Effects of Orchinol, Loroglossol, Dehydroorchinol, Batatasin III, and 3,4'- Dihydroxy-5-Methoxydihydrostilbene on Orchid Seedlings

Two naturally occurring orchid phytoalexins, orchinol and loroglossol; a synthetic analogue, dehydroor-chinol; a possible precursor of orchinol (3,4'-dihydroxy-5-methoxydihydrostilbene); and batatasin III (3,3'-dihydroxy-5-methoxydihydrostilbene) reduced the growth of Cattleya aurantiaca seedlings. These compounds had no effect on the length of the first leaves during early stages of development.


Introduction
Relatively little is known about the effects of phytoalexins, especially those from orchids, on higher plants (VANETTEN and PUEPPKE 1976;AR-DITTI 1979;STOESSL and ARDITTI 1984). Pisatin (a phytoalexin from Pisum sativum) represses the growth of wheat roots (CRUICKSHANK and PERRIN 1961) and is toxic to mammalian cells (OKu et al. 1976) and pea callus cultures (BAILEY 1970), possibly by injuring the plasma membrane (SHIRAISHI et al. 1975). Phaseollin, a phytoalexin from the Fabaceae, inhibits cell suspension cultures of kidney beans (GLAZENER and VANETTEN 1978). The cells were killed within 30 min by 32 Rg phaseollin/ml. Phaseollin blocks ATP formation in mitochondria of Cucumis sativus hypocotyls (VANETTEN and PUEPPKE 1976). A similar effect was reported for ipomeamarone, a phytoalexin from sweet potato, which prevents oxidative phosphorylation in mung bean mitochondria (URITANI et al. 1954). Seed germination and hypocotyl growth of clover are inhibited by trifolirhizin (CHANG et al. 1969). BAT from dormant bulblets of Dioscorea batatas induces dormancy in these organs (HASHIMOTo et al. 1974). Rishitin, a phytoalexin from potatoes, changes the electrical potential of cell membranes of Nitrella and Nitelopsis (VOROBIEv et al. 1975).
Orchid phytoalexins inhibit a number of fungi and bacteria at concentrations from 10-4 to 10-2 M. In Orchis militaris, ORC may reach a level of 0.5 X 10-2 M in tissues 8 days following infection with Rhizoctonia repens (NUESCH 1963). This concentration is well within the toxic range for some bacteria and fungi, but its effects on orchid seedlings-terrestrial or epiphytic, species from temperate or tropical regions-are not known.
Little is known about the effects of phytoalexins on (1) angiosperms in general, (2) the plants that produce them, and (3) species related to the source plants. This is regrettable because phytoalexins or related natural or synthetic compounds could be useful as fungicides, bactericides, or as anticontaminants for tissue or whole-plant cultures in vitro. Therefore, we determined the effects of LOR, BAT, ORC, its analogue DOR, and a possible precursor, DMS, on a model system consisting of germinating orchid seeds and developing seedlings.
Mature seeds of Cattleya aurantiaca were cultured (TAMANAHA et al. 1979). The effects of ORC, DOR, DMS, LOR, BAT, and the controls were evaluated by the growth index (SPOERL 1948) method, which measures seedling development (three determinations per concentration); leaf length measurements (TAMANAHA et al. 1979), using 100 seedlings per culture; and assays of chlorophyll content of entire seedlings (MACKINNEY 1941;AR-NON 1949; modified for use with orchids by HAR-RISON [1973]) in two samples per treatment. The data are averages of these.
Chlorophyll content (a, b, total) of seedlings on ORC was equal to or higher than on the two controls except that, on 0.078 X 10-4 M and 0.313 X i0-4 M, the total level was lower than on E ( fig.   3). On LOR and DOR all chlorophyll levels were equal to or higher than on the controls ( fig. 3). On DMS, levels of chlorophyll a varied, as did those of total chlorophyll ( fig. 3). Levels of chlorophyll b were similar to or slightly lower than in control seedlings ( fig. 3). Chlorophyll a levels on BAT were equal to or somewhat higher than the controls. Chlorophyll b content was lower than in the controls. Total chlorophyll concentration on BAT was equal to that in the controls.

Discussion
Germination of orchids is unlike that of other plants in that the embryo swells (stage 1), bursts through the testa (stage 2), and forms a conical protocorm (stage 3). A shoot tip develops on the protocorm and gives rise to leaves (stage 4) that increase in size (stage 5) and number (stage 6). Roots (stage 6) usually appear after the leaves (TAMA-NAHA et al. 1979). The growth index (SPOERL 1948) is based on the relative number of each stage in seedling populations and is a good method to measure growth of orchid seedlings because it quantifies development and does not merely record increases in size and mass of protocorms, leaves, and/ or roots.
A drawback of the growth index is that it does  Fig. 2, Leaf length of seedlings on KC medium (control) as well as media containing 2 ml 70% ethanol/liter (control), ORC, LOR, DOR, DMS, and BAT. Fig. 3, Chlorophyll content of seedlings on KC medium (control) as well as media containing 2 ml 70% ethanol/liter (control), ORC, LOR, DOR, DMS, and BAT. Explanation of symbols as in fig. 1 and as shown. Narrow lines are standard deviations. not measure leaf and root growth in terms of size increases or chlorophyll levels. Therefore, the effects of an agent that may not block formation of leaves and/or roots but does inhibit their elongation would remain undetected. To overcome these limitations, we measured the length of the first leaf of seedlings at stages 4 and 5. Since an agent may affect leaf size and number without influencing chlorophyll synthesis, we determined pigment levels. Measurement of leaf length ( fig. 2), when combined with the determination of chlorophyllwhich is found in protocorms, leaves, and roots of Cattleya seedlings ( fig. 3) and growth index (fig.  1), have proved useful in measuring orchid seed germination and seedling development (HARRISON 1973;ARDITTI 1979;TAMANAHA et al. 1979).
ORC is produced by Orchis militaris, Serapias species, Loroglossum hircinum, and L. iongibracteatum; L. hircinum is also the source of LOR (AR-DITTI 1979). These species are European terrestrial orchids that belong to the tribe Ophryoideae, subtribe Platanthereae. Cattleya aurantiaca, the assay plant, belongs to the tribe Kerosphaeroideae, Series A: Acranthae, subtribe Laelieae, and is an epiphytic species from Central America (SCHULTES and PEASE 1963). The assay plant and species that produce ORC and LOR are not closely related. Perhaps this distant relationship explains why some concentrations of ORC, LOR, and DOR are somewhat inhibitory to C. aurantiaca at concentrations lower than those found in fungus-infected Orchis plants. One reason may be that, in infected Orchis 19841 HILLS ET AL.  plants, the phytoalexins may enter or become bound to the fungus. A search for phytoalexins in extracts from infected roots and pseudobulbs of Cymbidium hybrids failed to detect phenanthrenes (STOESSL et al. 1980). A weak antifungal compound was isolated and identified as linoleic acid-i -monoglyceride (STOESSL et al. 1980). Recently, 2,7-dihydroxy-3,4,6-trimethoxy-9, 10-dihydrophenanthrene was isolated from Coelogyne ovalis (Kerosphaeroideae, Acranthae, Coelogyneae), a species more closely related to Cattleya than to Orchis (MAJUMDER and LAHA 1981). This suggests that phenanthrene phytoalexins may be more widespread among the Orchidaceae than believed until now. If so, it is reasonable to expect that these and chemically similar compounds would not be toxic to orchids. This assumption is supported by our findings because (1) reductions in growth indices in their presence, if and when they occurred, were small; (2) leaf length increased on LOR-containing media even at a concentration that inhibited the growth index; and (3) chlorophyll levels were not reduced by ORC and actually increased by LOR.
An obvious question pertains to whether the low solubility of ORC in an agar medium might suppress its biological effects: ORC effects on the seedlings indicated that it was taken up by the orchids because of solubilization by factors that may be present in the plant (GAUMANN and KERN 1959;STOESSL and ARDITTI 1984).
The effects of DMS on leaf length are generally similar to those of ORC. This suggests either that DMS (1) serves as a precursor to ORC or (2) has the same effects, regardless of whether it is a precursor of ORC.
BAT is reported to induce dormancy in yam bulbils (HASHIMOTO et al. 1974). If its effects on orchid seedlings were to be the same, it is reasonable to expect reduced growth, shorter leaves, and lower chlorophyll levels. This was not so in the concentration range screened by us. Orchid seedlings are not comparable to yam bulbils. Therefore, it is not surprising that a compound that induces dormancy in Dioscorea batatas (Dioscoreaceae) affects C. aurantiaca (Orchidaceae) differently.