2.2.10
ANALYSIS OF CERATOCYSTIS FIMBRIATA F. SP. PLATANI ITALIAN POPULATION BY AMPLIFICATION OF MINISATELLITE DNA

A. SANTINI1 and PAOLO CAPRETTI2

1 Istituto Patologia Alberi Forestali CNR, Firenze, Italy; 2 Istituto Patologia Zoologico Forestali e Agraria, Università di Firenze, P. le Cascine 28-50144 Firenze, Italy

Background and objectives

Ceratocystis fimbriata f. sp. platani, the causal agent of stain canker of plane tree, has invaded the Italian peninsula over the last 20 years [1-3]. It appeared at different times in geographically separate areas, causing considerable damage to Platanus acerifolia and P. orientalis. The aim of the present study was to examine whether the DAMD PCR (Direct Amplification of Minisatellite region DNA) technique [4-6] can be used to distinguish f. sp. platani from the strains of C. fimbriata that attack different hosts, and thus characterize the Italian population of C. fimbriata f. sp. platani and provide information about the genetic variability of the isolates collected in relation to their geographical source.

Materials and methods
To study the Italian population of C. fimbriata f. sp. platani, 46 isolates of f. sp. platani from various areas in Italy and five samples of formae specialis of C. fimbriata from the USA (belonging to the T. Harrington collection), that attack aspen and stone fruits were examined. Each isolate was inoculated into three 100-ml Erlenmeyer flasks containing 50 ml of 2.4% PDB liquid culture (DIFCO). The mycelium was collected after 21 days of incubation in darkness. The DNA was extracted according to the protocol of Hamelin et al. [7]. Amplifications were performed using 1 µl of genomic DNA and the core sequence of the minisatellite M-13, 5’-GAGGGTGGCGGTTCT-3’, as primer [6]. The cycling parameters were: pre-denaturation, 93°C, 3 min, followed by 45 cycles of 93°C denaturation for 1 min, 55°C annealing for 1 min, 72°C extension for 1 min and a 72°C final extension for 10 min. Amplification products were separated on 1.5% agarose gel in TAE buffer and stained with 0,1% ethidium bromide.

Results and conclusions
The electrophoretic profiles of the amplification products revealed bands of a molecular size between 200 and 800 bp. Polymorphism within f. sp. platani were not detected by M13 PCR amplification, while polymorphism between the various strains of C.  fimbriata was frequent.

Initially, the results indicate a high level of genetic homogeneity in the Italian population of C. fimbriata f. sp. platani, confirming Granata et al. [8] who, in an analysis of electrophoretic profiles of soluble proteins from a small number of C.  fimbriata samples, did not find any differences between proteins, leading them to conclude that the samples were very homogeneous. The results obtained in our study appear to confirm that the Italian population of C. fimbriata is 'clonal', with an identical fingerprint spread over an area that stretches for more than 1000 km. It is possible that after the pathogen was introduced into this country, it found favourable conditions for development and a particularly susceptible host population.

References
1. Panconesi A, 1972. Informatore Fitopatolico 22,10-13.
2. Panconesi A, 1981. European Journal of Forest Pathology 11, 385-395.
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6. Stenlid J., Karlsson J.O., Högberg N., 1994. Mycological Research 98, 57-63.
7. Hamelin RC, Lecours N, Hansson P, Hellgren M, Laflamme G, 1996. Mycological Research 100, 49-56.
8. Granata G, Parisi A, Caciolla SO, 1992. European Journal of Forest Pathology 22, 58-62.