Comparative and collaborative studies for the validation of a nested PCR for the detection of Xanthomonas axonopodis pv. dieffenbachiae from Anthurium samples

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires sensitive and reliable diagnostic tools. The European standard EN ISO 16140:2003 has been followed to compare a nested PCR assay (N‐PCR) to a reference method (isolation and serological identification of bacterial colonies) and to other alternative serological detection methods. The evaluation was performed in two steps: a comparative study and a collaborative study involving 15 European laboratories. Although inclusivity was maximal (100%) for all methods, a maximal exclusivity was obtained only with N‐PCR followed by an enzymatic restriction digestion of the amplicons. Exclusivity indices of 90·6, 88·7 and 47·2% were found for indirect ELISA, immunofluorescence and double antibody sandwich ELISA, respectively. An exclusivity of 92·5% was obtained with the reference method, further increased to 100% if pathogenicity tests were performed as a supplemental assay. The best level of sensitivity (relative detection level) was obtained with the reference method followed by the N‐PCR assay. The N‐PCR performance in terms of relative accuracy, accordance and concordance was very similar to that of the reference method. Moreover, N‐PCR had undeniable advantages compared to the reference method (less labour‐intensive and less time‐consuming). In addition, post‐test probabilities of infection were calculated to select the most appropriate detection scheme related to the prevalence of the pathogen. The N‐PCR assay has since been included in a revised version of the EPPO detection protocol.     

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。     

Molecular characterization of Xanthomonas species isolated from Araceae and the development of a triplex TaqMan assay for detection of Xanthomonas phaseoli pv. dieffenbachiae

European Journal of Plant Pathology - In total 58 Xanthomonas strains isolated from Araceae worldwide, together with 13 other phylogenetically-related Xanthomonas strains, were characterized using...     

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.     

Evaluation of triticale accessions for resistance to wheat bacterial leaf streak caused by Xanthomonas translucens pv. undulosa

The bacterium Xanthomonas translucens pv. undulosa (Xtu) causes bacterial leaf streak (BLS) on wheat and other small grains. Several triticale accessions were reported to possess high levels of resistance to wheat Xtu strains. In this study, a worldwide collection of triticale accessions as well as the major North Dakota hard red spring and durum wheat cultivars were evaluated for reaction to two local Xtu strains. All wheat cultivars showed a susceptible reaction but a wide range of reactions was observed among triticale accessions. Out of the 502 accessions tested, 45 and 10 accessions were resistant to the two virulent strains BLS‐LB10 and BLS‐P3, respectively, with five accessions, PI 428736, PI 428854, PI 428913, PI 542545 and PI 587229, being highly resistant to both strains. Statistical analysis showed significant difference among the accessions, strains, and the accession by strain interaction (P < 0.001). Bacterial population growth in resistant triticale was significantly slower than that in susceptible triticale. Molecular cytogenetic characterization in four representative triticale accessions confirmed the hexaploid level of the species and the presence of 12 or 14 rye chromosomes. The triticale accessions identified are valuable materials for developing wheat germplasm with high levels of BLS resistance.     

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.     

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.     

Molecular characterization,comparison of screening methods,and evaluation of cross‐pathogenicity of black rot (Xanthomonas campestris pv. campestris) strains from cabbage,choy sum,leafy mustard and pak choi from Taiwan

Choy sum (Brassica rapa var. parachinensis), leafy mustard (Brassica juncea) and pak choi (B. rapa var. chinensis) are highly nutritious components of diets in Taiwan and other Asian countries, and bacterial black rot caused by Xanthomonas campestris pv. campestris (Xcc) is a major biotic constraint in these crops. As very little was known about the Xcc strains from these crops in these regions, including their cross‐pathogenicity and aggressiveness on different hosts, Xcc strains were obtained from cabbage (Brassica oleracea var. capitata), choy sum, leafy mustard and pak choi crops in Taiwan. Two previously published PCR‐based assays reliably distinguished the Xcc strains from other Xanthomonas species and subspecies. Phylogenetic analysis based on repetitive sequence‐based PCR assays placed the Xcc strains in a clade distinct from other Xanthomonas species, and also showed host specificity. Although all of the Xcc strains from the different host species were pathogenic on all five Brassica test species in both a detached leaf assay and an intact plant assay, in the intact plant assay they showed differences in virulence or aggression on the different test hosts. The Xcc strains from leafy mustard and pak choi were consistently highly aggressive on all the test host genotypes, but the strains from choy sum and cabbage were less aggressive on leafy mustard and choy sum. The intact plant assay proved more discriminating and reliable than the detached leaf assay for comparing the aggressiveness of Xcc strains on different host genotypes, and so, with the new Xcc strains isolated in this study, will be useful for screening leafy brassica germplasm accessions for resistance to black rot.     

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.     

Characterization of Pseudomonas syringae pv. actinidiae (Psa) isolated from France and assignment of Psa biovar 4 to a de novo pathovar: Pseudomonas syringae pv. actinidifoliorum pv. nov.

Since 2008, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis) caused by Pseudomonas syringae pv. actinidiae (Psa) has resulted in severe economic losses worldwide. Four biovars of Psa can be distinguished based on their biochemical, pathogenicity and molecular characteristics. Using a range of biochemical, molecular and pathogenicity assays, strains collected in France since the beginning of the outbreak in 2010 were found to be genotypically and phenotypically diverse, and to belong to biovar 3 or biovar 4. This is the first time that strains of biovar 4 have been isolated outside New Zealand or Australia. A multilocus sequence analysis based on four housekeeping genes (gapA, gltA, gyrB and rpoD) was performed on 72 strains representative of the French outbreak. All the strains fell into two phylogenetic groups: one clonal corresponding to biovar 3, and the other corresponding to biovar 4. This second phylogenetic group was polymorphic and could be divided into four lineages. A clonal genealogy performed with a coalescent approach did not reveal any common ancestor for the 72 Psa strains. Strains of biovar 4 are substantially different from those of the other biovars: they are less aggressive and cause only leaf spots whereas Psa biovars 1, 2 and 3 also cause canker and shoot die‐back. Because of these pathogenic differences, which were supported by phenotypic, genetic and phylogenetic differences, it is proposed that Psa biovar 4 be renamed Pseudomonas syringae pv. actinidifoliorum pv. nov. Strain CFBP 8039 is designated as the pathotype strain.     

Development of a lateral flow device for in‐field detection and evaluation of PCR‐based diagnostic methods for Xanthomonas campestris pv. musacearum,the causal agent of banana xanthomonas wilt

Xanthomonas campestris pv. musacearum (Xcm) is the causal agent of banana xanthomonas wilt, a major threat to banana production in eastern and central Africa. The pathogen is present in very high levels within infected plants and can be transmitted by a broad range of mechanisms; therefore early specific detection is vital for effective disease management. In this study, a polyclonal antibody (pAb) was developed and deployed in a lateral flow device (LFD) format to allow rapid in‐field detection of Xcm. Published Xcm PCR assays were also independently assessed: only two assays gave specific amplification of Xcm, whilst others cross‐reacted with non‐target Xanthomonas species. Pure cultures of Xcm were used to immunize a rabbit, the IgG antibodies purified from the serum and the resulting polyclonal antibodies tested using ELISA and LFD. Testing against a wide range of bacterial species showed the pAb detected all strains of Xcm, representing isolates from seven countries and the known genetic diversity of Xcm. The pAb also detected the closely related Xanthomonas axonopodis pv. vasculorum (Xav), primarily a sugarcane pathogen. Detection was successful in both naturally and experimentally infected banana plants, and the LFD limit of detection was 10⁵ cells mL^?1. Whilst the pAb is not fully specific for Xcm, Xav has never been found in banana. Therefore the LFD can be used as a first‐line screening tool to detect Xcm in the field. Testing by LFD requires no equipment, can be performed by non‐scientists and is cost‐effective. Therefore this LFD provides a vital tool to aid in the management and control of Xcm.     

Genetic and phenotypic diversity of Mediterranean populations of the olive knot pathogen,Pseudomonas savastanoi pv. savastanoi

Pseudomonas savastanoi pv. savastanoi (Psav) is a member of P. syringae sensu lato, and causes olive knot disease, a disease first reported over 2000 years ago. Analysing 124 isolates of Psav from 15 countries by rep‐PCR, the population genetic structure of Psav was investigated. A total of 113 distinct fingerprints were detected. Cluster analysis revealed the existence of two clusters and four subclusters. These clusters were associated with the geographic origin of isolates, which in turn correspond to historic human migration events and trade routes across the Mediterranean Sea. In contrast, multilocus sequence typing (MLST) of 2788 bp of the gapA, gltA, gyrB and rpoD genes found only one variable site among 77 representative isolates. Virulence variation was observed within the Psav population, with the most virulent strains generating knots that had a weight that was 10‐fold greater than those generated by the least virulent strains. Taken together, these data suggest that today's Psav population is the result of clonal expansion of a single strain, that moderate migration of the pathogen occurred between countries, and that changes in virulence arose during its evolution.     

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.     

Species composition and genetic structure of Fusarium graminearum species complex populations affecting the main barley growing regions of South America

Members of the Fusarium graminearum species complex (FGSC), such as F. graminearum and F. asiaticum, are the main cause of fusarium head blight (FHB) of wheat and barley worldwide. In this study, 117 FGSC isolates obtained from commercial barley grain produced in Argentina (n = 43 isolates), Brazil (n = 35), and Uruguay (n = 39) were identified to species and trichothecene genotypes, and analysed using amplified fragment length polymorphism (AFLP) and sequence‐related amplified polymorphism (SRAP) markers. In addition, reductase (RED) and trichothecene 3‐O‐acetyltransferase (Tri101) were sequenced for a subset of 24 isolates. The majority of the isolates (n = 103) were identified as F. graminearum, which was the only species found in Argentina. In Uruguay, only one F. cortaderiae isolate was found among F. graminearum isolates. In Brazil, F. graminearum also dominated the collection (22/35), followed by F. meridionale (8/35), F. asiaticum (2/35), F. cortaderiae (2/35) and F. austroamericanum (1/35). Species were structured by trichothecene genotype: all F. graminearum were of the 15‐acetyldeoxynivalenol (ADON), F. meridionale, F. asiaticum and F. cortaderiae were of the nivalenol (NIV), and F. austroamericanum was of the 3‐ADON genotype. Both AFLP and SRAP data showed high levels of genetic variability, which was higher within than among countries. Isolates were not structured by country of origin. SRAP analysis grouped F. graminearum in a separate cluster from the other species within the complex. However, AFLP analysis failed to resolve the species into distinct clades with partial clustering of F. meridionale, F. austroamericanum, F. asiaticum and F. graminearum isolates.     

Isolation and Sequence of a Gene, celD Xo Involved in Cellobiose Utilization in Xanthomonas oryzae pv. oryzae

A genomic library of Xanthomonas oryzae pv. oryzae (X. o. pv. oryzae) T7174 was screened for 4-methylumbelliferyl β-D-glucoside-hydrolyzing (MUGase) activity. In subcloning of one of the MUGase-positive clones, an approximately 4.2-kb SacI-SphI fragment conferred not only MUGase activity but also 4-methylumbelliferyl β-D-cellobioside-hydrolyzing (MUCase) activity. Sequence analysis showed that the fragment contained an ORF of 2951 bp. The conceptual ORF product was significantly homologous with 1,4-β-D-glucan glucohydrolase D (CELD) from Pseudomonas fluorescens subsp. cellulosa, and was named CELD_Xo. Cell fractionation experiments suggested that CELD_Xo is localized in the cell-envelope fraction. We constructed a CELD_Xo-deficient mutant (74ΔCELD) from X. o. pv. oryzae. Little MUCase activity was detected in the cell-envelope fraction prepared from the mutant. The mutant 74ΔCELD did not grow in synthetic medium containing cellobiose as the sole sugar source. On the other hand, growth in rice leaves and pathogenicity of the mutant and the parental strain did not differ. These results suggested that CELD_Xo is involved in cellobiose utilization of X. o. pv. oryzae but that the gene is not required for bacterial growth in rice leaves. Received 16 February 2001/ Accepted in revised form 11 April 2001     

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.     

Characterization of the Xanthomonas sp. causing wilt of enset and banana and its proposed reclassification as a strain of X. vasicola

Comparative analyses were undertaken to characterize Xanthomonas campestris pv. musacearum, the causal agent of a wilt of enset and banana, and to assess its relatedness to other xanthomonads by fatty acid methyl esters, genomic fingerprinting using rep-PCR and partial nucleotide sequencing of the gyrase B gene. The results from all three analyses indicated that strains of X. campestris pv. musacearum are homogeneous and very similar to X. vasicola strains isolated from sugarcane and maize from Africa. Pathogenicity studies indicated that strains of X. vasicola pv. holcicola and X. vasicola from sugarcane induced no symptoms on banana, whereas X. campestris pv . musacearum produced severe disease. These data will support a future proposed reclassification of X. campestris pv. musacearum as X. vasicola pv . musacearum when more data are available.     

Genomics‐informed design of loop‐mediated isothermal amplification for detection of phytopathogenic Xanthomonas arboricola pv. pruni at the intraspecific level

The objective of this study was to develop a rapid, sensitive detection assay for the quarantine pathogen Xanthomonas arboricola pv. pruni, causal agent of stone fruit bacterial spot, an economically important disease of Prunus spp. Unique targets were identified from X. arboricola pv. pruni genomes using a comparative genomics pipeline of other Xanthomonas species, subspecies and pathovars, and used to identify specific diagnostic markers. Loop‐mediated isothermal amplification (LAMP) was then applied to these markers to provide rapid, sensitive and specific detection. The method developed showed unrivalled specificity with the 79 tested strains and, in contrast to previously established techniques, distinguished between phylogenetically close subspecies such as X. arboricola pv. corylina. The sensitivity of this test is comparable to that of a previously reported TaqMan^? assay at 10³ CFU mL^?1, while the unrivalled speed of LAMP technology enables a positive result to be obtained in <15 min. The developed assay can be used with real‐time fluorescent detectors for quantitative results as well as with DNA‐staining dyes to function as a simplified strategy for on‐site pathogen detection.     

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     

Development of a real‐time PCR method for the detection of the dagger nematodes Xiphinema index,X. diversicaudatum,X. vuittenezi and X. italiae,and for the quantification of X. index numbers

The ectoparasitic dagger nematodes Xiphinema index and Xiphinema diversicaudatum, often at low numbers in the soil, are vectors of grapevine nepoviruses, which cause huge agronomical problems for the vineyard industry. This study reports a method, based on real‐time PCR, for the specific detection of these species and of the closely related non‐vector species Xiphinema vuittenezi and Xiphinema italiae. Specific primers and TaqMan probes were designed from the ribosomal DNA internal transcribed spacer 1 (ITS1), enabling the specific detection of single individuals of each of the X. index, X. diversicaudatum, X. italiae and X. vuittenezi species whatever the nematode population. The specificity of detection and absence of false positive reaction were confirmed in samples of each species mixed with the three other Xiphinema species or mixed with nematodes representative from other genera (non‐plant‐parasitic Dorylaimida, Longidorus sp., Meloidogyne spp., Globodera spp. and Pratylenchus sp.). The method was shown to be valid for the relative quantification of X. index numbers through its use, from crude nematode extracts of soil samples, in a greenhouse assay of grapevine accessions ranging from highly susceptible to resistant. As an alternative to time‐consuming microscopic identification and counting, this real‐time PCR method will provide a fast, sensitive and reliable diagnostic and relative quantification technique for X. index nematodes extracted from fields or controlled conditions.