Growth of Glucose-uptake-deficient Mutant of Xanthomonas oryzae pv. oryzae in Rice Leaves

We isolated Xanthomonas oryzae pv. oryzae mutants deficient in the phosphoenolpyruvate : carbohydrate phos-photransferase system, a major glucose transport system in bacteria, using the glucose analogue 3-deoxy-3-fluoro-d-glucose (3FG). Glucose uptake by the mutants was decreased to 15–35% of the parental strain, and growth greatly decreased in synthetic media containing glucose as a sole sugar source. Growth of the mutants in rice leaves was, however, similar to the wild type. These findings suggest that glucose is not necessarily a major carbohydrate source for X. o. pv. oryzae in rice leaves. Received 11 August 2000/ Accepted in revised form 15 December 2000     

Growth Complementation of hrpXo Mutants of Xanthomonas oryzae pv. oryzae by Virulent Strains in Rice Cultivars Resistant and Susceptible to the Parental Strain

Xanthomonas oryzae pv. oryzae (X. o. pv. oryzae) T7174 is virulent on rice cultivar IR24 and avirulent on IR-BB2. From recent reports, some virulence and avirulence factors of plant pathogenic bacteria are transferred to plant cells through the hrp-dependent type III secretion system. In this study, we investigated the involvement of hrp genes in the compatible and the incompatible interactions between rice and X. o. pv. oryzae after co-inoculation with hrpXo mutants derived from T7174 and virulent strains. Growth of the mutants, named 74ΔHrpXo and 76ΔHrpXo, was repressed in IR24 when the mutants were applied alone. However, growth of the mutants was complemented by co-inoculation with virulent strains. Growth of bioluminescent hrpXo mutant 76ΔHrpXo in IR24 and its growth in IR-BB2 after co-inoculation with T7133, which is virulent on both cultivars, was equally complemented, as detected by bioluminescence from the mutant. On the other hand, only partial complementation of growth of T7174L76, which is a bioluminescent and pathogenic derivative of T7174, by T7133 was observed in IR-BB2. Thus, growth of the hrpXo mutant of X. o. pv. oryzae was complemented by virulent strains in both susceptible and resistant rice leaves with the parental strain. Received 21 July 2000/ Accepted in revised form 26 October 2000     

利用多重PCR诊断水稻白叶枯病和细菌性条斑病的复合发生

为准确检测水稻白叶枯病菌、细菌性条斑病菌及这两种病菌的复合发生,利用软件DNAStar分析比较这两种菌的部分核酸序列,设计了检测这两种病菌的特异性引物。引物Xoo F-Xoo R能特异性扩增出水稻白叶枯病菌中一条大小162 bp的条带;引物Xooc F1-Xooc R1和Xooc F2-Xooc R2能够分别特异性扩增出水稻细菌性条斑病菌中690 bp和945 bp的条带。通过优化PCR反应条件,成功建立了多重PCR技术,可以对不同国家的水稻白叶枯病菌和细菌性条斑病菌进行准确检测,对由这两种病菌引起的复合侵染实现了准确诊断。     

Notes on the Occurrence of Pathogenic Races of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae </Emphasis>Found in Hiroshima Prefecture in 1999

The pathogenic race of 59 cultures of Xanthomonas oryzae pv. oryzae, a pathogen of bacterial leaf blight of rice, isolated from six locations in the inland mountainous area of Hiroshima Prefecture in 1999, were determined by a set of traditional differentials. Four races—I, II, V and VII—were found across the area; however, we noticed the composition of the races as well as the dominant race in each location different. All races were avirulent on differential cultivar Te-tep. Races V and VII were new to Hiroshima. The rice cultivars infected with bacterial leaf blight in Hiroshima are thought to be grouped into the Kinmaze group, which does not have any resistance genes. Apparently, a variety of races occurred unexpectedly on the cultivars contrary to stabilizing selection theory. Received 25 February 2000/ Accepted in revised form 13 July 2000     

Baseline sensitivity of natural populations and resistance of mutants of Xanthomonas oryzae pv. oryzae to a novel bactericide,zinc thiazole

During 2009–2010, a total of 323 isolates of Xanthomonas oryzae pv. oryzae were obtained from rice with symptoms of bacterial leaf blight (BLB) in four provinces (Zhejiang, Jiangsu, Anhui and Hubei) in China. These isolates were tested for baseline sensitivity to zinc thiazole, a novel bactericide with strong antibacterial activity against Xanthomonas. The sampled pathogenic population had similar sensitivity to zinc thiazole (0·1–16·8 mg L^?1) in all four regions and over the whole two‐year study period. The baseline sensitivity was distributed as a unimodal curve with a mean EC₅₀ value of 6·79 ± 1·61 mg L^?1. The risk of mutation to resistance of zinc thiazole in X. oryzae pv. oryzae was further evaluated in vitro and in vivo. Twelve zinc thiazole‐resistant mutants were obtained through ultraviolet (UV) irradiation, culturing on zinc thiazole‐amended nutrient agar (NA) plates, and culturing on zinc thiazole‐treated rice plants. These zinc thiazole‐resistant mutants had resistance factors (RF = EC₅₀ value of a mutant / EC₅₀ value of the wildtype parent of this mutant) of 12·4 to 186·1 with a mean RF value of 44·1. Mutants obtained via UV irradiation, culturing on NA plates and culturing on rice plants had mean RF values of 51·8, 24·5 and 14·4, respectively. All mutants showed decreases in resistance to zinc thiazole after 20 successive transfers on bactericide‐free media or 10 successive inoculation–reisolations on bactericide‐free rice plants. No significant difference was found in bacterial growth and sensitivity to bismerthiazol between zinc thiazole‐resistant mutants and their parents. However, a significant decrease was observed in the pathogenicity of zinc thiazole‐resistant mutants compared with their parents, especially for mutants obtained via UV irradiation.     

Rapd Pcr-based differentiation of Xanthomonas campestris pv. phaseoli and Xanthomonas campestris pv. phaseoli var. fuscans

A RAPD PCR-based method was used to differentiate between isolates of Xanthomonas campestris pv. phaseoli and Xanthomonas campestris pv. phaseoli var. fuscans. Using random primer OP-G11, a single, high intensity band of 820 bp was amplified from DNAs of all X. c. pv. phaseoli var. fuscans isolates, while multiple amplification products of varying sizes were generated from X. c. pv. phaseoli DNAs. Whereas RAPD PCR differentiation gave an unambiguous result in under 4 h, standard differentiation by recording the production of a brown pigment by X. c. pv. phaseoli var. fuscans isolates took up to 7 days and showed variation both between isolates and between media. The unequivocal nature of the RAPD PCR method was demonstrated when isolate 408, originally classified as X. c. pv. phaseoli var. fuscans, failed to produce the 820 bp band typical of X. c. pv. phaseoli var. fuscans isolates, and after also failing to produce a brown pigment, was re-classified as X. c. pv. phaseoli.     

Bacterial Blight of Welsh Onion : A New Disease Caused by Xanthomonas campestris pv. allii pv. nov.

In June of 1998, a new bacterial disease was observed on Welsh onion in Okinawa Prefecture, Japan. Infected plants in nursery boxes were stunted with tip dieback, and heavily infected plants died. In fields, the disease appeared on leaves as irregular gray spots or elliptical spots with creases in the center. These spots enlarged and spread rapidly continued cloudy or rainy weather, and formed blight lesions on outer leaves. Yellow mucoid bacterial colonies were consistently isolated from these lesions. The causal bacterium was identified as a pathovar of Xanthomonas campestris on the basis of bacteriological properties. The bacterium was pathogenic to Welsh onion, onion, but nonpathogenic to chive, Chinese chive and hyacinth. Of Liliaceae plants, which contain Welsh onion and onion, only hyacinth has been reported as a host for the genus Xanthomonas, namely X. campestris pv. hyacinthi. However, strains of X. campestris pv. hyacinthi were not pathogenic against either Welsh onion or onion. From these results, the bacterium isolated from Welsh onion is considered to be a new pathovar of X. campestris, and the name of X. campestris pv. allii pv. nov. is proposed. A strain MAFF 311173 is designated as the pathotype strain. Received 29 March 2000/ Accepted in revised form 4 July 2000     

Bacterial Leaf Spot of Ivy Caused by Xanthomonas campestris pv. hederae

A bacterial leaf spot disease was observed on Hedera helix (English ivy) and H. canariensis (Algerian ivy) in Japan. The causal agent was identified as Xanthomonas campestris pv. hederae (Arnaud 1920) Dye 1978. Received 13 May 2002/ Accepted in revised form 3 July 2002     

Characterization of pathotypes among isolates of Xanthomonas axonopodis pv. manihotis in Colombia

Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis ( Xam ) is a destructive disease occurring in most cassava growing-areas. Although Colombian isolates of Xam differ in DNA polymorphism and pathogenicity, no suitable host differentials have been identified to demonstrate physiological specialization. A set of 26 Xam isolates from three edaphoclimatic zones (ECZs) in Colombia was selected for inoculation on a set of 17 potential cassava differentials. Leaf inoculation and stem puncture were used in order to detect possible specific interactions between cultivars and isolates. Cultivar × isolate interaction was highly significant ( P  < 0·001) after stem inoculation, but not after leaf inoculation. The stem inoculation technique was selected as a method for resistance screening of cassava cultivars for bacterial blight resistance. A highly significant interaction was also detected when cultivar behaviour was rated as area under the disease progress curve (AUDPC) after stem inoculation. Different pathotypes were defined among the 26 isolates and differential cultivars were proposed to define the pathotypic composition of Xam populations in three ECZs in Colombia. The results should help to improve selection of sources of resistance to cassava bacterial blight.     

Molecular characterization of the incitant of cowpea bacterial blight and pustule, Xanthomonas campestris pv. vignicola

Strains of Xanthomonas campestris pv. vignicola (Xcv), isolated from cowpea leaves with blight or minute pustules and collected from various geographic areas, were selected on the basis of pathological and physiological features. All strains were analyzed for genotypic markers by two methods: ribotyping with EcoRI endonuclease, and RFLP analysis with a plasmid probe (pthB) containing a gene required for pathogenicity from Xanthomonas campestris pv. manihotis. Ribotyping revealed a unique pattern for all the strains that corresponded to the previously described ribotype rRNA7. Based on polymorphism detected by pthB among Xcv strains, nine haplotypes were defined. The observed genetic variation was independent of the geographic origin of the strains and of pathogenic variation. Some haplotypes were widely distributed, whereas others were localized. In some cases, we could differentiate strains isolated from blight symptoms and pustules according to haplotypic composition. However, in most cases, no significant differences were observed. Our results and the previous pathogenic and biochemical characterizations suggest that the strains isolated from leaves with blight symptoms or minute pustules belong to the same pathovar. We provide information on pathogen diversity that can be used to identify and characterize resistant germplasm.     

Expression of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae hrp</Emphasis> Genes in XOM2, a Novel Synthetic Medium

To analyze the regulation of hrp expression and to detect and identify hrp-dependent secretion proteins of plant-pathogenic bacteria, an appropriate hrp-inducing medium is indispensable. In this study, two efficient hrp-inducing media for Xanthomonas oryzae pv. oryzae were designed by assaying the expression of a hrcU (the first gene of the hrpC operon) and a gus (β-glucuronidase) fusion gene. We modified XVM2, which is a hrp-inducing medium for X. campestris pv. vesicatoria, by adding 0.01% xylose in place of fructose and sucrose (0.18 and 0.34%, respectively) as a sugar source. The resulting medium induced approximately 15-fold more GUS activity from transformants containing a hrcU::gus gene than did XVM2. Moreover, a methionine-containing synthetic medium with 0.18% xylose as a sugar source was able to induce much stronger expression of HrcU::GUS, with GUS activity approximately 100-fold greater than that in XVM2. Induction depended on a regulator, HrpXo, and the PIP (plant-inducible-promoter) box, suggesting that HrcU::GUS was expressed in a hrp-dependent manner. The induction of operons hrpA to hrpF in XOM2 was also confirmed. These results suggest that both media, especially XOM2, are highly efficient hrp-inducing media for X. oryzae pv. oryzae. Received 7 October 2002/ Accepted in revised form 22 November 2002     

Notes on the occurrence of pathogenic races of Xanthomonas oryzae pv. oryzae found in Hiroshima Prefecture during 2000–2003

The pathogenic races of 450 cultures of Xanthomonas oryzae pv. oryzae, isolated from eight locations in the inland mountainous area of Hiroshima Prefecture during 2000 to 2003, were determined with a set of Japanese differentials. The rice cultivars infected with the bacterium are thought to be in the Kinmaze group, which does not have any resistance genes. Five Japanese races IA, IB, II, V, and VII occurred across the area, although the composition of the races in each location altered during the surveyed 4 years.     

Genetic Analysis of the Relationship between the Browning Reaction and Bacterial Blight Resistance Gene Xa3 in Rice

The relationship between bacterial blight resistance gene Xa3 and browning reaction was genetically analyzed using F₂ plants from the cross of rice cultivar Kuntulan with Kinmaze. Kuntulan harbors resistance Xa3 and developes a browning reaction to avirulent races of Xanthomonas oryzae pv. oryzae, whereas Kinmaze has a typical susceptible reaction to all known Japanese races of X. o. pv. oryzae. The F₂ plants were tested for their resistance to avirulent race II strain of X. o. pv. oryzae and the development of browning. Of 337 F₂ plants tested, 251 had resistance to the strain. In all the resistant plants, a browning reaction developed around the point of inoculation. The remaining 86 had a susceptible reaction to the strain without a browning reaction. The F₂ population of Kuntulan × Kinmaze had a clear-cut segregation ratio of 3R : 1S. These facts led to the conclusion that the browning reaction is a pleiotropic effect of Xa3. Received 29 January 2001/ Accepted in revised form 24 April 2001     

Movement of Xanthomonas campestris pv. vitians in the stems of lettuce and seed contamination

Xanthomonas campestris pv. vitians , the causal agent of bacterial leaf spot of lettuce (BLS), can be seedborne, but the mechanism by which the bacteria contaminates and/or infects lettuce seed is not known. In this study, the capacity of X. campestris pv. vitians to enter and translocate within the vascular system of lettuce plants was examined. The stems of 8- to 11-week-old lettuce plants were stab-inoculated, and movement of X. campestris pv. vitians was monitored at various intervals. At 4, 8, 12 and 16 h post-inoculation (hpi), X. campestris pv. vitians was recovered from 2 to 10 cm above (depending on stem length) and 2 cm below the inoculation site. Xanthomonas campestris pv. vitians was also recovered from surface-disinfested stem sections of spray-inoculated plants. Together, these results are consistent with X. campestris pv. vitians invading and moving systemically within the vascular system of lettuce plants. To investigate the mechanism of seed contamination, lettuce plants at the vegetative stage of growth were spray-inoculated with X. campestris pv. vitians and allowed to develop BLS. Seed collected from these plants had a 2% incidence of X. campestris pv. vitians external colonization, but no bacteria were recovered from within the seed.     

Genetic, phenotypic and pathogenic diversity of Xanthomonas arboricola pv. corylina strains question the representative nature of the type strain

A collection of 31 Xanthomonas arboricola pv. corylina strains isolated from Corylus maxima and C. avellana of different countries were assessed by means of repetitive PCR using ERIC, BOX and REP primer sets and analysis of whole-cell protein extracts; pathogenicity tests to three hazelnut ( C. avellana ) cultivars; and some key biochemical tests. From these studies, the X. arboricola pv. corylina strains were clustered into five and three groups by repetitive PCR and protein analysis, respectively, and by using UPGMA cluster analysis, with two strains forming an outlier group to these. The groups showed a high degree of similarity. Strain membership between the groups designed by the two methods exhibited a high degree of congruence, and diversity between the groups was low. Surprisingly, the two strains originating from C. maxima , that include the type strain NCPPB 935, formed the most distinctive group. No relationship to geographic origin of the strains was evident. All strains proved pathogenic towards three different hazelnut cultivars, although the strains obtained from C. maxima did not incite any significant symptoms on buds and twigs. No other relationships between rep-PCR and whole-cell protein groups and pathogenicity were evident. The distinctiveness of the C. maxima strains was supported further by atypical negative gelatin liquefaction test and reduced quinate metabolism results.     

The genetic characterization of Xanthomonas arboricola pv. juglandis,the causal agent of walnut blight in Poland

Leaves and fruits of walnut trees exhibiting symptoms of bacterial blight were collected from six locations in Poland. Isolations on agar media resulted in 18 bacterial isolates with colony morphology resembling that of the Xanthomonas genus. PCR using X1 and X2 primers specific for Xanthomonas confirmed that all isolates belonged to this genus. In pathogenicity tests on unripe walnut fruits, all isolates caused typical black necrotic lesions covering almost the entire pericarp. Results of selected phenotypic tests indicated that characteristics of all isolates were the same as described for the type strain of Xanthomonas arboricola pv. juglandis. Genetic analyses (PCR MP, ERIC‐, BOX‐PCR and MLSA) showed similarities between the studied isolates and the reference strain of X. arboricola pv. juglandis CFBP 7179 originating from France. However, reference strains I‐391 from Portugal and LMG 746 from the UK were different. MLSA analysis of partial sequences of the fyuA, gyrB and rpoD genes of studied isolates and respective sequences from GenBank of pathotype strains of other pathovars of X. arboricola showed that the X. arboricola pv. juglandis isolates consisted of different phylogenetic lineages. An incongruence among MLSA gene phylogenies and traces of intergenic recombination events were proved. These data suggest that the sequence analysis of several housekeeping genes is necessary for proper identification of X. arboricola pathovars.     

Survival of the Anthurium Blight Pathogen, Xanthomonas axonopodis pv. Dieffenbachiae, in Field Crop Residues

Bacterial blight, caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad), is a major threat to the anthurium cut flower industry worldwide. Two field trials in Hawaii evaluated the long-term persistence of Xad in artificially-infested crop residues. Xad survived in leaf, petiole, and root residues for as long as 4 months when tissues were left on the surface or buried 15cm deep. Survival was considerably shorter (approximately 20 days) outside of residues. Xad that was recovered from residues over a period of 4 months retained pathogenicity. Xad was isolated from living roots of naturally-infected plants which further suggests that roots left in the field after culling may be particularly important, but overlooked, inoculum source. This information is key to determining minimum fallow periods before replanting devastated fields.     

Genotype × environment Effects on Severity of Cassava Bacterial Blight Disease caused by Xanthomonas axonopodis pv. manihotis

Nine cassava genotypes were grown for three years at six sites representing three agro-ecological zones in Nigeria to study their reaction to cassava bacterial blight (CBB), investigate genotype × environment (G × E) interaction patterns for their reaction to CBB, and to identify genotypes with stability to the disease, using the additive main effects and multiplicative interaction (AMMI) statistical model. Environments, genotypes and G × E interactions accounted for 71.8%, 12.0% and 16.2% of the treatment sums of squares (SS), and were highly significant (P<0.0001) for the disease, indicating that genotypes responded differentially to CBB infection across environments. Clones 30555, 91934, U/41044, and 4(2)1425 showed the least CBB disease ratings. Other clones showed erratic and fluctuating reactions to CBB from environment to environment and were thus considered unstable to the disease. CBB was most severe in 1989 (with a mean score of 2.46) and least so in 1990 (with a score of 2.06). The sites with the most disease were Ibadan, Ilorin and Ubiaja (1989), Ibadan and Ubiaja (1990) and Mokwa (1991). Because of the favourable conditions for disease development at those sites, they could be appropriate for screening cassava genotypes for CBB resistance. The AMMI model selected AMMI1 as the best predictor for CBB because it had the smallest actual root mean square prediction difference (0.37646), and explained 90.7% of the G × E interaction for CBB. The AMMI model was successful in selecting the genotypes 30555, U/41044 and 4(2)1425 and the environments Ibadan 1989, Ilorin 1989 and Onne 1990 with stability of reaction to the disease.     

水稻中与白叶枯病菌hrf1基因同源序列克隆及功能分析

利用PCR技术从粳稻R109中克隆到与hrf1同源的序列,命名为hpfr1,与hrf1基因的同源性为92%。推导的氨基酸同源性为74.8%。hpfr1基因连接到含T7启动子的高表达载体pET30a（＋）上构建重组质粒pHOSJ4,并转化宿主菌BL21（DE3）产生表达菌株BL21（DE3）/pHOSJ4。hpfr1与hrf1基因在起始密码子ATG至225个碱基处完全相同。将ATG至225碱基的序列克隆,并构建表达载体pHOSJ4-225,表达产物注射烟草能引起过敏反应。BL21（DE3）/pHOSJ4和BL21（DE3）/pHOSJ4-225的蛋白抽提物诱导烟草抗烟草花叶病毒病均达极显著水平,浓度为60μl/ml时防效最显著,分别为96.35%、95.4%,与harpinXoo防效（95.9%）相近。     

Xanthomonas campestris pv. populi,the causal agent of bark necrosis in poplar

A bacterium was isolated from superficial bark necroses on young poplars and its pathogenicity demonstrated by inoculation experiments. The organism was identified asXanthomonas campestris. Cross-inoculations showed that a previously undescribed pathovar was involved. It is suggested to designate this organismX. campestris pv.populi.Samenvatting Uit een oppervlakkige bastnecrose bij jonge populieren werd massaal een bepaalde bacterie geïsoleerd. Met deze bacterie werden gezonde populieren in het veld geïnoculeerd via verwonding van de bast. Als gevolg van de inoculaties ontwikkelden zich bij ongeveer 40% van de geïnoculeerde bomen hetzelfde type bastnecrosen, terwijl bij de controleplanten geen enkele reactie optrad. Uit de kunstmatig verkregen necrosen werd dezelfde bacterie geïsoleerd.Identificatie met biochemische en serologische methoden toonde aan dat de bacterieXanthomonas campestris was.Vervolgens werden in de kas kruisinoculaties uitgevoerd met verschillende xanthomonaden op populier, wilg, kool en geranium. DeX. campestris isolaten uit populier tastten behalve populier ook wilg aan. De andere gebruikte stammen waren waardplant-specifiek, al bleven sommigen ervan minstens acht maanden in leven in een niet-waardplant, evenwel zonder symptomen te veroorzaken. Geconcludeerd wordt, dat de bastnecrosen zijn veroorzaakt door een nog niet beschreven pathovar vanX. campestris. Voorgesteld wordt om deze bacterieXanthomonas campestris pv.populi te noemen.