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.     

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.     

Survival of Xanthomonas campestris pv. campestris in the phyllosphere and rhizosphere of weeds

The phyllosphere and rhizosphere of weeds are important niches for phytobacterial survival. The absence of information in Brazil regarding Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, motivated this study. Twenty‐six weed species belonging to 14 botanical families were included in field experiments between August 2014 and October 2015. Lepidium virginicum and Raphanus raphanistrum (Brassicaceae) demonstrated great potential for survival of Xcc in the phyllosphere, with the bacterium isolated after 56 and 70 days, respectively. Low variation between maximum and minimum temperatures, high rainfall and high relative humidity at specific times of the year contributed to longer Xcc survival periods in the phyllosphere of some species. Xcc survived in the rhizosphere only in R. raphanistrum, where it was isolated for up to 28 days. No relation was found between climatic factors and survival in the rhizosphere. The data indicate that control of brassicaceous weeds will contribute to the control of black rot.     

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.     

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.     

XopN-T3SS effector modulates in planta growth of Xanthomonas axonopodis pv. punicae and cell-wall-associated immune response to induce bacterial blight in pomegranate

Bacterial blight caused by Xanthomonas axonopodis pv. punicae (Xap) is an important disease of pomegranate. Most phytopathogenic strains in the Genera Xanthomonas secrete effector proteins by the type III secretion system (T3SS) to suppress pathogen-associated molecular pattern (PAMP)-triggered plant immunity (PTI). The T3SS effectors, referred to as Xanthomonas outer proteins (Xops), are known to be key factors required for bacterial growth and colonization in distinct eukaryotic hosts. Xap contains six Xop-effectors, namely XopC2, XopE1, XopL, XopN, XopQ and XopZ. In this study we analyzed xopN, a conserved effector in Xanthomonas, with reference to sequence identity and its role in governing bacterial growth, and virulence. The xopN of Xap shared maximum sequence identity (98.6%) with pathovar citri. Overlapping extension PCR and double crossing over based homologous recombination strategy was employed to generate a xopN null mutant (Xap ΔxopN) of Xap. A kanamycin gene was used to replace the xopN gene. XopN was required for maximal Xap pathogenicity in its natural host pomegranate. The detailed image analysis on blight lesions revealed 3 fold reduction in watersoaked areas on leaves infiltrated with mutant Xap ΔxopN compared to that of with wild Xap. The in planta population count of Xap ΔxopN was reduced approximately 32-fold relative to the wild strain indicating that xopN is required for maximal growth of Xap in pomegranate. In addition, the Xap ΔxopN mutant induced more callose deposition in infected pomegranate leaves. Taken together, the present study shows that XopN governs Xap growth and modulates cell-wall associated immune response in pomegranate.     

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.     

Monitoring for resistant populations of <Emphasis Type="BoldItalic">Xanthomonas citri</Emphasis> subsp. <Emphasis Type="Italic">citri</Emphasis> and epiphytic bacteria on citrus trees treated with copper or streptomycin using a new semi-selective medium

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.     

Pathogenicity and virulence gene content of Xanthomonas strains infecting Araceae,formerly known as Xanthomonas axonopodis pv. dieffenbachiae

Bacterial leaf blight of aroids is caused by a heterogeneous group of xanthomonads listed as Xanthomonas axonopodis pv. dieffenbachiae (Xad) on the EPPO A2 quarantine list. Recently, Xad strains were shown not to belong to X. axonopodis but to the species X. citri, X. phaseoli and X. euvesicatoria. Here, to verify the pathovar designation, 11 representative strains were tested for pathogenicity on six aroid genera. They had overlapping host ranges and only the strain isolated from Syngonium showed host specificity. The X. citri strains, isolated from various hosts, showed dissimilarity in virulence to the tested aroid genera. The X. phaseoli strains, isolated from Anthurium and Syngonium, were generally more virulent and, additionally, induced systemic infections. The X. euvesicatoria strains, isolated from Philodendron, were scored as not pathogenic on the tested aroids. Four representative strains were genome sequenced and showed a variable virulence‐associated gene content. Pathogenicity to aroids was correlated with the presence of three specific T3 effector genes and with a T6SS gene sequence. Together, the phylogenetic and pathogenic differentiation among Xad strains justifies the installation of three pathovar epithets for the pathogens on aroids: X. phaseoli pv. dieffenbachiae comb. nov. for the strains isolated from Anthurium; X. phaseoli pv. syngonii comb. nov. for the strain isolated from Syngonium; and X. citri pv. aracearum comb. nov. for the strains isolated from Aglaonema, Xanthosoma and Dieffenbachia. It is proposed that phytosanitary regulations for xanthomonads on aroids are restricted to these three pathovars.     

Molecular characterization,comparison of screening methods,and evaluation of cross‐pathogenicity of black rot (Xanthomonas campestris pv. campestris) strains from cabbage,choy sum,leafy mustard and pak choi from Taiwan

Choy sum (Brassica rapa var. parachinensis), leafy mustard (Brassica juncea) and pak choi (B. rapa var. chinensis) are highly nutritious components of diets in Taiwan and other Asian countries, and bacterial black rot caused by Xanthomonas campestris pv. campestris (Xcc) is a major biotic constraint in these crops. As very little was known about the Xcc strains from these crops in these regions, including their cross‐pathogenicity and aggressiveness on different hosts, Xcc strains were obtained from cabbage (Brassica oleracea var. capitata), choy sum, leafy mustard and pak choi crops in Taiwan. Two previously published PCR‐based assays reliably distinguished the Xcc strains from other Xanthomonas species and subspecies. Phylogenetic analysis based on repetitive sequence‐based PCR assays placed the Xcc strains in a clade distinct from other Xanthomonas species, and also showed host specificity. Although all of the Xcc strains from the different host species were pathogenic on all five Brassica test species in both a detached leaf assay and an intact plant assay, in the intact plant assay they showed differences in virulence or aggression on the different test hosts. The Xcc strains from leafy mustard and pak choi were consistently highly aggressive on all the test host genotypes, but the strains from choy sum and cabbage were less aggressive on leafy mustard and choy sum. The intact plant assay proved more discriminating and reliable than the detached leaf assay for comparing the aggressiveness of Xcc strains on different host genotypes, and so, with the new Xcc strains isolated in this study, will be useful for screening leafy brassica germplasm accessions for resistance to black rot.     

Epiphytic Curtobacterium flaccumfaciens strains isolated from symptomless solanaceous vegetables are pathogenic on leguminous but not on solanaceous plants

In Iran, during 2013–16, 16 Gram‐positive corynebacteria‐like strains were recovered from the epiphytic parts of solanaceous vegetables including eggplant, pepper and tomato. The strains were recovered accidentally as a result of monitoring for other bacterial pathogens in solanaceous fields. The strains were phenotypically different from Clavibacter michiganensis strains. Although none of the strains were pathogenic on their host of isolation or on any other solanaceous plants, 12 out of 16 strains were pathogenic on common bean, cowpea, mung bean and soybean. Colonization by strains was observed on maize, zucchini, faba bean, honeydew melon, rapeseed, sugar beet and sunflower plants under greenhouse conditions. In PCR tests, the primer pair CffFOR2/CffREV4, specific for Curtobacterium flaccumfaciens pv. flaccumfaciens, enabled the amplification of the appropriately sized fragment in 12 out of 16 strains, and all 12 strains were pathogenic on dry beans. Phylogenetic analysis, using the gyrB and recA genes, showed all 16 bacterial strains clustered within several pathovars of C. flaccumfaciens. A nonpathogenic yellow‐pigmented strain (Xeu15) was clustered with the type strains of C. flaccumfaciens pv. betae and C. flaccumfaciens pv. oortii. Bacteriocin profiling assays revealed no significant differences among the pathogenic and nonpathogenic strains. Host range and population dynamics of four representative strains on 17 plant species showed population build‐up of the strains only on common bean, cowpea, wheat and red nightshade plants. The results provide important insights into the possible role of nonhost plants as reservoirs of plant pathogenic bacteria, which has important implications in plant disease epidemiology and management.     

Resistance and systemic dispersal of Xanthomonas fragariae in strawberry germplasm (Fragaria L.)

The angular leaf spot disease caused by Xanthomonas fragariae is an important plant disease with major impact for the strawberry nursery industry. Currently there is no plant protection product available for controlling the disease effectively. Planting of resistant cultivars seems to be promising, but all commercially used cultivars are susceptible and no donor with a high level of resistance has yet been found. Therefore, a total of 145 genotypes from the Fruit Genebank Dresden (Germany) were evaluated for resistance to X. fragariae by artificial inoculation. Six genotypes were classified as partly resistant, out of which only two (US4808 and US4809) are octoploid. Fragaria vesca f. alba, Fragaria nilgerrensis ‘Yunnan’, F. vesca ‘Illa Martin’ and F. moschata ‘Bauwens’ were also classified as partially resistant, but they are only of limited use for breeding because of their variable ploidy level. Fully resistant genotypes could not be detected. The systemic dispersal of the bacteria in strawberry plants was investigated after inoculation of leaves with X. fragariae strain XF3.9.C and the GFP‐tagged strain XF3.9.C_(pKAN). The systemic spread was evaluated after 3, 7, 14 and 28 days post‐inoculation (dpi) by nested PCR and fluorescence microscopy. After 3 dpi, X. fragariae could be found in all tissues tested including the inoculated leaf, its petiole, the rhizome, the heart bud up to the youngest fully expanded leaf and its petiole. The systemic spread was also detectable in partially resistant genotypes.     

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.     

Comparison of extraction procedures for detection of Xanthomonas axonopodis pv. phaseoli in common bean seed

Xanthomonas axonopodis pv. phaseoli (Xap) is an important seedborne pathogen of Phaseolus vulgaris. Accurate seed health testing methods are critical to protect seed quality and meet phytosanitary requirements. Currently employed selective media‐based methods include several variations in extraction procedures. In order to optimize pathogen extraction from seeds, the influence of different extraction steps on the sensitivity of Xap detection was assessed. Seeds were inoculated by vacuum infiltration with Xap to achieve inoculum levels from 10¹ to 10⁵ CFU per seed; one contaminated seed was mixed into 1000‐seed subsamples of uncontaminated P. vulgaris seeds. Thirty subsamples of 1000 seeds were tested using each different extraction procedure. These included soaking whole seeds in sterilized saline phosphate buffer, either overnight at 4°C or for 3 h at room temperature, with or without vacuum extraction, and either with or without concentrating the seed extract by centrifuging. Seed extract dilutions were cultured on semiselective agar media MT and XCP1. The percentages of positive subsamples were compared to measure the effects of each extraction step on detection sensitivity. Vacuum extraction and centrifugation of seed extracts increased sensitivity; the highest sensitivity was obtained with the 3 h vacuum extraction followed by centrifugation. These results were confirmed with naturally infested seeds; Xap was detected in 48 of 70 samples using the 3 h vacuum extraction with centrifugation, whereas only 35 of 70 field samples tested positive using overnight soaking, a significant difference. The results suggest that these steps would be valuable modifications to the current method approved by the International Seed Testing Association (ISTA).     

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.     

Development of Xanthomonas fragariae populations and disease progression in strawberry plants after spray‐inoculation of leaves

Xanthomonas fragariae is the causative agent of angular leaf spot disease of strawberry. Greenhouse experiments were conducted using a X. fragariae isolate tagged with a green fluorescent protein (GFP) for detailed population dynamic studies in and on leaves after spray‐inoculation. The GFP‐tagged bacteria were monitored with dilution plating of leaf washings and leaf extracts, and analysis of intact leaves using a non‐invasive monitoring system called PathoScreen, based on laser radiation of fluorescent cells in plant tissues and signal recording with a sensitive camera. PathoScreen was also used to monitor bacteria grown on an agar medium after leaf printing. During the first 3 days after inoculation, bacterial populations washed off leaves rapidly decreased by at least a factor of 1000, after which populations remained stable until 14 days post‐inoculation (dpi), when symptoms first started to appear. Thereafter, populations increased to a level of 10¹² colony‐forming units (CFU) g^?1 of leaf material or higher. Similarly, densities in leaf extracts were low during the first 3 days after inoculation, at a level of 100–1000 CFU g^?1 of leaf tissue. Gradually populations increased to a level of 10⁹–10¹² CFU g^?1 at 28 dpi. Higher densities of epiphytic populations were found on the abaxial side than on the adaxial leaf side during the first 2 weeks after inoculation. After spray‐inoculation of leaves, bacterial populations released from infected plants remained low until symptoms appeared, after which plants became highly infectious, in particular under high humidity.     

First record of the citrus red mite <Emphasis Type="Italic">Panonychus citri</Emphasis> in Cyprus

A new phytophagous mite species, Panonychus citri (McGregor) (Acari: Tetranychidae) (the citrus red mite), was found in Cyprus during spring 2008, infesting citrus plantations. Mites occurred in large numbers, mainly in the canopy of orange, lemon and mandarin trees. The adults and larvae preferred the upper surfaces of mature leaves of the above citrus species, although they occurred on both leaf surfaces. Orange, lemon and mandarin trees were greatly affected by this mite, as its high population densities under hot and dry conditions in the spring caused heavy leaf drop (firing) and twig dieback.     

Consequences of the taxonomic revision of Xanthomonas axonopodis pv. dieffenbachiae and of the analysis of its associated pathogenicity for the recommendation of regulation (EPPO A2 List)

Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of bacterial blight of Araceae (aroids), is a regulated pest in several countries and is included in the EPPO A2 List. Reference strains of Xanthomonas axonopodis pv. dieffenbachiae have recently been reclassified into the species Xanthomonas phaseoli, Xanthomonas citri and Xanthomonas euvesicatoria on the basis of different features, including multilocus sequence analysis, average nucleotide identity and homology in DNA–DNA hybridization analyses. Based on pathogenicity tests, Constantin et al. (2017) proposed naming the pathogens on aroids as X. phaseoli pv. dieffenbachiae, X. phaseoli pv. syngonii and X. citri pv. aracearum. Recommendations are made on how to deal with these changes for the group of pathogenic bacteria for Araceae. The name Xanthomonas axonopodis pv. dieffenbachiae on the EPPO List should be adjusted to the names proposed in the taxonomic study by Constantin et al. (2016). The current EPPO Diagnostic Standard is directed at strains pathogenic on Anthurium. They mainly belong to X. phaseoli pv. dieffenbachiae, but some also to X. citri pv. aracearum that are not detected by the EPPO Diagnostic Standard. Xanthomonas phaseoli pv. syngonii strains are also aggressive, but with a host range restricted to Syngonium. The pathogenicity specific to aroids of the bacterial isolates reclassified as Xanthomonas euvesicatoria was not confirmed and no pathovar epithet has been retained for these strains.