Seed Health Testing

Keshav Narayan PaiMSc II

DOS in BotanyManasagangotri

Content

• Introduction• Objectives• Seed Health Testing for Fungi• Detecting Seed Borne Bacterial Plant

Pathogens• Detecting Seed Borne Plant Viruses• Detection of Insects and Nematodes• Conclusion• References

Introduction

• Seed Health Testing is a Science of determining the presence or absence of disease causing agents, such as fungi, bacteria and viruses.

• Animal pests like eelworms, insects in the seed samples.

Objectives• To determine the health status of a seed lot which in

turn establishes the sanitary condition of the seed in commerce.

• To obtain objective proof of whether the seed lot meets the requisite certification standards or not.

• To obtain objective proof of whether the lot meets requisite quarantine requirements.

Methods of seed Heath Testing for Fungi

• Visual examination – with or without stereomicroscope.

• Inspected in dry state for the presence of impurities such as ergots or other sclerotia.

Determination of Karnalbunt (Neovassia indica) of wheat

• Infected seeds have a characteristic black powdery mass generally along with the suture.

• Severe infection – most of the endosperm together with pericarp and aleurone layer gets destroyed giving grains a boat like appearance.

Determination of bunt (Neovossia horrida) of Rice

•Seeds are opened with the help of the blade

•Whole grains transformed into black powdery mass

• Alternate procedure : Paddy seeds are soaked in 0.2% solution of sodium hydroxide for 24 hours at 18 -25°C .

• Swollen seeds are visually examined for shiny jet black colour.

Determination of Loose smut infection in Wheat

• Soak seeds for 24hours at 20°C in Sodium Hydroxide and Trypan Blue.

• Separate the embryos by passing the soaked material along with warm water (50-60°C)

• Embryos are washed in wire basket and dehydrated in 95% alcohol for 2min and later transferred to a mixture of lactophenol and water.

• Further separation of embryos can be made.• Embryos are placed in lactophenol and boiled.• Embryos are examined in microscope.• Infected fungus with loose smut fungus is present as hyphal

strands.• Golden brown thread like mycelium which is septate with non

uniform thickness and swellings are classified as infected embryos.

UV Examination

• Toxins in Fungi gives Fluoroscence appearance.

• This represents presence of Fungi

Blotter Method

•Seeds are placed on moistened blotters, filter papers atleast 20mm apart.•Blotters are placed in closed containers and incubated for certain number of days and later examined for pathogens

Agar Plate Method

•After pretreatment seeds are spaced on the surface of 2% malt extract sterilised agar.

•After plating dishes are incubated at 20-25°C for 5-8 days.

Wash Method

• Seeds are immersed in water with a wetting agent and shaked vigorously to remove fungal spores, hyphae etc.

• Excess liquid is removed and material is examined.

Examination of Imbibed Seeds

• Seeds are immersed in water, or other liquid to make fruiting bodies, symptoms more easily viable or to encourage the liberation of spores.

• After imbibition the seeds are examined with a stereoscopic microscope

PCR Method

• It is a kind of molecular method.• Specific primers are used in this method.• PCR is a promising tool for distinguishing

specific sequences from a complex mixture of DNA and therefore is useful for determining the presence and quantity of pathogen-specific or other unique sequences within a sample

Methods to detect seed borne bacterial pathogens

• Various methods are been developed.• Some of the methods are simple and some are

specialized namely serological methods.

Growing out test

The ‘growing out’ bioassay of a working seed sample involves the sowing of test seeds into seedlings under conditions optimal for the disease development in glass house or closed environmental chambers.

‘Growing out’ test has been successfully used for a large number of Xanthomonads and pseudomonad’s.

Indicator test

Working seed sample is sterilized with (2.6%) sodium-hypochlorite for 15 min, and rinsed with sterile water.

The seed sample is incubated for 18-24 h in sterile water.

The water suspension is inoculated by infiltration into the primary leaf node of 10 day old bean seedlings.

The appearance of lesions followed systemic necrosis is positive reaction.

Serological Technique Serological tests are based on ‘In vitro’ reactions between

antigens and antibodies.

This specific recognition of antigens by antibody has offered the basic principle for the development of various serological methods for detection and identification of phytobacteria.

The washing of the working seed samples are cultured for 36 hr using sterile distilled water.

The supernatant is tested with antiserum of the suspected pathogen.

Culture Method

• By culturing the samples on the particular media.

• Presence of bacteria can be observed on the media.

PCR Method

• PCR is a promising tool for distinguishing specific sequences from a complex mixture of DNA and therefore is useful for determining the presence and quantity of pathogen-specific or other unique sequences within a sample.

• It is a Molecular method and it used widely used in present day.

Methods to detect seed borne Plant Viruses

Biological Method

a) By growing seeds: The seeds are examined and abnormal seeds are separated.

b) Both normal looking and abnormal looking seeds are grown separately and the seedings are observed for the characteristic symptom of the virus disease.

c) Plants are kept free of insect vectors such as mites and white fly.

• Direct seed Testa) Seed are examined and abnormal seeds are

separated and handled separately.b) Seeds are soaked in aqueous medium and then

triturated.c) The slurries produced are then applied to

indicator or test plants.d) The slurries containing virus produce symptoms

typical for a given virus on the indicator plant.

Serological Tests

• Double diffusion testa) Seeds to be tested are soaked in tapwater.b) Seed or part (embryo) of it is triturated and

triturate is transferred to a well cut in a diffusion media(agar gel).

c) Antiserum specific for a suspect virus in a seed is placed in separate well.

• In time the virus particles(antigen) and antibody molecules diffuse towards one another.

• Since diffusion is in two direction it is called Double diffusion.

Radial Diffusion Test

• Procedure consists of charging the wells with the seed or seedling preparations being tested for the presence of virus.

• When virus molecules are present , they diffuse radially from the well surface they complex with antibody molecules and precipate in this region.

•Ring of hallow result around the charged well

Latex Flocculation test

Specifically Prepared seed extracts are placed in a pipette

Specified quantity of tagged latex is added

Pipette is then oscillated for about 15 minutes

Observed under microscopes

When Virus is present in the sample the latex suspension becomes flocculated or aggregated.

ELISA(Enzyme linked immunosobent assay)

• In this procedure , antiserum specific for a given virus is used to coat the polystyrene plate.

• Antibody molecules become absorbed.

• Then seed sample is added to the plate.

• It is followed by adding enzyme labelled specific antibody to the plate.

• The enzyme alkaline phosphates is conjugated to the antibody molecules specific for the virus under examination.

• Finally enzyme substrate is added to the plate.

• Hydrolyzed substrate is determined by measuring the extinction spectrophotometrically or by visual observation.

SSEM(Serologically specific electron microscopy)

• Ground seeds or homogenised preparation is applied to the electron microscope grid containing an antiserum specific for virus in question.

• Samples from infected seed lots reveal the presence of virus particle characteristics for the suspect virus.

Radio Immuno Assay (RIA)

• Antiserum is added to the radio labelled antigens and allowed to incubate in a centrifuge tube.

• After incubation , ammonium sulphate is added for precipitation of antigen antibody complex.

• After centrifugation the supernatent is discarded and the radio activity of the pallet is measured.

• The pallets showing radioactivity are counted as infected.

• PCR/ RT PCR is used inorder to detect the presence of Viruses

Detection of Insects and Nematodes

Detection of presence of Insects• Examined under magnification or stereoscopic microscope

• Result is mentioned as number of insects per weight of the sample

Detection of external insect injury• Working sample is Examined under magnification(10x) or

stereoscopic microscope.

• The absence of insects does not however guarantee that the seed lot is free from insect infestation, i.e internal injury to the seeds or internal infestation.

Alkali or Glycerine Method

• Seeds are boiled in 10% NaOH solution for 10min (depending upon the kind of seeds to make them transluscent)• Alternatively the seeds can also be made transluscent by lacto-

phenol solution.• Washed with water• Examined under magnification.• seeds with visible internal infestation are separated, cut opened

to confirm the presence of insect or its stage and counted.

Detection of Nematode Infestation

• Working sample is visually examined for the presence of ear cocke galls.

(hard, small, dark purplish- black colour structures)• Galls are separated and soaked in water for 30 minutes. and are cut in water in petridish for observing the release

of nematode larvae, galls releasing nematode are counted and result is reported in percentage.

Ear cockle of wheat (Anguina tritici)

Galls on wheat

Anguina tritici

Root knot Nematode of Sweet Potato (Meloidogyne incognita)

• Entire submitted sample is examined for visible symptoms of nematode infestation.

Galls on Sweet Potato

• Nematode infested tubers look ugly and disfigured.

• When tubers are cut across the knot , small glistening round pin head shaped bodies are seen and such tubers are separated and the results are reported.

References• Agrawal.R.L.2012.Seed Technology.2nd Edition.Oxford and IBH

Publishing CO.PVT.LTD. New Delhi.Pg no:591-610.• Srivastava, J.P. and Simarski, L.T. 1986. Seed Production

Technology. International Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria .Pg no:230-241.