Detection of plum pox potyvirus using monoclonal antibodies to structural and non-structural proteins1

Eleven monoclonal antibodies specific to plum pox potyvirus (PPV) coat protein were obtained by hybridoma technology from Spanish PPV isolates. In addition, two monoclonal antibodies specific for PPV cylindrical inclusions (CIP non-structural proteins) were obtained. The monoclonal antibodies specific for PPV coat protein were assayed by DASI ELISA against 81 PPV isolates. At least nine different epitopes were found and 21 distinct serological patterns of reaction (serogroups) were established using nine selected monoclonal antibodies against the collection of PPV isolates, indicating the high variability of coat protein among PPV isolates. Changes in epitope composition were observed after aphid and mechanical transmission, indicating the occurrence of mixtures of isolates in field trees. Monoclonal antibody 5B reacted with all PPV isolates assayed, with very high affinity, using DASI ELISA. This method was compared with immunocapture-PCR on field samples in spring, and showed very good coincidence of results. The efficiency of PPV detection can be slightly increased using monoclonal antibodies specific to cylindrical inclusions mixed with monoclonal antibodies against structural proteins, and using mixtures of monoclonal antibodies against different epitopes of coat protein. ELISA-I and immunoprinting-ELISA were able to detect CIP and PPV in extracts and tissue section, respectively, of woody plants. Two monoclonal antibodies offer the possibility of distinguishing between Marcus and Dideron PPV types (M or D). These D-specific monoclonal antibodies can be used in routine tests with high affinity.     

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.     

Spread and interaction of Pepino mosaic virus (PepMV) and Pythium aphanidermatum in a closed nutrient solution recirculation system: effects on tomato growth and yield

Pepino mosaic virus (PepMV) was shown to be efficiently transmitted between tomato plants grown in a closed recirculating hydroponic system. PepMV was detected in all plant parts after transmission via contaminated nutrient solution using ELISA, immunocapture RT‐PCR, RT‐PCR, electron microscopy, and by inoculation to indicator plants. Detection of PepMV in nutrient solution was only possible after concentration by ultracentrifugation followed by RT‐PCR. Roots tested positive for PepMV 1–3 weeks after inoculation, and subsequently a rapid spread from the roots into the young leaves and developing fruits was found within 1 week. PepMV was only occasionally detected in the older leaves. None of the infected plants showed any symptoms on fruits, leaves or other organs. Pre‐infection of roots of tomato cv. Hildares with Pythium aphanidermatum significantly delayed PepMV root infections. When mechanically inoculated with PepMV at the 2–4 leaf stage, yield loss was observed in all plants. However, only plants of cv. Castle Rock recorded significant yield losses when infected via contaminated nutrient solution. Yield losses induced by infection with PepMV and/or P. aphanidermatum ranged from 0·4 up to 40% depending on experimental conditions.     

Latent entry and spread of Colletotrichum acutatum (species complex) in strawberry fields

Anthracnose, caused by Colletotrichum acutatum (species complex), has become a troublesome problem in strawberry production worldwide. This paper reports (i) an optimized sampling method combined with a real‐time PCR technique to detect the latent presence of C. acutatum in cold‐stored strawberry plants used as planting material in several European countries, and (ii) a study of the spread of C. acutatum following a point inoculation under field conditions. Screening of different parts of planting material suggested that C. acutatum is most likely to be present on runners and old petioles. In addition, in seven out of nine batches of planting material from different nurseries, latent infection by C. acutatum was detected in at least one of five replicate samples. Field experiments in 2009 and 2010 showed extensive latent within‐field spread of the pathogen on strawberry leaves, with a within‐row dispersal distance up to at least 1·75 m in 1 week. A straw ground cover between the rows did not decrease C. acutatum spread, probably because introduced (and/or subsequent) inoculum was confined to the plant bed (within the row) and was not present between the beds. Moreover, the number of C. acutatum spores on the symptomless leaves, as estimated using a real‐time PCR method, was significantly (P < 0·05) correlated with the incidence of fruit rot at harvest and post‐harvest (r = 0·56–0·66). These results illustrate the importance of detecting latent infections in planting material and strawberry leaves in the field.     

Serendipitous identification of a new Iflavirus‐like virus infecting tomato and its subsequent characterization

The genomic sequence of a previously undescribed virus was identified from symptomless tomato plants (Solanum lycopersicum). The viral genome is a positive‐sense ssRNA molecule of 8506 nucleotides. It is predicted to encode a single polyprotein of 314·5 kDa that is subsequently processed into three coat protein components of 13·7, 17·9 and 13·5 kDa, and a viral replicase of approximately 207 kDa with conserved motifs for a helicase, a protease and RNA‐dependent RNA polymerase (RdRp). Pairwise analysis of the deduced amino acid sequence of the RdRp revealed that it shares closest identity with members of the family Iflaviridae, genus Iflavirus (19–47% identity). Evidence of replication in plants was detected by RT‐PCR of the viral replicative strand, and short interfering RNAs (siRNAs) matching the virus. The name Tomato matilda virus (TMaV) is proposed, and furthermore, that the genus Tomavirus (Tomato matilda virus) be created within the family Iflaviridae. This is the first report of a plant‐infecting virus resembling members of the Iflaviridae.     

Direct tuber testing for Potato Y potyvirus by real‐time RT‐PCR and ELISA: reliable options for post‐harvest testing?*

The method currently used for testing potato tubers for viruses following harvest involves a growing‐on test. This takes up to 6 weeks to complete, and there is therefore a demand for more rapid test results. The sensitivity and reliability of direct tuber testing by DAS‐ELISA and real‐time RT‐PCR (TaqMan) were compared with the growing‐on test. In addition, the reliability of all three methods for the detection of Potato Y potyvirus (PVY) in tubers was compared over post‐harvest intervals of 6, 10, 14 and 18 weeks. The test material came from plots of tubers (cv. ‘Maris Piper’) containing a primary infection of strains PVY^N and PVY^O, following aphid transmission from marked infector plants grown during the 2003 season. Sample material was homogenized and divided, to provide comparative test material for detection of PVY by ELISA and real‐time RT‐PCR. Tuber eye‐plugs were then taken and subjected to the growing‐on test. The remainder of the tuber was also grown on and tested, to ensure infection was not missed as a consequence of an uneven distribution of virus throughout the tuber material. The results obtained using the two methods for direct testing of the tubers, and those results obtained from the traditional growing‐on test, are compared. The advantages and disadvantages of each method are discussed.     

Virus transmission studies and diagnostic detection of four begomovirus isolates in selected crop hosts and in Bemisia tabaci biotype B*

Virus transmission studies were conducted under glasshouse conditions using the vector Bemisia tabaci biotype B to determine how effectively isolates of the begomoviruses Tomato yellow leaf curl virus (TYLCV) and Tomato leaf curl Bangalore virus (ToLCBV) could be transmitted to phaseolus bean, capsicum and tomato test plants, the latter host used as a positive control for transmission. Diagnostic detection of viruses in these host crops and vector was also evaluated. Polymerase chain reaction (PCR) detection of TYLCV in bean cv. Wade and capsicum cv. Bellboy was achieved 4 weeks after fumigation in asymptomatic plants. Detection of TYLCV in tomato controls was achieved 2 weeks after fumigation with improved frequency of detection at 4 weeks. PCR was found to be a more sensitive method than triple‐antibody sandwich enzyme‐linked immunosorbent assay (TAS‐ELISA) for the detection of TYLCV isolates in all hosts. ToLCBV was detected by PCR and TAS‐ELISA in bean. TYLCV was also detected by PCR in the vector, with a novel internal positive control. This work was carried out to facilitate the development of a diagnostic protocol for the begomoviruses causing tomato yellow leaf curl under the EU SMT programme project –‘Diagnostic protocols for organisms harmful to plants’ (DIAGPRO).     

Application of real‐time RT‐PCR for large‐scale testing of potato for Potato spindle tuber pospiviroid*

The real‐time RT‐PCR protocol of Boonham and coworkers performed extremely well in a recent ring test, comparing different methods for detection of Potato spindle tuber pospiviroid (PSTVd) in several laboratories. Since, in addition, real‐time PCR technology has proved suitable for high‐throughput testing, this method was chosen as the starting point for the development of a protocol for the large‐scale testing of potato. The initial experiments focused on the specificity of the primers and probes with regard to different isolates of PSTVd and other (pospi‐) viroids. Further experiments were performed with leaf material from both primarily and secondarily infected plants. The parameters studied were sampling position, growing‐on temperature and bulking rate. In addition, different grinding, nucleic‐acid extraction and disinfection methods were compared. To monitor false negatives and positives, different controls were included and tested in duplex and triplex formats. The final protocol was tested using a hundred samples from the Dutch potato‐monitoring programme. The results of this pilot experiment were promising. Future plans include the development of a protocol for direct tuber testing and inter‐laboratory ring testing of the protocols.     

Sensitive detection of Xanthomonas axonopodis pv. dieffenbachiae on Anthurium andreanum by immunocapture-PCR (IC-PCR) using primers designed from sequence characterized amplified regions (SCAR) of the blight pathogen

One of the most devastating Xanthomonas diseases affecting the Anthurium cut flower industry worldwide is the bacterial blight caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad). The disease can be spread through latently infected tissue-cultured plants that are used for the propagation of Anthurium worldwide. Current disease diagnostic techniques involve the use of semi-selective media and serological tests. This study describes the development of a PCR tool combined with a genus-specific monoclonal antibody for the sensitive detection of the pathogen directly from plants. It was demonstrated that the immunocapture PCR (IC-PCR) was more sensitive than the conventional PCR and even more sensitive than indirect ELISA for the detection of the pathogen. Latently infected plants could be positively screened for the presence of the pathogen. Three sets of primers were designed from DNA probes that were reported to show some specificity to the pathovar dieffenbachiae. The use of all three sets of primers in a single reaction successfully amplified the three individual loci when bacterial DNA was used as a template. The multiplex PCR generated PCR profiles that could differentiate between the reference strains of X. axonopodis pv. dieffenbachiae from other control bacteria. The new primers could therefore be used both for the diagnosis of Anthurium blight in single PCR reactions and also for the profiling of Xanthomonas. pv. dieffenbachiae strains using the multiplex PCR technique.     

Selection,optimization and characterization of molecular tests for the detection of Tobacco ringspot virus (TRSV)

The aim of the study was to select, optimize and characterize RT‐PCR tests for the detection of Tobacco ringspot virus (TRSV) in post‐entry quarantine. Among five different tests, the Poojari et al. (Journal of Virological Methods 235, 112–118) and Jossey & Babadoost (Plant Disease 90, 1361) conventionnal RT‐PCR tests were chosen and optimized for this purpose. Ten TRSV isolates, 17 healthy plants from the genera Vitis, Prunus and Malus, four other Nepoviruses or Secoviridae isolates and serial dilutions of three TRSV isolates were used for the complete characterization of the optimized tests. In the tested conditions, the Poojari and Jossey & Babadoost tests respectively showed 100% and 95% inclusivity, 100% and 100% exclusivity, 100% and 96.4% analytical specificity, 100% and 100% diagnostic specificity, [10^?7; 10^?1] and [10^?8; 10^?2] limit of detection dilution factors (analytical sensitivity), 91.1% and 90.0% repeatability, 90.3% and 91.1% reproducibility and 100% and 100% selectivity. Due to its higher inclusivity, the optimized Poojari test is recommended for the detection of TRSV isolates for post‐entry quarantine purposes, but can also be used for global surveys. The optimized Jossey & Babadoost test showed the best analytical sensitivity, suggesting that combining both tests can further enhance detection.     

PM 7/101 (1): ELISA tests for plant pathogenic bacteria

Specific scope

Enzyme Linked Immuno Sorbent Assay (ELISA) tests for bacteria may be used for screening of large numbers of samples as an alternative to immunofluorescence (IF) in certain cases (see PM 7/97 Indirect Immunofluorescence test for plant pathogenic bacteria). They can also be used as part of the identification of pure cultures. This standard describes how to perform an ELISA test for detection and/or identification in bacterial diagnostics using: (i) Indirect; (ii) Double antibody sandwich‐DAS; (iii) Double antibody sandwich indirect‐DASI also named triple antibody sandwich‐TAS; and (iv) Direct tissue‐print, squash or colony‐dot.

Specific approval and amendment

Approved as an EPPO Standard in 2010–09     

Transmission of ‘Candidatus Liberibacter solanacearum’ in carrot seeds

A protocol for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in carrot seeds using real‐time PCR was developed. The bacterium was detected in 23 out of 54 carrot seed lots from 2010 to 2014, including seeds collected from diseased mother plants. The average total number of ‘Ca. L. solanacearum’ cells in individual seeds ranged from 4·8 ± 3·3 to 210 ± 6·7 cells per seed from three seed lots, but using propidium monoazide to target live cells, 95% of the cells in one seed lot were found to be dead. Liberibacter‐like cells were observed in the phloem sieve tubes of the seed coat and in the phloem of carrot leaf midrib from seedlings. The bacterium was detected as early as 30 days post‐germination, but more consistently after 90 days, in seedlings grown from PCR positive seed lots in an insect‐proof P2 level containment greenhouse. Between 12% and 42% of the seedlings from positive seed lots tested positive for ‘Ca. L. solanacearum’. After 150 days, symptoms of proliferation were observed in 12% of seedlings of cv. Maestro. ‘Candidatus Liberibacter solanacearum’ haplotype E was identified in the seeds and seedlings of cv. Maestro. No phytoplasmas were detected in seedlings with symptoms using a real‐time assay for universal detection of phytoplasmas. The results show that to prevent the entry and establishment of the bacterium in new areas and its potential spread to other crops, control of ‘Ca. L. solanacearum’ in seed lots is required.     

The question of seed transmissibility of Plum pox virus

For many years, Plum pox virus (PPV) was considered to be transmissible by seed, increasing the fear of long-distance spread of the disease. In the late 1970s, it was claimed on the basis of biological transmission of the virus to herbaceous indicator plants and the development of serological diagnosis based on polyclonal antibodies, that PPV was seed-transmitted, with a different infection rate according to the plant species and part of the seed which was tested. In the 1990s, PPV was characterized into four different types, and specific monoclonal antibodies were produced for them. These new and more sensitive diagnostic techniques, together with RT-PCR with different sets of specific primers, were used to approach once again the problem of PPV transmission through seeds. The virus was detected in seed coats and cotyledons, but embryonic tissue and seedlings obtained from germinated seeds never showed symptoms, and gave negative results for PPV with both ELISA and PCR assays. No PPV isolate is currently recognized to be seed transmitted, so vertical transmission of PPV from infected mother plants to their progeny does not occur. Hypothetically, the only possibility of seed transmission would arise from a mutation in the helper component of the virus, associated with high susceptibility of the infected Prunus cultivar.     

PCR assays for the sugarcane rust pathogens Puccinia kuehnii and P. melanocephala and detection of a SNP associated with geographical distribution in P. kuehnii

Puccinia kuehnii and P. melanocephala cause orange and brown rust of sugarcane, respectively. Puccinia kuehnii has been confirmed in Asia, Australia and recently, the Caribbean basin, whereas P. melanocephala is distributed among the majority of sugarcane growing regions. Differentiating these two economically significant pathogens visually is problematic and limited to material exhibiting mature disease symptoms or spores. Partial ITS1, ITS2 and complete 5·8S sequences were generated from P. kuehnii and P. melanocephala isolates from around the world. PCR primers and dual labelled hydrolysis probes were designed for each pathogen for use in real‐time PCR and optimized using locked nucleic acids (LNA). The primers amplified DNA from their target pathogens and not from other species of Puccinia or fungal species isolated from sugarcane leaves. Optimized real‐time PCR conditions allowed the detection of 0·19 pg of P. kuehnii or P. melanocephala genomic DNA and differentiated the pathogens on sugarcane leaves prior to observing typical symptoms in the field. Primer‐introduced restriction analysis‐PCR (PIRA‐PCR) was used to detect a single nucleotide polymorphism (Pk ITS1 183A>G) in ITS1 of P. kuehnii. Allele 183A was observed in all samples, whereas 183G was detected in 52% of samples from Asia and Australia yet absent from all Caribbean basin samples. Long distance spore dispersal, dispersal through an intermediate location or improper movement of contaminated material could explain the introduction of P. kuehnii to the Western hemisphere. However, the current proliferation of the pathogen in the Americas is limited to isolates which contain only the 183A allele.