Discovery of Viruses and Virus-Like Pathogens in Pistachio using High-Throughput Sequencing.

Pistachio (Pistacia vera L.) trees from the National Clonal Germplasm Repository (NCGR) and orchards in California were surveyed for viruses and virus-like agents by high-throughput sequencing (HTS). Analyses of sequence information from 60 trees identified a novel virus, provisionally named "Pistachio ampelovirus A" (PAVA), in the NCGR that showed low amino acid sequence identity (approximately 42%) compared with members of the genus Ampelovirus (family Closteroviridae). A putative viroid, provisionally named "Citrus bark cracking viroid-pistachio" (CBCVd-pis), was also found in the NCGR and showed approximately 87% similarity to Citrus bark cracking viroid (CBCVd, genus Cocadviroid, family Pospiviroidae). Both PAVA and CBCVd-pis were graft transmissible to healthy UCB-1 hybrid rootstock seedlings (P. atlantica × P. integerrima). A field survey of 123 trees from commercial orchards found no incidence of PAVA but five (4%) samples were infected with CBCVd-pis. Of 675 NCGR trees, 16 (2.3%) were positive for PAVA and 172 (25.4%) were positive for CBCVd-pis by reverse-transcription polymerase chain reaction. Additionally, several contigs across multiple samples exhibited significant sequence similarity to a number of other plant virus species in different families. These findings require further study and confirmation. This study establishes the occurrence of viral and viroid populations infecting pistachio trees.

Description of a Novel Monopartite Geminivirus and Its Defective Subviral Genome in Grapevine.

A novel virus was detected in grapevines by Illumina sequencing during the screening of two table grape (Vitis vinifera) accessions, cultivars Black Beet and Nagano Purple, from South Korea. The monopartite circular ssDNA genome sequence was subsequently confirmed by rolling cycle amplification, cloning and Sanger sequencing. The complete viral genomic sequence from both accessions ranged from 2,903 to 2,907 nucleotides in length and contained the conserved nonanucleotide sequence TAATATT↓AC and other sequence features typical of the family Geminiviridae, including two predicted sense and four complementary-sense open reading frames. Phylogenetic analysis placed the novel virus in a unique taxon within the family Geminiviridae. A naturally occurring defective subviral DNA was also discovered. This defective DNA molecule carried a deletion of approximately 46% of the full-length genome. Both the genomic and defective DNA molecules were graft-transmissible although no disease is yet correlated with their occurrence in Vitis spp. The tentative names Grapevine geminivirus A (GGVA) and GGVA defective DNA (GGVA D-DNA) are proposed. PCR assays developed using primers designed in the coat protein gene led to the detection of GGVA in 1.74% of 1,262 vines derived from 15 grapevine cultivars from six countries across three continents.

Viral Diversity in Autochthonous Croatian Grapevine Cultivars.

A survey was conducted on nine autochthonous grapevine cultivars grown along the Croatian coastal region. In total, 48 vines (44 from germplasm collection, 4 from vineyards) originating from 23 sites were tested for 26 viruses using molecular methods. Results revealed high infection rates with Grapevine leafroll-associated virus 3 (GLRaV-3); Grapevine virus A (GVA, both 91.7%); Grapevine fleck virus (GFkV, 87.5%); and Grapevine rupestris stem pitting-associated virus (GRSPaV, 83.3%). Other detected viruses were: Grapevine fanleaf virus (GFLV); Grapevine leafroll-associated viruses 1, 2, and strains of 4 (GLRaV-1, GLRaV-2, GLRaV-4); Grapevine viruses B, D, F (GVB, GVD, GVF); Grapevine red globe virus (GRGV); Grapevine vein feathering virus (GVFV); Grapevine Syrah virus 1 (GSyV-1); and Grapevine Pinot gris virus (GPGV). No virus-free vine was found. Mixed infections were determined in all vines, the number of viruses in a single vine ranged from three to nine. GLRaV-3 variant typing confirmed presence of group I, II, and III. Four vines with leaf deformation and mottling were positive for GPGV. Seven viruses (GLRaV-4-like group, GVD, GVE, GVF, GRGV, GSyV-1, and GVFV) were detected for the first time in Croatia. This survey confirmed the deteriorated sanitary status of autochthonous Croatian grapevine cultivars.

Comparison of Next-Generation Sequencing Versus Biological Indexing for the Optimal Detection of Viral Pathogens in Grapevine.

A bioassay is routinely used to determine the viral phytosanitary status of commercial grapevine propagation material in many countries around the world. That test is based on the symptoms developed in the field by specific indicator host plants that are graft-inoculated from the vines being tested. We compared the bioassay against next-generation sequencing (NGS) analysis of grapevine material. NGS is a laboratory procedure that catalogs the genomic sequences of the viruses and other pathogens extracted as DNA and RNA from infected vines. NGS analysis was found to be superior to the standard bioassay in detection of viruses of agronomic significance, including virus infections at low titers. NGS was also found to be superior to the bioassay in its comprehensiveness, the speed of its analysis, and for the discovery of novel, uncharacterized viruses.

Detection of a New Luteovirus in Imported Nectarine Trees: A Case Study to Propose Adoption of Metagenomics in Post-Entry Quarantine.

In spring 2013, 5-year-old nectarine (Prunus persica) trees, grafted on peach rootstock Nemaguard, were found stunted in a propagation block in California. These trees had been propagated from budwood of three nectarine cultivars imported from France and cleared through the post-entry quarantine procedure. Examination of the canopy failed to reveal any obvious symptoms. However, examination of the trunks, after stripping the bark, revealed extensive pitting on the woody cylinder. To investigate the etiological agent, double-stranded RNA was extracted from bark scrapings from the scion and rootstock portions, and a cDNA library was prepared and sequenced using the Illumina platform. BLAST analysis of the contigs generated by the de novo assembly of sequence reads indicated the presence of a novel luteovirus. Complete sequence of the viral genome was determined by sequencing of three overlapping cDNA clones generated by reverse transcription-polymerase chain reaction (RT-PCR) and by rapid amplification of the 5'- and 3'-termini. The virus genome was comprised of 4,991 nucleotides with a gene organization similar to members of the genus Luteovirus (family Luteoviridae). The presence of the virus, tentatively named Nectarine stem pitting-associated virus, was confirmed in symptomatic trees by RT-PCR. Discovery of a new virus in nectarine trees after post-entry quarantine indicates the importance of including (i) metagenomic analysis by next-generation sequencing approach as an essential tool to assess the plant health status, and (ii) examination of the woody cylinders as part of the indexing process.

Grapevine Leafroll: A Complex Viral Disease Affecting a High-Value Fruit Crop.

Grapevine (Vitis spp.) is one of the most widely grown fruit crops in the world. It is a deciduous woody perennial vine for which the cultivation of domesticated species began approximately 6,000 to 8,000 years ago in the Near East. Grapevines are broadly classified into red- and white-berried cultivars based on their fruit skin color, although yellow, pink, crimson, dark blue, and black-berried cultivars also exist. Grapevines can be subject to attacks by many different pests and pathogens, including graft-transmissible agents such as viruses, viroids, and phytoplasmas. Among the virus and virus-like diseases, grapevine leafroll disease (GLD) is by far the most widespread and economically damaging viral disease of grapevines in many regions around the world. The global expansion of the grape and wine industry has seen a parallel increase in the incidence and economic impact of GLD. Despite the fact that GLD was recognized as a potential threat to grape production for several decades, our knowledge of the nature of the disease is still quite limited due to a variety of challenges related to the complexity of this virus disease, the association of several distinct GLD-associated viruses, and contrasting symptoms in red- and white-berried cultivars. In view of the growing significance of GLD to wine grape production worldwide, this feature article provides an overview of the state of knowledge on the biology and epidemiology of the disease and describes management strategies currently deployed in vineyards.

Complete genome sequence of a novel vitivirus isolated from grapevine.

A novel virus-like sequence from grapevine was identified by Illumina sequencing. The complete genome is 7,551 nucleotides in length, with polyadenylation at the 3' end. Translation of the sequence revealed five open reading frames (ORFs). The genomic organization was most similar to those of vitiviruses. The polymerase (ORF1) and coat protein (ORF4) genes shared 31 to 49% nucleotide and 40 to 70% amino acid sequence identities, respectively, with other grapevine vitiviruses. The virus was tentatively named grapevine virus F (GVF).

Gene from a novel plant virus satellite from grapevine identifies a viral satellite lineage.

We have identified the genome of a novel viral satellite in deep sequence analysis of double-stranded RNA from grapevine. The genome was 1,060 bases in length, and encoded two open reading frames. Neither frame was related to any known plant virus gene. But translation of the longer frame showed a protein sequence similar to those of other plant virus satellites. Other than in commonalities they shared in this gene sequence, members of that group were extensively divergent. The reading frame in this gene from the novel satellite could be translationally coupled to an adjacent reading frame in the -1 register, through overlapping start/stop codons. These overlapping AUGA start/stop codons were adjacent to a sequence that could be folded into a pseudoknot structure. Field surveys with PCR probes specific for the novel satellite revealed its presence in 3% of the grapevines (n = 346) sampled.

Association of a DNA virus with grapevines affected by red blotch disease in California.

In the Napa Valley of California, vineyards of 'Cabernet Franc' (CF) clone 214, 'Cabernet Sauvignon' clone 337, and 'Zinfandel' clone 1A (Z1A) with grapevines exhibiting foliar symptoms of red blotches, marginal reddening, and red veins that were accompanied by reduced sugar accumulation in fruit at harvest were initially suspected to be infected with leafroll-associated viruses. However, reverse-transcription polymerase chain reaction (PCR) tests were negative for all known leafroll-associated viruses, with the exception of Grapevine leafroll-associated virus 2 in Z1A. Metagenomic analysis of cDNA libraries obtained from double-stranded RNA enriched nucleic acid (NA) preparations from bark scrapings of dormant canes on an Illumina platform revealed sequences having a distant relationship with members of the family Geminiviridae. Sequencing of products obtained by PCR assays using overlapping primers and rolling circle amplification (RCA) confirmed the presence of a single circular genome of 3,206 nucleotides which was nearly identical to the genome of a recently reported Grapevine cabernet franc-associated virus found in declining grapevines in New York. We propose to call this virus "Grapevine red blotch-associated virus" (GRBaV) to describe its association with grapevine red blotch disease. Primers specific to GRBaV amplified a product of expected size (557 bp) from NA preparations obtained from petioles of several diseased source vines. Chip bud inoculations successfully transmitted GRBaV to test plants of CF, as confirmed by PCR analysis. This is the first report of a DNA virus associated with red blotch disease of grapevines in California.

Biological, molecular, and serological studies of a novel strain of grapevine leafroll-associated virus 2.

In California, a novel closterovirus was detected in "Redglobe" grapevine, associated with graft incompatibility and given a trivial name "Grapevine rootstock stem lesion associated virus (GRSLaV)." The biological properties of the putative virus were ascertained when asymptomatic yet infected Redglobe scion buds were graft-inoculated onto test plants of Cabernet Sauvignon propagated on 18 different rootstocks. It proved lethal on test plants growing on rootstocks 1616C, 5BB, 5C, 3309C, and 1103 P, whereas latent infections occurred on the remaining scion-rootstock combinations. In contrast, GLRaV-2 type (type strain) produced only typical leafroll symptoms. In a different experiment, GLRaV-2 type was successfully sap-transmitted to N. benthamiana, whereas sap transmission of GRSLaV was unsuccessful. Double-stranded RNA was extracted from infected Redglobe grapevines, cloned, sequenced, and determined a genome length of 16,527 nucleotides. Computer-assisted analysis of open-reading frames (ORFs) revealed a genome organization typical of monopartite viruses in the genus Closterovirus with nine ORFs (range 71-79% identity) with GLRaV-2 type, the closest similar virus species within the family Closteroviridae. Also the 3'-UTR of GRSLaV consisted of 223 nucleotides with an extended oligo(A) tract similar to that of GLRaV-2 type, Beet yellow stunt virus, and Beet yellows virus. Recombinant GRSLaV coat protein was expressed in E. coli, purified, and immunized a rabbit to produce polyclonal antiserum. Serological data matched the molecular data, whereby exposed plant tissue extracts of grapevines infected by both viruses (GRSLaV and GLRaV-2) reacted positively with homologous and heterologous viral antisera but not with healthy grapevine extracts in ELISA and Western blot tests. Based on the comparative sequence data and shared antigens, GRSLaV is now considered a strain of GLRaV-2 and redesignated as Grapevine leafroll associated virus-2 Redglobe (GLRaV-2RG). Primers specific for GLRaV-2RG were developed, which did not amplify GLRaV-2 type strain. When both sets of specific primers were used in assays of different grapevine collections, the incidence of the respective viruses varied considerably, e.g., 1.7 and 13.5%, respectively, for GLRaV-2RG and GLRaV-2 type.

Grapevine leafroll-associated virus 1 occurs as genetically diverse populations.

The genetic diversity of 34 isolates of Grapevine leafroll-associated virus 1 (GLRaV-1) from different wine, table, and ornamental grape cultivars in California, New York, and Washington States in the United States was investigated. Segments of the heat-shock protein 70 homolog (HSP70h) gene, coat protein (CP) gene, coat protein duplicate 2 (CPd2) gene, and open reading frame 9 (p24) were amplified by reverse-transcription polymerase chain reaction, cloned, and sequenced. A pairwise comparison of nucleotide sequences revealed intra- and interisolate sequence diversity, with CPd2 and HSP70h being the most and the least divergent, respectively, among the four genomic regions studied. The normalized values for the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site indicated different purifying selection pressures acting on each of the four genomic regions, with the CP and CPd2 being subjected to the strongest and weakest functional constraints, respectively. A global phylogenetic analysis of sequences from the four genomic regions revealed segregation of GLRaV-1 isolates into three major clades and a lack of clearly defined clustering by geographical origin. In contrast, only two lineages were apparent when the CP and CPd2 gene sequences were used in phylogenetic analyses. Putative recombination events were revealed among the HSP70h, CP, and p24 sequences. The genetic landscape of GLRaV-1 populations presented in this study provides a foundation for better understanding of the epidemiology of grapevine leafroll disease across grape-growing regions in the United States. In addition, this study will benefit grape clean plant programs across the country in improving the sanitary status of planting materials provided to nurseries and grape growers.

Rootstock influences the effect of grapevine leafroll-associated viruses on berry development and metabolism via abscisic acid signalling.

Grapevine leafroll-associated virus (GLRaV) infections are accompanied by symptoms influenced by host genotype, rootstock, environment, and which individual or combination of GLRaVs is present. Using a dedicated experimental vineyard, we studied the responses to GLRaVs in ripening berries from Cabernet Franc grapevines grafted to different rootstocks and with zero, one, or pairs of leafroll infection(s). RNA sequencing data were mapped to a high-quality Cabernet Franc genome reference assembled to carry out this study and integrated with hormone and metabolite abundance data. This study characterized conserved and condition-dependent responses to GLRaV infection(s). Common responses to GLRaVs were reproduced in two consecutive years and occurred in plants grafted to different rootstocks in more than one infection condition. Though different infections were inconsistently distinguishable from one another, the effects of infections in plants grafted to different rootstocks were distinct at each developmental stage. Conserved responses included the modulation of genes related to pathogen detection, abscisic acid (ABA) signalling, phenylpropanoid biosynthesis, and cytoskeleton remodelling. ABA, ABA glucose ester, ABA and hormone signalling-related gene expression, and the expression of genes in several transcription factor families differentiated the effects of GLRaVs in berries from Cabernet Franc grapevines grafted to different rootstocks. These results support that ABA participates in the shared responses to GLRaV infection and differentiates the responses observed in grapevines grafted to different rootstocks.

Mealybug transmission of Grapevine leafroll viruses: an analysis of virus-vector specificity.

To understand ecological factors mediating the spread of insect-borne plant pathogens, vector species for these pathogens need to be identified. Grapevine leafroll disease is caused by a complex of phylogenetically related closteroviruses, some of which are transmitted by insect vectors; however, the specificities of these complex virus-vector interactions are poorly understood thus far. Through biological assays and phylogenetic analyses, we studied the role of vector-pathogen specificity in the transmission of several grapevine leafroll-associated viruses (GLRaVs) by their mealybug vectors. Using plants with multiple virus infections, several virus species were screened for vector transmission by the mealybug species Planococcus ficus and Pseudococcus longispinus. We report that two GLRaVs (-4 and -9), for which no vector transmission evidence was available, are mealybug-borne. The analyses performed indicated no evidence of mealybug-GLRaV specificity; for example, different vector species transmitted GLRaV-3 and one vector species, Planococcus ficus, transmitted five GLRaVs. Based on available data, there is no compelling evidence of vector-virus specificity in the mealybug transmission of GLRaVs. However, more studies aimed at increasing the number of mealybug species tested as vectors of different GLRaVs are necessary. This is especially important given the increasing importance of grapevine leafroll disease spread by mealybugs in vineyards worldwide.

Real-time RT-PCR (TaqMan) assays for the detection of viruses associated with Rugose wood complex of grapevine.

Real-time TaqMan RT-PCR (TaqMan RT-PCR) assays were developed to detect the viruses associated with Rugose wood complex of grapevines. The viruses detected were Rupestris stem pitting-associated virus in the genus Foveavirus, Grapevine virus A, Grapevine virus B and Grapevine virus D in the genus Vitivirus. The coat protein was found to be the most conserved gene within the viral species, therefore, the primers and probes for TaqMan RT-PCR assays were designed from the multiple alignment of the coat protein sequence of various isolates of each virus. Comparisons were also made between the conventional one step RT-PCR and TaqMan RT-PCR for the detection of these viruses using four-fold serial dilutions of both purified RNA and crude extract prepared from grapevine tissue. Results showed that TaqMan RT-PCR was more sensitive and could detect viruses at 32- and 256-fold higher dilutions for purified RNA and crude extract, respectively, compared to RT-PCR. The use of an internal control (18S rRNA) allowed comparison of sample preparation protocols and amplification efficiencies between samples.

Comparison of low-density arrays, RT-PCR and real-time TaqMan RT-PCR in detection of grapevine viruses.

Low-density arrays (LDA) have been designed based on the real-time RT-PCR (TaqMan) assays for the specific detection of 13 viruses that infect Grapevines in addition to the housekeeping gene 18S rRNA. The viruses included in the study are Grapevine leafroll associated viruses 1, 2, 3, 4, 5, and 9, Grapevine leafroll associated virus-2 Redglobe (GLRaV-2RG) strain, Ruspestris stem pitting associated virus, Grapevine vitivirus A, Grapevine vitivirus B, Grapevine fanleaf virus, Tomato ringspot virus (ToRSV), and Grapevine fleck virus (GFkV). This study includes three new TaqMan RT-PCR assays that have been developed for GLRaV-2RG, GFkV and ToRSV and have been included in the TaqMan RT-PCR and LDA detection. The LDAs were evaluated against a wide range of isolates distributed geographically. Geographical locations included Africa, Europe, Australia, Asia, Latin America and the United States. High-throughput detection of these viruses using LDAs was compared to RT-PCR and real-time TaqMan RT-PCR. The efficiency of different RNA extraction methodologies and buffers were compared for use in low-density array detection. In addition improving the RNA extraction technique and testing the quality of the RNA using the 18S ribosomal RNA TaqMan assay as an RNA specific internal control proved to generate better diagnostic assays. This is the first report on the use of LDA for the detection of plant viruses.

Identification and Partial Characterization of a New Luteovirus Associated with Rose Spring Dwarf Disease.

A number of viruses in the genera Ilarvirus and Nepovirus have been shown to be associated with specific diseases in rose, but many graft-transmissible rose diseases still have unknown etiologies. One of these diseases originally was detected by grafting from nonsymptomatic roses to Rosa multiflora indicator plants. Double-stranded RNAs (dsRNAs) were recovered and used as templates for cDNA synthesis and generating a cDNA library. Analysis of deduced amino acid sequences clearly positioned this virus as a member of the family Luteoviridae. The name rose spring dwarf associated virus (RSDaV) is tentatively proposed for the novel virus because the symptoms of this virus on R. multiflora are consistent with previous descriptions of rose spring dwarf disease (RSD). Phylogenetic analysis revealed a close relationship of RSDaV with members of the genus Luteovirus. Aphid transmission studies identified the rose-grass aphid (Metapolophium dirhodum) and yellow rose aphid (Rhodobium porosum) as vectors for this new virus. Host range data showed that RSDaV has a host range including both monocots and dicots. A specific reverse-transcription polymerase chain reaction assay was developed and revealed the presence of the RSDaV in several rose cultivars. RSDaV-inoculated rose plants developed RSD symptoms, confirming its role in the etiology of the disease.

Characterization of grapevine leafroll-associated virus 3 genetic variants and application towards RT-qPCR assay design.

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world. Accurate identification of the virus is an essential component in the management and control of GLRaV-3; however, the diversity of GLRaV-3, coupled with the limited sequence information, have complicated the development of a reliable detection assay. In this study, GLRaV-3 sequence data available in GenBank and those generated at Foundation Plant Services, University of California-Davis, was used to develop a new RT-qPCR assay with the capacity to detect all known GLRaV-3 variants. The new assay, referred to as FPST, was challenged against samples that included plants infected with different GLRaV-3 variants and originating from 46 countries. The FPST assay detected all known GLRaV-3 variants, including the highly divergent variants, by amplifying a small highly conserved region in the 3' untranslated terminal region (UTR) of the virus genome. The reliability of the new RT-qPCR assay was confirmed by an enzyme linked immunosorbent assay (ELISA) that can detect all known GLRaV-3 variants characterized to date. Additionally, three new GLRaV-3 divergent variants, represented by four isolates, were identified using a hierarchical testing process involving the FPST assay, GLRaV-3 variant-specific assays and high-throughput sequencing analysis. These variants were distantly related to groups I, II, III, V, VI, VII and IX, but much similar to GLRaV-3 variants with no assigned group; thus, they may represent new clades. Finally, based on the phylogenetic analysis, a new GLRaV-3 subclade is proposed and named as group X.

Real-time RT-PCR (TaqMan) assays for the detection of Grapevine Leafroll associated viruses 1-5 and 9.

Real-time RT-PCR (TaqMan) assays were developed for the specific detection of Grapevine Leafroll associated viruses 1-5 and 9 (GLRaV-1-5 and -9). The assays were evaluated against a wide range of geographically distributed isolates. Geographical locations included South Africa, Europe, Australia, Asia, Latin America and the United States. Sequences were piled up from the most conserved regions of these geographically diverse isolates and TaqMan primers and probes were designed, targeting the regions with 100% sequence identity. Improving the RNA extraction technique and testing the quality of the RNA using the 18S ribosomal RNA TaqMan assay as an RNA specific internal control to validate the quality of the extracted RNA proved to generate better diagnostic assays. The real-time TaqMan RT-PCR assays were compared to the conventional RT-PCR assays for the detection of viruses using purified total RNA as well as crude extract. The data showed that when using total RNA extracted either by the Qiagen RNeasy method or by an ABI automated system more isolates were detected in comparison to crude extract. The optimum volume of crude extract prepared in GES for use in real-time TaqMan RT-PCR cocktail was determined to be 1 microl per reaction. In addition this report showed that TaqMan RT-PCR was more sensitive than conventional one-step RT-PCR for testing different isolates of these viruses either using RNA or crude tissue extract.

Pathogen testing and certification of Vitis and Prunus species.

Strategies to screen horticultural crops for graft-transmissible agents, particularly viruses and phytoplasmas, have advanced substantially over the past decade. Tests used for Vitis and Prunus are reviewed in detail, including both biological indexing procedures and laboratory-based assays. Despite advances in laboratory molecular-based detection techniques, a strong case is presented for the continued use of slower biological tests in programs requiring high levels of confidence in detection of pathogens that must be excluded from valuable germplasm.

Application of a spotting sample preparation technique for the detection of pathogens in woody plants by RT-PCR and real-time PCR (TaqMan).

An extraction technique for reverse transcription-PCR (RT-PCR) detection of plant pathogens including viruses, bacteria and phytoplasma is described. The total nucleic acid of these plant pathogens was obtained by direct spotting of crude sap derived from infected leaf, petiole or cambial tissue onto two different types of membranes, positively charged Hybond N(+) Nylon and FTA membranes, and processed for use in PCR. Thirteen different plant viruses, Xylella fastidiosa (causal agent of Pierce's disease) and phytoplasmas were included in the experiment. A thermal treatment (95 degrees C for 10 min) of the Hybond N(+) Nylon discs in a buffered solution improved the detection, but for FTA membrane discs the thermal treatment was not required and the discs were directly placed in the PCR reaction cocktail. Specific amplification of genomic or ribosomal RNA fragments of these pathogens was obtained by one-step RT-PCR except for X. fastidiosa in which a fragment of the genomic DNA was used for amplification. The same sample preparation methods also worked well for real-time RT-PCR (TaqMan). The sample preparation techniques reported here could be used to store samples for future PCR test or for long distance shipment to a detection laboratory.