Pathotype Strain Research Articles

No evidence of the Shiga toxin-producing E. coli O104:H4 outbreak strain or enteroaggregative E. coli (EAEC) found in cattle faeces in northern Germany, the hotspot of the 2011 HUS outbreak area

BackgroundRuminants, in particular bovines, are the primary reservoir of Shiga toxin-producing E. coli (STEC), but whole genome analyses of the current German ESBL-producing O104:H4 outbreak strain of sequence type (ST) 678 showed this strain to be highly similar to enteroaggregative E. coli (EAEC). Strains of the EAEC pathotype are basically adapted to the human host. To clarify whether in contrast to this paradigm, the O104:H4 outbreak strain and/or EAEC may also be able to colonize ruminants, we screened a total of 2.000 colonies from faecal samples of 100 cattle from 34 different farms - all located in the HUS outbreak region of Northern Germany - for genes associated with the O104:H4 HUS outbreak strain (stx2, terD, rfbO104, fliCH4), STEC (stx1, stx2, escV), EAEC (pAA, aggR, astA), and ESBL-production (blaCTX-M, blaTEM, blaSHV).ResultsThe faecal samples contained neither the HUS outbreak strain nor any EAEC. As the current outbreak strain belongs to ST678 and displays an en-teroaggregative and ESBL-producing phenotype, we additionally screened selected strains for ST678 as well as the aggregative adhesion pattern in HEp-2 cells. However, we were unable to find any strains belonging to ST678 or showing an aggregative adhesion pattern. A high percentage of animals (28%) shed STEC, corroborating previous knowl-edge and thereby proving the validity of our study. One of the STEC also harboured the LEE pathogenicity island. In addition, eleven animals shed ESBL-producing E. coli.ConclusionsWhile we are aware of the limitations of our survey, our data support the theory, that, in contrast to other Shiga-toxin producing E. coli, cattle are not the reservoir for the O104:H4 outbreak strain or other EAEC, but that the outbreak strain seems to be adapted to humans or might have yet another reservoir, raising new questions about the epidemiology of STEC O104:H4.

First Report of Pseudomonas syringae pv. actinidiae, the Causal Agent of Bacterial Canker of Kiwifruit in France.

In June 2010, young plants of kiwifruit growing in the French regions of Rhone-Alpes (Actinidia deliciosa 'Summer') and Aquitaine (A. chinensis 'Jintao') showed small, angular, necrotic leaf spots and cankers on some canes that was sometimes associated with production of a red exudate. Most of the affected canes died, and in a few cases after a few months, the entire plant died. Symptoms were consistent with Pseudomonas syringae pv. actinidiae, a bacterium that affects several species of Actinidia (A. deliciosa and A. chinensis, the two most important commercial species of kiwifruit). A recent outbreak of this disease is devastating the Italian kiwifruit industry. Bacterial colonies were isolated on King's medium B (KB) from leaf spots and infected canes. Three isolates from Aquitaine and 10 from Rhone-Alpes were retained for further characterization. The 13 isolates were gram negative, induced a hypersensitive reaction when infiltrated in tobacco plants, did not have a cytochrome c oxidase, an arginine dehydrolase or urease activity, did not hydrolyze esculin, starch, or gelatine, and did not induce ice nucleation. When plated on KB, these strains did not show strong fluorescence usually associated with strains of P. syringae. Complete lack of fluorescence reported for the pathotype strain ICMP 9817 has not been observed for those strains. They showed the same weak fluorescence as the strains of P. syringae pv. actinidiae recently isolated from Italy. Those characteristics match those of strains of P. syringae pv. actinidiae (3). Using total DNA of the 13 strains, the pathotype strain, and primers PsaF1/R2 (2), a 280-bp fragment was amplified by PCR, supporting the strains as being P. syringae pv. actinidiae. The amplicon from 6 of the 13 strains was sequenced and found to be 100% similar to the corresponding DNA fragment of the pathotype strain ICMP 9617 (GenBank AY342165). Partial sequences of 1,381 bp of the 16S rDNA gene of four of the six isolates, three strains isolated from Rhone-Alpes and one strain isolated from Aquitaine, were obtained by amplification with primers 27f and 1492r (1). Except for the sequence of strain 181, which was isolated from Aquitaine and had a 1 bp difference (GenBank JF323026), the other sequences were 100% identical to each other (GenBank JF323027 to JF323029). These four sequences were 99% identical to the 16SrDNA sequences of ICMP 9617, the pathotype strain of P. syringae pv. actinidiae (GenBank AB001431). These four strains and the pathotype strain were sprayed (1 × 109 CFU/ml) on leaves of four 6- to 8-week-old seedlings of A. chinensis each. After 4 days, small, necrotic, angular spots were found on all plants inoculated with those four strains and the pathotype strain. No symptoms were found on plants treated with water only. From those leaf spots, bacteria that had all the characteristics of P. syringae pv. actinidiae (as previously described) were isolated. Recently, two different haplotypes for the housekeeping gene cts were described for P. syringae pv. actinidiae (4), the strains isolated from France belong to the haplotype I. This is the same haplotype to which strains isolated from the recent Italian outbreak also belong. To our knowledge, this is the first report of bacterial canker of kiwifruit in France.

First Report in Burkina Faso of Xanthomonas citri pv. mangiferaeindicae Causing Bacterial Canker on Mangifera indica.

Bacterial canker of mango (or bacterial black spot) caused by Xanthomonas citri pv. mangiferaeindicae, is an economically important disease in tropical and subtropical areas (1). X. citri pv. mangiferaeindicae can cause severe infection on a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Fruit symptoms are black, star shaped, erumpent, and exude an infectious gum. A survey was conducted in Burkina Faso in May 2010 because budwood putatively associated with an outbreak of bacterial canker in Ghana had originated from Burkina Faso (3). Leaves and twigs with suspected bacterial canker lesions were collected from mango trees of the cvs. Amélie, Brooks, and Kent and from seedlings at five localities in Comoe and Houet provinces. Severe infections were observed on the sampled trees in Burkina Faso and leaf symptoms were typical of bacterial canker. Leaves were surface sterilized for 15 to 30 s with 70% ethanol, and nonpigmented, Xanthomonas-like bacterial colonies were isolated on KC semiselective agar medium (1). On the basis of an IS1595-ligation mediated PCR assay, 18 strains from Burkina Faso produced identical fingerprints and were identified as X. citri pv. mangiferaeindicae (4). The haplotype for strains from Burkina Faso was identical to that reported from Ghana (3). Three strains from Burkina Faso (LH127-2, LH130-1, and LH131-1) were compared by multilocus sequence analysis (MLSA) with the type strain of X. citri and the pathotype strain of several X. citri pathovars, including pvs. anacardii and mangiferaeindicae, targeting the atpD, dnaK, efp, and gyrB genes (2). Nucleotide sequences were 100% identical to those of the pathotype strain of X. citri pv. mangiferaeindicae, regardless of the gene assayed, but differed from any other X. citri pathovar assayed. Leaves of mango cv. Maison Rouge, taken from the youngest vegetative flush, were infiltrated (10 inoculation sites per leaf for three replicate leaves on different plants per bacterial strain) with the same three strains from Burkina Faso. Bacterial suspensions (approximately 1 × 105 CFU/ml) were prepared in 10 mM Tris buffer (pH 7.2) from 16-h-old solid cultures on YPG agar (7 g of yeast, 7 g of peptone, 7 g of glucose, and 18 g of agar per liter, pH 7.2). The negative control treatment consisted of three leaves infiltrated with sterile Tris buffer (10 sites per leaf). Plants were incubated in a growth chamber at 30 ± 1°C by day and 26 ± 1°C by night (12-h/12-h day/night cycle) at 80 ± 5% relative humidity. Typical symptoms of bacterial canker were observed for all assayed strains 1 week after inoculation; no symptoms were observed from negative control leaves. One month after inoculation, mean X. citri pv. mangiferaeindicae populations ranging from 2 × 107 to 8 × 107 CFU/leaf lesion were recovered, which was typical of a compatible interaction (1). The origin of inoculum associated with the bacterial canker outbreak in Burkina Faso is unknown. This report documents severe infections in Burkina Faso (including premature fruit drop due to severe fruit infections) and confirms the presence of bacterial canker in western Africa. A more extensive survey for the disease should be conducted in this region.

First Report of Bacterial Blight of Crucifers Caused by Pseudomonas cannabina pv. alisalensis in Australia.

In 1978 and 1979, Pseudomonas syringae pv. maculicola strains DAR 33362, DAR 33363, and DAR 33406 were isolated from diseased Brassica hirta, B. nigra, and B. napus var. napus, respectively, in Wagga Wagga and Armatree, NSW, Australia (2). Peters et al. (2) demonstrated that these strains were similar to P. syringae pv. maculicola ICMP 4326 (CFBP 1637), which was recently transferred to Pseudomonas cannabina pv. alisalensis (1). We evaluated these Australian strains to determine if they might also be P. cannabina pv. alisalensis. Amplification of DNA using the BOXA1R primer and PCR resulted in identical DNA fragment banding patterns for Australian strains DAR 33362 and DAR 33363 and P. cannabina pv. alisalensis ICMP 4326 and CFBP 6875. The third Australian strain, DAR 33406, was 90% similar to P. cannabina pv. alisalensis; in contrast, it was only 77% similar to P. syringae pv. maculicola. All strains of P. cannabina pv. alisalensis, including the pathotype strain (CFBP 6866) and all three Australian strains, were lysed by bacteriophage PBS1, which is specific for P. cannabina pv. alisalensis strains (1). To complete Koch's postulates, pathogenicity was evaluated on B. hirta, B. nigra, and B. napus var. napus. In two independent experiments, two plants of each species were inoculated with each Australian strain or a phosphate buffer control treatment. In separate experiments, pathogenicity was evaluated on the differential hosts radish (Raphanus sativus cv. Comet) and broccoli raab (Brassica rapa cv. Sorrento), and plants inoculated with the pathotypes of P. cannabina pv. alisalensis and P. syringae pv. maculicola served as additional control treatments. Inoculum was prepared by growing the bacteria on nutrient agar for 48 h (27°C), suspending the bacteria in 0.01 M phosphate buffer (pH 7.0), and adjusting each suspension to 0.6 OD at 600 nm (approximately 108 CFU/ml). Treatments were applied by spraying until runoff. DAR 33362, DAR 33363, and DAR 33406 caused typical bacterial blight symptoms on B. hirta, B. nigra, and B. napus var. napus. Infected leaves became yellow, followed by the development of small (<2 mm in diameter), angular, water-soaked, and eventually, shot-holed spots. Bacteria isolated from symptomatic tissue following surface disinfestation of tissue with sodium hypochlorite (0.525%) had identical characteristics (rep-PCR DNA fragment banding patterns and phage sensitivity) to the strains used to inoculate the plants. Additionally, DAR 33362, DAR 33363, and DAR 33406, as well as P. cannabina pv. alisalensis, caused symptoms on radish and broccoli raab while P. syringae pv. maculicola and the buffer control did not. These data support the transfer of the Australian crucifer strains, originally identified as P. syringae pv. maculicola, to P. cannabina pv. alisalensis. To our knowledge, this is the first report of a bacterial disease of crucifers caused by P. cannabina pv. alisalensis in Australia. Differentiation of these pathogens will inform crop rotation strategies for disease management.

Multilocus Sequence Typing of Pseudomonas syringae Sensu Lato Confirms Previously Described Genomospecies and Permits Rapid Identification of P. syringae pv. coriandricola and P. syringae pv. apii Causing Bacterial Leaf Spot on Parsley

Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.

First Report in Ghana of Xanthomonas citri pv. mangiferaeindicae Causing Mango Bacterial Canker on Mangifera indica.

Bacterial canker of mango (or bacterial black spot), caused by Xanthomonas citri pv. mangiferaeindicae, is an economically important disease in tropical and subtropical producing areas (1). X. citri pv. mangiferaeindicae can cause severe infection in a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Several months after infection, leaf lesions dry and turn light brown or ash gray. Severe leaf infection may result in abscission. Fruit symptoms appear as small water-soaked spots on the lenticels. These spots later become star shaped, erumpent, and exude an infectious gum. Often, a "tear stain" infection pattern is observed on the fruit. Severe fruit infections will cause premature fruit drop. Twig cankers are potential sources of inoculum and weaken resistance of branches to wind damage. Leaf lesions with suspected bacterial canker were collected in January 2010 from mango trees cv. Keitt in several blocks at the Integrated Tamale Fruit Company, Ghana. Non-pigmented Xanthomonas-like bacterial colonies were isolated on Kasugamycin-Cephalexin semiselective agar medium (3). On the basis of IS1595-Ligation Mediated-PCR data, 16 strains from Ghana produced identical fingerprints and were identified as X. citri pv. mangiferaeindicae (4). The haplotype corresponding to the Ghanaian strains had not been previously reported. On the basis of multidimensional scaling (4), this haplotype clustered together with a group of strains from multiple origins and the analysis was not informative as an aid for tracing back the outbreak. Five Ghanaian strains (LH2-3, LH2-6, LH2-8, LH2-11, and LH2-15) were compared by multilocus sequence analysis to the type strain of X. citri and the pathotype strain of several X. citri pathovars, including pvs. anacardii and mangiferaeindicae. This assay targeted the atpD, dnaK, efp, and gyrB genes as described previously (2). Nucleotide sequences were 100% identical to those of the pathotype strain of X. citri pv. mangiferaeindicae whatever the gene assayed, but differed from any other assayed X. citri pathovar. Mango cv. Maison Rouge leaves from the youngest vegetative flush were infiltrated (10 inoculation sites per leaf, three replicate plants) using inoculum of each of the same five Ghanaian strains made from suspensions in Tris buffer containing ~1 × 105 CFU/ml. Negative control treatments consisted of leaves infiltrated with sterile Tris buffer. Typical symptoms of bacterial canker were observed for all assayed strains a week after inoculation. No lesions were recorded from the negative control. One month after inoculation, mean X. citri pv. mangiferaeindicae population sizes ranging from 4 × 107 to 1 × 108 CFU/lesion were recovered from leaf lesions, typical of a compatible interaction (1). High disease prevalence was observed in Ghana, indicating the suitability of environmental conditions in this region for the development of mango bacterial canker. The budwood for these blocks was imported from Burkina Faso in 2002 and symptoms were observed in these blocks shortly after establishment. To our knowledge, this is the first report of mango bacterial canker in Western Africa.

The presence of double-stranded RNAs in Alternaria alternata Japanese pear pathotype is associated with morphological changes

Strains of the Japanese pear pathotype of Alternaria alternata were screened for double-stranded RNAs (dsRNAs). Four strains had several dsRNAs; strain N18 was associated with several dsRNAs and had impaired growth phenotypes such as irregular mycelium and abnormal pigmentation. We isolated dsRNA-cured isolates from strain N18 by single-conidium isolation. The dsRNA-cured isolates had recovered normal growth and pigmentation. Enlarged vesicles were observed in mycelial cells of the original dsRNA-carrying N18 strain. DAPI nuclear staining revealed regression of the nuclei in dsRNA-carrying N18 cells. These results indicate that the dsRNAs might have negative effects, such as apoptosis-like cell death, on the host fungus.

Genetic Diversity and Pathogenicity of Xanthomonas axonopodis Strains Inducing Citrus Canker Disease in Iran and South Korea

For the first time in 1989 citrus bacterial canker disease has seen on Citrusaurantiifolia in southern Iran. A total of 43 strains from affected citrus trees, ten strains from South Korea and representative from all known five pathotypes of Xanthomonas axonopodis pathogenic on citrus trees were used in this study. Isolated strains from Iran were indistinguishable by phenotypic, FAMEs, and SDS-PAGE analyses but showed different host range. First group were pathogenic on all tested citrus seedlings including C. aurantiifolia, C. limettioides, C. limon, C. jambhiri, Poncirus trifoliata X C. paradisi, C. aurantium, C. paradise, C. medica, P. trifoliate, C. grandis, C. sinensis, C. reticulate and C. sinensis X P. trifoliate. Pathogenicity of the second group were limited to C. aurantiifolia, C. limettioides, C. limon, C. jambhiri, P. trifoliata X C. paradis, and C. aurantium. Among the strains studied by AFLP fingerprinting six clusters were found. These clusters were: (1) strains of pathotype C; (2) strains of pathotypes B and D; (3) strains of pathotype A together with the main group of the Iranian strains; (4) strains isolated from Korea; (5) strains of pathotype E; and (6) seven strains from Iran which made a completely separate cluster. Strains from pathotypes B and D could not be differentiated by AFLP. The tested Iranian strains belongs to the two different groups and strains from Korea grouped as a subcluster from main cluster of Iranian strains belong to the pathotype A.

First Report of Bacterial Blight of Cabbage (Brassica oleracea var. capitata) Caused by Pseudomonas cannabina pv. alisalensis in California.

In 2008, field-grown cabbage (Brassica oleracea var. capitata L., cv. Grenader) from Monterey County, California showed symptoms on the wrapper leaves of immature plants that had formed heads. Initial symptoms consisted of small, brown, water-soaked flecks surrounded by chlorotic haloes. These flecks later coalesced into large, irregularly shaped, brown-black, necrotic lesions with chlorotic haloes visible on both adaxial and abaxial surfaces of the leaf. This disease resulted in lower quality and reduced marketability of the cabbage. Five gram-negative, blue-green fluorescing bacteria were isolated from separate lesions on different plants on King's medium B agar. The isolates were positive for levan formation and negative for oxidase and arginine dihydrolase. The isolates did not cause soft rot on potato slices but did induce a hypersensitive reaction in tobacco (Nicotiana tabacum L. cv. Samsun). These data indicated that the bacteria belonged to Lelliot's LOPAT group 1 (2). Repetitive extragenic palindromic sequence (rep)-PCR using the BOXA1R primer resulted in identical DNA fragment banding patterns for the cabbage isolates and the pathotype of Pseudomonas cannabina pv. alisalensis (formerly P. syringae pv. alisalensis). Additionally, both P. cannabina pv. alisalensis and the five cabbage isolates were sensitive to bacteriophage PBS1 while the pathotype strain of P. syringae pv. maculicola was not (1). Pathogenicity of the five cabbage isolates was evaluated in two independent experiments. Inoculum was prepared by growing the bacteria on nutrient agar for 48 h (27°C), suspending the bacteria in 0.01 M phosphate buffer (pH 7.0), adjusting each suspension to 0.6 OD at 600 nm (approximately 108 CFU/ml), and adding three to five drops of Tween 20. In each experiment, two cabbage, broccoli raab (Brassica rapa subsp. rapa cv. Sorrento), and oat (Avena sativa cv. Montezuma) plants were inoculated for each isolate by spraying until runoff. Positive control plants were inoculated with the pathotype strain of P. cannabina pv. alisalensis and negative control plants were treated with sterile 0.01 M phosphate buffer. The plants were placed in a mist chamber for 48 to 72 h and then in a greenhouse (20 to 25°C). After 7 to 10 days, foliar symptoms similar to symptoms observed on the original diseased cabbage plants developed on all inoculated plants including the positive control plants inoculated with P. cannabina pv. alisalensis. Additionally severe symptoms on broccoli raab and minor symptoms on oats developed on plants inoculated with cabbage strains or P. cannabina pv. alisalensis. For each experiment, bacteria reisolated from symptomatic tissue were identical to the bacteria used to inoculate the plants and to P. cannabina pv. alsialensis for rep-PCR DNA fragment banding pattern and sensitivity to phage PBS1. There were no symptoms on any of the cabbage and oat negative controls. Additionally, there were no symptoms on any broccoli raab negative controls in the first experiment; however, in the second experiment, a small (<1 mm) lesion was detected on one leaf of one plant. To our knowledge, this is the first report of P. cannabina pv. alisalensis causing bacterial blight of cabbage in California. This disease may have significant impact because of the large acreage of cabbage grown in California (approximately 5,666 ha annually).

First Report of Xanthomonas axonopodis pv. poinsettiicola Causing Bacterial Leaf Spot of Euphorbia pulcherrima in Slovenia.

In September 2009, water-soaked spots, 2 mm in diameter, surrounded by a pale yellow halo were observed on leaves of pot-grown poinsettia plants (Euphorbia pulcherrima L.) cv. Christmas Feeling in a commercial greenhouse in Slovenia. Several spots per leaf developed on 10% of 84 plants used for propagation and slowly progressed to necrotic brown spots. While all plants were watered by overhead irrigation until mid-September, and afterward by flooding, no symptoms were observed on parent plants of four other separately grown cultivars. Propagated cuttings were all grown together, and in addition to cv. Christmas Feeling, an estimated 90% of 315, 35% of 29, 10% of 240, and 5% of 840 plants of cvs. Crazy Marble Star, Crazy Christmas, Lemon Snow, and Cortez Red, respectively, developed leaf spots. Yellow, smooth, and butyrous colonies with entire margins were isolated from symptomatic leaves of poinsettia parent plants of cv. Christmas Feeling on yeast peptone glucose agar (YPGA). They were identified as a Xanthomonas sp. based on biochemical tests (oxidase negative, positive for hydrolysis of H2S, starch and tributiryn and acid production from sucrose) and the isolates caused a hypersensitive reaction in leaves of tomato cv. Moneymaker. Partial sequences of gyrase subunit B-like (gyrB) gene (2) from an isolate (Accession No. HQ215596, 676 bp) showed highest similarity to Xanthomonas axonopodis pv. poinsettiicola strain LMG 5401 (Accession No. GU144264.1, 99% identity, 98% coverage) and 98% identity with gyrB sequence of X. axonopodis pv. poinsettiicola pathotype strain LMG 849 (Accession No. GU144273.1, 99% coverage, 3 gaps). Repetitive BOX-PCR (3) revealed high similarity of our isolate to pathotype strain LMG 849 with one additional band of approximate size of 1,500 bp present in our isolate. The pathogenicity of two isolates from parent plants of cv. Christmas Feeling was confirmed on four young poinsettia plants each. Plants were inoculated with a bacterial suspension of approximate concentration of 106 CFU/ml by spraying on the under side and upper side of the leaves, some of which were pricked with a sterile needle (1). Plants were then incubated under high relative air humidity (minimum 85%), 12 h of daylight, and 25°C day and 20°C night temperature regimens. After 10 days, all inoculated plants developed faint leaf spots, consistent with mild symptoms observed in the greenhouse. Colonies isolated from the developed spots had identical morphology and BOX-PCR profile to original isolates. Mock inoculated, negative control plants did not develop characteristic symptoms and no colonies similar to X. axonopodis pv. poinsettiicola were isolated from them. Bacteria isolated from leaf spots of other poinsettia cultivars had the same biochemical characteristics and BOX-PCR profiles as the first isolate. Since no leaf blight was observed on poinsettias in the greenhouse in the previous season and no host plants were kept between the seasons, imported parent plants are the most likely source of infection. To our knowledge, this is the first report of X. axonopodis pv. poinsettiicola on poinsettia in Slovenia, providing further data on the occurrence and potential economic damage of leaf blight of poinsettia in Europe.

COMPREHENSIVE LIST OF NAMES OF PLANT PATHOGENIC BACTERIA, 1980-2007

The names of all plant pathogenic bacteria which have been effectively and validly published in terms of the International Code of Nomenclature of Bacteria and the Standards for Naming Pathovars are listed to provide an authoritative register of names for use by authors, journal editors and others who require access to currently correct nomenclature. Included are species, subspecies and pathovar names and details of type and pathotype strains reported from 1980 to 2007. An explanation of how to use this list is provided. In recent years the taxonomy of plant pathogenic bacteria has been extensively revised. For some taxa there are several valid synonyms. Unless otherwise stated, the most recently published name is used in this list as the reference name (in bold italic) to which all other synonyms are referred. This does not mean that the reference name is always to be preferred. A synonym, i.e., a previously published name for the same organism, may represent a classification considered by individual scientists or groups to give a more coherent taxonomy and may be used. This list is presented by the International Society of Plant Pathology Committee on the Taxonomy of Plant Pathogenic Bacteria, Carolee T. Bull Convener.

New Zealand strains of plant pathogenic bacteria classified by multi‐locus sequence analysis; proposal of Xanthomonas dyei sp. nov.

Strains of Xanthomonas isolated in New Zealand from diseased endemic and introduced plants, investigated in a multi‐locus sequence analysis (MLSA), were distributed primarily in two species. Strains from Dysoxylum, Hardenbergia, Liquidambar, Magnolia and Mahonia were members of Xanthomonas arboricola. Strains of Xanthomonas campestris pv. laureliae, X. campestris pv. eucalypti and strains from Aralia, Diospyros, Dysoxylum, Eriostemon, Eucalyptus, Metrosideros and Olea formed a population distinct from all known Xanthomonas spp. These latter strains have a unique fatty acid methyl ester profile and are considered to be members of a new Xanthomonas species, for which the name Xanthomonas dyei is proposed. Xanthomonas campestris pv. eucalypti and X. campestris pv. laureliae are considered to be pathovars of the new species and their reclassification as X. dyei pv. eucalypti and X. dyei pv. laureliae, respectively, is proposed. Strains previously identified as Pseudomonas syringae pv. dysoxyli from Dysoxylum spectabile, including the pathotype strain, were examined and found not to represent the pathogen originally described as P. dysoxyli. Other strains (proved to be pathogenic) from diseased D. spectabile, corresponding to the original description of P. dysoxyli, are members of X. dyei and the new combination X. dyei pv. dysoxyli is proposed. The geographic and evolutionary origins of X. dyei, apparently endemic to Australasia, and the adaptation of strains to the introduced host plants Aralia, Diospyros, Eriostemon, Metrosideros and Olea, are discussed. The origins of the X. arboricola strains isolated from Dysoxylum, Hardenbergia, Liquidambar, Magnolia and Mahonia pose a conundrum.

First Report of Bacterial Leaf Blight on Broccoli and Cabbage Caused by Pseudomonas syringae pv. alisalensis in South Carolina.

In May of 2009, leaf spot and leaf blight symptoms were observed on broccoli (Brassica oleracea var. italica) and cabbage (B. oleracea var. capitata) on several farms in Lexington County, the major brassica-growing region of South Carolina. Affected areas ranged from scattered disease foci within fields to entire fields. Initial infection symptoms on leaves of both crops included circular and irregular-shaped necrotic lesions that were 3 to 10 mm in diameter, often with yellow halos and water soaking. As the disease progressed, the lesions tended to coalesce into a general blight of the entire leaf. Diseased leaves from both broccoli and cabbage were collected from each of four fields at different locations in the county. Leaves were surface disinfested, macerated in sterile distilled water, then aliquots of the suspension were spread on King's medium B (KB) agar. All samples produced large numbers of bacterial colonies that fluoresced blue under UV light after 24 h of growth. In total, 23 isolates (13 from broccoli and 10 from cabbage) were collected. These isolates were gram negative, levan production positive, oxidase negative, pectolytic activity negative, arginine dihydrolase negative, and produced a hypersensitive response on tobacco, thus placing them in the Pseudomonas syringae LOPAT group (2). Two broccoli and two cabbage isolates were selected at random and tested for pathogenicity to cabbage cv. Early Jersey Wakefield, broccoli cv. Decicco, turnip cv. Topper, broccoli raab cv. Spring, collard cv. Hi-Crop, and oat cv. Montezuma in greenhouse tests. Bacteria were grown on KB agar for 24 h and a bacterial suspension was prepared and adjusted to an optical density of 0.1 at 600 nm. Three-week-old plants were spray inoculated to runoff and held at 100% relative humidity for 12 h after inoculation, prior to return to the greenhouse bench (4). P. syringae pv. maculicola strain F18 (4) and the pathotype strain of P. syringae pv. alisalensis BS91 were included as controls, along with a water-inoculated negative control. Plants were evaluated at 14 days postinoculation. The four unknown bacterial isolates and BS91 were pathogenic on all brassica plants tested, as well as on oat. In contrast, the P. syringae pv. maculicola strain F18 was not pathogenic on broccoli raab or oat. Symptoms produced by all isolates and strains tested were similar to those observed in the field. No symptoms were observed on water-inoculated plants. Comparative repetitive sequence-based (rep)-PCR DNA analysis using the BOXA1R primer (3) resulted in a DNA banding pattern of each of the isolates from the South Carolina fields (23 isolates), as well as those reisolated from inoculated plants, that was identical to P. syringae pv. alisalensis BS91 and differed from the P. syringae pv. maculicola F18 strain. On the basis of the rep-PCR assays and the differential host range (1), the current disease outbreak on broccoli and cabbage in South Carolina is caused by the bacterium P. syringae pv. alisalensis. Broccoli is a relatively new, albeit rapidly expanding, production vegetable in South Carolina; this disease may represent a limiting factor to future production.

First Report of Blossom Blight of Strawberry (Fragaria × ananassa) Caused by Pseudomonas marginalis.

In 2003, a new disease was observed on commercial strawberries (Fragaria × ananassa Duch.) grown in multiple fields in Watsonville, CA. Initial symptoms consisted of brown lesions on the undersides of the sepals of strawberry flowers. The lesions coalesced and spread to upper sepal surfaces and anther bases. No leaf symptoms were observed. Fields affected with this disease appeared to have a greater number of deformed fruit, though incidence data were not collected. A gram-negative, blue-green fluorescent pseudomonad was isolated from lesions on King's medium B agar from both sepals and anthers from 23 of 24 samples from three different fields. All isolates were levan, oxidase, and arginine dihydrolase positive. The strains did rot potato slices but did not induce a hypersensitive reaction in tobacco (Nicotiana tabacum L. cv. Sansun), indicating that the bacteria belonged to Lelliot's LOPAT group IVa, P. marginalis (3). Isolates from strawberry were compared with pathotype strains of Pseudomonas marginalis pv. marginalis, P. marginalis pv. alfalfae, and P. marginalis pv. pastinaceae. The 16S rDNA sequence of type strain of P. marginalis (Z76663) was 97 to 99% similar to the four strawberry isolates sequenced (GQ845121). Identity was further supported by analysis of fatty acid methyl esters (MIS-TSBA version 4.10; MIDI, Inc., Newark, DE). Polymerase chain reaction using BOX-A1R primers (repetitive sequence-based (rep)-PCR [1]) resulted in DNA fragment banding patterns that were identical among strawberry isolates. These banding patterns were different from the three distinct patterns of the P. marginalis pathotypes. Pathogenicity on strawberry (cv. Albion) was confirmed in three experiments using four strawberry isolates originally isolated from plants from three different fields and the P. marginalis pathotype strains. Inoculum was produced by growing bacteria in nutrient broth shake cultures for 48 h (24°C) and washing and suspending the cultures in 0.01 M phosphate buffer (pH 7.0). Three to five attached strawberry flowers on separate plants were dipped in the bacterial suspensions (106 CFU/ml) or sterile buffer for 1 min. To maintain high humidity, flower buds were enclosed in plastic bags for 36 to 48 h and then incubated in the greenhouse (24 to 26°C). After 7 days, approximately half of the flowers inoculated with the strawberry isolates had symptoms on sepals that were identical to symptoms seen in the field. Additionally, reisolates obtained from the symptomatic, inoculated flowers were identical to those used to inoculate the plants as confirmed by LOPAT reactions and rep-PCR, thus completing Koch's postulates. Flowers dipped in phosphate buffer or the P. marginalis pathotype strains did not develop symptoms and no bacteria were reisolated. To our knowledge, this is the first report of blossom blight of strawberry caused by P. marginalis and the first report of P. marginalis on strawberry in California. P. marginalis causes leaf bud rot of strawberry in Japan (2). Further research is needed to determine if the strawberry isolates represent a new or previously described pathovar of P. marginalis.

Horizontal Chromosome Transfer, a Mechanism for the Evolution and Differentiation of a Plant-Pathogenic Fungus

The tomato pathotype of Alternaria alternata produces host-specific AAL toxin and causes Alternaria stem canker on tomato. A polyketide synthetase (PKS) gene, ALT1, which is involved in AAL toxin biosynthesis, resides on a 1.0-Mb conditionally dispensable chromosome (CDC) found only in the pathogenic and AAL toxin-producing strains. Genomic sequences of ALT1 and another PKS gene, both of which reside on the CDC in the tomato pathotype strains, were compared to those of tomato pathotype strains collected worldwide. This revealed that the sequences of both CDC genes were identical among five A. alternata tomato pathotype strains having different geographical origins. On the other hand, the sequences of other genes located on chromosomes other than the CDC are not identical in each strain, indicating that the origin of the CDC might be different from that of other chromosomes in the tomato pathotype. Telomere fingerprinting and restriction fragment length polymorphism analyses of the A. alternata strains also indicated that the CDCs in the tomato pathotype strains were identical, although the genetic backgrounds of the strains differed. A hybrid strain between two different pathotypes was shown to harbor the CDCs derived from both parental strains with an expanded range of pathogenicity, indicating that CDCs can be transmitted from one strain to another and stably maintained in the new genome. We propose a hypothesis whereby the ability to produce AAL toxin and to infect a plant could potentially be distributed among A. alternata strains by horizontal transfer of an entire pathogenicity chromosome. This could provide a possible mechanism by which new pathogens arise in nature.

Factors Influencing The Attenuation of Serotype 1 Marek's Disease Virus by Serial Cell Culture Passage and Evaluation of Attenuated Strains for Protection and Replication

This study was carried out to better understand factors that influenced the process of attenuation of Marek's disease (MD) virus by serial passage in cell cultures. Three virulent (v) pathotype and three very virulent plus (vv+) pathotype strains were passed by three techniques up to 131 times, and the passage level at attenuation was determined. The 18 attenuated or partially attenuated viruses were evaluated for protection against challenge with virulent MD virus, and the virus load (latent infection) in blood lymphocytes at 14-21 days postvaccination was determined. Viral pathotype strongly influenced the rate of attenuation. The mean passage level at attenuation for v and vv+ strains was 74 and >109, respectively. Full attenuation was achieved for nine of nine passage series with v pathotype strains but for only four of nine passage series with vv+ pathotype strains. Time to attenuation was not significantly influenced by multiplicity of infection at passage or by cell type, although a possible advantage of alternate high- and low-multiplicity passage was noted. Protection was not significantly influenced by pathotype or time to attenuation. Protection varied from 50% to 95% for the 18 passaged virus preparations; six attenuated viruses provided high protection that did not differ from that of the prototype Rispens strain. Virus load was not influenced by pathotype or by passage strategy and showed no positive correlation with protection. In several cases the most protective vaccines had the least virus load. This finding differs from previous reports and warrants further study. Variation among different strains within the same pathotype was documented for attenuation rate, protection, and virus load. Also, variation was evident when the same strain was passaged by different strategies, probably reflecting random changes during serial passage. Strain 596A (v pathotype) was the first to become attenuated, provided the best protection, and had one of the lowest virus loads. In contrast, strain 617A (v pathotype) provided the least protection and had one of the highest virus loads. Such strains provide fertile opportunities for further study.

Polyphasic characterization of xanthomonads pathogenic to members of the Anacardiaceae and their relatedness to species of Xanthomonas

We have used amplified fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA) and DNA-DNA hybridization for genotypic classification of Xanthomonas pathovars associated with the plant family Anacardiaceae. AFLP and MLSA results showed congruent phylogenetic relationships of the pathovar mangiferaeindicae (responsible for mango bacterial canker) with strains of Xanthomonas axonopodis subgroup 9.5. This subgroup includes X. axonopodis pv. citri (synonym Xanthomonas citri). Similarly, the pathovar anacardii, which causes cashew bacterial spot in Brazil, was included in X. axonopodis subgroup 9.6 (synonym Xanthomonas fuscans). Based on the thermal stability of DNA reassociation, consistent with the AFLP and MLSA data, the two pathovars share a level of similarity consistent with their being members of the same species. The recent proposal to elevate X. axonopodis pv. citri to species level as X. citri is supported by our data. Therefore, the causal agents of mango bacterial canker and cashew bacterial spot should be classified as pathovars of X. citri, namely X. citri pv. mangiferaeindicae (pathotype strain CFBP 1716) and X. citri pv. anacardii (pathotype strain CFBP 2913), respectively. Xanthomonas fuscans should be considered to be a later heterotypic synonym of Xanthomonas citri.

Demystifying the nomenclature of bacterial plant pathogens

A unified approach to naming bacteria ensures accurate communication among scientists, regulators and the public. Rules for nomenclature, set out in the International Code of Nomenclature of Bacteria (ICNB), ensure that proposals for new names and combinations follow a logical and standardized progression that maintains the integrity of the established nomenclature while facilitating changes based on scientific inquiry into relationships among organisms. However, these Rules only apply to ranks at the level of subspecies and above and not to lower taxonomic ranks. The pathovar is one infraspecific rank that is widely used in the classification and nomenclature of plant pathogenic bacteria and is often included in legislation to provide statutory control of bacterial plant pathogens. Thus, phytobacteriologists must rely on two discontinuous but, complimentary systems: the Rules set forth in ICNB for naming down to subspecies level, and the Standards in the International Standards for Naming Pathovars of Plant Pathogenic Bacteria to name pathovars. A framework for determining the priority of names is provided by the Approved Lists of Bacterial Names, which gives genus, species and subspecies names and their corresponding type strains, and subsequent lists of validly published names appearing in the International Journal of Systematic and Evolutionary Microbiology. For pathovar names priority is based on the date of valid publication of legitimate names. A list of pathovar names and pathotype strains is maintained by the Committee on the Taxonomy of Plant Pathogenic Bacteria of the International Society of Plant Pathology. To help researchers avoid common pitfalls encountered when developing nomenclature for novel classification systems, this manuscript clarifies several key Rules and Standards. It aims to promote best practice, in that names developed to conform to the ICNB should also consider precedents set by previous nomenclatural designations as per the International Standards for Naming Pathovars of Plant Pathogenic Bacteria, thus ensuring continuity across the nomenclature of all phytopathogenic bacteria.

Molecular characterisation of infectious bursal disease viruses isolated in recent acute outbreaks in Solvenia

In 2004 and then in 2006 several outbreaks of infectious bursal disease (IBD) were reported in broiler and broiler breeder flocks in Slovenia. In this report ten recently emerged IBD viruses (IBDV) were characterised by sequence analysis of the VP2 hypervariable region and compared to previous Slovene IBDV strains from 1995/1996 and to some representative serotype 1 IBDV strains of different pathotypes. On the basis of nucleotide and amino acid identities, phylogenetic analyses and the presence of very virulent IBDV (vvIBDV) conserved amino acid substitutions, all Slovene isolates from recent outbreaks were identified as vvIBDV. Although some unique nucleotide exchanges and amino acid substitutions have been observed, the results of this study indicated that recent vvIBDV isolates are closely related with those from outbreaks in the 1990s. However, acute IBD has not been reported in commercial flocks in Slovenia for some years. This could lead to the conclusion that poor biosecurity and relaxed vaccination could be responsible for the re-emergence of vvIBDV.

Multiple copies of AMT2 are prerequisite for the apple pathotype of Alternaria alternata to produce enough AM-toxin for expressing pathogenicity

The apple pathotype of Alternaria alternata produces the cyclic depsipeptide AM-toxin and causes Alternaria blotch of apple. Previously, we cloned AMT2 from the apple pathotype as an orthologue of AFTS1, which is required for biosynthesis of the decatrienoic acid ester AF-toxin I of the strawberry pathotype. These genes were predicted to encode aldo-keto reductases involved in biosynthesis of a common precursor, 2-hydroxy-isovaleric acid, of AF-toxin I and AM-toxin. In this study, we analyzed the function of AMT2 in AM-toxin biosynthesis in the apple pathotype. DNA gel blot analysis of the apple pathotype strain IFO8984 with five restriction enzymes suggested that this strain has a single copy of AMT2 in the genome. However, gene disruption experiments showed that IFO8984 probably has three copies of AMT2. We made mutants having one or two copies of AMT2 disrupted. The single-copy mutants produced less AM-toxin than did the wild type and were still as pathogenic as the wild type. The two-copy mutants produced trace or undetectable amounts of AM-toxin and were markedly reduced in pathogenicity. Thus, AMT2 was verified to be required for AM-toxin biosynthesis and hence pathogenicity. The fact that the two-copy mutants have a remaining copy of AMT2 suggests that multiple copies of AMT2 are prerequisite for the pathogen to produce enough AM-toxin for full pathogenicity.