SEED TESTING METHOD FOR TRITICUM SPP.

Crop: Triticum spp (Wheat) http:///www.seedtest.org/upload/cms/user/7-014.pdf
Pathogen: Stagonospora nodorum Berk. = syn Septoria nodorum Berk., Perfect state: Leptosphaeria nodorum Mailer
Method Abstract
This method was originally published in the ISTA Handbook of Seed Health Testing in November 1964 as S.3. No. 19 and revised in 1984 by M. Kietreiber, Bundesanstalt für Pflanzenbau, Wien, Austria. The method was incorporated into the newly revised Annexe to Chapter 7 in 2002 from the 1999 edition of the ISTA Rules. The method was reviewed by the ISTA-Seed Health Committee in 2006 (Cockerell & Koenraadt, 2007) with the recommendation to accept for a further five years.
Summary of Validation Study:
Studied in International Comparative Testing: 1959, 1961, 1962, 1964 and 1979-81
Using potato dextrose in darkness, Hewett (1975) found a correlation coefficient of 0.95 between counts in the laboratory and the number of diseased seedlings in the field. Comparative tests organized by the ISTA Plant Disease Committee gave reasonable agreement between stations (Rennie, 1982).
International Rules for Seed Testing Effective from 1 January 2008
7-014: Detection of Septoria nodorum on Triticum aestivum (Wheat)
Annexe to Chapter 7: Seed Health Methods: 7-014-3
Safety Precautions
Ensure you are familiar with hazard data and take appropriate safety precautions, especially during preparation of media, autoclaving and weighing out of ingredients. It is assumed that this procedure is being carried out in 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 safety regulations.
Treated Seed
This method has not been validated for the determination of Septoria nodorum on treated seed. Seed treatments may affect the performance of the method.
(Definition of treatment: any process, physical, biological or chemical, to which a seed lot is subjected, including seed coatings. See 7.2.3)
Materials
Reference Material
The use of reference cultures or other appropriate material is recommended when ever possible.
Media
Malt Agar or Potato Dextrose Agar containing 100 ppm streptomycin sulphate.
Sodium hypochlorite solution
(1% available chlorine) for seed disinfection.
Petri dishes
When sowing density is given by a number of seeds per Petri dish, a diameter of 90 mm is assumed.
Incubator
Capable of operating in the range 20 ± 2 ºC.
Sample Preparation
The test is carried out on a working sample of 400 seeds as described in Section 7.4.1 of the International Rules for Seed Testing.
Method
1. Pretreatment
10 minutes in 1% (avilable chlorine) sodium hypochlorite.
2. Agar method
Malt agar or Potato Dextrose Agar containing 100 ppm streptomycin sulphate.
3. Incubation
7 days at 20 ºC in darkness.
4. Examination.
After 7 days examine each seed by naked eye for slow-growing circular colonies of dense white or cream mycelium that often covers infected seeds. The reverse of the colony is yellow/brown becoming darker with age. International Rules for Seed Testing Effective from 1 January 2008 7-014: Detection of Septoria nodorum on Triticum aestivum (Wheat) Annexe to Chapter 7: Seed Health Methods: 7-014-4
General Methods (common to many test procedures)
1. Checking tolerances
Tolerances provide a means of assessing whether or not the variation in result within or between tests is sufficiently wide as to raise doubts about the accuracy of the results. Suitable tolerances, which can be applied to most direct seed health tests, can be found in Tables 5B of Chapter 5 of the ISTA Rules, or in the Handbook of Tolerances and Measures of Precision for Seed Testing by S.R. Miles (Proceedings of the International Seed Testing Association 28 (1963) No 3, p 644).
2. Reporting Results
The result of a seed health test should indicate the scientific name of the pathogen detected and the test method used. When reported on an ISTA International Seed Analysis Certificate, results are entered under Other Determinations.
Preparation of Media and Solutions
1. Sodium Hypochlorite Solution
Sodium hypochlorite for pretreatment of seed can be prepared from commercial bleach diluted to 1% available chlorine. The concentration of chlorine in commercial bleach varies considerably. Use the formula Vstock = Vfinal x Cfinal / Cstock (where V= volume and C= % available chlorine) to calculate the volume of commercial bleach stock solution required to prepare sodium hypochlorite solutions for use in seed pretreatment.
To prepare a 1 liter solution of sodium hypochlorite containing 1% chlorine from a stock of commercial bleach containing 12% available chlorine:
Vstock = Vfinal x Cfinal / Cstock Vstock = 1 x 1/12 = 0.083
Thus add 83 mL of the 12% stock to 917 mL water.
2. Malt Agar
Compound
g/L
Malt Agar1
According to manufacturer's instructions
Distilled/de-ionized water
1000 mL
Streptomycin sulfate
1 mg
CCP 1 Malt agar constituents should be equivalent to those of the following manufacturers Difco, USA or Oxoid, UK.
Preparation
1. Weigh out ingredients into a suitable autoclavable container.
2. Add 1000 mL of distilled/de-ionized water.
3. Dissolve powdered Malt Agar in distilled/de-ionized water by stirring.
4. Autoclave at 15 p.s.i. and 121 °C for 15 min.
5. Allow agar to cool to approx. 50 °C.
6. Pour 15–22 mL of molten agar into 90 mm Petri plates and allow to solidify before use. International Rules for Seed Testing Effective from 1 January 2008 7-014: Detection of Septoria nodorum on Triticum aestivum (Wheat) Annexe to Chapter 7: Seed Health Methods: 7-014-5
Storage
Prepared plates may be stored at 4 ºC for up to 6 weeks.
Potato Dextrose Agar
Compound
g/L
Potato Dextrose Agar1
According to manufacturer's instructions
Distilled/de-ionized Water
1000 mL
Streptomycin sulphate
1 mg
CCP 1 PDA constituents should be equivalent to those of the following manufacturers Difco, USA or Oxoid, UK.
Preparation
1. Weigh out ingredients into a suitable autoclavable container.
2. Add 1000 mL of distilled/de-ionized water.
3. Dissolve powdered PDA in distilled/de-ionized water by stirring.
4. Autoclave at 15 p.s.i. and 121 °C for 15 min.
5. Allow agar to cool to approx. 50°C.
6. Pour 15–22 mL of molten agar into 90 cm Petri plates and allow to solidify before use.
Storage
Prepared plates may be stored at 4 ºC for up to 6 weeks.
Quality Assurance
Critical Control Points
Where the wording of the original Working Sheet suggests that an action is critical this has been marked with CCP.
References
The following references are extracted from the ISTA Handbook on Seed Health Testing, Working Sheet No. 19, M. Kietreiber, 1984.
Hewett, P.D. (1975). Septoria nodorum. Seedlings and stubble of winter wheat. Transactions of British Mycological Society, 65, 7-18.
Kietreiber, M. (1961). Die Erkennung des Septoria-Befalles von Weizenkornern