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.     

Characterization of a unique copper resistance gene cluster in <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> isolated in Trinidad,West Indies

Whole genome sequencing of a copper resistant (Cu^R) black rot strain of Xanthomonas campestris pv. campestris (Xcc) isolated from a broccoli plant in Trinidad revealed a unique operon for copper resistance. The cop genes of strain Xcc-BrA1 were determined to be present on a 160 to 180 kb plasmid shown to be non-conjugative with other xanthomonads. While nucleotide comparison of a putative 8.0 Kbp copLABMGF gene cluster identified in Xcc-BrA1 genome did not reveal any homologous region with other known Cu^R Xanthomonas strains from diverse origins, the comparison of the translated amino acid sequence indicated similarity with X. citri, X. c. pv. citrumelonis and X. vesicatoria Cop proteins. Cloning of the copLAB gene cluster from Xcc-BrA1 conferred copper resistance to other copper-sensitive xanthomonads. Although Xcc-BrA1 harbors copLAB genes with similar sizes and organization and is able to grow on Cu-amended medium as other Cu^R xanthomonads, the phylogenetic analysis of nucleotide sequences indicates that the cop cluster in Xcc-BrA1 is unique and distantly related to other copLAB genes from Xanthomonas and Stenotrophomonas. The origin of copper resistance genes in Xcc-BrA1 is likely a result of horizontal gene acquisition from a still unknown phylloplane cohabitant. The findings of this study have implications for the management of crop diseases caused by Cu^R xanthomonads. Future studies could focus on and determining the distribution, overall importance and appropriate control measures for strains harbouring these unique genes.     

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.     

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.     

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.     

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.     

HrpG regulates type II secretory proteins in Xanthomonas axonopodis pv. citri

HrpG, a two-component response regulator-like protein, is a key regulator of the type III secretion system (T3SS) in Xanthomonas spp. In X. campestris pv. vesicatoria, HrpG with a single amino acid substitution (HrpG*) gains the ability to induce the expression of T3SS-related genes even under nutrient-rich conditions. In this study, we investigated the role of HrpG in the synthesis of the secretory protein using HrpG* in strain NA-1 of X. axonopodis pv. citri (Xac NA-1), a causal agent of citrus canker. Eleven proteins secreted via a type II secretion system (T2SS) were induced by HrpG*. In proteomic analyses, six of the 11 proteins were identified as extracellular enzymes, and the others as a fimbrial biogenesis-related protein, a type IV-related protein, two hypothetical proteins, and a conserved hypothetical protein. Further analysis of these proteins revealed that the genes coding all 11 proteins were upregulated by HrpG*, even though they had different expression patterns for HrpXct-dependency. The data indicated that HrpG, a key regulator of T3SS, also acts as a positive regulator of certain proteins secreted via a T2SS in Xac NA-1.     

Narrow host range phages associated with citrus canker lesions in Florida and Argentina

Bacteriophages were isolated from naturally infected citrus canker lesions from diverse locations in Florida and Argentina and characterized for host range using a world-wide collection of Xanthomonas citri subsp. citri (Xcc) strains. Sixty-seven bacteriophages isolated from citrus canker lesions in Florida (37 bacteriophages) and Argentina (30 bacteriophages) revealed little diversity. All 30 phages isolated from four locations in Argentina had identical host ranges (group ARG), while 37 phages from Florida made up two groups (FLA and FLB). ARG and the 31 FLA phages produced clear plaques and had nearly identical host ranges as phage CP2 of Japan in that they only reacted with typical A strains and none of the atypical A strains (A^* and A^W) or other Xanthomonas spp. FLB phages had a different host range from the other strains and produced turbid plaques. We used phage typing, fatty acid analysis and riboprinter analysis to classify citrus-associated xanthomonads. Phage typing using 12 phages isolated from Xcc, X. fuscans subsp. aurantifolii (Xfa), X. alfalfae subsp. citrumelonis (Xacm), and other sources proved useful for classifying all major Xcc pathotypes and/or strains (A, A*, Miami (MI), Manatee (MA) and Wellington (A^W)), as well as B and C types of Xfa. X. citri subsp. citri strains from a worldwide collection were diverse in phage susceptibility. The majority of Xcc strains, which originated from different regions of the world and which were typical “A” pathotype strains based on pathogenicity characteristics, was sensitive to most phages (including CP2, FLA and ARG), and had nearly identical phage sensitivity profiles. MA strains were quite unique in that they reacted with none of the phages; furthermore, they were different from the putative progenitor MA strain, ATCC 49118, which reacted with a group of phages. Fatty acid analysis revealed considerable variation in Xcc-A, Xfa-B, Xfa-C and Xacm strains. Using riboprinter analysis, we identified a unique riboprinter pattern for strains isolated from an etrog tree (Citrus medica) in Florida that were “A” pathotype strains based on pathogenicity characteristics. Phage typing and fatty acid analysis were useful in corroborating that the etrog strains represent a unique new Xcc strain in Florida.     

柑桔溃疡病菌PCR快速检验检疫技术研究

 柑桔溃疡病是严重影响全世界柑桔生产的重大检疫性病害,根据柑桔溃疡病菌(Xanthomonas axonopodis pv. citri)新近公布的全基因组中独有的保守蛋白基因序列,设计筛选出一对种特异性引物(JYF5/JYR5),能专一地扩增检出柑桔组织表面所带溃疡病菌的DNA靶带(413 bp)。而柑桔叶面附生的非致病性黄单胞菌、野油菜黄单胞菌近缘种以及健康柑桔样品都不能扩增;靶细菌DNA检测下限1.56 pg/μL,靶细菌悬浮液检测下限10 cfu/μL;在不同PCR仪及各种控温方式下都能稳定地扩增出特征性靶带。这一特异、准确的柑桔溃疡病菌PCR检验技术和研制的预包被固相化PCR检测试剂盒已开始用于我国非疫生产区建设中柑桔苗木、果实的病害检疫检验。     

Improved PCR for identification of members of the genus Xanthomonas

A PCR-based system was developed to reliably and robustly identify group I and II members of the genus Xanthomonas. Primer sets developed from three gene targets namely fyuA, ITS and gumD were evaluated in the study. Primer sets were evaluated using DNA extracted from 45 Xanthomonas strains representing 25 species broadly covering the genus. Fifteen non-Xanthomonas strains of plant-associated bacteria including phylogenetically closely related species Stenotrophomonas maltophilia and Xylella fastidiosa were also tested. The primers targeting fyuA amplified DNA from all xanthomonads except X. theicola, while the ITS primers amplified a DNA fragment of 254 bp in all 45 Xanthomonas strains; whereas no amplification was observed for non-xanthomonads. The gumD primers allowed efficient amplification of DNA in 38 out of 39 isolates from Group II, whereas no or very weak amplification occurred with DNA from Group I members. Internal controls of primers targeting bacterial 16S rDNA or plant 26S mitochondrial rDNA were successfully applied in multiplex PCRs for testing bacterial cultures or plant tissue, respectively. The findings give us a PCR based approach that can reliably and effectively differentiate xanthomonads from non-xanthomonads as well as separating the strains belonging to the two described groups of the genus Xanthomonas. The study thus offers valuable tools for disease surveillance and management. It can effectively be applied in rapid assessment of new disease occurrences, for which no specific detection tools could be in place.     

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.     

Unstable green fluorescent protein for study of Xanthomonas citri subsp. citri survival on citrus

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.     

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.     

Epiphytic growth of Xanthomonas arboricola and Xanthomonas citri on non‐host plants

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.     

Short‐distance dispersal of splashed bacteria of Xanthomonas citri subsp. citri from canker‐infected grapefruit tree canopies in turbulent wind

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).     

A PCR-based assay for differentiating A- and A*-type strains of Xanthomonas citri subsp. citri, the causal agent of Asiatic citrus canker

By applying A- and A*-type strains of Xanthomonas citri subsp. citri (Xcc) in a repetitive sequence-based polymerase chain reaction (rep-PCR), two DNA amplicons, one unique to each strain, were evaluated as a probe against the DNA of Xcc strains. Two pairs of primers derived from these amplicons were tested in a PCR analysis. The results confirmed that primers Ms⁺/Ms^? are useful for differentiating A-type from A*-type strains of Xcc. Also, a multiplex PCR with both set of primers can be used to distinguish three groups in Xcc populations: A-type strains and two subgroups of A* strains including Iranian and Thai A* strains.     

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     

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.