Ectomycorrhizas of Cercocarpus ledifolius (rosaceae) Kelly R. McDonald, Jason Pennell, Jonathan L. Frank,and Darlene Southworth 學生 : 生科三甲 葉茱玫 教授 : 藍清隆

Background Woody species in the Rosaceae form ectomycorrhizal associations, but the fungal symbionts are unknown.

Aim To identified the fungi that form ectomycorrhizas with Cercocarpus ledifolius (curl-leaf mountain mahogany) Find out whether host plants are isolated from other ectomycorrhizal species in the plant community or linked with other trees through mycorrhizal networks.

Key words: Cercocarpus = mountain mahogany( 山紅木 ) is rose family( 科 ). Ectomycorrhizas 外生菌根 Geopora 類似松露的 fungi in the family Pyronemataceae Quercus garryana var.breweri the Garry oak, Oregon white oak or Oregon oak( 橡木 ) Rosaceae =rose family 薔薇科

Materials and Methods Ectomycorrhizal sampling Mycorrhizal morphotyping DNA analysis Data analysis

Materials and Methods Sites — Four sites were selected in southern Oregon: 1.Quarry Hill (42 ° 3 ′ N,122 ° 30 ′ W, 1585 m a.s.l.), 2.Hobart Bluff (42 ° 5 ′ N, 122 ° 28 ′ W, 1676 m a.s.l.), 3.Table Mountain (42 ° 11 ′ N, 122 ° 29 ′ W 1861 m a.s.l.), 4. Wagner Butte (42 ° 6 ′ N,122 ° 46 ’ W, 2011 m a.s.l.).

Ectomycorrhizal sampling Soil samples were taken from 15 trees of C. ledifolius and 8 trees of Q. garryana var. breweri in 1. fall late spring and summer mL of soil was sampled from each tree from the upper 15 – 20 cm of soil at the canopy edge in the four cardinal directions and combined Sampled trees were separated from each other by at least 8 m and from other woody plants by at least 5 m. Samples were stored at 4 ° C and processed within 14 d after collection.

Mycorrhizal morphotyping Roots were rinsed( 沖洗 ) with water over a 1-mm mesh screen. Ectomycorrhizas were sorted into morphotypes based on branching pattern( 分枝的形態 ) tip shape color texture( 質地 ) hyphal structure( 菌絲結構 ) mantle pattern( 覆蓋的型態 )

Photographed Mycorrhizas 1.with a Leica MZ75 dissecting microscope ( 解剖顯微鏡 ) 2.with a Leica DMLB compound microscope( 複合式顯微鏡 ) (root tips of mycorrhizal morphotypes were stored in buffer (0.1 M Tris, 0.3 M NaCl, 0.04 M EDTA) at 4 ° C for subsequent DNA extraction.)

DNA Analysis Extracted DNA (in 2% cetyltrimethyl ammonium bromide (CTAB) with chloroform ) Amplified( 放大 ) (polymerase chain reactions (PCR)) 1 min at 94 ° C → 35 cycles {of 30 s at 95 ° C → 40 s at 55 ° C → 1 min at 72 ° C }→ 5 min at 72 ° C ｛ using rbcL gene primer pairs rbcLa_F and rbcLa_R724,rbcLa- F and rbcL_ajf634R. ｝

DNA Analysis Purified PCR products( 純化 ) (with QIA quick PCR Purification kits prepared with BigDye Terminator Ready Reaction Mix v3.1 and sequenced in an ABI 310 Genetic Analyzer) Edited( 編輯 ) sequences (by sequencing the internal transcribed spacer (ITS) region,including ITS1, and ITS2, with forward primers ITS1F and ITS1, and reverse primer ITS4.) (with the program Chromas 1.45; contigs were assembled using the program Sequencher v4.7.)

DNA Analysis Compared sequences (with the program Clustal X and to fungal sequences in GenBank with the BLAST algorithm)

Data Analysis

Results Total of 97 sequences representing ( 相當於 )28 species were obtained from EM fungi(Table 1.) Some EM fungi occurred at multiple sites and on multiple hosts ( Table 1 ). 3 fungal species ( Cenococcum, Geopora,and Pezizomycotina1) were found at three or four sites. Geopora sp., the most frequently encountered mycobiont of C. ledifolius, was not found on Q. garryana var. breweri. Cercocarpus ledifolius shared 2 fungal species with Q. garryana var. breweri and 4 with Arctostaphylos spp.

Table 1. Genbank accession numbers of ectomycorrhizal taxa detected on roots of Cercocarpus ledifolius (Ce), Quercus garryana var. Breweri (Qu), and Arctostaphylos sp. (Ar) at four sites,Hbluff (H), Quarry hill (Q), Table mountain (T) and Wagner butte (W) in southern oregon, USA.

Results Based on rbcL gene sequencing, none of the ectomycorrhizas under C. ledifolius were roots of Q. garryana ; similarly, none of the ectomycorrhizas under Q garryana were roots of C. ledifolius. Fungi were identified as mycobionts 14 with C. ledifolius 12 with Q. garryana var. breweri 10 with Arctostaphylos species

Results Of 724 total characters in the phylogenetic alignment of ITS sequences, 401 characters were constant; 190 characters were considered parsimony-informative and 133 variable characters considered parsimony-uninformative. Both the parsimony and the maximum likelihood consensus trees generated from their ITS alignment unequivocally placed the ectomycorrhizal Geopora sp. in the genus( 種 ) Geopora nearest to clade 8 containing G. arenicola ( Fig. 3 ).

Fig. 3. Phylogenetic tree using parsimony for ITS data (with gen- bank numbers) showing placement of the ectomycorrhizal geopora sp. (GU205118) within the genus geopora with 1000 bootstrap replicates; bootstrap numbers greater than 50% are included above branches. Clade numbers are from tamm et al. (2010). the ectomycorrhizal Geopora sp

Results Fig.2 ectomycorrhizas of Cercocarpus ledifolius in southern oregon. (A) geopora. (B) genabea. (C) cenococcum. (D) tomentella- 1. (E) hebeloma. (F) laccaria. (G) piloderma. (H) cortinarius. (I) tricholoma Geopora was evenly( 均勻 ) brown with some pale( 淡色 ) tips ( Fig.2A ). Genabea ( Fig. 2B ) was lighter tan( 黃褐色 ) and slightly rusty( 鐵鏽色 ). The distinctive black Cenococcum morphotype had coarse( 粗糙 ) black hyphae ( 菌絲 )( Fig. 2C )

Results Tomentella was brown-black ( Fig. 2D ). Hebeloma and Laccaria were both white to pale tan( 黃褐色 ) and not easily distinguished ( Fig. 2E, F ).

Results Piloderma had the greatest amount of mycelial( 菌絲 ) strands, which obscured( 遮蔽 ) the actual EM root tip ( Fig. 2G ); Cortinarius species had nearly as many mycelia strands ( 菌絲體 束 ) ( Fig. 2H );Tricholoma had an even, white felty( 氈 ㄓㄢ 狀 ) covering ( Fig. 2I ).

Key results 16 species of fungi were identified from ectomycorrhizas of Cercocarpus ledifolius. The ectomycorrhizal community was distinguished( 顯著 ) by 1.the presence of a Geopora species situated( 位於 ) in the G. arenicola clade 2.and by the absence( 缺乏 ) of Rhizopogon, suilloids, and Sebacinales. 3.Of the species on C. ledifolius, 2 also occurred on trees of Quercus garryana var. breweri and 4 on Arctostaphylos sp.

DISCUSSIONc The most distinctive feature of the C. ledifolius EM community is the high frequency of a Geopora species. With only a 91% match to a vouchered sequence of G. arenicola in GenBank, it may be an undescribed species.

DISCUSSIONcussion The finding of generalist species in common with Q. garryana var. breweri and Arctostaphylos spp. Demonstrates( 表明 ) that C. ledifolius could link to other EM plants by mycorrhizal network The presence of fungal species in common with other ectomycorrhizal hosts shows that C. ledifolius, Q. garryana var. breweri, and Arctostaphylos species could be linked by a mycorrhizal network, allowing them to exchange nutrients or to share inoculum for seedling roots and new fine roots.

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