© 2007 Plant Management Network.
Desiccation at Ambient Temperature Effectively Preserves Plant Tissues Infected with Phytophthoras
Frances S. Ockels, 201 Kottman Hall, 2021 Coffey Road, Department of Plant Pathology, The Ohio State University, Columbus 43210; Matthew V. DiLeo, One Shields Avenue, Department of Plant Pathology, University of California, Davis 95616; and Pierluigi Bonello, 201 Kottman Hall, 2021 Coffey Road, Department of Plant Pathology, The Ohio State University, Columbus 43210
Ockels, F. S., DiLeo, M. V., and Bonello, P. 2007. Desiccation at ambient temperature effectively preserves plant tissues infected with Phytophthoras. Online. Plant Health Progress doi:10.1094/PHP-2007-0302-01-RS.
Conventionally, plant samples collected in the course of field surveys for Phytophthora spp. (e.g., in the context of the US Forest Service-sponsored national P. ramorum survey of forest environments) are either processed immediately or stored at low temperatures and processed as soon as possible for detection by molecular methods. In order to extend the useful life of the sample, a method involving tissue desiccation was explored for effectively storing Phytophthora-infected plant leaves. In one experiment, rhododendron leaves inoculated with an unknown Phytophthora sp. and desiccated for seven days yielded DNA of sufficient quality for species identification via sequencing of the ITS region. In a second experiment, P. ramorum was successfully detected by PCR in inoculated leaves of California bay laurel, California buckeye, bigleaf maple, rhododendron, and viburnum that were desiccated and stored at room temperature for four months. Therefore, desiccation might be a viable, reliable, and less expensive alternative to storing foliar samples at low temperature.
Phytophthora ramorum Werres, de Cock, and Man in’t Veld, the causal agent of Sudden Oak Death (7,9), continues to be a threat to US forests, having the potential to spread large distances on infected nursery stock. Despite intensive efforts to monitor plants for infection prior to shipping, nursery stock infected with P. ramorum has still been shipped from the west coast to other states in the US. In 2004, the USDA-APHIS national nursery survey found that 22 states received infected nursery stock, while seven states in 2005 and three states as of September 2006 received infected stock. Due to the continued threat of P. ramorum spread, the US Forest Service continued in 2005 and 2006 the national P. ramorum survey of forest environments, which was initiated in 2004. The objective of the survey was to examine forested areas around eastern nurseries receiving potentially infected stock from the West Coast, as well as other forest sites, for the presence of P. ramorum.
During the 2004, 2005, and 2006 national P. ramorum survey of forest environments, samples were stored in an ice chest on sealed coolant and shipped to the processing labs for PCR-based detection of P. ramorum within 72 h of collection. During these surveys the process of storing the samples on coolant and shipping every two days became cumbersome in terms of expense and convenience. Therefore, we decided to test the feasibility of using partial desiccation at ambient temperature to preserve infected tissue for analyses using Drierite (W.A. Hammond Drierite Co., Xenia, OH; 4 mesh). Previous research has demonstrated that plant tissue can be preserved by desiccation, but it remained to be determined if DNA extracted from the lesions caused by Phytophthora species, and more specifically by Phytophthora ramorum, on leaf tissue can be preserved and amplified by PCR after desiccation.
Experiment 1: Preservation of Rhododendron Leaves Inoculated with an Unknown Phytophthora sp.
Several Phytophthora cultures, including P. citricola Sawada, P. citrophthora (R.E. Sm. & E.H. Sm.) Leonian, P. cinnamomi Rands, and P. cactorum (Lebert & Cohn) J. Schrot, were kindly provided by Anne Dorrance, The Ohio State University. Additionally, an unknown Phytophthora isolate (SOD-OH-255-22) from a Rhododendron sp. was obtained from Nancy Taylor at The Ohio State University’s C. Wayne Ellett Plant and Pest Diagnostic Clinic. Cultures were maintained by transferring hyphal tips to a fresh dilute lima bean agar (LBA) plate every week (8). Each species was grown on dilute V-8 juice agar slants and submerged in distilled water for long-term storage at 10°C in the dark (8).
Two rhododendrons, cultivars Nova Zembla and P.J.M., were maintained in the greenhouse and inoculated with the Phytophthora spp., by wounding a leaf with a needle and then placing a plug of agar containing mycelium over the wound. The plug of agar was retained with tape to secure it and to protect the oomycete from drying out. Each species was inoculated on two leaves on each rhododendron. Infections were monitored for lesion development over a period of approximately 2 weeks. SOD-OH-255-22 was the most pathogenic isolate in our tests, particularly on the Nova Zembla rhododendron, and caused the largest lesions one week after inoculation (Fig. 1). Therefore, all subsequent inoculations were carried out with SOD-OH-255-22 and the Nova Zembla rhododendron.
Ten leaves were inoculated on 9 September 2005, with a plug of SOD-OH-255-22 isolate and ten leaves were inoculated with a plug of sterile dilute LBA as a control. The infection was monitored and allowed to develop for six days when a substantial lesion had formed. Subsequently, leaves with lesions and the control leaves were placed in separate plastic bags in the presence of a cheesecloth sachet containing approximately 30 g of activated Drierite. The plastic bags were placed in an air-tight plastic container and left for 10 days until leaves were crisp.
Plant samples were processed for DNA extraction according to USFS survey protocols (5), except that homogenized tissue was extracted using the Macherey-Nagel NucleoSpin Plant Kit (Macherey-Nagel, Easton, PA), following the manufacturer’s directions. PCR was performed with ITS6 and ITS4 primers. ITS6 is a universal primer designed to improve the amplification of part of the internal transcribed spacer (ITS) of the rDNA of Oomycota (2) while ITS4 is a universal primer for eukaryotic ITS (10). In addition to the experimentals containing DNA isolated from infected or uninfected tissue, each PCR set also contained negative controls with no added DNA template. Volume and composition of the PCR cocktail and PCR conditions were identical to those detailed online (2). Amplifications were carried out in a PTC-200 Peltier Thermal Cycler (MJ Research, Inc., Waltham, MA) and PCR products were separated on a 1.5% agarose gel containing EtBr in 1 X TAE buffer at 100 V for 30 min (5).
The PCR reactions from the inoculated leaves amplified with ITS6 and ITS4 yielded two DNA products. The DNA from the two bands was isolated from the agar using a Macherey-Nagel PCR clean-up and gel extraction kit (Macherey-Nagel, Easton, PA) and reamplified. The PCR products were purified with the same kit and sequenced using the ITS6 primer at the Plant-Microbe Genomics Facility at The Ohio State University. Identification of the sequenced samples was attempted using BLAST in Genbank.
DNA from Phytophthora isolate SOD-OH-255-22 was successfully extracted and amplified from desiccated Nova Zembla rhododendron using primers ITS6 and ITS4. To determine the species of isolate SOD-OH-255-22, hyphae from the culture were amplified by PCR and the product (band of ~900 kb) was purified and sequenced. The sequence most closely matched P. citrophthora (83% identity), and may represent a novel species. PCR of the DNA extracted from the margins of the lesions caused by SOD-OH-255-22 resulted in two products of differing sizes, approx. 900 kb and 700 kb (data not shown). Isolation of the bands of the two products and sequencing showed that the larger band most closely matched P. citrophthora (74% identity) and the smaller band was rhododendron (99% identity). The mock-inoculated leaves yielded one product (~700 kb) that was sequenced and identified as rhododendron DNA (99% identity).
Experiment 2: Preservation of Plant Leaves Inoculated with P. ramorum
This experiment was carried out in an approved laboratory under permit to handle P. ramorum. California buckeye (Aesculus californica (Spach) Nutt.), California bay laurel (Umbellularia californica (Hook. & Arn.) Nutt.), and bigleaf maple (Acer macrophyllum Pursh) leaves were collected from the California Foothill Collection of the UC Davis Arboretum. Rhododendron (Colonel Coen) and Sandankwa viburnum (Viburnum suspensum Lindl.) leaves were collected from residential properties in Davis, CA. Leaves were inoculated with P. ramorum on 12 April 2006 by submerging approximately a quarter of each leaf in beakers filled with 100 ml of a zoospore suspension with a final concentration of 4.5 × 104 zoospores/ml. The beakers were placed in 20°C incubator with lights for two days. Next, the leaves were transferred from the beakers to humid crispers. Lesions developed over a period of approximately one week (Fig. 2). For each species, five symptomatic leaves were placed in a sealable plastic bag and stored at -18°C, while five symptomatic leaves were placed in a sealable plastic bag in the presence of a Drierite sachet and stored in the dark at room temperature. The same storage strategy was used for asymptomatic, control leaves. After four months under the two treatments, DNA was extracted as described above.
DNA concentration in the extracts was quantified using a Thermo Spectronic BioMate 3 spectrophotometer against the NucleoSpin plant kit elution buffer (blank). DNA yields from infected and uninfected, frozen and desiccated tissue of the different species are shown in Table 1. ANOVA of DNA yields was carried out separately by infection status and tissue preservation method using SPSS 11.0 for Mac OS X. Across the five different species (used as replicates), DNA concentrations did not vary significantly by infections status (182 ± 35 ng/µl and 295 ± 63 ng/µl for control and infected tissue, respectively; F1,19 = 2.77, P = 0.114) or preservation method (234 ± 52 ng/µl and 243 ± 52 ng/µl for freezing and desiccation, respectively; F1,19 = 0.02, P = 0.896).
Table 1. DNA yields from Experiment 2.
P. ramorum was detected using the nested PCR protocol described in Hayden et al. (4,5). All extracts were diluted 1:10,000 in sterile de-ionized water prior to the initial round of PCR in order to overcome the effects of putative PCR inhibitors. Each experimental reaction set also contained negative controls with no added DNA template. All amplifications were carried out in a GeneAmp PCR system 9700 PCR thermocycler (Applied Biosystems, Foster City, CA).
Using this P. ramorum-specific assay, DNA was successfully amplified from lesions on California buckeye, California bay laurel, bigleaf maple, rhododendron, and viburnum leaves caused by P. ramorum. Both frozen and desiccated uninoculated leaves yielded no PCR products, while both frozen and desiccated inoculated leaves yielded a product of the expected size (291 bp, Fig. 3).
Leaf Desiccation and Amplification of Phytophthora DNA
This study demonstrated that (i) desiccation of symptomatic and asymptomatic leaf tissue for up to four months did not result in significant changes in total extractable DNA compared with freezing, and (ii) molecular detection of two different Phytophthora species in symptomatic tissue was possible despite an apparent slight decrease in DNA quality (as suggested by amplicon intensities in Fig. 3). Considering the logistical limitations of current survey protocols regarding plant sample preservation, which include the necessity to transport a number of personal coolers with sealed coolant (such as blue ice) to and from the field, we believe that using a desiccant like Drierite would be a significant practical improvement over the current guidelines. Tissue desiccation would also result in significant savings as it would not be necessary for surveyors to ship samples at the end of each day, but rather as a lump shipment at the end of the collection period, or as bulked periodic shipments via regular mail, rather than overnight courier service. Other studies support similar conclusions. For example, Liston et al. (6) collected plant material in a remote region of northwestern China by placing the samples, wrapped in tissue paper, in plastic bottles pre-filled to one-third capacity with Drierite. They found this to be an excellent preservation method for plant tissue used for DNA and isozyme analysis. The method was simple, inexpensive, reliable, and had potential for broader application. They observed degradation of samples only after several months of storage and suggested that DNA extraction be carried out relatively soon after samples reach the lab. Gitzendanner and Soltis (3) reported similar success in preserving plant tissue by desiccation with Drierite. DNA was successfully extracted from these desiccated samples and screened for sequence variation by amplified-fragment length polymorphisms (AFLPs) and single-strand conformation polymorphisms (SSCPs).
Desiccants other than Drierite may also work well. The use of silica gel (which we did not test) was investigated by Chase and Hill (1) to preserve leaf samples from a wide sampling of flowering plants in Argentina and Brazil. Samples became desiccated within 12 to 24 h. The authors concluded that the desiccation of plant tissue with silica gel is practical, reliable, and inexpensive. They found that DNA extracted from silica gel-dried samples was of good quality and could be used in restriction site studies and in PCR amplification in gene sequencing studies.
In summary, based on our results as well as previous work, preservation of foliar samples by desiccation appears to be a viable, practical, and less expensive alternative to freezing and immediate, or nearly immediate, processing for the purpose of detecting P. ramorum and other Phytophthora spp. in infected, symptomatic tissue using PCR-based protocols. We suggest that this procedure should also be tested for non-foliar tissue that is often collected in the course of surveys. If the tissue to be sampled is mainly internal (e.g., in the case of twigs, tree bark, large root material) it may be possible to directly embed the samples in activated Drierite or silica gel. Finally, we recommend that this method be tested in the course of an actual survey season by duplicating samples from selected sites and processing them using desiccation in conjunction with prescribed storage at low temperature.
In the course of field surveys for Phytophthora spp., for example in the national P. ramorum survey of forest environments sponsored by the US Forest Service, it is recommended that plant samples be collected and shipped, on ice or sealed coolant, to a processing lab within 72 h and stored at -20°C thereafter. To do that, field crews must carry coolers and sealed coolant, such as blue ice, to the field, and locate a shipping center near their field site to send the containers overnight to their assigned processing lab. Because this protocol is inconvenient and expensive, an alternate way of preserving leaf tissue at ambient temperatures for diagnostic assays was investigated. We explored the feasibility of desiccating foliar samples with Drierite as a way of preserving tissue at ambient temperature. In our study we found that desiccation with Drierite can preserve PCR-quality Phytophthora DNA in foliar tissue for at least four months, suggesting that this is a viable alternative to storing and immediately shipping field samples at low temperatures.
Funding provided by the US Forest Service (04-CA-11244225-432) and by State and Federal funds appropriated to the Ohio Agricultural Research and Development Center, the Ohio State University. Special thanks to Duan Wang, Nathan Kleczewksi, Emily Helliwell, and Justin Whitehill for technical help, and Alieta Eyles, and Brian McSpadden Gardner for helpful pre-submission comments on various drafts of the paper.
1. Chase, M. W., and Hills, H. H. 1991. Silica gel: An ideal material for field preservation of leaf samples for DNA studies. Taxon 40:215-220.
2. Cooke, D. E. L., and Duncan, J. M. 1997. Phylogenetic analysis of Phytophthora species based on ITS1 and ITS2 sequences of the ribosomal RNA gene repeat. Mycolog. Res. 101:667-677.
3. Gitzendanner, M. A., and Soltis, P. S. 2001. Genetic variation in rare and widespread Lomatium species (Apiaceae): A comparison of AFLP and SSCP data. Edinburgh J. Bot. 58:347-356.
4. Hayden, K. J., Rizzo, D., Tse, J., and Garbelotto, M. 2004. Detection and quantification of Phytophthora ramorum from California forests using a real-time polymerase chain reaction assay. Phytopathology 94:1075-1083.
5. Levy, L., and Mavrodieva, V. 2004. PCR detection and DNA isolation methods for use in the Phytophthora ramorum national program. USDA-ARS, Washington, DC.
6. Liston, A., Rieseberg, L. H., Adams, R. P., Do, N., and Ge-lin, Z. 1990. A method for collecting dried plant specimens for DNA and isozyme analyses, and the results of a field test in Xinjiang, China. Ann. Missouri Bot. Garden 77:859-863.
7. Rizzo, D. M., Garbelotto, M., Davidson, J. M., Slaughter, G. W., and Koike, S. T. 2002. Phytophthora ramorum as the cause of extensive mortality of Quercus spp. and Lithocarpus densiflorus in California. Plant Dis. 86:205-214.
8. Schmitthenner, A. F., and Bhat, R. G. 1994. Useful methods for studying Phytophthora in the laboratory. Ohio Agric. Res. and Dev. Center (OARDC) Spec. Circ. 143. Wooster, OH.
9. Werres, S., Marwitz, R., Veld, W., De Cock, A., Bonants, P. J. M., De Weerdt, M., Themann, K., Ilieva, E., and Baayen, R. P. 2001. Phytophthora ramorum sp nov., a new pathogen on Rhododendron and Viburnum. Mycolog. Res. 105:1155-1165.
10. White, T. J., Bruns, T. D., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. W. White, eds. Academic Press, Inc., San Diego, CA.