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Chapter - IV

DETECTION, ISOLATION A N D CHARACTERIZATION OF


THE PATHOGEN
*:s. Chapter Jk' Defection, Isolation and Characterization of the pathogen

Corynebacterium michiganensis subsp. michiganensis {Cmm) a canker causing

bacterium on tomato in tropical and temperate regions of the world. The quarantine

inspection for Cmm generally involves visual inspection for disease symptoms on crops.

But, visual observations sometimes fail to provide a satisfactory evidence for the

detection. Since, there are many reports of latent infection due to which the disease

spread is possible local as well as in international levels. Visual observations for

bacterial canker disease in field may sometime leads to confusion with that of vascular

wilt caused by Fusarium species and Verticillium species. In such conditions there is a

demand for rapid and specific method for the detection of Cmm from the infected plant

materials and seeds in the laboratory conditions.

Advancement in biotechnology and immunology since last years, several methods

are developed for the detection of Cmm in laboratory conditions. Some of them are; PCR

technique, RNA/DNA technology. Fatty Acid Profile, Serological methods using

Polyclonal or Monoclonal Antibodies (De Boer, 1994, 1998: Liu et. al, 2006: Kaneshiro

Wendy, 2006). However due to lack of sophisticated laboratory and skilled assistants the

practical applications of these methods still left behind. For the use of these advanced

techniques the pathogen has to be detected, isolated in pure form, which can be done only

by the conventional methods.

In the present study attempts are made to detect and isolate the bacterium from the

infected plant materials, collected from the fields and seed samples are different agro-

climatic zones of Kamataka. This chapter highlights detection of the pathogen by ooze

test, direct plating, liquid assay and ELISA (Mortensen, 1992); isolation and

characterization of the isolates based on the morphological, cultural, biochemical,

hypersensitive and pathogenesity tests.

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Chapter IV Detection, Isolation and Characterization of the pathogen

MATERIALS AND METHODS

Preparations of Media and Reagents

Media - A

Nutrient Broth Yeast Extract Agar Medium [NBY Medium) .

Peptone 5.00 gms


Beef Extract 3.00 gms
Yeast Extract 2.00 gms
Di-Potassium Hydrogen Ortho Phosphate 2.00 gms
Potassium di-Hydrogen Ortho Phosphate 0.5 gms
Distilled Water. 950 ml
Glucose 5.00 gms
Distilled Water 50.0 ml
Magnesium Sulphate Hepta Hydrate ml of IM (Make a 1 M solution by dissolving
2.46 gms in 10 ml distilled water)

Media - B

Growth Factor Media [GF Medium]

Potassium di-Hydrogen Ortho Phosphate 0.4 gms


Magnesium Sulphate Hepta Hydrate 0.05 gms
Sodium Chloride 0.1 gms
Ammonium di-Hydrogen Ortho Phosphate 0.5 gms
Ferric Chloride 0.01 gms
Yeast Extract 3.00 gms
Agar 20.00 gms
Distilled Water 1000 ml

Media - C

Semi Selective for C. mchiganensis subsp. michiganensis [SCM ]

Boric Acid 1.5 gms


Yeast Extract 0.1 gms
Di-Potassium Hydrogen Ortho Phosphate 2.00 gms
Potassium di-Hydrogen Ortho Phosphate 0.5 gms

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Chapter IV Oetection, Isolation and Characterization of the pathogen

Magnesium Sulphate Hepta Hydrate 0.25 gms


Sucrose 20.00 gms
Distilled Water 1000 ml

Autoclave and cool to 45''C - 50C and add (filtered sterilized)

Nicotinic Acid : 100 mg - 10 ml of aqueous solution containing 10 mg/ ml


Nalidixic Acid : 30 mg - 3 ml of solution containing 10 mg/ ml, dissolve in 0.1 N
NaOH
Cycloheximide : 200 mg - 2 ml of solution containing 100 mg/ml, dissolve in 75%
Ethanol
Potassium Telluric : 10 mg- 1 ml of Chapman K tellurite solution 1 % from difco.

Media - D

Yeast Extract Dextrose Calcium Carbonate Agar Media (YDC)

Yeast Extract 10.0 gms


Dextrose 20.0 gms
Calcium Carbonate 20.0 gms
Agar - Agar 20.0 gms

Media - E

Starch Medium

Soluble Potato Starch 2.0 gms


Peptone 5.0 gms
Beef Extract 3.0 gms
Agar - Agar 20.0 gms
Distilled Water 1000 ml
Dissolve the Nutrient agar powder in the water by heating. Dissolve the starch in
10ml distilled water and add to the molten agar.
Lugols Iodine

Iodine 5.0 gms


Potassium Iodide 10.0 gms
Distilled water 500 ml

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s;<^ Chapter IV Oetection, Isolation and Characterization of the pathogen

Dissolve by stirring in a closed container for several hours complete dissolution.

Media - F

Gelatin Medium

Yeast Extract 3.0gms


Peptone 5.0 gms
Gelatin 120.0gms
Distilled water 1000 ml
Allow the solids to stand in the water for 15 minutes and dissolve by heating.

Adjust pH to approximately 7.0, if necessary. Dispense into test tube to a depth of

approximately 5cm. Sterilized by autoclave at 121 C for 15 minutes.

Media - G

Nitrate Semi-solid medium

Peptone lO.Ogms
Sodium Chloride 5.0gms
Potassium Nitrate 2.0gms
Agar 3.0gms
Distilled Water 1000 ml
Adjust the pH 7.0 with 20% NaOH. Dissolve and dispense in a test tube.
Sterilized by autoclave at 121 C for 15 minutes.

Solution 1
Starch Iodide Solution
A Starch 0.4gms
Zinc Chloride 2.0gms
Distilled Water 100 ml
Dissolve the Zinc Chloride in 10 ml water. Boil and add the starch, dilute to 100
ml. Allow to stand for 1 week and filter.
B Potassium Iodide 0.2%

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sAswKis Chapter IV Detection, Isolation and Characterization of the pathogen

Mix A and B in equal volumes.

Solution 2

Hydrochloric acid Solution

Hydrochloric Acid (Concentrated) 16ml


Distilled water 86 ml.

Media - H

Levan Formation (Leiliot and Stead, 1987)

Sucrose Nutrient Agar Medium (SNA)

Peptone S.Ogms
Beef Extract 3.0gms
Agar 20.0gms
Sucrose SO.Ogms
Distilled Water 1000ml

Maintenance of Tobacco, Mirabilis and Tomato Plants

Tobacco {Nicotiana tobacum var. xanthi 'nc') Mirabilis jalapa and Tomato

Seedlings (S- 22, Pusa Ruby) were maintained in green house as well as at natural

condition in the experimental plots. These plants were used for hypersensitivity and

pathogenesity tests.

Washing and Sterilization

The plant materials like stem, leaf and seeds used for the test were subjected to

the process of washing and sterilization. The plant materials collected from the field

were washed thoroughly with running tap water to remove soil debris. They were surface

sterilized by dipping the plant materials into a beaker containing 100 ml of 1 % sodium

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mxsiffi^apterll^ Detection, Isolation and Characterization of the pathogen

hypo chlorite solution for 2 minutes. Then these materials were washed thoroughly in

sterile distilled water for five to six times to remove excess of surface sterilent. Washed

materials were blot dried and used for further assays.

Collection of Authentic Culture

Authentic culture of Corynebacterium michiganensis sub sp. michganensis was

obtained from Central Science Laboratory (NCPPB 2979) New Hutton, London. Thus

obtained culture was stored at 4C in a refrigerator in the department. Further the

pathogen was sub cultured on Yeast extract Dextrose Calcium Carbonate Agar (YDC)

medium for every two month to maintain virulence of the bacterial cell. This was used as

positive control for biochemical, hypersensitivity, pathogenesity and Elisa Test.

Collection of Infected Plant Materials and Seeds

Details of collection of infected plant materials and seed samples were listed in

the Table 3-5 and Table 19-27.

Detection of the Bacterium

All the plant materials and seed samples collected from field and markets during

the field survey were separately subjected for various detection methods.

Bacterial Ooze Test

The bacterium infected plant materials are known to produce milky ooze in the

liquid medium (Gross and Rudolph, 1987). In order to check this bacterial ooze test was

conducted. The suspected plant materials were cut into small pieces of two inches and

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Chapter IV Detection, Isolation and Characterization of the pathogen v

suspended into a test tube containing 10ml sterile distilled water and observed for the

milky ooze leaching from the cut end.

The leaf materials were also tested for the ooze. Five to six leaves were used for

this test. Washed, surface sterilized and blot dried leaf portion were cut into small bits on

a clean glass slide and a drop of sterile distilled water was added on to the slide and

observed under compound microscope for the streaming movement of bacteria from the

cut end of the leaf.

The seed materials collected from different sources were also tested for the

bacterial ooze. Approximately 400 seeds were suspended into 25 ml screw cap bottle

containing 10 ml of sterile distilled water, after surface sterilization and blotting.

Observations were made for the formation of bacterial ooze.

Bacterial ooze collected from all the plant parts were maintained separately as

stock solution and each stock solution was serially diluted up to 10"^. 50|il of the aliquot

from each dilution was separately streaked on to SCM (Medium C) with the help of

drigalski spatula. Four replicates were maintained. Plates were incubated at 22C for

about 48 hrs. in a BOD incubator (Servewell Instruments Inc., Bangalore) and

observation were made for the formation of bacterial colonies on the medium. Colony

forming units in all dilution were calculated using the formula: -

Number of colonies X Dilution X Dilution Factor X 2

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<m/fm&-^ Chapter H^'"-> --^^ Detection, Isolation and Characterization of the pathogen

Liquid Assay

One gram of stem was cut into one cm pieces and homogenized by adding 10ml

of sterile distilled water with the help of pestle and mortar under sterile condition. The

supernatant was decanted into a beaker and used as stock and serial dilution were made

up to 10"^. For seeds, one gram of each tomato seed sample were soaked in 10ml of

sterile distilled water in 25 ml screw cap bottle and agitated constantly using rotatory

shakers. Aliquot were drawn at an interval of 2, 6, 12, 36 and 48 hours. 50|al of aliquot

from stem homogenate, seed soaking, bacterial ooze collected were drawn from each

dilution and streaked onto the plates containing SCM with the help of drigalski spatula.

Four replicates were maintained for all the dilutions and plates were incubated at 22C in

a BOD incubator for 48 hours.

In another set seeds with and without surface sterilization were crushed separately

with the help of mortar and pestle in a aseptic condition. The seed powder was

suspended in 10 ml of sterile 20mM PBS and the supernatant was used as stock. Serial

dilutions were made up to 10" . 50|al of aliquot were placed on to the plates containing

SCM medium. Plates were examined for the appearance of bacterial colonies.

Direct Plating Method

Infected plant material like stem, leaf and seeds collected from different fields of

Kamataka during summer and winter season from various sources were subjected to

direct plating method. Surface sterilized, blot dried stem pieces and 400 seeds were

tested separately. Ten bits of stem portion and 25 seeds was placed equidistantly with the

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wmsmm Chapter IV >s^^-^^. Detection. Isolation and Characterization of the pathogen -ssmssms

help of sterile forceps. Four and eight replicates were maintained respectively for each

sample. The plates were kept under observation at 22^0 for 48 hours for the

development of bacterial colony.

Percent of seed infection was calculated using the formula

Percent of Number of Seed showed Pathogen X 100


Seed Infection ~ Total Number of Seeds plated

Identification and Maintenance of Isolate

Bacterial colonies developed around the infected plant materials and seed samples

separately isolated and maintained in the test tube containing YDC and SCM at 4C.

Thus obtained isolate were subjected to various tests for the identification of the target

pathogen. I

Identification of the Pathogen By ' ' '

Bacterial isolates were characterized based on their cultural, morphological,

biochemical, pathogenesity and hypersensitive reaction. For all these 24 to 48 hours

isolates cultured on YDC and SCM were used.

Cultural and Morphological Characters J'^^f^''Si-r.aari. SbaPK=>r^c h-.tt*

BIOCHEMICAL TESTS

Biochemical tests were performed as described by Mortenson (1992) using the

isolates of 48 hours grown on YDC and SCM and Xanothomonas axonopodis pv.

cyamopsidis used for the comparison along with the authentic culture characters. The

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Chapter IV befecfion. Isolation and Characterization of the pathogen

following are the various biochemical tests carried out during the confirmation of the

pathogen.

Grams Staining

On a clean glass slide, a uniformly spreaded thin film of bacteria was air dried and

fixed by flaming on the under side. The smear was flooded with crystal violet solution

for 1 min. and then washed with Lugol's iodine for 1 min. The smear was decolorized

with 95% ethyl alcohol for few seconds and then counter stained with safranine. The

slide was observed under microscope at lOOX magnification

KOH Solubility or Non - Gram Staining

On a clean glass slide, a drop of freshly prepared 3% Potassium hydroxide

solution was taken and a loop full of bacterium was mixed with the aid of toothpick and

the toothpick was raised few cm above the slide and observed for the development of

thread within ten seconds.

Starch Hydrolysis (Lelliot and Stead, 1987)

Plates containing starch medium (medium E) were streaked with 24 to 48 hours

pure colonies. Positive control was maintained along with the test sample at 30 C. The

plates were flooded with lugol's iodine solution after 24 hours to examine the hydrolysis

of starch.

Gelatin Liquifaction (Lelltiot and Stead, 1987)

Pure bacterial colonies were stab inoculated to the gelatin medium [medium F]

along with a un inoculated control. Observation was made after five days of inoculation

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;msmmf Chapter I\/>,-'-'- Detect/on, Isolation and Characterization of the pathogen

at 30 C. Keep the slants at 4C for thirty minutes and observe the liquefaction of the

medium

Nitrate Reduction (Fahy and Persley, 1983)

Nitrate semi solid medium (Medium G) was prepared and distributed in to the test

tubes at the rate of 5 ml per tube. The test tubes were sterilized and stab inoculated with

bacterium in duplicates and incubated for 5 days at 28 C. Care should be taken to avoid

shaking of tubes to prevent dissolution of oxygen, which inhibit the reaction. After the

incubation add 3 - 4 drops of starch iodine solution and Hydrochloric acid solution were

added and observed for the development of blue or black colour.

Lipase Activity

24 to 48 hours pure colonies of bacteria were inoculated to the plate containing

Tween - 80 agar and observed for the development of milky white precipitation around

the colony in the period of seven days which explains the ability of the bacteria to

hydrolysis the lipid Tween - 80.

Kovac's Oxidase Test

Three to four drops for freshly prepared 1% aqueous solution of Tetra methyl

Para phenylene diamine dihydrochloride was placed on the Whatmann filter paper no. 1

and a loop full of bacteria grown on YDC medium was streaked on the moist filter paper

and change in the colour was observed with in 10 seconds.

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Chapter IV Oetection, Isolation and Characterization of the pathogen ^ NS*

Levan Formation

Bacterial colonies grown on YDC were streaked on to the medium H. The culture

were incubated at 27 C for three days and observed for the formation of levan sucrose.

Action on Litmus Milk

Litmus milk (4%) was prepared with distilled water and poured into tubes and

sterilized. The tubes were inoculated with pure bacterial colonies and incubated for 48

hours. An uninoculated tube serves as control. The tubes observed for change in the

colour from lavender to pink. Depending upon the colour development the reaction was

classified as alkaline or acidic

Production of Hydrogen Sulphate

Peptone was supplemented with .02% (W/V) yeast extract was prepared by

dissolving peptone and yeast extract in distilled water. The medium was dispensed into

test tube of 5ml and sterilized at 121 lbs. for twenty minutes. Duplicate test tubes were

inoculated with test isolate and a positive control.

Sterile whatmann filter paper strips impregnated with saturated lead acetate

solution were introduced into each of the test tube aseptically using sterile forceps in such

a way that the lower end of the strips was about 5mm above the surface of the medium.

Test tubes were incubated at 26 C for seven days and observed for blackening of

filter paper due to the formation of lead sulphide.

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Chapter IV ^ Detection, Isolation and Characterization of the pathogen

Catalase Test

One ml of 3% hydrogen peroxide was allowed to flow over the surface of 24 to 48 hour

slant culture of the test bacterium and observed for the production of gas bubble.

Caesin Hydrolysis Test

Sucrose Calcium medium (Davis et. al., 1980) supplemented with 1% (W/V)

Casein was prepared and sterilized at 121 lbs. For 20 min. and poured into a sterilized

Petri plates. Out of 4 sectors two sectors were inoculated by streaking the test bacterium

and remaining two were inoculated with positive control. The plates were incubated at

26 C for three days and observed for clearing of medium around the bacterial growth

Pathogenesity Test

Bacterial isolates positive for gram staining, biochemical test and showing

coryneform rods were tested for pathogenesity following the method of Bolkan (1987)

Isolates sub-cultured on NBY broth and incubated at 26C for 24 hours on

shakers. The turbid culture was concentrated by centrifugation at low speed for five

minutes. The pellets thus obtained was washed with sterile distilled water and adjusted to

approximately 10^ CFU/ml by using spectrophotometer (.04 OD at 600nm)

Tomato seedlings were raised in sterile soil for 25 to 30 days up to 3-4 leaf stages.

Then the stem of the seedling were cut 5-10 cm above the cotyledon leaf using sterile

blade with the help of syringe a drop of bacterial suspension was placed on the excised

stem. In the same manner one of the seedlings was inoculated with sterile distilled water

and labeled as control. The pots were covered with polypropylene bag for 24 hours and

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Chapter IV detection. Isolation and Characterization of the pathogen

incubated under green house condition. Seedlings were observed for wilting of cotyledon

for 4 - 5 days.

Similarly young tomato seedlings of 4 to 6 week were also inoculated with the

help of sterile blade. Seedlings inoculated with sterile water serves as control. The

seedlings were incubated at 25 to 30C in a green house and observed for the wilting

symptoms up to one month

Host Plant Inoculation Test

48 hours old cultures grown on YDC/SCM were inoculated to one-month-old

susceptible Pusa Ruby tomato seedling with sterile syringe. For this root tip inoculation

was performed which is as follows; Tertiary roots of one-month-old tomato seedlings

were trimmed with sterile scissors and were dipped in a beaker containing 10^ CFU/ml

for one hour. Seedlings dipped sterile distilled water served as control. After root dip

inoculation seedlings were transplanted to the plot containing sterile soil and farmyard

manure in the ratio 3:1. They were maintained at 30 C and observed for the typical

symptoms. Pathogen were further re-isolated from symptom expressing plant and

confirmed the identity of the pathogen on specific media. Persistence of virulence of the

pathogen was determined by inoculating the re-isolated pathogen to susceptible tomato

seedlings.

Hypersensitivity Test

An expanded leaf of one-month old tobacco plant {Nicotiana tobaccum var.

xanthi 'nc') was hypodermically injected with bacterial suspension of 10 CFU/ml at

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sxsmsssx Chapter IV Detection, Isolation and Characterization of the pathogen

different sectors of the leaf Sterile water was also injected to the opposite sectors of the

same leaf serves as control. The plant was maintained at 33 C before inoculation to

speedup the reaction and subsequently maintained at 28 C after inoculation and observed

for the collapse and necrosis of the infiltrated tissue up to 48 to 72 hours.

Similarly a [eaf of Mirabilis jalapa (4 'O' clock plant) was also infiltrated with

the bacterial suspension and sterile water serves as control. Plants were incubated at 28

C for 48 hours and observed for the development of confluent necrosis in the infiltrated

region.

Enzyme Linked Immunosorbent Assay

Bacterium was detected by using polyclonal antibody raised against host specific

Cmm by conducting ELISA Test.

RESULTS

Authentic culture of Cmm NCPPB 2979 was received from Central Science

Laboratory, London. The obtained culture was orange-yellow mucoid, erumpent on

YDC and Blackish grey fluidal colony with internal flecks on SCM medium.

Detection of bacterium from infected plant materials and seeds

Collected plants showing typical wilting symptoms like marginal necrosis and

symptoms spreads from older leaf to the younger one. The plant parts such as stem and

leaves along with seeds subjected to different detection method revealed the following

results.

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Chapter IV Detection, Isolation and Characterization of the pathogen

Bacterial Ooze Test

A portion of the stem was examined for the bacterial ooze, soon after dipping the

stem in distilled water streaming movement of milky white bacterial ooze coming out

from the cut end. Similarly, leaf bits on the slide with a drop of water observed under

microscope showed streaming movement of bacteria from the cut end. Under

microscope the bacteria were rod shaped, oftenly with angular arrangement and non-

motile.

Table-12, shows the recovery of the pathogen on SCM. It is evident that the ooze

collected from the stem yielded more colonies. In leaf the numbers of colonies are less

when compared to the stem.

Liquid Assay Method

The aliquots collected, when plated on to the YDC and SCM medium showed

varied number of colonies with different morphology. In infected plant parts large

number of colonies were observed on YDC and indicating the presence of large number

of different colonies including Xanthomonas and SCM medium allowed to grow only the

Cmm with typical blackish gray colonies, whereas other bacterial colonies grew as white

colonies after four to five days of incubation. As SCM produces typical colonies

suggests that it is an ideal semi-selective medium for the detection of Cmm.

Seeds without sterilization showed slightly high number of colonies then

unsterilized seeds. The results suggests that infected tomato seeds may contaminated

with saprophytes, which can be effectively removed by surface sterilization and as the

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Chapter IV Detection, Isolation and Characterization of the pathogen

mode of infection is systemic some of the colonies are also present on the surface of the

seed coat and these colonies are also washed out during surface sterilization (Table - 13).

Direct Plating Method

Infected stem segments plated on to the semi selective medium gave positive

result with the formation of fluidal blackish gray colonies around the stem. On YDC

medium also produces Orange-yellow colonies but the other bacterium like Xanthomonas

and other saprophytes also grew on this medium.

Similarly, infected seeds plated on the same medium were also found to be

surrounded with similar type of colonies. Out of 1108 seed samples screened 474 seed

samples representing Davanagere, Chikkamagalur, Hassan, Mysore, Mandya,

Chamarajanagara, Bangalore and Kolar districts were found to be positive for direct

plating with varied degree of infection (Table - 14). Pusa Ruby, S-22, Madanapalli, S-

75, V-44 showed maximum incidence ranging from 1.5 -70% incidence. Table- 16,

summarizes the positivity of pathogen in different seed health testing methods.

Enzyme Linked Immunosorbent Assay (ELISA)

Bacterium isolated from infected stem, leaf and seeds were subjected for the

detection of the pathogen using Polyclonal Antibody raised against Cmm (Pah-Cmm).

The pathogen was readily detected by the Antibody. Figure - 04 explains the detection

of the pathogen by PAb -Cmm showing clear dose dependency.

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:;* * Chapter IV Detection, Isolation and Characterization of the pathogen

Identification and Maintenance of Isolate

The bacterial colonies isolated on the medium and confirmed by ELISA and

biochemical tests were identified as Cmm was supported by literature and seven different

isolates were obtained from infected plant parts and seeds materials and are maintained in

the departmental laboratory.

Identiflcation of the pathogen by

Cultural and Morphological Characters

The bacterium showed different colony morphology on four different media (Table- 17)

Gram Staining

The isolates sub cultured on YDC agar media showed the presence of Gram

positive, straight, oftenly curved rods of varying sizes. Some in pairs showing angular or

V- Shaped arrangement (Plate - 3, Fig. 1).

KOH Solubility test or Non-Gram staining

The isolates did not produce viscid strand when mixed with freshly prepared 3%

KOH solution. Hence they are negative for KOH solubility, i.e., they are gram positive.

BIOCHEMCIAL TESTS

Starch Hydrolysis

There was no formation of clear zone around the colonies of the test bacterium

when the culture was flooded with Lugol's Iodine. But there was clear zone just beneath

the colony. Hence, the isolate was weakly positive (Plate - 1, Fig. - 2 ).

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m Chapter IV defection. Isolation and Characterization of the pathogen

Gelatin Liquefaction

After incubation tlie medium in the uninoculated tube and in the tube inoculated

with the test bacterium solidified on holding the tube at 4C for 30 minutes indicating that

the isolates were negative for gelatin hydrolysis. Where as tube inoculated with

Xanothomonas axonopodis pv. cyamopsidis failed to solidify (Plate - 1, Fig. - 3).

Nitrate Reduction Test

There was no change in colour observed in the medium inoculated with the test

isolate and in control tube on addition of solution one and two. Colour of the medium

changed to blue on addition of Zinc Powder. Hence, the bacterium was considered

negative for nitrite production from nitrate.

Lipase Activity

The bacterium showed poor growth on the test medium and there was no

formation of white precipitate around the colonies. Hence, it negative for lipase or

esterase activity ((Plate - 3, Fig. - 4).

Levan Formation

The plates inoculated with the test bacterium showed the absence of white

doomed colonies. The bacterium grew as yellow, convex, circular colony ((Plate - 3,

Fig. - 5).

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Chapter IV Oetection, Isolation and Characterization of the pathogen

Kovac's Oxidase

The colour of the bacterial culture remained unchanged even after ten seconds of

rubbing on the filter paper impregnated with the Kovac's reagents or it failed to

formation of indole. Hence, it is oxidase negative (Plate - 4, Fig. - 3).

Action on Litmus Milk

The colour of the litmus milk inoculated with test bacterium gradually changes

from purple to pink indicating an acidic reaction ((Plate - 4, Fig. - I ).

Production of Hydrogen Sulphide

Blackening of Lead Acetate impregnated filter paper strips was seen the tubes

inoculated with test bacterium due to the production of hydrogen sulphide. No change in

the colour was observed in the control tube.

Catalase Activity

Formation of gas bubble was observed over the slant culture of the test bacterium.

Hence, the isolate were considered catalase positive.

Casein Hydrolysis

There was no formation of clear zone in sector streaked with the test bacterium.

Hence, the isolate was considered negative for casein hydrolysis. Clear zones were

formed around the Xanothomonas axonopodis pv. cyamopsidis ((Plate - 4, Fig. - 2 ).

Hypersensitivity Test

48 hours bacterial isolate inoculated to the Tobacco plant (Nicotiana tobacum var

xanthi 'nc') showed localized death of the cells at the region of infiltration after 24 hours.

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Chapter IV Detection, Isolation and Characterization of the pathogen

Similarly Mirabilis leaf also showed similar results after 24 hours. No symptoms was

expressed on the leaf sector inoculated with sterile saline (Plate - 5, Fig.-l).

Host Plant Inoculation Test

S-22 tomato seedlings inoculated with the bacterial suspension at ~10 cells/ml

showed wilting symptoms after four days of inoculation. The seedlings showed marginal

wilt of leaf let on unilateral axis. These once again plated on YDC and SCM media

revealed the development of bacterial colonies which are morphologically similar to that

of authentic culture (Plate - 7, Fig. - 2).

DISCUSSION

Detection, Isolation and identification of the pathogen is an important step in

diagnosis of any disease (Mortensen, 1992). Corynebacterium michiganensis subsp.

michiganensis a canker causing organism on tomato have been identified as quarantine

organism. For the detection and isolation of the pathogen classical methods are routinely

being used. Infected plant materials and seeds samples collected during field survey from

infected fields were used for the detection. The occurrence of the pathogen in tomato

seeds from Kamataka have been reported by Nedumaran and Vidyasekaran in 1982.

Present study, field survey also evidenced the presence of Cmm in tomato in different

regions of Kamataka nearly 15 to 40.62 percent.

Seed health testing methods of Bryan (1930), Mortensen (1992), Fatmi and

Schaad (1988) followed for the detection. The detection methods used were bacterial

ooze, direct plating, and liquid assay methods. Bacterial ooze test was found to be simple

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Chapter IV Detection, Isolation and Characterization of the pathogen

and rapid for the detection of bacteria. The infected stem and leaf showed bacterial ooze

coming out of the cut end showing positivity of infection by bacteria. Ooze collected

from stem and leaf separately serially diluted and plated onto the SCM medium. Plates

were incubated at 22C and showed typical blackish grey colonies with internal flecks.

Four different media incubated with test bacterium showed varied number of

colonies with different morphology. The morphological characters are compared (Table -

17). Colony morphology similar to authentic culture (NCPPB 2979) on SCM medium

were isolated and maintained as isolate.

Comparison of three different media for seed health testing indicated SCM

medium was best for the detection and isolation of the pathogen. It is observed that

Nutrient Broth Yeast Extract Agar medium and Yeast Extract Dextrose Calcium

Carbonate Agar medium supported the growth of target pathogen along with the other

saprophytes. On SCM the pathogen could be differentiated easily from the saprophytes

associated with the seeds. The colonies of Cmm was grey, speckeled, enlarging with time

and remained so even after prolonged incubation. Where as other bacterium darkened on

further incubation.

The pathogen either failed to grow or showed poor growth on Nutrient Broth

Agar medium. But it grew fairly on Nutrient Agar medium supplemented with Yeast

extract and glucose. The result are contradictory to those of Nedumaran and

Vidyasekaran (1982), who reported that they have isolated pathogen by direct plating

method on nutrient agar medium.

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^N &
;^^^^^ Chapter IV Detection, Isolation and Characterization of the pathogen

Direct plating and liquid assay methods detected the pathogen present in the

infected seeds and plant materials. And it showed that the bacterium infected seeds has

to be soaked for a minimum of 36 hours for leaching and enrichment of the medium for

the detection of the pathogen by plating on the medium as well as by ELISA.

Screened results of different seed samples showed the presence of Cmm only in

474 samples out of 1108 samples collected from different agro climatic conditions. Even

a single infected seed lots can contaminate a commercial nursery seedlings, which could

became a major source of infestation and spread to other places. Percent of seed infection

in the seed samples was found to be two to eighty percent. By considering soil borne

nature, high rate of seed transmission and ability of the pathogen to spread secondarily

even low percentage of infection can cause considerably.

Isolation and Characterization of the pathogen was carried out by following

procedure of Vidaver and Davis (1984). Results of pathogenesity test showed the similar

results with the Gitaits (1990). Schaad (1982) and Alarcon (1988)

Biochemical tests (Mortensen, 1992) performed (Table 18) along with the

authentic culture and Xanthomonas axonopodis pv. cyamopsidis and results of both

authentic and test isolates was similar to that of cited in the Bergey's manual of Systemic

Bacteriology.

Hyper sensitivity test were conducted by injecting the isolate to different sectors

of one month old Tobacco plant and Mirabilis jalapa, showed response after 48 hours

and 4 days respectively.

75
: JSJ*S Chapter IV Detection. Isolation and Characterization of the pathogen *

Pathogenesity test on tomato seedlings showed that one-month seedling

inoculated with bacterium by stem inoculation method developed wilting symptoms in

the cotyledons within seven to eight days, where as older plants requires five to six weeks

and these results agree with the results of Chang et. al.. 1992.

Hybrid seeds of tomato being imported constantly from other countries like USA,

where the pathogen has been a major constraint in tomato production. More over Cmm is

an EPPO A2 Quarantine organism.

The classical methods of detection and isolation of the bacterium is of more time

consuming and laborious. There is a need to develop rapid and easy detection technique

like Serological and molecular methods. New technologies have been developed which

may assist in the detection and identification of seedbome bacterial diseases. These

technologies include the use of antibody-based assays (IFAS, IFC, Affini-tips), DNA

based assays (PCR, real-time PCR, BIO-PCR) and a combination of the two (IMS-PCR).

The strengths of these assays are their ability to quickly screen out negative tomato lots

(Van Buren, 2006). Development of indigenous antibody (Polyclonal or Monoclonal)

against the pathogen will lead to easy, rapid and quick detection (chapter III).

76
Table -12: Detection of the pathogen from bacterial ooze on SCM medium at
different dilution

Ooze Colony forming units at various


Collected dilutions Colony forming units
from 10' 10^ 10"^ 10" 10-* 10" At 10 ^Dilution

Stem 2500 1400 500 121 50 21 28 X 10"'


Leaf 900 124 1 79 42 20 09 24 X 10"'

Table - 13: Detection of the pathogen from infected field samples by Liquid Assay
Method

Colony for ming units


SI.
Material Tested at 10 ^ di utions on
No.
YDC SCM
01 Stem Ooze 13 X 10"" 10 X 10""
02 Seed Extract 59 X 10"' 44 X 10"'
(Surface Sterilized)
03 Seed Extract 24 X 10"" 19x 10''
(Surface Unsterilized)

Table - 14: Detection of Cmm in infected tomato seeds by Direct Plating Method

No. of No. of
Percent
Seed seed
SI. of
Districts Place of Collection samples samples
No. Incidence
collected infected

Channapatna
Devanahalli
Doddaballapura
Bangalore 126 58 1.5- 12
01 Nelamangala
Ramanagara
Anekal
02 Belgam
Belgam 42 02 1.5-2
Athani
03 Chamarajanagara
Chamarajanagara Gundlupet 114 53 2-65
Kollegala
04 Chikkamagalore
Sakharayapatna
Chikkmagalore 127 62 2-57
Chikkagauja
Kadur
05 Davanagere
Honnali
Davanagere 87 44 1.5-23
Nyamathi

77
05 Davanagere
Honnali
Davanagere 87 44 1.5-23
Nyamathi

06 Dharwad
Dharwad Hubli 32 00 00
Shirahatti
07 Arasikere
Belur
Hassan Hassan 115 55 1.8-51
Holenarasipura
Channarayapatna
08 Bagepalli
Chikkaballapura
Kolara 93 43 1.5-18
Chintamani
Shidlaghatta
09 Mandya
Krishnarajapet
Mandya Malavalli 121 58 2-57
Pandavapura
Nelamangala
10 Mysore
Jyapura (H.D. Kote)
Mysore 132 88 3-70
Daripura (H.D. Kote)
Krishnarajasagara
11 Shimoga
Shimoga 39 00 00
Shikaripura
12 Chikkanayakanahalli
Kunigal
Tumkur Tiptur 80 11 1.8-15
Tumkur

Table - 15: Colony count at different time of incubation

Time in Colony count at 10''' Dilutions


Hours NBY YDC SCM
0 Nil Nil Nil
2 Nil Nil Nil
6 Nil Nil Nil
12 70 X 10' 88 X 10' 42 X 10'
24 85 X lO"" 90 X 10' 51 X 10'
36 321 X 10' 400 X 10' 93 X 10'

78
Table -16: Detection of Cmm from infected seeds by different Seed Health Testing
Methods

Seed Health Testing Methods


SI.
Cultivars Direct Liquid Seed ELISA
No.
Plating Assay Extract Reactivity
1. S-22 4- + + +
2. Abhinav - - - -
3. PED - - - -
4. LIHB + -t- + +
5. Madanapalli + + + +
6. Punjab Chhohara + + + +
7. Local -i- + + +
8. S-75 + + + +
9. PKM + -t- + +
10. Nandi + - + +
11. Surya + + + 4-
12. Arka Ashish + - - +
13. POC-96-2-G-7 - - - -

Table - 17: Colony Characters on different Media

Medium Form Elevation Margin Colour Nature


NA ~
NBY Circular Convex Entire Yellow Mucoid
YDC Circular Convex Entire Orange yellow Mucoid
SCM Irregular Convex Entire Blackish gray Fluidat
GF Circular Convex Entire White Mucoid

Table - 18: shows comparison of Cmm and Xac for various Biochemical Tests

X.axanopodis
SI. Test Authentic
Tests pv.
No. Isolate Culture
cyamopsidis
01 Starch Hydrolysis - ve
02 Gelatin Liquifaction - ve
03 Nitrate Reduction - ve - ve +ve
04 Lipase/Esterase - ve - ve +ve
05 Levan Formation - ve - ve +ve
06 Kovac's Oxidase - ve - ve +ve
07 Production of H2S - ve - ve +ve
08 Catalase Activity + ve +ve +ve
09 Caesin Hydrolysis - ve - ve +ve
10 Action on Litmus Milk Acidic Acidic Acidic
Reaction Reaction Reaction

79
Table - 19: Details of seed samples collection in different agro-climatic regions of
Karnataka state during 2002 - 03

SI. Place of No. of Seeds Variety/ Cultivar Source


NO. Collection Collected
Kharif Rabi
Local, S-22, PKM, PR, Farmers,
Pusa Ruby, Nandi TLB Retail Shop,
130, Madanapalli, Commercial
01 Bangalore 24 18
Marbglobe, PED, POC- Market
96-2-G-7 , Abhinav,
Avinash, Arka Vikas
Local, S-22, PKM, Pusa Farmers
Ruby, Madanapalli, Retail Shop
02 Belgam 05 06
Marbglobe, Abhinav, Commercial
Arka Vikas Market
Local, S-22, PKM, Pusa Farmers
Ruby, Madanapalli, Retail Shop
03 Chamarajanagara 20 19
Nandi , Arka Vikas Commercial
Market
Local, S-22, Farmers
Madanapalli, Retail Shop
04 Chikkamagalore 22 20
Abhinav,PKM Commercial
Market
Local, Nandi TLB 130, Farmers
S-22, Arka Vikas Retail Shop
05 Davanagere 19 13
Commercial
Market
Local, S-22, PKM, PR, Farmers
Abhinav. Retail Shop
06 Dharwad 05 07
Commercial
Market
Local, S-22, Pusa Ruby, Farmers
Abhinav Retail Shop
07 Hassana 25 18
Commercial
Market
S-22, Local, PKM Farmers
(Local), Retail Shop
08 Kolara 19 11
Commercial
Market
Local, S-75, S-22, PKM, Farmers
PR, Pusa Ruby, Retail Shop
09 Mandya 22 17
Madanapalli, Nandi TLB Commercial
130, Market
Local, S-75, S-22, PKM, Farmers
PR, Pusa Ruby, Retail Shop
10 Mysore 22 23
Madanapalli, Nandi TLB Commercial
130, LIHB, lAHNaveen Market

80
Farmers
Retail Shop
Local, S-22, Nandi TLB Commercial
11 Shimoga j 05 08
130.PKM, PR. Market
Local, S-22, Nandi TLB Farmers
130, PKM, PR Retail Shop
12 Tumkur 21 13
Commercial
Market
Table - 20: Details of seed samples collection in different agro-climatic regions of
Karnataka state during 2003 - 04

SI. Place of No. of Seeds Variety/ Cultivar Source


No. Collection Collected
Kharif Rabi
01 Local, S-22, PKM, PR, Farmers,
Pusa Ruby, Nandi TLB Retail Shop,
130, Madanapalli, Commercial
Bangalore 19 23
Marbglobe, PED, POC- Market
96-2-G-7, Abhinav,
Avinash, Arka Vikas
02 Local, S-22, PKM, Pusa Farmers
Ruby, Madanapalli, Retail Shop
Belgam 05 10
Marbglobe, Abhinav, Commercial
Arka Vikas Market
03 Local, S-22, PKM, Pusa Farmers
Ruby, Madanapalli, Retail Shop
Chamarajanagara 20 16
Nandi, Arka Vikas Commercial
Market
04 Local, S-22, Farmers
Madanapalli, Retail Shop
Chikkamagaiore 26 21
Abhinav,PKM Commercial
Market
Farmers
Local, Nandi TLB 130, Retail Shop
05 Davanagere 16 10
S-22, Arka Vikas Commercial
Market
Farmers
Local, S-22, PKM, PR, Retail Shop
06 Dharwad 05 07
Abhinav. Commercial
Market
Farmers
Local, S-22, Pusa Ruby, Retail Shop
07 Hassana 21 13
Abiiinav Commercial
Market
Farmers
S-22, Local, PKM Retail Shop
08 Kolara 18 18
(Local), Commercial
Market
09 Local, S-75, S-22, PKM, Farmers
PR, Pusa Ruby, Retail Shop
Mandya 25 17
Madanapalli, Nandi TLB Commercial
130, Market
10 Local, S-75, S-22, PKM, Farmers
PR, Pusa Ruby, Retail Shop
Mysore 22 16
Madanapalli, Nandi TLB Commercial
130, LIHB, lAHNaveen Market

82
Farmers
Local. S-22, Nandi TLB Retail Shop
Shimoga 09 08
11 130, PKM, PR Commercial
Market
Farmers
Local, S-22, Nandi TLB Retail Shop
Tumkur 12 08
12 130, PKM, PR Commercial
Market

83
Table - 21: Details of seed samples collection in different agro-climatic regions of
Karnataka state during 2004 - 05
No. of Seeds
SI. Place of
Collected Variety/ Cultivar Source
No. Collection
Kharif Rabi
Local, S-22, PKM, PR, Farmers,
Pusa Ruby, Nandi TLB Retail Shop,
130, Madanapalli, Commercial
01 Bangalore 19 23
Marbglobe, PED, POC- Market
96-2-G-7, Abhinav,
Avinash, Arka Vikas
Local, S-22, PKM, Pusa Farmers
Ruby, Madanapalli, Retail Shop
02 Belgam 06 10
Marbglobe, Abhinav, Commercial
Arka Vikas Market
Local, S-22, PKM, Pusa Farmers
Ruby, Madanapalli, Retail Shop
03 Chamarajanagara 21 18
Nandi, Arka Vikas Commercial
Market
Local, S-22, Farmers
Madanapalli, Retail Shop
04 Chikkamagaiore 22 16
Abhinav,PKM Commercial
Market
Local, Nandi TLB 130, Farmers
S-22, Arka Vikas Retail Shop
05 Davanagere 19 10
Commercial
Market
Local, S-22, PKM, PR, Farmers
Abhinav. Retail Shop
06 Dharwad 03 05
Commercial
Market
Local, S-22, Pusa Ruby, Farmers
Abhinav Retail Shop
07 Hassana 25 13
Commercial
Market
S-22, Local, PKM Farmers
(Local), Retail Shop
08 Kolara 10 17
Commercial
Market
Local, S-75, S-22, PKM, Farmers
PR, Pusa Ruby, Retail Shop
09 Mandya 21 19
Madanapalli, Nandi TLB Commercial
130, Market
Local, S-75, S-22, PKM, Farmers
PR, Pusa Ruby, Retail Shop
10 Mysore 29 20
Madanapalli, Nandi TLB Commercial
130, LIHB, lAHNaveen Market

84
Local, S-22, Nandi TLB Farmers
130, PKM, PR Retail Shop
11 Shimoga 05 04
Commercial
Market
Farmers
Local, S-22, Nandi TLB Retail Shop
12 Tumkur 15 11
130, PKM, PR Commercial
Market

85
Table - 22: Details of seed samples collection in different districts in Karnataka
State during Kharif season of 2002 - 03

No. of Percentage of
SI. Place of Collection Districts samples incidence
No.
collected [%1
01 Channapatna L^angalore 02 -Nil-
02 Devanahalli Bangalore 02 -Nii-
03 Doddabaliapura Bangalore 10 1.5-04
04 Nelamangala Bangalore 06 2 - 10
05 Ramanagara Bangalore 02 -Nil-
06 Anekal Bangalore 02 -Nil-
07 Athani Belgam 02 -Nil-
08 Belgam Belgam 03 1.5-2
09 Chamarajanagara Chamarajanagara 09 2-23
10 Gundlupet Chamarajanagara 04 -Nil-
11 Koilegala Chamarajanagara 07 8-65
12 Chikkamagaiore Chikkamagaiore 06 02-18
13 Sakharayapatna Chikkamagaiore 12 12-79
14 Chikkagauja Chikkamagaiore 02 04-17
15 Kadur Chikkamagaiore 02 2-8
16 Davanagere Davanagere 06 5-20
17 Honnali Davanagere 05 -Nil-
18 Nyamathi Davanagere 08 4 - 12
19 Dharwad Dharwad 01 -Nil-
20 Hubli Dharwad 02 -Nil-
21 Shirahatti Dharwad 02 -Nil-
22 Arasikere Hassana 04 -Nil-
23 Belur Hassana 09 7-79
24 Hassana Hassana 08 3-15
25 Holenarasipura Hassana 02 -Nil-
26 Channarayapatna Hassana 02 2.5-4
27 Bagepaili Kolara 03 -Nil-
28 Chikkaballapura Kolara 05 2-12
29 Chintamani Kolara 07 2.8-15
30 Shidlagatta Kolara 04 -Nil-
31 Mandya Mandya 04 3-18.5
32 Krishnarajapet Mandya 03 -Nil-
34 Malavalli Mandya 09 02-12.3
35 Pandavapura Mandya 02 -Nil-
36 Nagamangala Mandya 04 11-42
37 Mysore Mysore 04 3-26
38 Jayapura/H.D. Kote Mysore 08 8-70
39 Daripura/H.D. Kote Mysore 09 4.7-65
40 Krishnarajasagara Mysore 01 - Nil -
41 Shimoga Shimoga 02 -Nil-
42 Shikaripura Shimoga 03 -Nil-
43 Chikkanayakanahal 1 i Tumkur 02 -Nil-
44 Kunigal Tumkur 05 -Nil-
45 Tiptur Tumkur 06 -Mil-
46 Tumkur Tumkur 08 1.8-6
86
Table - 23: Details of seed samples collection in different districts in Karnataka state
during Rabi season 2002 - 03
No. of
SI. Percentage
Place of Collection Districts samples
No. of Incidence
collected
01 Channapatna Bangalore 02 -Nil-
02 Devanahalli Bangalore 02 1.9-3.4
03 Doddaballapura Bangalore 09 5-9
04 Nelamangala Bangalore 02 3.2-6
05 Ramanagara Bangalore 02 -Nil-
06 Anekal Bangalore 01 -Nil-
07 Athani Belgam 02 -Nil-
08 Belgam Belgam 04 -Nil-
09 Chainarajanagara Chamarajanagara 05 12-19
10 Gundlupet Chamarajanagara 08 -Nil-
11 Kollegala Chamarajanagara 06 2-16
12 Chikkamagalore Chikkamagalore 05 04-12
13 Sakharayapatna Chikkamagalore 09 21 - 4 8
14 Chikkagauja Chikkamagalore 04 2-8
15 Kadur Chikkamagalore 02 3-7
16 Davanagere Davanagere 02 3-11
17 Honnali Davanagere 04 1.7-6.8
18 Nyamathi Davanagere 07 8 -17.6
19 Dharwad Dharwad 02 -Nil-
20 Hubli Dharwad 04 -Nil-
21 Shirahatti Dharwad 01 -Nil-
22 Arasikere Hassana 03 -Nil-
23 Beiur Hassana 08 17-32
24 Hassana Hassana 03 13-24
25 Holenarasipura Hassana 02 -Nil-
26 Channarayapatna Hassana 02 2-7.1
27 Bagepalli Kolara 02 1.5
28 Chikkaballapura Kolara 03 2-12
29 Chintamani Kolara 04 2-6
30 Shidlagatta Kolara 02 -Nil-
31 Mandya Mandya 03 13-18
32 Krishnarajapet Mandya 03 -Nil-
34 Malavalli Mandya 06 2-8.3
35 Pandavapura Mandya 01 -Nil-
36 Nagamangala Mandya 04 5-21
37 Mysore Mysore 05 7.5-19
38 Jayapura/H.D. Kote Mysore 07 15-62
39 Daripura/H.D. Kote Mysore 08 11 -54.3
40 Krishnarajasagara Mysore 03 -Nil-
41 Shimoga Shimoga 03 -Nil-
42 Shikaripura Shimoga 05 -Nil-
43 Chikkanayakanahal 1 i Tumkur 02 -Nil-
44 Kunigal Tumkur 05 -Nil-
45 Tiptur Tumkur 03 1.9-3
46 Tumkur Tumkur 03 -Nil-

87
Table - 24: Details of seed samples collection in different districts in Karnataka state
during Kharif season 2003-04

No. of
SI. Percentage
Place of Collection Districts samples
No. of Incidence
collected
01 Channapatna Bangalore 02 -Nil-
02 Devanahaili Bangalore 02 1.5-3
03 Doddaballapura Bangalore 06 2.5-6
04 Nelamangala Bangalore 05 2.9 - 8
05 Ramanagara Bangalore 03 -Nil-
06 Anekal Bangalore 01 -Nil-
07 Athani Belgam 02 -Nil-
08 Belgam Belgam 03 - Nu-
09 Chamarajanagara Chamarajanagara 07 ll - 15
10 Gundlupet Chamarajanagara 04 -Nil-
11 Kollegala Chamarajanagara 09 2.9-10
12 Chikkamagalore Chikkamagalore 08 4.7-10.2
13 Sakharayapatna Chikkamagalore 12 15-30
14 Chikkagauja Chikkamagalore 04 2.9-14
15 Kadur Chikkamagalore 02 -Nil-
16 Davanagere Davanagere 05 2-7
17 Honnali Davanagere 03 -Nil-
18 Nyamathi Davanagere 08 10 - 14
19 Dharwad Dharwad 01 -Nil-
20 Hubli Dharwad 03 -Nil-
21 Shirahatti Dharwad 01 -Nil-
22 Arasikere Hassana 05 -Nil-
23 Belur Hassana 10 13-43
24 Hassana Hassana 04 21 - 2 8
25 Holenarasipura Hassana 01 -Nil-
26 Channarayapatna Hassana 01 -Nil-
27 Bagepalli Kolara 05 -Nil-
28 Chikkaballapura Kolara 07 3-8
29 Chintamani Kolara 04 3.7-11
30 Shidlagatta Kolara 02 -Nil-
31 Mandya Mandya 04 4-21
32 Krishnarajapet Mandya 03 -Nil-
34 Malavalli Mandya 08 1 2 - 18
35 Pandavapura Mandya 03 3-7
36 Nagamangala Mandya 07 7 - 12
37 Mysore Mysore 06 15-29
38 Jayapura/H.D. Kote Mysore 07 45-59
39 Daripura/H.D. Kote Mysore 08 31 - 5 7
40 Krishnarajasagara Mysore 01 - Nil -
41 Shimoga Shimoga 03 -Nil-
42 Shikaripura Shimoga 06 -Nil-
43 Chikkanayakanahai 1 i Tumkur 01 -Nil-
44 Kunigal Tumkur 04 -Nil-
45 Tiptur Tumkur 03 -Nil-
46 Tumkur Tumkur 04 -Nil-
88
Table - 25: Details of seed samples collection in different districts in Karnataka state
during Rabi season 2003 - 04

SI. No. of
Place of Collection Percentag e
Districts samples
No. of Incidence
collected
01 Channapatna Bangalore 04 -Nil-
02 Devanahaili Bangalore 03 -Nil-
03 Doddaballapura Bangalore 07 5-9.6
04 Nelamangala Bangalore 07 9-11.8
05 Ramanagara Bangalore 01 -Nil-
06 Anekal Bangalore 01 -Nil-
07 Athani Belgam 04 -Nil-
08 Belgam Belgam 06 -Nil-
09 Chamarajanagara C hamaraj anagara 05 14-28
10 Gundlupet Chamarajanagara 05 -Nil-
11 KoUegala Chamaraj anagara 06 19-24
12 Chikkamagalore Chikkamagalore 02 19-43
13 Sakharayapatna Chikkamagalore 07 28-45
14 Chikkagauja Chikkamagalore 09 2-9
15 Kadur Chikkamagalore 03 -Nii-
16 Davanagere Davanagere 03 1.5-9.6
17 Honnali Davanagere 03 -Nil-
18 Nyamathi Davanagere 04 9-12
19 Dharwad Dharwad 04 -Nil-
20 Hubli Dharwad 02 -Nil-
21 Shirahatti Dharwad 01 -Nil-
22 Arasikere Hassana 01 -Nil-
23 Beiur Hassana 05 23-44.3
24 Hassana Hassana 04 11-22
25 Holenarasipura Hassana 01 -Nil-
26 Channarayapatna Hassana 02 -Nil-
27 Bagepalli Kolara 03 -Nil-
28 Chikkaballapura Kolara 06 11-18
29 Chintamani Kolara 06 3-8
30 Shidlagatta Kolara 03 -Nil-
31 Mandya Mandya 05 14-25
32 Krishnarajapet Mandya 03 -Nil-
34 Malavalli Mandya 08 13-21
35 Pandavapura Mandya 02 -Nil-
36 Nagamangala Mandya 04 17-26
37 Mysore Mysore 04 11-32
38 Jayapura/H.D. Kote Mysore 06 55-62
39 Daripura/H.D. Kote Mysore 05 21 - 4 3
40 Krishnarajasagara Mysore 01 -Nil-
41 Shimoga Shimoga 03 -Nil-
42 Shikaripura Shimoga 05 -Nil-
43 Chikkanayakanahalli Tumkur 01 -Nil-
44 Kunigal Tumkur 03 -Nil-
45 Tiptur Tumkur 03 -Nil-
46 Tumkur Tumkur 01 -Nil-

89
Table - 26: Details of seed samples collection in different districts in Karnataka state
during Kharif season of 2004 - 05

No. of
SI. Percentage
Place of Collection Districts samples
No. of Incidence
collected
01 Channapatna Bangalore 02 -Nil-
02 Devanahalli Bangalore 03 2.1 - 5
03 Doddaballapura Bangalore 05 9-12
04 Nelamangala Bangalore 07 4.9-7.5
05 Ramanagara Bangalore 01 -Nil-
06 Anekal Bangalore 01 -Nil-
07 Athani Belgam 03 -Nil-
08 Belgam Belgam 03 -Nil-
09 Chamarajanagara Chamarajanagara 09 22-32
10 Gundlupet Chamarajanagara 04 -Nil-
11 Kollegala Chamarajanagara 08 17-29
12 Chikkamagalore Chikkamagalore 06 29-49
13 Sakharayapatna Chikkamagalore 08 15-57
14 Chikkagauja Chikkamagalore 05 - Nil -
15 Kadur Chikkamagalore 03 3.7- 14
16 Davanagere Davanagere 04 5 - 16
17 Honnali Davanagere 07 -Nil-
18 Nyamathi Davanagere 08 19 - 2 1
19 Dharwad Dharwad 02 -Nil-
20 Hubli Dharwad 01 -Nil-
21 Shirahatti Dharwad 02 -Nil-
22 Arasikere Hassana 05 -Nil-
23 Belur Hassana 08 32-43
24 Hassana Hassana 07 15-29
25 Holenarasipura Hassana 03 -Nil-
26 Channarayapatna Hassana 03 -Nil-
27 Bagepalli Kolara 02 -Nil-
28 Chikkaballapura Kolara 04 08-14
29 Chintamani Kolara 03 3.2-6.9
30 Shidiagatta Kolara 01 -Nil-
31 Mandya Mandya 04 11-32
32 Krishnarajapet Mandya 02 -Nil-
34 Malavalli Mandya 07 14-57
35 Pandavapura Mandya 02 -Nil-
36 Nagamangala Mandya 05 7-16
37 Mysore Mysore 05 11-32
38 Jayapura/H.D. Kote Mysore 11 35-70
39 Daripura/H.D. Kote Mysore 10 11-34
40 Krishnarajasagara Mysore 03 -Nil-
41 Shimoga Shimoga 03 -Nil-
42 Shikaripura Shimoga 02 -Nil-
43 Chikkanayakanahalli Tumkur 02 -Nil-
44 Kuniga! Tumkur 03 -Nil-
45 Tiptur Tumkur 07 -Nil-
46 Tumkur Tumkur 05 -Nil-
90
Table - 27: Details of seed samples collection in different districts in Karnataka state
during Rabi season 2004 - 05

Si i No. of
-,' i Place of Collection Percentage
Districts samples
No 1 of Incidence
collected
01 Channapatna Bangalore 02 -Nil-
02 Devanahalli Bangalore 05 -Nil-
03 Doddaballapura Bangalore 08 2.9-7
04 Nelamangala Bangalore 06 4.3-11
05 Ramanagara Bangalore 01 -Nil-
06 Anekal Bangalore 01 -Nil-
07 i Athani Belgam 03 -Nil-
08 Bel gam Belgam 07 -Nil-
09 Chamarajanagara Chamarajanagara 05 5.1 - 3 2
10 Gundlupet Chamarajanagara 05 3-7
11 Kollegaia Chamarajanagara 08 -Nil-
12 Chikkamagalore Chikkamagalore 03 21 - 3 9
13 Sakharayapatna Chikkamagalore 06 09-47
14 Chikkagauja Chikkamagalore 05 -Nil-
15 Kadur Chikkamagalore 02 7.1
16 Davanagere Davanagere 02 1.9- 18
17 Honnali Davanagere 04 -Nil-
18 Nyamathi Davanagere 04 12-23
19 Dharwad Dharwad 03 -Nil-
20 Hubli Dharwad 03 -Nil-
21 Shirahatti Dharwad 02 -Nil-
22 Arasikere Hassana 02 1.8-32
23 Beiur Hassana 05 11-39
24 Hassana Hassana 03 17-51
25 Holenarasipura Hassana 01 -Nil-
26 Channarayapatna Hassana 02 -Nil-
27 Bagepalli Kolara 03 -Nil-
28 Chikkabaliapura Koiara 05 3.9-10
29 Chintamani Kolara 06 2.6-17
30 Shidlagatta Kolara 03 -Nil-
31 Mandya Mandya 04 12-31
32 Krishnarajapet Mandya 03 -Nil-
34 Malavalli Mandya 06 11.2-29
35 Pandavapura Mandya 02 -Nil-
36 Nagamangala Mandya 04 6.5-25.6
37 Mysore Mysore 04 13-45
38 Jayapura/H.D. Kote Mysore 09 37-70
39 Daripura/H.D. Kote Mysore 05 20-59
40 Krishnarajasagara Mysore 02 -Nil-
41 Shimoga Shimoga 01 -Nil-
42 Shikaripura Shimoga 03 -Nil-
43 Chi kkanayakanahal 1 i Tumkur 01 -Nil-
44 Kunigal Tumkur 05 6.8-15
45 Tiptur Tumkur 04 -Nil-
46 Tumkur Tumkur 01 -Nii-