Mycologia, 103(4), 2011, pp. 703–709. DOI: 10.3852/10-296 # 2011 by The Mycological Society of America, Lawrence, KS 66044-8897
Diversity of culturable ericoid mycorrhizal fungi of Rhododendron decorum in Yunnan, China W. Tian
fungi colonize their fine roots, called hair roots, and produce hyphal complexes in epidermal cells (Smith and Read 2008). This association plays important roles in plant nutrition and ecological adaptation to harsh environments (Cairney and Meharg 2003, Rowe et al. 2008). Culturable fungi that have been isolated from ERM are ascomycetes (Harley and Harley 1987, Smith and Read 2008). Rhizoscyphus ericae-Scytalidium vaccini was the first isolated and identified ericoid mycorrhizal fungus (Read 1974, Bradley et al. 1981, Sharples et al. 2000, Vra˚lstad et al. 2002, Upson et al. 2007). Species in the anamorph genus Oidiodendron, especially O. maius, which is connected to the holomorph genus Byssoascus, have been found widely associated with different Ericaceae populations (Xiao and Berch 1999, Martino et al. 2007). Other fungi that form ERM associations with Ericaceae include species of ascomycetes in the Helotiales (McLean 1999) and Herpotriciellaceae, such as Capronia (Allen 2003), dark septate endophytes (DSE), such as Phialocephala fortinii (Vohnik et al. 2003), and some basidiomycetes, such as Clavaria (Peterson et al. 1980), and members of Sebacinales (Allen 2003, Selosse et al. 2007). Until now most work on ERM has focused on Vaccinium (Stribley et al. 1975, Martino et al. 2000, Villarreal-Ruiz et al. 2004) and Calluna (Bradley et al. 1981, Johansson 2001, von Oheimb et al. 2009). Rhododendron is the largest genus in the Ericaceae with more than 1000 species (Chamberlain et al. 1996) and plays a vital role in forest ecosystems, especially in the Himalayas. However only a few species of this genus have been studied for ERM fungi. ERM fungi R. ericae and O. maius, which were isolated from non-Rhododendron individuals, were demonstrated to form mycorrhizal associations with Rhododendron species (Piercey et al. 2002). ERM fungi of R. lochiae were identified by culture or cultureindependent molecular techniques (Bougoure and Cairney 2005b). The enzymatic release of complexes by ERM may increase nitrogen acquisition and contribute to the expasion of Rhododendron in the forest (Wurzburger and Hendrick 2007). In China fungal diversity of arbuscular mycorrhiza (AM) and ectomycorrhiza (ECM) in various ecosystems have been well studied (Liu 1989, Yu et al. 2007, Deng et al. 2009, Geng et al. 2009). However there have been few investigations of ERM, despite the dominance of many ecosystems by Rhododendron.
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China, 650204, and Graduate University of Chinese Academy of Sciences, Beijing, China 100049
C.Q. Zhang1 Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China 650204
P. Qiao Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China, 650204, and Graduate University of Chinese Academy of Sciences, Beijing, China 100049
R. Milne University of Edinburgh, Edinburgh EH9 3JH, United Kingdom
Abstract: The diversity of ericoid mycorrhizal fungi isolated from Rhododendron decorum Franch. in Yunnan, southwestern China, was examined for the first time. In total 300 hair-root samples were collected from 13 R. decorum individuals in two adjacent wild population sites and one cultivated population site. Two hundred eighteen slow-growing isolates were obtained; the ability of some to form ericoid mycorrhiza was tested in vitro. One hundred twenty-five isolates formed hyphal structures morphologically corresponding to ericoid mycorrhiza, and these were determined by morphological and molecular means to belong to 12 fungal species. There were hardly any differences in species among the three sampled populations. The sequences of several isolates were similar to those of Oidiodendron maius and ericoid mycorrhizal fungi from Helotiales, accounting respectively for 18.4% and 24.8% of the total culturable ericoid mycorrhizal fungi assemblage. Dark septate endophytes were detected in the sampled hair roots by microscopy. Key words: ericoid mycorrhiza, Oidiodendron maius, Rhizoscyphus ericae, Rhododendron decorum, southwestern China INTRODUCTION
Plants in the Ericaceae have a distinctive mycorrhizal association, the ericoid mycorrhiza (ERM), where Submitted 25 Sep 2010; accepted for publication 7 Dec 2010. 1 Corresponding author. E-mail:
[email protected]
703
704 TABLE I.
MYCOLOGIA Conditions of sampling sites and number of plants sampled
Sites
Location
Latitude and longitude
Altitude (m)
soil pH
Number of plants sampled
DHL
Haligu, Jade Dragon Snow Mountain, Lijiang, Yunnan Wenhai, Jade Dragon Snow Mountain, Lijiang, Yunnan Kunming Botanical Garden, Kunming, Yunnan
27u089N, 100u109E
3200
6.9
3
27u089N, 102u119E
3200
7.0
5
25u089N, 102u449E
1950
6.0
5
DWL DGK
Southwestern China is one of the most important distribution centers of Rhododendron in the world (Min and Fang 1979) with more than 400 species (Zhang 2003). One of the most common Rhododendron species in SW China is R. decorum, which grows mainly in Pinus yunnanensis forest at 2000–3200 m, especially at forest edges. It is an important ornamental and plays an important role in establishment of forests in harsh environments (Zhang 2003, 2008). It therefore is valuable to know the mycorrhizal associations of R. decorum in its native habitat. The aims of this research were (i) to investigate the mycorrhizal status of R. decorum, (ii) to isolate and identify ERM fungi associated with hair roots of R. decorum, (iii) to examine species diversity of the ERM fungi and (iv) to determine whether there are differences in ERM fungi between wild and cultivated populations. Three R. decorum populations in Yunnan, China, were investigated. Both cultivation-based and molecular methods were developed for this research. MATERIALS AND METHODS
Root sampling and microscopic examination.— Thirteen individuals of R. decorum were sampled from two adjacent wild populations in Haligu (DHL) and Wenhai (DWL), Lijiang, Yunnan, and from one cultivated population at Kunming Botanical Garden (DGK), Kunming, Yunnan (TABLE I). The distance between the two wild population sites, where R. decorum grows with Pinus yunnanensis, Quercus semecarpifolia and Q. pannosa (TABLE I), was about 1 km. The distance between the sampled plants was at least 1 m. The soil pH of the organic floor was tested with a Soil pH & Moisture Tester (DM-15). Sampling was carried out Sep 2005. Ten root samples were directly collected from hair roots of each of the 13 plants. They were washed with tap water 1 h, then 10 times with distilled water and cut into 1 cm segments. A portion of the segments from each plant were cleared in 10% KOH at 90 C for 30 min, rinsed with distilled water and then stained with 0.5% cotton blue in lactophenol for microscopic examination (Olympus BX50, Tokyo, Japan). The remaining root segments of each population were kept in FAA solution (30% formaldehyde: 50%
ethanol: acetic acid at 5: 90: 5 by volume) as vouchers, which are deposited in Kunming Institute of Botany, Chinese Academy of Sciences. Fungal isolation and culturing.— Three hundred fresh roots were sampled at random from each of the 13 plants (TABLE I). One hundred roots were taken from each of the 13 plants at each site. The roots were cleaned, cut into segments as above, surface sterilized with 0.1% mercuric chloride solution for 3–5 min, rinsed in sterile distilled water 7–8 times and dried with sterilized gauze. One surface-sterilized root tip was placed in a Petri dish with PDA medium (Hambleton et al. 1998), which was mixed with streptomycin (30 mg mL21), sealed with parafilm and incubated in the dark at 22–25 C. The plates were examined for macroscopic characters every 2 d during the first month and then weekly. Extended hyphae of fungal colonies were stained with 0.5% cotton blue in lactophenol and examined under the compound microscope (Olympus BX50, Tokyo, Japan). Based on the micro- and macromorphological characters of fungal colonies, they were grouped into morphotypes and one isolate selected from each morphotype were used for pure culture synthesis experiments. All isolates were subcultured on PDA medium as stock cultures for long-term preservation, and subcultured every 3–4 mo. Pure culture mycorrhizal synthesis.—Seeds of R. decorum were surface sterile with 0.1% mercuric chloride solution 1 min, rinsed in sterile distilled water 7–8 times, and 100 sterilized seeds were placed on sterilized Petri dishes with moistened filter paper (9 cm diam 3 1.2 cm deep). Petri dishes were sealed with parafilm and incubated at 22 C with a 16 h day: 8 h night photoperiod. Seedling roots were examined periodically under a compound microscope (10 3 100, Olympus BX50, Tokyo, Japan) to check for contamination. After 2 mo clean seedlings were transplanted into culture bottles with 0.5% water agar (five seedlings per bottle). In addition fungal liquid cultures were prepared in PDA medium and incubated 1 mo. Five bottles were inoculated with fungal mycelia of each isolate, and five bottles without inoculation were used as controls. The inoculated seedlings were incubated at 22 C with a 16 h day: 8 h night photoperiod, and their roots were aseptically sampled and examined for mycorrhization every 5 d after inoculation. When typical ERM hyphal coil structures were detected in the root epidermal cells the fungi were reisolated with the method described above and compared with the original isolates.
TIAN ET AL.: RHODODENDRON DECORUM TABLE II.
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Morphological characters of ERM fungi isolated from roots of R. decorum on PDA medium
Culturea
Color
Texture
Ridge and shape
Color of margin and shape
Exudation and color
FJ999650 FJ999645 FJ999640 FJ999646 FJ999655 FJ999653 FJ999648 FJ999639 FJ999654 FJ999651 FJ999652 FJ999642
Yellow white white brown brown brown white croci light brown gray light brown brown white yellow
felty fluffy fluffy floccose fluffy fluffy felty fluffy felty fluffy fluffy felty
radial cyclical, radial irregular — radial radial cyclical, radial cyclical, radial radial — radialized cyclic, radial
white, undulations white, undulations brown, branch white, peritrich white, peritrich white, peritrich light brown, crenate gray, peritrich white, undulations brown, peritrich white, lacerate white, undulations
— white yellow — yellow croci croci — gray — brown yellow —
a b
Conidiospore Growth and shape rateb spindly round long bacilliform long bacilliform — round — — — — — short bacilliform
0.5 0.5 4 4 4 1 0.5 0.5 0.5 4 2 1
Isolate number refers to root segment number if more than one produced an isolate. Growth rate refers to the growth of diameter of culture every day (mm/d).
DNA extraction, PCR amplification and sequencing.—DNA was extracted from the representative isolate of 20 morphotypes with the modified cetyltrimethylammonium bromide (43 CTAB) method. Primers ITS4 and ITS5 (White et al. 1990) were used to amplify the ITS region of ribosomal DNA. The PCR reaction mixtures (50 mL) contained 103 buffer (50 mM KCl, 10 mM Tris-HCl, 0.1% Trition X-100) (Tiangen, China), 25 pmol each primer, 25 mM MgCl2, 200 mM dNTP, 2.5 units Taq DNA polymerase (Tiangen, China) and 100 ng genomic DNA. Amplifications were performed with preliminary denaturation at 95 C for 5 min, 35 amplification cycles (94 C for 1 min, 48 C for 1 min, 72 C for 1 min), followed by a final extension at 72 C for 10 min. A negative control containing no DNA was included in the reactions. Amplification products were subjected to electrophoresis in 0.1% (w/v) agarose gels, stained with Goldview and viewed under UV light. ITS-PCR products were purified with a UNIQ-10 column PCR products purification kit (Sangon, China). The sequencing reaction was performed from both strands with the primers ITS4 and ITS5 using an ABI 3700 automated sequencer (Perkin Elmer). Fungal diversity analysis.— Sequences were edited and deposited in GenBank (accession numbers FJ999636– FJ999655). The sequences were assigned with BLAST in the NCBI nucleotide databases (http://www.ncbi.nlm.nih. gov/). The relative abundance of members of the isolated ERM fungi assemblage at each of the three sites was analyzed with Excel, determining the relative abundance (RA, percent) 5 species (isolates) number/total species (isolates) number 3100%.
RESULTS
The epidermal cells of hair roots were filled with hyphal coils (FIG. 1). A total of 218 slow-growing
isolates were obtained from the 300 root samples. Based on their morphology, the isolates were grouped into 65 morphotypes, from which 35 morphotypes were selected to perform the pure culture synthesis in vitro. Morphological characters of these ERM fungi were variable in color, ridges and growth rates, which were 0.5–4.0 mm daily (TABLE II). Twenty (representing 125 isolates in total) of the 35 isolates formed ERM coils in hair-root epidermal cells 1 mo after inoculation of R. decorum seedlings (FIG. 2), and 15 isolates failed. All inoculated plants grew well without any disease symptoms during synthesis. Intracellular hyphal coils were detected in synthesized mycorrhiza. The re-isolated fungi from the synthesized ERM associations produced the same colonies as that of the original isolates. Although DSE were detected in the hair-root samples under light microscopy, we could not obtain isolates. Amplified ITS sequences from the 20 isolate morphotypes were 440–670 bp long. BLAST queries of the sequences revealed that all the isolated fungi were ascomycetes and that they belonged to 12 species (TABLE III). Fungal isolates FJ999637, FJ999644, FJ999655 and FJ999641 were grouped together with Neonectria radicicola. FJ999638, FJ999646, FJ999643 and FJ999649 were almost identical to Bonectria ochroleuca. FJ999652 and FJ999648 showed almost 100% mutual similarity to sequences from Plectosphaerella cucumerina and the mycorrhizal ascomycete of Rhododendron. FJ999645 and FJ999636 had closest affinities to previously identified ERM fungi, Oidiodendron maius and Oidiodendron citrinum respectively. FJ999650 and FJ999647 were almost identical to uncultured fungi in Helotiales. FJ999651, FJ999653
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MYCOLOGIA
TABLE III. Putative taxonomic affinities of isolates obtained from roots of Rhododendron decorum as inferred from BLAST queries of ITS sequences in GenBank Culture
BLAST closest match
ITS-PCR products
Overlap (%)
Sequence matches (%)
FJ999637 FJ999644 FJ999655 FJ999641 FJ999638 FJ999646 FJ999643 FJ999649 FJ999650 FJ999647 FJ999651 FJ999639 FJ999636 FJ999645 FJ999652 FJ999653 FJ999640 FJ999642 FJ999648 FJ999654
Neonectria radicicola [AJ875336] Neonectria radicicola [AJ875337] Neonectria radicicola [AJ875336] Neonectria radicicola [AJ875336] Bionectria ochroleuca [AF106532] Bionectria ochroleuca [EU484314] Bionectria ochroleuca [AF106532] Bionectria ochroleuca [AF106532] Uncultured Helotiales [DQ182427] Uncultured ectomycorrhiza (Helotiales) [EF644169] Gliocladium cibotii [EF543846] Uncultured Hymenoscyphus ericae [AY394685] Oidiodendron citrinum [AF307762] Oidiodendron maius [AF307772] Plectosphaerella cucumerina [AM924165] Chaetomium sp. [EU035795] Uncultured ascomycete [AY969907] Chalara sp. [AY188359] Mycorrhizal ascomycete of Rhododendron [AB089663] Uncultured ascomycete isolate [AY969907]
573 530 460 539 569 599 583 534 467 469 481 670 541 462 437 592 485 499 520 490
97 95 97 100 98 100 98 99 100 99 95 99 86 100 100 95 65 99 100 70
99 93 99 99 94 93 99 99 97 97 82 76 93 93 97 91 84 87 96 84
and FJ999642 were close to Gliocladium cibotii, Chaetomium sp. and Chalara sp. respectively but with low affinity (82%, 91% and 87%, TABLE III). Of the 12 ERM fungal species detected, at least 11 were present at DWH and at least eight were present at the other two sites. This however is probably an underestimate of the true ERM diversity because only 35 of 65 detected morphotypes were chosen for synthesis. Furthermore some morphotypes might have represented multiple taxa. The most abundant ERM species overall was the Heliotales species (FJ999650), which occurred in 24 isolates, 19.2% of the total. Next most common was Oidiodendron maius (FJ999645), comprising 18.4% of all isolates. Three other taxa comprised . 10% of all isolates; these were ascomycete I (AY969907) (16%), Bionectria ochroleuca and Neonectria radicicola (13.6% each; TABLE IV). Two other taxa (Chaetomium sp. and ascomycete II) occurred in 5.6% of isolates each; all seven of these most frequent taxa were found in all three sites (TABLE IV). Of the remaining taxa Rhizoscyphus ericae was present in 4.6% of isolates overall but was not found in DGK whereas the last four taxa were found in just one isolate each and hence were detected from just one site each (TABLE IV). Comparing sites, the cultivated material in DGK differed most notably from the two wild sites in that O. maius was rare (5.1% of isolated as opposed to . 20% in both wild sites) whereas Heliotales was far more abundant (35.9% as opposed to , 14% at wild
sites). The apparent absence of R. ericae at DGK might be a further difference, although this species could have been present but undetected. Beyond this there were no clear differences between the sites (TABLE IV).
DISCUSSION
Rhododendron is an ecologically important genus across much of western China and the Himalayas, however until now nothing has been known about the mycorrhizal associations of any Rhododendron species within this area. Here, based on a small sample from two wild and one cultivated locality, we have detected a minimum of 12 distinct fungal species that occur as ERM associates of R. decorum, a common and ecologically relatively versatile species from SW China. At one wild site 11 ERM species were found in association with a sample of five R. decorum plants. These results revealed far greater mycrorhizal diversity than were detected for R. obtusum from Japan (Usuki et al. 2003), for which only four fungal species could be distinguished with RFLP data. Both studies however might have underestimated the true number of species involved. With DNA and culture-based detection, 15 ericoid mycorrhizal fungi were detected from roots of Gaultheria shallon from Cananda (Allen et al. 2003), including Sebacina-like and Capronia-like sequences, which were not detected here.
TIAN ET AL.: RHODODENDRON DECORUM
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TABLE IV. Frequency of occurrence and relative abundance (in parentheses) of 12 species of fungi on Rhododendron decorum roots at three sites and diversity information for each site Morphotype
Taxon
FJ999650 FJ999645 FJ999640 FJ999646 FJ999655 FJ999653 FJ999648 FJ999639 FJ999654 FJ999651 FJ999652 FJ999642 Number of species Number of isolates Simpson Index (1/D)a Shannon Index (H9)a
Heliotales Oidiodendron maius ascomycete AY969907 Bionectria ochroleuca Neonectria radicicola Chaetomium sp. ascomycete AB089663 Hymenoscyphus ericae ascomycete AY969907 Gliocladium cibotii Plectosphaerella cucumerina Chalara sp.
a
DHL 5 13 8 6 7 4 2 3 0 0 0 0
(10.4) (27.1) (16.7) (12.5) (14.6) (8.3) (4.2) (6.3) (0) (0) (0) (0) 8 48 7.48 6 1.67 7.41 6 1.23
DWH 5 8 5 4 6 2 2 3 1 0 1 1
(13.2) (21.1) (13.2) (10.5) (15.8) (5.3) (5.3) (7.9) (2.6) (0) (2.6) (2.6) 11 38 6.89 6 0.95 7.48 6 0.9
DGK 14 2 7 7 4 1 3 0 0 1 0 0
(35.9) (5.1) (17.9) (17.9) (10.3) (2.6) (7.7) (0) (0) (2.6) (0) (0) 8 39 6.93 6 0.67 7.68 6 0.65
Total 24 23 20 17 17 7 7 6 1 1 1 1
(19.2) (18.4) (16) (13.6) (13.6) (5.6) (5.6) (4.8) (0.8) (0.8) (0.8) (0.8) 12 125
Values given for diversity indices are mean 6 standard error (SE).
Among the 12 ERM fungal species the most common ericoid mycorrhizal fungi of R. decorum (four species) belong to Helotiales (TABLE IV). All 12 species formed hyphal coils in the hair roots, but only O. maius and a mycorrhizal ascomycete of Rhododendron were recorded previously (Usuki et al. 2003, Villarreal-Ruiz et al. 2004). Some of the species, such as N. radicicola, have been reported as a pathogen causing diseases (Houston 1994). We isolated fungi from the roots, synthesized mycorrhiza and re-isolated fungi, but we did not evaluate plant responses in plant growth parameters. Such tests are planned in future research. The results (TABLE IV) show that O. maius (FJ999645) is more abundant in the wild population. O. maius is a widespread ERM fungus, which has been isolated from roots of Rhododendron species in Ireland (Douglas et al. 1989), Woollsia pungens in Australia (Chambers et al. 2000) and other places (Addy et al. 2005). Eleven ERM fungal species were found in the DWL population site and eight fungi in its adjacent DHL population site. We selected only a few morphotypes in our synthesis trials and did not test for mutualistic effects between fungi and plant, so the result of isolated fungi cannot reflect the complete picture of the ERM fungal assemblage of R. decorum. However our research provided ERM fungi for us to investigate their function on hosts, which will be applied in cultivation and ecological protection. Southwestern China is a center of origin and dispersal of many Rhododendron species (Zhang 2003). From our preliminary study R. decorum has
higher ERM fungal diversity than we expected. Rhododendron plants are difficult to establish in some environments (Jansa and Vosa´tka 2000). The ecophysiological importance of Rhododendron hair roots in such environments is poorly understood. Unfortunately little attention was paid to their mycorrhizal partners when they were planted and managed. Further research therefore is needed to compare the diversity of ERM fungi among species of Rhododendron within and among populations and then to scrutinize the active role they play in host plants and entire ecosystems. This is crucial for Rhododendron sustainable management.
FIG. 1. Transverse section shows hyphae in the epidermal cells of root under light microscopy. Bar 5 20 mm.
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FIG. 2. The longitudinal section of mycorrhiza synthesized with FJ999653. Bar 5 100 mm. ACKNOWLEDGMENTS
We thank Professor Yun Wang, Professor Andrew (F.A.) Smith, Professor David Rankin and Professor Marc-Andre´ Selosse for valuable comments, Dr Shannon Berch for experimenal advice. This research was supported by the National Nature Science Foundation of China (30770139 and 2010DH011) and a project of the Ministry of Science and Technology China (2009GB2F300343). LITERATURE CITED
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