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THE USE OF PECTIC ENZYMES IN THE CHARACThRISATION OF ARMILLARIA EDIAS MWENJE
THE USE OF PECTIC ENZYMES IN THE CHARACTERISATION OF ARMILLARIA EDIAS MWENJE Dept of Applied Biol. and Biochem., National Univ. of Science and Technology, P.O. Box 346, Bulawayo, Zimbabwe. Background and objectives The genus Armillaria is well characterised in Europe with seven species known to occur. The taxonomy of the genus is based mainly on mating tests, although, the species are also known to differ in a number of morphological, physiological and biochemical characteristics. The rare occurrence of fruit bodies and the putative homothallic nature of Armillaria in Africa has imposed serious limitations on the use of mating tests. The absence of a haploid form in many African isolates means that the mating tests technique which was successful in determining biological species of temperate Armillariais not entirely transferable to the study of African Armillaria. The plasticity and interdependence of a number of morphological characteristics have also resulted in the search for new characteristics to aid in flingal systematics. Isozyme analysis is a relatively cheap and simple tool which can facilitate identification of organisms. The method has been applied at many different taxonomic levels but is successful at distinguishing species. Isozymes especially of pectic enzymes have proved successful in grouping isolates from Zimbabwe [1]. In the present study interrelationships of African Armillaria isolates have been studied using pectic enzymes. For comparative purposes 26 isolates of European Armillaria (already classified by mating tests) were included in the tests. Materials and methods European Armillaria isolates were collected from England, Scotland, France and Italy. The African isolates came from Zimbabwe, Kenya, Malawi, Congo, Cameroun and Reunion. For enzyme production cultures were grown under stationary conditions in 250 ml flasks containing 50 mivogel's medium supplemented with 1 g cell wall preparations [2]. Polygalacturonase activity was detected on gels as described by [1]. Pectin lyase (PL) and pectin methylesterases (PME) were detected after electrophoresis by incubating gels in 10 mM Cad2 for 10 mm at 5~C followed by incubation in 0.O2MTris-HCl pH 8.5 containing 10 mM Cad2 for 40 min at room temperature. Gels were stained in ruthenium red followed by washing in water. The computer program Clustan version 3.2 was used for the analysis of the electrophoretic data [2]. Results and conclusions The pectic enzyme zymograms of four European Armillaria species (A. tabescens, A. mellea, A. ostoyae, A. gallica) showed few similarities between species. However, isolates of the same species from different countries showed species-specific bands. Isolates of A. mellea sensu stricto previously identified through mating tests from different countries showed little or no variation in PL and PMF patterns. Overall the variation within species was much lower than between species for the European isolates. The present study indicates that Armillaria in Africa can be divided into at least four major groups based on pectic isozyme patterns. The differences observed in the patterns suggest that the groups could represent different species. Three of the groups (I, II, III) occur in Zimbabwe [1]. Isolates of group I are part of the large A. heimii group, being most clearly identifiable by their PL and PME isozymes. The fourth group is composed of isolates referred to as Armillaria mellea. In interpreting the results of this study, we are aware that only a limited number of isolates were used and a greater range of variability may have been revealed if sampling had been extended. The study strongly suggests that temperate A. mellea and tropical A. mellea are distinct species. The level of variation in PL and PME patterns between the two groups is as great as between many other pairs of species as observed in this study. The pectic enzyme patterns suggest that A. mellea sensu stricto may be closely related to some African Anmillaria species than other European Armillaria species. Pectic enzymes especially pectin lyase and pectin methylesterase can be used in grouping Armillaria species. References 1. Mwenje E, Ride JP, 1996. Plant Pathology 45, 1031-1051 2. Mwenje E, Ride JP, 1997. Plant Pathology 46, 341-354