Tricholoma matsutake and Rhizopogon roseolus form ectomycorrhizal (ECM) association with their host plant on natural habitats. The main objective of this study was to test mycelial growth, morphology, and host plant survival both in vitro and in vivo when treated with enriched media. Aseptically germinated seedlings of Pinus densiflora and P. thunbergii were inoculated with the strains of T. matsutake and R. roseolus, respectively. Under in vitro conditions mycelial growth rates performed best on pH 5 and were better on Modified-Melin-Norkrans-(MMN) based medium and Potato Dextrose Agar (PDA); addition of micronutrients and vitamins in MMN mycelial growth rates had 6–27% differences. Without ECM, plant survival rates on standard media were 30% to below 30% and by inclusion of elements they were 50% to 80%. On in vivo, soil containing different media with ECM allowed successful mycorrhizal association and increased seedling survival rates approximately 100%. Our findings confirm that MMN and PDA allowed higher mycelial growth but poor plant survival (<30%); however, enriched media supported 100% plant survival with successful ECM associations. The present method is advantageous in terms of giving objectivity for ECM by employing suitable media for strains and host plant, and making it possible for mass production of ECM-infected seedlings. 1. Introduction The development and survival of many forest trees and the success of a reforestation programme depend on the symbiosis involving host tree and ectomycorrhizae—their growth and establishment. Mycorrhizal symbiosis develops capabilities of the host root system by extending the plant’s ability to tolerate biological and environmental stresses such as phytopathogenic attacks, nutritional insufficiencies, pollution of heavy metal, extensive erosion, drought, and different pH [1]. These positive effects of the ectomycorrhizal symbiosis on the establishment and growth of forest plants have made the ectomycorrhizal inoculation a valuable technical tool for plant production in forestry [2]. Pinus densiflora and P. thunbergii have received extra attention due to their potential usage in pine forest reforestation programs. However, the pine forest in Japan has been under threat over the recent decades and now is facing a serious crisis to survive. The recent decline in pine forest has been aggravated by many interactive disfavourable growing conditions both for the host and the mycorrhizal fungi [2, 3]. This is ultimately reducing the production of edible mushrooms growing in pine forests which have significant
References
[1]
S. Smith and D. J. Read, Mycorrhizal Symbiosis, vol. 640, Academic Press, London, UK, 1996.
[2]
S. E. Smith and D. J. Read, Mycorrhizal Symbiosis, Academic Press, London, UK, 2008.
[3]
F. Islam and S. Ohga, “The response of fruit body formation on in situ condition Tricholoma matsutake by applying electric pulse stimulator,” ISRN Agronomy, vol. 2012, Article ID 462724, 6 pages, 2012.
[4]
S. T. Chang, “Global impact of edible and medicinal mushrooms of human welfare in the 21 century: non-green revolution,” International Journal of Medicinal Mushrooms, vol. 1, pp. 1–7, 1999.
[5]
G. C. Yen and C. Y. Hung, “Effects of alkaline and heat treatment on antioxidative activity and total phenolics of extracts from Hsian-tsao (Mesona procumbens Hemsl.),” Food Research International, vol. 33, no. 6, pp. 487–492, 2000.
[6]
I. C. F. R. Ferreira, P. Baptista, M. Vilas-Boas, and L. Barros, “Free-radical scavenging capacity and reducing power of wild edible mushrooms from northeast Portugal: individual cap and stipe activity,” Food Chemistry, vol. 100, no. 4, pp. 1511–1516, 2007.
[7]
L. Barros, R. C. Calhelha, J. A. Vaz, I. C. F. R. Ferreira, P. Baptista, and L. M. Estevinho, “Antimicrobial activity and bioactive compounds of Portuguese wild edible mushrooms methanolic extracts,” European Food Research and Technology, vol. 225, no. 2, pp. 151–156, 2007.
[8]
C. C. Weng, Taste quality of Grifola frondosa, Morcbella esculenta and Termitomyces albuminosus mycelia and their application in food application in food processing [M.S. thesis], National Chung-Hsing University, Taichung, Taiwan, 2003.
[9]
W. Yun, I. R. Hall, and L. A. Evans, “Ectomycorrhizal fungi with edible fruiting bodies 1. Tricholoma matsutake and related fungi,” Economic Botany, vol. 51, no. 3, pp. 311–327, 1997.
[10]
N. Ohnuma, K. Amemiya, R. Kakuda, Y. Yaoita, K. Machida, and M. Kikuchi, “Sterol constituents from two edible mushrooms, Lentinula edodes and Tricholoma matsutake,” Chemical and Pharmaceutical Bulletin, vol. 48, no. 5, pp. 749–751, 2000.
[11]
H. W. Lim, J. H. Yoon, Y. S. Kim, M. W. Lee, S. Y. Park, and H. K. Choi, “Free radical-scavenging and inhibition of nitric oxide production by four grades of pine mushroom (Tricholoma matsutake Sing.),” Food Chemistry, vol. 103, no. 4, pp. 1337–1342, 2007.
[12]
T. Ebina, T. Kubota, N. Ogama, and K. I. Matsunaga, “Antitumor effect of a peptide-glucan preparation extracted from a mycelium of Tricholoma matsutake (S. Ito and Imai) Sing,” Biotherapy, vol. 16, no. 3, pp. 255–259, 2002.
[13]
K. Matsunaga, T. Chiba, and E. Takahashi, “Mass production of Matsutake (Tricholoma matsutake) mycelia and its application to functional foods,” Bioindustry, vol. 20, pp. 37–46, 2003.
[14]
V. P. Cirillo, W. A. Hardwick, and R. D. Seeley, “Fermentation process for producing edible mushroom mycelium,” USA Patent no. 2, 928, 210; 1960.
[15]
A. Guerin-Laguette, L. M. Vaario, W. M. Gill, F. Lapeyrie, N. Matsushita, and K. Suzuki, “Rapid in vitro ectomycorrhizal infection on Pinus densiflora roots by Tricholoma matsutake,” Mycoscience, vol. 41, no. 4, pp. 389–393, 2000.
[16]
R. Molina and J. G. Palmer, “Isolation, maintenance, and pure culture manipulation of ectomycorrhizal fungi,” in Methods and Pricciples of Mycorrhizal Research, N. C. Schenck, Ed., pp. 115–129, American Phytopathological Socity, St Paul, Minn, USA, 1982.
[17]
R. L. Peterson and S. M. Bradbury, “Use of plant mutants, interspecific variants and non-hosts in studying mycorrhiza formation and function,” in Mycorrhiza: Structure, Function, Molecular Biology and Biotechnology, A. K. Varma and B. Hock, Eds., Springer, Berlin, Germany, 1995.
[18]
I. Langer, D. Krpata, U. Peintner, W. W. Wenzel, and P. Schweiger, “Media formulation influences in vitro ectomycorrhizal synthesis on the European aspen Populus tremula L.,” Mycorrhiza, vol. 18, no. 6-7, pp. 297–307, 2008.
[19]
L. Barros, P. Baptista, and I. C. F. R. Ferreira, “Influence of the culture medium and pH on the growth of saprobic and ectomycorrhizal mushroom mycelia,” Minerva Biotecnologica, vol. 18, no. 4, pp. 165–170, 2006.
[20]
M. C. Park, S. G. Sim, and W. J. Cheon, “Methods of preparing Tricholoma matsutake-infected young pine by coculturing aseptic pine seedlings and .T. matsutake,” US Patent no. 7,269,923 B2; 2007.
[21]
H. C. Chung, D. H. Kim, and S. S. Lee, “Mycorrhizal formation and seedling growth of Pinus densiflora by in vitro synthesis with the inoculation of ectomycorrhizal fungi,” “Mycobiology, vol. 30, no. 2, pp. 70–75, 2002, Copyright by The Korean Society of Mycology.
[22]
I. R. Hall and Y. Wang, “Methods for cultivating edible mycorrhizal mushroom,” in Mycorrhiza Manual, A. Varma, Ed., pp. 99–114, Springer, Berlin, Germany, 1998.
[23]
T. Hatakeyama and M. Ohmasa, “Mycelial growth characteristics in a split-plate culture of four strains of the genus Suillus,” Mycoscience, vol. 45, no. 3, pp. 188–199, 2004.
[24]
T. Lubbehusen, V. González Polo, S. Rossi et al., “Protein kinase A is involved in the control of morphology and branching during aerobic growth of Mucor circinelloides,” Microbiology, vol. 150, no. 1, pp. 143–150, 2004.
[25]
T. Hatakeyama and M. Ohmasa, “Mycelial growth of strains of the genera Suillus and Boletinus in media with a wide range of concentrations of carbon and nitrogen sources,” Mycoscience, vol. 45, no. 3, pp. 169–176, 2004.
[26]
M. Kusuda, M. Ueda, Y. Konishi, K. Yamanaka, T. Terashita, and K. Miyatake, “Effects of carbohydrate substrate on the vegetative mycelial growth of an ectomycorrhizal mushroom, Tricholoma matsutake, isolated from Quercus,” Mycoscience, vol. 48, no. 6, pp. 358–364, 2007.
[27]
L. L. Hung and J. M. Trappe, “Growth variation between and within species of ectomycorrhizal fungi in response to pH in vitro,” Mycologia, vol. 75, pp. 234–241, 1983.
[28]
E. J. Jokela, W. W. McFee, and E. L. Stone, “Micronutrient deficiency in slash pine: response and persistence of added manganese,” Soil Science Society of America Journal, vol. 55, no. 2, pp. 492–496, 1991.
[29]
A. Shalata and P. M. Neumann, “Exogenous ascorbic acid (vitamin C) increases resistance to salt stress and reduces lipid peroxidation,” Journal of Experimental Botany, vol. 52, no. 364, pp. 2207–2211, 2001.
[30]
J. V. D. Rousseau, D. M. Sylvia, and A. J. Fox, “Contribution of ectomycorrhiza to the potential nutrient-absorbing surface of pine,” New Phytologist, vol. 128, no. 4, pp. 639–644, 1994.
[31]
J. M. Trappe, “Selection of fungi for ectomycorrhizal inoculation in nurseries,”,” Annual Review of Phytopathology, vol. 15, pp. 203–222, 1977.
[32]
J. Parladé, J. Pera, and J. Luque, “Evaluation of mycelial inocula of edible Lactarius species for the production of Pinus pinaster and P. sylvestris mycorrhizal seedlings under greenhouse conditions,” Mycorrhiza, vol. 14, no. 3, pp. 171–176, 2004.
[33]
D. H. Marix, S. B. Maul, and C. E. Cordell, “Application of specific ectomycorrhizal fungi in world forestry,”,” in Frontiers in Industrial Mycology, G. F. Leatham, Ed., Chapman & Hall, New York, NY, USA; Kluwer Academic Publishers, Dordrecht, The Netherlands, 1992.
