SHORT COMMUNIGlTION PECTOLYTIC ENZYMES IN THE DEVELOPMENT OF COLLETOTRICHUM LEAF DISEASE IN RUBBER (HEVEA BRASILIENSIS)

of these enzymes in pathogenesis.3 We report here on the role played by pectolytic enzymes secreted by the fungus in the development of disease in leaves of H. brasiliensis.

Preparation of Inoculum: Fifteen ml of PDA medium was dispensed into 150 ml Erlenmeyer flasks and the medium inoculated with C. gloeosporioides, and incubated as described by Senaratna et a12. When the cultures were 7d old, 10 ml of sterile distilled water was added to each flask and shaken for 5-10 minutes. The spore concentration of the resultant suspension was adjusted to 2x10' spores ml-' using ,a haemocytometer and the suspensions were then filtered through four layers of muslin. The resulting filtrate was used to inoculate H. brariliensis leaves.
Inoculation: Young H. brariliensis leaves at the copper brown stage of development were inoculated by placing the fungal suspension (0.05 ml) on the abaxial surface. The cut ends of the petioles were then sealed with molten wax, and the leaves incubated at 30 + 1°C in moist, large petri dishes (15 cm in diameter). As controls, leaves were inoculated with sterile distilled water (0.05 ml).
Secretion of Polygalacturonase (PG): To obtain PG, the fungus was in the ammonium tartrateliquid medium described by Byrde and 13elding4 with citrus pectin as the main source of carbon, and inoculation, incubation and harvesting were carried out as described by Senaratna et Tlssue Extraction: The inoculated leaves were harvested at 24 h intervals for 6 days, and were stored at -4OC until used. Initially acetone treated leaf powder was prepared from leaf tissue according to the procedure of Wong et al? Thereafter the enzymes were extracted from the acetone powder with 0.01 M Tris-HC1 buffer (pH 7.6) as described by Batra and ~u h n .~ The extract was stored at -4OC and used to detect the presence of cell wall degrading enzymes. For some experiments the extracts were treated with 5% solution of NaCl in 0.01 M Tris-HCI (pH 7.6) to desorb enzymes bound to cell wall^.^^' Determination of Cell Wall Degrading Enzyme Activity: The agar plate method8 was used to determine PG activity. Activities were expressed relative to an aqueous solution of Pectin01 10 M (1.0 mg ml-l) which was defined as having 100 units of PG activity ml". PL was assayed using the thiobarbituric acid (TBA) method? One unit of enzyme activify is defined as that amount which produces 1 pmol of unsaturated uronide in 1 min based on the molar extinction coefficient for the product of 4600." Determination-of Molecular Weight: The approximate molecular weight of the pectolytic enzyme was determined by gel filtration," using a column of Sephadex Effect of Host Tissue on Polygalacturonase: The ability of both healthy and infected leaf extracts to inhibit the polygalacturonase produced by C gloeosporioi&s was examined as described by ~i e l d i n~l~. The enzyme used for the assay and the leaf extracts were prepared as described earlier. A mixture of enzyme and the potential inhibitor in the proportions 1:9; 3:7; 1:l; 73; 9:l (vlv) were added to wells in agar plates followed by incubation and development. In control experiments sterile distilled water was used instead of leaf extract.

RESULTS
Pectolytic Enzymes in Leaf 'Nssue: PL was the only pectolytic enzyme detected in extracts of Hevea leaves infected with the fungus. The PL activity was fust detected on the third day and the activity reached a maximum on the fifth day after inoculation ( Figure 1). PG was not detected at any stage of the infection. No pectolytic enzyme activity was detected in leaf extracts from healthy leaf tissue at any stage,. Leaf extracts treated with 5% NaCl also did not possess any PG activity.
~o l e c u l a r~e i~h t : PL detected in infected kaf tissuewas elutid afier cyclochro~e C on G-100 gel filtration suggesting a mol.wt. < 12,000 (data not shown).

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Effect of Leaf Tissue on PG Activiw Extracts of both healthy and infected leaf tissue, did not have any significant influence on the actiiity of P G produced by C. gloeosporioides (Table 1). Liyanage and de ,41wis13 showed that the entry of C. gloeosporioides into leaf tissue occurs about 9 h after inoculation and colonization takes place 48 h after inoculation.
Lesions are produced thereafter. The increase in PL activity in host tissue therefore coincides with fungal colonization and lesion formation. PL detected in infected tissue had a mol.wt. < 12,000 which is compatible with that of PL secreted by C. gloeosporioides in culture2. Since PL activity was not detected in healthy leaves the evidence strongly suggests that the PL detected in infected leaf tissue is of fungal origin.
Many plant pathogens secrete PG very early in infection? C. gloeosporioides secreted PG in culture but PG activity was not detected at any stage of the infection. A similar phenomenon occurs with the pathogen CoIlefotrichunt lindemufhianum14 and Cladosporium ~ucurnerinum.'~ Both C. lindemufhianum and C. ctrcumerinum produce PG in culture, but not in infected tissue. In both, binding of the enzyme to cell walls and the presence of a PG inhibitor in host tissue has been cited as the likely causes for the failure to detect PG activity.14 In the present investigation PG activity was not detected even after host tissue was treated with 5% NaCl and both infected and healthy host leaf tissue had no effect on the PG secreted by C. gloeosporioides in culture. Thus, binding of PG to host cell walls or the presence of a PG inhibitor in leaf tissue appears unlikely.
The results are consistent with the suggestion that the PL produced by C. gloeosporioides plays a major role in the development of the Colletotrichum leaf disease of H. brasiliensis.