RESUMEN
BACKGROUND: Xylella fastidiosa is a multi-host bacterium that can be detected in hundreds of plant species including several crops. Diseases caused by X. fastidiosa are considered a threat to global food production. The primary method for managing diseases caused by X. fastidiosa involves using insecticides to control the vector. Hence, it is necessary to adopt new and sustainable disease management technologies to control not only the insect but also the bacteria and plant health. We demonstrated that N-acetylcysteine (NAC), a low-cost cysteine analogue, is a sustainable molecule that can be used in agriculture to decrease the damage caused by X. fastidiosa and improve plant health. RESULTS: Using 15N-NAC we proved that this analogue was absorbed by the roots and transported to different parts of the plant. Inside the plant, NAC reduced the bacterial population by 60-fold and the number of xylem vessels blocked by bacterial biofilms. This reflected in a recovery of 0.28-fold of the daily sap flow compared to health plants. In addition, NAC-treated citrus variegated chlorosis (CVC) plants decreased the oxidative stress by improving the activity of detoxifying enzymes. Moreover, the use of NAC in field conditions positively contributed to the increase in fruit yield of CVC-diseased plants. CONCLUSION: Our research not only advances the understanding of NAC absorption in plants, but also indicates its dual effect as an antimicrobial and antioxidant molecule. This, in turn, negatively affects bacterial survival while improving plant health by decreasing oxidative stress. Overall, the positive field-based evidence supports the viability of NAC as a sustainable agricultural application. © 2024 Society of Chemical Industry.
Asunto(s)
Acetilcisteína , Enfermedades de las Plantas , Xylella , Xylella/efectos de los fármacos , Xylella/fisiología , Acetilcisteína/farmacología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Citrus/microbiología , Frutas/microbiologíaRESUMEN
Citrus sinensis and Citrus limonia were obtained by germination from seeds, and isotopic-labeling experiments using d-[1-13C]glucose were performed with the seedlings. After 60 days, the seedlings were analyzed by high-performance liquid chromatography-ultraviolet-solid-phase extraction-nuclear magnetic resonance, data and the 13C enrichment patterns of xanthyletin and seselin indicated that the pyran ring was formed by the methylerythritol phosphate pathway and that the coumarin moiety was derived from the shikimate pathway in both compounds. This information regarding the biosynthetic pathway can be used to increase resistance against phytopathogens, because xanthyletin and seselin are reported to have antimicrobial activity on the growth of Xylella fastidiosa, which causes citrus variegated chlorosis in orange.
Asunto(s)
Marcaje Isotópico/métodos , Piranocumarinas/metabolismo , Isótopos de Carbono , Cromatografía Líquida de Alta Presión , Citrus/metabolismo , Citrus sinensis/metabolismo , Espectroscopía de Resonancia Magnética , Estructura Molecular , Enfermedades de las Plantas/microbiología , Piranocumarinas/química , Piranocumarinas/aislamiento & purificación , Ácido Shikímico/metabolismo , Extracción en Fase Sólida , Espectrofotometría Ultravioleta , Xylella/efectos de los fármacosRESUMEN
Diketopiperazines can be generated by non-enzymatic cyclization of linear dipeptides at extreme temperature or pH, and the complex medium used to culture bacteria and fungi including phytone peptone and trypticase peptone, can also produce cyclic peptides by heat sterilization. As a result, it is not always clear if many diketopiperazines reported in the literature are artifacts formed by the different complex media used in microorganism growth. An ideal method for analysis of these compounds should identify whether they are either synthesized de novo from the products of primary metabolism and deliver true diketopiperazines. A simple defined medium (X. fastidiosa medium or XFM) containing a single carbon source and no preformed amino acids has emerged as a method with a particularly high potential for the grown of X. fastidiosa and to produce genuine natural products. In this work, we identified a range of diketopiperazines from X. fastidiosa 9a5c growth in XFM, using Ultra-Fast Liquid Chromatography coupled with mass spectrometry. Diketopiperazines are reported for the first time from X. fastidiosa, which is responsible for citrus variegated chlorosis. We also report here fatty acids from X. fastidiosa, which were not biologically active as diffusible signals, and the role of diketopiperazines in signal transduction still remains unknown.
Asunto(s)
Dicetopiperazinas/farmacología , Peptonas/química , Xylella/efectos de los fármacos , Carbono/química , Caseínas/química , Cromatografía Liquida , Dicetopiperazinas/síntesis química , Dicetopiperazinas/química , Peptonas/síntesis química , Peptonas/farmacología , Hidrolisados de Proteína/química , Espectrometría de Masa por Ionización de Electrospray , Xylella/crecimiento & desarrolloRESUMEN
The Xylella fastidiosa 9a5c strain is a xylem-limited phytopathogen that is the causal agent of citrus variegated chlorosis (CVC). This bacterium is able to form a biofilm and occlude the xylem vessels of susceptible plants, which leads to significant agricultural and economic losses. Biofilms are associated with bacterial pathogenicity because they are very resistant to antibiotics and other metal-based chemicals that are used in agriculture. The X. fastidiosa YcjZ-like (XfYcjZ-like) protein belongs to the LysR-type transcriptional regulator (LTTR) family and is involved in various cellular functions that range from quorum sensing to bacterial survival. In the present study, we report the cloning, expression and purification of XfYcjZ-like, which was overexpressed in Escherichia coli. The secondary folding of the recombinant and purified protein was assessed by circular dichroism, which revealed that XfYcjZ-like contains a typical α/ß fold. An initial hydrodynamic characterization showed that XfYcjZ-like is a globular tetramer in solution. In addition, using a polyclonal antibody against XfYcjZ-like, we assessed the expression profile of this protein during the different developmental phases of X. fastidiosa in in vitro cultivated biofilm cells and demonstrated that XfYcjZ-like is upregulated in planktonic cells in response to a copper shock treatment. Finally, the ability of XfYcjZ-like to interact with its own predicted promoter was confirmed in vitro, which is a typical feature of LysR. Taken together, our findings indicated that the XfYcjZ-like protein is involved in both the organization of the architecture and the maturation of the bacterial biofilm and that it is responsive to oxidative stress.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Proteínas Recombinantes/química , Factores de Transcripción/química , Xylella/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Cobre/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Datos de Secuencia Molecular , Estrés Oxidativo/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismo , Xylella/efectos de los fármacosRESUMEN
A high performance liquid chromatography-ultraviolet (HPLC-UV) method was developed for quantifying hesperidin and rutin levels in leaves and stems of Citrus limonia, with a good linearity over a range of 1.0-80.0 and 1.0-50.0 µg mL(-1) respectively, with r(2)>0.999 for all curves. The limits of detection (LOD) for both flavonoids were 0.6 and 0.5 µg mL(-1), respectively, with quantification (LOQ) being 2.0 and 1.0 µg mL(-1), respectively. The quantification method was applied to Citrus sinensis grafted onto C. limonia with and without CVC (citrus variegated chlorosis) symptoms after Xylella fastidiosa infection. The total content of rutin was low and practically constant in all analyses in comparison with hesperidin, which showed a significant increase in its amount in symptomatic leaves. Scanning electron microscopy studies on leaves with CVC symptoms showed vessel occlusion by biofilm, and a crystallized material was noted. Considering the difficulty in isolating these crystals for analysis, tissue sections were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to confirm the presence of hesperidin at the site of infection. The images constructed from MS/MS data with a specific diagnostic fragment ion (m/z 483) also showed higher ion intensities for it in infected plants than in healthy ones, mainly in the vessel regions. These data suggest that hesperidin plays a role in the plant-pathogen interaction, probably as a phytoanticipin. This method was also applied to C. sinensis and C. limonia seedlings, and comparison with the graft results showed that the rootstock had an increased hesperidin content â¼3.6 fold greater in the graft stem than in the stem of C. sinensis seedlings. Increase in hesperidin content by rootstock can be related to induced internal defense mechanisms.
Asunto(s)
Citrus/química , Hesperidina/análisis , Xylella/patogenicidad , Cromatografía Líquida de Alta Presión , Citrus/genética , Resonancia Magnética Nuclear Biomolecular , Hojas de la Planta/química , Tallos de la Planta/química , Rutina/análisis , Xylella/efectos de los fármacosRESUMEN
Xylella fastidiosa is a plant pathogen bacterium that causes diseases in many different crops. In citrus, it causes Citrus Variegated Chlorosis (CVC). The mechanism of pathogenicity of this bacterium is associated with its capacity to colonize and form a biofilm in the xylem vessels of host plants, and there is not yet any method to directly reduce populations of this pathogen in the field. In this study, we investigated the inhibitory effect of N-Acetylcysteine (NAC), a cysteine analogue used mainly to treat human diseases, on X. fastidiosa in different experimental conditions. Concentrations of NAC over 1 mg/mL reduced bacterial adhesion to glass surfaces, biofilm formation and the amount of exopolysaccharides (EPS). The minimal inhibitory concentration of NAC was 6 mg/mL. NAC was supplied to X. fastidiosa-infected plants in hydroponics, fertigation, and adsorbed to organic fertilizer (NAC-Fertilizer). HPLC analysis indicated that plants absorbed NAC at concentrations of 0.48 and 2.4 mg/mL but not at 6 mg/mL. Sweet orange plants with CVC symptoms treated with NAC (0.48 and 2.4 mg/mL) in hydroponics showed clear symptom remission and reduction in bacterial population, as analyzed by quantitative PCR and bacterial isolation. Experiments using fertigation and NAC-Fertilizer were done to simulate a condition closer to that normally is used in the field. For both, significant symptom remission and a reduced bacterial growth rate were observed. Using NAC-Fertilizer the lag for resurgence of symptoms on leaves after interruption of the treatment increased to around eight months. This is the first report of the anti-bacterial effect of NAC against a phytopathogenic bacterium. The results obtained in this work together with the characteristics of this molecule indicate that the use of NAC in agriculture might be a new and sustainable strategy for controlling plant pathogenic bacteria.
Asunto(s)
Acetilcisteína/farmacología , Agricultura , Antibacterianos/farmacología , Enfermedades de las Plantas/microbiología , Xylella/efectos de los fármacos , Acetilcisteína/química , Antibacterianos/química , Biopelículas , Hidroponía/métodos , Fenotipo , Hojas de la Planta/microbiología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/microbiología , Plantas/efectos de los fármacos , Plantas/microbiología , Polisacáridos Bacterianos/metabolismo , Xylella/fisiologíaRESUMEN
Investigations of biofilm resistance response rarely focus on plant-pathogenic bacteria. Since Xylella fastidiosa is a multihost plant-pathogenic bacterium that forms biofilm in the xylem, the behavior of its biofilm in response to antimicrobial compounds needs to be better investigated. We analyzed here the transcriptional profile of X. fastidiosa subsp. pauca in response to inhibitory and subinhibitory concentrations of copper and tetracycline. Copper-based products are routinely used to control citrus diseases in the field, while antibiotics are more widely used for bacterial control in mammals. The use of antimicrobial compounds triggers specific responses to each compound, such as biofilm formation and phage activity for copper. Common changes in expression responses comprise the repression of genes associated with metabolic functions and movement and the induction of toxin-antitoxin systems, which have been associated with the formation of persister cells. Our results also show that these cells were found in the population at a ca. 0.05% density under inhibitory conditions for both antimicrobial compounds and that pretreatment with subinhibitory concentration of copper increases this number. No previous report has detected the presence of these cells in X. fastidiosa population, suggesting that this could lead to a multidrug tolerance response in the biofilm under a stressed environment. This is a mechanism that has recently become the focus of studies on resistance of human-pathogenic bacteria to antibiotics and, based on our data, it seems to be more broadly applicable.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Cobre/farmacología , Tetraciclina/farmacología , Xylella/efectos de los fármacos , Xylella/genética , Antibacterianos/metabolismo , Biopelículas/crecimiento & desarrollo , Cobre/metabolismo , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica , Enfermedades de las Plantas/microbiología , Plantas/microbiología , Tetraciclina/metabolismo , Xylella/crecimiento & desarrollo , Xylella/metabolismoRESUMEN
Xylella fastidiosa is a xylem-limited bacterium responsible for important plant diseases, like citrus-variegated chlorosis (CVC) and grapevine Pierce's disease (PD). Interestingly, in vitro growth of X. fastidiosa in chemically defined media that resemble xylem fluid has been achieved, allowing studies of metabolic processes used by xylem-dwelling bacteria to thrive in such nutrient-poor conditions. Thus, we performed microarray hybridizations to compare transcriptomes of X. fastidiosa cells grown in 3G10-R, a medium that resembles grape sap, and in Periwinkle Wilt (PW), the complex medium traditionally used to cultivate X. fastidiosa. We identified 299 transcripts modulated in response to growth in these media. Some 3G10R-overexpressed genes have been shown to be upregulated in cells directly isolated from infected plants and may be involved in plant colonization, virulence and environmental competition. In contrast, cells cultivated in PW show a metabolic switch associated with increased aerobic respiration and enhanced bacterial growth rates.
Asunto(s)
Medios de Cultivo/farmacología , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Xylella/crecimiento & desarrollo , Xylella/genética , Xilema/metabolismo , Xilema/microbiología , Aerobiosis/efectos de los fármacos , Aerobiosis/genética , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/genética , Genes Bacterianos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Xylella/citología , Xylella/efectos de los fármacos , Xilema/efectos de los fármacosRESUMEN
In the xylem vessels of susceptible hosts, such as citrus trees, Xylella fastidiosa forms biofilm-like colonies that can block water transport, which appears to correlate to disease symptoms. Besides aiding host colonization, bacterial biofilms play an important role in resistance against antimicrobial agents, for instance antimicrobial peptides (AMPs). Here, we show that gomesin, a potent AMP from a tarantula spider, modulates X. fastidiosa gene expression profile upon 60 min of treatment with a sublethal concentration. DNA microarray hybridizations revealed that among the upregulated coding sequences, some are related to biofilm production. In addition, we show that the biofilm formed by gomesin-treated bacteria is thicker than that formed by nontreated cells or cells exposed to streptomycin. We have also observed that the treatment of X. fastidiosa with a sublethal concentration of gomesin before inoculation in tobacco plants correlates with a reduction in foliar symptoms, an effect possibly due to the trapping of bacterial cells to fewer xylem vessels, given the enhancement in biofilm production. These results warrant further investigation of how X. fastidiosa would respond to the AMPs produced by citrus endophytes and by the insect vector, leading to a better understanding of the mechanism of action of these molecules on bacterial virulence.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Biopelículas/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Xylella/efectos de los fármacos , Xylella/fisiología , Biopelículas/crecimiento & desarrollo , Análisis de Secuencia por Matrices de Oligonucleótidos , Enfermedades de las Plantas/microbiología , Nicotiana/microbiología , Virulencia/efectos de los fármacosRESUMEN
Citrus sinensis grafted on C. limonia produces a considerable number of compounds that are not common in both plants developed from germination of seeds. The chemical profile of scion and rootstock differ notably for absence in the form of flavonoids and coumarins containing C5 prenyl groups attached to the carbon atoms of aromatic and heterocyclic systems or to oxygen. Only linear pyranocoumarins xanthyletin and xanthoxyletin were found in scion. This observation indicates that the prenylated compounds once biosynthesized in the roots could have been translocated to other organs. Xylella fastidiosa colonizes the xylem of plants causing diseases on several economically important crops such as citrus variegated chlorosis (CVC). A number of flavonoids, coumarins, alkaloids, dihydrocinnamic acid derivative, anacardic acid, triterpenes, and limonoids were tested for in vitro activity on the growth of Xylella fastidiosa. Azadirachtin A was the most active. Hesperidin, which occurs in great amounts in cells of the mesophyll of the affected leaves with CVC, showed a moderate activity suggesting that it can act as a good barrier for small-size colonies from X. fastidiosa.
Asunto(s)
Antiinfecciosos/farmacología , Citrus sinensis/química , Citrus/química , Xylella/efectos de los fármacos , Xylella/crecimiento & desarrollo , Antiinfecciosos/análisis , Cruzamiento , Hesperidina/análisis , Hesperidina/farmacología , Limoninas/análisis , Limoninas/farmacología , Enfermedades de las Plantas , Hojas de la Planta/química , Raíces de Plantas/química , Tallos de la Planta/químicaRESUMEN
Xylella fastidiosa is the etiologic agent of a wide range of plant diseases, including citrus variegated chlorosis (CVC), a major threat to citrus industry. The genomes of several strains of this phytopathogen were completely sequenced, enabling large-scale functional studies. DNA microarrays representing 2,608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c were used to investigate transcript levels during growth with different iron availabilities. When treated with the iron chelator 2,2'-dipyridyl, 193 CDS were considered up-regulated and 216 were considered down-regulated. Upon incubation with 100 microM ferric pyrophosphate, 218 and 256 CDS were considered up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription-quantitative PCR. Several CDS involved with regulatory functions, pathogenicity, and cell structure were modulated under both conditions assayed, suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to functions of pili/fimbriae. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. The promoter regions of the strain 9a5c genome were screened for putative Fur boxes, and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X. fastidiosa, and they present novel evidence for iron regulation of pathogenicity determinants.
Asunto(s)
Bacteriocinas/genética , Fimbrias Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/genética , Genes Bacterianos/genética , Hierro/farmacología , Xylella/genética , 2,2'-Dipiridil/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriocinas/metabolismo , Quelantes/farmacología , Ensayo de Cambio de Movilidad Electroforética , Fimbrias Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulón/genética , Regulón/fisiología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Xylella/efectos de los fármacos , Xylella/crecimiento & desarrollo , Xylella/metabolismoRESUMEN
Xylella fastidiosa is a phytopathogen that causes diseases in different plant species. The development of disease symptoms is associated to the blockage of the xylem vessels caused by biofilm formation. In this study, we evaluated the sensitivity of biofilm and planktonic cells to copper, one of the most important antimicrobial agents used in agriculture. We measured the exopolysaccharides (EPS) content in biofilm and planktonic cells and used real-time reverse transcription polymerase chain reaction to evaluate the expression of the genes encoding proteins involved in cation/multidrug extrusion (acrA/B, mexE/czcA, and metI) and others associated with different copper resistance mechanisms (copB, cutA1, cutA2, and cutC) in the X. fastidiosa biofilm formed in two different media. We confirmed that biofilms are less susceptible to copper than planktonic cells. The amount of EPS seems to be directly related to the resistance and it varies according to the media where the cells are grown. The same was observed for gene expression. Nevertheless, some genes seem to have a greater importance in biofilm cells resistance to copper. Our results suggest a synergistic effect between diffusion barriers and other mechanisms associated with bacterial resistance in this phytopathogen. These mechanisms are important for a bacterium that is constantly under stress conditions in the host.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Cobre/farmacología , Farmacorresistencia Bacteriana , Xylella/efectos de los fármacos , Proteínas Portadoras/biosíntesis , Perfilación de la Expresión Génica , Viabilidad Microbiana , Polisacáridos Bacterianos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Xylella/fisiologíaRESUMEN
Xylella fastidiosa has a wide host range. Isolates of this bacterium that cause diseases in citrus (CVC) and grapes (PD) share 98% genome homology, and 95.7% amino acid identity. Drug resistance genes show a higher level of divergence and may be involved in the X. fastidiosa-host interaction. Antibiotic susceptibility of CVC and PD strains were compared utilizing the Etest strip method (AB Biodisk). Etest is applicable for fastidious slow-growing organisms due to its reproducibility. Results showed that the CVC strain was resistant to bacitracin, cefotaxime, and trimethoprim, and susceptible to chloramphenicol, erythromycin, gentamicin, kanamycin, streptomycin, and tetracycline. The PD strain was susceptible to all tested antibiotics, except kanamycin and trimethoprim. Both isolates produced a class C beta-lactamase. These data support previous antibiotic studies and gene discrepancies found in the sequencing data of PD and CVC strains. These results demonstrate the efficacy of utilizing Etest assays for X. fastidiosa strains.