Pathogenic and molecular comparison of Puccinia kuehnii isolates and reactions of sugarcane varieties to orange rust

Sugarcane orange rust, a disease caused by Puccinia kuehnii, was first reported in Brazil in 2009. There are no studies comparing the Brazilian P. kuehnii collections and the reaction of important sugarcane varieties under controlled conditions. This work compared the reaction of seven sugarcane varieties inoculated with six different P. kuehnii isolates from Brazilian sugarcane areas and verified the pathogenic and genetic variability of these isolates. The incubation (I) and latency (L) disease periods, disease severity (SEV), total number of lesions (TNL), total number of sporulating lesions (TNSL), and percentage of sporulating lesions (%SL) were evaluated. Furthermore, ITS1 and IGS ribosomal sequences of all P. kuehnii isolates used in this study were compared with pathogen sequences from 13 different countries. The disease incubation ranged from 7 to 10 days and the latency ranged from 10 to 21 days. SEV and TNL showed large variations and few significant differences between the reaction of the varieties to P. kuehnii, in contrast with the variables TNSL and %SL. The P. kuehnii isolates did not compose different virulent races, but the isolate from one site (Araras) was a more aggressive race. The ITS1 and IGS ribosomal sequences of six P. kuehnii isolates were identical with each other and to most P. kuehnii American sequences deposited at GenBank. The studied sequences of P. kuehnii isolates differed from the sequences from Asia, Tahiti and Oceania.     

Occurrence of two races of Puccinia kuehnii causing orange rust of sugarcane in Florida

Orange rust of sugarcane caused by Puccinia kuehnii was first reported in Florida in 2007. Since then, several sugarcane cultivars that were resistant during the initial epidemics became susceptible within a few years. These shifts in resistance were attributed to the evolution of the pathogen and appearance of new races. To study the variation in virulence of P. kuehnii, healthy leaf pieces of sugarcane cultivars susceptible to orange rust were brush inoculated with isolates of P. kuehnii collected from susceptible cultivars in the field. After inoculation, leaf pieces were placed in an incubator and disease severity based on the number of rust uredinia was determined 2 weeks postinoculation. Isolates of P. kuehnii collected from sugarcane cultivar CP 89-2143, which only showed severe symptoms of orange rust starting in 2011–2012, produced 300%–500% more uredinia on CP 89-2143 than the isolates collected from cultivar CL 85-1040 that has been susceptible since 2007. Sugarcane cultivar CL 85-1040 exhibited high and equivalent numbers of uredinia regardless of the inoculated isolate of the pathogen. These data support the occurrence of pathogenic specialization within P. kuehnii and the existence of at least two races of this pathogen in Florida. Analysis of amplified fragment-length polymorphism among isolates of P. kuehnii from cultivars CP 89-2143 and CL 85-1040 differing in resistance to orange rust revealed genetic variation among rust uredinia. However, this variation was not associated with a specific sugarcane cultivar, suggesting that pathogenic variation was not linked to major, but rather to small genetic changes within the genome of P. kuehnii.     

Distinguishing sporulating and nonsporulating lesions as a method for evaluating the resistance of sugarcane genotypes to orange rust

Sugarcane breeding programmes rank the resistance of genotypes to Puccinia kuehnii, causal agent of orange rust, according to levels of disease severity. However, during the screening stages, this method of assessment can lead to precipitous elimination of genotypes with promising agronomic traits but showing mild symptoms of rust such as flecks or lesions that do not produce spores. This study aimed to propose a new method to classify the resistance of sugarcane genotypes to orange rust by counting sporulating lesions. Five sugarcane varieties with different levels of resistance to P. kuehnii were inoculated with two pathogen populations under controlled conditions. The disease severity (SEV), total number of lesions (TNL), and total number of sporulating lesions (TNSL) were evaluated in a 20 cm leaf fragment from the most diseased leaf. The TNL and TNSL evaluations were performed at 11, 16 and 21 days after inoculation (DAI) and SEV at 21 DAI. The thresholds of 80% and 8% of sporulating lesions (SL) separated susceptible from the intermediate varieties and intermediate from the resistant ones, respectively. It is proposed that the method of counting sporulating lesions be used in screening genotypes for resistance to P. kuehnii in sugarcane breeding programmes.     

Phylogenetic Analysis of Sugarcane Rusts Based on Sequences of ITS, 5.8 S rDNA and D1/D2 Regions of LSU rDNA

Phylogenetic analysis of sugarcane rusts based on sequences of ITS and the 5.8 S rDNA revealed two highly divergent ITS groups among isolates of Puccinia sp. sensu Muta, 1987 and P. kuehnii specimens. Although there is sufficient divergence (exceeding normal intraspecific variation) between the ITS regions of the two groups to support separation into different species, unusually high homology of the ITS group I sequences with those of members of Cronartium and identical sequences of the D1/D2 regions of the LSU rDNA for all the isolates of “Puccinia sp.” and P. kuehnii that otherwise exhibited different ITS sequences, suggest that the two highly divergent sequences may have resulted from abnormal genetic events leading to non-orthologous, intraspeciflc polymorphisms. The other sugarcane rust, P. melanocephala and the grass rusts, P. miscanthi and P. rufipes, were separated from “Puccinia sp.” and P. kuehnii and from each other in D1/D2 region analyses, indicating that D1/D2 region sequences may more correctly reflect phylogenetic relationships in these rusts than do the ITS regions. Further studies to examine differences in patho-genicity or finer morphological features within P. kuehnii that may be correlated with the high divergence in ITS sequences and experiments to determine if these two sequence types represent intraspeciflc polymorphism are necessary. Received 11 October 2000/ Accepted in revised form 24 November 2000     

The development of a PCR-based method for detecting <Emphasis Type="Italic">Puccinia striiformis</Emphasis> latent infections in wheat leaves

Stripe rust of wheat caused by Puccinia striiformis f. sp. tritici is one of the most important diseases on wheat worldwide, especially in temperate regions with cool moist weather conditions. A rapid and reliable detection of the pathogen in latent infected wheat leaves during overwintering of the fungus in the dormant stage will contribute to determine the initial inoculum potential and thus to predict early outbreak and to improve effective management of the disease. To achieve this aim, a PCR-based method was developed for specific and sensitive detection of P. striiformis. Specific primers were designed according to a genome-specific sequence of P. striiformis. To evaluate the specificity of the primers, seven different isolates and races of P. striiformis as well as six other pathogens of wheat were tested. All isolates of P. striiformis yielded a distinct band of a fragment of 470 bp, while using DNA of the other wheat pathogens as a template no amplification product was detected. The sensitivity of the primers was tested using serial dilutions of total DNA from P. striiformis; the limit of detection was 10 pg of DNA. Using extracts from P. striiformis-infected wheat leaves, the fungus could be determined in the leaves before symptoms appeared. The stripe rust could also be detected in the dormant stage by the PCR assay in samples of wheat leaves taken during the winter season. The application of the PCR assay may be useful for rapid and reliable detection of P. striiformis in latent infected leaves of overwintering wheat plants.     

Molecular identification and prevalence of Acidovorax avenae subsp. avenae causing red stripe of sugarcane in China

Red stripe caused by the bacterium Acidovorax avenae subsp. avenae (Aaa) is a disease of sugarcane that is distributed worldwide. In this study, 108 sugarcane leaf samples were collected in 2013–2016 from nine sugarcane‐growing regions in China. Aaa was detected by PCR with specific and novel primers from the 16S–23S rDNA internal transcribed spacer region in 81 of 84 (96%) leaves with red stripe symptoms and in 20 of 24 (83%) leaves without symptoms. Furthermore, Aaa was detected in all nine sampling locations representing six sugarcane‐producing provinces in China. The 101 amplified fragments were cloned and sequenced. The size of the nucleotide sequences varied from 436 to 454 bp and the sequence identity ranged from 89.2% to 100%, suggesting a significant genetic variation among Aaa strains from China. Five major restriction fragment length polymorphism (RFLP) profiles were obtained by in silico and polyacrylamide gel electrophoresis analyses of the PCR products digested with HindIII and EcoRI. The causal agent of sugarcane red stripe was also successfully isolated from a diseased plant and its pathogenicity confirmed by inoculation of healthy sugarcane plantlets and reproduction of disease symptoms. The data showed that Aaa is currently widespread in China, suggesting that control methods should be implemented to limit the impact of red stripe on sugarcane production.     

小麦条锈病菌和白粉病菌多重TaqMan Real-time PCR方法的建立

<正>小麦条锈病(Puccinia striiformis f.sp.tritici,Pst)和小麦白粉病(Blumeria graminis f.sp.tritici,Bgt)是我国小麦生产上的重要病害。条锈病主要发生在西北、华北、长江中下游和西南各省、自治区;白粉病则在西南各省和河南、山东、湖北、江苏、安徽等省发生较重,且西北、东北麦区也日趋严重[1]。条锈病菌依靠夏孢子造成小麦初侵染和再侵染并随气流远距离传播导致大区流行,白粉病菌则依赖于分生孢子或子囊孢子进行初侵染和再侵染。二者作为典型的气传病害,空中的接种体在     

Detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. palmivora</Emphasis> in citrus roots using PCR-RFLP in comparison with other methods

Phytophthora nicotianae and P. palmivora are the most important soil-borne pathogens of citrus in Florida. These two species were detected and identified in singly and doubly infected plants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS) regions of ribosomal DNA. The sensitivity of the PCR-RFLP was analyzed and the usefulness of the method evaluated as an alternative or supplement to serological methods and recovery on semi-selective medium. In a semi-nested PCR with universal primers ITS4 and ITS6, the detection limit was 1 fg of fungal DNA, which made it 1000× more sensitive than a single-step PCR with primers ITS4 and DC6. The sensitivity of detection for P. nicotianae was shown to be ten-fold lower than for P. palmivora, limiting its detection with restriction profiles in plants infected by both fungal species. Phytophthora nicotianae was detected with species-specific primers in all samples inoculated with this species despite the absence of species-specific patterns in RFLP. In contrast, the incidence of detection of P. palmivora in the presence of P. nicotianae was considerably lower using plating and morphological detection methods. Due to its high sensitivity, PCR amplification of ribosomal ITS regions is a valuable tool for detecting and identifying Phytophthora spp. in citrus roots, provided a thorough knowledge of reaction conditions for the target species is established prior to the interpretation of data.     

PCR‐based identification of Pythium spp. causing cavity spot in carrots and sensitive detection in soil samples

On the basis of ITS sequences PCR primers were designed for the identification of the five Pythium species found to be most important for the development of carrot cavity spot in Norway: P. intermedium, P. sulcatum, P. sylvaticum, P. violae and P. ‘vipa’. The P. ‘vipa’ isolates had a unique ITS sequence, differed morphologically from all other Pythium isolates, and thus probably represent a new species. The PCR primers were species‐specific with no cross‐reaction to other Pythium species or to fungal isolates from carrot tested. The detection limits varied for the different primer pairs. The two most sensitive assays allowed detection of as little as 5 fg DNA. All five Pythium species could be detected in lesions from diseased carrots. Weak positive signals were obtained from some carrot samples without symptoms. PCR assays allowed detection of pathogens in soil. In samples of soil known to produce cavity spots on cropped carrots, strong signals were obtained. In several soil samples more than one of the five Pythium species could be detected. The utilization of this diagnostic PCR assay in analysis of field soil and carrot tissue might in the future be exploited to reduce the incidence of this serious carrot disease.     

Usefulness of single copy genes containing introns in Phytophthora for the development of detection tools for the regulated species P. ramorum and P. fragariae

Introns are generally highly polymorphic regions within genes and were proven to be of great interest for discriminating among phylogenetically-close Phytophthora species. Phytophthora ramorum and P. fragariae are considered as quarantine pathogens by the European Union and accurate detection tools are therefore necessary for their monitoring. From introns located in different single copy genes (GPA1, RAS-like, and TRP1), we developed a series of PCR primers specific to P. ramorum and P. fragariae. The specificity of these primers was successfully checked with a wide collection of Phytophthora isolates and a protocol was developed to detect both pathogens directly in infected plant tissues. These genes should be of particular interest for the development of additional species-specific detection tools within the Phytophthora genus.     

Puccinia psidii in Queensland,Australia: disease symptoms,distribution and impact

Puccinia psidii has long been considered a significant threat to Australian plant industries and ecosystems. In April 2010, P. psidii was detected for the first time in Australia on the central coast of New South Wales (NSW). The fungus spread rapidly along the east coast and in December 2010 was found in Queensland (Qld) followed by Victoria a year later. Puccinia psidii was initially restricted to the southeastern part of Qld but spread as far north as Mossman. In Qld, 48 species of Myrtaceae are considered highly or extremely susceptible to the disease. The impact of P. psidii on individual trees and shrubs has ranged from minor leaf spots, foliage, stem and branch dieback to reduced fecundity. Tree death, as a result of repeated infection, has been recorded for Rhodomyrtus psidioides. Rust infection has also been recorded on flower buds, flowers and fruits of 28 host species. Morphological and molecular characteristics were used to confirm the identification of P. psidii from a range of Myrtaceae in Qld and compared with isolates from NSW and overseas. A reconstructed phylogeny based on the LSU and SSU regions of rDNA did not resolve the familial placement of P. psidii, but indicated that it does not belong to the Pucciniaceae. Uredo rangelii was found to be con‐specific with all isolates of P. psidii in morphology, ITS and LSU sequence data, and host range.     

Specific and Sensitive Detection of Phytophthora nicotianae By Simple and Nested-PCR

Phytophthora nicotianae Breda de Haan is one of the most important soil-borne plant pathogens. The identification of this pathogen based on morphological or physiological characters is time-consuming and labour-intensive and requires comprehensive knowledge of fungi. Molecular analysis of the internal transcribed spacer (ITS) regions of rDNA is a novel and very effective method of species determination. Based on this concept, conventional and single closed tube nested-PCRs were developed for the specific and sensitive detection of P. nicotianae. Two new specific primers, designed from the spacer regions ITS1 and ITS2, internal to the nucleotide sequence flanked by universal primers ITS4 and ITS6, were used. To evaluate the specificity of the method, 36 morphologically characterized isolates were tested. A positive reaction, characterized by an amplification product of 737 bp, was shown by all P. nicotianae isolates and two P. nicotianae/cactorum hybrids. No amplification product was observed when other Phytophthora species and genera were assayed. The sensitivity of this method was analysed by serial dilutions of a defined amount of fungal DNA in a healthy root extract. Nested-PCR was at least 1000 times more sensitive than conventional PCR. In addition, samples from different infection sites, origins and crops, samples from nutrient solution, water and the rockwool used in hydroponic cultures, were analysed to validate this method.     

Genetic Relatedness among Pseudomonas avellanae, P. Syringae pv. Theae and P.s. pv. Actinidiae, and their Identification

A total of 37 strains of Pseudomonas avellanae, P. syringae pv. theae and P.s. pv. actinidiae, including pathotype and reference strains, obtained from all the countries where these pathogens have been reported, were compared by means of ARDRA, repetitive PCR using ERIC, BOX and REP primer sets, whole-cell protein analysis, biochemical and nutritional tests, and pathogenicity tests. P. syringae pathovar type strains representing six genomospecies sensu Gardan et al. (1999), were also included for comparison in UPGMA cluster analysis of repetitive PCR data and SDS-PAGE of protein extracts. Among the 12 endonucleases used in ARDRA, only Tru 9I differentiated P. avellanae from P.s. pv. theae and P.s. pv. actinidiae. UPGMA cluster analysis of repetitive PCR genomic fingerprints showed 65% similarity between P.s. pv. theae and P. avellanae and 50% between the latter species and P.s. pv. actinidiae. Strains of P.s. pv. actinidiae could be grouped according to their geographic origin. Similar results were obtained with SDS-PAGE cluster analysis. PCR amplification using primers PAV 1 and PAV 22 that were developed to detect P. avellanae in apparently healthy and visibly infected hazelnut specimens yielded a band of 762bp from all strains of P. avellanae, P.s. pv. theae and P.s. pv. actinidiae. All strains lacked the syrB gene. Based on these data, we suggest that P.s. pv. actinidiae should be included in the genomospecies 8 together with P. avellanae and P.s. pv. theae. Selected biochemical and nutritional tests could differentiate these groups of strains. Pathogenicity tests clearly indicated that each group is specifically pathogenic only on the host plant species from which it was originally isolated.The author is staff member of the Istituto Sperimentale per la Patologia Vegetale, Roma, Italy temporarily assigned to ISF.     

A Phytophthora conserved transposon‐like DNA element as a potential target for soyabean root rot disease diagnosis

A transposon‐like element, A3aPro, with multiple copies in the Phytophthora sojae genome, was identified as a suitable detection target for this devastating soyabean root rot pathogen. The PCR primers TrapF1/TrapR1 were designed based on unique sequences derived from the transposon‐like sequence. A 267‐bp DNA fragment was amplified using this primer pair, the specificity of which was evaluated against 118 isolates of P. sojae, 72 isolates of 25 other Phytophthora spp., isolates of Pythium spp. and isolates of true fungi. In tests with P. sojae genomic DNA, detection sensitivities of 10 pg and 10 fg DNA were achieved in standard PCR (TrapF1/TrapR1) and nested PCR (TrapF1/TrapR1 and TrapF2/TrapR2), respectively. Meanwhile, PCR with TrapF1/TrapR1 primers detected the pathogen at the level of a single oospore, and even one zoospore. These primers also proved to be efficient in detecting pathogens from diseased soyabean tissues, residues and soils. In addition, real‐time quantitative PCR (qPCR) assays coupled with the TrapF1/TrapR1 primers were developed to detect and quantify the pathogen. The results demonstrated that the TrapF1/TrapR1 and TrapF2/TrapR2 primer‐based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in plants and in production fields.     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

Detection of Phytophthora nicotianae and P. citrophthora in Citrus Roots and Soils by Nested PCR

The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B–Pn6 and Pc2B–Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2–ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1pgl^–1 DNA for all the primer pairs (Ph2–ITS4, Pn5B–Pn6, and Pc2B–Pc7). In nested PCR, with primers Ph2–ITS4 in the first round, the detection limit was of 1fgl^–1 for both the primer sets (Pn5B–Pn6 and Pc2B–Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2–3h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2–ITS4 in the first round. In the second round the primer pairs Pn5B–Pn6 and Pc2B–Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.     

Temperature adaptation in Australasian populations of Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the major fungal pathogens of wheat. A new pathotype was introduced to Australia in 2002 and several derivative pathotypes were detected in subsequent seasons. It has been suggested that the severity of stripe rust outbreaks in Australia since 2002 could be as a result of traits other than virulence in the pathogen population. This study was conducted to investigate the hypothesis that the stripe rust pathogen population dominant in Australia since 2002 was better adapted to warm temperature conditions compared to previous pathogen populations. Sixteen pathotypes were selected to examine the influence of two contrasting temperature regimes during the 24 h incubation (10°C and 15°C) and the subsequent post‐inoculation (17°C and 23°C) periods on latent period and infection efficiency on four susceptible wheat cultivars. In addition, the effect of two contrasting incubation temperatures on urediniospore germination was examined. The results indicated that pathotypes of P. striiformis f. sp. tritici detected after 2002 did not show evidence of adaptation to high temperatures, which suggests that other factors contributed to the observed increased aggressiveness.     

Detection of botryosphaeriaceous species in environmental samples using a multi‐species primer pair

Botryosphaeriaceous species are significant grapevine trunk pathogens worldwide, which can be difficult to identify to species level using conventional morphological methods. This study developed and optimized a quick, reliable molecular identification method that could facilitate investigations into the epidemiology of these diseases in vineyards. The multi‐species primers, BOT100F and BOT472R, amplified a 371–372 bp portion of the rRNA gene region from the six botryosphaeriaceous species commonly found in New Zealand vineyards. In silico analysis indicated that they would amplify DNA from six of the 12 lineages of the Botryosphaeriaceae, including all of the main species pathogenic to grapevines. A detection sensitivity of 1 and 0·1 pg DNA in standard and nested PCR, respectively, was achieved and this was calculated as equivalent to 2·5 conidia. Validation of the primers for environmental samples showed that their specificity was not compromized by the presence of competing DNA templates extracted from wood and soil. Single stranded conformational polymorphism (SSCP) analysis of the amplicons could resolve Neofusicoccum australe, N. luteum, Diplodia mutila and D. seriata, but did not differentiate between N. parvum and N. ribis. The optimized PCR‐SSCP was used to identify botryosphaeriaceous species present in rainwater traps collected over 1 year in a vineyard known to contain infected vines. It could detect multiple species in individual samples and demonstrated differences in the dispersal patterns of conidia from different species. Given the specificity and sensitivity of this method it could prove useful in epidemiology studies involving the numerous botryosphaeriaceous species that infect a wide range of host species.     

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.