Ex situ symbiotic seed germination, isolation and identification of effective symbiotic fungus in Cymbidium mannii (Orchidaceae)

doi:10.3724/SP.J.1258.2012.00859

Abstract

Abstract:

Aims The in situ and ex situ seed baiting techniques, which have been developed in recent years, are effective methods to study the compatible mycorrhizal fungi of orchids. Our aim was to obtain the compatible mycorrhizal fungi of epiphytic Cymbidium mannii using the ex situ seed baiting technique.
Methods Bark, moss, litter and humus around the roots of adult plants of C. mannii were collected as substrate to cultivate C. mannii seeds in the laboratory. The fungi were isolated from developed protocorms and identified by morphological and molecular characteristics. The effects of different mycorrhizal fungi and light on seed germination were examined by in vitro symbiotic germination.
Important findings The isolated fungus was identified as a species of the genus Epulorhiza and was named as FCb4. After 58 days cultivation, seeds inoculated with FCb4 strain and FDaI7 strain (Tulasnella, isolated from Dendrobium aphyllum) had a high germination ratio, whereas seeds without the fungus failed to germinate. There was no significant difference between the germination ratios of seeds inoculated with FCb4 and FDaI7 strains, but FCb4 was significantly superior for seedling formation and development compared with FDaI7 in light. This indicated a lower degree of symbiotic fungal specificity on seed germination than the protocorm development stage in C. mannii. The fungus we obtained was effective for seed germination, and protocorm and seedling development for C. mannii. Germination and protocorm production were higher in the dark (0/24 h light/dark) than the light condition (12/12 h light/dark), whereas the subsequent protocorm development was better with light. These findings will aid in seedling production and reintroduction of C. mannii.

Key words: Cymbidium manii, ex situ symbiotic seed germination, orchid conservation, protocorm, symbiotic fungi, Tulasnella

SHENG Chun-Ling, LEE Yung-I, GAO Jiang-Yun. Ex situ symbiotic seed germination, isolation and identification of effective symbiotic fungus in Cymbidium mannii (Orchidaceae)[J]. Chin J Plant Ecol, 2012, 36(8): 859-869.

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URL: https://www.plant-ecology.com/EN/10.3724/SP.J.1258.2012.00859

https://www.plant-ecology.com/EN/Y2012/V36/I8/859

Figures/Tables 5

Table 1 Different germination stages of Cymbidium mannii seeds

萌发阶段 Germination stage	描述 Description
0	未萌发的种子 Ungerminated seed
1	种胚膨大, 产生根状物(视为萌发) Enlarged embryo, production of rhizoid(s) (= germination)
2	种胚继续膨大, 突破种皮(形成原球茎) Continued embryo enlargement, rupture of testa ( = protocorm formation)
3	出现原生分生组织(原球茎发育阶段) Appearance of protomeristem (protocorm development)
4	长出第一片叶片及后续生长(幼苗发育初期) Emergence of first leaf and continue development (early stage of seedling development)

Fig. 1 Seed, protocorm and seedling of Cymbidium mannii in different germination stages after 133 days of ex situ symbiotic seed germination. A, Stage 0, ungerminated seed. B, Stage 1, enlarged embryo, production of rhizoid(s) (= germination). C, Stage 2, continued embryo enlargement, rupture of testa (= protocorm formation). D, Stage 3, protocorm enlarges with appearance of protomeristem. E, Stage 4, seedling.

Fig. 2 Cross-section of seed and protocorm of Cymbidium mannii. A, Ungerminated seed without fungi infection. B, Protocorm with fungi infection on the base. C, Protocorm forms the first thrust (arrow), and fungi infect the base of protocorm. D, Protocorm enlarges with appearance of protomeristem (arrow) with lots of pelotons (arrowhead). E, Partial enlarged drawing of Fig. C, arrow indicates loosened pelotons. F, Partial enlarged drawing of Fig. D, arrow indicates digested pelotons, and arrowhead indicates loosened pelotons.

Table 2 Seed germination percentage, protocorm ratio and the proportion of geminated seeds, protocorms and seedlings in different germination stages in different treatments after 58 days cultivation for Cymbidium mannii (mean ± SE)

处理 Treatment	光照 Photoperiod (h Light/Dark )	重复 Replication	萌发率 Germination (%)	原球茎比率 Protocorm ratio (%)	不同阶段萌发的种子、原球茎或幼苗的比率 Proportion of geminated seeds, protocorms and seedlings in different stages (%)
处理 Treatment	光照 Photoperiod (h Light/Dark )	重复 Replication	萌发率 Germination (%)	原球茎比率 Protocorm ratio (%)	阶段1 Stage 1	阶段2 Stage 2	阶段3 Stage 3	阶段4 Stage 4
FCb4菌株 FCb4 strain	12/12	7	73.64 ± 6.14	60.0 ± 12.08	13.63 ± 9.95	5.05 ± 4.05	29.30 ± 8.82	25.67 ± 9.27
FCb4菌株 FCb4 strain	0/24	7	85.04 ± 5.15	83.89 ± 5.42	2.48 ± 1.06	17.7 ± 12.85	66.6 ± 13.05	0.35 ± 0.35
FDaI7菌株 FDaI7 strain	12/12	10	69.82 ± 3.80	45.11 ± 8.74	24.47 ± 7.94	28.56 ± 7.35	13.51 ± 6.75	3.04 ± 2.27
FDaI7菌株 FDaI7 strain	0/24	10	75.20 ± 5.49	66.13 ± 8.56	9.48 ± 6.43	13.38 ± 4.72	50.56 ± 8.88	2.42 ± 1.00
对照 CK	12/12	10	0	0	0	0	0	0
对照 CK	0/24	10	0	0	0	0	0	0

Fig. 3 Proportion of Cymbidium mannii seeds in different germination stages after 58 days cultivation (mean ± SE). A, Proportion of geminated seeds, protocorms and seedlings in different germination stages in the treatment inoculated with FCb4 strain under different light conditions. B, Proportion of geminated seeds, protocorms and seedlings in different germination stages in the treatments inoculated with FCb4 and FDaI7 strains under the light condition (12/12 h light/dark). * indicates the significant difference between different treatments within the same stage (α = 0.05).

References 45

[1]	Arditti J (1967). Factors affecting the germination of orchid seeds. The Botanical Review, 33, 1-97.
[2]	Arditti J, Ernst R (1984). Physiology of germination orchid seeds. In: Arditti J ed. Orchid Biology: Reviews and Perspectives, Vol. 3. Cornell University Press, New York. 177-222.
[3]	Arditti J, Michaud JD, Oliva AP (1981). Seed germination of North American orchids. I. Native California and related species of Calypso, Epipactis, Goodyera, Piperia, and Platanthera. Botanical Gazette, 142, 442-453.
[4]	Bai YQ (白毓谦), Fang SK (方善康), Gao D (高东), Shi AH (施安辉) (1987). Microbiology Experiment Technology (微生物实验技术). Shandong University Press, Ji’nan. (in Chinese)
[5]	Batty AL, Brundrett MC, Dixon KW, Sivasithamparam K (2006). In situ symbiotic seed germination and propagation of terrestrial orchid seedlings for establishment at field sites. Australian Journal of Botany, 54, 375-381.
[6]	Batty AL, Dixon KW, Brundrett M, Sivasithamparam K (2001). Constraints to symbiotic germination of terrestrial orchid seed in a mediterranean bushland. New Phytologist, 152, 511-520.
[7]	Brundrett MC (2007). Scientific approaches to Australian temperate terrestrial orchid conservation. Australian Journal of Botany, 55, 293-307.
[8]	Brundrett MC, Scade A, Batty AL, Dixon KW, Sivasithamparam K (2003). Development of in situ and ex situ seed baiting techniques to detect mycorrhizal fungi from terrestrial orchid habitats. Mycological Research, 107, 1210-1220. DOI URL
[9]	Currah RS, Zelmer CD, Hambleton S, Richardson KA (1997). Fungi from orchid mycorrhizas. In: Arditti J, Pridgeon AM eds. Orchid Biology: Reviews and Perspectives, VII. Kluwer Academic Publishers, Dordrecht. 117-170.
[10]	Dearnaley JDW (2007). Further advances in orchid mycorrhizal research. Mycorrhiza, 17, 475-486.
[11]	Dixon K (1987). Raising terrestrial orchids from seed. In: Harris WK ed. Modern Orchid Growing for Pleasure and Profit. Orchid Club of South Australia, Inc., Adelaide. 44-100.
[12]	Duan CF (段春芳), Li ZL (李枝林), Fang F (方飞), Yang GH (杨根华), Chen SS (陈思思), Hong QY (洪群艳) (2010). Isolation and identification research of mycorrhizal isolated from several orchids in Yunnan Province. Southwest China Journal of Agricultural Sciences (西南农业学报), 23, 756-759. (in Chinese with English abstract)
[13]	Ernst R (1982). Orchid seed germination and seedling culture―a manual: Paphiopedilum. In: Arditti J ed. Orchid Biology: Reviews and Perspectives. Cornell University Press, New York. 350-353.
[14]	Godo T, Komori M, Nakaoki E, Yukawa T, Miyoshi K (2010). Germination of mature seeds of Calanthe tricarinata Lindl., an endangered terrestrial orchid, by asymbiotic culture in vitro. In Vitro Cellular and Developmental Biology―Plant, 46, 323-328.
[15]	Hollick PS (2004). Mycorrhizal Specificity in Endemic Western Australian Terrestrial Orchids (Tribe Diurideae): Implications for Conservation. PhD dissertation, Murdoch University, Perth.
[16]	Hu T (胡陶), Li LB (李潞滨), Yang K (杨凯), Tang Z (唐征), Liu ZJ (刘振静), Zhuang CY (庄彩云), Peng ZH (彭振华) (2008). Isolation and identification of Cymbidium mycorrhizae of China. Journal of Beijing Forestry University (北京林业大学学报), 30, 132-135. (in Chinese with English abstract)
[17]	Ke HL (柯海丽), Song XQ (宋希强), Tan ZQ (谭志琼), Liu HX (刘红霞), Luo YB (罗毅波) (2007). The technique of orchid seeds baiting in situ and its applications. Scientia Silvae Sinicae (林业科学), 43, 125-129. (in Chinese with English abstract)
[18]	Liu HX, Luo YB, Liu H (2010). Studies of mycorrhizal fungi of Chinese orchids and their role in orchid conservation in China―a review. The Botanic Review, 76, 241-262.
[19]	Liu ZJ, Chen XQ, Cribb PJ (2009). Cymbidium Swartz. In: Wu ZY, Raven PH, Hong DY eds. Flora of China, Vol. 25. Science Press & Missouri Botanical Garden Press, Beijing & St. Louis. 264.
[20]	Masuhara G, Katsuya K (1994). In situ and in vitro specificity between Rhizoctonia spp. and Spiranthes sinensis (Persoon) Ames. var. amoena (M. Bieberstein) Hara (Orchidaceae). New Phytologist, 127, 711-718.
[21]	McKendrick S (2000). In Vitro Germination of Orchids: a Manual. Ceiba Foundation for Tropical Conservation. http://orchideenvermehrung.at/downloads/seed%20sowing%20manual.pdf . Cited 10 April 2012.
[22]	Moore RT (1987). The genera of Rhizoctonia-like fungi: Ascorhizoctonia, Ceratorhiza gen. nov., Epulorhiza gen. nov., Moniliopsis, and Rhizoctonia. Mycotaxon, 29, 91-99.
[23]	Rasmussen HN (1992). Seed dormancy patterns in Epipactis palustris (Orchidaceae): requirements for germination and establishment of mycorrhiza. Physiologia Plantarum, 86, 161-167.
[24]	Rasmussen HN (1995). Terrestrial Orchids from Seed to Mycotrophic Plant. Cambridge University Press, Cambridge, UK.
[25]	Rasmussen HN, Whigham DF (1993). Seed ecology of dust seeds in situ: a new study technique and its application in terrestrial orchids. American Journal of Botany, 80, 1374-1378.
[26]	Smith SE, Read DJ (1997). Mycorrhizal Symbiosis. Academic Press, New York.
[27]	Smith ZF, James EA, Mclean CB (2010). Mycorrhizal specificity of Diuris fragrantissima (Orchidaceae) and persistence in a reintroduced population. Australian Journal of Botany, 58, 97-106.
[28]	Stewart SL (2008). Orchid reintroduction in the United States: a mini-review. North American Native Orchid Journal, 14, 54-59.
[29]	Stewart SL, Kane ME (2007). Orchid conservation in the Americas―lessons learned in Florida. Lankesteriana, 7, 382-387.
[30]	Stewart SL, Zettler LW, Minso J, Brown PM (2003). Symbiotic germination and reintroduction of Spiranthes brevilabris Lindley, an endangered orchid native to Florida. Selbyana, 24, 64-70.
[31]	Swarts ND, Batty AL, Hopper SD, Dixon KW (2007). Does integrated conservation of terrestrial orchids work? Lankesteriana, 7, 219-222.
[32]	Tsi ZH (吉占和), Chen SC (陈心启) (1995). Miscellaneous notes on orchids in Xishuangbanna of Yunnan, China. Acta Phytotaxonomica Sinica (植物分类学报), 33, 281-296. (in Chinese with English abstract)
[33]	Vujanovic V, St-Arnaud M, Barabé D, Thibeault G (2000). Viability testing of orchid seed and the promotion of colouration and germination. Annals of Botany, 86, 79-86.
[34]	Wang H, Fang HY, Wang YQ, Duan LS, Guo SX (2011). In situ seed baiting techniques in Dendrobium officinale Kimuraet Migo and Dendrobium nobile Lindl.: the endangered Chinese endemic Dendrobium (Orchidaceae). World Journal of Microbiology and Biotechnology, 27, 2051-2059.
[35]	Wang D (王娣), Jia SH (贾书华), Zhang ZX (张兆轩), Cai YP (蔡永萍), Lin Y (林毅) (2007). Isolation and culture of an endophytic fungus associated with Dendrobium huoshanense and its effects on the growth of plantlets. Journal of Fungal Research (菌物研究), 5, 84-88. (in Chinese with English abstract)
[36]	Weising K, Nybom H, Wolff K, Meyer W (1995). DNA Fingerprinting in Plants and Fungi. CRC Press, Boca Raton, USA.
[37]	White TJ, Bruns T, Lee S, Taylor J (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ eds. PCR Protocols: A Guide to Methods and Applications. Academic Press, New York. 315-322.
[38]	Williamson B, Hadley G (1970). Penetration and infection of orchid protocorms by Thanatephorus cucumeris and other Rhizoctonia isolates. Phytopathology, 60, 1092-1096.
[39]	Wu JR, Ma HC, Lü M, Han SF, Zhu YY, Jin H, Liang JF, Liu L, Xu JP (2010). Rhizoctonia fungi enhance the growth of the endangered orchid Cymbidium goeringii. Botany, 88, 20-29.
[40]	Yamazaki J, Miyoshi K (2006). In vitro asymbiotic germination of immature seed and formation of protocorm by Cephalanthera falcata (Orchidaceae). Annals of Botany, 98, 1197-1206.
[41]	Yeung EC (1999). The use of histology in the study of plant tissue culture systems―some practical comments. In Vitro Cellular and Developmental Biology―Plant, 35, 137-143.
[42]	Zelmer CD, Currah RS (1997). Symbiotic germination of Spiranthes lacera (Orchidaceae) with naturally occurring endophyte. Lindleyana, 12, 142-148.
[43]	Zettler LW, Piskin KA, Stewart SL, Hartsock JJ, Bowles ML, Bell TJ (2005). Protocorm mycobionts of the federally threatened eastern prairie fringed orchid, Platanthera leucophaea (Nutt.) Lindley, and a technique to prompt leaf elongation in seedlings. Studies in Mycology, 53, 163-171.
[44]	Zhang JH, Cao M (1995). Tropical forest vegetation of Xishuangbanna, SW China and its secondary changes, with special reference to some problems in local nature conservation. Biological Conservation, 73, 229-238.
[45]	Zhou X, Lin H, Fan XL, Gao JY (2012). Autonomous self- pollination and insect visitation in a saprophytic orchid, Epipogium roseum (D. Don) Lindl. Australian Journal of Botany, 60, 154-159.