DIFFERENCES BETWEEN THREE PORZA HYPOUTERITlA ISOLATES IN SRI LANKA.

Three isolates of Pona hypolarcritia, the fungus causing the red root disease in the tea plant, were obtained from three climatically different areas of Sri Lanka and examined for differences in growth and secretion of enzymes involved in pathogenesis. Significant differences behveen the growth rates of the isolates were seen in many growth media. All isolates showed optimum growth at 25' C, but the optimum pH for growth differed among the isolates. All three isolates secreted a single form of polygalacturonase. Two of the isolates secreted three forms of B-glucosidase that differed in molecular weights while one.isolate secreted only two forms. The results show that significant differences exist among the three isolates of the fungus. a role in pathogenecity: examined. The isolates were maintained on potato dextrose agar at 4O during the period of study (about 3 months).


INTRODUCTION
Poria hypolateritia causes the red root disease of tea. Red root is a serious root disease of tea in Sri Lanka.and the disease occurs at elevati,ons between about 750. m and 2,000 m.' The elevations between 750 m and 2,000 m ihclude regions with dilferent climatic conditions. Differences among isolates of fungal pathogens of rubber obtained from different regions of Sri Lanka have been reported.29 A knowledge of the variation in pathogenic fungi is useful for disease control, development of new clones or cultivars and in , cultivation programmes. There is no information on variation in R hypolateritia in Sri Lanka. This investigation was initiated to determine whether significant differences exist among isolates of I! hypolateritia originating from regions with different climatic conditions. Isolates of R hypolateritia were obtained from .three such regions and the growth of the isolates and secretion of two enzymes viz. polygalacturonase and glucosidase, that have a role in pathogenecity: examined.

METHODS AND MATERIALS
Fungal Isolates -Three isolates of R hypolateriria obtained from the Tea Research Institute, Talawakalle, were used in this investigation. The isolates were T 1 ( from Hettikanda, Ratnapura; altitude 1,066 m; soil pH 4.3), T 2 ( from Densford, Nanu-Oya; altitude 1,505 m; soil pH 4.4) and T 3 ( from Madulkelle, Kandy, altitude 1,371 m; soil pH 5.1). The isolates were maintained on potato dextrose agar at 4O C during the period of study (about 3 months).
Media -(a) Solid Media. Potato dextrose agar (PDA), Malt extract agar (MEA), Corn meal agar (CMA), Czapek-Dox agar (CDA) and Root extract agar (REA) were used. REA was made from 35 g of powdered, dried, healthy tea roots and 15g agar in 1 1 distilled water. Fifteen ml of each medium was dispensed in 9 cm diameter petri dishes. Effect of Temperature on Growth -To examine the temperature effect the isolates were grown on PDA and incubated' at 15O, 20°, 2S0, 30' and.3S0 C and growth assessed as above.
Effect of pH on Growth -The isolates were grown on Czapek-Dox liquid medium buffered at pH values 3.0, 3.5, 4.5 , 5.5 and 6.5. To assess growth cultures were harvested at 48 h intervals for 16 d by filtering through Whatman no.1 filter paper and the residual mycelium was dried to a constant weight at 8 0 '~ in a hot air oven for 6-8 h and the weight was determined.2

. .
Determination of Enzyme Activity -The liquid media were harvested 12 d .after inoculation. by filtration through Whatman no.1 filter paper. The culture filtrates . were dialysed against distilled water at 4' C for 48 h and concentrated ten fold by freeze drying . The dialysed, concentrated culture filtrates were used for enzyme . . . .

assays.
Polyg$acturonase (PG): Polygalacturonase activity was determined using the agar plate method and by the release of reducing sugars from 0.1% (w/v) solutions of polygalacturonic acid in 0.1M sodium acetate buffer (pH 5.0)~. In the agar plate method a sodium polypectate agar gel was used and activities were determined relative to an aqueous solution (1 mglml) of pectin01 10M (Rhom & Hass, USA). p-Glucosidase: /3-Glucosidase activity was measured by the hydrolysis of the chromogenic substance p-nitrophenyl / 3 -~-~l u c o~~r a n o s i d e .~ The p-nitrophenol released were estimated by measuring the absorbance at 403 nm.

RESULTS
On REA and CMA the growth rate of T 1 was significantly different from the other two isolates and on MEA and CDA the growth rates of all isolates differred significantly from each other (Table 1). There was no significant difference among the growth rates of the three isolates on PDA.  The. isolate T 1 had optimum growth at pH 4.0, T 2 at pH 3.5 and T 3 at pH 5.0 when grown in Czapek-dox liquid media (Fig. 1). The growth was optimum at 25" C for all isolates. There was a rapid decrease of growth above 30° C and below 20" C in all three isolates (Fig. 2).
The elution profiles of the enzymes are given in Figure 3.

DISCUSSION
The growth rate of T 1 was always significantly different from T 2 and T 3, except when grown on PDA. The cause for the variation in growth rates in different media is likely to be the availability of nutrients. Synthetic media such as PDAare rich in nutrients and the nutrients are easily available to the fungus, hence the-higher rates of growth. In REA where the rates of growth were slower the availabilty of nutrients is relatively low.
The isolates had differnt pH optimas for growth. It is of interest to note that in isolates T 1 and T 3, the pH of the soil from which the isolates were obtained and their pH optimas for growth match ; T 1 pH optima 4.5 (soil pH 4.3), T 3 pH optima 5.5 (soil pH 5.1). This could be an adaptation of the isolates to their environments. However, this was not observed in T 2 where the pH optima was 3.5 and soil pH 4.4. The temperature optimum for growth did not differ among the isolates.
The molecular weights of the enzymes suggest that, (a) All isolates secrete a similar form of polygalacturonase. (b) The isolates T 2 and T 3 secrete three similar forms of b-glucosidase whereas T1 secrete only two of these forms.
It can be concluded that significant differences exist among the three isolates studied. The isolate T 1, which was obtained from the lowest elevation, differred most ie. in the rate of growth, pH optima and secretion of p-glucosidase. The isolates T 2 and T 3 differed from each other in the rate of growth on two media and in pH optima.