The PPV Lab at NYSAES works in conjunction with NYSDAM and USDA-APHIS to screen New York State Prunus trees for the presence of Plum plum virus (PPV).


Sample collection

On a daily basis, the PPV Lab in Geneva tests approximately 2,500 to 3,000 leaf samples.

Trees are tagged with a barcode label.

Sampling of leaves from Prunus trees in commercial orchards and homeowner properties in New York for testing of Plum pox virus (PPV) is handled by NYSDAM and USDA-APHIS, respectively. Surveys are most tightly centered on and around locations where positive trees have been found the previous year.

Within 1 mile of a site that tested positive, all trees have one sample taken (8 leaves per tree). Out to 5 miles from former sites of infection, every tree is sampled at 4 leaves per tree, two trees per sample. Beyond 5 miles, all the orchards are sampled and each orchard has 25% of the trees sampled at 4 leaves per tree.

Each sample consists of eight leaves taken from multiple locations on a tree, as distribution of PPV in infected trees is not uniform.  This strategy increases the probability that the virus will be detected if present.

Leaf samples are placed in plastic bags, barcode labeled, and stored on ice until shipped to the PPV Lab at NYSAES in Geneva. Once orchard and homeowner samples are delivered to the PPV Lab, they are scanned into a database and tracked by their barcode number.  No information other than the barcode is available to NYSAES.  In other words, the PPV Lab performs blind tests with no information on orchard location and history, as well as owner’s name.

Sample processing

Leaf samples are stacked with the petioles aligned and 0.5 grams of tissue is cut from the base of the leaves, avoiding petioles and midribs.  Leaf tissue weights are checked every 10 samples or whenever leaf types change or a new Prunus species is tested.Upon delivery at NYSAES, leaf samples are unpacked, scanned into a database and tracked by a barcode assigned to them by the collection team.  Duplicate barcode labels are then printed and stored with the samples in a cold room until processing.

Excised leaf material is placed in a plastic bag, which is labeled with the corresponding barcode.  Grinding buffer (5 ml) is added to the bag and the sample is ground with a tissue homogenizer.  Crude leaf extracts are then tested for PPV by Enzyme-Linked ImmunoSorbent Assay (ELISA).

(Left) Excision of leaf tissue from orchard or homeowner samples to be processed for PPV by ELISA. (Middle) Leaf tissue is placed in grinding bags and grinding buffer is added. (Right) Leaf samples before and after grinding.

Leaf Grinder

Grinding leaf samples with a tissue homogenizer.

Enzyme-Linked ImmunoSorbent Assay

For ELISA, microtiter plates consist of 96 wells, 6 of which are designated as controls (positive, i.e. PPV-infected material, negative, i.e. healthy plant material, and grinding buffer).  Every sample is replicated, so that a total of 45 samples can be run on one microtiter plate.Remaining leaf material is stored in a cold room until the sample has gone through the entire ELISA procedure with no indication of PPV infection.

 

ELISA plates

A microtiter ELISA plate loaded with ground leaf samples (top) and showing a colorimetric reaction for PPV-infected samples (bottom).

Loading Diagram

A loading diagram (left) and the corresponding microtiter plate (right).


Sample testing is a three-day process.

 

On the first day, 96-well microtiter plates are coated with an antibody specific to PPV and incubated overnight in a cold room, allowing the antibodies to adhere to the surface of the wells. On the second day, microtiter plates are rinsed, loaded with ground leaf samples, and incubated overnight in a cold room.  If PPV is present in the leaf sample to be tested, virus particles will adhere to the antibodies coated on the microtiter plate wells.

 

On the third day, microtiter plates are rinsed and treated with an antibody specific to PPV that has an enzyme tag.  If the coating antibody captured PPV, the second antibody will adhere to it, sandwiching the PPV particle between the two antibodies.  Microtiter plates are rinsed and a solution that colorimetrically reacts with the enzyme tag on the secondary antibody is added.  After one hour of incubation in the dark, any well, in which the virus is present, will turn yellow.  In contrast, wells that do not contain the virus will remain colorless.  Plates are scanned on a microplate reader and any sample that reads 2.0 times higher than the negative control is flagged as a positive suspect.  An overview of the ELISA procedure is illustrated below.

 

Test reports with the status of samples assayed are filed with NYSDAM and USDA-APHIS on a weekly basis.  If the PPV Lab at NYSAES identifies a positive suspect, NYSDAM and USDA-APHIS are immediately notified.  In that case, remaining leaf tissue of the suspect sample will be shipped to the National Plant Germplasm and Biotechnology Laboratory, USDA-APHIS, in Beltsville, MD for confirmation.  Concurrently, NYSDAM or USDA-APHIS will re-sample to corresponding tree that is suspect, as well as neighboring trees.  If leaf re-samples are found infected by PPV, the grower or homeowner will be contacted directly by NYSDAM and USDA-APHIS for removal of infected trees.

 

Loading Diagram

(1) Coating antibody specific to PPV adheres to microtiter plate wells. (2) Ground leaf material is added and virus particles, if present, adhere to antibodies. (3) Antibody with an enzyme tag is added. (4) Substrate reactive with enzyme added. (5) Color develops.