2.2.74
AFLP DNA FINGERPRINTING OF PLANTPATHOGENIC FUNGI

P BONANTS, M HAGENAAR-DE WEERDT, G KEMA, P VAN DEN BOOGERT and C WAALWIJK

Research Institute for Plant Protection (IPO-DLO), PO BOX 9060, 6700 GW Wageningen, The Netherlands

Background and objectives
AFLP (amplified fragment length polymorphism) [1] is a powerful DNA fingerprinting technique to identify fungi and bacteria and to study their genetic variation [2]. At IPO-DLO this method is applied to answer different research questions. Population dynamics are studied of different AGs of Rhizoctonia solani. In Septoria tritici AFLP are used to construct a genetic map. In Phytophthora spp. and Fusarium oxysporum markers linked to race specific avirulence genes will be identified using AFLPS.

Results and conclusions
(1) Thanatephorus cucumeris.T. cucumeris (anamorph Rhizoctonia solani) is an important soil pathogen, of which several AGs (anastomosis groups) exist. We focus our attention on AG2-2 and particularly those isolates which are pathogenic to sugarbeet. With AFLP we look at the genetic variation within fields and between fields in the Netherlands. We found that within a single field there is little or no genetic variation. Populations from two different fields were also comparable. However, isolates from a third field formed another group. Individual isolates from several different fields grouped within either of these clusters.
(2) Mycosphaerelia graminicola. M. graminicola (anamorph Septoria tritici) is pathogenic to wheat. A cross was made between two isolates with different virulence patterns. From 68 progeny isolates of this cross, after prescreening with 64 primercombinations, AFLP DNA fingerprints were generated with 1:1 primer combinations (EcoRI+2/MspI+2). A total of 317 polymorphic markers were scored. Based on these results a preliminary genetic map was constructed with JoinMap. Markers were found that cosegregated both with the mating type gene as well as a complex locus conferring aviruience towards three unrelated differentials. Those markers will be used to isolate these genes.
(3) Phytophthora spp. In Phytophthora research AFLP has been used to study the genetic variation of a worldwide collection of P. fragariae isolates. This pathogen of strawberry is a quarantine organism, of which several races are known. Correlation of polymorphic AFLP markers will be made with inoculation experiments on a differential set of strawberry cultivars to associate AFLP markers to avirulence genes. Crosses of individual isolates will be made to prove correlation of AFLP markers and avirulence genes.
(4) Fusarium oxysporum. Fusarium oxysporum is pathogenic to more than 100 different crops and F. oxysporum f. ;sp. dianthi is the causal agent of wilt in carnation. AFLPs employed on F. oxysporum f. ;sp. dianthi have demonstrated that VCGs (vegetative compatibility groups) are homogeneous groups. However, the relationship between different VCGs within a forma specialis is not stronger than between VCGs from different specialized forms.
(5) AFLP database. AFLP DNA fingerprints of plantpathogenic fungi will be generated with one primer-combination. Homologeous bands of different isolates will be saved into a database. In this way unknown isolates can be identified easily. Analysis of the AFLP DNA fingerprints, which have been generated with an automatic DNA sequencer (ALFexpress), will be done with software.

References

1. Vos et al. 1995. Nucleic Acids Research 23, 4407-4414.
2. Majer et al. 1996. Mycological Research 100, 1107-1111.