Endophytic Mycobiota in Aucuba Japonica in Sagami-Nada and Its Adjacent Area, Central Japan Based on Molecular Identification
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国立科博専報，（47）： 387–403，2011年4月15日 Mem. Natl. Mus. Nat. Sci., Tokyo, (47): 387–403, April 15, 2011 Endophytic Mycobiota in Aucuba japonica in Sagami-nada and Its Adjacent Area, Central Japan Based on Molecular Identification Yousuke Degawa1, Mikio Nishimura2, Yumiko Hirayama3 and Tsuyoshi Hosoya3 1 Laboratory of Mycology, Sugadaira Montane Research Center, University of Tsukuba, 1278–294 Sugadaira Kogen, Ueda-shi, Nagano 386–2204, Japan E-mail: [email protected] 2 Syonandai, Fujisawa-shi, Kanagawa 252–0804, Japan 3 Department of Botany, National Museum of Nature and Science, 4–1–1 Amakubo, Tsukuba-shi, Ibaraki 305–0005, Japan Abstract. To elucidate factors affecting the distribution of mycobiota in Sagami sea and its ad- jacent areas, endophytic mycobiota in Aucuba japonica was examined. Leaves of A. japonica were collected from seven points in Sagami sea and its adjacent areas, and 141 endophytes were isolated by surface sterilization. Based on ITS-5.8S ribosomal RNA sequence, 36 taxa were rec- ognized. The most frequently detected fungus was Glomerella cinguilata (anamorph: Colletotri- chum gloeosporoioides), followed by Diaporthe (anamorph: Phyllosticta), sodarialean fungi, xy- lariaclean fungi. Most of the obtained endophytes belonged to asocmycetes, but few basidiomy- cetes were included. No clear difference of mycobiota was observed based on part of leaves and isolation media. However, mycobiota were found to be affected by the altitude, suggesting the host condition influenced by the environmental factors surrounding the host. Key words: altitude, biogeographic distribution, endophyte, environment, fungal diversity. Stone et al. 2004). However, the procedure is Introduction time consuming and costly, and covering all the The kingdom Fungi is presently known to em- fungi in the given area may be virtually impossi- brace some 97,000 species (Kirk et al., 2008). ble. More systematic method to estimate mycobi- However, it is estimated to embrace 1.5 millions ota is required. Detection of mycobiota by isola- of species (Hawksworth, 1991). The Fungi is one tion from the same substrate using the same of the largest groups of organisms. Due to their method is one of the efficient comparisons. How- diverse ecology, small size of vegetative struc- ever, several problems arise in such methods. tures and production of large numbers of One of the problems is that identification of the propagules, fungi distribute almost everywhere in isolate may not be completely possible, because nature, even in microhabitat. Distribution of fungi some isolates may not produce spores required is primarily delimited by mode of life (saprophyt- for the identification. The problem can be solved ic, symbiotic, or parasitic). Additionally, host or by adopting barcoding method using the sequenc- substrate selectivity (host, habitat, degree of de- es of the barcoding region. The barcoding method composition of the substrates, etc.) are important is to distinguish fungal species using the barcod- factors for the distribution of parasitic fungi ing sequence, namely internal transcribed spacer (Wicklow, 1981). Environmental factors, such as 1, 2 and 5.8s ribosomal RNA regions (ITS-5.8S) temperature, precipitation, etc. are also important of extracted DNA. By referring the data cumulat- factors. ed in GenBank, it is sometimes possible to identi- To elucidate fungal diversity in a given area, fy the given isolate at the specific level. Even if conventional collecting based on observations by the obtained sequence was not identical with the naked eyes is effective (Rossman et al. 1998; previously known sequences, it is still possible to 388 Yousuke Degawa et al. distinguish the isolates to some extent (Stone et Materials and Methods al., 2004; Seifert, 2009). Another problem is the concern that all the Collection of the materials fungi may not be isolated. Isolation is an artificial The leaves of A. japonica were collected in process that is affected by isolation media, tem- March of 2010 by altering the altitude from the perature, sterilization techniques, etc. However, following seven sites (Figs. 1, 2). 1) Manazuru, constant results are expected by following the Manazuru-machi (alt. 5 m). Population of A. same procedure. japonica distributed along the shore, accompa- The Sagami Sea and its adjacent areas are nied with evergreen broadleaf forest. The popula- known to embrace diverse organisms due to the tion facing the shore had few leaves, and healthy typical natural environment in central Japan. The looking leaves were even more rare. However, authors studied the mycobiota in these areas by more rich populations were found apart from the conventional method and illustrated remarkable shore. 2) Shiroyama, Odawara-shi (alt. 50 m). mycobiota (Degawa et al., 2006; Hosoya et al., Population of A. japonica from the urban area ac- 2006). However, to reveal the factors that contrib- companied with evergreen broad leaved trees. ute to fungal community in these areas, more sys- The population was rich in healthy looking tematic approach is desired. leaves, but a number of unhealthy looking leaves To delimit the target fungal group, we poured were also found. 3) Miyashita, Yugawara-machi our attention to fungal endophytes. Fungal endo- (alt. 50 m). The population was well preserved phytes here is interpreted in their broad sense: because of suburban shrine forest. The population fungi that colonize living plant tissue without was rich in healthy looking leaves. 4) Hatajuku, causing any immediate, overt negative effect Hakone-machi (alt. 560 m). The population was (Hirsch & Braun, 1992). Fungi with parasitic, along with walking trail in broad leaved decidu- commensal, or mutualistic relationship to plants ous forest. The majority of the leaves were are included. A number of studies have been con- healthy looking. 5) Miyagami, Yugawara-machi ducted for endophytes of the tree leaves in Japan (alt. 550 m). Major population with healthy (e.g. Osono, 2009). leaves was accompanied with broad leaved decid- Aucuba japonica (Cornaceae) is one of the well uous forest along with Tsubaki line (motor road). known and widely distributed plants in these ar- 6) Ubako, Hakone-machi, (alt. 880 m). The popu- eas. It is an evergreen shrub distributed in low- lation with many variegated leaves was accompa- land to mountains mainly in south-west, but al- nied with broad leaved deciduous forest. Aucuba most all over Japan (Satake et al., 1989). Aucuba japonica occured in clumps. 7) Komagatake, Ha- japonica is also known to be endemic to Japan, kone-machi (alt. 890 m). The population was and the genus is endemic to east Asia. In Sagami found at the margin of Cryptomeria japonica Sea and its adjacent areas, A. japonica is distrib- plantation, along with motor road. The population uted at high frequency from evergreen broadleaf was minor. forest in lowland to deciduous forest in Hakone and Tanzawa mountains at > 500 m altitude. Au- Isolation cuba japonica is one of the suitable plants to In each site, four healthy looking leaves were compare endophytic mycobiota in different envi- collected and named A, B, C, and D for practical- ronments. ity. The leaves were washed by running tap water To elucidate the factors affecting mycobiota in to remove surface contaminants, and cut into Sagami Sea and its adjacent areas, comparisons three parts (apical, middle, and basal). Each part of the endophytic mycobiota in A. japonica in was immersed in 70% ethanol for 1 min, followed various site with different altitude was conducted. by 2 min. in 1% Sodium hypochlorite for surface sterilization. The leaf pieces were rinsed by steril- Mycobiota in Aucuba 389 Fig. 1. Collection sites. A, map of Japan showing the Sagami Sea and its adjacent areas studied in the present study (B); B, the Sagami Sea and its adjacent areas including the collection sites (C); C-1, Manazuru; C-2, Shiroyama; C-3, Miyashita; C-4, Hatajuku; C-5, Miyagami; C-6, Ubako; C-7, Komagatake. ized DW, and dried over night on sterilized filter facturer’s instruction. To identify the obtained paper. Before dried, two sets of a 5 mm leaf piec- isolates by molecular technique, internal tran- es from apical and basal parts and two from mid- scribed spacer 1, 2 and 5.8s ribosomal RNA re- dle leaf blade were dissected by a sterilized knife gions (ITS-5.8S) were sequenced. Primers ITS1 so that costa were included (four pieces in total and ITS4 (White et al. 1990) were used to ampli- from a single leaf). The leaf pieces were inoculat- fy the ITS-5.8S region. DNA was amplified using ed onto two plates of half diluted potato dextrose 40 ml PCR reactions, containing 0.2 mM each agar (1/2PDA, Nissui) and cornmeal agar (CMA, primer, 1 U TaKaRa Ex Taq DNA polymerase Nissui), respectively, so that each plate contained (TaKaRa, Tokyo, Japan), and a deoxynucleoside all the four pieces from the leaf. The plates were triphosphate (dNTP) mixture containing 2.5 mM sealed with parafilm, and incubated at room tem- each dNTP and ExTaq buffer containing 2 mM perature. The occurring hyphae was isolated dur- Mg2+. PCR was carried out using a Gene Amp ing the following 6 weeks of observation. The PCR system 9700 (Applied Biosystems, Foster isolates were kept in PDA slants. City, CA, USA). DNA was denatured for 3 min at 95 ºC, followed by 30 cycles of denaturation at Molecular anaysis 95 ºC for 30 s, annealing at 50 ºC for 30 s, and The isolates were cultivated in 2 ml of 2% malt extension at 72 ºC for 2 min, followed by final extract for 2 weeks, and the mycelia was harvest- extension at 72 ºC for 10 min. ed and frozen at –80ºC. About 50 mg of myceli- PCR products were purified using an ExoSAP- um was mechanically lysed by a Quagen Tis- IT purification kit (USB, Cleveland, OH, USA). sueLyser, using ceramic beads.