Calculation of diagnostic parameters of advanced serological and molecular tissue-print methods for detection of Citrus tristeza virus: a model for other plant pathogens.

Citrus tristeza virus (CTV) is one of the most important virus diseases that affect citrus. Control of CTV is achieved by grafting selected virus-free citrus scions onto CTV-tolerant or -resistant rootstocks. Quarantine and certification programs are essential for avoiding the entry and propagation of severe strains of CTV. Citrus nurseries in Spain and central California (United States) maintain zero-tolerance policies for CTV that require sensitive, specific, and reliable pathogen-detection methods. Tissue-print (TP) real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay was compared with the validated TP enzyme-linked immunosorbent assay (ELISA), using the CTV-specific monoclonal antibodies 3DF1 and 3CA5, for CTV detection. In total, 1,395 samples from healthy and CTV-infected nursery and mature tree plants were analyzed with both methods. The total agreement between both detection methods was substantial (Cohen's kappa index of 0.77 ± 0.03). The diagnostic parameters of each technique (i.e., the sensitivity, specificity, and likelihood ratios) were evaluated in a second test involving 658 Citrus macrophylla nursery plants. Mexican lime indexing was used to evaluate samples with discrepant results in the analysis. For TP-ELISA, a sensitivity of 0.8015, a specificity of 0.9963, and a positive and negative likelihood ratio of 216.42 and 0.199, respectively, were estimated. For TP real-time RT-PCR, a sensitivity of 0.9820, a specificity of 0.8519, and a positive and negative likelihood ratio of 6.63 and 0.021, respectively, were estimated. These diagnostic parameters show that TP real-time RT-PCR was the most sensitive technique, whereas TP-ELISA showed the highest specificity, validating the use of the molecular technique for routine CTV-detection purposes. In addition, our results show that the combination of both techniques can accurately substitute for the conventional biological Mexican lime index for the detection of CTV. The calculation of diagnostic parameters is discussed, as a necessary tool, to validate detection or diagnostic methods in plant pathology. Furthermore, assessment of the post-test probability of disease after a diagnostic result and CTV prevalence allows selection of the best method for accurate and reliable diagnosis.

First Report on Almond in Europe of Bacterial Spot Disease of Stone Fruits Caused by Xanthomonas arboricola pv. pruni.

Symptoms characteristic of bacterial spot disease of Prunus spp. (4) were observed on almond trees (Prunus dulcis (Mill.) Webb) in 14 localities of Comunidad Valenciana (eastern Spain) and Aragón (northeastern Spain) between 2006 and 2009. Symptoms were first noted in the spring and were observed until leaf fall. Initial infections began on leaves as small, angular, water-soaked spots, which mainly developed toward the tip and along the leaf margins. These water-soaking lesions were surrounded by chlorotic tissue, although chlorosis did not extend more than a few millimeters. Subsequently, the lesions turned light brown, necrotic, and sometimes the necrotic spots fell out. When the lesions coalesced, they produced large necrotic areas. Sometimes premature leaf drop of infected leaves was observed in severely affected trees. Infected fruits initially displayed sunken, corky lesions that oozed gum, which later became raised when the mesocarp dehydrated. Infected fruits either dropped prematurely or remained on trees after harvest. Cankers typical of bacterial spot disease of stone fruit trees were observed on branches and shoots. Isolations from diseased leaves and fruits yielded Xanthomonas-like colonies on YPGA medium (yeast extract, peptone, and glucose agar), which were subsequently purified and characterized. All strains were gram-negative rods, oxidase negative, and strictly aerobic and showed typical biochemical characteristics of the Xanthomonas genus (3). A collection of 70 strains were further identified by PCR with primers Y17CoF/Y17CoR (1) as Xanthomonas arboricola pv. pruni by comparison with reference strains ISPaVe B4 and ISPaVe B6 isolated from Prunus salicina in Italy. A selection of 46 strains were also analyzed by immunofluorescence (IF) and ELISA using commercial polyclonal antibodies from NEOGEN Europe Ltd. (Ayrshire, Scotland, UK) and SEDIAG S.A.S. (Longvic, France), respectively), although ELISA antibodies proved to be not specific for X. arboricola pv. pruni. Pathogenicity was confirmed by inoculation of 70 almond strains and the reference strains on leaves of potted almond trees and/or on detached leaves (2) with bacterial suspensions (107 CFU per ml). One leaf was inoculated at 8 to 10 sites per strain. Characteristic bacterial spot disease symptoms (4) appeared on all inoculated leaves after 1 week of incubation at 25°C and high humidity, but not on the negative controls infiltrated with sterile distilled water. The original pathogen was reisolated from lesions of inoculated leaves and confirmed by biochemical tests, IF and PCR. As observed in Spain, the disease produces serious damage on the most susceptible almond cultivars like Antoñeta, Guara, Marta, Mas Bovera, and Vayro and can be very harmful, with severity of infection depending upon the relative cultivar susceptibility and environmental conditions. Appropriate eradication measures were taken after the causal agent was confirmed as X. arboricola pv. pruni. This pathogen was previously reported on almond in Japan and New Zealand (4). To our knowledge, this is not only the first report on almond in Spain but also in Europe. References: (1) M. C. Pagani. Ph. D. thesis, North Carolina State University, Raleigh, 2004. (2) P. S. Randhawa and E. L. Civerolo. Phytopathology 75:1060, 1985. (3) L. Vauterin et al. Int. J. Syst. Bacteriol. 45:472, 1995. (4) J. M. Young. N. Z. J. Agric. Res. 20:105, 1977.

Role of boost radiotherapy for local control of pure ductal carcinoma in situ after breast-conserving surgery: a multicenter, retrospective study of 622 patients.

PURPOSE: To evaluate the effect of boost radiotherapy on ipsilateral breast tumor recurrence (IBTR) for ductal carcinoma in situ (DCIS) after breast-conserving surgery and whole breast radiotherapy (WBRT) with or without boost. METHODS AND MATERIALS: Retrospective, multicentre study of 622 patients (624 tumors) diagnosed with pure DCIS from 1993-2011. RESULTS: Most tumors (377/624; 60.4%) received a boost. At a median follow-up of 8.8 years, IBTR occurred in 64 cases (10.3%). A higher percentage of patients with risk factors for IBTR received a boost (p < 0.05). Boost was not associated with lower rates of IBTR than WBRT alone (HR 0.75, 95% CI 0.42-1.35). On the univariate analyses, IBTR was significantly associated with tumor size (11-20 mm, HR 2.32, 95% CI 1.27-4.24; and > 20 mm, HR 2.10, 95% CI 1.14-3.88), re-excision (HR 1.76, 95% CI 1.04-2.96), and tamoxifen (HR 2.03, 95% CI 1.12-3.70). Boost dose > 16 Gy had a protective effect (HR 0.39, 95% CI 0.187-0.824). Multivariate analyses confirmed the independent associations between IBTR and 11-20 mm (p = 0.02) and > 20 mm (p = 0.009) tumours, and re-excision (p = 0.006). On the margin-stratified multivariate analysis, tamoxifen was a poor prognostic factor in the close/positive margin subgroup (HR 4.28 95% CI 1.23-14.88), while the highest boost dose ( > 16 Gy) had a significant positive effect (HR 0.34, 95% CI 0.13-0.86) in the negative margin subgroup. CONCLUSIONS: Radiotherapy boost did not improve the risk of IBTR. Boost radiotherapy was more common in patients with high-risk disease. Tumor size and re-excision were significant independent prognostic factors.

Graft transmission efficiencies and multiplication of 'Candidatus Liberibacter americanus' and 'ca. Liberibacter asiaticus' in citrus plants.

In Brazil 'Candidatus Liberibacter asiaticus' and 'Ca. L. americanus' cause huanglongbing (also known as greening), the most destructive citrus disease. A shift in pathogen prevalence was observed over time, with a disproportional increase in 'Ca. L. asiaticus' occurrence. Graft transmission experiments were used for a comparative study of both species using budsticks from symptomatic branches of field-affected trees as inoculum. The plants were inoculated with 'Ca. L. asiaticus' or 'Ca. L. americanus' alone, or simultaneously with both species. Symptom manifestation and conventional and quantitative real-time polymerase chain reaction were used for plant evaluations. 'Ca. L. americanus' was detected mainly in symptomatic plants and 'Ca. L. asiaticus' was detected in symptomatic plants as well as in infected plants prior to symptom manifestation. Transmission percentages varied from 54.7 to 88.0% for 'Ca. L. asiaticus' and 10.0 to 45.2% for 'Ca. L. americanus' in two experiments. In co-inoculated plants, 12.9% contained 'Ca. L. americanus' only, 40.3% contained 'Ca. L. asiaticus' only, and 19.3% contained both species. Average bacterial titers for 'Ca. L. asiaticus' and 'Ca. L. americanus', in log cells per gram of leaf midrib, were 6.42 and 4.87 for the experimental plants and 6.67 and 5.74 for the field trees used as the source of inoculum. The higher bacterial populations of the 'Ca. L. asiaticus'-infected plants provided an explanation for the disproportional increase in field prevalence of this species over time, based on the greater likelihood for pathogen transmission by the insect vector.

First Report of Pepper as a Natural Host for Pelargonium zonate spot virus in Spain.

Pelargonium zonate spot virus (PZSV) was first reported on Pelargonium zonale (L.) L'Hér. ex Aiton and later on tomato in Italy, Spain, France (1), and the United States (2). In Spain, PZSV was first detected in 1996 in tomato plants of cv. Royesta from greenhouses in Zaragoza Province (3) and subsequently in tomato in the Catalonia and Navarra areas. In April 2006, symptoms of PZSV were found at high incidence on tomato in a greenhouse in Huesca, Aragón (northeastern Spain). Randomly distributed pepper plants (Capsicum annuum L.) of cv. Estilo F1 growing in the same greenhouse showed severe foliar chlorotic ringspots and line patterns similar to those observed in tomato. Samples from symptomatic peppers and tomatoes and one asymptomatic weed of Rubia tinctorum L. tested positive by double-antibody sandwich (DAS)-ELISA using polyclonal antibodies against PZSV (Agdia Inc., Elkhart, IN and DSMZ, Braunschweig, Germany) as did a Spanish PZSV isolate used as a positive control (3). Sap extracts from two tomatoes, three peppers, and the single R. tinctorum plant were mechanically inoculated to 22 indicator species, including pepper and tomato. On 17 of 22 species inoculated, sap from symptomatic tomatoes and peppers elicited local or systemic symptoms similar to those reported earlier for PZSV isolates (3). Systemic symptoms were mainly mosaic, chlorotic, and necrotic line patterns and ringspots on leaves of most indicator species, closely resembling those observed on the greenhouse pepper and tomato plants. Symptoms on inoculated tomatoes also included stem necrosis and death. Reactions of indicator species did not indicate the presence of any other pepper- or tomato-infecting viruses. Both field infected and mechanically inoculated plants of pepper cvs. Yolo Wonder and Doux des Landes were maintained in the greenhouse until the development of fruit symptoms. Only fruits of cv. Yolo Wonder showed dark green and slightly depressed circles on their surface. Local and systemic infection by PZSV was confirmed by DAS-ELISA in most inoculated plants. Total RNA from leaves of field or inoculated plants was used as template for amplification by reverse transcription (RT)-PCR with primers R3-F and R3-R that are specific for the PZSV 3a gene (2), and amplicons were sequenced directly. The sequences of 697 nt from pepper and tomato isolates from the same greenhouse were identical (GenBank Accession Nos. CQ178217 and CQ178216, respectively) and had 96.1% identity to nucleotides 384 to 1,080 in PZSV RNA-3 (NC_003651). Our results confirm the natural infection of pepper plants in Huesca by PZSV. To our knowledge, this is the first report of pepper as a natural host for PZSV, a significant finding considering the potential risks of PZSV dispersion whenever tomato and pepper coexist, particularly in greenhouses and nurseries. References: (1) M. Finetti-Sialer and D. Gallitelli. J. Gen. Virol. 84:3143, 2003. (2) H. Y. Liu and J. L. Sears. Plant Dis. 91:633, 2007. (3) M. Luis-Arteaga and M. A. Cambra. Plant Dis. 84:807, 2000.

Liberibacters Associated with Citrus Huanglongbing in Brazil: 'Candidatus Liberibacter asiaticus' Is Heat Tolerant, 'Ca. L. americanus' Is Heat Sensitive.

In São Paulo State, Brazil, 'Candidatus Liberibacter americanus' and 'Candidatus Liberibacter asiaticus' are associated with huanglongbing (HLB). Affected municipalities occur mainly in the central and southern regions, where the annual number of hours above 30°C is two to five times lower than that in the extreme northern and western regions. The influence of temperature on sweet orange trees infected with 'Ca. L. asiaticus' or 'Ca. L. americanus' was studied in temperature-controlled growth chambers. Symptom progression on new shoots of naturally infected and experimentally graft-inoculated symptomatic sweet orange trees was assessed. Mottled leaves developed on all infected trees at 22 to 24°C, but not on any 'Ca. L. americanus'-infected trees at 27 to 32°C. Quantitative, real time-PCR was used to determine the liberibacter titers in the trees. After 90 days, 'Ca. L. asiaticus'-infected trees had high titers at 32 and 35°C, but not at 38°C, while 'Ca. L. americanus'-infected trees had high titers at 24°C, but at 32°C the titers were very low or the liberibacters could not be detected. Thus, the multiplication of 'Ca. L. asiaticus' is not yet affected at 35°C, while a temperature of 32°C is detrimental to 'Ca. L. americanus'. Thus, 'Ca. L. americanus' is less heat tolerant than 'Ca. L. asiaticus'. The uneven distribution of these two liberibacters in São Paulo State might be in relation with these results.

Management of breast ductal carcinoma in situ in Catalonia, Spain: Results from the Grup Oncologic Calalà-Occità-Catalonia survey with 9-year follow up.

INTRODUCTION: Reliable data on DCIS incidence and management are not available in many countries. The present study describes the management of DCIS in Catalonia, Spain in the year 2005 and compares these findings to data obtained in France. Local recurrence and late toxicity rates from 2005 through the end of 2014 are reported. MATERIALS AND METHODS: Observational survey of patients with pure DCIS (n = 270) diagnosed during 2005. A written questionnaire, the same as used in the French survey, was completed by 14 doctors at 12 cancer centres in Catalonia, Spain. RESULTS: Median patient age was 55 years (range, 29-89). Diagnosis was mammographic in 225 cases (83.3%). Treatment approaches included: mastectomy (10.4% of cases), breast-conserving surgery (BCS) alone (3.7%), and BCS plus radiotherapy (RT) (85.5%). Sentinel node biopsy and axillary dissection were performed in 27.4% and 5.6% of patients, respectively. Hormonotherapy was prescribed in 45.2% of cases. Tumour nuclear grade was as follows: low (16.7% of cases), intermediate (23%), and high (55.6%). Excision was complete (margins ≥1 mm) in 75% of patients treated with BCS alone vs. 95.7% for BCS+RT. The treatment approach varied widely: mastectomy rates ranged from 7.1% to 26.7% of centres, BCS+RT from 55.5% to 87.8%, and hormonotherapy from 3.3% to 83.3%. At a median follow-up of 102.6 months, 14 patients (5.6%) presented ipsilateral breast tumour recurrence. CONCLUSIONS: These findings on DCIS management in Catalonia are consistent with previous international reports. The inter-centre differences observed are similar to those reported in other international surveys during the same period.

Field Trials of Plum Clones Transformed with the Plum pox virus Coat Protein (PPV-CP) Gene.

Transgenic clones C2, C3, C4, C5, C6, and PT-6, of plum (Prunus domestica L.) transformed with the coat protein (CP) gene of Plum pox virus (PPV), PT-23 transformed with marker genes only, and nontransgenic B70146 were evaluated for sharka resistance under high infection pressure in field trials in Poland and Spain. These sites differed in climatic conditions and virus isolates. Transgenic clone C5 showed high resistance to PPV at both sites. None of the C5 trees became naturally infected by aphids during seven (Spain) or eight (Poland) years of the test, although up to 100% of other plum trees (transgenic clones and nontransgenic control plants) grown in the same conditions showed disease symptoms and tested positively for PPV. Although highly resistant, C5 trees could be infected artificially by chip budding or via susceptible rootstock. Infected C5 trees showed only a few mild symptoms on single, isolated shoots, even up to 8 years post inoculation. These results clearly indicate the long-term nature and high level of resistance to PPV obtained through genetically engineered resistance.

Incidence and epidemiology of Citrus tristeza virus in the Valencian community of Spain.

The first outbreak of citrus tristeza disease in Spain caused by Citrus tristeza virus (CTV) was recorded in 1957 in the Valencian Community (VC). In total c. 40 million trees, mainly of sweet orange and mandarin grafted on sour orange rootstocks, declined due to CTV. Large-scale surveys in different municipalities of the VC indicated that the disease spread very fast. Incidence increased from 11% in 1989 to 53% in 1998. Toxoptera aurantii and Aphis spiraecola (inefficient aphid vectors of CTV) predominated before 1985-87. Since then the relatively efficient vector Aphis gossypii has become dominant and induced an epidemic that has been modelled. The large number of A.gossypii that visited each clementine tree (estimated to exceed 97000 per year) explained the difference between the temporal pattern of spread of CTV in clementine which followed the Gompertz model and that in sweet orange (logistic model). The susceptibility of the different citrus species to CTV infection by aphids seems to depend on the number of young, succulent shoots produced. The epidemiological data allowed specific recommendations to be made to growers in order to facilitate a change to a modern citrus industry based on the use of selected varieties grafted on tristeza-tolerant rootstocks produced within a certification scheme. This has been done already in almost 90% of the VC citrus-growing area. The tristeza problem has been solved unless more aggressive isolates are introduced and become prevalent.

A simple imprint-hybridization method for detection of viroids.

An imprint-hybridization method has been designed to simplify the processing of samples during routine viroid indexing. The method requires minimal sample manipulation and has been evaluated for detection of viroids in 11 viroid-host combinations including 4 viroids (CEVd, CSVd, HSVd, ASBVd) and 7 hosts (chrysanthemum, citron, cucumber, Gynura, tomato, peach and avocado). The method is fast and sensitive, and provides additional information on the sites of viroid accumulation.

Detection of double-stranded RNA by ELISA and dot immunobinding assay using an antiserum to synthetic polynucleotides.

An antiserum against polyinosinic-polycytidylic acid (In-Cn) was used to detect double-stranded RNA (dsRNA) by several serological techniques. DsRNA was readily detected by indirect ELISA (ELISA-I) and dot immunobinding assay (DIA). Addition of the antigen to poly-L-lysine-precoated plates and blocking with uncreamed milk powder allowed detection levels of 100 pg.ml-1 In-Cn by ELISA-I. Concentrations as low as 1 ng.ml-1 were detected by DIA using polyvinyliden difluoride (PVDF) membranes. Detection capacity with nitrocellulose membranes was 1000 times lower than with PVDF. ELISA-I and DIA enabled detection of dsRNA in enriched fractions from cucumber mosaic virus (CMV)- and citrus tristeza virus (CTV)-infected plants and from virus-infected Penicillium chrysogenum mycelium. These techniques showed similar or higher sensitivity for detection of dsRNA than separation by polyacrylamide gel electrophoresis and silver staining.

Differentiation of citrus tristeza virus isolates by serological analysis of p25 coat protein peptide maps.

A procedure was developed to purify rapidly and easily a sufficient quantity of native p25 coat protein (CP) to allow comparison of five isolates of citrus tristeza virus (CTV) by serological analysis of peptide maps, using monoclonal and polyclonal antibodies. CTV particles were concentrated by centrifugation and purified by agarose gel electrophoresis. The CP was extracted from gel slices riched in virions and protein yields were about three times greater than those obtained previously and of comparable purity. The purified CP was partially digested with either V8 or papain endo-protease, and the peptides generated were separated and electroblotted to a membrane. Protein blots were tested with four monoclonal antibodies and one source of polyclonal antibodies. The serological maps generated by papain allowed differentiation of all the isolates examined, and those generated by V8 endoprotease allowed discrimination of four of the five isolates tested. Some of these isolates had been indistinguishable based on their reactivity in DASI-ELISA, dsRNA pattern and biological characterization. Serological analysis of peptide maps, as described below, allowed accurate comparison of CTV isolates with minimum amounts of p25 CP and proved superior to other techniques for discriminating CTV isolates.

Single-step multiplex RT-PCR for simultaneous and colourimetric detection of six RNA viruses in olive trees.

A single-step multiplex RT-PCR was developed for the simultaneous and colourimetric detection of six RNA viruses (Cucumber mosaic virus, Cherry leaf roll virus, strawberry latent ringspot virus, Arabis mosaic virus, Olive latent-1 virus and Olive latent-2 virus) which infect olive trees. Six compatible primer set for one-step RT-PCR amplification in a single closed-tube and 3' digoxigenin labelled probes were designed in optimal, specific and conserved regions. The method has been assessed with 195 Spanish field olive trees, suggesting that approximately 1.5% of the tested material was infected by Cucumber mosaic virus and 0.5% by Cherry leaf roll virus. This method saves time and reagent costs compared with monospecific RT-PCR which needs several reactions for the same number of tests. Using colourimetric detection, it is possible to analyse many samples, it increases sensitivity 10-fold, and whilst facilitating the interpretation of results, it avoids the use of gels and the toxic ethidium bromide. The method could be used routinely for sanitary and certification programmes.

Simultaneous detection and typing of plum pox potyvirus (PPV) isolates by heminested-PCR and PCR-ELISA.

Two techniques for simultaneous detection and typing of plum pox potyvirus (PPV) isolates belonging to the D or M serotypes, heminested PCR (H-PCR) and PCR-ELISA, have been developed. Ten PPV isolates typed using PPV-D and PPV-M specific monoclonal antibodies by ELISA-DASI were used to validate these two methods. The results obtained show a complete coincidence of the nucleic acid-based techniques with the serological data. When serial dilutions of infected plant extracts were assayed, H-PCR and PCR-ELISA were found to be 100 times more sensitive than the more conventional immunocapture-PCR (IC-PCR) assay. Testing of 228 PPV-infected fruit tree samples coming from different hosts and locations indicated that so far only PPV type D appears to be present in Spain and in Chile. Coupled with print-capture sample preparation (Olmos et al., Nucl. Acids Res. 24, 2192-2193, 1996) the increased sensitivity provided by heminested-PCR allowed the detection of PPV targets of D and M types, in wingless individuals of the aphid vector Aphis gossypii.

A fast one-step reverse transcription and polymerase chain reaction (RT-PCR) amplification procedure providing highly specific complementary DNA from plant virus RNA.

Reverse transcription and polymerase chain reaction (RT-PCR) are being used increasingly for detection and typing RNA viruses. For this purpose, metal block thermal cyclers (MBTC) are considered to provide higher DNA yield, whereas air thermal cyclers (ATC) allow PCR amplification in a much shorter time. A fast ATC protocol (0 s denaturation, 0 s annealing, and 4-8 s elongation) was developed to amplify genomic segments from two RNA viruses, which allowed increasing the number of cycles without a parallel increase of non-specific DNA fragments. Under these conditions, 80-90 cycles with the ATC provided a DNA yield close to that of a standard 40-cycles MBTC protocol in about half the time. The DNA synthesised by the new procedure was highly specific and could be cloned readily.

Biotechnological aspects of plum pox virus.

Plum pox potyvirus (PPV), the causal agent of a devastating disease that affects stone fruit trees, is becoming a target of intense studies intended both to fight against viral infection and to develop practical applications based on the current knowledge of potyvirus molecular biology. This review focuses on biotechnological aspects related to PPV, such as novel diagnostic techniques that facilitate detection and typing of virus isolates, strategies to implement pathogen-derived resistance through plant transformation, the potential use of genetic elements derived from the virus, and the recent development of PPV-based expression vectors.

Fully "Recombinant Enzyme-Linked Immunosorbent Assays" Using Genetically Engineered Single-Chain Antibody Fusion Proteins for Detection of Citrus tristeza virus.

ABSTRACT Recombinant single-chain variable fragment antibodies (scFv) that bind specifically to Citrus tristeza virus (CTV), which cause the most detrimental viral disease in the citrus industry worldwide, were obtained from the hybridoma cell lines 3DF1 and 3CA5. These scFv were genetically fused with dimerization domains as well as with alkaline phosphatase, respectively, and diagnostic reagents were produced by expressing these fusion proteins in bacterial cultures. The engineered antibodies were successfully used for CTV diagnosis in plants by tissue print enzyme-linked immunosorbent assay (ELISA) and double antibody sandwich-ELISA. The fully recombinant ELISAs were as specific and sensitive as conventional ELISAs performed with the parental monoclonal antibodies, showing the usefulness of recombinant antibodies for routine detection of a virus in woody plants for the first time.

Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the d and m serotypes of plum pox potyvirus.

ABSTRACT Plum pox potyvirus (PPV) isolates may be divided into four groups separated by serological, molecular, and epidemiological differences. Monoclonal antibodies specific for the two major groups of isolates, represented by the D and M serotypes of the virus, have been obtained. Polymerase chain reaction (PCR)-based assays allowing the direct detection and differentiation of PPV isolates have also been developed. We now report on a large-scale comparison of these two typing approaches. The results obtained show an overall excellent correlation between the results obtained in indirect double-antibody sandwich enzyme-linked immunosorbent assay using PPV-D- and PPV-M-specific monoclonal antibodies and those derived from either specific PCR assays or restriction fragment length polymorphism analysis of PCR fragments. Without exception, all isolates reacting positively with the PPV-M-specific monoclonal antibody were found to belong to the M serotype using the PCR-based assays, while 51 out of 53 isolates recognized by the D-specific monoclonal antibodies belonged to the D serotype according to the PCR typing results. However, failure to react with a specific monoclonal antibody did not prove as effective a predictor of the serotype of the isolate analyzed. In a few cases, the results obtained with the various techniques diverged, indicating low level variability of the epitopes recognized by the serotype-specific monoclonal antibodies. Isolates belonging to the two minor groups of PPV (El Amar and Cherry) also gave divergent results, indicating that the current typing assays are not suited for the analysis of such isolates.

Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus.

The coat protein gene of citrus tristeza virus (CTV) has been introduced into Mexican lime (Citrus aurantifolia Swing.) plants by using an improved Agrobacterium-mediated genetic transformation system. Internodal stem segments from greenhouse-grown seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pBI 121/CTV-CP in a medium rich in auxins that provided the explant cells with the proper treatment to shift them to a competent state for transformation. The transformation frequency was enhanced, and this allowed us to recover 42 transgenic plants from 1200 explants. Regenerated shoots were identified as transformants by performing ß-glucuronidase (GUS) assays and subsequently by PCR amplifications of the CTV-CP transgene. Southern analyses revealed that at least one copy of the CTV-CP gene was integrated in all PCR positive plants. Interestingly, 70% of them had linked T-DNAs arranged at one locus. Copy number of the CTV-CP gene varied from one to six among the transgenic lines. Half of them showed truncated T-DNAs in which the left border was lost. Expression of the CTV-CP transgene was demonstrated in 38 out of 42 plants by western analysis and DASI-ELISA. No correlation was found between coat protein expression and transgene copy number or integration pattern.

Traumatic rupture of the pericardium. Case report and literature review.

Rupture of the pericardium due to blunt thoracic trauma is a rare pathology with a range of mortality between 30 and 64% according to different authors. We review 40 cases which have been reported in the literature in the last decade and report a case of our own. We have found that 82% of the patients with traumatic rupture of the pericardium were men with a mean age of 45 years. In 80% of the cases the cause was a motor vehicle accident, 17% were due to falls and only 1 case was associated with a crush. The commonest location of the tear was the left pleuropericardium (62%) followed by the diaphragmatic portion of the pericardium (22%). In 80% of the cases the diagnosis was achieved in the course of a surgery performed for associated lesions. None of the cases was diagnosed in a post-mortem study. The traumatic rupture of the pericardium is a disease which often remains undiagnosed, especially when one does not have a high index of suspicion. Nevertheless, this is a disease which can threaten the life of the patient and we should keep it in mind to diagnose and treat it as soon as possible. It is known that an early and aggressive management of these patients implies a much better prognosis with a significant reduction of the mortality. In this article we want to give useful clues to allow a preoperative diagnosis and an early and adequate management.