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X-disease phytoplasma (Candidatus Phytoplasma pruni) Sampling Recommendations for Nurseries

Written by Scott Harper, WSU Plant Pathology, Tianna DuPont, WSU Extension. Last updated July 22, 2022.

Nursery Mother Trees

 Where to sample

  • Trees with symptoms: Sample from symptomatic limbs.
  • Trees with no symptoms: Sample from each leader or compass point (N,E,S,W) of 1/4″-1/2″ (pencil diameter wood) with leaves. Sample from the lower limbs of the tree as close to the trunk as possible.

Material to sample

  • Collect four five-inch cuttings of 1/4-1/2″ (pencil diameter) diameter limb which include flower buds, fruit stems, or leaves depending on time of year (Table 1).
  • Trim off excess tissue as needed (Figure 1).
  • Avoid areas with dead or dying tissue.

 Table 1. The recommended tissue for testing cherry  for other stonefruit tissue for Ca. P. pruni varies depending on the time of year or season, the growth state of the plant, and how advanced the infection is. Sequential testing (Wright et al. 2022) of heavily infected trees over the course of three years suggested the following tissues are most optimal:

Time of Year Growth Stage Best Tissue Secondary Tissues
February -March Bloom Either second-year or current year wood (if late season and lignified / hardened, not green), from symptomatic limbs if possible. Flower Buds
April Shuckfall Flower/Fruit Buds
April-May Preharvest Fruit Stem (Pedicel)
June-July Harvest Fruit Stem (Pedicel)
July-August Postharvest Leaf Midrib & Petiole
September-October Pre-dormancy None
October-January Dormancy Not Recommended Not Recommended
Figure 1. Collection of woody stem tissue at harvest. Both the trimmed cutting and pedicel (fruit stem) will be submitted together for testing. Leaves are suboptimal at this time and are discarded. The fruit itself should not be submitted, as it is difficult to work with, bulky for storage, and prone to fungal growth that can contaminate the sample.
Figure 1. Collection of woody stem tissue at harvest. Both the trimmed cutting and pedicel (fruit stem) will be submitted together for testing. Leaves are suboptimal at this time and are discarded. The fruit itself should not be submitted, as it is difficult to work with, bulky for storage, and prone to fungal growth that can contaminate the sample.

When to sample

  • The week before harvest to mid-August is standard for commercial orchard trees.
  • Target sampling in mid-to-late June for mother trees. Early in the season the concentration of the pathogen in the upper parts of the tree is low and may provide false negatives. June timing will allow for results before June budding. Do not sample after mid-August when phytoplasma concentration in the tree begins to drop.
  • Sample when temperatures are below 90 F. Elevated temperatures can suppress virus and phytoplasma titer, leading to false negative results. After collection and bagging, samples should be directly enclosed in a cooler with an ice pack.

If the tree is dormant, sampling is not recommended as phytoplasma replication and titer drops with lower temperature, and there is little to no active movement of solutes or the phytoplasma in the phloem. It may be detectable in large lower limbs and/or the trunk, however the probability of false negative test results is high. The phytoplasma can be detected in primary and secondary root tissues of infected trees, often prior to the phytoplasma becoming detectable in the limbs of the tree. However, sampling of roots of planted, actively growing orchard or mother trees is difficult and not recommended for routine testing. Check with your diagnostic lab whether they can extract DNA from roots successfully before testing this tissue. A longer description of the seasonal titer and in planta distribution of Ca. P. pruni at different stages of infection can be found in: Wright et al. (2022) Titer and Distribution of Candidatus Phytoplasma pruni in Prunus avium. Phytopathology, https://doi.org/10.1094/PHYTO-11-21-0468-R.

 Sampling sanitation

  • Clean tools with 10% bleach between each tree. Sodium hypochlorite (bleach) denatures both viral coat proteins and the phytoplasma cell membranes.
  • 70% or greater ethanol or isopropanol can also be used but are less effective than bleach against viruses.
  • The chance of mechanically transmitting LChV-2 or Ca. P. pruni is minimal, but there is always a risk of transmitting other pathogens such as Prune dwarf virus or Prunus necrotic ringspot virus which are common in the Pacific Northwest.

 Packaging and sending samples

  • Keep tissue moist and cool (with ice packs).
  • Old or dried tissue or tissue which has gotten hot is likely to have false negatives.
  • Send to labs with current proficiency test results. For certification program choose a University or WSDA lab:

Labs testing for Little Cherry Virus and X-disease Phytoplasma

  • Shipping on a Monday or Tuesday to get to labs on time is recommended.

Dormant, Bare-Rooted or ‘Production’ Trees

The sampling of bare-rooted trees for sale, either immediately prior to, during, and after dormancy is more difficult than sampling larger, mature trees because sampling is destructive, and these small trees lack branching limbs that can be safely removed without harming the tree. Also, as the young trees enter dormancy, phytoplasma titer and distribution becomes primarily focused on the roots.

Material to sample

  • One 3-4″ cutting from the tip of the tree including and buds + one 3-4″ cutting from the first order lateral roots (at least ¼” thick), or from the taproot/root core should it be present (Figure 2). Diagnostic laboratories should process these two tissues together.
Figure 2. Collection of root and stem tissue from a dormant rootstock. Both the trimmed stem cutting and root will be submitted together for testing.  Samples should be as free of dirt as possible to aid the laboratories in sample processing.
Figure 2. Collection of root and stem tissue from a dormant rootstock. Both the trimmed stem cutting and root will be submitted together for testing.  Samples should be as free of dirt as possible to aid the laboratories in sample processing.

When to sample

  • Sampling the young dormant trees after digging them out of the ground, or once they are in cold storage and easily accessible, is recommended.

Sampling sanitation

  • Clean tools with 10% bleach between each tree. Sodium hypochlorite (bleach) denatures both viral coat proteins and the phytoplasma cell membranes.
  • 70% or greater ethanol or isopropanol can also be used but are less effective than bleach against viruses.
  • The chance of mechanically transmitting LChV-2 or Ca. P. pruni is minimal, but there is always a risk of transmitting other pathogens such as Prune dwarf virus or Prunus necrotic ringspot virus which are common in the Pacific Northwest.

Packaging and sending samples

  • Keep tissue moist and cool.
  • Old or dried tissue or tissue which has gotten hot is likely to have false negatives.
  • Send to labs with current proficiency test results. For certification program choose a University or WSDA lab:

Contacts

Tianna DuPont, WSU Extension

(509) 713-5346

tianna.dupont@wsu.edu

 

Scott Harper, Department of Plant Pathology, Washington State University

(509) 786-9230

scott.harper@wsu.edu