Methods for the detection of seed-borne pathogens: Seed-born pathogens are one of the important factors for crop health and productivity. Detecting this seed-borne pathogen is essential for preventing the spread of diseases in crops. Different methods have been developed to identify seed-borne pathogens. This article explains some of the important techniques.
Methods for the Detection of Seed-Borne Pathogens
A. Examination of seed without incubation
i) Examination of dry seeds:
- Visual Examination: The seed sample is first examined by the naked eye to identify any visible signs of contamination or abnormality like weed seeds, or inert matter, on the seed.
- Microscopic Examination: Then under a stereo binocular microscope to record observations on the mixture of seed, discolorations, malformations, sclerotia galls, bunt balls, fungal bodies like Acervuli, Pycnidia, Perithecia, etc.
- Mechanical damage: Mechanical damage to seeds is also recorded as they act as a suitable site for the entry of pathogens.
ii) Examination of seed washing:
- Seed Washing Process: This method shows the presence of the pathogen in a seed sample. A few seeds of a seed sample are kept in sterilized water and shaken in a mechanical shaker. The washing is centrifuged and lactophenol is added to the sediment after removing the supernatant.
- Microscopic Analysis: After shaking thoroughly slides are prepared to identify the spores, conidia, etc, under a microscope, and the spore load of seed-borne fungi is quantified by using a Haemocytometer.
- Spore count= NxVx10000/ Wt of sample
- Where N is the number of spores/square and V is the volume of mounting fluid.
B. Incubation methods
i) Standard blotter method: This method was developed by Doyer in 1938. It is one of the most common and widely used techniques.
Procedure:
- Three pieces of blotting paper of 90 mm size were moistened with distilled water and placed in 90 mm sterilized Petri plates.
- Excessive water is drained off, and untreated seeds are placed in a Petri plate (25 seeds per Petri plate), spaced equally.
- The Petri plates are incubated at room temperature under alternating cycles of 12 hours of darkness and 2 hours of near-ultraviolet (NUV) light.
Observation:
- After 8 days of incubation, the seeds were examined under a stereoscopic binocular microscope for the associated fungi and they were identified based on habit characteristics. This is the most convenient and efficient of all incubation methods.
Also, visit the List of Seed born diseases, Seed storage
ii) Deep freezing blotter method:
Procedure:
- 400 seeds were placed at a rate of 25 seeds per plate on moistened blotters similar to the standard blotter method.
- The petri plates were incubated at 25 o C for 24 hours under alternate cycles of 12 hours of NUV light and darkness.
- For the next 24 hours, the plates are incubated at -20 o C in the dark and then kept back under original conditions for the next 6 days.
Observation: After 8 days of incubation the seeds were examined under a stereo binocular microscope. Deep freezing allows better growth of certain fungi.
iii) Agar plate method:
Procedure:
- Seeds were surface sterilized with 1 % sodium hypochlorite solution for 1-2 minutes and then washed with sterile water 2-3 times.
- Sterilized seeds were placed at the rate of 10 seeds per petri plate containing 20 ml of potato dextrose agar or nutrient agar.
- The petri plates were incubated for 7 days similar to the standard blotter method.
Observation: After 7 days of incubation the fungal growth was examined under a microscope. This method is particularly useful for specific fungi that grow well on nutrient media.
C. Molecular Techniques
- Polymerase Chain Reaction (PCR) and Loop-Mediated Isothermal Amplification (LAMP): This method is used to detect specific DNA or RNA sequences of pathogens.
D. Serological Methods
- Enzyme-linked immunosorbent Assay (ELISA) and Immunofluorescence.
E. Biochemical Methods
- Isoenzyme Analysis and Fatty Acid Methyl Ester (FAME) Analysis.
