Diagnosis and Quantification of Strawberry Vein Banding Virus Using Molecular Approaches Ali Mahmoudpour Department of Plant Pathology, University of California, Davis, CA, 95616, USA Current Address: Department of Plant Protection University of Tabriz Tabriz, 51664 Iran Keywords: Limits of PCR inhibition/detection; nucleic acid extraction; virus diagnosis Abstract PCR and gel analyses were used for detection and quantification of Strawberry vein banding virus. A viral DNA isolation procedure was optimized to allow quick isolation of total nucleic acids prior to PCR. Different aged leaves, petioles, flowers and roots from infected strawberry plants including a virus indicator and three commonly grown cultivars were used to determine the most suitable tissues for virus testing and evaluate different diagnostic procedures. Nucleic acid preparations were diluted 10-10,000 fold in sterile water to determine the detection limits of PCR and the relative virus titer of samples based on dilution end point assay. The highest virus titer was in old symptomatic leaves and the lowest was in petioles. However, virus titer varied greatly among source plants used. To determine the PCR inhibition limits, concentrated preparations (up to 100x higher than those used in routine diagnosis) were used as templates. False negative results were interpreted as inhibition of Taq DNA polymerase activity by host derived contaminants with the highest levels in terminal roots and the lowest in young terminal leaves. These data were used to design a framework in mass screening field samples by pooling multiple samples. Colorimetric PCR and dot blot hybridization assay were used to confirm the above findings. INTRODUCTION Strawberry vein banding virus (SVBV) is a member of the Caulimoviridae. Several SVBV detection methodologies have been developed for SVBV diagnosis using molecular approaches. Using a high-titer indicator plant (Fragaria vesca semperflorens ) in a dot blot hybridization assay, Stenger and co-workers (1988) reported that the virus could be detected from a minimum of 100 µg fresh tissue. The use of non-radioactive probes and dot blot analysis was reported (Mráz et al., 1997) but again, the sensitivity of this method (150 µg tissue from indicator strawberries) was not suitable for reliable detection of SVBV in infected cultivars. They further modified the procedure by first using PCR for amplification of SVBV DNA and then blotting the PCR products on a membrane and detecting amplified viral DNA with chemoluminescent in a dot blot assay (Mráz et al., 1997). The objectives of this investigation were: 1) to develop PCR based methodologies for SVBV detection, 2) to develop a simple and reliable SVBV DNA extraction procedure for testing strawberry plants, 3) to determine optimal tissue for sampling and dilution of extracted DNA. METHODS AND MATERIALS Virus Sources and Maintenance Fragaria vesca L. UC-5 infected with a California isolate of SVBV were used as the main source of infected tissue in this study. These plants were inoculated originally by leaf-grafting and subsequently propagated vegetatively. F. ananassa Carlsbad, Pacifica, and Seascape were inoculated by agroinfection according Mahmoudpour Proc. X th IS on Small Fruit Virus Diseases Ed. R. R. Martin Acta Hort 656, ISHS 2004 69
(2000, 2003). Non-infected samples were obtained from virus free-plants that were generated by heat-treatment and meristem-tip culture. All these plants were tested for virus infection by indexing and maintained in a greenhouse. Sample Preparation A sample preparation procedure was developed by modifying Method 4 of Rowhani et al. (1995) and used in all experiments to evaluate the inhibition and detection limits (Mahmoudpour, 2000). Briefly, 250 mg of fresh strawberry tissue was homogenized in 5.0 ml grinding buffer (100 mm sodium or potassium phosphate, 2% polyvinylpyrrolidone-40, 50 mm benzoic acid, 0.2% β- mercaptoethanol, ph 7.0). The homogenate was clarified by centrifugation at 1,300 X g for 3 min. One ml of the supernatant was further centrifuged at 16,800 X g for 30 min at 4 C, and the pellet was suspended in 0.5 ml of disruption buffer (50 mm Tris, 10 mm EDTA, 2.0% SDS, 0.2% β-me, ph 8.0). The suspension was incubated at 65 C for 30 min, and then chilled at 10 C after adding 1/3 volume of 4.0 M potassium acetate (KAc), ph 5.5. The supernatant was recovered after centrifugation at 16,800 X g for 10 min, and DNA was precipitated by addition of an equal volume of isopropanol followed by chilling at 80 C for 15 min. The nucleic acids were recovered by centrifugation at 16,800 X g for 20 min at 4 C. The pellet was rinsed with 70% ethanol, air-dried, dissolved in 50 µl of sterile water (equivalent of 1 mg tissue/µl), and stored at 20 C to be used for PCR. DNA samples were diluted 10, 100, 1,000 or 10,000 fold with sterile water. One, 3 and 10 µl of the original preparation, and 1 and 3 µl of each of the diluted samples were used in a 20-µl PCR reaction. Rather than amount of DNA used in each reaction the amount of tissue represented by that DNA will be given per 20 µl PCR. Diagnosis and Quantification PCR reactions were performed in final volumes of 20 µl with 1-10 µl of template using a primer pair that resulted in a 944-bp amplicon of the coat protein gene (sense 5 - ATGGTAAGCAGAAGAGAAAGA-3, position 1890-1910, and complementary sense 5 -GGACAACACATATTTCTACGTA-3, position 2833-2811). The entire 20-µl PCR reaction was analyzed by electrophoresis in 1.0% agarose gel using Tris-acetate-EDTA buffer (TAE) for 15-20 min at 100 volts. Gels were stained with EtBr (1 µg/ml), and destained before photographing with a Kodak digital camera at an exposure time of 2.0 sec. The brightness of each band was rated visually as described by Mahmoudpour (2000). Colorimetric PCR conducted according Rowhani et al. (1998) was performed to validate the visual rating of DNA fragments in agarose gels. The presence of inhibitory compounds and detection limits of PCR was determined for seven types of tissues: symptomatic lower (old) leaves, middle-aged leaves, young terminal leaves, petioles from middle-aged leaves, runner tips, flowers (clipped from peduncle), and terminal roots obtained from strawberry cultivars. In this analysis, three UC-5 plants and three selected cultivars were examined. Three samples of the seven tissue types per each plant were tested. All raw data from PCR analysis of inhibition/detection limits were used in a statistical analysis. In PCR analysis of pooled samples, 50 mg of symptomatic young leaves of SVBV-infected UC-5 plants was mixed with 4.95 g of tissue from an uninfected plant. After homogenization, three sub-samples of clarified supernatant including 1.0 ml, 1.0 ml, and 70 ml (scaled up) were used for DNA isolation. Further dilutions were made in sterile water and PCR was performed as described before. RESULTS Consistent data were obtained in all experimental replicates of virus distribution in different tissues and tissue-specific PCR inhibition. Each experiment was repeated 3-10 times by sampling tissues from different UC-5 plants. No significant variation was observed among these replicates (P = 0.05). The highest rate of virus detection was observed in old symptomatic leaves and the lowest in leaf petioles. Inhibition was greatest 70
in root samples and old leaves. The minimal inhibition was observed from young leaves and petioles using template concentration of 10-100x of what used in routine detection. The consistency of these data were confirmed by extending the survey to three commonly grown strawberry cultivars. Tissue-specific inhibition (using concentrated DNA templates in PCR) by cultivars is shown in Fig. 1. Similar analyses were conducted to determine detection limits or relative virus titer by using diluted DNA templates in PCR. Consistent trend of tissue-specific virus titer with considerable variation among the hosts was observed (Fig. 2). Inhibition and detection ranges of terminal leaves in different host types as averaged from 3 replicates are illustrated in Fig. 3. PCR analysis indicated that the highest and lowest SVBV titers were in older symptomatic leaves and petioles, respectively, and in UC-5 and Seascape, respectively. These findings, albeit with 30,000 fold lower sensitivity, were confirmed by dot blot analysis (data not shown). Detection of the PCR-amplified SVBV DNA fragment in pooled samples is shown in Fig. 4. The PCR-amplified virus DNA was detected after pooling samples from infected UC-5 with 1:100 uninfected tissue and diluting the preparation up to 300 fold. DISCUSSION Faint or lack of amplified DNA fragments at high concentrations of sample DNA indicated inhibitors were present and at low concentrations of sample DNA indicated the limit of detection for the PCR. The signal strength obtained with the colorimetric PCR was comparable with visual ratings based on band intensity in agarose gels Quantification of PCR analysis allowed for the determination of the limits of confidence in SVBV detection. Positive results were easy to interpret, while negative results were more difficult because false negative results could be due to PCR inhibition, low virus titer, or non-infected samples. The optimal amount of tissue represented in a PCR reaction to allow for reliable detection of SVBV and best tissue source for testing were determined. The optimal sample preparation method used the equivalent of 50 mg of tissue from older leaves suspended in 500 µl of sterile water after extraction. The maximum levels of PCR inhibitors were found in preparations from root samples followed by those from older leaves. Consistency of tissue-dependent inhibition was demonstrated across different host genotypes. Young leaves followed by petioles and runner tips had the lowest amounts of PCR inhibitors, whereas middle-aged leaves were intermediate. Preparations obtained from old symptomatic leaves provided the best source of DNA for detection of SVBV, and were significantly better than other types of tissue. Furthermore, PCR detection of SVBV was correlated with symptoms, i.e., symptoms were usually observed in older and lower leaves, which also produced higher signals at the lower dilutions compared to other tissues. These results indicated that a virus indicator (Fragaria vesca UC-5 ) and a newly released cultivar Carlsbad, which expressed strong vein banding symptoms, provided higher levels of PCR-amplified SVBV DNA in any given tissue compared with the same tissue in the other genotypes tested. In contrast, symptomless older leaves produced low PCR signals. The lowest PCR signals were observed in petiole samples. Therefore, low PCR signal from petioles or runners (without leaves) may reflect the absence of significant amount of virus in these tissues. Inhibition and detection limits identified best tissues and dilutions to optimize detection of SVBV in strawberry tissues. This information and the mixing of infected tissue with healthy tissue to determine sensitivity of the assay will be useful if pooling samples for large scale testing. As demonstrated in Fig. 4, SVBV was detected when the extract from a 50-mg-leaflet of UC-5 was diluted 100-fold with extract from uninfected tissue prior to precipitation and washing of the DNA. Detection was still possible after further dilution to 300-fold with sterile water. As shown in this analysis, the level of inhibition increased at least 30 fold in pooled samples compared with sample DNA not diluted with healthy DNA as shown in Fig. 1. This increased level of inhibition is due to an increased ratio of inhibitory molecules to viral template or to diluting the virus DNA with 100X inhibitors during sample preparation. On the other hand, the detection limit of pooled samples (3.0 µg tissue diluted 100-fold with non-infected tissue) was similar to 71
those observed for symptomatic leaves (30 ng) as seen in Fig. 2. Finally, these tests were all carried out using greenhouse grown plants and an evaluation of inhibitors and virus titer in field plants needs to be done before the technique can be used for large scale testing. It is quite possible that field grown plants will have a lower virus titer and a higher concentration of inhibitors compared to greenhouse grown plants. Also as shown in this study, all cultivars to be tested by this method need to be evaluated as there are significant differences in virus titer and inhibitor concentrations between cultivars. ACKNOWLEDGMENTS This study was accomplished during 1996-2000 at the Department of Plant Pathology and Foundation Plant Materials Service (FPMS), University of California, Davis. The author appreciates the critical review made by Dr. Robert R. Martin. Literature Cited Mahmoudpour, A. 2003. The infectivity of recombinant strawberry vein banding virus DNA. J. Gen. Virol. 84:1377-1381. Mahmoudpour, M.M.A. 2000. Strawberry vein banding caulimovirus: biology and characterization. PhD dissertation. University of California, Davis. Mráz, I., Honetšlegrová, J. and Šip, M. 1996. Diagnosis of strawberry vein banding virus by a non- radioactive probe. Acta Virologica 40: 130-141. Mráz, I., Petrzik, K., Fránová-Honetšlegrová, J. and Šip, M. 1997. Detection of strawberry vein banding virus by polymerase chain reaction and dot blot hybridization. Acta Virologica 41:241-242. Rowhani, A., Biardi, L., Routh, G., Daubert, S.D. and Golino, D.A. 1998. Development of a sensitive colorimetric-pcr assay for detection of viruses in woody plants. Plant Disease 82:880-884. Rowhani, A., Meanings, M.A., Lile, L.S., Daubert, S.D. and Golino, D.A. 1995. Development of a detection system for viruses of woody plants based on PCR analysis of immobilized virions. Phytopathology 85:347-352. Stenger, D.C., Mullin, R.H. and Morris, T.J. 1988. Isolation, molecular cloning, and detection of strawberry vein banding virus DNA. Phytopathology 78:154-159. 72
Figures Fig. 1. Rate of PCR inhibition in different tissues/hosts as determined by visual rating of PCR-amplified SVBV DNA fragments in EtBr-stained agarose gels. Values in Y axis represent mean for three replicates at six concentration (equivalents of 10 mg, 3.0 mg, 1.0 mg, 300 µg, and 100 µg fresh tissue weight). Fig. 2. PCR detection limits in different tissues/hosts based on PCR-amplified SVBV DNA fragments in EtBr-stained agarose gels. Each value represents mean of 3 replicates at 7 concentrations (equivalents of 30 µg, 10 µg, 3.0 µg, 1.0 µg, 300 ng, 100 ng, and 30 ng fresh tissue weight). 73
Fig. 3. PCR detection limits in terminal leaves from 4 different hosts as determined by visual rating of PCR products in EtBr-stained agarose gel. Each value was obtained from averaging three replicates. Fig. 4. PCR analysis of pooled samples. DNA was isolated from pooled samples (50 mg of UC-5 symptomatic young leaf pooled with 100 fold non-infected tissue) subsequently used in PCR after serial dilution. Templates represented as equivalent tissue weight used for isolation of DNA. Sample preparation was repeated 3 times, and each template was analyzed in 4 concentrations. The target DNA fragment was 944 bp. 74