Fatima Osman

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.

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.

Vitis californica and Vitis californica × Vitis vinifera Hybrids are Hosts for Grapevine leafroll-associated virus-2 and -3 and Grapevine virus A and B

Vitis and non-Vitis spp. surrounding nine Napa Valley vineyards were surveyed for Grapevine leafroll-associated virus (GLRaV)-1 to -5 and -9, Grapevine virus A (GVA), Grapevine virus B (GVB), and Grapevine virus D (GVD). Vitis spp. from three riparian areas not adjacent to vineyards were also included. DNA fingerprinting and probability analyses indicated that the Vitis samples consisted primarily of Vitis californica followed by V. californica × V. vinifera hybrids. Single and mixed infections of GLRaV-2, -3, GVA, or GVB were detected by conventional or quantitative reverse-transcription polymerase chain reaction in 6 of the 66 V. californica and 11 of the 19 V. californica × V. vinifera hybrids. GLRaV-1, -4, -5, -9, and GVD were not detected. Phylogenetic analysis of GLRaV-2 and -3 partial coat protein gene nucleotide sequences indicated that the isolates from V. californica and V. californica × V. vinifera hybrids were closely related to isolates from V. vinifera.

Comparative procedures for sample processing and quantitative PCR detection of grapevine viruses.

In this study different instruments and methods used for tissue homogenization, RNA extraction and quantitative PCR (qPCR) based detection of grapevine RNA viruses were evaluated. Semi-automated and automated homogenization techniques were compared to process samples from grapevine petioles and cambial tissue. Four different high throughput automated nucleic acid extraction platforms were compared with the RNeasy plant extraction kit for their capacity and efficiency of extracting viral RNA from grapevine infected tissues. The RNA prepared from each extraction platform was then used as template for a comparative analysis of qPCR by One Step RT-qPCR, Two Step RT-qPCR and low density array (LDA) detection. This study showed that a thorough homogenization of grapevine tissues using the Tissue Lyser as well as DNase digestion of the purified RNA prior to cDNA synthesis improved the virus detection and yielded the lowest quantitation cycle (Cq) values in RT-qPCR. Comparison of different RNA extraction methods showed that methods implementing the magnetic bead-based technology were superior to other methods used. Comparing different qPCR detection methods, One Step RT-qPCR showed the lowest Cq values for the same sample tested compared to Two Step RT-qPCR and LDA.

Development and validation of a multiplex quantitative PCR assay for the rapid detection of Grapevine virus A, B and D

A single real-time multiplex quantitative PCR (qPCR) assay for the simultaneous detection of Grapevine virus A, B and D (GVA, GVB and GVD) was developed, using three different fluorescently labeled minor groove binding probes. This multiplex RT-qPCR was compared to singleplex RT-qPCR designed specifically for each virus and a conventional multiplex RT-PCR. The capacity of the multiplex RT-qPCR assay in detecting the three vitiviruses in mixed infections from a range of virus concentrations in the host was assessed. A series of cDNA derived from 48 different grapevine cultivars obtained from diverse geographical regions infected with various isolates and strains of GVA, GVB and GVD were subjected to singleplex, multiplex RT-qPCR, and conventional multiplex RT-PCR testing. The results showed that the developed multiplex RT-qPCR assay was a cost-effective diagnostic tool that could streamline the testing of grapevine vitiviruses, and replace the singleplex RT-qPCR assays, thus reducing time and labor while retaining the same sensitivity and specificity. In particular, no significant differences in detection limits were found between singleplex and multiplex RT-qPCR and specificity was not affected by the inclusion of the three primer/probe combinations within a multiplex RT-qPCR. Comparing the viral load for each virus using singleplex and multiplex RT-qPCR assays revealed no significant differences between the two assays in detecting GVB and GVD. However, while in detecting GVA using singleplex RT-qPCR assay, viral load was higher. Finally, the multiplex RT-qPCR assay was also more sensitive and time efficient than the conventional multiplex RT-PCR that was designed using degenerate primers to detect GVA, GVB and GVD. This multiplex RT-qPCR method could detect viruses in 95.83% of mixed infected samples as compared to 77.08% for multiplex RT-PCR.

Detection of Grapevine leafroll-associated virus 7 using real time qRT-PCR and conventional RT-PCR

Nine isolates of Grapevine leafroll-associated virus 7 (GLRaV-7) from diverse geographical regions were sequenced to design more sensitive molecular diagnostic tools. The coat protein (CP) and heat shock protein 70 homologue (HSP70h) genes of these nine isolates were sequenced. Sequences were then used to design more sensitive molecular diagnostic tools. Sequence identity among these isolates ranged between 90 to 100% at the nucleotide and amino acid levels. One RT-PCR and two qRT-PCR assays were used to survey 86 different grapevines from the University of California, Davis Grapevine Virus Collection, the Foundation Plant Services collection and the USDA National Clonal Germplasm Repository, Davis, CA with primers designed in conserved regions of the CP and HSP70h genes. Results revealed that qRT-PCR assays designed in the HSP70h gene was more sensitive (29.07% positives) than that designed in the CP gene (22.09% positives) and both qRT-PCR assays proved to be more sensitive than RT-PCR.

Evaluation of the phytosanitary status of the Prunus species in the National Clonal Germplasm Repository in California: survey of viruses and viroids.

The USDA National Clonal Germplasm Repository (NGCR) at the University of California, Davis is recognized as one of the richest sources of Prunus species material in the US. The repository maintains more than 2450 trees representing 96 taxa collected from around the world. However, the phytosanitary status of the NCGR Prunus collection has not been thoroughly evaluated. In this study, a comprehensive screening of a selected subset of the collection for virus and virus-like diseases affecting Prunus trees has been completed. Two factors were considered for selecting the trees: the country of origin and the observation of suspicious disease symptoms. A total of 221 trees representing 185 different cultivars of cherry, almond, peach, apricot and plum were sampled. Conventional RT-PCR detection was used to test for 13 different viruses and two viroids. The pathogens included in the survey were Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Plum bark necrosis stem pitting-associated virus (PBNSPaV), American plum line pattern virus (APLPV), Cherry virus A (CVA), Cherry leafroll virus (CLRV), Cherry rasp leaf virus (CRLV), Cherry green ring mottle virus (CGRMV), Cherry necrotic rusty mottle virus (CNRMV), Apple chlorotic leafspot virus (ACLSV) Tomato ringspot virus (ToRSV), Little cherry virus 1 (LChV-1), Little cherry virus 2 (LChV-2), Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). Though the majority of these trees were asymptomatic, all tested pathogens were detected except for ToRSV and CRLV. Two of the viruses detected (ACLSV and LChV-2) had never been reported from California. Incidence of PNRSV in tested trees was the highest (55 trees) followed by the two viroids (PLMVd and HSVd), with 33 and 32 infected trees, respectively. The incidence for the rest of the pathogens ranged between 0 to 19 trees. The infection rate of all tested samples ranged from 0.5% to 24.9%.

Improved detection of ilarviruses and nepoviruses affecting fruit trees using quantitative RT-qPCR.

Reverse transcription quantitative PCR (RT-qPCR) assays were developed for the detection of the ilarviruses Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Apple mosaic virus (ApMV), and American plum line pattern virus (APLPV), and the nepoviruses Tomato ringspot virus (ToRSV) and Cherry leafroll virus (CLRV). These viruses affect various stone fruits such as apricots, cherries, peaches, plums, and almonds. The goal of this work was to improve the RT-qPCR detection of PNRSV, PDV, and ApMV in addition to developing three new RT-qPCR assays for the detection of APLPV, ToRSV and CLRV. Primers for conventional RT-PCR as well as primers and probes for RT-qPCR assays were designed after aligning coat protein (CP) gene sequences of geographically diverse isolates with the corresponding CP gene sequences from GenBank, targeting regions with 100% sequence identity. The efficiency of each RT-qPCR assay, as well as the intra- and inter-assay variability, were determined. These conventional RT-PCR and RT-qPCR assays were validated using purified total RNAs from 221 trees from the USDA Clonal Germplasm Repository orchards. The data showed that more isolates were detected by RT-qPCR than by RT-PCR.

Virus distribution and seasonal changes of grapevine leafroll-associated viruses

A time-course study was performed over two years to investigate seasonal changes in copy numbers of different Grapevine leafroll associated virus (GLRaV) in infected grapevine (Vitis vinifera) in North America. We examined GLRaV-1, -2, -3, -4, and GLRaV-2RG in 104 grapevines that previously tested positive for one or more of these GLRaVs. Vines were selected from the Davis grapevine virus collection and the USDA National Clonal Germplasm Repository in northern California. Samples were collected from the beginning of the growing season (late April) until February; from April to November, mature leaf petioles were collected, and from December to February, cambial scrapings were taken from dormant cuttings. All samples were tested by RT-PCR and RT-qPCR; RT-qPCR was found to be the more sensitive technique. GLRaVs were more reliably detected in samples collected between August and February. Grapevines infected with GLRaV-2 were evenly detected throughout the year, while infection with GLRaV-1, -3 -4, and -2RG was best observed from November to February for both experimental years. Virus titer was lowest early in the growing season (April/May), and viral load differed among the GLRaVs. The viral load of GLRaV-3 remained unchanged, regardless of the season tested. Viruses associated with leafroll disease in grapevines were not uniformly distributed in both leaf petioles and cambium of grapevines; GLRaV-2 was the most evenly distributed virus in the grapevine, followed by GLRaV-1, GLRaV-3, and finally by GLRaV-4, which had the most uneven distribution.

One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts

A one-step multiplex reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) based on species-specific minor groove binding (MGB) probes, was developed for the simultaneous detection, identification, and quantification of three citrus viroids belonging to different genera. Citrus exocortis viroid (Pospiviroid), Hop stunt viroid (Hostuviroid), and Citrus bark cracking viroid (Cocadviroid) cause a variety of maladies in agriculturally significant crops. Therefore, reliable assays for their detection are essential tools for various government and industry organizations implementing disease management programs. Singleplex qPCR primers and MGB probes were designed individually for the detection of the three targeted viroids, and subsequently combined in a one-step multiplex RT-qPCR reaction. A wide host range of woody plants, including citrus, grapevines, apricots, plums and herbaceous plants such as tomato, cucumber, eggplant and chrysanthemum different world regions were used to validate the assay. Single, double and triple viroid infections were identified in the tested samples. The developed multiplex RT-qPCR assay was compared with a previously reported SYBR Green I RT-qPCR for the universal detection of citrus viroids. Both assays accurately identified all citrus viroid infected samples. The multiplex assay complemented the SYBR Green I universal detection assay by differentiating among citrus viroid species in the positive samples. The developed multiplex RT-qPCR assay has the potential to simultaneously detect each targeted viroid and could be used in high throughput screenings for citrus viroids in field surveys, germplasm banks, nurseries and other viroid disease management programs.