Monoclonal Antibodies Specific for Xanthomonas campestris Bacteria Pathogenic on Wheat and other Small Grains, in Comparison with Polyclonal Antisera

4 hybridoma cell lines (named F1-AA9-D9, F1-AB3-B6, F1-BC7-C1 and F2-CA7-F11) secreting monoclonal antibodies to Xanthomonas campestris pv. undulosa were produced by fusing splenocytes from immunized Lou rats with IR983F myeloma cells. Whole cells were used both as immunogen and as antigen in ELISA and indirect immunofluorescence tests.
The monoclonal antibodies produced reacted positively with X. c. pv. undulosa (38 strains), pv. translucens (3), pv. hordei (3), pv. cerealis (2) and pv. secalis (1).
Strains from other pathovars ( X. c. pv. arrhenatheri, pv. graminis, pv. manihotis, pv. oryzicola, pv. poae and pv. pruni ) and from other species ( X axonopodis, X. ampelina ) and genus ( Pseudomonas, Erwinia, Clavibacter , wheat saprophytic strains) gave a negative reaction. In comparison, seven polyclonal rabbit antisera showed to be less specific: they reacted with unrelated X. campestris pathovars as well as with Pseudomonas strains. Nevertheless, the use of phenol-treated cells in Ouchterlony double immunodiffusion could reduce the effect of cross-reaction for antisera.
The detection of X. c. pv. undulosa by indirect immunofluorescence on infected wheat seed lots has already been applied with success.     

Phylogenetic Analysis of Phaeosphaeria Species Using Mating Type Genes and Distribution of Mating Types in Iran

Phaeosphaeria species are pathogenic on wheat, barley and a wide range of wild grasses. To analyze mating type loci of the Phaeosphaeria species and investigate mating type distribution in Iran, we sequenced mating type loci of 273 Phaeosphaeria isolates including 67 isolates obtained from symptomatic leaves and ears of wheat, barley, and wild grasses from two wheat-growing region in Iran as well as 206 isolates from our collection from other regions in Iran which were isolated in our previous studies. Mating type genes phylogeny was successfully used to determine the species identity and relationships among isolates within the Phaeosphaeria spp. complex. In this study, we reported seven new host records for Phaeosphaeria species and the Phaeosphaeria avenaria f. sp. tritici 3 group was first reported from Iran in this study. Mating type distribution among Phaeosphaeria species was determined. Both mating types were present in all sampling regions from Iran. We observed skewed distribution of mating types in one region (Kohgiluyeh va Boyer-Ahmad) and equal distribution in the other region (Bushehr). However, when considering our entire dataset of 273 Iranian Phaeosphaeria isolates, the ratio of mating types was not deviated significantly from 1:1 suggesting possibilities for isolates of opposite mating type to interact and reproduce sexually, although the sexual cycle may infrequently occur in some regions especially when the climatic conditions are unfavorable for teleomorph development.     

Genetic Diversity among Xanthomonas campestris Strains Pathogenic for Small Grains

A collection of 51 Xanthomonas campestris strains from throughout the world was studied to detect and assess genetic diversity among pathogens of small grains. Isolates from barley, bread wheat, bromegrass, canary grass, cassava, maize, orchard grass, rice, rough-stalked meadow grass, rye, timothy, and triticale were analyzed by pathogenicity tests on bread wheat cv. Alondra and barley cv. Corona, indirect immunofluorescence, and restriction fragment length polymorphism (RFLP). Three probes were used for the RFLP analysis. They were an acetylaminofluorene-labelled 16S+23S rRNA probe from Escherichia coli and two (sup32)P-labelled restriction fragments from either plasmidic (pBSF2) or chromosomal (pBS8) DNA of X. campestris pv. manihotis. Strains clustered in 9 and 20 groups with the rRNA probe and the pBSF2 DNA probe, respectively. Strains of X. campestris pv. graminis, X. campestris pv. phleipratensis, and X. campestris pv. poae are shown to be related but are also distinguishable by RFLP patterns, serology, and pathogenicity on bread wheat. Strains pathogenic only for barley and not for wheat grouped together. Another group is temporarily designated deviant X. campestris pv. undulosa. These South American isolates from bread wheat did not react by indirect immunofluorescence and produced atypical lesions in pathogenicity tests. The results stress the need to perform pathogenicity tests before strains are named at the pathovar level. The importance of the different probes used for epidemiological studies or phylogenetic studies of closely related strains is underlined.     

Differentiation between Xanthomonas campestris pv. oryzae, Xanthomonas campestris pv. oryzicola and the bacterial 'brown blotch' pathogen on rice by numerical analysis of phenotypic features and protein gel electrophoregrams

Thirty-five Xanthomonas campestris pv. oryzae, fourteen X. campestris pv. oryzicola strains and six 'brown blotch' pathogens of rice, all of different geographical origin, were studied by numerical analysis of 133 phenotype features and gel electrophoregrams of soluble proteins, %G + C determinations and DNA:rRNA hybridizations. The following conclusions were drawn. (i) The Xanthomonas campestris pathovars oryzae and oryzicola display clearly distinct protein patterns on polyacrylamide gels and can be differentiated from each other by four phenotype tests. (ii) Both pathovars are indeed members of Xanthomonas which belongs to a separate rRNA branch of the second rRNA superfamily together with the rRNA branches of Pseudomonas fluorescens, Marinomonas, Azotobacter, Azomonas and Frateuria. (iii) 'Brown blotch' strains are considerably different from X. campestris pv. oryzae and oryzicola. They are not members of the genus Xanthomonas, but are more related to the generically misnamed. Flavobacterium capsulatum, Pseudomonas paucimobilis, Flavobacterium devorans and 'Pseudomonas azotocolligans' belonging in the fourth rRNA superfamily. (iv) No correlation was found between the virulence, pathogenic groups or geographical distribution of X. campestris pv. oryzae or oryzicola strains and any phenotypic or protein electrophoretic property or clustering.     

Differential expression of conserved protease genes in crucifer-attacking pathovars of Xanthomonas campestris.

Strains of Xanthomonas campestris pathovars armoraciae and raphani, which cause leaf spotting diseases in brassicas, produce a major extracellular protease in liquid culture which was partially purified. The protease (PRT 3) was a zinc-requiring metalloenzyme and was readily distinguishable from the two previously characterized proteases (PRT 1 and PRT 2) of X. campestris pv. campestris by the pattern of degradation of beta-casein and sensitivity to inhibitors. PRT 3 was produced at a low level in the vascular brassica pathogen X. campestris pv. campestris (five strains tested), in which PRT 1 and PRT 2 predominate. In contrast, expression of PRT 1, a serine protease, could not be detected in the six tested strains of the leaf spotting mesophyll pathogens. However, all these strains had DNA fragments which hybridized to a prtA probe and which probably carry a functional prtA (the structural gene for PRT 1). The structural gene for PRT 3 (prtC) was cloned by screening a genomic library of X. campestris pv. raphani in a protease-deficient X. campestris pv. campestris strain. Subcloning and Tn5 mutagenesis located the structural gene to 1.2 kb of DNA. DNA fragments which hybridized to the structural gene were found in all strains of the crucifer-attacking X. campestris pathovars tested as well as in a number of other pathovars. Experiments in which the pattern of protease production of the pathovars was manipulated by introduction of cloned genes into heterologous pathovars suggested that no determinative relationship exists between the pattern of protease gene expression and the (vascular or mesophyllic) mode of pathogenesis.     

Pathogenicity, Non-Host Hypersensitivity and Host Defence Non-Permissibility Regulatory Gene hrpX is Highly Conserved in Xanthomonas Pathovars

Xanthomonas campestris pv. campestris and X. oryzae pv, oryzae contain the 1428 base pair hrpX gene whose product is involved in the regulation oi hrp genes required for pathogericity, non-host hypersensitivity and non-permissibility of compatible host defence responses. Previous Southern blot hybridization studies have suggested that hrpX is conserved in several X. campestris pathovars and X. oryzae. strains. We have confirmed and extended these findings using hrpX gene amplification by polymerase chain reaction, coupled with Southern blot hybridization analyses. Sixteen distinct pathovars of X. campestris and 12 strains of X. oryzae pv, oryzae were shown to contain homologs of hrpX which were not apparent in heterologous bacteria such as Agrobacterium tumefaciens, A. rhizogenes, Erwinia carolovora ssp. carotovora, Pseudomonas syringae pv, glycinea. P. syringae pv, labaci , and Escherichia coli. The hrpX gene is therefore highly conserved among Xanthomonas species and its gene product strongly resembles positive regulatory proteins of the AraC protein family,     

Characterization of Xanthomonas campestris pv. pruni strains from different hosts by pathogenicity tests and analysis of whole-cell fatty acids and whole-cell proteins

Strains of Xanthomonas campestris pv. pruni obtained from Prunus armeniaca. P. domestica, P. persica and P. salicina in different geographical areas were compared for pathogenicity, fatty acid and wholecell protein analysis. Four strains, one per each host plant, were inoculated at the same time, on the foliage of P. armeniaca, P. avium, P. persica and P. salicina cultivars . Mean content of fatty acids of X.c. pv. pruni strains were also compared with those of many strains of X.c. pv. campestris , pv. graminis , pv. hyacinthii , pv. pelargonii and pv. vasculorum. Strains showed a remarkable homogeneity in fatty acids content and whole-cell protein profiles and principal component and cluster analysis did not reveal any grouping according to original host or geographical origin. However, X.c . pv. pruni strains can be grouped apart from the other X. campestris pathovars. There appears to be no pathogenic specialization among the strains tested, however, they varied in aggressiveness to host plants and host plant in susceptibility. The most of the strains were able to cross-infect species other that from where they were originally isolated, although, P. avium did not show any symptom of disease. P. persica cv. Sentry and P. salicina cv. Globe Sun, recently licensed as resistant to X.c. pv. pruni. were infected, although to a lesser extent, by some strains.     

Comparison of the nucleotide sequences of wheat dwarf virus (WDV) isolates from Hungary and Ukraine

Wheat dwarf virus (WDV) is the most ubiquitous virus in cereals causing huge losses in both Hungary and Ukraine. The presence of barley-and wheat-adapted strains has been confirmed, suggesting that the barley strain is restricted to barley, while the wheat strain is present in both wheat and barley plants. Five WDV isolates from wheat plants sampled in Hungary and Ukraine were sequenced and compared with known WDV isolates from GenBank. Four WDV isolates belonged to the wheat strain. Our results indicate that WDV-Odessa is an isolate of special interest since it has originated from wheat, but belongs to the barley-adapted strain, providing novel data on WDV biology and raising issues of pathogen epidemiology.     

Differentiation of Xanthomonas campestris pvs. pelargonii and hederae by Polymerase Chain Reaction

Geranium isolates of Xanthomonas campestris pv. pelargonii ( Xcp ) and English ivy isolates of X. campestris pv. hederae ( Xch ) were tested by polymerase chain reaction (PCR) to determine whether the two pathovars could be discriminated using amplification conditions developed to identify and detect Xcp in infected geraniums. Using PCR, other workers reported that the genomes of Xcp and Xch were indistinguishable. The objective of this study was to determine whether the two pathovars have sufficient sequence diversity to allow them to be distinguished by molecular means. Three primer pairs were used for PCR amplification. Two of the primer pairs (REP and XcpM1/XcpM2) were able to distinguish between Xch and Xcp , whereas amplification with the third primer pair (ERIC) did not allow discrimination between the two pathovars. Based on PCR amplification, Xcp and Xch are distinctly different pathovars. Additionally, all three primer pairs showed discrimination between Xcp and Acidovorax , a bacterial pathogen that induces leaf spot on geranium.     

Phenotypic Switching Affecting Chemotaxis, Xanthan Production, and Virulence in Xanthomonas campestris

The chemotaxis towards sucrose and yeast extract of nine strains of Xanthomonas campestris representing pathovars campestris, armoraciae, translucens, vesicatoria, and pelargonii was analyzed by using swarm plates. Unexpectedly, each of these strains formed small or reduced swarms typical of nonmotile or nonchemotactic bacteria. With time, however, chemotactic cells appeared on the swarm plates as blebs of bacteria. These cells were strongly chemotactic and were concomitantly deficient in exopolysaccharide production. The switch from the wild type (exopolysaccharide producing and nonchemotactic) to the swarmer type (exopolysaccharide deficient and chemotactic) appeared irreversible ex planta in bacteriological medium. However, in radish leaves swarmer-type strains of X. campestris pv. campestris were able to revert to the wild type. Swarmer-type derivatives of two X. campestris pv. campestris wild-type isolates showed reduced virulence and growth in the host plants cauliflower and radish. However, exocellular complementation of X. campestris pv. campestris Hrp (nonpathogenic) mutant was achieved by coinoculation with a swarmer-type strain.     

Structure of the O polysaccharides and serological classification of Pseudomonas syringae pv. porri from genomospecies 4.

Strains of Pseudomonas syringae pv. porri are characterized by a number of pathovar-specific phenotypic and genomic characters and constitute a highly homogeneous group. Using monoclonal antibodies, they all were classified in a novel P. syringae serogroup O9. The O polysaccharides (OPS) isolated from the lipopolysaccharides (LPS) of P. syringae pv. porri NCPPB 3365 and NCPPB 3364T possess multiple oligosaccharide O repeats, some of which are linear and composed of l-rhamnose (l-Rha), whereas the major O repeats are branched with l-rhamnose in the main chain and GlcNAc in side chains (structures 1 and 2). Both branched O repeats, which differ in the position of substitution of one of the Rha residues and in the site of attachment of GlcNAc, were found in the two strains studied, O repeat 1 being major in strain NCPPB 3365 and 2 in strain NCPPB 3364T. [formula: see text]. The relationship between OPS chemotype and serotype on one hand and the genomic characters of P. syringae pv. porri and other pathovars delineated in genomospecies 4 on the other hand is discussed.     

Proteins in intercellular washing fluid from noninoculated and rust-affected leaves of wheat and barley

Proteins in intercellular washing fluid (IWF) from wheat (Triticum aestivum) and barley (Hordeum vulgare) leaves were separated by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue (CBB) or silver. Intracellular protein from the cut ends of leaves accounted for only a small proportion of total protein in IWF from wheat leaves. When these were heavily infected with the stem rust fungus (Puccinia graminis f. sp. tritici) and grown at 19°C, four infection-related CBB-stainable proteins were detected in IWF.

To compare IWF proteins from wheat and barley leaves infected with the same pathogen, conditions were established that permitted luxuriant growth of stem rust of wheat in barley (exposure to chloroform before inoculation and maintenance at 25°C thereafter). Under these conditions, at least 10 infection-related silver-stainable proteins were detected in IWF from infected wheat in addition to the more than 50 that were of host origin. The electrophoretic properties of 8 of the infection-related proteins were the same as those of 8 infection-related proteins in IWF from barley.

IWF from wheat and barley grown under these conditions was analyzed for Concanavalin A-binding glycoproteins immobilized on nitrocellulose membrane replicas made from gels. Of the many infection-related glycoproteins that were detected in IWF from stem rust-affected wheat, approximately 20 occupied the same positions as those from stem rust-affected barley. The glycoprotein pattern of IWF prepared from wheat leaves grown at 19°C and infected with the leaf rust fungus (P. recondita f. sp. tritici) was markedly different to that of IWF from the same host infected with the stem rust fungus. We conclude that IWF from rust-affected cereal leaves may be a useful source of surface or extracellular proteins from the parasitic mycelium.

     

Differentiation between Xanthomonas campestris pv. graminis (ISPP List 1980), pv. phleipratensis (ISPP List 1980) emend., pv. poae Egli and Schmidt 1982 and pv. arrhenatheri Egli and Schmidt 1982, by Numerical Analysis of Phenotypic Features and Protein Gel Electrophoregrams

A numerical analysis of 257 phenotypic features of 45 bacterial isolates from grasses, revealed three phenons corresponding to (i) X. campestris pv. graminis (ISPP List 1980), (ii) X. campestris pv. phleipratensis (ISPP List 1980) and (iii) X. campestris pv. poae Egli and Schmidt 1982 and X. campestris pv. arrhenatheri Egli and Schmidt 1982. In each phenon, the strains clustered together regardless of the geographical origin of the isolates orthe year of isolation. Polyacrylamide gel electrophoresis of soluble proteins and host range studies, revealed four groups corresponding to the pathovars mentioned above. The four pathovars constitute definite biological entities that can be differentiated by phenotypic, gel electrophoretic and host range features.     

Deoxyribonucleic acid relatedness of 21 strains of Xanthomonas species and pathovars

The relationship of 17 Xanthomonas campestris pathotype strains, three additional X. campestris strains, and the type strain of Xanthomonas albilineans were examined by DNA-DNA hybridization tests. The results coupled with those of a previous study (Hildebrand et al. 1990) support the hypothesis that X. campestris does not constitute a single bacterial species. There were low levels of DNA-DNA reassociation among many of the different pathovars examined. Six clusters of related pathovars were discerned. In addition, four of the pathovars were only distantly related to each other and to the six clusters. Xanthomonas albilineans was not closely related to any of the other xanthomonads tested.
Mapping and superimposing the botanical families of the host plants upon a three-dimensional genomic distance matrix of the xanthomonads confirms previous observations that pathovars that infect plants of the same botanical family do not necessarily belong to the same genomic group. Six legume-infecting pathovars cluster within one genomic group, but one pathovar, X. cam. pv. pisi is only distantly related to this group. There was also no genomic relationship between X. cam. pv. oryzicola and X. albilineans both of which infect Gramineae. Consequently, pathogenicity toward members of the same plant family is not a good indicator of the genomic relationships among xanthomonads nor is it a good taxonomic determinant.     

Outer Membrane Proteins and Lipopolysaccharides in Pathovars of Xanthomonas campestris

Variations in the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of 54 isolates belonging to 16 different pathovars of Xanthomonas campestris were characterized. OMP samples prepared by sarcosyl extraction of cell walls and LPS samples prepared by proteinase K treatment of sonicated cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 4 M urea. In general, the OMP and LPS profiles within each pathovar were very similar but different from the profiles of other pathovars. Heterogeneity in OMP and LPS profiles was observed within X. campestris pv. campestris, X. campestris pv. translucens, and X. campestris pv. vesicatoria. LPSs were isolated from six X. campestris pathovars, which fell into two major groups on the basis of O antigenicity. The O antigens of X. campestris pv. begoniae, X. campestris pv. graminis, and X. campestris pv. translucens cross-reacted with each other; the other group consisted of X. campestris pv. campestris, X. campestris pv. pelargonii, and X. campestris pv. vesicatoria. A chemical analysis revealed a significant difference between the compositions of the neutral sugars of the LPSs of those two groups; the LPSs of the first group contained xylose and a 6-deoxy-3-O-methyl hexose, whereas the LPSs of the other group lacked both sugars.     

Isozyme Variation and Genetic Diversity of the European Barley Powdery Mildew Population

Isozyme and virulence analyses of Erysiphe graminis bordei were performed with samples collected from different sites from nearly all over Europe. Isozymes and unspecific proteins extracted from conidia were separated by starch gel electrophoresis and isoelectric focusing, respectively, and the resulting isozyme banding patterns were compared with the corresponding virulence data. One isozyme phenotype prevailed in all samples. Only 7.9% of 280 isolates showed divergent banding patterns. Expected frequencies of isolates with divergent banding patterns were calculated for each subsample. In the Italian subsample, isolates with divergent banding patterns were significantly more frequent than expected. At the same time, isolates from Italy had significantly fewer virulence factors than those from N.W. Europe, indicating weaker selection by host resistance genes. It is suggested that isozyme uniformity in the homogeneous north-western European barley powdery mildew population has arisen from strong selection pressures for specific virulence genes. The direction of this selection, acting upon a mainly asexually reproducing population, has changed over space and time due to the introduction of new resistance genes, forcing local populations through bottlenecks. This may have led to random loss of genetic variation (genetic drift) in the barley powdery mildew gene pool.     

Infection of cereals and grasses by isolates of Polymyxa graminis (Plasmodiophorales)

The host range of isolates of Polymyxa was tested in mono-fungal sand cultures. Fourteen isolates of P. graminis, obtained from barley, wheat, oats or Poa annua and from several different countries, all infected barley and all but one infected wheat. Rye was also a good host, whereas oats (nine cultivars), Lolium multiflorum and Poa pratensis became only slightly infected. Wheat cultivars differed in susceptibility, with Galahad much more resistant than Avalon. Several common weed and pasture grasses were not infected by the two isolates tested. A range of wild Hordeum spp. were mostly susceptible to P. graminis and/or barley mild mosaic virus, which it transmits. An isolate of P. betae, used for comparison, caused slight infection on oats but not on other cereals. The variation within and between species of Polymyxa needs more detailed investigation.     

Characteristics of a PCR-Based Assay for In Planta Detection of Xanthomonas campestris pv. pelargonii

Polymerase chain reaction (PCR) amplification was carried out with a primer pair targeting a sequence in the genome of Xanthomonas campestris pv. pelargonii , the causative agent of bacterial blight in geraniums. PCR amplification with the primer pair XcpMl/XcpM2 using total nucleic acid preparations from 22 geographicallydiverse isolates of X. campestris pv. pelargonii generated a major 197 bp DNA product. In contrast, no major amplification products were consistently generated from 12 other pathovars of X. campestris or from 19 isolates representing 10 different plant pathogenic bacteria, including two other bacterial pathogens of geraniums, Corynebacterium fascians and Pseudomonas cichorii . After PCR using this primer pair, between 1380 and 13800 copies of the X, campestris pv. pelargonii bacterial DNA target as template were detected by ethidium bromide staining of agarose gels, and between 13.8 and 138 copies by blot hybridization to a pathovar-specific biotinylated probe. Similarly, between 630 and 6300 colonyforming units (CFU) of X. campestris pv. pelargonii could be detected after ethidium bromide staining of agarose gels, and between 63 and 630 CFU after blot hybridization. The PCR-based assay was used to identify X. campestris pv. pelargonii in diseased geraniums; whereas discrete amplification products were not obtained with healthy plants.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates.

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.