Quite frequently water samples consist of samples from a nutrient solution, taken before and after a disinfection system that is based on UV or slow sand filtration. Such samples are taken to verify the operation of the disinfection system. However, the protocol listed below is applicable to all types of water samples.
- Take an empty 1.5 or 2 liters plastic water bottle (do not use bottles that contained soda).
- Disinfect the bottle with a small volume of ethanol (± 50ml) and shaking vigorously. As long as the bottle is kept closed, the timing is not relevant. Relatively inexpensive technical or denatured ethanol can be used.
- Determine the sampling points:
- ‘post disinfection’ samples need to be taken as close as possible after the disinfection installation. Disinfect the sampling point. This should be heat based in case of an inox tap. If heat can not be used, disinfect with ethanol.
- ‘pre disinfection’ samples should also be taken as close as possible to the disinfection installation. This can contain recycled drain water as well as mixed-in rain or well water. Also disinfect the sampling point.
If the point of the analysis is only to evaluate the number of propagules in the drain water then of course the sample should be taken before other sources of water are mixed in. The choice of the sample point thus depends on the objective of the analysis and the available sampling points.
- if the only available sampling point is a water reservoir, then the sample can be taken with a small bucket connected to a stick or a rope and the sample can be poured in the water bottle. Disinfect the bucket first with a small amount of ethanol, for example using a few pieces of paper towel. In order to obtain a representative sample, use the bucket to mix the water in the reservoir before the actual sample is taken.
- if there is no certainty about the proper pre- and post-disinfection sampling points, contact the company that installed the disinfecting unit in order to avoid the analysis of inappropriate samples.
- After location and disinfection of the sampling point, let a few liters of water run away (e.g. in a bucket) before the actual sample is taken. This will rinse the water pipe and cool the tap in case it was heat-disinfected.
- Pour the ethanol out of the sample bottle and rinse it at least twice with a about 100 ml of sample water. There should be no ethanol residue in the bottle before the sample is taken.
- Take the sample (a full bottle) and close the bottle. Write the date and the nature of the sample on the bottle (company name, pre- or post-disinfection).
- Deliver the sample to the lab. Preferentially, keep and transport the bottles in a cooler, especially during warm outdoor temperatures.
Leaf bait test
If the only purpose of the test is to determine the presence or absence of Pythium and/or Phytophthora in a water reservoir then the rhododendron leaf bait test is most fit. The grower can place the leaves in the water reservoir himself.
How to prepare the Rhododendron leaves for the Pythium and Phytophthora leaf bait test?
- Rhododendron leaves. Usually leaves of Rhododendron "Cunningham White" are chosen because of the sensitivity and availability of this cultivar. It is best to take leaves of plants that have been on the premises for a while. Newly bought plants may have been treated with fungicides, which would reduce the effectiveness of the bait.
- Nylon mesh. This is used to make a bag which will contain the leaves. Use relatively strong mesh such as that used for screen windows, available in hardware stores (mesh size 1 to 2 mm). One bag needed per reservoir.
- Piece of styrofoam (one per reservoir). This is used as a floating device, to control the depth at which the leaves are suspended.
- Twine (to connect the nylon mesh bag with the piece of styrofoam and to retrieve the bait from the reservoir). Preferentially made from non-degradating material (such as nylon).
How to make the nylon mesh bags
- Cut pieces of 12x30 cm out of the nylon mesh. Fold it and close the sides with staples so that a 12 by 15 cm bag is obtained. Leave the top side open to place and remove the bait leaves.
- Make a hole near the top side (e.g. with a Phillips screwdriver) to attach the twine (and close the bag).
Protocol to set up the bait
- Bait leaves should be of appropriate age. If they are too old then they may have a substantial amount of natural damage (tears, spots, etc.). If they are too young their wax layer may still be too thin. This layer is important for the specificity of the test. Do not take leaves with symptoms of a disease, as contamination of the reservoir should be avoided (see remark at the bottom).
- Use one normal size or two small-sized leaves per bag. Carefully wash the leaves in tap water, cut them in two pieces with a scissors and place them in the bag, avoiding overlap. If the bait is not immediately used, place the nylon mesh bag containing the leaves in a plastic bag inside the fridge. Preferentially, do not store it this way for longer than one day.
- Connect the mesh bag with the styrofoam block using a piece of twine of approximately 20 cm. Connect a longer piece of twine to the styrofoam block. Place the Styrofoam block in the reservoir so that the mesh bag is hanging approximately 15 to 20 cm below the water surface. Connect the longer piece of twine to an object outside the reservoir, allowing easy recovery of the mesh bag at the end of the baiting period.
- Leave the bait in the water for approximately 4 days. If we provided the mesh bags then place the mesh bag + leaves in a labeled (date, company, reservoir number) plastic bag and return them to our lab. It is not advisable to send these via mail in case of warm outside temperatures.
If you provided the mesh bags then remove the bait leaves from the bags and only send the leaves to our lab (in a labeled plastic bag). The mesh bags can be reused but should be disinfested (e.g.with ethanol or commercial bleach) and rinsed thoroughly in tap water in such case. The styrofoam block may also benefit from some disinfecting.
- In the lab the leaves will be verified for lesions. Those will be plated on a semi-selective nutrient medium and resulting colonies will be analysed microscopically.
Remark: Given that the bait leaves should be asymptomatic and are washed prior to use, chances are very small that pathogens would be introduced in the reservoir via the bait leaves. Fungi and oomycetes that could be latently present in the rhododendron leaves are also unlikely to be pathogenic to strawberry, tomatoes, bell pepper or cucumber. Although they could be pathogenic to azalea and Rhododendron, chances of contamination from these leaves is negligible.