DETECTION AND CHARACTERIZATION OF FUNGAL-INFECTIONS OF AMMOPHILA-ARENARIA (MARRAM GRASS) ROOTS BY DENATURING GRADIENT GEL-ELECTROPHORESIS OF SPECIFICALLY AMPLIFIED 18S RDNA

Marram grass (Ammophila arenaria L.), a sand stabilizing plant species in coastal dune areas, is affected by a specific pathosystem thought to include both plant-pathogenic fungi and nematodes, To study the fun gal component of this pathosystem, we developed a method for the culti vation-independent detection and characterization of fungi infecting p lant roots based on denaturing gradient gel electrophoresis (DGGE) of specifically amplified DNA fragments coding for 188 rRNA (rDNA), A nes ted PCR strategy was employed to amplify a 569-bp region of the 188 rR NA gene, with the addition of a 36-bp GC clamp, from fungal isolates, from roots of test plants infected in the laboratory, and from field s amples of marram grass roots from both healthy and degenerating stands from coastal dunes in The Netherlands, PCR products from fungal isola tes were subjected to DGGE to examine the variation seen both between different fungal taxa and within a single species, DGGE of the 18S rDN A fragments could resolve species differences from fungi used in this study yet was unable to discriminate between strains of a single speci es, The 18S rRNA genes from 20 isolates of fungal species previously r ecovered from A. arenaria roots were cloned and partially sequenced to aid in the interpretation of DGGE data, DGGE patterns recovered from laboratory plants showed that this technique could reliably identify k nown plant-infecting fungi. Amplification products from field A. arena ria roots also were analyzed by DGGE, and the major bands were excised , reamplified, sequenced, and subjected to phylogenetic analysis, Some recovered 18S rDNA sequences allowed for phylogenetic placement to th e genus level, whereas other sequences were not closely related to kno wn fungal 18S rDNA sequences, The molecular data presented here reveal fungal diversity not detected in previous culture-based surveys.