2.6.3
PCR-BASED DETECTION OF SOIL-BORNE AND ROOT-COLONIZING ACREMONIUM ENDOPHYTES INDUCING FUSARIUM-WILT RESISTANCE IN LINSEED AND TOMATO PLANTS

G GRUNEWALDT-STOECKER, T KRASKA and E MAISS

Institute of Plant Diseases and Plant Protection, University of Hannover, Herrenhauser Strasse 2, D-30419 Hannover, Germany

Background and objectives
Acremonium soictum (A.s.) and A. ochraccum (A.o.) are soil-borne mutualistic symbionts restricted to the root cortex of several host plants. They have no effect on plant growth. Applied as biocontrol agents via spore suspensions to seedlings prior to pathogen infection or insect attack, they (1) confer induced resistance towards fungal wilt diseases and (2) changed host suitability to phloem-sucking insects, e.g. Viffite fly. In contrast to the endophytic Acremonium species of turf grasses, such as A. coenophialum and A. lolii, which live in the intercellular spaces of shoots and are distributed mainly by seeds, these root endophytes do not synthesize antibiotic or alkaloid substances which directly stop growth of pathogenic Fusarium oxysporum in plate assays. The interaction mechanisms between endophytes and host plants appears to be different from those of the turf grass Acremonium spp. To discover the mechanisms behind induced systemic effects of delayed and reduced Fusarium-wilt symptoms and reduced pathogen spread, histological and physiological investigations of stem were performed on host plants: in endophyte-inoculated linseed and tomato plants the auxin content in shoots were reduced. In the basal stem of tomato, tyloses occurred early in the period of secondary xylem differentiation, serving as morphological barriers for subsequent spread of Fusarium spread. Entomological studies on host preferences and insect development revealed that A.o. impaired moulting processes of Trialeurodes vaporariorum by systemic alterations in the phytosterol composition of tomato shoots. A severe problem in all experiments was detecting these Acremonium spp. in roots with reliable and sufficient certainty to correlate physiological and histological results with infection rates. This is especially of interest for their elucidation, whether the endophytes are necessary to act over a longer time period or only to initiate a cascade of physiological reactions leading to the observed resistance effects. Because reisolation techniques, microscopy of stained roots and preparations of antibodies for aerological ELISA-tests failed, selective DNA-probes for A.s. and A.o. were developed.

Materials and methods
A RAPD pattern was generated using the decamer 5'-CGGGATCCGC-3' and purified DNA from A.s. and A.o. mycelium grown in biomalt liquid culture. Random PCR fragments of approximately 500 to 700 bp were eluted from a low-melting-point agarose gel, cloned and sequenced. Subsequently, specific oligonucleotides for A.s. and A.o. were synthesized and used to detect the fungi in roots of linseed after DNA preparation.

Results and conclusions
The suitability and specificity of the designed oligonucleotides were first tested with purified DNA of A.o. and A.s. as well as with DNA from Pythium ultimum, Rhizoctonia solani, Fusarium oxysporum f. sp. lini, F. lycopersici, f. sp. cyclaminis and Trichoderma harzianum. PCR fragments of the expected size were only detected with the Acremonium species, but not with the other fungi. With the establishment of oligonucleotides specific for the detection of A.s. and A.o., a modified method for DNA preparation from root extracts and an optimized PCR programme, it is now possible to selectively detect each of the applied endophytes in aliquots of freeze-dried root material of linseed plants grown in sand-peat-mixtures under greenhouse conditions. The calculated limit of detection is 0.01% (0.005 ng/gl purified DNA), representing at least one infection site per root system of one 2-week-old plant. In current trials, the PCR-based tests provide reliable information on the infection level, infection progress and endophyte distribution in the root system within a time course. In accordance with reisolation tests, the endophytes are easily detectable in the second week after inoculation, while later samples show less pronounced bands, indicating a slow colonization rate in fast-growing roots. These findings will be used now to reveal more about the principles of Acremonium-induced alterations in host plant metabolism of linseed as well as tomato plants and Fusarium-wilt resistance.