Observatree Volunteer Sampling Guide

Compiled by S. Sancisi-Frey (FR)

Contents

Page

Equipment list for sampling 1 Taking good photos 2 Bark sampling 3 Bark sampling procedure 3 Foliage sampling 7 Leaf litter sampling 7 Soil sampling 8 Sampling for insects 8 Acute Oak Decline Swab Sampling 9 Lateral Flow Device (LFD) testing for Phytophthora Diseases 10 Carrying out an LFD test 11 Summary of LFD Test 12 Labelling your Sample Bag 13 Where to send samples 13

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As Observatree volunteers you may be asked to follow up Tree Alert reports by taking samples from, or around, potentially diseased trees. This is so that symptoms can be observed first hand by the Tree Health Diagnostic and Advisory Service (THDAS) and so that the pathogen causing the disease can be isolated from the sample and /or identified by lab testing. The purpose of this guidance is to help you take samples safely and effectively and will cover bark; foliage (shoots and leaves); soil; litter and insect sampling, as well as sampling methods for specific diseases. The equipment that you may need is listed below: Mallet Chisel (preferably 1 or 2 different sizes) Folding Pruning saw Pruning knife Trowel Secateurs Rubber gloves (provided within your biosecurity kit) Sample bags of different sizes (provided at training days) Sample tubes for insects (provided at training days) Tweezers/forceps Black/blue permanent markers Binoculars Hand lens (provided at training days) Map/GPS device Camera/camera phone Disinfectants for hands and tools (provided within your biosecurity kit) Paper towels/tissue (provided within your biosecurity kit) Recording form (download from Observatree website) Weather writer for bad weather, but better not to sample in bad weather Acute Oak decline sample swabs (available on request) Phytophthora lateral flow devices for rapid early stage diagnosis (available on request) Bag specifically for samples Plus a waterproof rucksack/bag to carry it all in Please note we can only provide the items specified; any of the other items will need to be sourced by you. However, please don’t feel that you should to go out and buy all of this equipment. It is unlikely that you will need all of it at any one time

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and we will do our best to help you if there is a particular item that you need for sampling and cannot obtain. Please ensure that you have your biosecurity equipment and a first aid kit with you when you are out sampling.

Between samples, always remove mud/plant debris from tools and disinfect them, and clean your hands with hand gel. Taking good photos Remember that taking photos is essential prior to sampling, especially if only 1 lesion is present. Include: tree in context, symptom in context and close up of symptoms (please see below)

tree in context symptom in context close-up of symptoms It is important that photos are of a fairly high quality so that they can be zoomed in on without becoming pixelated. Further information on how to take good photos can be found on the Observatree website (www.observatree.org.uk)

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Bark sampling

Bark sampling for pathogens is a destructive process so try to keep the amount of material sampled to the minimum required for isolation. Take two or three inner bark panels of around 6 x 4 cm from different positions on the lesion/tree.

For a pathogen to be isolated from bark, the sample needs to be fresh and moist. Please avoid any dry or obviously old areas of lesion.

Lesion material is usually darker than healthy tissue, with fresher/newer areas of the lesion appearing more moist and slightly lighter than those that are older.

The sample should be taken from an area of the lesion where healthy tissue meets dead/infected tissue (dead-live junction) because this is the area of the lesion where the pathogen is the most active and provides us with the best chance of isolating it in the lab.

Once the outer layers of the bark are removed to expose inner areas, the material can oxidise quickly to become a darker colour. This makes it difficult to see what areas are healthy but oxidised, or dead and dying because of a disease lesion. Please ensure that the sample does not have all of the outer bark removed completely, so that the underlying tissue is protected thereby allowing healthy and diseased tissue to be differentiated during lab testing.

Please send your sample into the lab (details later) as soon after sampling as possible (ideally within 24 hrs.) because contamination of the sample will increase with time and other organisms will colonise the sample making it more difficult to isolate the pathogen of interest.

Bark Sampling Procedure

Locate a lesion on the branch or stem (bleeding/weeping or areas where the tissue is raised/ sunken/discoloured or cracked or just looks different in some way from the rest of the bark on the tree).

Explore the diseased bark area with the corner of a sterilised chisel or with a strong pruning knife, searching for changes in the colour of the inner bark. Once you have found the darker coloured diseased tissue, work outward until you come to the dead/live junction (See photo below).

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Look for the dead/live Junction

Using a mallet and chisel (4cm /1.5" chisel) carefully remove the outer bark to reveal the dead live junction and active lesion in the inner bark (phloem) and photograph the area (See photo below).

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Cut and remove an inner bark panel of approximately 6 x 4 cm, down to the wood surface (See photos below). An ideal sample panel would consist of around 70-80% lesion material and 20-30% healthy bark. If stain in the underlying wood is evident remove a panel of this too.

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Where possible keep some the outer bark attached to the sample to protect it from oxidising and drying out.

Place bark samples in separate sample bags. Add a dense, folded piece of wet

paper/tissue (not soaking wet) to maintain moisture. Remove excess air and seal bag tightly. Label as show below.

For bark lesions on smaller branches you may need to just remove the part of the branch containing the lesion with a pruning saw. Ensure that the branch piece contains the dead/live junction by sawing at least 2 – 5 cms beyond the lesion in both directions. Trim off any unwanted twigs or foliage to make packaging easier.

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Foliage sampling

Don’t take foliage samples in the rain as wet foliage can become colonised by contaminants and decompose rapidly.

Take samples of diseased foliage including adjoining shoots and small branches. This is particularly important if leaves are turning uniformly brown or wilting as this can indicate a problem within the shoots or small branches.

If lesions are present on the shoots take the samples 2-5 cms beyond the lesion into the healthy tissue to ensure a dead/live junction.

Please provide a fair amount of foliage sample including some healthy material for comparison (a small to medium bunch of flowers).

Ensure that all stages of foliar symptoms are included within the sample if possible.

Look carefully for any fruiting bodies on the leaves or shoots and if present include these within the sample.

Foliage samples need to be fresh in order for pathogens to be isolated so it is particularly important to send them in as quickly as possible.

The sample bag can be slightly inflated, or tissue paper can be used to protect delicate leaf samples.

Leaf litter Sampling

Remove the very uppermost layer of litter (where depth of litter allows) and discard.

Take a disinfected trowel and remove a volume of litter similar to about a quarter of a bag of sugar from each of the 4 sampling points. Transfer the litter from the 4 sampling point to a single sample bag.

Try not to include soil

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Soil Sampling

Don’t take soil samples in the rain.

Remove and discard the uppermost layer of soil/litter (about an inch; as this is not suitable for sampling) from 4 points around the tree of interest. The 4 sample points should be like the 4 corners of a square with the tree in the centre.

Take the soil from around the roots, but for large trees you can extend out to up to a metre from the trunk.

Take a disinfected trowel and remove a volume of soil similar to about a quarter of a bag of sugar from each of the 4 sampling points. Transfer the soil from the 4 sampling point to a single sample bag.

Try not to include stones, plant roots or vegetation from plants that are not being sampled. Small roots from the tree being sampled can be included.

Sampling for Insects

For Observatree volunteers sampling for insect pests will mainly involve photographic evidence of larval cavities or other insect related signs and symptoms, or bark samples showing exit holes. It is unlikely that you will be asked to sample for the insects themselves; but if you are, here are a few tips:

Catch the insect (rubber gloves/plastic forceps can be used)

Put it in a small labelled tube and seal on the lid tightly

Bark removal and exposure of larval galleries may be required to obtain wood boring beetle larvae e.g. the Great Spruce Bark Beetle.

Branch beating and tray collection may be required to catch foliage feeding insects such as oak and plane lace bugs

Place insects in a labelled sample bag and post off. Refrigerate if the sample cannot be posted on the day of sampling.

Do not attempt to collect the oak or pine processionary moth larvae as they are a public health risk!

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Acute Oak Decline Swab Sampling

Take swab and break the seal at the top of the tube by holding the lid in one hand and twisting the tube until the seal tears. A large cotton bud is attached to the lid.

Roll the bud around in the bleeding/seeping fluid until it is completely covered in sample.

Replace the cotton swab in the tube and seal it tightly.

Label the tube with the date, site and contact details.

Place in a labelled sample bag and post in a padded bag to address below (See later). Please post as quickly as possible and if there is any delay in posting please refrigerate.

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Lateral Flow Device (LFD) testing for Phytophthora Diseases

If you suspect a Phytophthora infection you can use a lateral flow device (LFD) to assist in early stage diagnosis.

The LFDs can be used to test foliage, bark and roots.

Included in an LFD test kit: A plastic pipette, a bottle containing buffer and beads and the lateral flow device.

LFDs are affected / damaged by low temperatures and should be kept at room temperature if possible.

Used LFDs should be kept in a separate bag from the physical plant material sample in order to keep it clean.

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Carrying out an LFD test

Take a few small /thin slivers of bark/foliage and insert into the bottle containing buffer and beads.

Shake the bottle vigorously for 1-2 minutes. Avoid adding resinous material as this may disrupt the reaction.

Let the liquid settle then use the pipette provided to take out some of the buffer. Put 2 drops onto the membrane in the circular reception well on the LFD. Take care not to flood the well.

Place the LFD on a flat surface and wait for the liquid to move across the rectangular window (takes up to 30 seconds). If the liquid does not move across the window after 30 seconds; add one more drop.

Leave the test to develop for no longer than 10 mins.

If a line appears under the ‘C’ (Control) then the LFD is working; if not, then discard the LFD. If a line appears under the ‘T’ (Test) as well as the C then the sample is positive for Phytophthora. Both lines need to be present on the LFD for the sample to be positive. If no line appears under the T, then the sample does not contain Phytophthora.

Label the LFD with date and sample identifier.

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Summary of LFD Test

A positive LFD result

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Labelling your Sample Bag

A correctly labelled sample bag

Label with black/blue (not red or green) permanent marker pen. Include the date, your name, the tree species, tree/sample identifier, site location and 10 figure national grid reference of the tree.

Please keep samples as cool as possible (not frozen).

Try to collect samples early in the week to allow time for mailing and lab isolation. If collected later in the week, samples may spend time unnecessarily in the post or in the lab before they can be processed and this will reduce the chances of culturing any pathogens that might have originally been present.

Mail in padded/jiffy bags or similar and send Special Delivery if possible to: Tree Health Diagnostic and Advisory Service Forest Research Alice Holt lodge Farnham Surrey GU10 4LH

Or as directed when you are asked to take the samples.