N‐acetylcysteine interferes with the biofilm formation,motility and epiphytic behaviour of Xanthomonas citri subsp. citri

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.     

Description of copper tolerant Xanthomonas citri subsp. citri and genotypic comparison with sensitive and resistant strains

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 (Cu^R). However, the study of copper sensitivity of X. citri strains from Paraná state, Brazil, did not reveal the existence of Cu^R, but copper tolerant (Cu^T) strains. The aim of this study was to describe for the first time the existence of Cu^T X. citri and compare the genetic determinants that differentiate the Cu^T strains from the sensitive (Cu^S) and Cu^R strains. Cu^T strains supported intermediate concentrations of copper in comparison to Cu^S and Cu^R. Cu^T strains lack the gene clusters copLAB or copABCD responsible for copper resistance in Cu^R 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. Cu^T strains differed from Cu^S strains by the higher expression of the homologous chromosomal genes cohA and cohB in the presence of copper. Cu^T X. citri strains are not precursors of Cu^R 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.     

Genetic structure analysis of strains causing citrus canker in Iran reveals the presence of two different lineages of Xanthomonas citri pv. citri pathotype A*

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 (R_ST = 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.     

Biofilm formation and motility of Xanthomonas strains with different citrus host range

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, A^w 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.     

Development of a simplified NASBA protocol for detecting viable cells of the citrus pathogen Xanthomonas citri subsp. citri under different treatments

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.     

Detection of Xanthomonas citri subsp. citri from field samples using single‐tube nested PCR

A single‐tube nested PCR was developed for detection of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. The assay targets the pthA gene of Xcc and utilizes different annealing temperatures for the two primer pairs. It reliably detected as few as 1·0 × 10² Xcc cells, and was unaffected by the presence of PCR inhibitors. It was 10‐fold and 8500‐fold more sensitive than standard PCR and ELISA, respectively. Increased sensitivity was also achieved via the use of a washing method for DNA extraction, as opposed to direct extraction from leaf tissue. When evaluated for Xcc detection in 90 samples collected from affected pomelo orchards, the single‐tube nested PCR was superior to standard PCR, detecting the pathogen in 67 vs. 54 samples. It was also able to detect Xcc from samples with and without symptoms. This assay can be used as a rapid and sensitive technique for routine Xcc detection in field samples for surveillance of citrus canker.     

Bacteria from the citrus phylloplane can disrupt cell–cell signalling in Xanthomonas citri and reduce citrus canker disease severity

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.     

Copper resistance genes from different xanthomonads and citrus epiphytic bacteria confer resistance to Xanthomonas citri subsp. citri

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 (Cu^R) strains of other Xanthomonas species and citrus epiphytic bacteria pose a risk for the development of copper resistance in Xcc. Cu^R 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. Cu^R 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 Cu^R strains from citrus, an epiphytic Xanthomonas ssp. and a strain of Stm, harboured homologs of the copper resistance genes found in Cu^R 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.     

Detached leaf inoculation of germplasm for rapid screening of resistance to citrus canker and citrus bacterial spot

A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xanthomonas citri subsp. citri, Xcc) and citrus bacterial spot (Xanthomonas alfalfae subsp. citrumelonis, Xac) was developed to evaluate limited quantities of leaf material. Bacterial inocula of Xcc or Xac at 10⁴, 10⁵, or 10⁸ cfu ml⁻¹ were injection-infiltrated into the abaxial surface of disinfested, immature leaves of susceptible and resistant genotypes. Inoculated detached leaves were placed on the surface of 0.5% water agar plates and incubated at 28°C under a 12 h photoperiod. Likewise, inocula were infiltrated into attached leaves of greenhouse plants. At high inoculum concentrations of Xcc or Xac (10⁸ cfu ml⁻¹), resistant cultivars of kumquat developed a hypersensitive-like reaction within 3 days post inoculation (dpi). At 10⁵ cfu ml⁻¹, populations 14 dpi were <10⁴ per inoculation site. In canker-susceptible Citrus spp. (‘Duncan’ grapefruit and ‘Rough’ lemon), water-soaked areas occurred by 3 dpi and typical canker lesions developed by 7 to14 dpi. Concentration of Xcc recovered from inoculation sites was approximately 10⁵ cfu ml⁻¹ by 14 dpi. In citrus bacterial spot-susceptible citrus (‘Swingle’ citrumelo and grapefruit), symptoms developed within 7 dpi. Populations of Xac after inoculation at 10⁵ cfu ml⁻¹ were comparable to Xcc in susceptible hosts 14 dpi (>10⁵). The detached leaf assay is useful for the characterization and differentiation of lesion phenotype for each Xanthomonas pathogen permitting rapid screening of germplasm resistance based on the quantification of number of lesions and bacterial concentration.     

Spirodiclofen and spirotetramat bioassays for monitoring resistance in citrus red mite, Panonychus citri (Acari: Tetranychidae)

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     

Pathogenic and non‐pathogenic Agrobacterium tumefaciens,A. rhizogenes and A. vitis strains form biofilms on abiotic as well as on root surfaces

Pathogenic and non‐pathogenic Agrobacterium tumefaciens, A. rhizogenes and A. vitis strains growing in minimal liquid medium adhered to different abiotic surfaces, forming biofilms at initial stages of development. Agrobacterium tumefaciens and A. vitis strains were able to attach to both polystyrene and polypropylene materials, whereas the A. rhizogenes strains only bound to polystyrene surfaces. Strains of the three species were also able to form biofilms on borosilicate coverslips. It is concluded that their ability to adhere to and form nascent biofilms on abiotic surfaces is dependent on the Agrobacterium species (biovar), surface material and growth conditions. Furthermore, tumorigenic A. tumefaciens and A. vitis strains, and the biological control agent A. rhizogenes strain K84, bound tightly to and formed complex biofilms on the surface of tomato root tips ex planta. More importantly, in planta assays confirmed that all three Agrobacterium spp. strains efficiently colonized tomato seedlings and also formed biofilms on roots. These complex structures, as revealed by scanning electron microscopy, were composed of numerous bacterial cells arranged in different ways: either dense and continuous carpets, large aggregates embedded in extra‐cellular material or globular mushrooms traversed internally by channels. Confocal laser scanning microscopy, using GFP‐marked derivative strains, corroborated the presence of live, three‐dimensional and thick green fluorescent structures attached to plant material. This study illustrates that besides A. tumefaciens, strains of the species A. rhizogenes and A. vitis are also able to build biofilms on abiotic as well as on root surfaces.     

Involvement of carbon catabolite repression on regulation of endopolygalacturonase gene expression in citrus fruit

 The Acpg1 gene of Alternaria citri encodes an extracellular endopolygalacturonase that is important for virulence in citrus fruits. Expression of Acpg1 is regulated by substrate induction and carbon catabolite repression. A green fluorescent protein (GFP) gene was employed as a reporter gene to define 813 bases upstream of the translation start site comprising the Acpg1 promoter. This upstream sequence contains five putative binding sequences of catabolite repressive element A (CreA), a cis-acting zinc finger repressor involved in carbon catabolite repression. We constructed each CreA-binding site-deleted Acpg1 promoters with GFP reporter gene and transformed them to A. citri. The construct PGPDL4 deleted from −401 to −813 showed both substrate induction and catabolite repression, whereas PGPDL5 additionally deleted from −1 to −84, including one putative CreA-binding site, resulted in a loss of catabolite repression function. Green fluorescence of PGPDL4 was induced by pectin in the peel but was repressed completely in the juice sac area of citrus fruit. However, green fluorescence of PGPDL5 was induced in both the peel and juice sac area, indicating that repression of Acpg1 in the juice sac area is likely accomplished by carbon catabolite repression. Received: October 4, 2002 / Accepted: October 31, 2002 Acknowledgments The authors thank Drs. D. Cullen, A. Van den Wymelenberg, and J. Andrews, University of Wisconsin, for providing pTEFEGFP containing GFP and Dr. T. Tsuge, Nagoya University, for providing transformation vector pSH75. The nucleotide sequence data of Acpg1 promoter region in the DDBJ, EMBL, and GenBank sequence databases is under accession number AB047543. This research was supported in part by grants to K.A. from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.     

Rate, placement and timing of aldicarb applications to control Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), in oranges

BACKGROUND: The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a cosmopolitan insect pest of citrus and vectors the bacterium Candidatus Liberibacter asiaticus, a suspected causal organism of citrus greening or ‘huanglongbing’ disease. Aldicarb 150 g kg^?1 GR (Temik^® 15 G) was evaluated at three rates, two placements and three timings for ACP control in orange trees. RESULTS: Application of aldicarb at 5.6, 2.8 and 1.4 kg AI ha^?1 in March 2006 reduced adults by 58–66%, 45–46% and 25–37% respectively compared with untreated controls in two separate trials. No difference was observed in placement (one versus two sides of the tree) or tree size (8 years old versus 12 years old). Application at 5.6 kg ha^?1 in January 2007 reduced adults by 86% and shoot infestation by 77% in spring, and was generally better than the November and especially February applications. Even more striking results were evident on adults caged on treated plants for 25 days in March. Spiders and ladybeetles were equally abundant in treated and untreated trees. CONCLUSION: Aldicarb application at 5.6 kg ha^?1 to the bed side of mature citrus trees 2–3 months before spring growth can suppress ACP through spring without a direct effect on principal psyllid natural enemies. Copyright © 2008 Society of Chemical Industry     

Antifeedant and sublethal effects of imidacloprid on Asian citrus psyllid,Diaphorina citri

BACKGROUND: Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, transmits the causal bacteria of the devastating citrus disease huanglongbing (HLB). Because of the variation in spatial and temporal uptake and systemic distribution of imidacloprid applied to citrus trees and its degradation over time in citrus trees, ACP adults and nymphs are exposed to concentrations that may not cause immediate mortality but rather sublethal effects. The objective of this laboratory study was to determine the effects of sublethal concentrations of imidacloprid on ACP life stages. RESULTS: Feeding by ACP adults and nymphs on plants treated daily with a sublethal concentration (0.1 µg mL^?1) of imidacloprid significantly decreased adult longevity (8 days), fecundity (33%) and fertility (6%), as well as nymph survival (12%) and developmental rate compared with untreated controls. The magnitude of these negative effects was directly related to exposure duration and concentration. Furthermore, ACP adults that fed on citrus leaves treated systemically with lethal and sublethal concentrations of imidacloprid excreted significantly less honeydew (7–94%) compared with controls in a concentration‐dependent manner suggesting antifeedant activity of imidacloprid. CONCLUSIONS: Sublethal concentrations of imidacloprid negatively affect development, reproduction, survival and longevity of ACP, which likely contributes to population reductions over time. Also, reduced feeding by ACP adults on plants treated with sublethal concentrations of imidacloprid may potentially decrease the capacity of ACP to successfully acquire and transmit the HLB causal pathogen. Copyright © 2009 Society of Chemical Industry     

Over-expression of the citrus gene CtNH1 confers resistance to bacterial canker disease

Citrus canker is a devastating disease, caused by Xanthomonas axonopodis pv. citri (Xac). It is well established that the NPR1 gene plays a pivotal role in systemic acquired resistance (SAR) in Arabidopsis. In this study, we report the isolation and characterization of an NPR1 homolog from citrus, namely Citrus NPR1 homolog 1 (CtNH1). Sequence alignment and phylogenetic analysis indicate that CtNH1 is closely-related to the Arabidopsis NPR1 gene and its orthologs from rice, grapevine, and cacao. When over-expressed in citrus, CtNH1 confers resistance to Xac and leads to constitutive expression of the pathogenesis-related (PR) gene chitinase 1 (Chi1), suggesting that CtNH1 is orthologous to NPR1.     

Distribution of canker lesions on the surface of diseased grapefruit

The objective of this study was to describe and quantify the distribution of citrus canker lesions (caused by Xanthomonas citri subsp. citri) on the surface of grapefruits, and provide evidence for splash‐driven infection of fruit. Based on fruit diameter, each fruit was sliced in four by taking three horizontal planes across the vertical axis such that each horizontal zone (Z1–Z4, from peduncle to flower scar) had the same vertical height. Each zone had equal surface area. Lesion number was counted on each zone of the fruit. Although lesion number among fruits was variable, both lesion number and percentage of total lesions showed a decline from the uppermost zone (lesion number: 10·5, 7·0, 3·4 and 2·4; percentage: 44·6, 30·5, 14·4 and 10·5 on Z1–Z4, respectively). General linear modelling (GLM) using a Poisson distribution with a log‐link function demonstrated a significant effect of zone and cultivar on lesion number, and significant differences between all zones and both cultivars. An analysis of covariance showed no effect of lesion number on fruit size, although cultivars differed in total lesion count. Canker‐susceptible citrus fruit is susceptible for a prolonged period after fruit set and minimizing inoculum and reducing wind speed and splash, and use of copper sprays during that period, will help reduce disease on fruit, particularly on the upper surfaces that appear to be more prone to infection.     

Laboratory Evaluation of Citrus Cultivars Susceptibility and Influence of Fruit Size on Fortune Mandarin to Infection by Alternaria alternata pv. citri

Young leaves of 62 citrus cultivars were inoculated with conidia of three Spanish isolates of Alternaria alternata pv. citri, the causal agent of brown spot of citrus. Hybrids with Dancy mandarin, King mandarin or their derivates as a parent, grapefruit cultivars and the mandarin cultivars Guillermina, Emperor, Clemenpons and Esbal were highly susceptible to the pathogen. Satsuma cultivar Clausellina and orange cultivars, with the exception of Sanguinelli, were slightly susceptible. Lemon and lime cultivars were not susceptible, with the exception of Mexican lime (Citrus aurantifolia), which was slightly susceptible. Although this study shows a range of potential hosts for this pathogen, to date the only affected cultivars in Spain are Fortune and Nova mandarins, and Minneola tangelo. The susceptibility of Fortune fruits decreased as diameter increased, being susceptible through the whole season. This was confirmed with field observations in autumn where fruit infections have been detected when the diameter reaches 6–7 cm.