Handbook of Vanilla Science and Technology

Colonization of Vanilla roots by mycorrhizal fungi was variable. A technique was developed to expedite the search for mycorrhizae in extensive Vanilla root systems: the velamen was stripped off the roots, and areas with mycorrhizae could be seen as brown spots on the vascular cylinder (Figure 16.2; Porras and Bayman 2003).

Roots in soil had significantly more mycorrhizae than roots on tree bark (Porras-Alfaro and Bayman 2007). In V. planifolia, a few roots attached to rough-barked trees were heavily colonized, but most roots on bark had no mycorrhizal pelotons. In V. poitaei, no mycorrhizae were found in roots on bark. Vanilla roots on tree bark are important for support and contribute to plant nutrition through photosynthesis, but the lack of mycorrhizae suggests that their contribution to mineral nutrition is limited. In the forests of Puerto Rico, we have seen healthy V. planifolia and V. poitaei plants dangling from branches with no roots in contact with any substrate, either bark or soil. These unattached roots have no mycorrhizae, suggesting that adult plants do not require mycorrhizae to survive.

Differences in mycorrhizal fungi between V. phaeantha and V. aphylla, and differences between countries where samples were collected, could not be evaluated because of limitations in sample size and distribution.

16.5 FUNCTIONAL SPECIFICITY OF MYCORRHIZAL FUNGI OF VANILLA

16.5.1 Seedling germination experiments

The fungi identified in roots of adult Vanilla plants are not necessarily the same as those that stimulate seed germination. The three genera described in Section 16.3 may differ in functional significance for the host plant. To relate phylogenetic specificity to function, we compared symbiotic germination of Vanilla planifolia seeds inoculated with different mycorrhizal fungi (Porras-Alfaro and Bayman 2007). The growth medium contained cellulose as the sole carbon source; cellulose can be used as a carbon source by fungi but not by plants, which obliges the seeds to get their carbon from the fungi (Otero et al. 2004). Five Ceratobasidium isolates and three Tulasnella isolates from Vanilla were tested, along with three Ceratobasidium isolates obtained from the epiphytic orchid Ionopsis utricularioides in Puerto Rico (Otero et al. 2005). Thanatephorus isolates from Vanilla could not be included because of their recalcitrant growth in culture. Control plates had V. planifolia seeds without fungi; in some controls a fungicide was added to reduce contamination (Porras-Alfaro and Bayman 2007).

Fig. 16.5 Symbiotic germination of Vanilla planifolia seeds and contamination of cultures inoculated with different mycorrhizal fungi. (A) Percent germination of V. planifolia seeds after two months' incubation; (B) contamination of seed germination plates with bacteria; (C) contamination of seed germination plates with fungi. Bar shading indicates the fungi used: Tulasnella, Ceratobasidium isolated from Vanilla, Ceratobasidium isolated from I. utricularioides, and control seeds without fungi. Controls: CA = cellulose agar, CAF = cellulose agar + fungicide, Knudson F = Knudson C agar + fungicide. Error bars = s.e. N = 5 petri plates for all samples. From Porras-Alfaro 2004.

Five of eight Ceratobasidium isolates stimulated seed germination (Figure 16.5A). Phylogeny was associated with germination success: isolates from one clade of Ceratoba-sidium did not stimulate germination, and the two other clades differed significantly. Germination frequency was highest with Ceratobasidium isolates from the epiphytic orchid Ionopsis utricularioides rather than with isolates from Vanilla itself. Seeds inoculated with Tulasnella and control seeds without fungi did not germinate.

Inoculation of seed germination plates with Ceratobasidium also eliminated contamination by bacteria and greatly reduced contamination by fungi (Figures 16.5B and C). In contrast, plates with Tulasnella and control plates without fungi had more than 50% contamination after 2 months’ growth. Orchid seed germination and seedling growth are slow, and in tropical environments it is hard to avoid contamination when petri plates are incubated for long periods; fungus mites often invade cultures, carrying bacteria and fungi.