Method for the Detection of Septoria apiicola on Celery and Celeriac Seed Crop: Pathogen: Celery (Apium graveolens) and Celeriac (Apium graveolens var. rapaceum) Septoria apiicola Date: Version 1.1, July 2017 Sample and sub-sample size The minimum recommended sample size is 10,000 seeds and the maximum sub-sample size is 2,500 seeds. Principle Seeds are shaken in a buffer solution and aliquots of the seed wash solution assessed under a microscope. Healthy, susceptible celery or celeriac plants are inoculated with the seed wash solution with putative spores. The inoculated plants are assessed visually for disease symptoms (see Annex 1). The method is able to discriminate between viable and dead spores. Restrictions on Use o This test method is suitable for untreated seed. o This test method is suitable for seed that has been treated using physical (hot water) or chemical (e.g. calcium or sodium hypochlorite) processes with the aim of disinfestation/disinfection provided that any residue, if present, does not influence the assay. It is the responsibility of the user to check for such antagonism and/or inhibition by analysis, sample spiking or experimental comparisons. o This test method is not suitable for seed that has been treated with protective chemicals or biological substances. If the user chooses to test such treated seed using this method, it is the responsibility of the user to determine empirically (i.e. through analysis, sample spiking, or experimental comparisons) whether the protective chemicals or biological substances have an effect on the method result. Validation This method has been validated in two ISHI-Veg comparative tests using celery seed (Oosterhof, 2015). As celeriac (Apium graveolens var. rapaceum) is a variety of celery cultivated for its edible roots, hypocotyl and shoots, the method is also considered appropriate for celeriac seeds. Method description 1. Growing indicator celery plants 1.1 Sow healthy celery seeds of a susceptible cultivar (e.g. Imperial ) in clean trays containing well-watered peat cubes or potting soil. Note: The section Principle has been updated
1.2 After sowing cover the seeds with vermiculite or a Styrofoam sheet, or both, for 4 days at 15 C. Do not water and keep the seeds covered so that they germinate in the dark. 1.3 Place the trays under normal greenhouse conditions at 15 C and 12-14 hours light. 1.4 Grow the celery plants until there are 3-4 expanded leaves (± 4 weeks). Do NOT spray any pesticides on the seedlings during the entire test. 1.5 Transplant the seedlings in clean trays (ca. 30 x 45 cm) containing potting soil with no more than 10 uniformly healthy celery plants per tray (see Annex 1, Picture 4). As some celery seed may not germinate ensure there are a minimum of 6 uniformly healthy celery plants per subsample and per tray. In case there are not enough plants indicate the number of plants transplanted per tray on the data sheet. 1.7 Label the trays with numbers to indicate the subsamples. Ensure that 2 trays are labelled Positive and Negative controls. 2. Preparation of the seed wash, Positive and Negative control suspensions for counting S. apiicola spores 2.1 Use 100 ml volume Erlenmeyer flasks. Label the flasks with the subsample numbers and as the Positive and Negative controls. 2.2 Place the seeds (2500 maximum) of each subsample into the corresponding flask. 2.3 Add 8 ml demineralized water to each flask and vortex for 5 seconds to assure good separation and washing of the seeds. 2.4 Prepare the Positive and Negative controls: 2.4.1 If the Positive control is a spore suspension, add it to the flask labelled Positive control together with 8 ml demineralized water. 2.4.2 If the Positive control is an infected seed sample, these seeds should be added to the flask labelled Positive control together with 8 ml demineralized water. 2.4.3 The Negative control is demineralized water only and should be added to the flask labelled Negative Control. 2.5 Incubate the flasks for 2-3 hours at room temperature on a shaker at 60 rpm. 2.6 After incubation take approximately 10 µl from each seed-wash suspension to verify the presence of and to quantify the number of S. apiicola spores per ml using a haemocytometer to estimate the infection level. 2.7 If no suspect spores are found the result of the assay is negative and the test is over. 2.8 If suspected spores are found, continue with the seed wash inoculation assay. 3. Preparation of the seed wash, Positive and Negative control suspensions for inoculating celery indicator plants 3.1 Prepare 0.1% freshly boiled and cooled water-agar: Add 0.1 g agar to 100 ml demineralized water, boil briefly in a microwave before cooling the mix down to room temperature. Shake until the agar and demineralized water are mixed thoroughly. 3.2 To each suspect subsample in the flasks along with control subsamples (Positive and Negative) add 1 ml of 0.1% freshly boiled and cooled water-agar. 2
4. Inoculation of indicator celery plants 4.1 Dust all the indicator celery plant leaves (3-4 expanded leaves) with carborundum powder. 4.2 Start by inoculating the plants in the trays labelled with the subsample numbers with the corresponding suspect seed subsample suspensions, then inoculate the plants in the Positive control tray with the Positive control suspension, and finally the plants in the Negative control tray with the Negative control (demineralized water). 4.3 Inoculate the leaves of the plants in the tray (a minimum of 6 and no more than 10 per subsample) with the corresponding suspension as follows: 4.3.1 Place the sponge on the opening of the Erlenmeyer flask (see Annex 1, Picture 1). 4.3.2 Turn the Erlenmeyer flask so that the seed-wash suspension is absorbed by the sponge (Annex 1, Picture 2). Be careful not to spill any of the seed-wash suspension. Do not remove any seeds remaining on the sponge (Annex 1, Picture 3). 4.3.3 Rub the leaves smoothly with the seed-wash suspension dampened sponge (see Annex 1, Picture 3). 4.4 Change the sponge and the gloves (to prevent cross contamination) between each subsample. Do not water the plants using a spray or sprinkler after inoculation! Also do not use any pesticides until the test is completed. 4.5 Incubate the plants in a climate chamber/greenhouse for 4 days at 18 C, 100% humidity and 16/8 hours light/dark. The required humidity can be achieved by covering the plants with a plastic tent. 4.6 On the 5th day after inoculation, reduce the humidity to 70% (e.g. by opening the plastic tent) but maintain temperature and light at the same levels and continue to incubate plants. 5. Evaluation of inoculated plants 5.1 Evaluate all plants 2-3 weeks after inoculation for typical symptoms of S. apiicola infection: grey-brownish spots with black pycnidia (see Annex 1, Picture 5 and 6). 5.2 To confirm the results look for the presence of black pycnidia in the typical spots using a magnifying glass (see Annex 1, Picture 7). Compare the symptoms to Negative and Positive control plants. 5.3 If infected plants are found the seed lot tested is infected with viable S. apiicola. Record the number of plants showing typical S. apiicola symptoms out of the total number of inoculated plants per subsample/tray to estimate the infection level. References Champion, R. (1997). Identifier les champignons transmis par les semences. Techniques et Pratiques. Paris, FRA: INRA Editions, 398 p. (http://prodinra.inra.fr/record/52346) Oosterhof, J. (2015) Report of the two comparative tests by ISHI-Veg for the detection of Septoria apiicola in Celery and Celeriac seeds using the seed wash inoculation method. ISHI-Veg Research Report, Nyon, Switzerland: International Seed Federation. 3
Annex 1 Pictures 1 to 4: Inoculating healthy plants Picture 1 Picture 2 Picture 3 Picture 4 Pictures 5 and 6: Grey-brownish sports with black pycnidia: symptoms of Septoria apiicola on the leaves of indicator celery plants Picture 5 Picture 6 4
Picture 7: 80x magnification of a grey-brown spot with black pycnidia. 5