Classification and Identification of Xanthomonas translucens Isolates, Including Those Pathogenic to Ornamental Asparagus

ABSTRACT In order to confirm and refine the current classification scheme of Xanthomonas translucens and to identify novel strains from ornamental asparagus, a collection of field and reference strains was analyzed. Rep-polymerase chain reaction (PCR) genomic fingerprint profiles were generated from 33 isolates pathogenic to asparagus as well as 61 X. trans-lucens reference strains pathogenic to cereals and grasses. Amplified ribo-somal gene restriction analysis profiles were obtained from most of these and 29 additional Xanthomonas reference strains. Rep-PCR genomic fingerprint profiles of all strains were compared with those in a large Xanthomonas database using computer-assisted analysis. Rep-PCR ge-nomic fingerprinting facilitated the characterization and discrimination of X. translucens, including the pathovars arrhenatheri, graminis, phlei, phleipratensis, and poae, as well as a number of strains received as X. translucens pv. cerealis. Strains received as pathovars hordei, secalis, translucens, undulosa, and other cerealis strains were grouped in two subclusters that correspond to the recently redefined pathovars X. trans-lucens pvs. undulosa and translucens. All 33 novel isolates from ornamental asparagus (tree fern; Asparagus virgatus) were identified as X. translucens pv. undulosa. Moreover, a unique amplified small subunit ribosomal gene MspI/AluI restriction profile specific for all X. translucens strains tested, including those pathogenic to asparagus, allowed discrimination from all other Xanthomonas spp. Although phage tests were inconclusive, the classification of the asparagus strains within the X. translucens complex was supported by pathogenicity assays in which all the isolates from ornamental asparagus induced watersoaking on wheat. Surprisingly, several X. translucens reference strains affected asparagus tree fern as well. That the novel asparagus isolates belong to X. translucens pv. undulosa is extraordinary because all hosts of X. translucens pathovars described to date belong only to the families Gramineae and Poaceae, whereas asparagus belongs to the phylogenetically distant family Liliaceae.     

Xanthomonas translucens from Small Grains: Diversity and Phytopathological Relevance

ABSTRACT Sixty-eight presumptive Xanthomonas translucens strains isolated from 15 small grains or grass species were studied by pathogenicity tests on barley, bread wheat, oat, and bromegrass species, and also by AFLP, analysis of fatty acid methyl esters (FAME), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein extracts. The X. translucens strains were divided into three pathogenicity types based on differences observed on barley and bread wheat. Two unspeciated strains producing atypical symptoms formed a fourth pathogenicity type. Pathogenicity on oat and bromegrass species varied within these types. Clusterings observed by AFLP analysis and, to a lesser extent, by FAME analysis were consistent with these pathogenicity groupings. The current results, as well as those of previous restriction fragment length polymorphism analyses of the same strains, support the recent reclassification of X. translucens pv. translucens and X. translucens pv. hordei as true synonyms. X. translucens pv. cerealis, X. translucens pv. translucens, and X. translucens pv. undulosa cluster in different groups by AFLP and FAME analyses. Even though distinction by simple biochemical tests is not clear-cut, the data indicate that the pathovars cerealis, translucens, and undulosa correspond to true biological entities.     

Pathogenic and genetic diversity of Xanthomonas translucens pv. undulosa in North Dakota

Bacterial leaf streak (BLS), caused by Xanthomonas translucens pv. undulosa, has become more prevalent recently in North Dakota and neighboring states. From five locations in North Dakota, 226 strains of X. translucens pv. undulosa were collected and evaluated for pathogenicity and then selected strains were inoculated on a set of 12 wheat cultivars and other cereal hosts. The genetic diversity of all strains was determined using repetitive sequence-based polymerase chain reaction (rep-PCR) and insertion sequence-based (IS)-PCR. Bacterial strains were pathogenic on wheat and barley but symptom severity was greatest on wheat. Strains varied greatly in aggressiveness, and wheat cultivars also showed differential responses to several strains. The 16S ribosomal DNA sequences of the strains were identical, and distinct from those of the other Xanthomonas pathovars. Combined rep-PCR and IS-PCR data produced 213 haplotypes. Similar haplotypes were detected in more than one location. Although diversity was greatest (≈92%) among individuals within a location, statistically significant (P ≤ 0.001 or 0.05) genetic differentiation among locations was estimated, indicating geographic differentiation between pathogen populations. The results of this study provide information on the pathogen diversity in North Dakota, which will be useful to better identify and characterize resistant germplasm.     

Testing for Pseudomonas syringae pv. atrofaciens and Xanthomonas campestris pathovars on cereals in Italy

During recent years, recurrent attacks of basal glume rot/leaf blight attributed to Pseudomonas syringae pv. atrofaciens have been observed on cultivars of durum wheat grown in the Po valley (IT). The main aim of this study was to test for this pathogen and Xanthomonas campestris pathovars in commercial seed lots of cereals produced in different regions of Italy, as well as in symptomatic plants collected in the field. None of the analyses led to the detection of xanthomonads. In testing for P.s. atrofaciens , representative bacterial isolates were selected and characterized; this was done by combining conventional identification tests and computerized densitometric analysis of electrophoretic patterns of cell proteins obtained using the SDS-PAGE technique. P.s. atrofaciens was detected in seed samples (barley, wheat and durum wheat) and symptomatic plants (durum wheat) grown in northern and central Italy. The possible pathogenic role of other unrelated fluorescent pseudomonads in this interaction was also examined. Difficulties in discriminating pvs syringae and atrofaciens of P. syringae are discussed and the uncertain taxonomic relationship between these two pathovars is emphasized.     

Sensitivity of different methods for the detection of Ralstonia solanacearum in potato tuber extracts

The sensitivities of various methods for the detection of Ralstonia solanacearum following dilution in healthy potato tuber tissue macerate were compared. Estimated pathogen populations in undiluted macerates, from samples of 200 heel-end vascular cores each containing a single diseased and 199 healthy tubers, ranged from 1.2 × 10⁶–7.4 × 10⁷ colony-forming units per ml. Following concentration by high-speed centrifugation and resuspension in phosphate buffer, the pathogen was detected by all methods studied, including culture on semi-selective media, enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent-antibody staining (IFAS) of fixed cells, immunofluorescent colony staining (IFCS), detection of specific DNA sequences following amplification by the polymerase chain reaction (PCR) and bioassay in tomato seedlings. Both ELISA and PCR methods were improved by pre-enrichment of samples in semi-selective broth prior to testing. A nested PCR method was evaluated which could detect fewer than 10 cells per ml in the potato extracts. Of the other methods only dilution plating on semi-selective medium and tomato bioassay could detect fewer than 10⁴ cells per ml. In order to combine ease and speed of use with sensitive detection, it was recommended that a series of methods be used for routine screening of potato tuber stocks for infection by R. solanacearum.     

Detection and identification of seed-borne pathogenic bacteria of imported tomato seeds in Egypt

Imported tomato seed lots of different cultivars were assayed for the presence of seed-borne bacterial pathogens. The liquid assay method was used for detection of the bacteria, and seed extracts were plated on different semi-selective media. Pseudomonas corrugata and Xanthomonas campestris pv. vesicatoria were detected in 14.7% and 12% of the seed samples tested respectively. These pathogens were identified by means of biochemical, physiological and pathogenicity tests as well as the Biolog GN Microplate System for X. campestris pv. vesicatoria. Both P. corrugata and X. campestris pv. vesicatoria were more easily identified on Tween B and CKTM media than on other media. This is the first report of the occurrence of these important pathogens on tomato seeds in Egypt.     

Evaluation of proposed amended names of several pseudomonads and xanthomonads and recommendations

ABSTRACT In 1980, over 90% of all plant-pathogenic pseudomonads and xanthomonads were lumped into Pseudomonas syringae and Xanthomonas campestris, respectively, as pathovars. The term "pathovar" was created to preserve the name of plant pathogens, but has no official standing in nomenclature. Proposals to elevate and rename several pathovars of the genera Pseudomonas and Xanthomonas to the rank of species has caused great confusion in the literature. We believe the following changes have merit and expect to adopt them for publication in a future American Phytopathological Society Laboratory Guide for Identification of Plant Pathogenic Bacteria. Upon review of published data and the Rules of The International Code of Nomenclature of Bacteria, we make the following recommendations. We reject the proposal to change the name of P. syringae pvs. phaseolicola and glycinea to P. savastanoi pvs. phaseolicola and glycinea, respectively, because both pathogens are easily differentiated phenotypically from pv. savastanoi and convincing genetic data to support such a change are lacking. We accept the elevation of P. syringae pv. savastanoi to the rank of species. We accept the reinstatement of X. oryzae to the rank of species with the inclusion of X. oryzicola as a pathovar of X. oryzae and we accept the species X. populi. We agree with the elevation of the pvs. cassavae, cucurbitae, hyacinthi, pisi, and translucens to the rank of species but not pvs. melonis, theicola, and vesicatoria type B. We recommend that all type A X. vesicatoria be retained as X. campestris pv. vesicatoria and all type B X. vesicatoria be named X. exitiosa. We reject the newly proposed epithets arboricola, bromi, codiaei (poinsettiicola type B), hortorum, sacchari, and vasicola and the transfer of many pathovars of X. campestris to X. axonopodis. The proposed pathovars of X. axonopodis should be retained as pathovars of X. campestris.     

Grouping of Xanthomonas campestris pathovars of cereals and grasses by fatty acid profiling1

Fatty acid profiles were prepared for a range of strains representing all 14 Xanthomonas campestris pathovars from the Gramineae. Profiles were complex, containing up to 40 acids, most of which were iso- and anteiso-branched acids. Grouping of profile types generally correlated with pathovar, although pvs translucens, hordei, cerealis, secalis and undulosa could not be differentiated. Pvs graminis, arrhenatheri, poae and phlei formed another profile type although there were some differences between the pathovars. These graminis and translucens groups had similar profiles but could usually be differentiated by the ratios of 15:0 iso/16:0 iso and 15:0 iso/15:0 anteiso fatty acid methyl esters. The other pathovars each had a very different profile type. Pvs oryzae and oryzicola had profiles which were very different from all others. Pv. vasculorum comprised at least two distinct profile types. This did not correlate with host species or geographic distribution. Fatty acid profiling can be used to identify strains of X. campestris pathovars from Gramineae.     

PCR-based detection of Xanthomonas campestris pathovars in Brassica seed

Xanthomonas campestris is a seedborne bacterium that causes black rot of crucifers. Substantial crop losses may result from the rapid spread of the bacteria under favourable conditions, especially those occurring during seedling production. A PCR-based method has been developed for the rapid and sensitive detection of the pathovars of X. campestris that affect crucifers. Primers were designed to specifically amplify a 619 bp fragment of the hrpF gene from X. campestris . Amplification products were not detected from other Xanthomonas species, or from other pathogenic or epiphytic bacteria occurring on these plants. To avoid false-negative results arising from the presence of amplification inhibitors in plant extracts, primers targeting a 360 bp section of the internal transcribed spacer (ITS) region from Brassica spp. were included in a multiplex PCR. The assay readily detected X. campestris pv. campestris infections in diseased plants and from bacterial colonies isolated on growth media, and was more sensitive and specific than traditional plating methods and a commercially available ELISA. A seed-washing protocol was optimized to allow the detection of a single artificially infected seed among 10 000 healthy seeds using the multiplex PCR.     

Differentiation of three species of Xanthomonas and Stenotrophomonas maltophilia using cellular fatty acid analyses

Five hundred eighty-eight strains, representing Xanthomonas albilineans, X. fragariae, ten pathovars of X. campestris, and Stenotrophomonas maltophilia from ornamentals, were subjected to fatty acid methyl ester (FAME) analyses. Quantitative variance among FAME profiles enabled identification of the four species with 100% accuracy. Dendrogram cluster analysis placed strains of X. albilineans remotely from those of the other two Xanthomonas species and S. maltophilia. Whereas some profiles of pathovars of X. campestris were distinct, strains within X. albilineans, X. fragariae, and S. maltophilia were homogeneous by their conserved FAME ratios. Pathovars of X. campestris that had conserved profiles were fittonia, hederae, malvacearum, pelargonii, and zinniae. FAME profiles of X. campestris pathovars begoniae, dieffenbachiae, fici, maculifoliigardeniae, and poinsettiicola were, however, quantitatively diverse. These pathovars did not form discrete subgroups, and intercalated randomly with one another on the dendrogram. Certain species or pathovars of X. campestris which have homogeneous FAME profiles can easily be identified with fatty acid analysis; however, pathovars of X. campestris with heterogeneous profiles are not readily identified by fatty acid analysis.     

Bacterial leaf streak or black chaff of cereals

The literature related to bacterial leaf streak of cereals, due to various pathovars of Xanthomonas campestris , is confusing and dispersed. This review presents a synthesis of the knowledge that has been accumulated on the disease in cereals since the early reports on barley and wheat. The aim is to present the current status on geographical distribution of the disease, to explain possible confusions in symptoms, and to show the limited quantitative information available on losses and on host/pathogen interactions. Disease etiology and means of control are also reviewed. The objective is to define the present situation of black chaff in wheat, barley and triticale, and to delineate the major research needs in these crops. Difficulties in controlling bacterial leaf streak arise as a result of limited epidemiological knowledge and of the absence of satisfactory seed treatment permitting the eradication of the bacterium. There is an urgent need for a seed certification scheme and for more information on genetic resistance, with a view to limiting outbreaks of this sporadic but devastating disease.     

Bacterial leaf streak of Gramineae in Iran

Bacterial leaf streaks of wheat and barley in Kerman province (Iran) were found to be distinct in etiology. The barley strains were limited in host range and could only infect barley. The wheat strains were pathogenic to barley, wheat, rye, triticale, Bromus spp., Agropyron spp., Elymus junceus, Lolium multiflorum , and Phalaris tuberosa , but not Dactylis glomerata, Cynodon dactylon, Phleum pratense , and oat. Based on host reactions and electrophoretic patterns of cell proteins, the barley and wheat strains were identified as Xanthomonas campestris pv. hordei and pv. cerealis , respectively.     

Protein electrophoresis and DNA:DNA hybridizations of xanthomonads from grasses and cereals1

More than 120 Xanthomonas campestris strains pathogenic for grasses and cereals were compared by polyacrylamide gel electrophoresis (SDS-PAGE) of their whole-cell proteins. Genotypic relationships between representative strains of the electrophoretic groups were determined by DNA:DNA hybridizations. Two major groups of bacteria were delineated. The first included X. campestris pv. graminis, pv. arrhenatheri and some isolates from Bromus, which could be differentiated from each other by their protein fingerprints, and also the following pathovars which it was impossible to differentiate by SDS-PAGE: cerealis, hordei, poae, secalis, translucens and undulosa. DNA:DNA hybridizations indicated that significant degrees of DNA-binding (>60% D) exist between all these pathovars. In the second group, strains of X. campestris pv. holcicola, pv. vasculorum and pv. oryzae were related at 40–45% DNA-binding, while strains of pv. oryzae and pv. oryzicola were genotypically highly related (85% D). All the pathovars of this second group could be differentiated from each other by their protein electrophoretic fingerprints.     

Biocontrol of seedborne Botrytis cinerea in chickpea with Gliocladium roseum

An isolate of Gliocladium roseum proved highly antagonistic to Botrytis cinerea . Sporulation of B. cinerea on chickpea seed naturally infected or inoculated with B. cinerea was suppressed by seed treatment with conidial suspensions of G. roseum at 10⁷ and 10⁸ conidia/mL, respectively. Establishment of healthy seedlings in punnets (small trays) 5 weeks after sowing with inoculated seed was increased from 29.2% to 59.7% by treatment with G. roseum at 3×10⁷ conidia/mL, and from 1.4% to 69.4% with G. roseum at 3×10⁸ conidia/mL, the latter being equivalent to disease control by Thiram. There was no significant effect of Rhizobium on disease suppression by G. roseum , and treatment with G. roseum at 10⁸ conidia/mL did not reduce nodulation. Amendment with culture filtrates of G. roseum did not affect the growth rate of B. cinerea on potato dextrose agar, indicating that constitutive production of an antibiotic is not involved in biocontrol. A selective medium was developed to enumerate propagules of G. roseum on seed recovered from soil. There was no significant change in the population of G. roseum on seed after incubation for 4 weeks in soil to which the isolate of G. roseum was indigenous.     

A comprehensive species to strain taxonomic framework for xanthomonas

ABSTRACT A comprehensive classification framework was developed that refines the current Xanthomonas classification scheme and provides a detailed assessment of Xanthomonas diversity at the species, subspecies, pathovar, and subpathovar levels. Polymerase chain reaction (PCR) using primers targeting the conserved repetitive sequences BOX, enterobacterial repetitive intergenic consensus (ERIC), and repetitive extragenic palindromic (REP) (rep-PCR) was used to generate genomic fingerprints of 339 Xanthomonas strains comprising 80 pathovars, 20 DNA homology groups, and a Stenotrophomonas maltophilia reference strain. Computer-assisted pattern analysis of the rep-PCR profiles permitted the clustering of strains into distinct groups, which correspond directly to the 20 DNA-DNA homology groups(genospecies) previously identified. Group 9 strains (X. axonopodis) were an exception and did not cluster together into a coherent group but comprised six subgroups. Over 160 strains not previously characterized by DNA-DNA hybridization analysis, or not previously classified, were assigned to specific genospecies based on the classification framework developed. The rep-PCR delineated subspecific groups within X. hortorum, X. arboricola, X. axonopodis, X. oryzae, X. campestris, and X. translucens. Numerous taxonomic issues with regard to the diversity, similarity, redundancy, or misnaming were resolved. This classification framework will enable the rapid identification and classification of new, novel, or unknown Xanthomonas strains that are pathogenic or are otherwise associated with plants.     

Detection of Xanthomonas campestris pv. pelargonii in geranium and greenhouse nutrient solution by serological and PCR techniques

Specificity of a new monoclonal antibody, 2H5, to Xanthomonas campestris pv. pelargonii, causal agent of geranium bacterial blight, was determined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence tests on 14 strains of X. c. pelargonii, 12 strains of other X. campestris pathovars, 3 strains of other Xanthomonas spp., 3 strains of other plant pathogens, and 43 saprophytic bacteria isolated from geranium. X. c. pelargonii was detected in tissue from symptomatic and asymptomatic geraniums sampled from commercial growers, and artificially inoculated plants, by monoclonal antibody-based tests. The intensity of response in ELISA was only moderately correlated (r = 0.56) with symptom severity, while symptom severity was not correlated (r = 0.16) with the number of fluorescing cells in immunofluorescence. The bimodal frequency distribution of ELISA and immunofluorescence results served to validate arbitrarily chosen positive/negative threshold values. Most positive ELISA and immunofluorescence test results were confirmed by the polymerase chain reaction (PCR) using published primers (Manulis et al., 1994. Appl. Environ. Microbiol 60, 4094-4099). In contrast to plant tissue, the bacterium was detected in greenhouse nutrient solution with greater sensitivity by immunofluorescence and PCR than by ELISA. Sensitivity of detection was enhanced 100-fold by concentration of the bacteria by centrifugation.     

Incidence of bacteria inhibitory to Erwinia amylovora from blossoms in New Zealand apple orchards

Populations of total bacteria and Erwinia herbicola were monitored on apple blossom in selected orchards at two different geographical regions in New Zealand (Canterbury and Hawke's Bay). In all four orchards surveyed E. herbicola populations remained negligible (less than 50cfu/blossom) throughout flowering, increasing rapidly at petal drop to reach levels of 1 × 10³ cfu/blossom (Hawke's Bay) to 1 × 10⁵ cfu/blossom (Canterbury). Total bacterial populations increased 100-fold at petal drop in both locations. Pseudomonas populations were predominant in Canterbury throughout all flowering stages and in Hawkes Bay after early flowering. When screened for inhibitory activity to E. amylovora , 26% of all isolates from Canterbury showed some inhibition in vitro , but none of the isolates from Hawke's Bay showed inhibition of E. amylovora in vitro. Two Canterbury isolates with strong in vitro inhibitory activity also inhibited E. amylovora in immature pear fruit. One isolate was identified as E. herbicola and one isolate as Pseudomonas fluorescens.     

芽孢杆菌JPC-2的营养竞争测定及其对小麦根部病害的防治效果

对营养竞争拮抗测定方法进行了改进,对接法将芽孢杆菌JPC-2与小麦纹枯病菌接种于大豆麦麸(SWB)培养基上,JPC-2可在SWB培养基上迅速扩展并完全抑制小麦纹枯病菌的菌丝生长。田间试验表明,芽孢杆菌JPC-2液体菌剂(107cfu/mL,1∶100)处理种子对小麦纹枯病的冬前期防效高于扬花期防效,防治效果为61.9%,高于3%苯醚甲环唑包衣处理(用量为种子质量的0.3%);对小麦根腐病的防治效果可达46.8%,与空白对照差异显著(p<0.05);并使小麦增产73.5%,高于3%苯醚甲环唑种衣剂。芽孢杆菌JPC-2固体菌剂(106cfu/mL,225kg/hm2)沟施于土壤,再播种液体菌剂包衣(107cfu/mL,药种比1∶30)的小麦种子,对小麦纹枯病菌的冬前期防效为68.9%,扬花期防效为59.7%,小麦增产38.9%,与0.2%戊唑醇种衣剂(1∶50)增产效果相当。     

Identification and sensitive endophytic detection of the fire blight pathogen Erwinia amylovora with 23S ribosomal DNA sequences and the polymerase chain reaction

Two short sequences, situated in the bacterial 23S rDNA gene, were used as primers for the PCR detection of Erwinia amylovora bacteria. All 34 E. amylovora strains tested, coming from different geographical and host plant origins and of different virulence, produced a 565-bp PCR fragment. The E. amylovora bacteria could be discriminated from all other phytobacteria with which no PCR product was observed. Only Escherichia coli bacteria were cross-recognized by the production of a weaker PCR band of similar size to E. amylovora . In a fast PCR protocol, where two temperatures were cycled, E. amylovora in pure culture could be detected on gel at concentrations as low as 3 × 10² cfu mL^–1. This corresponds to a detection limit of 1.5 bacteria per PCR. However, reliable PCR detection in woody host plant tissue was only obtained with PVP/PVPP-treated sample extracts. Using E. amylovora -spiked plant extracts and extracts of fruit tree shoots artificially infected with E. amylovora , the PCR detection sensitivity was determined to be 6.6 × 10² cfu mL^–1 of extract. Starting from the plant samples, the PCR detection results were visualized on gels within 5 h.     

枯草芽孢杆菌NJ-18和氟酰胺联合拌种防治小麦纹枯病研究

研究了枯草芽孢杆菌NJ-18菌株的芽孢制剂(109 cfu/g)与20%氟酰胺可湿性粉剂(WP)联合拌种对小麦纹枯病的防治作用。结果表明:氟酰胺抑制小麦纹枯病菌Rhizoctonia cerealis菌丝生长的平均EC50值为(0.34±0.06) μg/mL;NJ-18发酵液(108 cfu/mL)及其滤液稀释1 000倍时对小麦纹枯病菌菌丝生长的抑制率分别为96.38%和93.97%;氟酰胺对NJ-18的菌体生长和芽孢存活无抑制作用,具有很好的相容性;在温室条件下,NJ-18芽孢制剂(109 cfu/g)300 g分别与20%氟酰胺WP 50 g和100 g混合拌种处理100 kg小麦种子,在小麦拔节期对纹枯病的防效分别为47.96%和64.58%,显著高于二者同剂量单用处理的防效;在大田条件下,二者混合拌种处理也能显著提高对小麦纹枯病的田间防效,NJ-18芽孢制剂(109 cfu/g) 300 g与20%氟酰胺WP 200 g混合拌种处理100 kg小麦种子,对拔节期小麦纹枯病的防效高达74.83%,且能显著提高小麦千粒重,同时对小麦生长安全。