2.9.16
LATENT PROPAGULES IN BIRCH TREES: WHERE ARE THEY?

A DANBY1, L BODDY1 and D LONSDALE2

1School of Pure and Applied Biology, University of Wales, Cardiff CF1 3TL, UK; 2Forest Research, Alice Holt, Farnham, Surrey GU10 4LH, UK

Background and objectives
Latent endophytic propagules are the suspected origin of early fungal development in dysfunctional tissues of many angiosperm tree species. Their precise location within the tree was investigated in three ways: by sectional incubation, microscopy and molecular analysis. Betula pubescens and B. ;pendula were used as a study case.

Materials and methods
Billet incubation. Whole living Birch stems were felled and immediately cut into 25-cm billets. These billets were incubated under a range of drying rates and destructively sampled at 0, 5, 10 and 15 ;weeks. Fungi arising from wood chip isolations and direct-incubated billets were cultured and paired for vegetative compatibility, so as to test for the presence of identical individuals within different zones of tissue.
Microscopy. Discs of functional Birch sapwood (4 ;mm thick and 5 ;mm diameter) were inoculated with various fungal species suspected to have a latent strategy in this tissue. The discs were then stored under sterile water in cryovials to simulate the high water content of living birch sapwood. Discs were fixed and microtomed, then examined by light microscopy and TEM.
Molecular analysis. Ribosomal DNA was extracted from various fungi suspected to be latent colonizers of birch. The DNA underwent PCR, using four primers targeting ITS regions. The resulting sections were sequenced.

Results and conclusions
For the billet incubation, identical individuals were isolated from different regions of the same tree, derived from genetically identical propagules previously latent in host tissues. One of the species exhibiting this strategy was Piptoporus betulinus, a major decay fungus in Betula spp. Upon microscopy, propagules were found to have formed within the wood vessels. Molecular analysis revealed novel sequences for the species under investigation.

Endophytes are widespread in Betula spp. and in many other species of plant. They can be located on a large scale by incubation of stem billets and, more precisely, by microscopy. Accuracy may be especially important in identifying points where fungi enter before forming latent propagules. The development of novel sequences from the molecular work should enable gene probes to be produced to allow further investigation of the distribution and frequency of propagules in apparently healthy trees, without felling them.