Botanical Studies (2007) 48: 445-451.

*

Corresponding author: E-mail: hyha@ntu.edu.tw; Tel. &

Fax: +886-2-33664845.

INTRODUCTION

Anoectochilus formosanus (Orchidaceae) is a native

perennial and terrestrial orchid plant grown in the forests

of Taiwan. It is an orchid with beautiful netted-veins

on the upper surface and purple-red color on the lower

leaf surface. It has come to be known as a jewel orchid

(Teuscher, 1978) and is regarded as the "king medicine"

by the aboriginal population of Taiwan. It has been used

to cure diseases, such as cancer, high blood pressure,

diabetes, snake bite, and even liver, heart and lung diseases

(Yen et al., 1996). It has been also used as a special feed

for doves to prolong their stamina during long distance

races (Shiau et al., l995; Liu et al., l987). In recent years,

wild Anoectochilus plants have been intensively collected

and have thus become scare, resulting in a market value

as high as 3-4 times the price of greenhouse cultivated

plants. For the profit in the medicine market, many florists

have started to grow them on a large scale by using

micropropagated seedlings (Shiau et al., 1995; Tang et al.,

1996). Now, in 2006, the greenhouse-cultivated products

are still around 120 USD/kg on a fresh weight basis.

This is mainly due to the fact that the plants are highly

susceptible to Fusarium, Pythium spp, and mites. Also the

growing of this orchid has to be restricted to those areas

with temperatures around 20¢XC or lower. Additionally,

fungicides and pesticides must be applied intensively

to assure good growth. Since this plant is used as an

herb, minimizing the use of chemicals has been of great

importance.

The germination and growth of this orchid could

be stimulated by the inoculation of specific isolates of

OMF (R01, R02 or R04) (Tsai, l997; Chou and Chang,

2004). Differences in enzyme activities and in some

component contents between the non-mycorrhizal control

and the mycorrhizal plants of this orchid are lacking in

the literature and were thus presented in this study. Our

goal was to evaluate the benefits of using OMF in the

commercial production of this orchid.

Growth responses, enzyme activities, and component

changes as influenced by Rhizoctonia Orchid mycorrhiza

on Anoectochilus formosanus Hayata

Doris Chi-Ning CHANG

1,

*

and Ling-Chin CHOU

2

1

Department of Horticulture, National Taiwan University, Taipei, Taiwan, 10617, ROC

2

Department of Life Sciences, National Science Council, Taipei, Taiwan, 10622, ROC

(Received November 6, 2006; Accepted August 8, 2007)

ABSTRACT.

Rhizoctonia spp. of binucleate R02 and multi-nucleate R04 (Rhizoctonia solani, AG-6) of

orchid mycorrhizal fungi (OMF)¡Xcollected and isolated from terrestrial orchid roots in Taiwan, including

Anoectochilus formosanus Hayata, an orchid native to Taiwan¡Xenhanced the growth of A. formosanus plants

to a high degree, both in vitro and ex vitro. OMF (R02 or R04) inoculation used oatmeal agar (OMA) in vitro,

in which mycorrhizal seedlings were larger than non-mycorrhizal controls on Hyponex # 3 agar medium.

Both Rhizoctonia isolates (R02, R04) inoculated alone or mixed could significantly enhance plant grown

ex vitro in terms of plant height, leaf number, root length, and fresh weight. Light and scanning electron

microscopy showed that the infection of OMF on A. formosanus was a type of tolypophagy, with hyphal coils

and pelotons (mycelial masses) formed in the cortical region of the roots. Mycorrhizal plants showed higher

enzyme activities (superoxide dismutase; SOD in leaf; acid and alkaline phosphatases in roots) and markedly

higher constituent contents, like ascorbic acid, polyphenols, and flavonoids, which made the mycorrhizal plants

a better source of antioxidants, and of polysaccharides and phosphates indicating a more potent medicinal

value. Mycorrhizal plants were also susceptible to diseases and mites, so a plastic bag cultivation method was

applied and showed itself to be a very effective and labor saving way to grow fungicide- and pesticide-free A.

formosanus plants.

Keywords: Inoculation; Pelotons; Plastic bag cultivation method; Rhizoctonia spp.; Tolypophagy.

Abbreviations: OMF, orchid mycorrhizal fungi; R02, Rhizoctonia sp.; R04, Rhizoctonia solani; AG-6, Anas-

tomosis group 6.

pHySIOlOGy

446

Botanical Studies, Vol. 48, 2007

MATERIAlS AND METHODS

Usually 20 to 60 plants were grown for each experiment

to assure that at least 12 replicates for each of the follow-

ing treatments could be performed.

Inoculum of Orchid Mycorrhizal Fungi

Orchid mycorrhizal fungi (OMF) of R02 and R04

grown in crushed peatmoss of Rhizoctonia spp. were

used as the so-called solid form of inoculum (Chang and

Chou, 2001; Chou and Chang, 2004). R02 was binucleate

and R04 (Rhizoctonia solani, Anastomosis group 6; Lee,

2001a) was multinucleate Rhizoctonia sp. About 0.2-0.3 g

of fresh solid form inoculum was applied under the roots

of seedlings ex vitro on Sunshine growth medium, which

was a peat mixture.

light microscopy and SEM observation of

orchid mycorrhiza

Root segments, surrounded by mycelia, were selected

for mycorrhizal samples. After being stained with 0.05%

of aniline blue in lactoglycerin for 15-20 min, they were

examined by light microscopy (Koske and Gemma, l989).

For SEM observation, roots were collected and sterilized,

cut and fixed by 3% glutaraldehyde for 1-2 days, then

were dehydrated with acetone series, and critical point

dried (CPD) by liquid carbon dioxide. Finally, the root

segments were coated with gold for 90 s by an Biorad Ion

Coater (Dawes, l971). The SEM samples were examined

by ABT-60 of SEM and digital images were recorded.

Growth of seedlings in vitro and ex-vitro

For symbiotic germination, seeds were collected

in a lamilar flow hood and sprinkled on the following

growth media (Warcup, l973): In vitro symbiotic growth

seedlings were cultivated on oatmeal agar (OMA; 2.5 g l

-1

of oat meal and 11.5 g Difco agar). No-symbiotic growth

seedlings were on: 1. Hyponex # 3 agar, containing 3 g

of Hyponex # 3, 3 g tryptone, 30 g sucrose, and 8 g agar

(modified from Kano, l968); 2. WM medium (including

0.3 g NaNO

3

, 0.2 g KH

2

PO

4

, 0.1 g MgSO

4

¡P7H

2

O, 0.1 g

KCl, 10 g cellulose powder, 0.1 g malt extract and 8 g

Sigma agar in 1 liter); and 3. WY medium (including

0.3 g NaNO

3

, 0.2 g KHPO

4

, 0.1 g MgSO

4

¡P7H

2

O, 0.1 g

KCl, 10 g cellulose powder, 0.1 g yeast extract, and 8 g

Sigma agar in 1 liter). One week after being sown, seeds

were inoculated with OMF (R02 or R04 isolate). Only

one isolate was inoculated for each treatment. Growth

responses of mycorrhizal and non-mycorrhizal seedlings

were compared 6 months after inoculation in vitro.

Ex vitro growth of seedlings were cultivated on

Sunshine growth medium for 4 months in a greenhouse at

Puli in central Taiwan. Rhizoctonia spp. of OMF, namely

R02, R04 and a mixture of R02+R04 were used for the

inoculation of OMF.

A plastic bag cultivation method for A. formosanus

(Lee, 2001a) was applied to the cultivation of fungicide

and pesticide-free plants. By this method, polyethylene

or polypropyrene plastic bags were used to cover the

container with the ex vitro grown A. formosanus plants as

protection against diseases and mites. Field capacity water

(i.e. squeezing the growth medium, yielded only a few

drops of water) and slow-release fertilizer were applied to

the growth medium. The plastic bag was then sealed with

a paper clip.

Estimation of enzyme activities

For the activity of superoxide dismutase (SOD), the

method of Hung (2002) was used. Sigma enzyme kits for

both acid and alkaline phosphatases were obtained, and

methods described in the Sigma technical bulletins # 386

(for acid phosphatase) and # 85 (for alkaline phosphatase)

were applied. Naphthol AS-BI phosphoric acid and

naphthol AS-BI phosphoric alkaline were used as the

substrates for both enzymes, respectively.

Estimation of several component contents

Ascorbic acid, nitrate (Cheng, 1997) and phosphate

were measured by a Merck, RQ flex spectrophotometer.

Also flavonoids (Chang, 2000), polyphenols (Kujala et al.,

2000) and polysaccharides (Lee, 2001b) were estimated

and compared between non-mycorrhizal and mycorrhizal

tissues in root, stem, and leaves.

RESUlTS AND DISCUSSION

The morphology and flower of A. formosanus is shown

in Figure 1A & B. This orchid has netted veins on the

upper surface and red surfaces on its leaves and stems.

The OMF inocula, namely R02 and R04, were Rhizoctonia

spp. (Tsai, l997), isolated from wild grown terrestrial

orchid roots, including A. formosanus of Taiwan. The

chosen isolates R02 and R04 enhanced the growth of this

orchid more than R01 (Chou and Chang, 2004). Thus,

R02 and R04 inocula were used for subsequent symbiotic

germination and growth responses.

Light microscopy showed that the hyphae of OMF

could infect the orchid roots either by root hairs or through

root epidermal cells (Figure 1C) and formed pelotons

in the cortical region of the roots (Figure 1D). SEM

observation showed that typical orchid mycorrhiza were

formed in the cortex region of A. formosanus roots (Figure

2). In mycorrhizal root, hypha could penetrate the cell

wall (Figure 2A); also noted was the presence of young

and old pelotons in the cortex cells of the roots (Figure

2B-D), indicating that it was a tolypophagy-type infection,

in which definite layers of host cells and digestion cells

occurred. This is a common relationship characteristic of

the majority of orchids (Hadley, 1982). It should be noted

that there were connections between the pelotons in the

adjacent cells across cell walls (Figure 2C). The size of the

connections showed that they were hyphae (H).

Growth of plantlets in either Murasgige & Skoog (MS)

medium or other common micropropagated media would

CHANG and CHOU ¡X Mycorrhizal

Anoectochilus formosanus

447

Figure 1. Morphology (A) and flower (B) of Anoectochilus formosanus Hayata. The Rhizoctonia hyphae (H) of orchid mycorrhizal

fungi (C) could infect either through root hair (RH) or root epidermal cells (E) and then formed pelotons (P) in cortex (D) of root.

Figure 2. Orchid mycorrhizal development processes as revealed by scanning electron microscopy. Note the hypha (H) could pass

through cell wall (CW) in A and form peletons (P) in B-D in the cortex cells of orchid roots. Later the peleton in D would be digested.

448

Botanical Studies, Vol. 48, 2007

cause the overgrowth of the OMF hyphae, and result

in the death of seedlings or plantlets. Thus, OMA was

chosen for the growth of mycorrhizal plantlets in vitro

(Chou, 1997, 2004) while the growth of non-mycorrhizal

plantlets was better in Hyponex # 3 agar (H3A) medium

(Figure 3; Chou and Chang, 2004). After inoculation with

Rhizoctonia sp. isolate and 4 months of ex vitro growth,

results showed that both isolates (R02 and R04) could

enhance plant height, number of leaves, root length, and

fresh weight (Figure 4). The mixed inoculum of R02+R04

showed higher growth enhancement for strains L1 and P

of A. formosanus than the single kind of inoculum (R02 or

R04). Thus the mixed inoculum of Rhizoctonia spp. could

be considered for practical use. The inoculation of R02 or

R04 for plantlets ex vitro, was tested for more than 10,000

plantlets both in Taipei and at Puli (central Taiwan), and

all showed enhanced growth. Many growers asked for a

continuous supply of OMF inoculum in their commercial

production of A. formosanus. Thus R02 or R04 isolates

are recommended for such production of A. formosanus

ex vitro. A commercial supply of pathogen-free OMF

inoculum would become a practical trend in the near

future. Also the plastic bag cultivation method had proven

to be an effective and labor-saving method of cultivating

fungicide and pesticide-free plants against disease- and

mite-susceptible A. formosanus plants grown ex vitro. If

field capacity water and a small amount of slow release

orchid fertilizer were applied before sealing the bag with a

paper clip, then watering or applying fertilizer to the plants

in the plastic bag was unnecessary for 6-8 months.

Figure 5. Acid (A & B) and alkaline (C & D) phosphatas es enzymatic histochemistry in non-mycorrhizal (A & C) and orchid

mycorrhizal roots (B & D) of Anoectochilus formosanus Hayara, Mycorrhizal roots (B & D) showed higher enzyme activities than the

non-mycorrhizal control (A & C). Note that the alkaline phosphatase activity was only shown in fungal structures (D).

Figure 4. Growth of five Anoectochilus formosanus Hayata lines

(C, L1, L3, P & T) in greenhouse as cultivated in plastic bag

and inoculated with Rhizoctonia sp. of orchid mycorrhizal fungi

(R02, R04) for 4 months ex vitro.

Figure 3. Growths of Anoectochilus formosanus Hayata in H3A,

WM, WY and OMA media of non-mycorrhizal control (NM)

and inoculated with orchid mycorrhizal fungi (R02 or R04) for 6

months in vitro.

CHANG and CHOU ¡X Mycorrhizal

Anoectochilus formosanus

449

Changes in SOD activity and acid and alkaline

phosphatases as well as some component contents in non-

mycorrhizal and mycorrhizal tissues were estimated.

Results showed that in root: acid and alkaline phospha-

tases activities (Figure 5); in leaf: SOD activity and ascor-

bic acid, flavonoid, polyphenol (Figure 6), phosphate and

polysaccharide contents (Figure 7), in stem: polyphenol

and polysaccharide contents and meanwhile ascorbic acid,

polyphenol and polysaccharides contents (Figures 6 & 7)

all increased significantly more in the mycorrhizal tissues

than in the non-mycorrhizal control. The only exception

was the nitrate content, which showed no difference be-

tween non-mycorrhizal and mycorrhizal tissues (Figure

7). The alkaline phosphatase activity was present only in

the fungal structures (Figure 5-D). This meant that this

enzyme was brought in by the OMF, indicating a more

effective way for phosphate metabolism. The results indi-

cated that mycorrhizal tissues were better antioxidants than

the non-mycorrhizal control, with higher SOD activity and

more ascorbic acid, polyphenol, and flavonoids (Figure

6). Thus, as a medicine, mycorrhizal plants would become

more effective.

There are six important findings in this paper: 1. An in -

vestigation of the mycorrhizal literature indicates that this

is the first paper to report on the application of Rhizoctonia

spp. to the successful commercial-scale cultivation for A.

Figure 6. Superoxide dismutase (SOD) activity and chemical

components of non-mycorrhizal (NM) and mycorrhizal (R02,

R04) Anoectochilus formosana Hayata.

Figure 7. Chemical components of non-mycorrhizal (NM) and

mycorrhizal (R02, R04) Anoectochilus formosana Hayata.

formosonus orchids; 2. Previously we were told that only

bi-nucleated Rhizoctonia spp. were useful in enhancing

the growth of orchids. Our data clearly indicated that both

bi-nucleate (R02) and multi-nucleate (R04) Rhizoctonia

isolates can enhance the germination (Chou and Chang,

2004) and growth of A. formosonus (Chou, 2004); 3. The

Hyponex #3 agar medium was very easy to prepare and

resulted in the highest A. formosonus growth rate in our

study. Previously some scientists used only Hyponex #1

agar medium (Kano, l968; Liu et al., 2001) while others

used more complex agar media, such as MS medium for

orchids; 4. Our data clearly demonstrated the applicabil-

ity of single or mixed isolates of Rhizoctonia spp. to the

practical horticulture production of orchids, especially for

the inoculation of outplanted seedlings ex vitro; 5. This is

the first report to compare the differences between non-

mycorrhizal and mycorrhizal tissues for their enzyme

activities and component contents, and to prove that the

mycorrhizal tissues of A. formosanus become more effec-

tive as a medicine; 6. Before disease and mite-resistant A.

formosanus strains are found, the plastic bag cultivation

method developed by our group, has proved to be a very

effective and labor saving way to grow fungicide- and

pesticide-free A. formosanus plants.

To date, we have tested more than five commercial

strains of A. formosanus and concluded that both R02 and

R04 (R solani, Anatomosis group 6) definitely enhance

their growth. However, mycorrhizal plants were also

susceptibe to diseases and mites. Thus the plastic bag

cultivation method was developed to cultivate fungicide-

and pesticide-free A. formosanus plants.

CONClUSIONS

It is highly recommended that H3A medium be used

for the asymbiotic growth and OMA for mycorrhizal

plantlets of A. formosonus in vitro. The inoculation of

R02 or R04 enhances the growth of A. formosonus in vitro

and ex vitro. We also strongly recommend inoculating

OMF (R02 or R04) during outplanting of the plantlets for

practical production of A. formosanus. The mycorrhizal A.

formosanus plants would be a better antioxidants than the

non-mycorrhizal control plants. The plastic bag cultivation

method is a very effective and labor saving way to grow

fungicide- and pesticide-free A. formosanus plants.

Acknowledgments. This research was financially

supported by NSC grants: NSC- 88-2313-B-002-060 and

NSC - 88- 2317- B-002-001. The orchid mycorrhizal fungi

(R02 and R04) were isolated by Ms. Ching-Yi Tsai and

were highly appreciated.

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CHANG and CHOU ¡X Mycorrhizal

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451