Sabine Werres and Thomas Schröder
Reliable detection of a pathogen in nurseries requires good knowledge of the pathogen itself, epidemiology and disease development, and the different types of nursery management. Last, but not least, a well-trained staff with enough specialists is necessary for monitoring and survey.
If the disease symptoms are not characteristic, samples must be taken to the laboratory for detailed analysis. The quality of the sampling method and the sample is as important for reliable monitoring and surveys as the specificity and sensitivity of the detection method.
Important questions to consider include the following:
- Where to take samples on a big field with seedlings, on a container stand, and so on?
- Which part of the plant should be sampled?
- How many samples must be taken?
- When is the optimal season to take samples?
Of course, statistical rules can be used to calculate the optimal number of samples and to optimize the sampling procedure in the field. They are, of course, very helpful in determining where the pathogen is present in the field. To detect Phytophthora on plants, it is best to look at dips/depressions/swales in the field or at a container stand. In these dips the soil moisture is much higher and persists longer than in the surrounding area because the water accumulates there after rainy periods or after irrigation. And since P. ramorum (like all other Phytophthora species) needs water to develop and to infect, latently infected plants or disease symptoms will be first found in very moist places.
Which part of the plant should be sampled depends on the plant species. On Rhododendron, P. ramorum was detected from leaves and twigs. But experience with plant material from nurseries has shown that the detection rate can be higher from twigs than from leaves (Werres, unpublished data). That may be due to application of fungicides (see below). Concerning Viburnum, only samples from the stem base enable sufficient detection, not the wilting twigs. To summarize the experience with sampling in nurseries: If it is not certain whether the disease symptom really is caused by P. ramorum, it is always better to take the whole plant to the laboratory if possible, rather than just twigs or leaves.
It is very difficult to say how many samples must be collected for adequate monitoring, but it helps if samples are taken from different parts of the plant. Samples from different plants within a diseased area should be examined. The detection rate can be higher with younger than with old nursery plants, so more samples should be collected for monitoring old plants.
The best season for sampling in nurseries is late spring to autumn. Observations in nurseries have shown that, especially at high temperatures in combination with heavy rainfall, disease symptoms can occur within a few days after infection. Very often the assortment of plants in the nursery changes within a season. Therefore, more than one sampling date is needed within a season.
Detection methods for monitoring or surveys should be specific for P. ramorum, sensitive to detect latent infection, and robust toward influencing factors from the nurseries. In addition they should give rapid results and be easy to handle.
Initial diagnosis of P. ramorum infection in nurseries is usually based on disease symptoms. A reliable diagnosis by disease symptoms is possible only when:
- the kind of symptom(s) that can develop is known;
- the symptom itself is characteristic for the pathogen.
P. ramorum causes different symptoms depending on the plant species (see http://cemarin.ucdavis.edu/symptoms.html). Presently, disease symptoms caused by natural infection with P. ramorum in European nurseries are only known from Rhododendron, Viburnum, and in 2002 in United Kingdom two findings from Pieris and one finding from Camellia (http://www.defra.gov.uk/planth/publicat/sudden.pdf). Status on P. ramorum findings in Europe is available on http://www.eppo.org. Symptoms on other possible hosts in nurseries are mainly known from artificial inoculation of a single part of the plant. On Rhododendron, P. ramorum causes mainly a twig and shoot dieback (figure 1a, b). However, brown to black discoloration of single non-wooden twigs is characteristic not only for P. ramorum, but also for other Phytophthora species like P. citricola, P. cactorum, and P. syringae (figure 2a, b). So diagnosis solely by disease symptoms is not sufficient for P. ramorum. The wilting symptoms on Viburnum are very unspecific. To verify the diagnosis P. ramorum for this host, it is necessary to look for cambial necrosis at the stem base.
Figure 1: Twig dieback (a) and wilting (b) caused by Phytophthora ramorum
Figure 2a: Twig dieback on Rhododendron caused by Phytophthora cactorum
Figure 2b: Twig dieback on Rhododendron caused by Phytophthora citricola
Further detection methods for P. ramorum are discussed in detail on the Web site: http://cemarin.ucdavis.edu/symptoms.html. None of the methods show all criteria to be optimal. Based on the present knowledge from experiments in scientific laboratories and in laboratories of the plant protection services, combinations of different methods seem to be most effective.
Factors that can influence the detection rate
In many nurseries, specific fungicides are used for control of P. ramorum. Fungicides can suppress the development of disease symptoms, possibly leading to false-negative results and more difficult sampling. Furthermore, fungicides can influence the detection rate in plant or soil samples depending on the method used. For example, those methods that are based on the detection of living propagules, like baiting and microbiological techniques, will give false- negative results if the fungicide has damaged or killed P. ramorum. On the other hand, these methods can prove whether chemical control was successful. Residues of chemicals and/or ingredients can interact with the detection method itself so that false positive or false negative results are possible. Therefore, it is always very important to know the cultivation methods used in the commercial nurseries being monitored.
Monitoring of P. ramorum is done according to the definition of the IPPC Standard No. 6 (1997) for a "detection survey" (to conduct a survey in an area to determine if pests are present). A survey in this case is "an official procedure conducted over a defined period of time to determine the characteristics of a pest population or to determine which pest species occur in an area." In Europe, for example, the obligation to monitor the occurrence of P. ramorum is laid down in the EU-Directive 2002/757/EG (2002), so each of the EU member states has to provide data to the EU. Those data are also the background and the justification of regulatory measures for both import and export of host plants of the corresponding harmful organism.
The problems or preconditions for monitoring are described below. These apply not only to P. ramorum but also to all "new" harmful organisms.
At the beginning of monitoring in some cases, information on the biology of the organism is lacking so the following criteria have to be analyzed thoroughly to concentrate the usually limited amount of workers, laboratory capacity, and financial resources: confirmed host plants, potential host plants, risk areas, detection methods, and scientific data to develop a "survey plan." Of course, it is necessary to adapt monitoring according to new available information. For example, in Europe the monitoring of P. ramorum was first focused on Viburnum and Rhododendron species in nurseries, but now it has to be enlarged to public green areas. More data on potential host plants of P. ramorum are available as the result of artificial inoculation trials, so it has to be decided which plants should be included in the monitoring.
Monitoring usually is carried out concerning a new organism. Therefore, the results depend on the qualifications of the staff carrying out the monitoring. In summary, a well-trained staff, following good surveillance and record keeping practices is essential for effective monitoring.
Brasier, C.D., Rose, J., Kirk, S.A., and Webber, J.F. 2002.. Pathogenicity of Phytophthora ramorum isolates from North America and Europe to bark of European Fagaceae, American Quercus rubra and other forest trees. Proceedings of the Sudden Oak Death Symposium, Dec. 17.-18., 2002, Monterey, California. 30-31.
Proceedings of the Sudden Oak Death Symposium, Dec. 17.-18., 2002, Monterey, California. Session "Survey/Monitoring," 23-27, 88-93.