3.3.27
GENOMIC VARIABILITY IN COFFEE ISOLATES OF COLLETOTRICHUM SPP. ASSESSED BY PCR-BASED METHODS

T COELHO1, V VARZEA2 and R TENREIRO1

1Departamento de Biologia Vegetal and Centro de Genetica e Biologia Molecular, Universidade de Lisboa, Portugal; 2 Centro de Investigação das Ferrugens do Cafeeiro, Oeiras, Portugal

Background and objectives
Colletotrichum is one of the major plant pathogenic fungi in tropical and subtropical regions, where C. gloeosporioides is responsible for huge damages in coffee and other tree fruits. Although this species is recognized as the causative agent of brown blight in ripe coffe berries, the anthracnosis of green berries (coffee berry disease - CBD) is attributed to a different species, C. kahawae, and represents the major coffee disease with production losses high as 60-70%[1]. The low correlation level of usual CBD identification methods (pathogenicity tests in hypocotils and inoculation of green berries produced in greenhouses) asks for more reliable and fast procedures and so molecular typing becomes advisable.

Results and conclusions
In the presented work, a group of Colletotrichum isolates from coffee (20 of C. kahawae, 11 of C. gloesporioides and 2 of C. acutatum), with African and Asiatic origin, was analysed by restriction profiling of genomic and mitochondrial rDNA obtained by PCR [2]. Six conjugations of primers were used to amplify 3’-sequences of 18S rDNA, 5.8S rDNA, internal transcribed spacers and 5’-sequences of 28S rDNA from genomic DNA; three other conjugations of primers were used to amplify rrs and rrl regions of mitochondrial rDNA.
Analysis of restriction fragments produced by 4 bp- and 6 bp-recognizing enzymes was performed, using the Dice coefficient and the UPGMA clustering method, in order to evaluate the genomic variability of coffee isolates. Although a general homogeneity was found among C. kahawae isolates, some variation was observed within the amplified 3’-sequence of 18S rDNA that may be used to discriminate among groups of isolates. Furthermore, the differences between C. kahawae isolates and the other species enable the use of the PCR approach as a diagnosis method.

References
1. Sreenivasaprasad S, Brown AE, Mills PR, 1993. Mycological Research 97, 995-1000.
2. Mitchell JI, Roberts PJ, Moss ST, 1995. Mycologist 9, 67-75.