SEED TESTING METHOD FOR DAUCUS CAROTA

Crop: Daucus carota (carrot)
Pathogen: Alternaria dauci
.Background
This method was originally published in the ISTA Handbook of Seed Health Testing in
November 1964 as S.3. No. 4 and revised in 1987 (Gambogi, 1987). Safety Precautions
Ensure you are familiar with hazard data and take appropriate safety precautions. It
is assumed that this procedure is being carried out in a microbiological laboratory by
persons familiar with the principles of Good Laboratory Practice, Good Microbiological
Practice, and aseptic technique. Dispose of all waste materials in an appropriate way
(e.g. autoclave, disinfect) and in accordance with local health, environmental and safety
regulations.
Treated seed
Seed treatments may affect the performance of this test. It should only be performed on
untreated seed.
Materials
Reference material - The use of reference cultures or other appropriate material is
recommended.
Substrate - Blotters or fi lter papers, 9.0 cm, circular (e.g. Whatman No
1 or equivalent), free from micro-organisms and inhibitors (3
per plate).
Plates - 9.0 cm sterile Petri dishes (one per ten seeds).
Incubator - Operating at 20 ± 2°C, equipped with timer-controlled nearultraviolet
lights (NUV, peak at 360 nm, e.g. colour number
08, Philips; BLB, Sylvania).
Freezer - Operating at –20 ± 2°C.
Sample Preparation
1. It is vital to exclude any possibility of cross-contamination between seed samples.
This can be achieved by swabbing/spraying equipment and gloved hands with 70%
ethanol.
2. The test is carried out on a working sample as described in Section 7.4.1 of the
International Rules for Seed Testing.
Method
[Critical control points are indicated by CCP]
1. Place three 9.0 cm fi lter papers in each plate and soak with sterile distilled/de-ionised
water. Drain away excess water.
2. Aseptically place 10 seeds, evenly spaced (CCP), on the surface of the fi lter paper in
each plate.
3. Incubate for 3 d at 20 ± 2°C in the dark.
4. Transfer plates to freezer and maintain at –20 ± 2°C for 24 h.
5. After freezing, incubate for 6 d at 20 ± 2°C with alternating 12 h periods of darkness
and NUV light (ISTA,1984; Tempe, 1968). Plates should be approx. 25 cm below the
lights and should not be stacked.
6. Examine seeds under a stereoscopic microscope at x30 for fungal growth and up
to x80 magnifi cation for identifi cation of conidia. Conidiophores are simple or slightly
branched (Fig. 1), arising singly or in small groups from the surface of the seed or on
aerial mycelium. Conidia are usually solitary, obclavate, up to 450 μm long (including
beak), pale olivaceous brown at fi rst, becoming brown with age, with a long pale
Annexe to Chapter 7: Seed Health Methods: 7-001a-4
International Rules for Seed Testing Effective from 1st January 2003
7-001a (2003): Alternaria dauci on Daucus carota (Blotter)
beak up to 3 times the length of the body (Ellis, 1971). Groups of sunken conidia are
sometimes visible by the emerging clusters of their bright long beaks (Fig. 1, bottom
left). Record the number of infected seeds in each plate (CCP).
General Methods
1. Checking Tolerances
Tolerances provide a means of assessing whether or not the variation in result within or
between tests is suffi ciently wide as to raise doubts about the accuracy of the results. A
tolerance table, which can be applied to most direct seed health tests, can be found in
Table 5.1 of Annexe 16 of the International Rules for Seed Testing or in Table G1 of the
Handbook of Tolerances and Measures of Precision for Seed Testing (Miles, 1963)
2. Reporting Results
The result of a seed health test should indicate the scientifi c name of the pathogen,
and the test method used. When reported on an ISTA Certifi cate, results are entered
under Other Determinations.
In the case of a negative result (pathogen not detected), the results should be
reported in terms of the tolerance standard (e.g. infection level less than 1% with 95%
probability). The tolerance standard depends on the total number of seeds tested, n,
and is approximately 3/n (P=0.95) (see Roberts et al., 1993).
In the case of a positive result the report should indicate percentage of infected
seeds.
Quality Assurance
Specifi c Training
This test should only be performed by persons who have been trained in fungal
identifi cation or under the direct supervision of someone who has.
Critical Control Points
[identifi ed by CCP in the methods]
Spreading hyphae may lead to contamination of other seeds. Seeds must therefore be
spaced at least 20 mm from each other with not more than 10 seeds per 9.0 cm Petri dish
(Step 2).
Samples may be diffi cult to examine due to the growth of contaminants, especially
Alternaria tenuis, and/or A. radicina. Experience and great care is required for the
detection of all occurrences (ISTA 1984) (Step 6).
Supplementary examination may be required 13-14 d after plating of seeds to allow
hampered or deeper inoculum to exceed contaminants by its fructifi cation (Hewett, 1964)
(Step 6)