Biological control of pythium root rot of chrysanthemum in small-scale hydroponic units

The capacity of several strains of root-colonizing bacteria to suppressPythium aphanidermatum, Pythium dissotocum and root rot was investigated in chrysanthemums grown in single-plant hydroponic units containing an aerated nutrient solution. The strains were applied in the nutrient solution at a final density of 10⁴ CFU ml⁻¹ and 14 days later the root systems were inoculated withPythium by immersion in suspensions of 10⁴ zoospores ml⁻¹ solution. Controls received no bacteria, noPythium, or one of thePythium spp. but no bacteria. Strain effectiveness was estimated based on percent roots colonized byPythium and area under disease progress curves (AUDPC). In plants treated respectively withPseudomonas (Ps.)chlororaphis 63-28 andBacillus cereus HY06 and inoculated withP. aphanidermatum, root colonization by the pathogen was 83% and 72% lower than in the pathogen control, and AUDPC values were reduced by 61% and 65%. ForP. dissotocum, the respective strains reduced root colonization by 87% and 91%, and AUDPC values by 70% and 90%. In plants treated respectively withPs. chlororaphis Tx-1 andComamonas acidovorans C-4-7-28, root colonization byP. aphanidermatum was 84% and 80% lower than in the controls and AUDPC values were reduced by 66% and 57%; these strains did not suppressP. dissotocum. Burkholderia gladioli C-2-74 andC. acidovorans OCR-7-8-38, respectively, suppressed colonization of roots byP. dissotocum by 74% and 86%, and reduced AUDPC values by 60% and 70%, but were ineffective againstP. aphanidermatum. C. acidovorans OCR-7-8-39 reduced colonization and AUDPC values ofP. aphanidermatum by 57% and 42%, respectively.Pseudomonas corrugata 13,Ps. fluorescens 15 and JZ12, and three additional strains ofC. acidovorans were weakly or nonsuppressive againstP. aphanidermatum. Strains that reduced AUDPC values forP. aphanidermatum orP. dissotocum when applied at 10⁴ CFU ml⁻¹ were 11%–39% less effective at 10³ CFU ml⁻¹. Four tested strains (Ps. chlororaphis 63-28,Ps. chlororaphis Tx-1,B. cereus HY06, andB. gladioli C-7-24) in most instances suppressed root colonization and lowered AUDPC values ofP. aphanidermatum when applied at 14, 7 or 0 days before inoculation, but reduction of the respective variables was generally greater when the strains were applied at 14 days (63%–87% and 75%–78%) or 7 days (44%–47% and 31%–88%) than at 0 days (14%–31% and 23%–62%) before inoculation.Ps. chlororaphis Tx-1,Ps. chlororaphis 63-28 andB. cereus HY06 significantly suppressedP. aphanidermatum whether the temperature of the nutrient solution was high (32°C) or moderate (24°C). Taken together, the observations suggest thatPs. chlororaphis 63-28,B. cereus HY06,Ps. chlororaphis Tx-1,B. gladioli C-2-74 andC. acidovorans OCR-7-8-38 have the potential for controlling Pythium root rot in hydroponic chrysanthemums. http://www.phytoparasitica.org posting Jan. 24, 2007.     

Problems of detecting phytopathogenic bacteria by ELISA1

Xanthomonas ampelina can be detected with the same sensitivity by ELISA double-antibody sandwich (ELISA-DAS) or indirect ELISA (ELISA-I) but the latter is preferred for its greater simplicity. The two methods also gave similar sensitivity results for soft-rot erwinias, but their application is complicated by the large number of erwinia serotypes. ELISA-I was unable to distinguish strains of Agrobacterium tumefaciens from A. radiobacter. The cross reactions with other bacteria of antisera against X. fragariae were tested by ELISA-I. The problems of applying these methods are discussed.     

Structural conservation of the <Emphasis Type="Italic">hrp</Emphasis> gene cluster in <Emphasis Type="Italic">Xanthomons oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

The clustered hrp genes encoding the type III secretion system in the Japanese strains MAFF301237 and MAFF311018 of Xanthomonas oryzae pv. oryzae were sequenced and compared. The strains differ in their pathogenicity, location, and year of isolation. A 30-kbp sequence comprising 29 open reading frames (ORFs) was identical in its structural arrangement in both strains but differed from X. campestris pv. campestris, X. axonopodis pv. citri, and X. axonopodis pv. glycines in certain genes located between the hpaB-hrpF interspace region. The DNA sequence and the putative amino acid sequence in each ORF was also identical in both X. oryzae pv. oryzae strains as were the PIP boxes and the relative sequences. These facts clearly showed that the structure of the hrp gene cluster in X. oryzae pv. oryzae is unique.     

Recent research progress on Xanthomonas ampelina1

Bacterial blight of grapevine (caused by Xanthomonas ampelina) occurs in Greece, France, Spain, Italy, Turkey, Portugal, USSR and South Africa. It is a serious, chronic vascular disease which is widespread in some areas of grape production, affecting commercially important cultivars. There is evidence that the disease is presently spreading into new areas in some countries. The actual distribution of blight may be much wider, since its symptoms may be confused with those of other diseases. It is therefore essential to confirm the presence of X. ampelina by isolation and identification. X. ampelina is an anomalous member of the genus Xanthomonas which is readily distinguished on the basis of a few tests. The pathogen may also be rapidly detected in grapevine sap using the indirect immunofluorescence technique. The susceptibility of shoots to infections, under Cretan conditions, is highest from November to late January and very low during February and March. It has been found that up to 50% of apparently healthy canes obtained from diseased vineyards were latently infected. Transmission of the pathogen through soil and roots during the control of Daktulosphaira vitifolii or frost damage by flooding has been demonstrated in France. Several of the more widely grown cultivars were found to be very susceptible to the pathogen while no completely resistant cultivar has been detected. Control trials based on four sprays with a number of chemicals were unsuccessful. Control policy should be directed: (a) to preventing spread of the disease, by exclusive use of certified material; (b) to minimizing the disease in affected areas by suitable sanitary, cultural and preventive measures.     

Differentiation of Xanthomonas species Pathogenic to Sugarcane by PCR-RFLP Analysis

The PCR-RFLP of the 16S-23S rDNA spacer region was used to differentiate Xanthomonas species pathogenic to sugarcane. Strains of X. albilineans, X. campestris pv. vasculorum Types A and B, X. sacchari and Xanthomonas sp. from Trinidad, South Africa and India were examined. The amplification products were digested with Alu I, Hae III, Hpa II and Mbo I and the results showed that the different groups of bacterial strains exhibited distinct RFLP patterns for each tested endonuclease, except X. albilineans and X. sacchari which could only be differentiated from each other by the digestion with Hpa II. The results also allowed the separation of X.c. pv. vasculorum Type A from X.c. pv. vasculorum Type B and strongly suggested that the analyzed Xanthomonas sp. strains belong to X. sacchari. Nine X. campestris (pv. not determined) strains included in this study showed identical profiles to X.c. pv. vasculorum Type A group and DNA–DNA hybridization experiments confirmed these results. PCR-RFLP of the 16S-23S rDNA spacer region could be applied as a reliable method for differentiating the xanthomonads pathogenic to sugarcane.     

Gene Flow Analysis of Phytophthora porri Reveals a New Species: Phytophthora brassicae Sp. Nov.

Isozyme analysis and sequence analysis of the internal transcribed spacer regions (ITS-1 and ITS-2) and the 5.8S subunit of the ribosomal DNA gene repeat were used to examine whether isolates of Phytophthora porri from Allium and Brassica represent a single homogeneous species. Twenty-six strains of P. porri, 16 strains isolated from the genus Allium, and 10 strains isolated from the genus Brassica, were analyzed using malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH) and lactate dehydrogenase (LDH), represented altogether by four putative loci (Mdh-2, Idh-1, Idh-2, and Ldh-2). Isozyme analysis revealed that strains isolated from Allium contained five private alleles at three isozyme loci (Ldh-2 ⁸³, Ldh-2 ¹⁰⁴, Idh-1 ¹⁰⁸, Idh-1 ¹¹², and Idh-2 ⁹⁸), whereas six different alleles were observed at four isozyme loci (Ldh-2 ⁸⁵, Ldh-2 ¹⁰⁰, Ldh-2 ¹¹⁴, Idh-1 ¹⁰⁰, Idh-2 ¹⁰⁰, and Mdh-2 ¹¹¹) in strains obtained from Brassica. The heterozygosity at the Ldh-2 locus, differing in allele composition, however, between strains from Allium and Brassica, was present in all strains, indicating that it is probably fixed. Sequence analysis of the ITS regions and the 5.8S subunit showed consistent differences between isolates from Allium and isolates from Brassica. Based on isozyme data, ITS sequence analysis and formerly published differences in restriction enzyme patterns of mitochondrial DNA, morphology and pathogenicity, it was concluded that the isolates of P. porri Foister did not represent a homogeneous species. Isolates from Brassica constitute a distinct species which is described here as P. brassicae sp. nov. It was inferred from isozyme patterns, which were in no case intermediate between the two species, that P. porri and P. brassicae do not hybridize and are reproductively isolated by barriers to gene flow.     

Characterization and PCR-based Typing of Xanthomonas campestris pv. vesicatoria from Peppers and Tomatoes in Serbia

During the last two decades bacterial strains associated with necrotic leaf spots of pepper and tomato fruit spots were collected in Serbia. Twenty-eight strains isolated from pepper and six from tomato were characterized. A study of their physiological and pathological characteristics, and fatty acid composition analysis revealed that all of the strains belong to Xanthomonas campestris pv. vesicatoria. Being non-amylolytic and non-pectolytic, pathogenic on pepper but not on tomato, containing lower amounts of fatty acid 15 : 0 ante–iso, the pepper strains were designated as members of the A group of X. campestris pv. vesicatoria. However, the tomato strains hydrolyzed starch and pectate, caused compatible reactions on tomato but not on pepper, had higher percent of 15 : 0 ante–iso fatty acid, and were classified into B phenotypic group and identified as X. vesicatoria. PCR primers were developed which amplified conserved DNA regions related to the hrp genes of different strains of X. campestris pv. vesicatoria associated with pepper and tomato. Restriction analysis of the PCR product resulted in different patterns and enabled grouping of the strains into four groups. When xanthomonads isolated from pepper and tomato in Serbia were analyzed, they clustered into two groups corresponding to the grouping based on their physiological and pathological characteristics. According to the reaction of pepper and tomato differential varieties, the strains from pepper belong to races P7 and P8 and tomato strains belong to the race T2. All strains were sensitive to copper and streptomycin. Advantages and disadvantages of various bacterial spot management practices are discussed.     

Genome-based PCR Primers for Specific and Sensitive Detection and Quantification of Xylella fastidiosa

Xylella fastidiosa is an important pathogen of many commercial crops. Detection of X. fastidiosa is difficult due to low concentrations of the bacteria in insects and asymptomatic plant tissue, and non-uniform distribution in infected plants. A dual purpose conventional PCR and quantitative PCR (TaqMan™) system was developed for the generic detection of X. fastidiosa strains. Primers HL5 and HL6, designed to amplify a unique region common to the sequenced genomes of four Xylella strains, amplified a 221 bp fragment from strains associated with Pierce’s disease of grapes, almond leaf scorch, and oleander leaf scorch disease and from DNA from an Xf strain associated with citrus variegated chlorosis. Standard curves were obtained using concentrations of Xylella ranging from 5 to 10⁵ cells per reaction in water and grape extracts and 10–10⁵ cells in insect DNA. Regression curves were similar, with correlation coefficients of r ² > 0.97. In quantitative PCR, C_t values ranged between 20 and 36 cycles for 5–10⁵ bacterial cells per reaction. No amplicons were obtained with several non-Xf bacterial strains tested including related plant pathogenic, grape endophytic bacteria and endosymbiotic bacteria isolated from glassy-winged sharpshooters. The method was evaluated for clinical diagnosis of Xf in grapes, almonds and insect vectors. The procedure described is reliable for detection of the pathogen with a high degree of sensitivity and specificity.     

Development of a specific molecular tool for detecting Xanthomonas campestris pv. musacearum

A specific and rapid diagnostic tool has been developed to detect Xanthomonas campestris pv. musacearum, the causal agent of bacterial wilt of banana. PCR primers were developed from intergenic regions of X. campestris pv. musacearum following its partial sequence. A total of 48 primers were tested for specificity to X. campestris pv. musacearum strains collected from various regions in Uganda. These were also tested for specificity against related Xanthomonas species from the vasicola group, Xanthomonas species pathogenic to other crops, and against those existing saprophytically on banana plants. Seven primer sets (Xcm12, Xcm35, Xcm36, Xcm38, Xcm44, Xcm47 and Xcm48) were found to be very specific to X. campestris pv. musacearum. These primer sets directed the amplification of the expected product for all 52 strains of X. campestris pv. musacearum collected from different locations in Uganda. No amplification products were obtained with unrelated phytopathogenic bacteria or endophytic/epiphytic bacteria from banana. A detection limit of 10³ CFU mL^?1 corresponding to about four cells per PCR reaction was observed when X. campestris pv. musacearum cells were used for all the seven primer sets. The DNA samples from symptomless plant tissues also tested positive with primer set Xcm38. The specific PCR method described here is a valuable diagnostic tool which can be used to detect the pathogen at early stages of infection and monitor disease.     

Aetiology of anthracnose on grapevine shoots in Brazil

Anthracnose is an important disease in vineyards in south and southeast Brazil, the main grape‐producing regions in the country. This study aimed to identify the causal agents of grapevine anthracnose in Brazil through multilocus phylogenetic analyses, morphological characterization and pathogenicity tests. Thirty‐nine Elsinoë ampelina and 13 Colletotrichum spp. isolates were obtained from leaves, stems and berries with anthracnose symptoms collected in 38 vineyards in southern and southeastern Brazil. For E. ampelina isolates, the internal transcribed spacer (ITS), histone H3 (HIS3) and elongation factor 1‐α (TEF) sequences were analysed. HIS3 was the most informative region with 55 polymorphic sites including deletions and substitutions of bases, enabling the grouping of isolates into five haplotypes. Colonies of E. ampelina showed slow growth, variable colouration and a wrinkled texture on potato dextrose agar. Conidia were cylindrical to oblong with rounded ends, hyaline, aseptate, (3.57–) 5.64 (?6.95) μm long and (2.03–) 2.65 (?3.40) μm wide. Seven species of Colletotrichum were identified: C. siamense, C. gloeosporioides, C. fructicola, C. viniferum, C. nymphaeae, C. truncatum and C. cliviae, with a wide variation in colony and conidium morphology. Only E. ampelina caused anthracnose symptoms on leaves, tendrils and stems of Vitis vinifera and V. labrusca. High disease severity and a negative correlation between disease severity and shoot dry weight were observed only when relative humidity was above 95%. In this study, only E. ampelina caused anthracnose symptoms on grapevine shoots in Brazil.     

Description of copper tolerant Xanthomonas citri subsp. citri and genotypic comparison with sensitive and resistant strains

The copper-based products widely used for control of citrus canker may lead to the development of Xanthomonas citri subsp. citri (X. citri) resistant to copper (Cu^R). However, the study of copper sensitivity of X. citri strains from Paraná state, Brazil, did not reveal the existence of Cu^R, but copper tolerant (Cu^T) strains. The aim of this study was to describe for the first time the existence of Cu^T X. citri and compare the genetic determinants that differentiate the Cu^T strains from the sensitive (Cu^S) and Cu^R strains. Cu^T strains supported intermediate concentrations of copper in comparison to Cu^S and Cu^R. Cu^T strains lack the gene clusters copLAB or copABCD responsible for copper resistance in Cu^R strains and the large plasmids (c. ≥200 kb) that normally carry these genes. The nucleotide sequences of chromosomal homologous genes cohLAB, involved in copper homeostasis, were 100% similar in strains of all phenotypes. Cu^T strains differed from Cu^S strains by the higher expression of the homologous chromosomal genes cohA and cohB in the presence of copper. Cu^T X. citri strains are not precursors of Cu^R strains and do not pose a threat to the efficient use of copper-based bactericides for management of citrus canker in citrus orchards. Copper resistance and tolerance are distinct phenotypes and should not be used as synonyms. The proper characterization of the sensitivity to copper leads to a more confident monitoring of the distribution of copper resistant populations of X. citri and adoption of containment measures only when necessary.     

Specific characters of 16S rRNA gene and 16S–23S rRNA internal transcribed spacer sequences of Xylella fastidiosa pear leaf scorch strains

Pear leaf scorch, the only Xylella fastidiosa-induced disease reported from Taiwan, was found in area where the variety Hengshan (Pyrus pyrifolia) was grown. Strains of pear leaf scorch Xyl. fastidiosa (XF-PLS) shared similarities to strains of other host origins in the requirement of complex medium and the exhibition of rippled cell walls, however, recent serological and molecular biology studies showed difference among them. Five strains of XF-PLS were compared with 20 other strains originally isolated from almond, oleander, pecan, plum, peach, mulberry, grapes, citrus, coffee, and sycamore by sequence analyses of the 16S rRNA gene and 16S–23S rRNA internal transcribed spacer region (ITS). When sequences of 16S rRNA gene based on fragment size of 1,537–1,540 bp were compared, the similarity index among 5 XF-PLS strains was 99.3–99.8%, whereas it was 97.8–98.6% between XF-PLS strains and strains from other hosts. When sequences of 16S–23S rRNA ITS based on fragment size of 510–540 bp were compared, the similarity index among 5 XF-PLS strains was 99.0–100%, whereas it was 80.7–82% between XF-PLS strains and strains from other hosts. Multiple sequence alignments led to the identification of 5 polymorphic nucleotides in the 16S rRNA gene among the 25 Xyl. fastidiosa strains, and there were considerable variations in the nucleotide sequences of 16S–23S rRNA ITS between XF-PLS and the other 20 Xyl. fastidiosa strains. The phylogenetic trees revealed that XF-PLS strains were separated from strains of other hosts. Strains of other hosts were divided into four subgroups: strains from (1) oleander, (2) grape, almond M23 and mulberry, (3) citrus and coffee, and (4) pecan, peach, plum, sycamore and almond M12. Results indicate that XF-PLS strains were not closely related to the above-mentioned strains from other hosts and could possibly belong to a new subspecies of Xyl. fastidiosa.     

<Emphasis Type="Italic">Pseudomonas syringae </Emphasis>Strains Are Classified into Five Groups by Comparing DMA Homology at the <Emphasis Type="Italic">hrp </Emphasis>Neighboring Regions

Previously, we classified Pseudomonas syringae strains into at least three groups (I, II and U) by comparing DNA homology at the hrp cluster and its neighboring regions (Inoue and Takikawa 1999). However, heterogeneous strains remained in the undetermined group (group U). We further classify group U, using pvs. syringae and coronafaciens as references. Comparison of restriction sites for regions of each pathovar revealed distinct differences. By using probes from the two pathovars, comparisons of DNA homology at the regions separated two additional distinct groups (III and IV) from group U. Therefore, P. syringae strains are classified into at least five groups. Received 4 November 1999/ Accepted in revised form 27 January 2000     

Xanthomonas arboricola pv. fragariae: what's in a name?

Polyphasic analysis exposed important heterogeneity between bacterial strains catalogued as Xanthomonas arboricola pv. fragariae (Xaf) from different culture collections. Two draft whole‐genome sequences revealed pathogenicity related genes of the type‐three secretion system in strain LMG 19146, whereas none were found in the Xaf pathotype strain LMG 19145. Also, considerable sequence divergence was observed in the phylogenetic marker genes gyrB, rpoD, dnaK and fyuA. Further study of 16 Xaf culture‐collection strains showed that co‐classification is not justified. Partial 16S rRNA gene and gyrB sequencing demonstrated that 12 strains belonged to X. arboricola, but that they did not form one homogeneous group within the species. The four remaining strains were identified as Xanthomonas fragariae and Xanthomonas sp. All sequence‐based identifications were confirmed by MALDI‐TOF MS fingerprinting. Also, the pathogenicity genes hrcQ and avrBs2 were detected in only three of the 12 analysed X. arboricola strains. The X. arboricola and Xanthomonas sp. strains showed pectolytic activity, and upon inoculation in strawberry none of the strains reproduced the leaf blight symptoms reported for Xaf. This study demonstrates that (i) no clear criteria exist for the identification of strains as Xaf, (ii) the name Xaf is currently used for a genetically diverse assortment of strains, and (iii) the species X. arboricola holds many undetermined plant‐associated bacteria besides the described pathovars.     

Bacterial pathogens of rice in the Kingdom of Cambodia and description of a new pathogen causing a serious sheath rot disease

A study of rice diseases in Cambodia from 2005 to 2007 showed widespread occurrence of diseases caused by Acidovorax avenae subsp. avenae, Burkholderia gladioli, B. cepacia and Pantoea ananatis. This is the first report of these pathogens in Cambodia. Additionally, a pseudomonad causing a widespread disease similar to sheath brown rot (caused by Pseudomonas fuscovaginae) was isolated. The studied strains were pathogenic to rice cvs Sen Pidau and IR 66, producing similar, though slightly less severe, symptoms to those observed in the field. Based on comparative 16S rDNA gene sequence analysis, combined with cell wall fatty acid analysis and metabolic profiles, the isolated strains were allocated to the genus Pseudomonas. The novel species were differentiated from Pseudomonas fuscovaginae and P. putida by their inability to metabolize d ‐fructose, d ‐galactose, d ‐galactonic acid lactone, d ‐galacturonic acid, d ‐glucosaminic acid, d ‐glucuronic acid, p‐hydroxy phenylacetic acid, d ‐saccharic acid and urocanic acid. The major fatty acids were C_16:0, summed feature 3 (C_16:1ω7c and C_16:1ω6c) and summed feature 8 (C_18:1ω7c), representing 80% of the total. Partial 16S rRNA gene sequences (1460 bp) were identical, except for two nucleotide changes amongst the six strains. Alignment of the causal strains within type‐culture databases revealed similarities of 99·7% with Pseudomonas parafulva AJ 2129^T, 99·2% with P. fulva IAM 1592^T, 98·9% with P. plecoglossicidia FPC 951^T, and 98·1% with P. fuscovaginae MAFF 301177^T. On the basis of data from this polyphasic study, it is proposed that the unknown strains isolated from rice represent a novel species of the genus Pseudomonas.     

Genetic Diversity of Xylella fastidiosa Strains from Costa Rica, São Paulo, Brazil, and United States

ABSTRACT The diversity of 42 Xylella fastidiosa strains from Costa Rica, S?o Paulo, Brazil, and the United States were analyzed using the sequence of the 16S rRNA gene by variable number of tandem repeat (VNTR) fragment analysis and by restriction fragment length polymorphisms (RFLP) of a specific polymerase chain reaction (PCR)-amplification product using enzyme CfoI. Limited variability in the sequence of the 16S rRNA gene was observed and, although the separation was not absolute, most strains from Costa Rica clustered with strains from the United States and not with strains from S?o Paulo. The PCR-RFLP produced different patterns of DNA bands. The same pattern was shared by strains from Costa Rica, the United States, and two coffee strains from S?o Paulo, but a different pattern was observed in six coffee and orange strains from Brazil. In all, 32 amplification products were scored in the VNTR fragment analysis. The total variation observed among the X. fastidiosa strains had significant (P < 0.001) contributions from both geography and host origin as inferred by Nei's values of genetic diversity and WINAMOVA statistics. The strains from Costa Rica were isolated from diseased grapevines, coffee, and sweet orange and these strains grouped together and could be distinguished from strains from grapevine from the United States or from either coffee or sweet orange from S?o Paulo. The strains tested from Costa Rica are most likely of local origin, although the possibility that they have been introduced along with horticultural crops cannot be excluded. In either case, they are examples of independent selection of strains of X. fastidiosa affecting coffee and sweet orange. Greater genetic similarity was observed between strains from Costa Rica and the United States than with those from S?o Paulo.     

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.     

Assessment of Subtractive Hybridization to Select Species and Subspecies Specific DNA Fragments for the Identification of Xylophilus ampelinus by Polymerase Chain Reaction (PCR)

Eighteen Bsp143I digested DNA fragments specific to Xylophilus ampelinus were cloned from a library enriched for X. ampelinus obtained after a subtractive hybridization step. It was also possible to clone specific DNA sequences directly after DNA digestion with Bsp143I probably because X. ampelinus is a unique bacterium. Nucleotidic sequences of four cloned specific fragments were determined. They did not share any homology with other DNA sequences in the EMBL/GeneBank database. Four primer sets were designed and tested for specificity to X. ampelinus. One primer set (Xamp 1.27) was a good candidate for a species-specific reagent in a procedure of identification of X. ampelinus using PCR. One primer set detected only Greek strains isolated from Vitis vinifera cv. Sultana. Genetic diversity within the X. ampelinus species can be used in further epidemiological studies on the bacterial necrosis of grapevine.