GENETIC VARIATION AND PHYTOTOXIN PRODUCTION AMONG CYLINDROCLADIUM . QUINQUESEPTATUM ISOLATES OF HEVEA BRASILIENSIS AND EUGENIA CARRYOPHYLLATA

Cylindrocladium quinqueseptatum (Boedijn & Reitsma 1950) is one of the major plant pathogenic fungi that infects a wide range of plants in humid tropics. The prevailing strains in Sri Lanka cause the foliage and shoot blight diseases and severe defoliation of Hevea brasiliensis (rubber) and Eugenia carryophyllata (clove). The isolates obtained from rubber and clove plants growing in Rathnapura district, in Sabaragamuwa province and Kalutara district, in Western province were evaluated for their toxin activity on hosts and genetic characterization was done using random amplified polymorphic DNA Polymerase Chain Reaction (RAPD-PCR grouping). The analysis of the results using ‘Wilcoxon signed-rank test’ revealed no correlation among the isolates based on their host or geographical origins. But a relationship was observed between the toxin activity and the RAPD grouping of the isolates. The time course experiment reveled that the minimum toxin activity recorded after three days of inoculation and maximum toxin activity was recorded after nine days of inoculation in Modified Fries Medium (MFM) for all the isolates. Based on the toxin activity and genetic characterization data, the isolates were categorized into two groups. The close genetic relationship  Corresponding author Email: jayarat@kln.ac.lk


INTRODUCTION
Cylindrocladium quinqueseptatum is one of the most common plant pathogen responsible for epidemic plant diseases in several countries including Australia, India, Vietnam, Laos and Thailand (Old et al. 2003).It causes damping off, root rot, crown canker, seedling, leaf and shoot blight, extensive defoliation and dieback on a wide variety of hosts including ornamental and forest plants and the fungus is widely distributed in the humid tropics (Old et al. 2003, Radziah et al. 1998).
In Hevea brasiliensis (rubber), Cylindrocladium quinqueseptatum leaf spot disease was first reported in Peninsular Malaysia in 1972 as a new disease affecting budwood nurseries (Anon 1972).However it remained insignificant until 1990, when it caused defoliation of several clones in budwood nurseries and plantations.The symptoms included shrivelling, blackening and falling off of young leaves, numerous chlorotic pin head like leaf spots on mature leaves and purplish and brown leaves surrounded by a prominent yellow halo (Radziah et al. 1988).
In Sri Lanka the fungus Cylindrocladium quinqueseptatum was first recorded as a pathogen in 1982 on Eugenia carryophyllata from Ratnaupra district.The fungus was reported to cause considerable mortality of E. carryophyllata seedlings and severe defoliation in young plants (Jayasinghe & Liyanage 1982).First report of the natural occurrence of Cylindrocladium leaf spot disease of rubber in Sri Lanka was in the year 2003 on the clone RRISL 206 in Kalutara district (Jayasinghe et al. 2003).
The first report on the toxic effect of culture filtrates of C. quinqueseptatum is from India on the toxin activity of the Eucalyptus isolate (Anahosur et al. 1976).Subsequently, Kaushik & Guptha (1991)

Isolation of the pathogen
Clove and rubber leaves showing typical symptoms of C. quinqueseptatum infection, collected from randomly selected rubber and clove plantations in different agro-climatic regions were used for the isolation of the pathogen.A total of 20 diseased leaf samples including ten from each host grown in Kalutara and Rathnapura were used for the isolation of the pathogen into Czepek Dox Agar (CDA) medium.The isolates were cultured on CDA medium at 28 ± 2 °C incubation.The conidial and conidiophores morphology of these isolates were used for the identification of C. quinqueseptatum grown in CDA.Single spore isolates obtained into Bacto Agar of these cultures were inoculated on CDA and incubated at 28 ± 2 °C under normal light and dark regime for six days were used for this investigation.

Extraction of the toxin
Erlenmeyer flasks each containing 100 ml of Modified Fries Medium (MFM) (Luck & Wheeler, 1955) were inoculated with three mycelial plugs, 5 mm diameter taken from six day old colony of the test fungus grown on CDA.The cultures were incubated at 28 ± 2°C under normal light and dark regime as stationary cultures.After nine days of incubation the cultures were harvested by filtering through 0.22 µm Millipore filters.The resulting culture filtrates were used as the source of the toxin.

Detection of the toxin activity
Detection of the toxin activity and the determination of the time course of toxin production were carried out according to the leaf wilt bio assay technique (Breton et al. 2000).Detached leaflets of eight H.For this experiment, nine replicates from each clone were used.The toxin activity was assessed by the intensity of wilting, which was quantified by the estimation of water loss using the following formula.
The time course of toxin production was assessed at 72 h intervals of incubation for all the isolates.The results were analyzed using the nonparametric procedure NPARIWAY, available in statistical package SAS (SAS, 1987).The statistical method employed was the Kruskal Wallis test and the results were presented in the form sum of ranks.Subsequent paired comparisons were made using Wilcoxon 2sample test.

Analysis of RAPD
DNA products were analysed by scoring 1 for the presence of major bands and 0 for their absence.Faint bands that were not clearly resolved were not considered.The discrete data set of 56 bands thus generated was analysed using 'RAPD distance' programme by Nei & Lei (1979) and a neighbour joining tree was produced (Figure 7).UPGMA (Unweighed Pair Group Method with Arithmetic Mean) method was used to develop the neighbour joining tree.

Variation among the isolates of C. quinqueseptatum from H. brasiliensis & E. carryophyllata in toxin production
Time course of toxin production: Toxin production was first detected after 72 h of incubation in all isolates.All the isolates had the maximum toxin activity after 9 days of incubation (Figure 1).Hence the extraction of the crude toxin was done after 9 days of incubation to maintain the evenhandedness in the toxin activity assays.A total of 56 DNA fragments were scored and out of these 30 showed polymorphism (Figure 3).

CONCLUSION
The results reveal that C. quinqueseptatum isolates Cr produced a high mean wilting intensity has a close genetic association with the rubber isolates Rk-206 and Rk-210.Consequently it can be predicted that this strain of C. quinqueseptatum isolated from clove can be a potential pathogen for rubber clones.The RAPD-PCR grouping approach using 20 isolates obtained from two different hosts seems to be a useful rapid molecular biological technique for the identification of virulent strains of the pathogen before reaching them to epidemic levels in rubber and clove plantations.
reaffirmed the findings of Anahosur et al.In 1996 Jayasinghe et al. suggested that the toxin might be considered as a host specific toxin.The present study was undertaken to investigate the variation of phytotoxin production among the isolates of C. quinqueseptatum from H. brasiliensis & E. carryophyllata and to differentiate the isolates using RAPD -PCR (Randomly Amplified Polymorphic DNA -Polymerase Chain Reaction) analysis.
photoperiod.Filtrates obtained from un-inoculated media served as controls.

Figure 2 :
Figure 2: Variation of toxin activity among the isolates of C. quinqueseptatum on Hevea clones