Media And Culture Conditions

Published Date: 02 Nov 2017

Four Botrytis cinerea isolates were recovered from infected plants of chickpea, strawberry, rose and marigold. Mycelium suspected to be B. cinerea was identified by microscopic examination of spores and conidiophores. The isolates were characterized in terms of cultural, morphological and biochemical traits. Genetic identification among the isolates was also carried out by using B. cinerea specific primers.

3.2 Media and culture conditions

For sporulation and morphological observations all the four isolates were grown on Potato Dextrose Agar (PDA), Potato Carrot Agar (PCA) and Malt Extract Agar (MEA) in 9-cm plastic petri dishes. The isolates were also stored in 20 % glycerol at -20 ï‚°C for further use.

3.3 Morphological Characterization

Morphology of isolates of B. cinerea was ascertained in terms of conidial length, breadth and pattern of sclerotia on Potato Dextrose Agar (PDA), Potato Carrot Agar (PCA) and Malt Extract Agar (MEA) media.

3.3.1. Conidia

Conidia were sampled randomly from 14-d old colonies by flooding PDA, PCA and MEA plates with 10 ml of sterile water containing a drop of Tween 20 and gently rubbing the colony surface with a sterilized glass rod. Slides were prepared using lactophenol cotton blue.

3.3.2. Sclerotia pattern

Pattern of sclerotia and their arrangement were observed on PDA, MEA and PCA amended petri plates after 21 days of incubation in BOD incubator at 20 °C under dark conditions.

3.4 Cultural Characterization

Growth pattern of different isolates of B. cinerea was investigated on following growth media: Potato Dextrose Agar (PDA), Malt Extract Agar (MEA) and Potato Carrot Agar (PCA)

A 5 mm stub of an actively growing culture of each isolate was inoculated in the centre of a 90 mm diameter petri-plate containing desired media and incubated at 20 °C in BOD incubator for 10 days. Cultural features of B. cinerea isolates were observed for colony colour, colony type, diameter, margin and texture.

3.4.1. Determination of fungal spore density

The spore density was calculated by counting B. cinerea spores in a haemocytometer. It had a depth of 0.1 mm. The single large square was subdivided into 16 smaller squares, which were further divided into 16 mini squares each (0.0025mm2). The external supports were moistened with distilled water and the cover glass was pushed gently onto the counting chamber from the front. The formation of interference lines (Newton rings) between the external support and the cover glass showed that the cover glass was correctly positioned. A drop of a diluted spore suspension was placed into the counting chamber and covered by cover glass. The counting chamber was placed under a light microscope, an appropriate magnification was selected and spores were counted in 4 small squares. Spores crossing the factor border of a square were counted on two sides only for each square. The spore concentration was calculated as:-

Spores per ml = total spore count in 4 small squares X 2500 X dilution.

3.5 Biochemical Characterization

Spectrophotometric quantification of oxalic acid, pectinases (polygalacturonase and pectin methyl esterase) was performed on B. cinerea isolates.

3.5.1. Quantification of Oxalic acid

B. cinerea isolates were grown in 100 ml flasks containing 15 ml of potato dextrose broth (PDB). Medium was autoclaved at 121 oC for 15 min. Flasks were statistically incubated for 10 days at room temperature. Cultures were vacuum filtered and the supernatant was used for oxalic acid estimation. Oxalic acid was determined in the culture filtrate following Xu and Zhang (2000) with few modifications. Reaction mix contained 0.2 ml of culture filtrate (or standard oxalic acid solution), 0.11 ml of bromophenol blue (BPB, 1 mM), 0.198 ml of sulfuric acid (1 M), 0.176 ml of potassium dichromate (100 mM) and 4.8 ml of distilled water. The reaction mix was placed in a water bath at 60 °C and quenched by adding 0.5 ml sodium hydroxide solution (0.75 M) after 10 min. The absorbance was measured at 600 nm by means of a spectrophotometer (UV-VIS, Shimadzu) and PDB was used as the blank control. The concentration of oxalic acid was calculated and was expressed as μg oxalic acid/ml of the culture filtrate. Assay was run in triplicate.

3.5.2. Quantification of hydrolytic enzymes

The activities of hydrolytic enzymes, polygalacturonase (PG) and pectin methyl esterase (PME) were quantified. Sterile minimal medium supplemented with pectin was used for the growth of isolates. Flasks were incubated in BOD incubator for ten days at 20 °C. After incubation, the mycelium was filtered through autoclaved muslin cloth and the supernatant was used as enzyme extract.

Polygalactouronase (PG)

Enzyme extract (0.8 ml) was added to two ml of assay buffer (2 mg/ml pectin in 0.1 M citrate buffer, pH 5.0) and test tubes were incubated at 37°C for 1 h. To 1 ml of reaction mixture, 5.5 ml of phenol sulphuric acid reagent (PSA; 0.5 ml 80% phenol in 5 ml sulphuric acid) was added. Absorbance was measured at 480 nm. One reducing group unit corresponded to 1 µM of reducing sugar liberated from substrate in one hour at 37 oC with a standard calibration curve obtained with galacturonic acid as reducing sugar.

Pectin methyl esterase (PME)

To 0.5 ml of enzyme extract, 7 ml of assay buffer (10 mg/ml pectin in 0.02 M Tris-HCl buffer, pH 8.0) was added. The pH of the reaction mixture was adjusted to 8.0 using 0.05 M NaOH. The reaction was then incubated at 37 oC in water bath for 1 h. After incubation the pH was checked and noted down as initial reading. Further, the pH was adjusted to 8.0 using titration against 0.02 N NaOH containing 5 mM sodium azide and noted down to bring back the initial pH of 8.0 as final reading. The amount of enzyme utilized was calculated as 1 µM/h NaOH to maintain the pH 8.0.

3.6. In vitro Pathogenicity Assay

In vitro pathogenicity assay was performed on chickpea leaves as described by Gourgues et al., (2004). Conidial suspension (1 x 105spores/ml) of all the isolates were deposited on the adaxial surface. The petri-plates were incubated at 22 oC in a humid environment for four days.

3.7 Fungicide Assay

Different classes of Botrytis specific fungicides i.e. ‘botryticides’ were screened for their resistance against four isolates of B. cinerea. The fungicides used were of Sigma grade and were provided as wettable powder formulation. Stock solutions of fungicides were prepared in organic solvents and were maintained at -20 °C. All five fungicides were tested at varying concentrations ranging from 10 - 500 μg/ml in order to ascertain inhibitory doses of fungicides which ranged from 50 – 200 μg/ml. Thereby fungicide assay of isolates were performed at three concentrations of sampled botryticides: 50, 100 and 200 μg/ml. PDA plates were inoculated with isolates and were incubated at 20 °C in BOD incubator till they attain radial growth of approx. 2-3 cm. Plates were inoculated with sterile discs (HiMedia) impregnated with varying concentrations of fungicides ( 50, 100 and 200 μg/ml) by dipping for 2 minutes. One disc impregnated with sterile water was placed onto each plate which served as control. Plates were incubated at 20 °C for six days in BOD incubator and zone of inhibition was measured with measuring scale.

Table 6.1: Details of fungicides and organic solvents used for making of stock solutions

Fungicide

Stock solution(mg/ml)

Organic solvent