PCR-Mediated Detection of Endophytic and Phytopathogenic Fungi from Needles of the Japanese Black Pine, Pinus thunbergii

DOI: 10.4236/ojf.2015.54037   PDF   HTML   XML   3,665 Downloads   4,469 Views   Citations


A specific and sensitive polymerase chain reaction (PCR) assay based on the internal transcribed spacer (ITS) region of rDNA sequences was developed to detect endophytic and phytopathogenic fungi from needles of the Japanese black pine, Pinus thunbergii. Sequences of the ITS regions of Lophodermium conigenum, Lecanosticta acicola, Pestalotiopsis neglecta, Rhizosphaera kalkhoffii, and Septorioides pini-thunbergii were compared, and each specific primer pair for these species was designed. First, the designed primer pairs were tested for their specificity to detect each species. A PCR product was amplified only each combination of species and its specific primer pair, confirming the specificity of the designed primer pairs. These primer pairs were also tested on DNA extracted from the needles of P. thunbergii. The PCR products were amplified not only in needles with lesions but also in healthy needles without symptoms. Furthermore, several endophytic and phytopathogenic fungi could be simultaneously detected from the same region in a needle. The PCR-mediated detection method developed in this study will be a valuable tool for the detection of the endophytic and phytopathogenic fungi, not only as a rapid diagnostic tool for early detection but also for monitoring variations in both the quality and quantity of the endophytic and phytopathogenic fungi in needles in Japanese black pines.

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Kihara, J. , Ueno, M. and Arase, S. (2015) PCR-Mediated Detection of Endophytic and Phytopathogenic Fungi from Needles of the Japanese Black Pine, Pinus thunbergii. Open Journal of Forestry, 5, 431-442. doi: 10.4236/ojf.2015.54037.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Begoude, B. A. D., Slippers, B., Wingfield, M. J., & Roux, J. (2011). The Pathogenic Potential of Endophytic Botryosphaeriaceous Fungi on Terminalia Species in Cameroon. Forest Pathology, 41, 281-292.
[2] Broders, K. D., & Boland, G. J. (2010). Molecular Diagnostic Assay for Detection of the Butternut Canker Pathogen Sirococcus clavigignenti-juglandacearum. Plant Disease, 94, 952-958.
[3] Chan, P. (2014). Japanese Black Pine, Kuro Matsu. In P. Chan (Eds.), The Bonsai Bible (pp. 88-89). London: Mitchell Beazley, a Division of Octopus Publishing Group.
[4] Choi, Y. W., Hyde, K. D., & Ho, W. H. (1999). Single Spore Isolation of Fungi. Fungal Diversity, 3, 29-38.
[5] Ganley, R. J., Brunsfeld, S. J., & Newcombe, G. (2004). A Community of Unknown, Endophytic Fungi in Western White Pine. Proceedings of the National Academy of Sciences of the United States of America, 101, 10107-10112.
[6] Ganley, R. J., & Newcombe, G. (2006). Fungal Endophytes in Seeds and Needles of Pinus monticola. Mycological Research, 110, 318-327.
[7] Ganley, R. J., Sniezko, R. A., & Newcombe, G. (2008). Endophyte-Mediated Resistance against White Pine Blister Rust in Pinus monticola. Forest Ecology and Management, 255, 2751-2760.
[8] Ghignone, S., Tamietti, G., & Girlanda, M. (2003). Development of Specific PCR Primers for Identification and Detection of Rhizopycnis vagum. European Journal of Plant Pathology, 109, 861-870.
[9] Goh, T. K. (1999). Single-Spore Isolation Using a Hand-Made Glass Needle. Fungal Diversity, 2, 47-63.
[10] Hata, K., & Futai, K. (1995). Endophytic Fungi Associated with Healthy Pine Needles and Needles Infested by the Pine Needle Gall Midge, Thecodiplosis japonensis. Canadian Journal of Botany, 73, 384-390.
[11] Ito, K., & Zinno, Y. (1972). Preliminary Information about Dothistroma Needle Blight of Pines in Japan. Forest Protection, 21, 86-89. (In Japanese)
[12] Ito, K., Zinno, Y., & Suto, Y. (1975). Dothistroma Needle Blight of Pines in Japan. Bulletin of the Government Forest Experiment Station, No. 272, 123-140.
[13] Kaneko, S., Fujioka, H., & Zinno, Y. (1989). A New Species of Septoria on Japanese Black Pine. Transactions of the Mycological Society of Japan, 30, 463-466.
[14] Korkama-Rajala, T., Müeller, M. M., & Pennanen, T. (2008). Decomposition and Fungi of Needle Litter from Slow- and Fast-Growing Norway Spruce (Picea abies) Clones. Microbial Ecology, 56, 76-89.
[15] Langrell, S. R. H. (2011). Nested Polymerase Chain Reaction-Based Detection of Dothistroma septosporum, Red Band Needle Blight of Pine, a Tool in Support of Phytosanitary Regimes. Molecular Ecology Resources, 11, 749-752.
[16] Lin, Z., Xu, S., Que, Y., Wang, J., Comstock, J. C., Wei, J., McCord, P. H., Chen, B., Chen, R., & Zhang, M. (2014). Species-Specific Detection and Identification of Fusarium Species Complex, the Causal Agent of Sugarcane Pokkah Boeng in China. PloS ONE, 9, e104195.
[17] Lovic, B. R., Martyn, R. D., & Miller, M. E. (1995). Sequence-Analysis of the Its Regions of rDNA in Monosporascus spp. to Evaluate Its Potential for PCR-Mediated Detection. Phytopathology, 85, 655-661.
[18] Malvick, D. K., & Impullitti, A. E. (2007). Detection and Quantification of Phialophora gregata in Soybean and Soil Samples with a Quantitative, Real-Time PCR Assay. Plant Disease, 91, 736-742.
[19] Min, Y. J., Park, M. S., Fong, J. J., Quan, Y., Jung, S., & Lim, Y. W. (2014). Diversity and Saline Resistance of Endophytic Fungi Associated with Pinus thunbergii in Coastal Shelterbelts of Korea. Journal of Microbiology and Biotechnology, 24, 324-333.
[20] Müller, M. M., Valjakka, R., Suokko, A., & Hantula, J. (2001). Diversity of Endophytic Fungi of Single Norway Spruce Needles and Their Role as Pioneer Decomposers. Molecular Ecology, 10, 1801-1810.
[21] Osono, T., & Hirose, D. (2011). Colonization and Lignin Decomposition of Pine Needle Litter by Lophodermium pinastri. Forest Pathology, 41, 156-162.
[22] Popov, A. P., Tsvetkov, I. L., Belov, A. A., Konichev, A. S., Ivanushkina, N. E., Kochkina, G. A., & Ozerskaya, S. M. (2010). Molecular Genetic Identification of the Phytopathogenic Fungus Cryphonectria parasitica. Microbiology, 79, 223-228.
[23] Pravi, V., Jeeva, M. L., & Archana, P. V. (2014). Rapid and Sensitive Detection of Sclerotium rolfsii Associated with Collar Rot Disease of Amorphophallus paeoniifolius by Species-Specific Polymerase Chain Reaction Assay. Molecular Biotechnology, 56, 787-794.
[24] Qadri, M., Rajput, R., Abdin, M. Z., Vishwakarma, R. A., & Riyaz-Ul-Hassan, S. (2014). Diversity, Molecular Phylogeny, and Bioactive Potential of Fungal Endophytes Associated with the Himalayan Blue Pine (Pinus wallichiana). Microbial Ecology, 67, 877-887.
[25] Rigano, L. A., Malamud, F., Orce, I. G., Filippone, M. P., Marano, M. R., Morais do Amaral, A., Castagnaro, A. P., & Vojnov, A. A. (2014). Rapid and Sensitive Detection of Candidatus Liberibacter Asiaticus by Loop Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick. BMC Microbiology, 14, 86.
[26] Romeralo, C., Santamaría, O., Pando, V., & Diez, J. J. (2015). Fungal Endophytes Reduce Necrosis Length Produced by Gremmeniella abietina in Pinus halepensis Seedlings. Biological Control, 80, 30-39.
[27] Sakalidis, M. L., Hardy, G. E. S., & Burgess, T. I. (2011). Endophytes as Potential Pathogens of the Baobab Species Adansonia gregorii: A Focus on the Botryosphaeriaceae. Fungal Ecology, 4, 1-14.
[28] Sakuyama, T. (1993). Physiological Characteristics of Two Pine Needle Cast Fungi, Lophodermium iwatense and Lophodermium pinastri. Journal of the Japanese Forest Society, 75, 273-277. (In Japanese with English Abstract)
[29] Seo, S. T., Park, M. J., Park, J. H., & Shin, H. D. (2012). First Report of Brown Spot Needle Blight on Pinus thunbergii Caused by Lecanosticta acicola in Korea. Plant Disease, 96, 914.
[30] Stanosz, G. R., Blodgett, J. T., Smith, D. R., & Kruger, E. L. (2001). Water Stress and Sphaeropsis sapinea as a Latent Pathogen of Red Pine Seedlings. New Phytologist, 149, 531-538.
[31] Stenstrom, E., & Ihrmark, K. (2005). Identification of Lophodermium seditiosum and L. pinastri in Swedish Forest Nurseries Using Species-Specific PCR Primers from the Ribosomal ITS Region. Forest Pathology, 35, 163-172.
[32] Suto, Y. (2000). Septoria pini-thunbergii: A Fungus Produced on Dead Needles of Pinus thunbergii and P. ponderosa. Applied Forest Science, 9, 163-164. (In Japanese)
[33] Suto, Y., & Ougi, D. (1998). Lecanosticta acicola, Causal Fungus of Brown Spot Needle Blight in Pinus thunbergii, New to Japan. Mycoscience, 39, 319-325.
[34] Takahashi, K., & Kobayashi, T. (1998). Pestalotia Diseases of Pinus spp. and Picea glehni Caused by Pestalotiopsis spp. Journal of Tree Health, 2, 9-15. (In Japanese)
[35] Takahashi, K., & Kobayashi, T. (1999). Pestalotia Diseases of Pinus spp. Caused by Pestalotiopsis spp. Journal of Tree Health, 3, 21-30. (In Japanese with English Abstract)
[36] Tanaka, K., & Chiba, O. (1971). On a Needle Blight of Pine Caused by Rhizosphaera kalkhoffii Bubak: Life History, Physiological Characteristics and Pathogenicity of the Causal Fungus. Journal of the Japanese Forestry Society, 53, 279-286. (In Japanese)
[37] Townsend, A. M., & Kwolek, W. F. (1987). Relative Susceptibility of Thirteen Pine Species to Sodium Chloride Spray. Journal of Arboriculture, 13, 225-228.
[38] Tsukahara, H., Kozlowski, T. T., & Shanklin, J. (1985). Tolerance of Pinus densiflora, Pinus thunbergii, and Larix leptolepis Seedlings to SO2. Plant and Soil, 88, 385-397.
[39] White, T. J., Bruns, T., Lee, S., & Taylor, J. W. (1990). Amplification and Direct Sequencing of Fungal Ribosomal Rna Genes for Phylogenetics. In M. A. Innis, D. H. Gelfand, J. Sninsky, & T. J. White (Eds.), PCR Protocols: A Guide to Methods and Applications (pp. 315-322). San Diego, CA: Academic Press.
[40] Yamamoto, M., Yasumori, H., & Suto, Y. (1964). Studies on the Pine Needle Cast (1) on the Pathogens of Pine Needle Cast. Journal of the Japanese Forest Society, 46, 347-354. (In Japanese with English Abstract)
[41] Yoo, J. J., & Eom, A. H. (2012). Molecular Identification of Endophytic Fungi Isolated from Needle Leaves of Conifers in Bohyeon Mountain, Korea. Mycobiology, 40, 231-235.
[42] Yuan, Z., & Chen, L. (2014). The Role of Endophytic Fungal Individuals and Communities in the Decomposition of Pinus massoniana Needle Litter. PLoS ONE, 9, e105911.
[43] Zhang, J. X., Fernando, W. G. D., & Remphrey, W. R. (2005). Molecular Detection of Apiosporina morbosa, Causal Agent of Black Knot in Prunus virginiana. Plant Disease, 89, 815-821.
[44] Zhu, J., Gonda, Y., Yu, L., Li, F., Yan, Q., & Sun, Y. (2012). Regeneration of a Coastal Pine (Pinus thunbergii Parl.) Forest 11 Years after Thinning, Niigata, Japan. PLoS ONE, 7, e47593.

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