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Virus Testing

Written by Tom Auvil, Tree Fruit Research Commission

Preparing for Virus Testing of Fruit Trees

General instructions for sample collection

  1. Immediately place the sample in a sealed plastic bag (e.g. Zip-Loc). Label bag (tree, row, grower, GPS etc. and barcode the bags (if possible) for more detailed identification within large volume sampling).
  2. Do not add additional moisture in the bag.
  3. Do not allow the bag to sit in the sun (the samples heat very quickly).
  4. Keep the samples cool and shaded.
  5. If you use ice packs, do not place the bags with leaf samples directly against the ice pack. This will freeze the tissue and the lab will not be able to test the sample reliably. Use several layers of paper or bubble wrap to insulate the leaves.

NOTE: The virus distribution in plants is uneven, particularly with recent infections, and the virus concentration changes from season to season. The type of tissue and season are the critical factors influencing reliable results. Dormant bud sticks are often one of the best tissues for virus detection. Generally the virus concentration is reduced in the hot summer season (with exceptions). Suggestions for collecting different sample tissues are provided below for testing major stone and pome fruit viruses/diseases.

Stone Fruits
Sample collection for testing Ilar (PDV, PNRSV, APLPV), poty (PPV), Nepo (CLRV, ToRSV, TRSV), Chera (CRLV), Tricho (ACLSV, CMLV), Fovea (ApLV), Capillo (CVA, ASGV) and CGRMV, CRMaV, CNRMV, CTLaV viruses.
Type of sample
Collection time
Sample Description
Dormant bud sticks
End of Jan. until bud break 8 – 10 inches at the base of the 1-year-old wood. Samples close to leader generally give more consistent results. Collect one stick each from all major scaffold limbs and package together in a zip lock bag.
Flower and leaf buds
Bud swell to bud popcorn stage Collect sample from all major scaffolds and limbs. Each individual sample must have at least 6 buds. Package individual samples together in a zip lock bag.
Branches
2 weeks after full bloom until end of Aug. 8 to 10 inches of 1-year-old wood or the base of the current season’s growth (with leaves attached) are the best samples. Samples close to leader generally give more consistent results. Collect sample from all the symptomatic limbs. For non-symptomatic trees collect a sample from each major scaffold limb and package together. For testing Ilar viruses, collect the samples prior to summer (from full bloom until mid-June)

Sample collection for testing Little cherry disease (Little Cherry Virus and Western X Phytoplasma). The results are more reliable when samples are collected near or after commercial harvest. During growing season, flag limbs with suspicious symptoms for sample collection later in the season.

Type of sample
Collection time
Sample Description
Dormant bud sticks
End of Jan. until bud break 8 – 10 inches at the base of the 1-year-old wood. Samples close to leader generally give more consistent results. Collect one stick each from all major scaffold limbs and package together in a zip lock bag.
Flower and leaf buds
Bud swell to bud popcorn stage Collect sample from all major scaffolds and limbs. Each individual sample must have at least 6 buds. Package individual samples together in a zip lock bag.
Branches
Near to harvest or after

commercial harvest

8 to 10 inches of 1-year-old wood or the base of the current season’s growth (with leaves attached) are the best samples. Samples close to leader generally give more consistent results. Collect sample from all the symptomatic limbs. For non-symptomatic trees, collect sample from each major scaffold limb and package together. The results are more reliable when samples are collected near or after commercial harvest. During the growing season, flag limbs with suspicious symptoms for sample collection later in the season.

Pome Fruits
Sample collection for testing Ilar (ApMV), Nepo (ToRSV), Chera (CRLV), Tricho (ACLSV), Fovea (ASPV) and Capillo (ASGV) viruses.
Type of sample
Collection time
Sample Description
Dormant bud sticks
End of Jan. untilbud break 8 – 10 inches at the base of the 1-year-old wood. Samples close to leader generally give more consistent results. Collect one stick each from all major scaffold limbs and package together in a zip lock bag.
Flower and leaf buds
Bud swell to bud popcorn stage Collect sample from all major scaffolds and limbs. Each individual sample must have 6 buds at least. Package individual samples together in a zip lock bag.
Branches
2 weeks after full bloom untilend of June 8 to 10 inches of 1-year-old wood or the base of the current season’s growth (with leaves attached) are the best samples. Samples close to leader generally give more consistent results. Collect sample from all the symptomatic limbs. For non-symptomatic trees collect a sample from each major scaffold limb and package together.

PDV- Prune Dwarf Virus, PNRSV- Prunus Necrotic Ring Spot Virus, APLPV- American Plum Line Pattern Virus, PPV- Plum Pox Virus, CLRV- Cherry Lead Roll Virus, ToRSV- Tomato Ring Spot Virus, TRSV- Tobacco Ring Spot Virus, CRLV- Cherry Rasp Leaf Virus, ACLSV- Apple Chlorotic Leaf Spot Virus, CMLV- Cherry Mottle Leaf Virus, ApLV- Apricot Latent Virus, CVA- Cherry Virus A, ASGV- Apple Stem Grooving Virus, CGRMV- Cherry Green Ring Mottle Virus , CRMaV – Cherry Rusty Mottle associated Virus , CNRMV- Cherry Necrotic Rusty Mottle Virus, CTLaV- Cherry Twisted Leaf associated Virus, ApMV- Apple Mosaic Virus, ASPV- Apple stem Pitting Virus


Lab-based diagnostic methods for fruit tree virus/virus-like agents

Type of Test
Source material
Advantages
Limitations
ELISA
Crude tissue extract Quick, simple, low cost, suitable for high-volume testing Low sensitivity; antibodies are limited for detecting few viruses; lack of resolution to identify closely related strains of viruses; false positive/ negatives; narrow sample collection window; mostly single virus per test
Isothermal Amplification (RPA and LAMP)
Crude tissue extract and Nucleic acid Quick, can be used for in-field sample testing, sensitivity comparable to PCR Requires expertise to design probes and primers; probes and primers are available for the detection of very few virus/virus-like agents
PCR/RT-PCR
Nucleic acid Very sensitive, several different platforms are available with varying sensitivity, multiple viruses or groups of viruses can be tested in a single reaction High sensitivity can easily lead to false   positive results from   contamination; trained professionals are required for the assays
Nucleic acid hybridizations
Nucleic acid Very sensitive and accurate More time-consuming, skilled labor required; labelled probes available for detection of only a few viruses
Arrays (micro/macro arrays)
Nucleic acid Sensitive and highly reproducible Expensive; difficult to detect subtle virus presence, requires confirmation testing
Next Generation sequencing (Deep sequencing/High throughput sequencing)
Nucleic acid Highly sensitive and reproducible, will be able to detect all known/unknown viruses/virus-like agents present in the plants. Expensive; highly trained professionals are required for sample preparation for sequencing and data analysis; relatively high turnover time, identification of unknown virus/s leads to regulatory concerns

ELISA – Enzyme Linked Immunosorbent Assay, RPA- Recombinase Polymerase Amplification, LAMP- Loop mediated isothermal Amplification, PCR- Polymerase Chain Reaction, RT- PCR- Reverse Transcriptase Polymerase Chain Reaction


Virus testing at Clean Plant Center Northwest, Washington State University, Prosser

Contact information

Call the lab before collecting and shipping samples, to ensure that the samples can be analyzed and that the samples are expected.

PCR lab: 509-786-9372 or 509-786-9206

ELISA lab: 509-786-9382

Shipping instructions

  1. Include a packing list that clearly indicates the tests being requested, billing information and contact information in case questions arise about the tests. Ship by overnight express to:
  2. Clean Plant Center Northwest – PCR/Elisa lab
  3. WSU– IAREC, 24106 North Bunn Road
  4. Prosser, WA 99350
  5. Telephone: 509-786-9372 or 206-786-382
  1. Ship early in the week (Monday through Wednesday) so that the samples are not stranded over the weekend. In extremely hot weather, it is best to ship the samples in an insulated container with ice packs (but do not allow leaves to freeze).
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