Growth and development of Phaius tankervilleae ( Banks ) Blume when inoculated with orchid mycorrhizal fungi

1 Department of Horticulture and Landscape Architecture, National Taiwan University, Taipei, Taiwan 10617, Republic of China. 2 Herbarium (TAI), College of Life Science, National Taiwan University, Taipei, Taiwan 10617, Republic of China. 3 Taoyuan District Agricultural Research and Extension Station, Council of Agriculture, Executive Yuan, Taoyuan, Taiwan 327, Republic of China. 4 Department of Agronomy, National Taiwan University, Taipei, Taiwan 10617, Republic of China.


INTRODUCTION
Phaius tankervilleae (Banks) Blume is a large terrestrial orchid native to Taiwan that is distributed from India, Sri Lanka and Southern China, through Thailand and Malaysia to Indonesia, Australia and the Pacific islands.In Taiwan, it grows in broad leaved forests at elevations below 1000 m and throughout Lanyu Island.The orchid favors high temperatures and wet environments, and is not cold-resistant (Su, 2000).P. tankervilliae is a robust plant that blooms easily.Plant height 60 to 200 cm, without slender stems, fleshy pseudo bulbs ovoid-elliptic and internode very close, alternate leaves, ovatelanceolate or broad lanceolate 3 to 5, 40 to 60 cm long, leaves papery, surface wrinkle.Flower stalk thick 60 to 100 cm long, formation from the side of pseudo bulb, 6 to 20 flowers of one inflorescence, flower diameter 8 to 10 cm.Sepals white outside, white to yellowish white and tinged with red-brown inside, petals red-brown or purplish, lip yellowish white, tinged with red and decorated with dark purple spots (Bechtel et al., 1986;Su, 1990;Yeh et al., 2007).Horticulturally, the flowers are large and beautiful and flower from April to July.A single flower lasts about 20 days; the inflorescence itself lasts up to 45 days (Chang and Jian, 2010).P. tankervilliae has slender leaves, graceful greenery, and can be used as a potted plant, a garden plant, or as cut flowers or leaves (Mukherjee, 1979;Lee, 1989;Chang and Jian, 2010).P. tankervilleae has been overharvested in the wild and is on the verge of extinction (Su, 1989).The flower of P. tankervilleae is big and beautiful with great ornamental and economic value (Mukherjee, 1979;Lee, 1989;Chang and Jian, 2010).Only a small amount of P. tankervilleae could be found in the flower market of Taiwan.Su (1989) reported that the wild population of P. tankervilleae was gradually reduced (Su, 1989).It is urgent to preserve this endangered orchid.
The sexual reproduction of P. tankervilleae is accomplished through seed germination with fungal symbiosis and in vitro seed germination to obtain a seedling.In asexual reproduction, the top of the stem or flower stalk are used to culture an in vitro seedling, reproductive ramets and the flower stalk are buried in the soil and a seedling forms on the node of flower stalk (Lee, 1989;Wu 1991;Tsai, 2010).Previous studies demonstrated that inoculation with mycorrhizal fungi markedly improved vegetative and reproductive growth in Orchidaceae plants (Rasmussen et al., 1990;Chu, 2000;Takahashi et al., 2006;Chang, 2008;Smith et al., 2009;Wu et al., 2011).Orchid mycorrhizal fungi (OMF)-inoculated mother and daughter Oncidium sp.showed increased plant height and pseudo bulb length and width compared to the nonmycorrhizal control; leaf quantity, thickness, fresh weight, surface area, chlorophyll and soluble sugar content were also greater (Chu, 2000).Leaf length and fresh weight of Paphiopedilum delenatii Guillaumin orchids was greatly increased by OMF R04 and PaR2-inoculation (Tsai, 2003).Phalaenopsis amabilis (L.) Blume var.Aphrodite (Reichb.f.) Ames inoculated with R01 showed higher stalk length, and R02-inoculated plants showed increased floral diameter and higher bolting rates (Lan, 2001).Doritaenopsis Luchia Davis × Doritaenopsis Taisuco Firebird seedlings inoculated with R02 had a 40% flowering rate compared to 0% in the non-mycorrhizal control kept at the natural temperature in Taipei (Lan, 2001).Haemaria discolor var.dawsoniana showed greater stalk length and flower number when inoculated with R01 (Chou, 2004).
Orchidaceae Phaius species can be produced through in vitro seeding or tissue culture propagation, but plantlet survival rates decrease during the transplantation (personal communication).Since there is relatively little data on P. tankervilliae, identifying the effects that some OMF have on its growth and development was the main focus in selecting the appropriate fungi to improve its Cheng et al. 5645 survival rate during the transplant period, and on enhancing the vegetative and reproductive growth of P. tankervilleae.We also investigated the effects of OMF on orchid growth during the seedling stage, flowering period regulation and flower production promotion.This horticultural research can be useful for mass production of the seedling, regulating flowering periods, and improving the yield and quality of P. tankervilleae flowers.It can show the ornamental value and economic potential of this native Taiwanese orchid and promote its preservation, restoration and development as an ornamental plant.

Mycorrhizal inocula
In this experiment, we isolated four strains of Rhizoctonia spp.including R01 (Rhizoctonia solani), R02 (Rhizoctonia sp., teleomorph = Ceratobasidium sp.; AG-A), R15 (Rhizoctonia sp., teleomorph = Ceratobasidium sp.; AG-Fb) and R19 (Rhizoctonia sp., teleomorph = Ceratobasidium sp.; AG-G), identified through DNA analysis, that were cultured in our laboratory for more than seven years.They all proved to be non-pathogenic (Chang, 2008).They were cultured on a medium prepared by mixing peat with 20% V8 juice to reach field capacity (Stevens, 1974).All P. tankervilleae plants used in this experiment were inoculated with a single fungal strain.All OMF used in this research are stored in the Bioresource Collection and Research Center at the Food Industry Research and Development Institute in Hsinchu, Taiwan.

Medium and culturing method
The tissue culture container seedlings of P. tankervilliae were obtained and removed from glass containers, then planted in 7.5 cm-diameter black plastic pots with the mix media at a 1:1:1 (v:v:v) (volume) ratio of peat moss, coconut fiber and tree fern fiber.Samples were divided into five groups of 36 seedling each, including the non-mycorrhizal (NM) control and mycorrhizal treatment (Rhizoctonia isolates R01, R02, R15 and R19).
Treatments were arranged in a completely randomized design (CRD).The roots of each seedling received 0.2 g of fungal inocula.
As the plants became larger, they were transplanted with same medium into 18 cm-diameter plastic pots.Each pot initially received 2 g of Osmocote (14N-5.2P-11.6K)and was watered once every week.Water-soluble fertilizer (Peters, Scotts, Marysville, USA) was applied every two weeks, changing from 0.5 g·L -1 of 30N-4.3P-8.3Kwater-soluble fertilizer (Peters, Scotts, Marysville, USA) at the vegetative stage to 0.5 g·L -1 of 10N-12.9P-16.6Kwater-soluble fertilizer at the reproductive stage.The experimental site was a plastic greenhouse on the National Taiwan University campus (10 m above sea level), the average value of the photosynthetic photon flux (PPF) was 200 to 250 µmol·m -2 ·s -1 at noon, and the average values of the highest and lowest temperatures were 32.21 and 15.21°C respectively.

Carbohydrate content measurement
The analytical method for measuring carbohydrate content was modified from the method described in the study of Luchsinger and Cornesky (1962), Yoshida et al. (1972) and Lindsay (1973).The third and fourth leaves from the distal end and pseudo bulbs were taken from matured P. tankervilliae plants after 26 months of cultivation and the dried sample pulverized for testing.To evaluate starch content, the following steps were taken: (i) the sample was extracted with 80% EtOH and distilled water, (ii) 20 mM (pH 6.9) of sodium phosphate buffer was added to the precipitate, and then the sample was heated in a boiling water bath for 15 min.0.5 ml αamylase was then added and the sample placed in a 37°C water bath for 16 h. 1 ml crude extract was obtained to which was added 2 ml DNSA (3,5-dinitrosalicyclic acid reagent) before returning to a boiling water bath for 10 min, cooled to room temperature and 2 ml distilled water was then added.Absorbance was then (iii) measured by a spectrophotometer (HITACHI, 2800 JP, Japan) at A570 wavelength and the value estimated.To evaluate total soluble sugar content, we took the following steps: (i) the sample was extracted twice with 80% EtOH; (ii) the extract was heated in the boiling water bath to remove ethanol, (iii) then chloroform and distilled water were added; (iv) anthrone solution was slowly added in an ice bath and then boiled in the water bath for 7 min 30 s before (v) measuring the absorbance by a spectrophotometer (HITACHI, 2800 JP, Japan) at A630 wavelength to estimate the value.

Photosynthetic rate estimation
The photosynthetic rate for matured P. tankervilliae plants after 26 months growth was detected on a 6 cm 2 leaf area using LI-6400 portable photosynthesis system (LI-COR Inc., Nebraska, U.S.A) (Jiang, 1996).The photosynthetic rate was measured at the second leaf of the daughter pseudo bulb from 5 to 10 cm above the pseudo bulb from 9 to 11 AM.

Plant growth and development
Plant height (leaf tip), number of leaves, leaf length and width, pseudo bulb diameter, number and length of flower stalks and number of flowers were measured for each P. tankervilliae plant.
Each treatment involved 36 plants, 15 from each of these groups of 36 were randomly selected for statistical analyses, using ANOVA (analysis of variance) and LSD (least significant difference) test to compare the effects of treatments.

Anatomic observation of orchid mycorrhizal
We collected roots of P. tankervilleae with or without OMF inoculation and cleaned and stained them with aniline blue in acidic glycerol (Koske and Gemma, 1989) for optical microscopy (Olympus BH-2 USA).For scanning electron microscope (SEM) observations, the collected roots were dissected into sections, fixed with 2.5% glutaraldehyde for 10 to 12 h, dehydrated in an acetone series, critical point dried (SPI # 13200-AB Manual CPD) by liquid carbon dioxide, coated with gold for 90 s by a Biorad Ion Coater (Dawes, 1971), then examined and digitally recorded using the SEM Topcon-60.

RESULTS AND DISCUSSION
The survival rates (SR%) of P. tankervilleae seedlings were significantly increased for 22 months after inoculation with R02, R15, R19 when compared to those of the non-mycorrhizal (NM) control plants.The SR% of P. tankervilleae seedlings after treatments with the R15 and R02 isolates were 79 to 81% and 78 to 80%, respectively, a 27 to 31% higher SR% compared with 48 to 54% for the NM control plants (Figure 1).Plant height and leaf length were greatly increased in P. tankervilleae plants inoculated with R02 or R15 for 22 months.Plant height and leaf length were lower in R01-treated plants as compared with the non-mycorrhizal (NM) control plants (Table 1).The NM control, R02, and R15-inoculated plants were thus used to measure the carbohydrate content and photosynthetic rates.Results showed that when plants were inoculated with the R02 isolate, the rate of net CO 2 fixation increased significantly as compared with that for plants inoculated with R15 or NM control group (Figure 2).Oncidium orchids typically have large pseudo bulbs that store the carbohydrates necessary for flowering (Yong and Hew, 1995;Chang and Lee, 1999).Cymbidium orchids only produce flowers when the amount of carbohydrates stored in their pseudo bulbs is sufficient (Komori, 1986).Since P. tankervilleae plants also have large pseudo bulbs (Tsai and Chang, 2009), we wanted to determine their carbohydrate content during the early stage of flower bud differentiation.Analytical results indicated that 80% EtOH-soluble sugars in leaves and starch content in pseudo bulbs of plants at 26 months after inoculation with R02 were higher than those in NM control plants (Table 2).Total carbohydrate content in leaves and pseudo bulbs of OMF (R02)-inoculated plants was also higher than in NM control plants.
Furthermore, P. tankervilleae plants inoculated with Rhizoctonia spp.flowered early (Figure 3), and plants inoculated with R02 or R19 isolates flowered 5 days earlier than did NM control plants (Table 3 and Figure 3).Other than greater height as compared to the NM control, no other vegetative growth differences, such as number of leaves and pseudo bulbs, occurred between the R01inoculated plants and the control.Compared to the NM control plants, plants inoculated with R01, R02, R15 or R19 isolates for 28.5 months had significantly increased flower stalk length, with R19 isolates also significantly increasing the number of flowers (Table 4 and Figure 3).
The NM P. tankervilleae plants did not have any pelotons in their root cortex cells (Figure 4A), while plants infected with R02 had pelotons built by a number of mycelia (Figure 4B).Although, SEM showed no pelotons in the cortex cells of control group roots (Figure 5A), they were present in the R02-infected plant roots (Figure 5B).
Figure 5C gives details on the infected cells that formed pelotons while Figure 5D shows digested hyphae.The mycelia extended into the surrounding non-infected cells after the pelotons had degraded into amorphous clumps, Cheng et al.         after which pelotons formed until the entire root was infected.This particular infection process is known as tolypophagy.Plants infected with R15 or R19 also produced pelotons, the digested amorphous clumps also can be observed (Figure 5E and F).The Mycorrhizal structures formed by different Rhizoctonia species in P. tankervilleae roots were all similar.The advantages of using mycorrhizal P. tankervilleae include an increased transplant survival rate for seedlings grown in glassware.Tsai (2003), Jin et al. (2009) and Smith et al. (2009) had similar results with other Orchidaceae species.Inoculation with an appropriate OMF (R19) was thus beneficial for P. tankervilleae reproductive growth, earlier flowering, increased number of flower stalks, and increased flower count.Similar results were obtained by Lee et al. (1997), Chu (2000), Takahashi et al. (2006) and Smith et al. (2009) with other Orchidaceae plants.Plants inoculated with the R02 isolate of Rhizoctonia sp. had higher total carbohydrate content and more flower stalk length than did NM control plants; those inoculated with R02 also had a higher CO 2 fixation rate (higher rate of photosynthesis).Inoculation with orchid mycorrhizal fungi increased chlorophyll content (Chu, 2000;Chou, 2004).The fungi also help turn plant cellulose into carbohydrates that are easily absorbed, and can act as a carbon source for orchids (Smith, 1966).Furthermore, OMF provides minerals, vitamins, and other nutrients for plants (Bernard, 1911;Hadley, 1984;Alexander and Hadley, 1985), promotes the absorption of nitrogen, phosphorus and potassium for seedlings (Alexander and Hadley, 1984;Zhao et al., 1999), and promotes plant growth and development, which finally leads to better reproduc-tive growth than in NM control plants.These results lead to the conclusion that the symbiotic relationship between OMF and P. tankervilleae enhances the photosynthetic rate, seedling viability, and repro-duction growth.Many researches focused on the Orchidaceae symbiosis with mycorrhizal for resto-ration of orchids (Batty et al., 2001;Beltran-Nambo et al., 2010).Further application of OMF may preserve and restore the endangered orchid species for future commercial production.

Conclusion
In conclusion, the inoculation of OMF was done with just a small amount (about 0.3 g/plant) of inoculum under the roots of orchid; hence, it was very simple.However, this treatment could highly increase the survival rate, growth and development of this orchid.Hopefully, this practice will help to preserve and restore more of the endangered orchid species.

Figure 2 .
Figure 2. Net CO2 uptake rate for Phaius tankervilleae seedlings after inoculation with orchid mycorrhizal fungi for 26 months.NM indicates non-mycorrhizal control.

Figure 3 .
Figure 3. Vegetative and reproductive growth of Phaius tankervilleae (Banks) Blume seedlings after inoculation with various Rhizoctonia spp. of orchid mycorrhizal fungi for 30.5 months.NM indicates non-mycorrhizal control.
non-mycorrhizal control; R01, R02, R15, R19 = orchid mycorrhizal fungi.Each treatment had 15 replicates.The effects of mycorrhizal infection of Phaius tankervilleae were tested by ANOVA.The F values of ANOVA are presented in the table.Means in each column followed by different letters were significantly different (P=0.05) as determined by LSD test.

Figure 5 .
Figure 5. SEM observation of structural changes by hyphal infection in the cortex cells of Phaius tankervilleae (Banks) Blume roots after inoculation with Rhizoctonia spp. of orchid mycorrhizal fungi R02 (A-D), R15 (E), or R19 (F) for 22 months.A, Control.B, Root cortex cells of P. tankervilleae (Banks) Blume as inoculated with R02.C, Root cortex cells of P. tankervilleae (Banks) Blume as inoculated with R02 and the formation of peloton (P).D, Later stages of pelotons (P), which were digested in the roots of P. tankervilleae (Banks) Blume as inoculated with R02.E, Later stages of pelotons (P), which were digested in the root of P. tankervilleae (Banks) Blume as inoculated with R15.F, Later stages of pelotons (P), which were digested in the root of P. tankervilleae (Banks) Blume as inoculated with R19.

Table 1 .
Vegetative growth for seedlings of Phaius tankervilleae after inoculation with various Rhizoctonia spp. of orchid mycorrhizal fungi for 22 months.NM = non-mycorrhizal control; R01, R02, R15, R19 = orchid mycorrhizal fungi.Each treatment had 15 replicates.The effects of mycorrhizal infection of Phaius tankervilleae were tested by ANOVA.The F values of ANOVA are presented in the table.Means in each column followed by the different letters were significantly different (P=0.05) as determined by LSD test. *

Table 2 .
Carbohydrate content of leaf and pseudo bulb for seedlings of Phaius tankervilleae after inoculation with orchid mycorrhizal fungi (R02, R15) ex vitro for 26 months.
*NM = non-mycorrhizal control; R02, R15 = orchid mycorrhizal fungi.Each treatment had 9 replicates.The effect of mycorrhizal infection of Phaius tankervilleae was tested by ANOVA.The F values of ANOVA are presented in the table.Means in each column followed by different letters were significantly different (P=0.05) as determined by LSD test.

Table 3 .
Days before flowering in Rhizoctonia spp.-inoculatedPhaius tankervilleae.The effects of mycorrhizal infection of Phaius tankervilleae were tested by ANOVA.The F values of ANOVA are presented in the table.Means in each column followed by different letters were significantly different (P=0.05) as determined by LSD test.

Table 4 .
Comparison of growth and development of Rhizoctonia spp-inoculated Phaius tankervilleae seedlings at 28.5 months.