In vitro seed germination and seedling growth of an endangered epiphytic orchid, Dendrobium officinale, endemic to China using mycorrhizal fungi (Tulasnella sp.)

Scientia Horticulturae 165 (2014) 62–68

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In vitro seed germination and seedling growth of an endangered epiphytic orchid, Dendrobium officinale, endemic to China using mycorrhizal fungi (Tulasnella sp.) Xiao Ming Tan a,b , Chun Lan Wang a , Xiao Mei Chen a , Ya Qin Zhou b , Yun Qiang Wang c , An Xiong Luo a , Zhi Hua Liu a , Shun Xing Guo a,∗ a Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing 100193, China b Guangxi Botanical Garden of Medicinal Plant, Guangxi Key Laboratory of Medicinal Resources Conservation and Genetic Improvement, Nanning 530023, China c Botanical Garden of Xishuangbanna South Medicine, Yunnan 666100, China

a r t i c l e

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Article history: Received 15 April 2013 Received in revised form 20 October 2013 Accepted 27 October 2013 Keywords: Mycorrhizal fungi Tulasnella Epiphytic orchid Seed germination Plant growth

a b s t r a c t To date, tropical orchids appear to commonly associate with mycorrhizal fungi assignable to Tulasnella (Basidiomycota) for their seed germination and developmental needs in situ. In this study, two Tulasnella strains (JC-02 and JC-05) isolated from roots of an endangered species, Dendrobium nobile Lindl (Orchidaceae) collected from Yunnan province in China, were identified using the nuclear ribosomal internal transcribed spacer (ITS) and 5.8S rDNA sequences. Seed germination and plant growth were evaluated up to 11 weeks and 7 weeks after interaction with Tulasnella fungi, respectively. The results revealed that the two isolates could promote seeds germination up to stage 5 after sowing for 11 weeks, and the rates of germination were 98.47% and 99.05%, respectively, higher than that of control (81.05%). Without fungi, seed development was arrested at stage 2. After inoculating Tulasnella isolate to seedlings for 49 days, it was found that mycelium formed pelotons in the cortical cells of roots in the form of intact and degenerate pelotons. Significant differences were detected between the control group and the experimental group treated with Tulasnella isolates in dry weight and fresh weight of plants respectively. The study firstly demonstrated that two different strains of Tulasnella vary in their ability to facilitate germination/development of D. officinale in vitro. © 2013 Published by Elsevier B.V.

1. Introduction The association with a suitable mycorrhizal fungi, which can provide carbon sources, is essential to the seed germination of all orchids in nature (Yam and Arditti, 2009). Due to limited seed reserves, epiphytic orchids rely on mycorrhizal fungi as a carbon source to facilitate seed germination and/or seedling development to a photosynthetic stage under natural conditions (Dressler, 1990; Rasmussen, 2002; Porras-Alfaro and Bayman, 2007). In both photosynthetic and achlorophyllous orchids, mycotrophy is now thought to be retained well into maturity as a source of carbon, even for epiphytic species, such as Cephalanthera longifolia and Cephalanthera damasonium (Leake, 1994; Gebauer and Meyer, 2003; Julou et al., 2005; Abadie et al., 2006; Rasmussen and Rasmussen, 2007). Moreover, mycorrhizal association have played a more and more important roles in the conservation and horticultura of orchids

∗ Corresponding author. Tel.: +86 010 62819871; fax: +86 010 62819871. E-mail address: [email protected] (S.X. Guo). 0304-4238/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.scienta.2013.10.031

(Rasmussen, 2002; Zettler et al., 2007; Swarts and Dixon, 2009; Nontachaiyapoom et al., 2010). Tulasnella fungi are one of the major group of mycorrhizal fungi forming symbiotic association with orchids worldwide (Pereira et al., 2005). Mycorrhizal fungi of this genus had been isolated from roots of many photosynthetic species, e.g., Amerorchis rotundifolia (Zelmer et al., 1996), Acianthus caudatus (Warcup, 1981), Dactylorhiza majalis (Kristiansen et al., 2001), Diuris spp. (Warcup, 1971), Goodyera oblongifolia (Zelmer et al., 1996), Microtis parviflora (Perkins et al., 1995), Neuwiedia veratrifolia (Kristiansen et al., 2004), Platanthera hyperborean (Zelmer et al., 1996), Spiranthes sinensis (Masuhara and Katsuya, 1994), and others (McCormick et al., 2004; Shefferson et al., 2005). However, few reports have surfaced that document Tulasnella fungi was isolated from Dendrobium species (Nontachaiyapoom et al., 2010). Dendrobium is one of the largest genera of orchid families worldwide, with over 1000 species (Lavarack et al., 2000), and about 74 species in China (Tsi, 1999). Some members of this genus have medicinal value e.g., Dendrobium officinale and Dendrobium nobile (Li et al., 2009). However, more and more Dendrobium species have

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2. Materials and methods

34 cycles at 94 ◦ C for 30 s, 60 ◦ C for 40 s, 72 ◦ C for 30 s, with a final extension at 72 ◦ C for 10 min. The primers ITS1-OF-1, ITS1OF-2 and ITS4-OF used in this study were referred to Taylor and McCormick (2008). The 5 ␮l PCR products were electrophoresed in 1% (w/v) agarose gels and stained with ethidium bromide. After visual inspection assisted by a UV light, 45 ␮l PCR products were purified and sequenced at the Bejing GENEWIZ Biological Engineering Technology & Services. The sequences obtained in this study were submitted to GenBank and the accession numbers of sequences were JN863900 and JN863903.

2.1. Sample collection

2.4. Phylogenetic analysis

Whole shoots of D. nobile (Fig. 1a) and mature capsules (Fig. 1b) of D. officinale were collected from Yunnan Province of South Western China in August 2011. Samples were placed in polyethylene bags, labeled, transported to the laboratory and placed in a refrigerator at 4 ◦ C. The mature capsules were surface-sterilized in 70% (v/v) ethanol for 1 min, 2.5% (v/v) NaClO 15 min and rinsed three times in sterile distilled water, opened using a sterile scalpel and the axenic seeds were used for the test of germination and culture of seedlings.

All sequences were compared with ITS sequences closest downloaded from the GenBank database via BLAST search (Altschul et al., 1990). The sequences were aligned using Clustal X 1.83 with default settings visually (Thompson et al., 1997). The Neighbor-joining (NJ) phylogenetic trees were inferred using heuristic search with 1000 replicates of random sequence addition by using Molecular Evolutionary Genetics Analysis (MEGA 4) (Tamura et al., 2007). Branches corresponding to partitions reproduced in less than 70% bootstrap replicates were collapsed. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option) (Mysore et al., 2011).

been introduced into commercial trade and some members, including D. officinale, are newly extirpated in situ, and are now cataloged as endangered in the Chinese Plant Red Book (Fu, 1992). Therefore, the present report aimed to investigate the mycorrhizal fungi associated with Dendrobium and determine their benefit for seed germination and plant growth. The results from this report are helpful for the actual propagation and conservation of Dendrobium plants and other orchids.

2.2. Isolation, purification and conservation of mycorrhizal fungi Mycorrhizal fungi were isolated using an improved process as used by Nontachaiyapoom et al. (2010). Briefly, hand sections of roots (Fig. 1c) were observed for the presence of pelotons with a light microscope. Then colonized roots were surface-sterilized in 70% (v/v) ethanol for 40 s, 2.5% (v/v) NaClO 2 min and rinsed three times in sterile distilled water. Ten sections per root, obtained with a sterile blade, were placed on the potato dextrose agar (PDA) containing 50 ␮g/ml streptomycin, and 100 ␮g/ml tetracycline. Samples were incubated in the darkness at 25 ◦ C, and observed daily for 30 days under stereomicroscope. Hyphal tips of fungi were transferred to fresh PDA for purification. All isolates growing on the PDA for one week were inoculated into distilled 10% glycerol and placed in ambient temperature for 4 days and then deposited in the ultra cold storage freezer (−80 ◦ C) of Laboratory of Mycology, Biotechnology Center, Institute of Medicinal Plant Development (IMPLAD), and Chinese Academy of Medical Sciences (CAMS). 2.3. Extraction of DNA and PCR amplification of ITS rDNA Isolates in this study were incubated on the fresh PDA in the darkness at 25 ◦ C for 10 days, then the actively growing mycelium on PDA plate were scraped using a sterile pipette tips. The DNA were extracted using the E.Z.N.A.TM Fungal DNA Mini Kit (Omegabiotek, Norcross, USA) referring to the manufacturers’ instructions. PCR amplification reaction of 50 ␮l was as follows: Taq PCR Master Mix 25 ␮l (Qiagen, Bejing), 2 ␮l (5 ␮M) each primer, 19 ␮l H2 O and 2 ␮l genomic DNA. The PCR condition was 95 ◦ C for 2 min,

2.5. Effects of Tulasnella isolate on germination of D. officinale seeds Tulasnella isolates were grown on PDA in darkness at 25 ◦ C. After 10 days, five PDA agar plugs (diameter 2 mm) with active mycelium from the colony margin were inoculated to a petri dish (diameter 9 mm) containing 15 ml sterile oat meal agar (OMA: oat 2.5 g l−1 agar 12 g l−1 , the pH measured at 5.2 prior to autoclaving) with five nylon clothes (1 cm × 1 cm), and grown at 25 ◦ C in dark. After 1 week, approximately 150 axenic seeds of D. officinale (Fig. 1d) were sown on the surface of each cloth. Each treatment was replicated on five plates. The treatment without fungi was used as a control. All treatments were placed in tissue culture chamber in 12 h light at 25 ◦ C for 77 days. To assess and record the seed germination as well as protocorm development, a stereomicroscope (LAEICA S8APO) was used. Stewart et al. (2003) had divided the seed germination and protocorm development of orchids into 6 stages (0–5) just as Table 1 showing, which used as a reference for assessing the percentage of seed germination and protocorm development of D. officinale. 2.6. Effects of Tulasnella isolate on the growth of D. officinale seedlings Axenic seeds of D. officinale were sown onto 80 ml N6 medium (Chu et al., 1975) in 250 ml Erlenmeyer flasks for 60 days at 25 ◦ C under cool-white fluorescent lamps for 12 h photoperiod. After 60

Fig. 1. (a) Shoots of D. nobile (arrow), bar = 2.5 mm; (b) mature capsules of D. officinale, bar = 10 mm; (c) root of D. nobile (arrow), bar = 5 mm; (d) seed of D. officinale, bar = 10 ␮m.

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Table 1 Seed germination and protocorm development in D. officinale. Stage

Description

0 1 2

No germination, viable embryo Enlarged embryo, production of rhizoid(s) (=germination) Continued embryo enlargement, rupture of testa, further production of rhizoids Appearance of protomeristem Emergence of first leaf Elongation of first leaf

3 4 5

Adapted from Stewart et al. (2003).

days, the seedlings were transferred to 80 ml of 1/2 MS medium in 250 ml Erlenmeyer flasks (Murashige and Skoog, 1962), and subcultured for two months in the same condition. Then, one axenic seedlings and two colonized agar plugs (diameter 12 mm) from the edge of fungal colony, which grown on PDA in dark for 10 days, were transferred to a sterile cylindrical glass bottle having rotted wood and 10 ml deionized water, covered with a plastic sheet and autoclaved for 2.8 h in 121 ◦ C. The agar plugs (diameter 12 mm) transferred from PDA plate without fungi were inoculated as the control. All cultures were grown at 25 ◦ C and 1500 lux under cool-white fluorescent lamps for 12 h photoperiod. 7 weeks after inoculation, the fresh weight was measured for per plant, and then the plants were dried to constant weight for the dry weight of plant at 50 ◦ C. The initial fresh weight and dry weight were assessed using the mean of 30 plants randomly selected before inoculation. Roots of some seedlings were fixed in FAA (50% ethanol: acetic acid: formaldehyde, 18:1:1, by vol.) and stored at 4 ◦ C.

2.7. Morphology observation To observe the morphological characteristics of the interaction between fungi and seed, squashes of the protomeristem were prepared according to the process of Brundrett et al. (1984). The roots fixed in FAA were taken out and then dehydrated in a graded ethanol series, embedded in paraffin and 12 ␮m sections taken. Sections were stained either with safranine T and fast green, sealed by neutral balsam, observed and photographed with a light microscope (ZEISS Axio Imager A1, Germany).

2.8. Data collection Eleven weeks after sowing, the seeds germination and the development stage were assessed using the process described by Stewart et al. (2003). The percentages of seeds germination for per stage were calculated using the following formula: Percentage of seeds germination =

The number of seeds in per germination stage × 100 The total number of viable seeds in the sample

Seven weeks after inoculated, all seedlings were harvested and the fresh weigh were measured. The seedlings were then dried at 50 ◦ C for three consecutive days and the dry weight of the seedlings was measured. 2.9. Statistical analyses The SPSS Statistics software was used for statistical analyses. Per treatment containing 30 samples and all tests were performed in a complete randomized block design. The data of all tests repeated 3 times were analyzed by the one-way analysis of variance (ANOVA). The significant differences between per treatment was performed with the Duncan’s multiple-range test (P ≤ 0.05). 3. Results 3.1. Fungal isolation and identification Based on morphological characteristics and phylogenetic analysis of the internal transcribed spacer (ITS) sequences, two isolates collected from roots of D. nobile were identified as the members of the genus Tulasnella. The colonies of the two fungi grew at the rate of 5–6 mm/day. Three weeks after inoculated on PDA, colonies of the two isolates were white to cream-colored with regular margin (Fig. 2a). Monilioid cells are spherical to ellipsoidal, 6–10 ␮m × 7–20 ␮m, in branched (Fig. 2b and c). Result of the BLAST searches showed that the internal transcribed spacer (ITS) and 5.8S rDNA sequences of two isolates had 98% identity with the internal transcribed spacer (ITS) and 5.8S rDNA sequences of Tulasnella sp. Neighbor-joining (NJ) phylogenetic tree analysis using the internal transcribed spacer (ITS) and 5.8S rDNA sequences showed the two fungal endophytes clustered together with the Tulasnella genus with a bootstrap support of 100% (Fig. 3).

Fig. 2. Cultural and morphological characters of Tulasnella isolate. (a) Culture of isolate JC-02 at 10 days on PDA, bar = 1 cm. (b) Monilioid cells of isolate JC-02, bar = 10 ␮m. (c) Monilioid cells of isolate JC-05, bar = 20 ␮m.

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100 100 100

100

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Tulasnella calospora DQ388044 Tulasnella calospora GU166421 JC-05 JC-02 Tulasnella sp. AY373264 Epulorhiza sp. EF393625 Tulasnella sp. AY373266 Tulasnella sp. AY373281 Epulorhiza sp. GQ856214 Tulasnella calospora DQ388041 Tulasnella deliquescens AY373291 Sebacina vermifera DQ983816 Sebacina sp. DQ974770 Ceratobasidium sp. DQ093647 Ceratobasidium sp. DQ102431 Ceratobasidium sp. DQ102430 Armillaria sinapina FJ495039

Fig. 3. Neighbor-joining phylogenetic tree of ITS rDNA sequences of endophytic fungi from roots of Dendrobium nobile. Armillaria sinapina was used as the outgroup. Bootstrap values (calculated from 1000 resamplings) ≥50% are shown at each branch.

Fig. 4. Symbiotic seed germination and protocorm developmental stages of D. officinale using mycorrhizal fungi Tulasnella sp., JC-02, cultured on oat meal agar 11 weeks after sowing; (a) stage 0; (b) arrow show stage 1 and star show stage 2; (c) arrow show stage 3 and star show stage 4; (d) stage 5 of protocorm developments; note elongation of first leaf. e peloton structures and coiled hyphae in the cortical cells of JC02+ protomeristem stained with 0.05% (w/v) trypanblue in lactoglycerol; (f) D. officinale plants after 49 days of inoculation, note: the JC02+ plants (left) and the control (right). Bar: (a) 0.25 mm, (b) 0.5 mm, (c) 0.25 mm, (d) 1 mm, (e) 20 ␮m and (f) 2 cm.

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98.51%, 99.59%, respectively, significantly more than the control (Fig. 6A and B). 3.4. Observation of mycorrhizal fungi colonization

Fig. 5. Effects of mycorrhizal fungi on seed germination of D. officinale 77 days after sowing. Note: the results show the mean of five replicates with bars indicating the standard error. Different letters denote significant differences according to Duncan’s multiple-range test at the P ≤ 0.05 level.

3.2. Effects of Tulasnella isolate on germination of D. officinale seeds After sowing for 14 days, all seeds of D. officinale harbored swollen embryos, broken testa and/or initiated rhizoid. Two Tulasnella isolates (JC-02 and JC-05) promoted seed germination of D. officinale up to elongation of first leaf after sowing for 11 weeks (Fig. 4a–d). The typical characteristics e.g., peloton and coiled mycelium (Fig. 4e) of orchid’s mycorrhizal fungi were observed in the cortical cells of symbiotic seed treatments after sowing for 77 days, stained with 0.05% (w/v) trypanblue with a light microscope. Percent of seed germination was 98.47% and 81.05%, respectively, significantly higher than that of control (81.05%) (Fig. 5). Without symbiotic fungi, seed development of D. officinale was arrested at stage 2.

3.3. Effects of Tulasnella isolate on the growth of D. officinale seedlings Although white mycelium grew over the rotted wood and up to the wall of bottle 49 days after inoculation, hyphae appeared to penetrate the roots of D. officinale without covering the stems and leaves. JC-02+ plants and JC-05+ plants appeared more vigorous compared to the control. Plant fresh weight and plant dry weight of JC-02+ plants and JC-05+ plants increased 174.51%, 114.60% and

Base on the positive effects of Tulasnella isolates on the growth of D. officinale seedlings, transverse sections of roots infected by JC02 and JC-05, respectively, were observed under a light microscopy after cultivation for 7 weeks. The fungal hyphae were observed in the velamen, exodermal passage cells, exodermis and cortex, but not in the endodermis and vascular bundle sheath. Some hyphae had formed pelotons and coils mycelium in the cortex of roots of JC02+ plant and JC-05+ plant (Fig. 7A–C). Transverse sections of roots (control) confirmed the lack of mycelium in the velamen, exodermal passage cells, exodermis and cortex as well as the endodermis and vascular bundle sheath (Fig. 7D). 4. Discussion The neighbor joining phylogenetic tree of the internal transcribed spacer (ITS) and 5.8S rDNA sequences of two isolates and other 13 known sequences from GenBank database indicated that the Tulasnella isolates (JC-02 and JC-05) associated with D. nobile formed a clade with orchid mycorrhizal fungi from other epiphytic orchids and terrestrial orchids (McCormick et al., 2004; Suarez et al., 2006; Taylor and McCormick, 2008; Nontachaiyapoom et al., 2010; Chutima et al., 2010). Tulasnella fungi are a major group of mycorrhizal fungi forming symbiotic association with orchids in the tropics (Currah et al., 1997; Ma et al., 2003; Pereira et al., 2005). Tulasnella has also been reported to occur in soil and bark (Taylor and McCormick, 2008). Based on the molecular data, Shefferson et al. (2005) found that the species of Tulasnellaceae are the major fungal group forming mycorrhizas with Cypripedium spp. by direct PCR amplification of the fungal genes in root tissue. Many investigations have revealed that the infection of mycorrhizal fungi, which can provide the carbon sources, is essential to the seed germination of all orchids in the nature (Yam and Arditti, 2009). Without the association of mycorrhizal fungi, the small size of orchid seeds cannot carry out germination due to insufficient food reserves contained within the seed (Dressler, 1990; Rasmussen, 2002; Porras-Alfaro and Bayman, 2007). Therefore, it is very important to assess whether some single fungus can stimulate seed germination of orchids in vitro or not to confirm the symbiotic relationships between the two livings. Moreover symbiotic germination is also an useful and convenient way to propagate orchids in the experiment condition as well as reintroduce orchids in the

Fig. 6. Effects of mycorrhizal fungi on the growth of D. officinale plants 49 days after inoculation. Bars show mean change in plant fresh weight (A) and in plant dry weight (B). Bars show standard errors (X ± SE n = 20 plants per fungi and n = 30 for controls). Mean values with the different letter are significantly different (ANOVA and means were compared by Duncan’s multiple test at P ≤ 0.05 level).

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Fig. 7. Light micrographs of interactions between roots of D. officinale and mycorrhizal fungi JC02 or JC-05. (A and B) Transverse section across a mycorrhizal root of JC-02+ plant showing hypha (arrow) in the cells of velamen, the presence of intact (fp) and degenerate (d) pelotons in the cortical tissue; (C) enerate (d) pelotons in the cortical tissue as well as the hypha (arrow) and nucleus (star) in the exodermal passage cells; (D) transverse section of D. officinale root (noninoculated control). Annotation: VE, velamen; P, exodermal passage cell; EX, exodermis; CO, cortex; VS, vascular bundle sheath; N, nucleus.

ecology. In this paper, Tulasnella isolates (JC-02 and JC-05) from roots of D. nobile seedlings showed a significant, positive effect on seeds germination of D. officinale. The rates of seed germination were 98.47% and 99.05%, respectively, significantly higher than that of the control (81.05%). The rate of seed germination was enhanced by 21.49% and 22.21% respectively with infection of fungal isolates JC-02 and JC-05. Moreover, the two Tulasnella isolates not only promoted the highest final percent germination, but also promoted seeds development up to stage 5. Without symbiotic fungi, asymbiotic treatments can only develop to stage 2. Wang et al. (2011) have previously reported symbiotic seed germination of D. officinale in vitro. In their study, seeds of D. officinale were cultured on the oat meal agar (OMA) with an isolate (SHH44) from the roots of wild D. officinale, and the result indicated that germination rate was 33.4%, and only 1.8% higher than asymbiotic group (control). The fungal isolate originating from the roots of wild D. officinale was identified as belonging to Sebacina genus (Wang et al., 2011), which was known to have a positive effect on seed germination of some orchids (Batty and Dixon, 2001). Interestingly, Tulasnella isolates appear to be more suited at facilitating germination in D. officinale than Sebacina isolates reported previously (Wang et al., 2011). Porras-Alfaro and Bayman (2007) also observed the same result that the mycorrhizal fungi isolated from Ionopsis utricularioides can improve seed germination of Vanilla better than the isolates form Vanilla itself. This finding is also supported by other studies (e.g., Otero et al., 2004; McCormick et al., 2004), and may prove useful for future orchid conservation studies that select mycorrhizal fungi for propagation.

Moreover, the two Tulasnella isolates had significantly positive effects on plant fresh growth and plant dry weight. This result was morphologic supported by the observation of mycorrhizal fungi colonization. Differences between JC-02 and JC-05 in effect on the plant height, number of new roots and plant diameter of D. officinale were less pronounced after 7 weeks. This result suggests that D. officinale plants might need longer time to acclimatize the infection of fungi. Although the two Tulasnella isolates can significantly promote the seed germination and plant growth under laboratory culture conditions, it is necessary to assess the interaction between orchids and fungi during different stages in nature. Therefore, further studies are essential to confirm the relationship between the two Tulasnella isolates and D. officinale in situ. Acknowledgments This work was supported by the National Natural Science Foundation of China (30830117, 31170314), and the International Science and Technology Cooperation Projects of China (2011DFA31260, 2009DFA32250). References Abadie, J.C., Puttsepp, U., Gebauer, G., Faccio, A., Bonfante, P., Selosse, M.A., 2006. Cephalanthera longifolia (Neottieae Orchidaceae) is mixotrophic: a comparative study between green and non-photosynthetic individuals. Can. J. Bot. 84, 1462–1477.

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