Research and Application of Orchid Mycorrhiza in Taiwan

D.C.N. Chang
Department of Horticulture
National Taiwan University
Taipei, 10617
Taiwan

Keywords: Rhizoctonia, Anoectochilus, Haemaria, Dendrobium, Doritaenopsis,

Paphiopedilum, Phalaenopsis

Abstract
Orchid mycorrhizal research was conducted on various orchid genera, such as
Anoectochilus formosanus, Haemaria discolor var. dawsoniana, Dendrobium,
Doritaenopsis, Paphiopedilum and Phalaenopsis, which were inoculated with mainly
one of three Rhizoctonia spp. of orchid mycorrhizal fungi (OMF) isolated in Taiwan.
Results showed that germination and transplanting survival rate, vegetative and
reproductive growths were highly enhanced by the inoculation of OMF. Activities of
acid and alkaline phosphatases and superoxide dismutase, contents of polysaccharides,
polyphenols, flavonoids, ascorbic acid and phosphate were markedly higher in the
mycorrhizal tissues of A. formosanus. OMF inoculation resulted in Phalaenopsis having
more flowers and increased flower size. OMF-inoculated orchids were observed to
have less disease infection. The infection by the beneficial Rhizoctonia spp. was tolypo-
phagy in all of the inoculated orchids. The growth of Bletilla formosana, Oncidium
Gower Ramsey, Dendrobium amethystoglossum, Dendrobium tosaense, Dendrobium
Chao Praya Garnet, and Dendrobium Snow Flake seedlings was enhanced by
Rhizoctonia spp. and other OMF as reported by others in Taiwan.
INTRODUCTION
The orchid-fungus relationship is one of many known forms of mycorrhiza (the
term means fungus-root), a common form of symbiosis between fungi and plants (Hadley,
1982). The association of orchid roots and the infected beneficial fungi (orchid
root-fungus) was called orchid mycorrhiza, while the beneficial fungi that infected orchid
roots were termed orchid mycorrhizal fungi (OMF). Burgeff (1959) studied seed
germination of orchids and recognized that in general, and particularly under natural
conditions, successful germination was virtually impossible in the absence of a fungus. But
symbiotic germination in vitro was difficult for the commercial grower and resulted in a
low success rate (Hadley, l982; Zettler and Hofer, 1998). On the contrary, the success of
orchid seed germination by Knudson (1922) on a medium containing only sugars and
mineral salts and then the non-symbiotic methods are successful with many growers
(Hadley, l982). Nowadays in Taiwan, these non-symbiotic seed germination methods are
well established in the orchid industry. The author suggests that growth of the micro-
propagated orchids in vitro can be promoted by the inoculation of OMF as well as ex
vitro, thus many efforts have been made to isolate the OMF from wild and greenhouse
cultivated orchids. In Taiwan, growth stimulation by OMF was also reported for Bletilla
formosana (Su, 1995), Oncidium sp. (Chu, 2000) and Dendrobium sp. (Lin, 2002). This is
a brief report on the three Rhizoctonia spp. (R01, R02 and R04) of OMF isolated in
Taiwan that enhanced the growth and development of various commercially available
orchids.
Three Rhizoctonia spp. isolated from greenhouse-grown Haemaria sp. and wild
Calanthe sp. and Anoectochilus formosanus var. dawsoniana in Taiwan, namely R01
(Chou, 1997), R02 and R04 (Tsai, 1997), were used as inocula. R01 and R02 were
unidentified binucleate isolates. R04 was the multinucleate Rhizoctonia solani, one of the
AG-6 OMF (Lee, 2001). Solid powder form of OMF grown on crushed sphagnum moss
was used as inoculum. For the commercially available Phalaenopsis orchid plants with

Proc. XXVII IHC-S5 Ornamentals, Now!

Ed.-in-Chief: R.A. Criley 299
Acta Hort. 766, ISHS 2008
leaf spans of 12, 18 and 28 cm, 0.1, 0.3 or 0.5 g of powdered OMF inoculum, respectively,
was placed under the roots of orchids during transplanting. Then, more growth medium
was added.

SEED GERMINATION AND SEEDLING GROWTH OF ANOECTOCHILUS

FORMOSANUS AND HAEMARIA DISCOLOR VAR. DAWSONIANA
Seeds of A. formosanus germinated on agar medium in vitro (Fig. 1A) and on peat
ex vivo in the presence of OMF (Fig. 1B). In nature, orchid seeds germinating under the
parent plants might indicate the presence of OMF in the surrounding soil. The growth of
both A. formosanus and H. discolor was enhanced in vitro (Figs. 2A and 2C) and ex vitro
(Figs. 2B and 2D) by the inoculation of OMF (Fig. 2). Germination (Fig. 2A) and
seedling growth (Figs. 2A-2D) of both orchids were highly enhanced by the inoculation
of OMF (Figs. 1 and 2). The key point for the success of in vitro inoculation of OMF is to
use oat meal agar medium (2.5 g of oat meal and 8 g of Sigma agar in 1 L of water) and to
inoculate the OMF inoculum grown on PDA, as less as possible in the test tube or flask.

GROWTH ENHANCEMENT OF BLETILLA, DENDROBIUM, ONCIDIUM,

PAPHIOPEDILUM AND OTHER ORCHIDS BY OMF
Vegetative growth of Dendrobium houshanense (Kang, 2004; Tseng, 2001; Fig.
3A), Phalaenopsis Tai Lin Redangel V31 (Fig. 3B), Oncidium Gower Ramsey (Fig. 3C;
Chu, 2000) and Paphiopedilum delenatii (Fig. 3D) was also promoted by Rhizoctonia spp.
of OMF (Chen, 2005). The combinations of OMFs for improving the growth of various
orchids were summarized in Tables 2 and 3. All the results showed growth enhancement
by the inoculation of Rhizoctonia spp. on six genera of orchids (Table 3), including
Dendrobium cumulatum and D. macrophyllum (Tseng, 2002); D. candidum and D.
moniliforme (Kang, 2004); D. houshanense (Chang, 2006); D. amethystoglossum, D.
Snow Flake, D. tosaense and D. Chao Praya Garnet (Lin, 2002); Bletilla formosana (Su,
1995) and Oncidium Gower Ramsey (Fig. 3C; Chu, 2000), but with some degree of
specificity.

STRUCTURAL CHANGES OF ORCHID MYCORRHIZAE AS INFECTED BY

RHIZOCTONIA SPP.
There were two types of orchid mycorrhizal infections: (a) Tolypophagy: showing
one layer of host cells adjacent to the epidermis and two layers of digestion cells, (b)
Ptyophagy: showing two layers of passage cells and the phagocyte layer with hyphae
liberating the cytoplasm, which is absorbed into spherical ptysomes (Hadley, l982). Only
tolypophagy type of infection was found in various roots of orchids as infected by either
one of the three Rhizoctonia spp. (Figs. 4A and 4B; Chang, 2005). The OMF once utilized
the nutrients from the host plants to develop pelotons (Fig. 4A), but later the peloton
would be self-digested (Fig. 4B) and would release all of their contents to the host, thus
real symbiosis for the host- plant occurred.

ENZYME ACTIVITY AND COMPONENT CHANGES OF A. FORMOSANUS

Chou (2004) reported that superoxide dismutase activity in leaf, and acid and
alkaline phosphatases activities in roots of A. formosanus and many other constituents
such as ascorbic acid, acid, flavonoid, phosphate, polyphenol and polysaccharide in stem
or leaf were significantly increased by the inoculation of OMF (Table 1). These results
indicated that mycorrhizal A. formosanus plants had higher antioxidant ability than
non-mycorrhizal control, and thus might be more powerful for their medicinal uses.

ENHANCEMENT OF VEGETATIVE AND REPRODUCTIVE GROWTH BY OMF

Tables 1-3 show the growth effects which were significantly increased by the
OMF inoculations. Survival rates of Doritaenopsis hybrids and Phalaenopsis species and
cultivars and their vegetative and reproductive growth were highly promoted by the
inoculation of proper OMF (Table 2; Chang, 2005). OMF could be inoculated to orchids

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of various sizes, and some growth enhancement could be detected 3-4 months later (Lan,
2001; Wang, 2005). Vegetative growth such as leaf number, leaf span and leaf width, and
reproductive growth including flower diameter and flower number, were enhanced (Table
2; Chang, 2005).
Reduced disease infection (less than 1%) of Phalaenopsis for the mycorrhizal-
inoculated plants was observed, while the NM control usually had about 3% or more
diseased plants in the greenhouse. Table 3 shows that various orchids manifested some
degrees of specificity for the OMF. It is recommended that the combination of the
specificity between a host and OMF be tested before large scale inoculation with OMF is
conducted. The author wants to emphasize that the R04 used here, one of the AG-6
Rhizoctonia solani (Lee, 2001), is different from the pathogenic AG-1, 2, 3, 4, 5, 7 and 8
(Pope and Carter, 2006), and until now it has only shown some growth enhancement for
several orchids, and no harmful effects were observed.

PHYSIOLOGICAL EFFECTS OF ORCHID MYCORRHIZA AND THEIR

APPLICATION
There are several beneficial effects of OMF. (1) To enhance symbiotic seed
germination in vitro and ex vitro; (2) to increase the survival rates for the micro-
propagated plantlets or seedlings ex vitro; (3) to enhance both vegetative and reproductive
growths of orchid plants; (4) to result in earlier flowering and increase flower quality; (5)
to increase enzymatic activities, such as acid and alkaline phosphatases, superoxidase
dismutase; and to promote antioxidant abilities by increasing contents of ascorbic acids,
flavonoids, polyphenols and polysaccharides in A. formosanus; and (6) to lower disease
infection rates.

ACKNOWLEDGMENT
The work being reported in this paper was financially supported by the National
Science Council (NSC-91-RD-201; 92-2317-B002031 & 93-2317-B002-020), Taiwan
Salt Company (2002-2004) and Council of Agriculture, Taiwan, 89-Tech.-1.1-Food-69
(09); 91-Agri. Sci. -1.1.1-Food 27(4); 92- Agri. Sci. -1.1.1-Food-Z1(3).

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Tables

Table 1. Mycorrhizal Anoectochilus formosanus contained significantly higher enzyme

activities or component contents than the non-mycorrhizal control (modified from
Chou, 2004).

Plant part Enzyme activity Component contents

ascorbic acid, flavonoid, phosphate,
Leaf superoxide dismutase polyphenol and polysaccharide
Stem - polyphenol and polysaccharide
ascorbic acid, polyphenol, and
Root acid & alkaline phosphatases polysaccharide

Table 2. Significant growth enhancement of three beneficial Rhizoctonia spp. on the

growth of Doritaenopsis Hos Happy Auckland Song and an unknown clone of
Phalaenopsis Sogo Manager four months after inoculation. The growthenhancement
was from high to low efficacy, respectively.

Growth enhancement Doritaenopsis Hos Phalaenopsis

Happy Auckland Song Sogo Manager
Vegetative growth
Leaf no. R02, R01 R01, R02
Leaf span R02, R01, R02
Leaf width R02,
Reproductive growth
Total flower no. R02, R04, R01 R02, R04, R01
Flower diameter R02, R04, R01

Table 3. Combinations of orchid hosts and Rhizoctonia spp. which could result in
significant differences in growth. The mycorrhiza combinations for growth enhance-
ment are listed from high to low efficacy.

Host Orchid mycorrhizal Vegetative Reproductive

fungi (OMF) growth growth
Anoectochilus formosanus R02, R04
Haemaria discolor R01, R02
Dendrobium spp. R01, R04
Doritaenopsis
Hos Happy Auckland Song R02
R02, R01, R04
Paphiopedium delenatii R02, R04
Phalaenopsis amabilis R02
Phalaenopsis Sogo Manager R01, R02, R04
Phalaenopsis Sogo Manager R04, R02

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Figurese

Fig. 1. Faster and effective in vitro (on agar medium, 1A) and in vivo (on peat, 1B)
germination of Anoectochilus formosamus seeds was resulted by mycorrhiza (+M)
than the non-mycorrhizal control (NM).

Fig. 2. In vitro (2A & 2C) and ex vitro (2B & 2D) growth of mycorrhizal (+M; R02, R04)
Anoectochilus formosamus (2A & 2B) and Haemaria discolor var. dawsoniana
(2C & 2D) seedlings had bigger sizes than the non-mycorrhizal control (NM).

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Fig. 3. Ex vitro growth of Dendrobium houshanense (3A), Phalaenopsis Tai Lin
Redangel V31 (3B), Oncidium Gower Ramsey (3C, Chu, 2000) and
Paphiopedium delenatii orchids plants (3D) was highly increased by mycorrhiza
(+M; R01, R02, R04, GD, BF2) than the non-mycorrhizal control (NM).

Fig. 4. SEM micrograph of orchid mycorrhiza as infected by Rhizoctonia spp. (R02) of

beneficial fungi, showed that it was tolypophagy type of infection, i.e., the hyphae
formed peloton (P) in context cells (4A & 4B) of orchid root. Later the peloton
would be digested (4B). CW: cell wall.

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