Seed health testing


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Seed health testing-A lecture note for L-4, S-1 students of BAU by K. M. Golam Dastogeer
Bangladesh Agricultural University, Mymensingh, Bangladesh

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Seed health testing

  1. 1. STUDY OF SEED HEALTH TESTINGSeed healthSeed health concerns the overall condition of seeds. It includes pathogenic infection of seed, insectinfestation, morphological and physiological disorder, inert matter etc.Seed health standardSeed health standard is the maximum acceptable limit of the presence of a given pathogen in a givenseed lot. This is also referred to as "Pathogen tolerance level "or "Seed standard" for pathogen.Field health standardField health standard is the maximum acceptable limit of a given seed borne disease present in a givenseed crop field. This is also referred as "Disease Tolerance level" or "Seed Standard" for disease ofseed crop.Seed health testingSeed health testing is a procedure by which can be determined whether tile seed is healthy or diseasedor it is a procedure by which the presence of absence of seed borne pathogen(s) in a seed lot can bedetermined.Seed borne pathogenThe establishment of a pathogen in, on and with the seed implies that the pathogen is seed borne.Objectives of Seed health testing1. Seed health testing is necessary for the improvement of seed stock in certification scheme.2. It is necessary to satisfy quarantine requirement of a country.3. It is done to know the planting value of a given seed lot in order to forecast the field emergence and predict the health f the mature crop.4. It is necessary to know the storage quality or feeding value of a seed lot.5. It is necessary for checking the advisability of treatment.6. It is done to know the efficacy of seed treating chemicals.Methods of seed health testingl. Dry inspection method2. Microscopic examination of suspension obtained by
  2. 2. a. Washing test methodb. Whole embryo count method3. Incubation tests a. Blotter method b. Agar plate method c. Deep freezing blotter method d. Water agar plate method e. Test tube agar method4. Seedling symptom test - Hiltners bricks stone method - Sand methods - Standard soil method - Test tube agar method5. Growing on test6. Serological tests7. Indicator plant test8. Electron microscopyBasic requirements or considerationsRoutine methods for seed health testing must fulfill the following demands of efficiency and economy:1. A test must give reliable information pertaining to field performance and quarantine requirements.2. The results must be reproducible within the statistical limit3. The time, labor and equipment for carrying through a test must be kept within economic limits.4. The tests requiring incubation must be able to give the result quickly.Equipment and chemicals required for seed health testing in the Laboratory 1. A hand lens. 2. A steriobinocular microscope. 3. A compound microscope. 4. An oven 5. A Refrigerator
  3. 3. 6. An autoclave 7. A centrifuge 8. Plastic/glass petridishes and/or earthen dishes 9. Filter papers or blotters/newsprints 10. Small equipments like forceps, glassware like-glass slides, cover slips, beakers, flasks, test tube etc. 11. Chemicals and medium like-agar, dextrose, Nutrient agar, mounting fluid (lactophenol /Cotton blue ctc,) chemicals for pretreatment like sodium hypochlorite (Clorox) and Hgc12 (mercuric chloride) solution. 12. Potatoes, cheesecloth, pencils, markers ctc , 13. Sampling equipments like treys, seed triers, dividers etc.Seed lotA lot is a specified quantity of seed physically identifiable in respect of which an International orNational Analysis Certificate may be issued.Seed samplingPrimary sampleA primary sample is a small portion taken from one point of the seed lot.Composite sampleThe composite sample is formed by combining and mixing all the primary samples taken from the lot.Submitted sampleA submitted sample is a sample which is submitted to the testing laboratory for analyses of the qualityof a seed lotWorking sampleThe working sample is a sub-sample taken from the submitted sample in the laboratory, on which thequality analyses will be done.Dry inspection methodIt is a very simple and preliminary method for testing the seed health.
  4. 4. ProcedureThe dry seed samples were examined for impurities such as a. Inert matter: It includes plant debris, spotted, unfilled & chaffy grains, sclerotia, galls, smut balls, insects etc. It should also be incubated either on blotters or, agar media and examined, after a standard period of infection. b. Symptoms: Such as (Discoloration, staining, necrosis, malformation and similar indications of infections, including fruiting bodies of fungi, resting hyphae on the seed-surface, spore or bacterial masses on the seed as well as mechanical damage.Seed samples were examined first by naked eye and the observed under a stereobinocular microscopefor confirmation of the above, impurities in, proper way. The inert matters need to be incubated for thedetection of the pathogens associated with these:Advantages/Usefulness of dry inspection1. Dry inspection method provides quick information.2. It does not require much equipment.3. It helps to take first hand measureDisadvantages/Limitations of dry inspection1. Only those diseases giving external, symptoms and signs can be detected by this method.2. Information pertaining to viability of the seeds cannot be obtained.3. It is not more reliable because badly infected seeds may look healthy.The following samples were Supplied letr dry inspectionl. Discolored and spotted rice grains: the spikelets are dark brownish in color, upper surface showingblack velvety appearancxe with brown spots. Some spots are spindle or eye shaped.a. Associated pathogens: Bipolaris oryzae Pyricularia oryzae Curvularia oryzae
  5. 5. Curvularia lunata Cladosporium oryzae Nigrospora oryzaeb. Brick pieces2. Black point of wheat: The embryonic point of wheat seed become black and the brains are alsoshriveled.The main causal organism associated: Bipolaris sorokinianaOther organisms may associate as saprophytes: Alternaria tenuis Curvularia spp. Fusarium spp.3. Discolored and shriveled light weight jute seeds: Due to attack of several pathogens, the seedsare discolored and shriveled.Causal organisms: Macrophomina phaseolina Botryodiplodia theohromae Colletotrichum corchori4. Discolored and shriveled seeds of mustard: Due to attack of seed-borne pathogens, the seedsbecome discolored and shriveled.Causal organisms: Alternaria brassicae Alternaria brassicicola Aspergillus spp. Penicillium spp. Phoma lingam5. Purple stained soybean seeds:Purple stain of soybean: The diseased seeds became purple in color in one side and other side isnormal.Causal organism associated: Cercospora kikuchi6. Discolored and shriveled okra seeds: Dark brown in color rather than normal healthy seed and
  6. 6. most of the seeds arc shriveled.Causal organisms may associate: Colletotrichum dematium Macrophomina pharseolina Aspergillus spp. Penicillium spp. Fusarium spp.7. Discolored and shriveled seeds of country beanThe diseased seeds become discolored and shriveled. Causal organisms may associate: Colletotrichum lindemuthianum Uromyces phaseoli Cercospora spp. Aspergillus spp. Alternaria spp. Fusarium spp. Xanthomonus campestris pv. phaseoli8. Discolored and shriveled seeds of mungbean: Causal organisms may associate: Colletotrichum dematium Mungbean golden mosaic virus Cercospora canescens Mungbean mosaic virus Macrophomina pharseolina Mungbean mosaic virus Botryodiplodia theohromae Fusurium spp. Phoma spp.9. Discolored seeds of white gourd Pathogens may associate: Colletotrichum spp. Aspergillus spp. Alternaria spp.10. Discoloured seeds of sweet gourdPathogens may associate:
  7. 7. Colletotrichum spp. Aspergillus spp. Alternaria sppBlotter method/testThe blotter test is a combination of the in vitro and the in vivo principles of investigation. In this method,the seeds are sown in petridishes other suitable containers on moistened absorbent blotting paper,usually three layers to provide enough moisture for duration of the test.Materials required1. Plastic petridishes 2. Blotting paper 3. Seeds4. Sterilized water 5. Forceps 6. Stereoscopic microscope7. Compound microscope 8. Cotton 9. Spirit10. Mounting fluid, slide and cover slip etc.Procedures 1. Required number of plastic petridishes was taken. 2. The plates/petridishes were then rinsed with methylated spirit and were dried. 3. The petridishes were left for a while to allow the spirit to be given off. 4. Required numbers of blotting papers were soaked in sterilized water and three sterilized blotting papers were then placed on each petridish. 5. Then 400 seeds were taken randomly from a working sample.Surface sterilization of seed lot: Then the seed lot or working sample were sterilized with 0.001%HgCI2 solution or 10% Clorox solution for detection of internal seed-borne pathogens. For this, the seedlot was soaked into the 10% Clorox solution in a petridish with repeated shaking for about one minuteand then the chemical solution were drained out from the petridish carefully. The seed lot was washedwith sterilized distilled water for 3 times to remove the chemical from the seed.6. 5 or 10 or 25 seeds (with and without surface sterilized) were placed on the wet blotting papers keptat the bottom per petridish or pyrex glass petridish or earthen dish (depending on the size of the seeds)maintaining equal distance among the seeds.7. Then the petridishes were kept into an incubation chamber at 20"C ± 2"C in 1 2/ 12 hours alternating
  8. 8. cycles under ultraviolet (UV) light and darkness for 7 days.ObservationAfter a week, incubated petridishes containing seedlings and non-gerininated seeds were carefullyobserved under stereobinocular microscope. The identification and the frequency of different categoryof fungi associated with the seeds were performed by observing the color, growth habit andmorphological features. The data were recorded as shown in the following table Table: Blotter method of Seed Health Testing Nature of %Germ % Pre- % Post No. of Pathogen(s) disinfection ination emergence emergence colonies associated death death Without surface sterilizedWith surface sterilizedThe percentage of healthy seed in the sample tested=% Germination- (% pre-emergence death + % post emergence death)Remarks:From the above results, it was observed that --------% healthy seed was present in the seed lot. So thehealth status of the seed sample is good/poor. Therefore, the tested seed sample can be/ can not besuggested for sowing at farmer’s level.Advantages/Usefulness of blotter test 1. Pathogens can be detected quickly by observing their growth characters. 2. It is economic 3. Pathogenic potentials of the associated fungi can be determined by observing the signs and symptoms produced by them on the germinating seeds and seedlings. 4. It can be applied for detecting wide range of fungal pathogens from all different kinds of seeds. 5. Results obtained by it, is more reliable because it is the combination of in vitro and in vivo. 6. Blotter method is widely used while agar plate method is impracticable.Disadvantages/Limitations of Blotter method 1. Examination may be hampered due to the fast growth of certain Fungi over the slow growing ones.
  9. 9. 2. Pathogenic bacteria can not be detected. 3. It is time consuming. 4. Pathogenicity cannot be detected. 5. Symptoms may not be detected.Earthen dish newsprint methodThis method is, in fact, the modification of f3lc3ttcr microflora of seed health testing (in this methodearthen dish and old newspaper are used in stead of petridish and filter papers, respectively.Procedures:1. The earthen dish was washed and the newspaper was then placed in tile centre of the dish.2. Then 5 or 10 or 25 seeds (depending on the sire of the seeds) were placed on the wet newsprint papers kept the bottom earthen dish maintaining equal distance among the seeds.3. Then the dishes were covered with thin polythene sheet and were incubated in the incubation chamber for at least seven days (depending on the crop species) .4. Finally the data on the prevalence of seed borne pathogens, per cent infected seed, per cent pre and post emergence death and per cent germination were recorded.Agar plate methodThis method is the modification of blotter incubation method. In this method, nutrient agar, malt extractagar or potato dextrose agar (PDA) are used in place of blotting papers. But generally PDA is used forthe growth of fungal pathogens and Nutrient agar is used for bacteria.Materials required 1. Seeds sample 2. Medium: PDA, NA etc. 3. Stereobinocular microscope 4. Compound microscope 5. Petridish 6. Needle 7. Forceps 8. Sterile water 9. Cotton 10. Spirit 11. Marker 12. Beaker 13. Mounting fluid, Slide and cover glass etc.Procedure
  10. 10. 1. Required numbers of petridishes were taken. 2. The petridishes were then sterilized with methylated spirit and were dried. 3. The petridishes were left for while to allow the spirit to be given off 4. 400 seeds were taken randomly from a working sample. 5. Seeds were surface sterilized with 10% Clorox solution or 0.001% HgCI2 for 1 minute. 6. Then 5 or 10 or 25 seeds (depending on the sized) with and without surface sterilized were placed on PDA containing petridish with the help of forceps. 7. Then the petridishes were incubated at 20i2"C for an incubation period of from 5 to 7 days with 12 hours darkness alternating with 12 hours NUV light.ObservationAfter incubation, the incubated petridishes containing seedling and non-germinated seeds werecarefully observed macroscopically by naked eyes. The identification and the frequency of differentcategory of fungi associated with seeds were performed by observing the color, growth habit andmorphological features. The whole mounts were also prepared and observed under a compoundmicroscope for the identification of different fungal species associated with the seeds. The data wererecorded as shown in the following table. Table: Agar plate method of Seed Health Testing Nature of %Germination % Pre- % Post No. of Pathogen(s) disinfection emergence emergence colonies associated death death Without surface sterilized
  11. 11. With surface sterilizedThe percentage of healthy seed in the sample tested=% Germination- (% pre-emergence death + % post emergence death)RemarksFrom the above results, it was observed that ---------------% healthy seed was present in the seed lot.So the health status of the seed sample is good/pour. Therefore, the tested seed sample can be/ cannot be suggested for sowing at farmerAdvantages/Usefulness of Agar plate method 1. This method is useful only for selective pathogens. . 2. Fruiting structures of fungi can develop quickly on agar. 3. The fungi that not identified by blotter method, can easily be identified by agar plate method. 4. Pathogenic bacteria can be detected by this method. 5. Agar plate method provides adequate conditions for the mycelial growth, sporulation and symptoms on seedlings and seeds than blotter method.Disadvantages 1. The method is costly, laborious and time consuming. 2. Slow-growing fungi can be suppressed by the rapidly growing ones. 3. Pathogenic potentials of the associated organisms cannot be determined as no disease symptoms are produced oil the poorly germinated seeds and seedling on agar. 4. The analysts need experience to perform the test.Growing-on testCertain categories of seed-borne pathogens require a longer period of incubation to be detected than isprovided by the usual incubation procedures such as the blotter method, tile agar plate method and theseedling symptom tests. This test is highly applicable for bacterial and viral pathogens. Also systemicinflections of fungi may be detected in growing on .test.Conditions: 1. The seeds are to be sown in sterilized soil in suitable containers and subjec.ted to suitable conditions of temperature (21-30°C) and humidity. 2. The containers (Earthen pot; polythene bags, plastic tray etc.) must be kept in a glasshouse or controlled environment cabinet (insect proof) protected against contamination.
  12. 12. Procedure 1. The pots or trays were tilled with sterilized soil/sand/saw dusts. 2. Then 25 or 100 seeds were sown in each pot/tray depending on the size of pot or tray. 3. The pots or trays were then placed in greenhouse/glasshouse at 21- 23% temperature for 2 to 3 weeks to allow the development of symptoms in plants. -Observation 5. The seedlings/plants were examined for disease symptoms produced. 6. The number of infected seedlings was counted and expressed in percentage.Advantages 1. It is of great importance in quarantine control as exemplified in the import inspection of seed samples for breeding purposes. 2. The seed harvested from the plants grown under this test can be retested for health in the laboratory before they are released for final surveillance under field conditions. 3. It is only method for detection of fungi, bacteria and viruses where other methods of seed health testing are not available.Limitations 1. The importance of growing-on tests is underestimated and the procedure is little used in routine testing because it does require considerable space, greenhouse facilities or environmental controlled rooms and also more time than laboratory procedures. 2. Growing-on test must be adopted when certain organisms are not detectable by available laboratory procedures. 3. It does not provide reliable information because many seed transmitted pathogens may be carried in a symptom less manner or may only produce vague symptoms.