Genetic diversity of the coat protein of olive latent virus 1 isolates.

The CP gene variability among 21 olive latent virus 1 (OLV-1) isolates obtained from different hosts and locations and at different times was assessed. Amplicons obtained by RT-PCR were cloned, and at least 10 sequences from each isolate were analyzed and compared. OLV-1 sequences available in GenBank were included. The encoded CPs consisted of 270 amino acids, except those of isolates G1S and C7 (269 aa) and G6 (271 aa). Comparison of CP genomic sequences of the isolates under study showed very low values of nucleotide diversity, 0.02, and maximum nucleotide distances between (0.087) or within isolates (0.001). Although very few nucleotide sequence differences were observed among the isolates, olive isolates exhibited lower diversity (0.012). In addition, at position 158 (157 in C7 and G1S and 159 in G6) of the deduced aa sequences, an alanine residue was found to be conserved among the olive isolates. In citrus and tulip isolates, a threonine residue was present at position 158, whereas a valine was present at this same position in tomato isolates. Phylogenetic analysis indicated that OLV-1 isolates clustered in five groups according to original host. However, G6, originally recovered from olive but repeatedly inoculated and maintained in N. benthamiana plants for 8 years in our laboratory, was separated from other isolates. This may be attributable to adaptation to the experimental host over time. There was no correlation of phylogenetic grouping of isolates based on geographical location or year of collection. Strong negative selection may have contributed to the low diversity among the OLV-1 CP isolates.

Development of an asymmetric PCR-ELISA typing method for citrus tristeza virus based on the coat protein gene.

The coat protein gene of isolates of citrus tristeza virus (CTV) from 20 citrus-producing regions around the world was amplified by RT-PCR, TA cloned, and characterized by SSCP. Haplotypes that produced different patterns within each geographic region were sequenced and a database of 153 accessions of CTV was assembled. Phylogenetic analysis revealed the existence of seven well-defined clusters (Coefficient of differentiation 0.78). An asymmetric PCR-ELISA typing (APET) assay was developed in the frame of this clustering pattern using a set of eight hybridisation probes. The membership of any unknown haplotype is determined by comparing its pattern of reaction against the whole set of probes and not, as previously done in hybridisation assays, in an all-or-nothing basis. Interpretation of the results is objective and done through a visual basic application that compares the rates of hydrolysis of the ELISA substrate of an assayed isolate to a matrix of rates of hydrolysis obtained from standard haplotypes. This assay was validated and showed a better ability to resolve haplotypes than other assays to which it was compared experimentally. It may be automated to the same extent as any ELISA.

Occurrence of genetic bottlenecks during citrus tristeza virus acquisition by Toxoptera citricida under field conditions.

In this study, we address the involvement of T. citricida in strain segregation and genetic bottleneck events by comparing the nucleotide diversity of CTV coat protein (CP) gene variants present in field-grown trees with that of variants retrieved from single apterous aphids. Plant material and aphids were collected in orange orchards in the northern part of Portugal. Shoots from two trees that were found to be positive using ELISA and twenty-four apterous aphids from these same trees were selected for individual molecular assays. CTV was detected in 60% of the aphids by amplification of a 417-bp fragment of the CP gene. Analysis of molecular variance (AMOVA) of this fragment revealed that most of the variation of the virus was found among individual aphids (FSC: 0.766) within each location. Nucleotide diversity comparison between the pool of sequences obtained from a given shoot and sequences obtained from individual aphids present on that shoot showed a reduction of more than one order of magnitude in most cases. Computer simulations of random virus acquisition by single aphids showed that in 54% of the cases only a single CP gene phylogenetic group was acquired. However, a small number of aphids (e.g. 6) was enough to acquire the full complement of phylogenetic groups present.

Rupestris stem pitting associated virus isolates are composed by mixtures of genomic variants which share a highly conserved coat protein.

Broad spectrum primers were used to amplify a fragment comprising the CP gene and putative ORF6 by RT-PCR from ds-RNA templates originating from 46 Portuguese varieties, totalling 190 samples, including some wild Vitis ssp sylvestris vines, and 2 vines from Slovenia. SSCP analysis was used as a preliminary screen to avoid cloning and sequencing very similar variants. Four groups of variants were recognized. In pair wise comparisons between nucleotide sequences the minimal homology found was 81%. In case of the cultivated varieties, no relationship could be seen between the phylogenetic groups and geographic origin or grape variety. Several isolates were found harbouring mixed infections with genomic variants from different groups, but the mixing did not lead to an extensive recombination between them. The deduced amino-acid sequences revealed a conserved CP subjected to strong purifying selection pressure. Analysis of the selection pressure operating on the putative ORF6 suggests that this ORF does not exist. Previously produced polyclonal antiserum raised against the recombinant CP of RSPaV expressed in Escherichia coli was shown to be able to detect all four groups of variants of RSPaV included in this study, which might enable the diagnosis of the virus on a serological basis.

Genomic variability of prune dwarf virus as affected by agricultural practice.

Twelve new sequences of the coat protein gene of Prune dwarf virus (PDV) variants, obtained from almond trees, are presented. Comparison with previously reported sequences of the same region, obtained from other hosts (plum, cherry and peach) revealed not only the existence of a wider range of variants of PDV than formerly predicted, but also the frequent presence of a mixture of variants in each sample. In spite of the heterogeneity found in almond, the amino acid composition of the domain at the N terminus of the coat protein maintained the potential to form an amphipathic helix, and hence the capacity to serve the previously suggested function of binding the viral RNA during particle formation. Except for synonymous substitutions, measures of nucleotide diversity calculated for the two groups, respectively 13 sequences from almond and 14 sequences from other hosts, were found to be significantly different, with the almond group showing a much higher variability. Analysis of the dendrogram constructed based in all 27 PDV CP sequences did not reveal host specificity, in agreement with previous findings. However, a clear divergence between almond and other hosts sequences could be found. It is discussed that the observed differences between almond and other hosts variants may derive from differences in agricultural practices.

Occurrence of Stem-Pitting Strains of Citrus tristeza virus in Croatia.

Citrus is grown in Croatia (approximately 1,500 ha of citrus groves) on the Dalmatian Coast and Islands between 42 and 43°30'N. The major species, Citrus unshiu Marc. (Satsuma mandarin), is grafted on trifoliate rootstock. The presence of Citrus tristeza virus (CTV) in Satsumas in the Neretva Valley Region was previously reported (3). During the course of a biomolecular characterization of isolates from Croatia, 15 budsticks were collected from field-infected, enzyme-linked immunosorbent assay (ELISA)-positive sources during the autumn of 2003 near Kastela, Split, Metkovic (Neretva Valley), and on the island of Vis. Isolates were propagated by graft transmission to Madam Vinous sweet orange (SwO) and maintained in an insect-proof greenhouse at 21 to 33°C. Eight months later, the bark of terminal twigs was peeled off, and the wood was examined for the occurrence of pits. Typical tristeza stem-pitting (SP) was observed in four isolates originating from cvs. Fukumoto navel, Washington navel, and Ichimaru Satsuma and C. wilsonii. The bark from the infected sources was analyzed using immunocapture reverse transcription-polymerase chain reaction (RT-PCR) with primers CTV1 and CTV10 (1), targeting the whole coat protein (CP) gene. The PCR products of the expected size (669 nucleotides) were obtained and TA cloned (pGEM-T Easy Vector; Promega, Madison, WI) in E. coli cells. Thirty-two clones harboring the CTV CP gene were sequenced. Two of the SP isolates contained four genomic variants that differed an average of 2.0% from the severe SwO SP strains SY568 and Nuaga (4) from California and Japan, respectively. The other two SP isolates contained four variants that differed as little as 1.6% from the severe SwO SP from India, CTV-Puna, and CTV-Bangalore (2). The net average distance between these two clusters of sequences is 5.2%. One sequence from each of the four SP isolates was deposited in GenBank (Accession Nos. AY791841 to AY791844). These findings were confirmed by direct observation of SP symptoms in a Satsuma orchard in the Neretva Valley during the spring of 2004. No other conspicuous symptoms that could be attributed to CTV were observed in the field. Most Satsumas were introduced to the Neretva Region from Japan between 1964 and 1984. Together with the fact that the related Nuaga strain was also isolated from Satsumas in Japan (4), our results suggest that SwO SP strains were introduced into Croatia at the same time and have been spreading for several decades. It has been generally believed that this kind of CTV strains either do not exist in the Mediterranean basin or, when found (e.g., Spain), are immediately eradicated. The findings reported here suggest that the epidemiological scenario for the Mediterranean Basin requires revision. References: (1) G. Nolasco et al. Eur. J. Plant Pathol. 108:293, 2002. (2) A. Roy et al. Arch. Virol. 148:707, 2003. (3) A. Saric and I. Dulic. Agric. Conspectus Sci. 55:171, 1990. (4) G. Suastika et al. J. Gen. Plant Pathol. 67:73, 2001.

First Report of Citrus tristeza virus in the State Union of Serbia and Montenegro.

Citrus production in the State Union of Serbia and Montenegro has a strategic importance to the agricultural sector. Approximately 400,000 trees are now grown in the major citrus producing region, which is the Montenegrin Coastal Region. Satsuma mandarins and lemons grafted on Poncirus trifoliata are the most cultivated varieties. In December 2003, eight samples taken from the coastal region close to the towns of Bar and Ulcinj were analyzed using enzyme-linked immunosorbent assay (ELISA) with SP7 antibodies produced at Universidade do Algarve, Portugal (3). Further analysis was done using immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) targeting the entire coat protein (CP) gene (forward primer CTV1: 5'- ATGGACGACGAAACAAAGAA-3' and reverse primer CTV10: 5'-ATCAACGTGTGTTGAATTTCC-3'). Using both techniques, seven of eight samples analyzed were found to be infected by Citrus tristeza virus (CTV), including samples from five trees that exhibited chlorosis, gummosis, and fruit deformation, and two trees that were symptomless. When analyzed using agarose gel electrophoresis, PCR products from the positive samples consisted of a single amplicon of the expected size for the CP gene compared with a positive control. The PCR products of two samples were TA cloned (pGEM-T Easy Vector; Promega, Madison, WI) in E. coli cells and the CP inserts were analyzed using single-strand conformation polymorphism (SSCP) and DNA sequencing. In both cases, the SSCP analysis of several clones showed a variety of different patterns, suggesting the occurrence of infections with a mixture of genomic variants. Sequence analysis of different variants showed a CP gene with 669 nucleotides having greater than 90% nucleotide identity to most CTV CP gene sequences available in GenBank. A genomic variant (GenBank Accession No. AY764154) was closely related (98.5% nucleotide identity) to the T30 mild strain from Florida (GenBank Accession No. AF260651). However, other sequences obtained showed only 93% nucleotide identity with this variant and were closely related to other CP gene sequences obtained from Croatian isolates. A previous report (1) refers to the existence of CTV-infected Satsuma plants illegally introduced in Italy from the former Yugoslavia. The presence of CTV in the former Yugoslavia was later confirmed (2) but in a region that became part of the Croatian Republic. To our knowledge, this is the first report of CTV in the State Union of Serbia and Montenegro, although a relationship with Croatian isolates cannot be excluded. Although a very small number of samples were analyzed in this study, CTV appears to be very common in the Satsuma orchards. This could be due to the traditional use of the trifoliate rootstock that prevents the appearance of tristeza decline, thus enabling the unnoticed propagation of infected material. Because the kind of symptoms observed in five trees are not typical of CTV and two infected trees were symptomless, the virus is probably not responsible for the symptoms observed in the field. References:(1) M. Davino et al. Pages 8-13 in: Proc. Conf. Int. Organ Citrus Virol. IOCV, Riverside, 1988. (2) A. Saric and I. Dulic. Agric. Conspectus Sci. 55:171, 1990. (3) Z. Sequeira and G. Nolasco, Phytopathol. Mediterr. 41:552, 2002.

Identification to the species level of the plant pathogens Phytophthora and Pythium by using unique sequences of the ITS1 region of ribosomal DNA as capture probes for PCR ELISA.

The ribosomal internal transcribed spacer 1 region was sequenced for 10 species of Pythium and eight species of Phytophthora. Alignment of the sequences revealed considerable sequence microheterogeneity, which was utilized to prepare a capture probe of unique sequence for each species. The capture probes were tested by PCR ELISA, combining the sensitivity and specificity of the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The probes were entirely species specific, enabling the detection and identification of the amplified DNA of species from individual cultures or from mixed samples of the DNAs of two different species. This approach to species identification, which provides a molecular technology to process large numbers of samples and still identify the fungi with a high level of confidence, may greatly reduce the resources and the time of highly trained specialists currently needed to identify these important species of plant pathogenic fungi.

Progress on strain differentiation of Citrus tristeza virus and its application to the epidemiology of citrus tristeza disease.

Citrus tristeza virus (CTV) occurs in most citrus producing regions of the world, and it is the most serious viral pathogen of citrus. With the recent establishment of the brown citrus aphid, Toxoptera citricida, its most efficient vector, on Madeira Island (Portugal) and in Florida (USA) and the countries of the Caribbean Basin, the impact of CTV is likely to increase in these regions. Since there are many strains of CTV and CTV infections frequently occur as mixtures of several strains, it is necessary to be able to distinguish the strains for regulatory purposes, disease management and epidemiology. We describe the evolution of techniques developed to detect CTV and to differentiate the individual strains, and present the results of tests using these latest methods on CTV isolates from mainland Portugal, Madeira Island and Florida. Mild and decline-inducing strains of CTV were detected in mainland Portugal and mild, decline-inducing and severe stem pitting strains on Madeira Island. In Florida we demonstrated the presence of infections that reacted with probes made against stem pitting strains not previously detected there. It is concluded that CTV presents a significant threat to citrus production in mainland Portugal, on Madeira Island and in the neighbouring countries of the Mediterranean Basin, as well as in Florida, elsewhere in the USA and throughout the Caribbean Basin, especially following the widespread establishment of T. citricida throughout the region.

Cucurbit yellow stunting disorder virus (Genus Crinivirus) Associated with the Yellowing Disease of Cucurbit Crops in Portugal.

During autumn 1998, chlorotic mottling, yellowing, and stunting symptoms were observed on cucumber (Cucumis sativus L.) plants in an experimental plot, in Algarve, southern Portugal. The first symptoms appeared 3 weeks after planting, associated with a heavy infestation of Bemisia tabaci (Gennadius). Plants affected early produced few and small fruits. Analysis of double-stranded RNA (dsRNA) extracted from symptomatic cucumber leaves revealed the presence of two dsRNAs of about 8 and 9 kbp, not present in healthy cucumber plants. Reverse-transcription polymerase chain reaction (RT-PCR) using dsRNA or total RNA extracts as template and the oligonucleotide primers 410L and 410U (1), specifically amplified a fragment of expected size of the HSP70-homolog gene of Cucurbit yellow stunting disorder virus (CYSDV). The RT-PCR-amplified fragment was sequenced (Acc. No. AF287474) and showed 99% sequence identities with the corresponding sequences (GenBank accessions AJ223619 and U67170) from two CYSDV isolates belonging to group I (2), confirming CYSDV detection. CYSDV was also detected in samples of cucumber, melon (Cucumis melo L.) and watermelon (Citrullus lanatus [Thunb.] Matsun.) collected during the summer of 1999 in commercial greenhouses. CYSDV is an emerging and important virus of cucurbits in the Middle East and Mediterranean Europe (2). This is the first report of CYSDV infecting cucurbit crops in Portugal. References: (1) A. Célix et al. Phytopathology 86:1370, 1996. (2) L. Rubio et al. Phytopathology 89:707, 1999.

Direct PCR detection of foot-and-mouth disease virus.

A PCR assay for the detection and characterization of foot-and-mouth disease virus was developed. The procedure allows RT-PCR amplification following direct adsorption of viral suspensions to microtiter plates, avoiding previous steps of phenol-extraction or heating. Using this procedure, FMDV-specific (based on 3D gene sequences), as well as serotype-specific (based on VP1 gene sequences) amplification were achieved for viral samples of serotypes A, O and C, either from cell culture supernatants or from lesions of infected animals. The assay allowed detection of around 15 PFU, being 500-fold more sensitive than a conventional indirect ELISA. This new method constitutes a simple, rapid and efficient alternative for the diagnosis and characterization of FMDV by PCR.

A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogens.

A method for the detection of RNA viral and subviral plant pathogens was developed that combines pathogen partial purification by solid-phase adsorbed antibodies, reverse transcriptional-polymerase chain reaction (RT-PCR) and quantitation of the amplified products by fluorescence. The reverse transcription of the RNA is performed directly on the retained material without any previous thermal or chemical disruption of the virus particles. The whole procedure can be carried out in a microtiter plate. Its validity has been successfully confirmed for the detection of bean yellow mosaic virus, cherry leafroll virus, cucumber mosaic virus, citrus tristeza virus, grapevine fanleaf virus, potato leafroll virus, pepper mild mottle virus, and tomato spotted wilt virus, as well as the satellite RNA of cucumber mosaic virus and potato spindle tuber viroid. In this procedure virus-specific antibodies can be replaced by monoclonal antibodies against double-stranded RNA, thus offering the possibility of detection when no specific virus antibodies are available, or immunological methods are difficult to use (i.e., subviral pathogens like satellite-RNAs or viroids). The method described has the typical sensitivity of assays based on the polymerase chain reaction, it is not more laborious than ELISA, and an equivalent degree of automation is possible.