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1.
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2.
Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a disease that affects almost all types of citrus crops. Production of particular Xcc pathogenicity factors is controlled by a gene cluster rpf, which encodes elements of a cell–cell communication system called quorum sensing (QS), mediated by molecules of the diffusible signal factor (DSF) family. Interference with cell–cell signalling, also termed quorum quenching, either by signal degradation or over‐production, has been suggested as a strategy to control bacterial disease. In this study, three bacterial strains were isolated from citrus leaves that displayed the ability to disrupt QS signalling in Xcc. Pathogenicity assays in sweet orange (Citrus sinensis) showed that bacteria of the genera Pseudomonas and Bacillus also have a strong ability to reduce the severity of citrus canker disease. These effects were associated with alteration in bacterial attachment and biofilm formation, factors that are known to contribute to Xcc virulence. These quorum‐quenching bacteria may represent a highly valuable tool in the process of biological control and offer an alternative to the traditional copper treatment currently used to treat citrus canker disease.  相似文献   

3.
Nucleic acid sequence based amplification (NASBA) is a method of amplifying RNA, for the detection of RNA viruses and human pathogenic bacteria. Recently, NASBA has also been employed for the detection of plant diseases caused by viruses and quarantine bacteria. A major citrus pathogen, Xanthomonas citri subsp. citri (Xcc), causal agent of citrus bacterial canker, is being studied in depth due to its economic importance, with recent focus concentrating on its viability and survival under different stress conditions and control treatments. In this work, a NASBA protocol using primers for gumD mRNA has been developed to assess the viability of this pathogen under different bacteriocidal treatments. This method is rapid, specific and sensitive, and is able to detect viable bacterial cells, using a hybridization device which allows the visualization of the results in only 30 min. The usefulness of the method has been confirmed with bacterial suspensions subjected to different heat treatments and to sodium orthophenylphenate.  相似文献   

4.
Genetic exchange is considered to be an important process in the selective adaptation of microorganisms to shifting and challenging environmental conditions. As a consequence of the copious use of copper bactericides, many species of plant pathogenic bacteria, including Xanthomonas citri subsp. citri (Xcc), have developed resistance to copper. This study assesses whether copper resistant (CuR) strains of other Xanthomonas species and citrus epiphytic bacteria pose a risk for the development of copper resistance in Xcc. CuR epiphytic bacteria were isolated on MGY agar from citrus leaves collected in two citrus groves treated with copper bactericides in Florida. Horizontal gene transfer of copper resistance genes was investigated within different Xanthomonas species and from citrus epiphytic bacteria to Xanthomonas. CuR epiphytic bacteria from citrus were screened for the presence of copper resistance genes homologous to copL, copA and copB genes from Xcc and characterized regarding tolerance to copper. Copper resistance determinants from a citrus epiphytic strain of Stenotrophomonas maltophilia (Stm) were cloned and expressed in Xcc and other Xanthomonas strains. Copper resistance genes in Xcc were determined to be present on a large (~300?kb) conjugative plasmid. Cu resistance was transferred via conjugation from two copper resistant citrus strains, Xcc and X. alfalfae subsp. citrumelonis (Xac), and two tomato pathogens, X. euvesicatoria (Xe) and X. perforans (Xp), to Xcc. PCR analysis revealed that two CuR strains from citrus, an epiphytic Xanthomonas ssp. and a strain of Stm, harboured homologs of the copper resistance genes found in CuR Xcc. The introduction of copLAB gene cluster from Stm into different xanthomonads conferred copper resistance to sensitive strains of Xcc, Xac, Xe and Xp. Based on these results there is a low, but significant, likelihood of horizontal gene transfer of copper resistance genes from other xanthomonads or epiphytic bacteria to Xcc in nature.  相似文献   

5.
Xanthomonas citri subsp. citri (Xcc) strain A is the causal agent of citrus bacterial canker (CBC) on most Citrus spp. and close relatives. Two restricted host range strains of CBC, Aw and A*, from Florida and southwest Asia, respectively, infect Mexican lime. Several studies have linked biofilm formation by Xcc to bacterial colonization prior to and after plant ingress, but none have evaluated connections between biofilm formation and the behaviour of different strains of Xcc on citrus hosts and non‐hosts. In this study biofilm formation and swimming motility were evaluated for citrus pathogenic xanthomonads including wide and restricted host range strains of Xcc, X. alfalfae subsp. citrumelonis (Xac) (the causal agent of citrus bacterial spot) and X. campestris pv. campestris (Xc). Differential biofilm formation was observed in vitro and in planta among the Xanthomonas strains assayed. Minimal medium XVM2 increased biofilm formation, especially for those strains with a host range restricted to Mexican lime. In planta, strains produced more biofilm on leaves or fruits of their host than on non‐hosts. Scanning electron microscopy of biofilms on leaf and fruit surfaces revealed differences in structure of bacterial aggregates with respect to the strain's host range. In addition, swimming motility varied widely depending on the host range of the strain. It was concluded that biofilm formation in vitro and in planta for strains of Xcc and Xac was related to their host range, as these processes affect colonization at the early stages of the infection process.  相似文献   

6.
Citrus canker is caused by Xanthomonas citri subsp. citri. Bacterial biofilm formation is important in the development of this disease because it is a factor in epiphytic bacterial survival on leaves and in infection. N‐acetylcysteine (NAC), in addition to having antibacterial properties, reduces biofilm formation by a variety of bacteria and was therefore tested for impairing biofilm formation by X. citri. Copper is currently the antimicrobial compound most commonly applied in agriculture to control citrus canker. Therefore, this study also evaluated a possible synergistic effect between NAC and copper to improve the strategy for controlling this phytopathogen. NAC was found to decrease biofilm formation, the production of extracellular polysaccharides and bacterial stickiness. Motility was also affected in the presence of NAC. The best combination of NAC and copper for controlling X. citri was application of NAC followed by copper 48 h later. The concentrations of 6 mg mL?1 of NAC and 3·5 μg mL?1 of copper were able to kill X. citri. NAC inhibited the epiphytic behaviour of X. citri on leaves, altering cell growth and the bacterial ability to form biofilms. The addition of copper to cells previously treated with NAC enhanced its bactericidal activity. In conclusion, NAC has antibacterial properties against X. citri, interfering with bacterial growth, motility and biofilm formation. Under epiphytic conditions, NAC made the cells more susceptible to copper by affecting X. citri biofilm formation. This study opens new possibilities for the use of NAC in combination with copper, possibly resulting in more sustainable management of citrus canker.  相似文献   

7.
The copper-based products widely used for control of citrus canker may lead to the development of Xanthomonas citri subsp. citri (X. citri) resistant to copper (CuR). However, the study of copper sensitivity of X. citri strains from Paraná state, Brazil, did not reveal the existence of CuR, but copper tolerant (CuT) strains. The aim of this study was to describe for the first time the existence of CuT X. citri and compare the genetic determinants that differentiate the CuT strains from the sensitive (CuS) and CuR strains. CuT strains supported intermediate concentrations of copper in comparison to CuS and CuR. CuT strains lack the gene clusters copLAB or copABCD responsible for copper resistance in CuR strains and the large plasmids (c. ≥200 kb) that normally carry these genes. The nucleotide sequences of chromosomal homologous genes cohLAB, involved in copper homeostasis, were 100% similar in strains of all phenotypes. CuT strains differed from CuS strains by the higher expression of the homologous chromosomal genes cohA and cohB in the presence of copper. CuT X. citri strains are not precursors of CuR strains and do not pose a threat to the efficient use of copper-based bactericides for management of citrus canker in citrus orchards. Copper resistance and tolerance are distinct phenotypes and should not be used as synonyms. The proper characterization of the sensitivity to copper leads to a more confident monitoring of the distribution of copper resistant populations of X. citri and adoption of containment measures only when necessary.  相似文献   

8.
Streptomycin has been tested as an alternative to copper bactericides, which are routinely used for the control of citrus canker (Xanthomonas citri subsp. citri, Xcc) in citrus producing areas where the disease is endemic. A major concern is that excessive use of copper as a bactericide may lead to development of copper-resistant strains of Xcc. In this study, we developed a semi-selective medium to recover copper or streptomycin-resistant strains of Xcc from citrus leaves. The newly developed semi-selective medium was used to monitor the effect of a 21-day-interval copper or streptomycin spray program on Xcc for three consecutive seasons and on citrus epiphytic bacterial populations for two seasons in a commercial grapefruit grove. Although, no copper- or streptomycin-resistant strains of Xcc were isolated after three seasons, we observed a significant increase over time in the frequency of citrus epiphytic bacteria resistant to these chemicals. Overall, the proportion of epiphytic bacteria resistant to streptomycin on treated and untreated leaves was proportionally lower than the copper-resistant bacterial population. When application of each bactericide was suspended for the season, the proportion of bactericide-resistant bacteria in the epiphytic population decreased to that of the non-treated bacterial population. Availability of an alternative bactericide, such as streptomycin, to integrate into a copper-based program would reduce the amount of each bactericide sprayed in citrus orchards and possibly lower the selection pressure for bacterial resistance to these chemicals.  相似文献   

9.
West Asia has been recognized as a major centre for the diversification of Xanthomonas citri pv. citri, a citrus quarantine pathogen of considerable economic importance. However, little genotyping data is available mainly due to the paucity of microbial resources in this region. Using a comprehensive strain collection, several genotyping techniques and a pathogenicity assay, the status of strains causing Asiatic citrus canker in Iran, an internationally significant citrus‐producing country, was clarified. All strains were genetically related to X. citri pv. citri pathotype A* (i.e. strains with a host range restricted to Mexican lime and related species) but not to pathotype A (i.e. strains with a wide host range among rutaceous species). The findings were based on discriminant analysis of the principal components of MLVA‐31 data and were further confirmed by pathogenicity data. Two genetically, geographically and pathologically separate groups of strains in Iran were identified. One of the groups had never been previously reported anywhere in the world. A very strong genetic structure was found (RST = 0·938), consistent with their geographical isolation. Strains from these two groups also differed in terms of their type III effector repertoire. The atypical host range of one of these groups could explain why some Iranian strains had previously been mistakenly identified as pathotype A. This study suggests the absence of invasive pathotype A strains in Iran (known as DAPC 1), which account for most of the economically important outbreaks internationally.  相似文献   

10.
The effects of copper sprays on annual and polyetic progress of citrus canker, caused by Xanthomonas citri subsp. citri, in the presence of the Asian citrus leafminer (Phyllocnistis citrella), were evaluated in a study conducted in a commercial orchard in northwest Paraná state, Brazil, where citrus canker is endemic. Nonlinear monomolecular, logistic and Gompertz models were fitted to monthly disease incidence data (proportion of leaves with symptoms) for each treatment for three seasons. The logistic model provided the best estimate of disease progress for all years and treatments evaluated and logistic parameter estimates were used to describe polyetic disease dynamics. Although citrus canker incidence increased during each of the seasons studied, it decreased over the whole study period, more so in copper‐treated trees than in water‐sprayed controls. Copper treatment reduced disease incidence compared with controls in every year, especially 2004–2005, when incidence was ca. 10‐fold higher in controls than in treated plots (estimated asymptote values 0·82 and 0·07, respectively). Copper treatment also reduced estimated initial disease incidence and epidemic growth rates every year.  相似文献   

11.
The population dynamics of Xanthomonas arboricola pv. pruni (Xap) and X. citri subsp. citri (Xcc) was assessed on over three dozen plant species/genotypes under field and greenhouse conditions. Both Xap and Xcc multiplied on red nightshade, black nightshade, bindweed, Chenopodium, common bean and wheat up to 20 days post‐inoculation (dpi) under greenhouse conditions. A high bacterial growth rate was observed on all (alfalfa, bindweed, Chenopodium, field mustard, millet and prickly lettuce) but one (liquorice) plant species tested under field conditions. Xap successfully proliferated on both lemon and sweet lemon up to 140 dpi, attaining a population density even higher than that of Xcc. The latter showed an increased growth rate on GxN, GF677, Ghisella 6 and Mariana 2624 rootstocks up to 140 dpi. While Xap and Xcc did not grow on pomegranate and common fig, they had a steady population growth on apple and pear plants up to 140 dpi, although the final population sizes were smaller than those observed on lemon and sweet lemon plants. The results suggest that a large number of non‐host plant species could support epiphytic populations of Xap or Xcc, which may have implications for plant disease epidemiology.  相似文献   

12.
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13.
BACKGROUND: Citrus red mite, Panonychus citri (McGregor), is one of the most important pesticide‐resistant pests in China. In order better to understand its resistance status, six populations of the mite were collected from Chinese citrus orchards for monitoring of resistance to spirodiclofen and another five acaricides. RESULTS: All the samples collected in the field in 2006 were susceptible to spirodiclofen. However, the LC50 values in populations sampled in 2009 ranged from 3.29 to 418.24 mg L?1 spirodiclofen, a 127‐fold difference between the least and most sensitive populations. Compared with a susceptible strain, 50‐fold and 90.8‐fold resistance to spirodiclofen was detected in populations sampled from Pinghe and Fuzhou in 2009, as well as cross‐resistance to spirotetramat. The LC50 values for abamectin, fenpropathrin, hexythiazox and pyridaben in the collected samples ranged from 0.041 to 3.52 mg L?1, from 23.91 to 696.16 mg L?1, from 13.94 to 334.19 mg L?1 and from 48.90 to 609.91 mg L?1 respectively. CONCLUSION: Great variations in resistance to the tested acaricides were observed among the sampled populations. The Pinghe population developed resistance to all the acaricides tested. The Jianning population was susceptible to most acaricides tested, except pyridaben. Resistance management strategies were conducted on the basis of these observations. Copyright © 2010 Society of Chemical Industry  相似文献   

14.
Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus bacterial canker, an important disease for the citrus industry. Studies of Xac survival in environments outside of the lesion performed in the past may have underestimated the viable population because the recovery was based on the ability of the bacterium to grow on culture media. This study monitored survival of Xac that express green fluorescent protein (GFP) in two different forms: the native protein, and a protein that is unstable due to a specific oligopeptide tail targeted by proteases within the bacterium. Transformed strains of Xac were verified to be stable in their expression of GFP and to show no differences in virulence and fitness compared to wild type strains. Evaluation of protein stability confirmed that strains with unstable GFP only expressed and fluoresced in metabolically active cells, and not in dead bacteria. Fluorescence of unstable GFP strains under confocal microscopy was used to track bacterial survival and biofilm formation on leaf and fruit surfaces. After spray inoculation, aggregates of fluorescing cells of unstable GFP strains formed biofilms on leaves and fruit. Bacterial cells that aggregated on the surfaces only survived when protected from desiccation. Aggregation of viable bacteria in biofilms confirms their role in pathogen survival outside of lesions and protection from bactericide treatments in the field or in the fruit disinfection process.  相似文献   

15.
Citrus canker, caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc), is a serious leaf and fruit spotting disease affecting many important citrus cultivars including grapefruit and certain sweet oranges. Currently, efficacious and economical disease control measures for highly susceptible citrus cultivars are lacking. Development of commercial cultivars with greater field resistance to citrus canker is the optimum strategy for effective disease management. In this study, we generated transgenic ‘Duncan’ grapefruit (DG) and ‘Hamlin’ sweet orange (Ham) expressing the Arabidopsis NPR1 gene (AtNPR1), which is a key positive regulator of the long-lasting broad-spectrum resistance known as systemic acquired resistance (SAR). Our results indicate that over-expression of AtNPR1 in citrus increases resistance to citrus canker and that the resistance is related with the expression levels of AtNPR1 in the transgenic plants. The line (DG 42-2) with the highest expression level of AtNPR1 was also the most resistant, which developed significant fewer lesions accompanied by a ten-fold reduction in Xcc population. The lesions developed on DG 42-2 were smaller and darker than those on the control and lacked callus formation. These lesion phenotypes resemble those on canker resistant kumquats and canker susceptible citrus trees treated with SAR-inducing compounds. Therefore, over-expression of AtNPR1 in citrus is a promising approach for development of more resistant cultivars to citrus canker.  相似文献   

16.
Citrus canker (Xanthomonas citri subsp. citri (Xcc)) can cause yield loss and trade restrictions. The pathogen is dispersed in rain splash and spread is promoted by wind. The goal of this study was to gain some insight into the properties of short‐distance splash dispersal of Xcc from ～1·5 m‐tall cankered grapefruit canopies in turbulent wind, common during rainstorms in Florida. Turbulent wind up to 19·9 m s?1 was tested in five experiments. Bacteria flux density (BFD, bacteria cm?2 min?1) was quantified at heights of 30, 70, 110, 130 and 180 cm above ground, and at four horizontal points (17, 51, 85 and 119 cm) at each height across the direction of the wind 1 m downwind. BFD varied among experiments, but the lowest BFDs were consistently detected at the greatest sample height. Despite differences between experiments, the relationship between log BFD and sample height was consistently described by a linear function (P = 0·06–<0·0001, R2 = 0·75–>0·99). The BFD collected at the horizontal points across the wind path was variable. BFDs collected were sometimes significantly different, but no relationship was discernible. Stronger, turbulent wind resulted in greater BFD, with a linear function describing the relationship between log BFD and wind speed (P = 0·2–0·02, R2 = 0·94–0·96). Multiple regression analysis demonstrated predictability of the proportion of total bacteria collected (F = 141, P < 0·0001, d.f. = 3, R2 = 0·53).  相似文献   

17.
BACKGROUND: Citrus red mite, Panonychus citri (McGregor), is a key pest of San Joaquin Valley California citrus. Spirodiclofen was registered for mite control in 2007, and spirotetramat for scale control in 2008. Because of the potential for resistance to spirodiclofen to develop in spider mites, and cross‐resistance to spirotetramat used for other citrus pests, bioassay methods for resistance monitoring were developed. RESULTS: The responses of four populations of adult female, egg and larval stages of P. citri to spirodiclofen were compared to determine the most robust bioassay method for this pesticide. Adult females responded with a higher LC99 and larval stages exhibited higher control mortality and a lower slope of response compared with the egg stage. Thus, the egg stage was found to be the most suitable stage for testing. Egg production and egg shape were significantly affected by spirodiclofen treatment of adult female mites. Bioassays with the related compound spirotetramat revealed that P. citri egg hatch was less affected by this compound, requiring the assessment of mortality to be extended to 11 days after treatment when the hatched larvae succumbed to the pesticide. Discriminating concentrations of 10 ppm for spirodiclofen and 31.6 ppm for spirotetramat in an 11 day bioassay were tested against eight field populations of P. citri, and 99–100% mortality resulted. CONCLUSION: These results provide a baseline for the response of P. citri to spirodiclofen and spirotetramat that will aid resistance management in California citrus. Copyright © 2011 Society of Chemical Industry  相似文献   

18.
Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, ‘Candidatus Liberibacter asiaticus’, ‘Candidatus Liberibacter africanus’ and ‘Candidatus Liberibacter americanus’, is essential for the preventive control of the disease. Real‐time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR. Universal detection of ‘Ca. Liberibacter’ species was achieved by direct tissue‐printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with TaqMan chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real‐time PCR. This simplified method was validated and could detect HLB‐liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB‐infected trees. The use of direct assays as template showed good agreement with use of purified DNA (κ = 0·76 ± 0·052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina citri that fed on leaves of HLB‐infected trees with or without symptoms. A commercial ready‐made kit based on this technology showed 96% accuracy in intra‐laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies.  相似文献   

19.
Citrus canker (caused by the bacterial pathogen Xanthomonas citri subsp. citri, Xcc) can cause severe damage to citrus. It is endemic in Florida, and occurs in other citrus growing regions. The bacterium is dispersed predominantly in rain splash. To simulate dispersal in splash, and to investigate the effect of wind speed on infection, young plants of Swingle citrumelo were exposed to sprayed inoculum at different wind speeds. Wind was generated using an axial fan, and a pressurized sprayer delivered the inoculum spray. In the five experiments, higher wind speeds (>10 m s−1) consistently resulted in higher incidence and severity of citrus canker developing. By 15 ms−1, there was a dramatic increase in disease. Visible injury to leaves of Swingle citrumelo due to wind was evident at wind speeds ≥ 13 m s−1. The relationship between wind speed and disease, and wind speed and injury was described by a logistic model. More disease was associated with visible injury as the wind speed increased, and disease not associated with visible injury also increased with wind speed. The petiole-leaflet junction was more often infected at higher wind speeds (≥17 m s−1). The concentration of the Xcc inoculum increased the incidence and severity of citrus canker in all experiments. Reducing wind speed in citrus groves with the aid of wind breaks may contribute to a reduction in the severity of an epidemic by reducing dispersal and infection events.  相似文献   

20.
The pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains containing the avrBs2 avirulence gene in susceptible pepper and tomato. The avrBs2 gene is highly conserved in the Xanthomonas genus and when bacteria lack this gene their growth in a susceptible host is diminished, indicating that the avrBs2 gene product could confer an adaptive advantage to the pathogen. The avrBs2 of Xanthomonas citri subsp. citri (Xcc), cause of citrus canker, shares 96% homology with avrBs2 of Xcv. To evaluate if Bs2 could recognize avrBs2 of Xcc in citrus plants and thereby activate plant defence mechanisms to increase resistance to canker, transient expression experiments were conducted using Agrobacterium tumefaciens in lemon plants subsequently challenged with wildtype Xcc. The results showed that transient expression of Bs2 reduced canker formation in lemon and induced plant defence mechanisms, as shown by callose deposition and PR‐1 expression. Moreover, when an avrBs2 mutant of Xcc was used, no decrease in disease symptoms was observed. This work shows that the Bs2 gene from Solanaceae is functional in lemon, a member of the Rutaceae family. Therefore, Bs2 is a potential candidate gene for stable expression in transgenic citrus plants in order to improve resistance to canker disease.  相似文献   

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