3 Techniques for Detection of Fungal Pathogens in Plants

The following points highlight the top three techniques for detection of fungal pathogens. The techniques are: 1. Examination of Seeds without Incubation 2. Incubation Method 3. Seedling Symptoms Test.

Technique # 1. Examination of Seeds without Incubation:

(i) Dry Seed Examination:

Inspection of dry seed can be applied to detect seed- borne pathogen which when present in the seed may cause discoloration of seed coat or changes in the seed size and shape.

The seed sample is first examined by naked eyes, then under stereoscopic binocular microscope to record observation on the mixture of seeds, weed seeds, plant parts, inert matter, discoloration, malformations, sclerotia, galls, bunt balls, bacterial ooze, fungal bodies like, acervuli, Pycnidia, Perithecia, hyphae, spore masses etc. Mechanical damage of seed is also recorded as they act suitable site for the entry of pathogen.

According to the rules of the International seed testing Association (ISTA), the inert matter fraction such as soil, sand and stones, various types of plant debris, including nematode galls, fungal bodies such as ergot sclerotia and smut balls, are of great pathological importance.

The inspection of dry seed in seed health testing is a qualitative test for which no standard working sample size has been worked out. However, it may be suggested to use a sample equal in size to the sample employed in the purity analysis in seed certification.

All parts of a seed sample are examined carefully by naked eye or with the help of hand lens. During the examination, emphasis is laid on galls, sclerotia and smut balls; the technique is simple and gives quick information about the health status of the seed lot.

Procedure:

a) Take 400 seeds at random in a Petri-plate.

b) Note down the weight of the seeds.

c) Examine the seed samples with the help of stereoscopic binocular microscope.

d) Make suitable record out of 400 seed.

(ii) Washing Test:

The washing test is a seed health testing method which is used solely to test seeds for externally seed borne pathogens, the inoculum of which is present loosely on the seed surface.

The washing test is a qualitative test for which no standard working sample has been approved so far by the ISTA. The washing test as mentioned here is generally used to detect presence or absence of fungal prop gules.

Procedure:

a) Two gm of seed is taken in a test tube with 10 ml of water and shaken for 10 minutes on a mechanical shaker.

b) The suspension is examined as such or the suspended spores are concentrated by centrifuging at 3000 rpm for 15-20 minutes.

c) The suspension is discarded and the spores are again suspended in 2 ml of lacto phenol (a mixture of lactic acid, phenol, water and glycerol in the ratio of 1:1:1:2).

d) This suspension is then examined under the microscope for the presence of spores, conidia and other fructifications.

e) Rice (1939) suggested the use of haemocytometer for the semi-quantitative estimation, where the spores load/gm of seed can be calculated with the help of the following formula:

(0.0001 being the value of fluid in Central Square of the haemocytometer)

where, N is the number of spores in the central square.

V is the value of mounting fluid added to the sediment

W is the weight of seeds

So, the spore’s load/g of seed will be:

(iii) Viability Test:

To test the viability of the spore obtained from the washing test, the suspension containing spores is spread on 2 per cent plain agar. The viability can also be checked by streaking the spore’s suspension on potato dextrose agar medium. Incubate the Petriplates containing plain agar or PDA for 5 days at 25 ± 1°C (Fox 1993).

Technique # 2. Incubation Method:

In this method the seeds are examined after examined after incubating them on some suitable media or substrata.

(i) Standard Blotter Method:

The blotter method was developed by Doyer in 1938 which was later included in the International seed Testing Association (ISTA) rules of 1966.

Fungi found associated with seeds are carefully examined and identified based on “Habit characters”. Habit characters of fungi can only be learnt with practice. In the beginning, an analyst has to make slide preparation of fruiting structures, such as Conidia borne on conidiophores, spores held together in spore masses, sporodochia and acervuli, Pycnidiospores in Pycnidia and ascospores in Perethecia.

Examine them at higher magnifications of compound microscope for confirming their identity with the help of an experienced analyst.

The blotter method is widely used for detecting fungi which are able to produce mycelial growth and fruiting structures under the incubation conditions available in the test. All kinds of seeds, cereals, vegetables, legumes, ornamentals and forest seeds are test by this method.

It is the most widely practiced seed health testing method. Many laboratories use it as the first screening test for seed health testing method. Blotter teat is a combination of in vitro and in vivo principles of investigations.

Obligate parasites can’t be observed by this method.

Procedure:

1. Three layers of blotters moistened with tap water are placed in Petri plates either of plastic or of Pyrex glass.

2. Usually 400 seeds/samples were taken.

3. 25 seeds in each Petridish are placed following 15/16 seeds in the outer ring, 9/8 in the middle and one in the centre.

4. Incubate the dishes at 20-22°C for 7 days in alternating cycles of 12 hours darkness and 12 hours light. The common source of light used at present is the near ultraviolet (NUV) supplied by black light tubes or day light provided by cool, white fluorescent tubes.

In either case, light should be supplied by two tubes hanging horizontally, 20 cm apart from each other and the distance between the light tubes and the dishes should be 40 cm.

5. After 7 days’ incubation, start examination of seeds under a stereoscopic microscope with magnification of at least 50 or 60 times. All seeds of the outer ring must be examined first, then the seeds of the second ring and finally the seed in the centre of the dish.

6. Green pencils, which can write on wet blotters, should be preferred for making the observations.

7. The first seed to be examined is the one which lies on the right hand side of the line in the outer circle. Examine thoroughly the whole seed at different magnifications before proceeding to the next. Once the examination of the first seed is finished rotate the dish gently clockwise with the middle finger and the thumb of the left hand while still looking into the microscope.

Follow this procedure in moving from one seed to the other. Whenever identifiable growth of a fungus is seen on a seed, mark the infected seed by writing abbreviations such as “Py” for Pyriculoria oryzae near to it.

8. Sometimes, it is difficult to identify the growth of a fungus under stereomicroscope. In such cases, make slide preparations and identify under compound microscope. If the fungus is identified write its abbreviation near the infected seed.

9. Count seeds infected with different fungi in different dishes.

10. Note the names of the fungi and number of seed infected.

(ii) Rolled Paper Towel Method:

The seeds are placed on moist paper towel and covered with another moist paper towel and rolled carefully. The rolled paper towels containing seeds are incubated in dark at suitable temperature for a fixed period of time. The seeds are examined after incubation for the presence of micro-organisms and germination. (This method is used for the detection of Fusarium spp. in cereals and Ascochyta diseases in pea).

(iii) 2, 4-D Method:

The method has been recommended by ISTA (1966) as a standard test for a number of seed borne pathogens. The use of 2, 4-D in the blotter test was first introduced by Neergaard (1973) while testing cabbage seeds for Phoma lingam, although Hagborg (1950) first used it in agar medium for the detection of colletotrichum lindemuthianum on bean seeds. However, it gives a lower count as compared to standard blotter.

Procedure:

1. Instead of water the blotters are soaked in 0.2% solution of 2, 4-D (Dichlorophenoxy acetic acid).

2. Incubated the seed in incubation chamber at 28±1°C with cycles of 12 hours light and 12 hours darkness.

3. Examination of seed under stereoscopic binocular microscope at 50 x magnification after 8 hours.

4. Since 2, 4-D retards seed germination and seedling growth facilitating the examination quick and easy.

(iv) Deep Freeze Method:

This method is used for seed health investigation. The seeds are plated as in blotter method and incubated for 24 hours under usual conditions. Seeds are placed on blotters moistened with a solution containing 0.1% (100 pg/ml) streptomycin sulfate or terramycin, incubated for three days at 10±1°C then frozen at -20°C over night and further incubated for 5-7 days at the required temperature (20-25°C).

This allows better growth of certain fungi as the imbibed seeds on moist blotters are killed by deep freezing and the enclosed nutrients in seeds are utilized by fungi. Deep freezing does not affect the fungi associated with the seed.

Procedure:

1. Take 400 seeds at random.

2. Plate the seeds on moist blotters as per techniques described under blotter method.

3. Incubate the plates in deep freeze at -20°C for 24 hours.

4. Again transfer the plates in original incubator under 12 hours light and darkness at 28°C for remaining 5 days.

5. Examine the seeds on 8^th day.

6. Record the growth characters of the fungi as well as the percentage of infection.

(v) Agar Plate Method:

This method is used for identification and detection of micro­organisms associated with seed based on the growth and colony characteristics on a nutrient agar. In Northern Ireland Muskett and Malone (1941) first time used this method for seed health testing of flax seeds. Most commonly used media are Potato dextrose agar and Czeapak’s dox agar.

The use of selective media for detection of pathogens is more reliable than blotter or agar method. E.g. Mangan (1971) modified agar plate method to isolate Phoma betae and named the method as “Hold fast method”. The method helps to observe hold fast like structures or hyphal clums of P. betae.

Fungus Fusarium moniliformae in various crop seed is not easily detected in the blotter test and it is also not able to complete with other fast growing fungi when tested on plain Potato dextrose agar (PDA) medium. Therefore, a selective medium modified Czpeck’s dox agar medium (MC_ZA) is employed when testing seed for F. moniliformae.

Similarly, specific Graiacol-Agar method has been developed by Kulik in 1973 for detection of Pyricularai Oxyzae from paddy seeds. The seed may or may not be surface sterilized with various surface sterilizing agents before planting.

Procedure:

1. Prepare P.D.A. medium.

2. Sterilize P.D.A. and Petri-plates.

3. Pour 20 ml P.D.A. medium in each Petri-plate.

4. Take 200 seeds at random.

5. Treat the seeds with 0.1% mercuric-chloride for two minutes.

6. Wash the seeds three times with sterilized water.

7. Place 10 seeds per plate at equal distance.

8. Incubate the plates at 28 ± 1°C with 12 hours alternating cycles of light and darkness.

9. Examine the plates after 8 days of incubation.

10. Note characteristics of fungal colonies from top and reverse.

11. Prepare the slides and examine them under compound microscope.

12. Record percentages of infection of different fungi.

The agar plate method provides an efficient tool for quick identification of specific seed infection ones the analyst has become familiar with the colony characteristics of the fungi in question. \

Colony characteristics are likely to change if another agar medium is employed or if the incubation conditions are changed. Considering the price of agar plate method may only be used in routine seed health testing for the detection of fungi which are not easily recorded in the blotter test.

Technique # 3. Seedling Symptoms Test:

The seeds are sown in autoclaved soil, sand, gravel or similar material under standard conditions of temperature and moisture. The infected seeds rot and seedlings exhibit symptoms as in field. This procedure helps to see the field performance of the seed lot in relation to seed borne seedling diseases.

(i) Hiltner’s Brick Stone Method:

It was developed by Hiltner in 1917. Sterile crushed brick stone with a maximum grain size of 3-4 mm is used filled in plastic pots up to ¾ th of their capacity. The crushed brick stone in the pot is saturated with water and seeds are placed one cm deep.

The pots are kept in darkness at room temperature and observations for disease symptoms are recorded after two weeks by removing the seedlings. It is a good method for filed performance test giving information on seedling symptoms. It is also used for testing treated seed.

(ii) Sand Method:

Similar to Hiltner’s method except that instead of sterile crushed brick stone, sterilized sand is used.

(iii) Standard Soil Method:

A pre-sterilized uniform soil mixture containing 4 parts clay, 6 parts peat and essential amount of fertilizer is filled in plastic multi pot-trays. After planting the seed they are covered with a polyethylene bag to retain the moisture. The symptoms are observed after incubation for 2-4 weeks depending on the kind of seed and temperature. This method was by Karlberg (1974).

(iv) Test Tube Agar Method:

This method is developed by Khare, Mathur and Neergaard in 1977. It is used for the detection of Septoria nodorum in wheat grains.

Procedure:

1. 10 ml water agar is taken in each 16 mm diameter rimless test tube, sterilized and solidified with slight slant.

2. One seed is sown in each test tube.

3. The tubes are placed for incubation at 20°C under artificial day light tubes, 12/12 h cycle.

4. To retain moisture they are covered individually or in groups by a plastic sheet or aluminum foil which is removed when the seedlings have reached the cover.

5. Seedlings are examined after 14 days for the typical symptoms of disease in the coleoptiles.

6. The symptoms can be easily studied being visible on roots as well as on green parts.

This method is useful for testing the efficiency of fungicides and also in quarantine stations as diseased seedlings of valuable crops can be destroyed and healthy one can be saved.

(v) The Embryo Count Method:

Special method to separate the embryo has been devised by Hewett (1970) and Agarwal 1978.

Procedure:

1. Soak 2000 seeds in 5 per cent sodium hydroxide solution containing 0.02 per cent try pan-blue for 24 h at room temperature (25-40°C).

2. Pass the soaked material through 10 mesh sieve and retain the material in a 20 mesh sieve along with showers of tap water.

3. Collect the extracted embryos in a beaker.

4. Dehydrate the embryos in rectified spirit for 5-10 minutes.

5. Take the dehydrated embryos along with the chaff etc. in a beaker containing 50 ml solution of lactic acid and glycerol (1:1).

6. Add to above beakers 100 ml water and stir it.

7. Allow the material to stand for 5 minutes to settle the chaff at bottom.

8. Collect the floating embryos in another beaker containing 25ml fresh solution of lactic acid and glycerol (1:1).

9. Boil the above material for 2 minutes.

10. Pour the embryos in Petri dish and arrange in lines along with some solution of lactic acid and glycerol (1:1).

11. Observe the embryos under stereo binocular microscope for the presence of mycelium.

12. Mycelium appears as blue thread like knotted structure in the scutellum of the embryo.

13. Examine as far as possible all the extracted embryos.

14. Count the total number of embryos, including infected ones.

15. Calculate embryos and report the result of infection in percentage up to two decimal places and record the data.