during the stationary phase. KatE was the most active catalase in this phase of growth. At this stage cells were more resistant to hydrogen peroxide as was determined by the analysis of CFU after the exposition to different H(2)O(2) concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H(2)O(2) resistance after treatment with sub-lethal concentrations of the oxidant. In the plant-like medium XVM2 the expression of KatE was strongly induced and in this medium Xac was more resistant to H(2)O(2). A XackatE mutant strain was constructed by insertional mutagenesis. We observed that catalase induction in stationary phase was lost meanwhile the adaptive response to peroxide was maintained in this mutant. Finally, the XackatE strain was assayed in planta during host plant interaction rendering a less aggressive phenotype with a minor canker formation. CONCLUSIONS: Our results confirmed that in contrast to other Xanthomonas species, Xac catalase-specific activity is induced during the stationary phase of growth in parallel with the bacterial resistance to peroxide challenge. Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides. The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process. Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.

Construction of a BAC library and a physical map of a major QTL for CBB resistance of common bean (Phaseolus vulgaris L.).

A major quantitative trait loci (QTL) conditioning common bacterial blight (CBB) resistance in common bean (Phaseolus vulgaris L.) lines HR45 and HR67 was derived from XAN159, a resistant line obtained from an interspecific cross between common bean lines and the tepary bean (P. acutifolius L.) line PI319443. This source of CBB resistance is widely used in bean breeding. Several other CBB resistance QTL have been identified but none of them have been physically mapped. Four molecular markers tightly linked to this QTL have been identified suitable for marker assisted selection and physical mapping of the resistance gene. A bacterial artificial chromosome (BAC) library was constructed from high molecular weight DNA of HR45 and is composed of 33,024 clones. The size of individual BAC clone inserts ranges from 30 kb to 280 kb with an average size of 107 kb. The library is estimated to represent approximately sixfold genome coverage. The BAC library was screened as BAC pools using four PCR-based molecular markers. Two to seven BAC clones were identified by each marker. Two clones were found to have both markers PV-tttc001 and STS183. One preliminary contig was assembled based on DNA finger printing of those positive BAC clones. The minimum tiling path of the contig contains 6 BAC clones spanning an estimated size of 750 kb covering the QTL region.

Synthesis and antimicrobial activity of 1-benzhydryl-sulfonyl-4-(3-(piperidin-4-yl) propyl)piperidine derivatives against pathogens of Lycopersicon esculentum: a structure-activity evaluation study.

Several 1-benzhydryl-sulfonyl-4-(3-(piperidin-4-yl)propyl)piperidine derivatives 8(a-j) were prepared by the treatment of substituted benzhydryl chlorides with 4-(3-(piperidin-4-yl)propyl)piperidine followed by N-sulfonation with sulfonyl chlorides in the presence of dry methylene dichloride and triethyl amine. The synthesized compounds were characterized by (1)H-NMR, IR, and elemental analysis. All the synthesized compounds were evaluated in vitro for their efficacy as antimicrobial agents by artificial inoculation technique against standard strains of two important bacterial viz., Xanthomonas axonopodis pv. vesicatoria and Ralstonia solanacearum as well as and two fungal pathogens namely Alternaria solani and Fusarium solani of tomato plants. We have briefly investigated the structure-activity relation studies and reveal that the nature of substitutions on benzhydryl ring and sulfonamide ring influences the antibacterial activity. Among the synthesized new compounds 8b, 8d, 8g, 8h, 8i, and 8j were showed significant potent antimicrobial activities compared to the standard drugs chloramphenicol, mancozeb.

Isolation and characterization of antagonistic bacteria against bacterial leaf spot of Euphorbia pulcherrima.

AIMS: To investigate the inhibition potential of leaf-associated bacteria against the pathogen of bacterial leaf spot of Euphorbia pulcherrima. METHODS AND RESULTS: Seven out of 200 bacterial strains were effective antagonists by in vitro screening and the two strains PAB241 and PAB242 significantly reduced the disease incidence and severity as foliar treatments of E. pulcherrima. The two effective strains, PAB241 and PAB242, were both identified as Bacillus amyloliquefaciens by a polyphasic approach including phenotypic feature, carbon source utilization profile, fatty acid methyl esters and analysis of 16S rRNA gene sequence. In addition, the suspensions of B. amyloliquefaciens PAB241 and PAB242 showed antibacterial activities against the pathogen of bacterial leaf spot of E. pulcherrima under different treatments. CONCLUSIONS: The leaf-associated bacteria, B. amyloliquefaciens PAB241 and PAB242, markedly inhibited the growth of X. axonopodis pv. poinsettiicola under different treatments and protected E. pulcherrima from pathogen infection in growth chamber conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that showed B. amyloliquefaciens from plant leaves was a potential bactericide against bacterial leaf spot of E. pulcherrima.

The copper resistance operon copAB from Xanthomonas axonopodis pathovar citri: gene inactivation results in copper sensitivity.

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker and the completion of the Xac genome sequence has opened up the possibility of investigating basic cellular mechanisms at the genomic level. Copper compounds have been extensively used in agriculture to control plant diseases. The copA and copB genes, identified by annotation of the Xac genome, encode homologues of proteins involved in copper resistance. A gene expression assay by Northern blotting revealed that copA and copB are expressed as a unique transcript specifically induced by copper. Synthesis of the gene products was also induced by copper, reaching a maximum level at 4 h after addition of copper to the culture medium. CopA was a cytosolic protein and CopB was detected in the cytoplasmic membrane. The gene encoding CopA was disrupted by the insertion of a transposon, leading to mutant strains that were unable to grow in culture medium containing copper, even at the lowest CuSO(4) concentration tested (0.25 mM), whereas the wild-type strain was able to grow in the presence of 1 mM copper. Cell suspensions of the wild-type and mutant strains in different copper concentrations were inoculated in lemon leaves to analyse their ability to induce citrus canker symptoms. Cells of mutant strains showed higher sensitivity than the wild-type strain in the presence of copper, i.e. they were not able to induce citrus canker symptoms at high copper concentrations and exhibited a more retarded growth in planta.