4 Diagnosis of Seed Borne Pathogens
Transcript
Hello!! Welcome to the fourth talk of the week that is detection of seed borne pathogens. Seed borne pathogens we now that it has greater impact on agriculture as well as seed industries and detection of seed borne pathogens is very much essential as the seed borne inoculums me give rise to severe disease investigation in the field condition and severe loss to the growers. There are conventional methods as well as molecular methods that is deployed for the detection of diagnosis of seed borne pathogens. Some of them are like:
Blotter method
Agar method
Paper towel method
Embryo Extraction method
De-hulling and embryo extraction
Extraction and Agar plating method
Extraction and Polymerase Chain Reaction method
ELISA method
These are some of the common seed borne methods applied for seed health testing for detection and diagnosis of seed borne pathogens. Let us talk about the Blotter method: Say for example Alternaria dauci that causes disease in Daucus carota (carrot) and it’s a seed borne pathogen. It can be diagnosed with Blotter method. In this particular method we take 3 layers of blotting paper on both the lids of petriplate and the we moisten it and then we place the seeds at uniform distances and then we incubate it for period and then once pathogens grow outside the seed we can visualize the pathogens under microscope at different magnifications. And then by looking at the morphology of the fungal pathogen associated with it we can very well identify the pathogenic nature of and the pathogens associated with the carrot seeds. So, in short three layers of 90mm filters are placed in both the lids after soaking with sterilized distilled water. We have to drain away the excess water.
Then we should place atleast 10 seeds on the plate, evenly placed, on the surface of the filter pare, then incubate it for 3days at 20oC in the dark. Then transfer the plate to freezer and maintain a temperature of -20oC for 24 hours and after freezing incubate for 6 days at 20oC with alternating 12hour period of darkness and near NUV lights and plates should be approximately 25cm below the lights and should not be stacked. Then we can examine as we have shown in the previous slide, the fungal spores or fungal growth on the seed surface and we can establish the relationship between the inherent thing pathogen like fungi that is on the carrot seeds following this particular method.
The same Blotter method is also used for detecting seed borne pathogens such as Alternaria radicina in carrot, Botrytis cinerea in sunflower seeds. Then Alternaria radicina in carrot can also be established through malt agar method. Malt agar method can also be diagnosed through Leptosphaeria maculans and Plebdomus biglobosus in Brassica seeds, then Ascochyta pisi in Pisum species and the method is basically Aseptically place around 10 seeds evenly spaced on the agar surface of malt agar plate. Then incubate it for 10days under 20oC with altering 12hour periods of darkening and near UV light (NUV). The similar way plate should be approximately 25cm below the lights and plate should not be stacked. Subculture of reference culture to a malt agar plate at the same time of the seeds are plated and incubated in the test plates so that we can have a comparative idea between the actual culture and the fungal growth that is taking place on the seed surface. Then examine the plates visually under stereoscopic microscope to establish the morphological characteristics of the associated fungi.
Colletotrichum lindemuthianum in bean then Bipolaris oryzae in rice these are some of the other pathogens that are also used in agar plate method for detection as seed borne pathogens. Then the next method is Rolled paper towel method where seeds are placed between two paper towels and paper towels were moistened and the seeds were allowed to germinate and grow as seedlings and by looking at the seedling health one can establish from the germinating seeds of infected and healthy seeds then looking at the fungal spores or fungal colony strata associated with the infected seeds to establish the relationship between fungi that is causing certain seed borne diseases.
Embryo Extraction method – it is used for Ustilago nuda in case of barley. So the method basically as such we have to place the seeds 1 litre of freshly prepared 5% aqueous solution of sodium hydroxide and maintain at 20oC for 24hours. After soaking, the entire sample should be transferred to a suitable container and washed in warm water to separate the embryos, which appear through the softened pericarps. Then collect the embryos in a sieve of 1 mm mesh. Additional sieve of larger mesh can be used to collect pieces of endosperm and chaff. Then transfer the embryos to a mixture of equal quantities of glycerol and water in which further separation of embryos and chaff can be made and transfer the embryos to a beaker containing 50ml of lactic acid solution and clear them by maintaining the lactic acid solution at boiling point for approximately 5min in a fume cupboard. Then transfer the embryos to fresh glycerol for examination. The scutellum becomes more transparent when embryos are left in glycerol for 1-2 hours making the examination much easier. So, this is how we can go for Embryo Extraction method for detection of Ustilago nuda in barley seeds. Then we have to examine it under microscope for presence of the Ustilago nuda pathogen.
Ustilago nuda in barely seeds can also be done by dehulling and embryo extraction method. Here, the embryo extraction method is the same but the dehulling method has to be preceded the embryo extraction method. In case for dehulling place the working sample in glass beaker with 25-37% sulphuric acid until the seeds are covered. Incubate in an oven at 75oC for 50min or until the seeds turn a medium brown colour. Carefully pour of the sulphuric acid solution, rinse seeds by pouring water into the beaker, gently mix and pour off the water and add new
water and remove loosened hulls by stirring robustly with a rod. Remove hulls by carefully removing the water. If hulls remain, add new water and either use an hand mixer at low speed or continue stirring. Repeat the procedure until all hulls are removed. Be careful not to lose any kernels i.e. seeds without hulls. So, after dehulling we can go for embryo extraction method as it is mentioned earlier.
The next stage Extraction and Polymerase Chain Reaction method – So, this is used for detection of Xantomonas campestris, pathogonas campestris in Brassica seeds. Here the pathogen is first cultured on petriplates and then DNA is extracted from the bacterial cultures and PCR amplification is done using specific primers for detecting the pathogens. Presence of specific bans on the cultures confirms whether the pathogen is of the targeted ones or and other microbes that is associated with. The detail about Polymerase Chain Reaction method will be discussed in subsequent talks. Similarly, Elisa method is used for detection of certain viral diseases of that are seed borne in nature for example squash mosaic virus, cucumber green mottle mosaic virus and melon necrotic spot virus in cucurbit seed.
So, another test that is known as Grow-out test. So this infected seeds are grown on sample pots and they are allowed to grow in a way that disease symptoms are manifested on the leaves. So, this is another confirmatory test which we call it as Grow-out test. It can be also perform for Squash mosaic virus where the symptoms are very much evident after growth of the seedling to a certain stage and by doing this test by looking at the symptoms one can confirm whether the pathogen is associated with the seeds or not. So, these are some of the basic methods for detection and diagnosis of seed borne pathogens and with the time different molecular tools have been deployed for detection of seed borne pathogens more accurately along with the races and strains that are associated with the disease seed lot and finally we can conclude that with this methods we can certainly able to establish a seed lot whether it is affected or not with certain fungal bacterial or viral pathogens.
Thank You very much.
