1 title: identity, diversity and molecular phylogeny of ...(n 22°04 -22°17 ; e 100°32 -100°44 )...

1

Title: Identity, Diversity and Molecular Phylogeny of the Endophytic Mycobiota in 1

Rare Wild Rice Roots (Oryza granulate) from a Nature Reserve in Yunnan, China. 2

Running title: Endophytic fungi associated with wild rice. 3

4

Zhi-lin Yuan1, 2, Chu-long Zhang1*, Fu-cheng Lin1*, Christian P. Kubicek3 5

6

1 State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, 7

310029, China 8

2 Institute of Subtropical Forestry, Chinese Academy of Forestry, 311400, China 9

3 Institute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, 10

Vienna University of Technology, 1060 Vienna, Austria 11

* Corresponding author: [email protected]; [email protected] 12

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01911-09 AEM Accepts, published online ahead of print on 28 December 2009

on August 27, 2020 by guest

http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

2

Rice (Oryza sativa L.) is-on a global scale-one of the most important food crops. Although 13

endophytic fungi and bacteria associated with rice have been investigated, little is known about 14

the endophytic fungi of wild rice (Oryza granulate) in China. Here we studied the root 15

endophytic mycobiota residing in roots of O. granulate using an integrated approach consisting 16

of microscopy, cultivation, ecological indices and direct PCR. Microscopy confirmed the 17

ubiquitousness of dark septate endophytes (DSEs) and sclerotium-like structures in root tissues. 18

Isolations from 204 root segments from 15 wild rice plants yielded 58 isolates, in which 31 19

ITS-based genotypes were recorded. The best BLAST match indicated that 34.5 % of all taxa 20

encountered may represent hitherto undescribed species. Most of the fungi were isolated with a 21

very low frequency. Calculation of ecological indices and estimation of taxon-accumulation 22

curves indicated a high diversity of fungal species. A culture-independent approach was also 23

performed to analyze the endophytic fungal community. Three individual clone libraries were 24

constructed. Using a threshold of 90 % similarity, 35 potentially different sequences 25

(phylotypes) were found among 186 positive clones. Phylogenetic analysis showed that 26

frequently detected clones were classified into Basidiomycota, and 60.2% of total analyzed 27

clones were affiliated with unknown taxa. Exophiala, Cladophialophora, Harpophora, Periconia 28

macrospinosa and Ceratobasidium/Rhizoctonia complex may act as potential DSEs groups. A 29

comparison of the fungal communities characterized by the two approaches demonstrated 30

distinctive fungal groups and only a few taxa overlapped. Our findings indicate a complex and 31

rich endophytic fungal consortium in wild rice roots, thus offering a potential bioresource for 32

establishing a novel model of plant-fungal mutualistic interactions. 33

Key words: wild rice roots, dark septate endophytes, ITS, clone library sequencing, symbiosis 34


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

3

The majority of terrestrial plant roots are intimately associated with mycorrhizal fungi, and many 35

aspects of the ecological roles played by these mycorrhizal fungi are well understood. In recent years, 36

however, endophytic fungi are gaining increasing interest. There is accumulating evidence that plant 37

roots usually harbor both mycorrhizal as well as endophytic fungi (29, 30, 34, 39, 52, 63). Dark 38

septate endophytes (DSEs), which are characterized by dark pigmented hyphae and sclerotium-like 39

structures, are believed to represent the primary non-mycorrhizal root-inhabiting fungi (23). In some 40

cases, DSEs are even more frequent than mycorrhizal fungi (68). 41

Endophytic fungi have frequently been reported to be associated with crop plants including 42

wheat (Triticum aestivum), wild barley (Hordeum brevisubulatum, H. bogdanii), soya bean (Glycine 43

max) and maize (Zea mays) (6, 9, 11, 13, 21, 26, 27, 33, 36, 67). Some of the endophytic fungi in 44

these crops conferred resistance of the plant to insect or fungal pathogens. 45

Domesticated from the wild grass Oryza rufipogon 10,000 to 14,000 years ago, rice is today the 46

main staple for more than 3 billion people (i.e. half of the world’s population). Its consumption 47

exceeds 100 kg per capita annually in many Asian countries and is the principal food for most of the 48

world’s poorest people, particularly in Asia. The association of arbuscular mycorrhizal fungi and 49

endophytic bacteria with rice plants has been well documented (15, 32, 35, 44, 53, 56, 60). Less 50

however is known about its fungal endophytes. Fungal endophytes have been detected in cultivated 51

rice (Oryza sativa L.) (12, 14, 37, 61, 70), and an antagonistic or plant growth-stimulating properties 52

have been claimed for some of these isolates. For example, endophytic Fusarium spp. from 53

cultivated rice roots proved to be effective in bio-control of root-knot nematode (28). The 54

co-occurrence of mycorrhizal and endophytic fungi in variety of rice cultivars has also recently been 55

reported (63). 56

Non-domesticated, wild plant species may live in symbiosis with a unique and rich mycoflora 57

that may have been lost during breeding of the cultivars used in agriculture (20, 59). The purpose of 58


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

4

this research was to characterize the endophytic fungal community of rare (nearly extinct) wild rice 59

(Oryza granulate) roots from a Nature Reserve in Yunnan, China. Our results showed that arbuscular 60

mycorrhizal fungi were apparently absent from wild rice roots. This finding was confirmed by 61

standard roots staining techniques and molecular detection using the AM-specific primer pairs (69). 62

The characterization of root endophytes in wild rice as reported in this study will improve our 63

knowledge concerning the ecology and evolution of plant-mutualistic fungal interactions. 64

65

MATERIALS AND METHODS 66

Site of study. The site of study was located in Xishuangbanna Nature Reserve, Yunnan Province, 67

southwest of China. (N 22°04’-22°17’; E 100°32’-100°44’) 68

Sampling. We sampled a total of fifteen O. granulate plants within an approximate 50 m radius 69

of the georeference point in September 2008. Due to the endangered species status of wild rice, 70

additional sampling in other sites was not permitted. The O. granulate plants grew in latosol (acidic 71

red soil, pH<6.0) at an altitude of 650 m within a bamboo forest. Usually, they grew with two 72

bamboo species including Pleioblastus amarus and Oxytenantheca nigrociliata. Upon collection, 73

healthy and intact wild rice plants with bulk soil were carefully packed into a box and transported to 74

a laboratory within 48 hours. 75

Microscopic analysis of roots. To detect the presence of fungal developmental structures such as 76

arbuscular mycorrhizal fungi or root endophytes, roots were stained using a modification of a 77

previously described protocol (42). Specifically, roots were cleaned with sterile deionized water and 78

fixed in 50% (v/v) ethanol for 24h. They were then rinsed three times with deionized water and 79

placed in 5% (w/v) KOH for 2 h at 90 °C. After further rinsing with deionized water, the roots were 80

submerged in 2% (w/v) lactic acid for 2 min, stained with 0.05% (w/v) trypan blue at 50 °C for 5 h, 81


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

5

and de-stained in 50% (v/v) glycerin for 24 h. Squash preparations of the root segments in 50% (v/v) 82

glycerol were examined by light microscopy (Olympus BX51, Japan). 83

Isolation and identification of endophytic fungal cultures. Visual and microscopic inspection 84

was first performed to ensure that roots were free of obvious lesions. Healthy roots were rinsed with 85

tap water immersed in ethanol (75%, v/v) for 40 s, then in sodium hypochlorite 1% (v/v) for 4 min 86

and finally rinsed three times in sterile distilled water. Equal numbers of old and young roots from 87

the 15 sampled plants were cut into 0.5 cm lengths for a total of 204 segments (root tips were not 88

included because of low frequency of fungal colonization with previous microscopic staining 89

examination) and transferred to plates with 2% (w/v) MEA (malt extract agar, 20 g malt extract+20 g 90

agar/L) medium supplemented with chloromycetin (50 mg/l) to prevent bacteria growth. Six 91

segments per plate were distributed to MEA. A total of 34 plates were sealed with Parafilm to avoid 92

desiccation and cultured at 25 °C in darkness. Hyphae emerging from segments were sub-cultured 93

onto fresh PDA (potato 200 g+ glucose 20 g/L) for purification of isolates. The remaining root 94

samples were used for extraction of total DNA. To ensure that the surface-sterilization had removed 95

all hyphae and chlamydospores externally adhering to the roots, they were placed in MEA agar 96

plates and incubated. Only roots, which were negative in this test, were used for isolation of 97

endophytes. 98

All fungal isolates were initially identified to the genus and/or species using cultural and 99

morphological characters, which included colony appearance, conidia morphology and 100

conidiophore/conidial structures. Some isolates sporulated readily on PDA media after one week 101

inoculation in darkness at 25 °C. The microscopic characteristics of strains were based on light 102

microscopy (Olympus BX51, Japan) and/ or cryo-scanning electron microscopy (cryo-SEM, 103

HITACHI S-3000N, Japan). Specimens for light microscopy were mounted in 3% KOH or sterile 104

distilled water for observation. Remaining sterile fungal isolates were subjected to molecular method 105


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

6

of identification. 106

Fungal ITS amplification and sequencing. Fungal DNA was extracted from 58 pure culture 107

isolates using the Multisource Genomic DNA Miniprep Kit (Axygen Incorporation, China) following 108

the manufacturer's instructions. Primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 109

(5’-TCCTCCGCTTATTGATATGC-3’) (66) were used for amplification of the fungal rDNA 110

internal transcribed spacer (ITS) regions 1 and 2 of all isolates. The PCR reaction (50 µl total volume) 111

contained 5µl 10×PCR buffer, 4µl 25mM Mg2+

, 2µl 10 mM dNTP, 1µl of each primer (10µM), 2µl 112

original template, 1µl Taq polymerase and ddH2O 34 µl. 35 cycles were run, each cycle consisting of 113

a denaturation step at 94 °C (40 s), an annealing step at 54 °C (60 s), and an extension step at 72 °C 114

(60 s). After the 35th cycle, a final 10 min extension step at 72 °C was performed. The reaction 115

products were separated in 1.0% (w/v) agarose gel, the amplicons purified using a gel band 116

purification kit (Axygen Incorporation) and sequenced in ABI 3730 sequencer (Applied Biosystems, 117

USA) using the ITS1 and ITS4 primers. 118

Direct amplification of fungal ITS sequences from roots. All root samples were stored at -70°C 119

until used in DNA extraction. For extraction of DNA, all root slices were pooled together and 120

grouped into three batches which were treated independently. 100 mg of root material was cut into 121

sections of approximately 1 cm length and ground to a fine powder in liquid nitrogen. Total DNA 122

was subsequently extracted using the Multisource Genomic DNA Miniprep Kit (Axygen 123

Incorporation, China) following the manufacturer's instructions. The fungal specific primers ITS1-F 124

(5’-CTTGGTCATTTAGAGGAAGTAA-3’), ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) were 125

used for amplification of the ITS 1 and 2 region of ascomycetes and basidiomycetes (30). The PCR 126

reaction (50 µl total volume) contained 5µl 10×PCR buffer, 7µl 25mM Mg2+

, 2µl 10 mM dNTP, 2µl 127

of each primer (10µM), 4µl original template, 1µl Taq polymerase and ddH2O 27µl. Amplification 128


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

7

conditions were 35 cycles of 94 °C for 40 s, an annealing step at 55 °C for 50 s, and an extension 129

step at 72 °C for 60 s. After the 35th cycle, a final extension step at 72 °C for 10 min was performed. 130

The reaction products were then separated and purified as above. The products from three individual 131

PCR reactions (R1, R2 and R3) were ligated into pGEM-T Easy (Promega, USA) respectively and 132

transformed into Escherichia coli JM109 (Promega) following the manufacturer’s instructions, 133

resulting in three individual clone libraries. The transformants were plated on LB agar plates 134

containing 50 µg/ml ampicillin and X-Gal/IPTG. A total of 214 positive clones were randomly 135

selected and subjected to sequencing. Primer M13F was used for sequencing. The flow diagram of 136

detailed procedure was presented in supplementary Fig.1. 137

Phylogenetic analysis. Vector sequences of sequenced fungal clones were removed using 138

VecScreen (http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html). To remove potential chimeric 139

sequences, the sequences were first manually inspected for the presence of signature shifts, and then 140

subjected to analysis by Bellerophon (http://foo.maths.uq.edu.au/~huber/bellerophon.pl) (19). Only 141

28 of 214 positive clones were chimeric and thus removed. The frequency of unique phylotypes was 142

determined by assembling sequences with 90% similarity threshold (2) using Sequencher ver. 4.1.4 143

(http://www.genecodes.com/ ). For culture-based method, the sequences were then aligned and those 144

with ≥ 99% sequence identity over the whole amplicon length were defined as one genotype. 145

The final sequence sets were then submitted to BLAST analysis and identities ≥ 99% considered 146

as conspecific. Sequences obtained from cultures and clones were deposited at GenBank (accession 147

numbers FJ752597-FJ752627 and FJ524295-FJ524304; FJ524306; FJ524308- FJ524311; FJ524313- 148

FJ524316; FJ524318; FJ524320- FJ524321; FJ524324- FJ524329; FJ882005-FJ882011, 149

respectively). To verify the phylogenetic position of DSEs genotypes, they and corresponding best 150

BLAST hits were aligned by Clustal X and manually corrected in GENEDOC. Maximum parsimony 151

analysis was performed in PAUP* 4.0 b 10, using the heuristic search option with TBR branch 152


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

8

swapping; stability of clades was tested using 1000 bootstrap replications. For inferring the 153

phylogeny of all detected phylotypes in the clone libraries, the 5.8 S rDNA analysis was performed 154

with sequences retrieved in BLAST searches. NJ (Neighbor-joining) trees were built using a Kimura 155

two-parameter (K2P) model in PAUP * 4.0 b 10. The robustness of the internal branches was also 156

assessed with 1000 bootstrap replications. 157

Calculation of ecological indices and estimation of taxon accumulation curves for quantifying 158

fungal biodiversity. The rates of colonization and isolation were calculated by the following formula: 159

160

To quantify fungal diversity of rice roots, Fisher’s alpha (α), Shannon diversity index (H), Simpson's 161

diversity index (1-D) and Margalef’s richness index (R1) were calculated (18, 57, 58) by the 162

following equations, respectively: 163

164

Therein S is the number of taxa (ITS genotype or phylotype), N is the number of individuals (defined 165

by numbers of isolates or sequenced clones), and i is the proportion of species relative to the total 166

number of species (Pi). Taxon-accumulation curves and bootstrap estimates of total species richness 167

based on recovered fungal isolates and sequenced clones were generated in EstimateS V7.5 (8) using 168

50 randomizations of sample order (http://viceroy.eeb.uconn.edu/EstimateS). 169

RESULTS 170

Microscopic detection of endophytic fungi in wild rice roots. To assess the presence of fungal 171

endophytes in rice roots, we first examined the roots microscopically. The presence of dark septate 172


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

9

endophytes (DSEs) in root tissues can be seen by the presence of intraradical microsclerotia ( Fig. 1). 173

Large sclerotium-like structures were detected inside of the root cortex, some occupying the whole 174

cortical cell volume. Other small sclerotium-like structures also co-existed with dark septate hyphae, 175

suggesting that they represent different stages of development of DSEs or different DSEs species 176

(Fig. 2-B). In addition, we found extensive colonization by other fungal endophytes whose hyphae 177

stained blue (Fig. 2-A, D, E). Further, some chlamydospore-like structures similar to those found in 178

Piriformospora indica-infected maize roots (24), were also observed,. In most cases, the dark and the 179

blue hyphae grew along the epidermis or cortex in parallel to the longitudinal axis of the roots (Fig. 180

2-C, D, E). Also, the root epidermis contained a dense hyphal network which was infrequent in the 181

cortex layers (Fig. 2-C). In the early stage of development, the formation of microsclerotia was 182

apparent by the detection of dark hyphal fragments (Fig. 2-F). 183

Isolation and identification of endophytic fungi. A total of 58 fungal isolates were recovered 184

and purified from 204 root tissue samples. A proportion of isolates were identified to genus and/or 185

species level based on morphology of conidia, conidiophore and unique phenotypic characters (Fig. 186

3). The identified fungi belonged to the Ascomycota with the exception of one basidiomyceteous 187

Rhizoctonia-like isolate. To confirm the reliability of morphological identification, all 58 isolates 188

were subjected to molecular identification based on rDNA ITS sequence analysis. In total, 31 189

distinctive genotypes were detected at a 99% sequence similarity threshold (Fig. 3), which 190

corresponded well with morphological differences between these fungal cultures. This allowed the 191

placement of these isolates into several ascomycete lineages representing at least seven orders 192

(Hypocreales, Diaporthales, Eurotiales, Xylariales, Microascales, Capnodiales and 193

Magnaporthales). In addition, the Rhicotonia-like fungus was identified as an anamorphic species of 194

Ceratobasidium (anastomosis group: AG-G) (Fig. 6). Nine genotypes, however, could not be 195

assigned to any genus or species as their ITS sequences did not resemble any described species in the 196


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

10

Genbank database, and thus likely represent novel fungal lineages (Fig. 3). Molecular phylogeny 197

assigned all nine to the class Dothideomycetes (Ascomycota) group (data not shown). 198

To characterize the biodiversity of our samples, we calculated Fisher’s alpha, Shannon diversity 199

index, Simpson's diversity index and Margalef’s richness index. The values obtained by these tests 200

(34.39, 3.14, 0.94 and 7.39 based on cultured isolates; and 232.87, 2.85, 0.90 and 6.51 based on 201

sequenced clones; respectively) indicate that the biodiversity of fungal endophytes in wild rice roots 202

is very high. On the other hand, unlike in other perennial herbaceous and woody plants, the 203

colonization rate and isolation rate in root tissues of O. granulate were low (23% and 28.4%, 204

respectively). Nineteen fungal genotypes were recovered only once (61.3%) (Fig. 3). With the 205

exception of the fungi exhibiting the ITS type 13, for which 9 conspecific isolates were found, no 206

other genus or species dominated. This suggests that the ITS type 13 fungi are potentially important 207

root symbionts in wild rice. These findings would be in accordance with the hypothesis that 208

horizontally transmitted endophytes re-infect annual grasses yearly and accumulate seasonally, 209

while- in contrast- they accumulate in older tissues for evergreen plants and perennial grasses (5, 49). 210

Estimated species and phylotype richness accumulation curves were generated using EstimateS 211

(8). Two “taxon-based” accumulation curves showed a declining rate of accumulation of ITS 212

genotypes or phylotypes (Fig. 4). The relative steepness of the curves and the high number of 213

estimated total richness implied that more endophytes are waiting to be discovered. Bootstrap 214

estimates of species richness exceeded the observed species richness. The observed species richness 215

fell within the 95 % confidence intervals of the estimated richness, indicating that the sampling 216

method used was effective in recovering the fungal species of the endophyte community. It was also 217

shown that the estimated richness curve based on sequenced clones (SC) increased more quickly 218

with few clone numbers (Fig. 4-B) than the curve based on cultured isolates (CI). However, with 219

increasing numbers of analyzed isolates or clones, the SC curve gradually flattened and the CI curve 220


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

11

did not reach saturation level. The final value of the abundance-based estimator (ACE) for CI and SC 221

was 71.4 and 54.5, respectively. 222

Endophytic fungal community evaluation via environmental PCR. In order to learn whether 223

the culturable endophytes would represent the actual biodiversity of fungi in rice roots, we analyzed 224

which fungal sequences would be retrieved by an analysis of direct PCR from the total root DNA. 225

186 clones were obtained from the three individual clone libraries, sequenced and subjected to 226

phylogenetic analysis. BLAST and NJ analysis placed the cloned sequences into various groups of 227

Ascomycota and Basidiomycota (Fig. 7). In contrast to the results obtained by the cultivation-based 228

approach, the endophytic fungal community in roots was dominated by basidiomycetes (63 %) 229

(Table 1). 60.2 % of the clones had sequences that were close – but not identical - to currently 230

unknown taxa (Table 1). The most frequently detected phylotype was genetically close to an 231

uncultured basidiomycetes fungus (FN296244). Four phylotypes were closely related to the 232

basidiomycetes Trichosporon, Wallemia, Marasmius and Mycena (Table 1). All these data provide 233

strong evidence that roots act as large reservoir for colonization of unexplored endophytic fungi. 234

235

DISCUSSION 236

Fungal symbionts, mainly comprising mycorrhizal fungi and fungal endophytes, are ubiquitously 237

distributed in terrestrial plant roots. They act beneficially on the plant by modulating host nutrition, 238

metabolites and stress response (4, 45-47). Based on previous investigations, it can be concluded that 239

the colonization rate of these two types of fungi is highly variable and dependent on habitat, host and 240

seasonal fluctuations in the climate (31). 241

In this study, we show that roots of wild rice exhibit a very high biodiversity of endophytic but 242

not mycorrhizal fungi. Recently, 16 fungal genera were recovered from the roots of O. sativa from 243


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

12

Bhadra River Project Area, Karnataka (India), and only 6 genera were found in rice varieties 244

cultivated in Italy and in Guangdong province, China (37, 61, 63). Results of these studies involved 245

culture-based approaches, not environmental PCR methodology. Moreover, they did not look for the 246

occurrence of dark septate endophytes. The difference in the results from these and the present study 247

may be due to the fact that these authors investigated cultivated rice. It appears plausible to assume 248

that wild rice roots host more and novel endophytes relative to cultivated plants. 249

Among root associated fungi, dark septate endophytes (DSEs) are ubiquitous and cosmopolitan 250

and found in a wide range of plant species. While the abundance of DSEs in arctic, alpine and 251

temperate habitats has been widely investigated (31, 39, 51, 52), their role in tropical ecosystems is 252

still poorly understood. The melanised hyphae, typical for these fungi, are considered to be of 253

importance for the host to survive stress conditions because cell wall melanin can trap and eliminate 254

oxygen radicals generated during abiotic stress (48). Therefore, the dominant colonization of wild 255

rice by DSEs may confer tolerance to a variety of environmental stress factors. Surprisingly, even 256

though light microscopy revealed that DSE fungi were ubiquitous in rice roots, sequence analysis 257

indicated that only 4 of 186 sequenced clones matched potential DSE fungi, i.e. 3 clones of 258

Exophiala pisciphila, and one clone of Cladophialophora chaetospira. Likely, our total DNA 259

extraction protocol was not well suited for efficient recovery of genomic DNA from dark hyphae 260

and/or sclerotia. The genus Exophiala is phylogenetically close to Phialophora (23) and recent 261

experimental data confirm that Exophiala sp. is responsible for DSE appearance (22, 71). Also, some 262

species of Cladophialophora are morphologically and phylogenetically similar to Heteroconium 263

chaetospira (38), a recognized DSE which however is phylogenetically different from most DSE 264

taxa. Interestingly, none of the endophytes isolated by cultivation could be identified as Exophiala or 265

Cladophialophora, indicating that the methods for disinfection and/or cultivation media may be 266

inappropriate for some DSE species. Alternatively, some unculturable DSE fungi may also exist 267


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

13

within the roots. We propose that the application of high-throughput cultivation methods should be 268

applied in the future to complement the drawback of traditional isolation methods. The so called 269

“dilution-to-extinction” culture method has recently been successfully used to recover more diverse 270

endophytic fungi species from leaf tissues than segment plating method (7, 62). 271

The genera Phialocephala and Phialophora are typical DSEs and frequently observed in roots 272

(54, 65). Phialocephala fortinii preferentially colonizes roots of woody plant species, while 273

Phialophora spp. usually live in herbaceous plant roots as hosts, especially in Gramineae (54). The 274

ITS type 10 sequence was 96% identical to Phialophora sp. (anamorphs of Gaeumannomyces, now 275

widely named as Harpophora sp.), although only one strain was isolated in culture (Fig. 3). In vitro 276

inoculation testing has verified that this isolate is capable of living endophytically in cultivated rice 277

(Oryza sativa L.) roots and formed dark brown hypha in the tissues. After 30 days co-culture under 278

controlled aseptic conditions, Harpophora sp. significantly promoted the growth and biomass of rice 279

seedlings (data not shown). Further characterization of colony and conidia morphology confirmed 280

the isolate to be a member of the genus Harpophora, but its ITS sequence was not conspecific with 281

any of the so far described species of Harpophora (Fig. 5). Harpophora graminicola (previously 282

referred to Phialophora graminicola), was also shown to be a beneficial dark endophyte in grass 283

roots (40). 284

In addition, both BLAST and phylogenetic analysis revealed that the ITS genotype 22 was 285

closely related to Periconia macrospinosa (98% sequence identity; Fig. 5). P. macrospinosa was 286

recently reported as a unique DSE that inhabits in various plant roots (31). Furthermore, one isolate 287

(ITS type 31) was morphological and phylogenetically identified as a member of the 288

Ceratobasidium/Rhizoctonia complex (Fig. 6). This genus comprises a diverse group of soil 289

inhabitant fungi that includes important crop pathogens, and orchid mycorrhizal symbionts. Most 290

Rhizoctonia orchid mycorrhizae are members of anamorphs of Tulasnella, Ceratobasidium and 291


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

14

Thanatephorus (41). Members of the Ceratobasidium/Rhizoctonia complex may also act as fungal 292

symbiont in non-orchid hosts, as recently suggested for Fagopyrum esculentum and F. tataricum 293

(30). 294

Only five ascomycete genera (Cylindrocarpon, Fusarium, Xylaria, Phomopsis and Penicillum) 295

were detected both by direct isolation from the roots and by direct PCR analysis. This observation 296

could indicate a potential technical bias in examining fungal diversity (1). The direct PCR method 297

effectively recognized endophytic fungi belonging to Basidiomycota, while pure-culture isolation 298

preferentially detected those fungi within the Ascomycota (25). Thus these results highlight the 299

importance of integrating multiple approaches for analyzing endophytic microbial biodiversity in 300

plants (25). 301

Some of our isolates were identified as being members of commonly observed genera of soil 302

fungi, e.g. Fusarium, Penicillium, Trichoderma and Paecilomyces. Representatives of these genera 303

have been identified as endophytes in cultivated and wild rice roots (37, 61, 63). These fungi are 304

characteristically free-living saprophytes that can also be opportunistic root symbionts (3, 17). 305

Our data demonstrate that roots of wild rice are associated with a surprisingly rich endophyte 306

community. The combination of microscopy, isolation of pure cultures, ecological analysis and clone 307

sequencing yielded comprehensive information about the identity, diversity and phylogeny of fungal 308

endophytes. Our results also provide additional evidence that endophyte diversity in gramineous 309

grass roots may be as rich as other perennial grasses and woody plants (49). In comparison to 310

previous studies, 49 and 51 fungal phylotypes and operational taxonomic units (OTUs) were found in 311

the grasses Arrhenatherum elatius and Bouteloua gracilis, respectively (43, 64). It must be noted that 312

these authors (43, 64) actually targeted root-associated fungi (endophytes, epiphytes and some 313

rhizosphere soil fungi), and therefore probably have also detected Chytridiomycota and Zygomycota . 314

Most of the endophytic lineages belong to the Ascomycota clade and some belong to the 315


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

15

Basidiomycota. Currently, direct clone library sequencing (also called “environmental PCR”) has 316

been applied in studying foliar and root endophytic fungi diversity (2, 10, 16, 58). It must be 317

admitted that the sterilization of roots as described in this paper technique will certainly kill all the 318

microorganisms in root surface, but the dead cells may still contain DNA that becomes extracted and 319

thus amplified. Yet we consider the extraction of total endophytic fungal DNA still as reliable 320

because some epiphytic fungi may also penetrate the cortex tissue and live endophytically (50). 321

Furthermore, none of our clones matched any sequences of “lower fungi”, which indicates that the 322

sterilization procedure in this study was effective in degrading the DNA of most epiphytic and 323

rhizosphere soil fungi. There was no indication of overestimation of endophytic fungal diversity. 324

In summary, we obtained consistent results (microscopy, culturing and molecular detection) that 325

non-mycorrhizal fungi - including several lineages of ascomycetes and basidiomycetes - may 326

constitute the dominant fungal consortium in wild rice roots. This highlights the similar contributions 327

of mycorrhizal fungi and endophytic fungi to modulating host growth and development. It should 328

also be conceded that the limited sample size (15 wild rice roots collected in one site) in this study 329

may bias conclusions. Analyzing additional samples from multiple locations will be necessary to 330

determine whether the endophytic fungal lineages found in samples from the Yunnan site reflect a 331

pattern common to wild rice from other regions. 332

The specific DSE fungi also represent a novel system for exploring mutualistic plant-fungal 333

interactions. Further work is needed to elucidate the roles these co-habiting colonizers play in plant 334

performance and stress response. Considering the global importance of rice plant for food production, 335

examination of endophyte-mediated plant growth promoting and/or disease resistance will aid in the 336

production of this crop. 337

338

339


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

16

Acknowledgements 340

This work was supported by National Natural Science Foundation of China (Grant No. 341

30600002 and 30970097) to Chu-long Zhang. We would also like to express our great appreciation 342

to Dr. Yang Yun for collecting samples. 343

344

REFERENCE 345

1. Allen, T.R., T. Millar, S.M. Berch, and M.L. Berbee. 2003. Culturing and direct DNA 346

extraction find different fungi from the same ericoid mycorrhizal roots. New Phytol. 160: 347

255–272. 348

2. Arnold, A.E., D.A. Henk, R.L. Eells, F. Lutzoni, and R. Vilgalys. 2007. Diversity and 349

phylogenetic affinities of foliar fungal endophytes in loblolly pine inferred by culturing and 350

environmental PCR. Mycologia 99: 185–206. 351

3. Bacon, C.W., and I.E. Yates. 2006. Endophytic root colonization by Fusarium species: 352

histology, plant interactions, and toxicity, p. 133–152. In B.J.E. Schulz, C.J.C. Boyle, and T.N. 353

Sieber (ed.), Microbial root endophytes. Springer-Verlag, Berlin. 354

4. Baldi, A., A. Jain, N. Gupta, A.K. Srivastava, and V.S. Bisaria. 2008. Co-culture of 355

arbuscular mycorrhiza-like fungi (Piriformospora indica and Sebacina vermifera) with plant 356

cells of Linum album for enhanced production of podophyllotoxins: a first report. Biotechnol. 357

Letts. 30: 1671–1677. 358

5. Carroll, G.C. 1986. The biology of endophytism in plants with particular reference to woody 359

perennials, p. 205–222. In N. Fokkema., and J. van den Heuval (ed.), Microbiology of the 360

phyllosphere. Cambridge University press, UK. 361

6. Clement, S.L., A.D. Wilson, D.G. Lester, and C.M. Davitt. 1997. Fungal endophytes of wild 362


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

17

barley and their effects on Diuraphis noxia population development. Entomol. Experiment. 363

Appl. 82: 275–281. 364

7. Collado, J., G.Platas, B. Paulus, and G.F. Bills. 2007. High-throughput culturing of fungi 365

from plant litter by a dilution-to-extinction technique. FEMS. Microbiol. Ecol. 60: 521–533. 366

8. Colwell, R.K. 2005. EstimateS: Statistical estimation of species richness and shared species 367

from samples. Version 7.5. User's Guide and application published at: 368

http://purl.oclc.org/estimates. 369

9. Crous, P.W., O. Petrini, G.F. Marais, Z.A. Pretorius, and F. Rehder. 1995. Occurrence of 370

fungal endophytes in cultivars of Triticum aestivum in South Africa. Mycoscience 36: 105–111. 371

10. Dearnaley, J.D.W., and A.F. Le Brocque. 2006. Molecular identification of the primary root 372

fungal endophytes of Dipodium hamiltonianum. Aust. J. Bot. 54: 487–491. 373

11. Dingle, J., and P.A. McGee. 2003. Some endophytic fungi reduce the density of pustules of 374

Puccinia recondita f.sp. tritici in wheat. Mycol. Res. 107: 310–316. 375

12. Fisher, P.J., and O. Petrini. 1992. Fungal saprobes and pathogens as endophytes of rice 376

(Oryza sativa L.). New Phytol. 122: 137–143. 377

13. Fisher, P. J., O. Petrini, and H. M. Lappin-Scott. 1992. The distribution of some fungal and 378

bacterial endophytes in maize (Zea mays L.). New Phytol. 122: 299–305. 379

14. Fisher, P.J., and J. Webster. 1992. A Trematosphaeria endophyte from rice roots and its 380

Zalerion anamorph. Nova Hedwigia 54: 77–81. 381

15. Glassop, D., R.M. Godwin, S.E. Smith, and S.W. Smith. 2007. Rice phosphate transporters 382

associated with phosphate uptake in rice roots colonized with arbuscular mycorrhizal fungi. Can. 383

J. Bot. 85: 644–651. 384

16. Gotz, M., H. Nirenberg, S. Krause, H. Wolters, S. Draeger, A. Buchner, J. Lottmann, G. 385

Berg, and K. Smalla. 2006. Fungal endophytes in potato roots studied by traditional isolation 386


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

18

and cultivation-independent DNA-based methods. FEMS. Microbiol. Ecol. 58: 404–413. 387

17. Harman, G.E., C.R. Howell, A. Viterbo, I. Chet, and M. Lorito. 2004. Trichoderma 388

species-opportunistic, avirulent plant symbionts. Nat. Rev. Microbiol. 2: 43–56. 389

18. Hoffman, M., M. Gunatilaka, J. Ong, M. Shimabukuro, and A.E. Arnold. 2008. Molecular 390

analysis reveals a distinctive fungal endophyte community associated with foliage of Montane 391

oaks in southeastern Arizona. J Arizona-Nevada Academy of Science 40: 91–100. 392

19. Huber, T., G. Faukner, and P. Hugenholtz. 2004. Bellerophon: a program to detect chimeric 393

sequences in multiple sequence alignments. Bioinformatics 20: 2317-2319. 394

20. Hung, P.Q., S.M. Kumar, V. Govindsamy, and K. Annapurna. 2007. Isolation and 395

characterization of endophytic bacteria from wild and cultivated soybean varieties. Biol. Fertil. 396

Soils. 44: 155–162. 397

21. Istifadah, N., and P.A. McGee. 2006. Endophytic Chaetomium globosum reduces development 398

of tan spot in wheat caused by Pyrenophora tritici-repentis. Austral. Plant. Path. 35: 411–418. 399

22. Julou, T., B. Burghardt, G. Gebauer, D. Berveiller, C. Damesin, and M.A. Selosse. 2005. 400

Mixotrophy in orchids: insights from a comparative study of green individuals and 401

nonphotosynthetic individuals of Cephalanthera damasonium. New Phytol. 166: 639–653. 402

23. Jumpponen, A., and J.M. Trappe. 1998. Dark septate endophytes: a review of facultative 403

biotrophic root-colonizing fungi. New Phytol. 140: 295–310. 404

24. Kumar, M., V. Yadav, N. Tuteja, and A.K. Johri. 2009. Antioxidant enzyme activities in 405

maize plants colonized with Piriformospora indica. Microbiology 155: 780–790. 406

25. Kwasna, H., G. L. Bateman, and E. Ward. 2008. Determining species diversity of 407

microfungal communities in forest tree roots by pure-culture isolation and DNA sequencing. 408

Appl. Soil Ecol. 40: 44–56. 409

26. Larran, S., A. Perelló, M. R. Simón, and V. Moreno. 2006. The endophytic fungi from wheat 410


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

19

(Triticum aestivum L.). World J. Microbiol. Biotechnol. 23: 565–572. 411

27. Larran, S., C. Rollan, H. A. Bruno, H. E. Alippi, and M. I. Urrutia. 2002. Nota corta: 412

Endophytic fungi in healthy soybean leaves. Invest. Arg. Prod. Prot. Veg. 17: 173–178. 413

28. Le, H.T.T., J. L. Padgham, and R. A. Sikora. 2009. Biological control of the rice root-knot 414

nematode Meloidogyne graminicola on rice using endophytic and rhizosphere fungi. Int. J. Pest 415

Manag. 55: 31–36. 416

29. Li, L.F., A.N. Yang, and Z.W. Zhao. 2005. Seasonality of arbuscular mycorrhizal symbiosis 417

and dark septate endophytes in a grassland site in southwest China. FEMS. Microbiol. Ecol. 54: 418

367–373. 419

30. Likar, M., U. Bukovnik, I. Kreft, N.K. Chrungoo, and M. Regvar. 2008. Mycorrhizal status 420

and diversity of fungal endophytes in roots of common buckwheat (Fagopyrum esculentum) and 421

tartary buckwheat (F. tataricum). Mycorrhiza 18: 309–315. 422

31. Mandyam, K. 2008. Ph. D. Thesis. Kansas State University, Manhattan, USA. Dark septate 423

fungal endophytes from a tallgrass prairie and their continuum of interactions with host plants. 424

32. Mano, H., and H. Morisaki. 2008. Endophytic bacteria in rice plant. Microbes Environ. 23: 425

109–117. 426

33. Marshall, D., B. Tunali, and L.R. Nelson. 1999. Occurrence of fungal endophytes in species 427

of wild Triticum. Crop Sci. 39: 1507–1512. 428

34. Mathew, A., and R.M. Malathy. 2008. The evidence of mycorrhizal fungi and dark septate 429

endophytes in roots of Chlorophytum borivillianum. Acta. Bot. Croat. 67: 91–96. 430

35. Mattos, K.A., V.L. Pádua, A. Romeiro, L.F. Hallack, B.C. Neves, T.M. Ulisses, C.F. Barros, 431

A.R. Todeschini, J.O. Previato, and L. Mendonça-Previato. 2008. Endophytic colonization 432

of rice (Oryza sativa L.) by the diazotrophic bacterium Burkholderia kururiensis and its ability 433

to enhance plant growth. An. Acad. Bras. Cienc. 80: 477–493. 434


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

20

36. Monfort, E., L.V. Lopez-Llorca, H.B. Jansson, J. Salinas, J.O. Park, and K. 435

Sivasithamparam. 2005. Colonisation of seminal roots of wheat and barley by egg-parasitic 436

nematophagous fungi and their effects on Gaeumannomyces graminis var. tritici and 437

development of root-rot. Soil Biol. Biochem. 37: 1229–1235. 438

37. Naik, B.S., J. Shashikala, and Y.L. Krishnamurthy. 2009. Study on the diversity of 439

endophytic communities from rice (Oryza sativa L.) and their antagonistic activities in vitro. 440

Microbiol. Res. 164: 290-296. 441

38. Narisawa, K., S. Hambleton, and R.S. Currah. 2007. Heteroconium Chaetospira, a dark 442

septate root endophyte allied to the Herpotrichiellaceae (Chaetothyriales) obtained from some 443

forest soil samples in Canada using bait plants. Mycoscience 48: 274–281. 444

39. Newsham, K.K., R. Upson, and D.J. Read. 2009. Mycorrhizas and dark septate root 445

endophytes in polar regions. Fungal. Ecol. 2: 10–20. 446

40. Newsham, K.K. 1999. Phialophora graminicola, a dark septate ungus, is a beneficial associate 447

of the grass Vulpia ciliata ssp. ambigua. New Phytol. 144: 517–524. 448

41. Otero, J.T., J.D. Ackerman, and P. Bayman. 2002. Diversity and host specificity of 449

endophytic Rhizoctonia-like fungi from tropical orchids. Am. J. Bot. 89:1852–1858. 450

42. Peterson, R.L., H.B. Massicotte, and L.H. Melville. 2004. Mycorrhizas: Anatomy and cell 451

biology. NRC Research Press, Ottawa, Ont. 452

43. Porras-Alfaro, A., J. Herrera, R.L. Sinsabaugh, K.J. Odenbach, T. Lowrey, and D.O. 453

Natvig. 2008. Novel root fungal consortium associated with a dominant desert grass. Appl. 454

Environ. Microbiol. 74: 2805–2813. 455

44. Prayitno, J., J. Stefaniak, J. McIver, J.J. Weinman, F.B. Dazzo, J.K. Ladha, W. Barraquio, 456

Y.G. Yanni , and B.G. Rolfe. 1999. Interactions of rice seedlings with bacteria isolated from 457

rice roots. Aust. J. Plant Physiol. 26: 521–535. 458


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

21

45. Rai M., D. Acharya, and A. Singh. 2001. Positive growth responses of the medicinal plants 459

Spilanthes calva and Withania somnifera to inoculation by Piriformospora indica in a field trial. 460

Mycorrhiza 11: 123–128. 461

46. Rai, M.K., A. Varma, and A.K. Pandey. 2004. Antifungal potential of Spilanthes calva after 462

inoculation of Piriformospora indica. Mycoses 47: 479–481. 463

47. Redman, R.S., K.B. Sheehan, R.G. Stout, R.J. Rodriguez, and J.M. Henson. 2002. 464

Thermotolerance generated by plant / fungal symbiosis. Science 298: 1581. 465

48. Richier. S., P. Furla, A. Plantivaux, P.L. Merle, and D. Allemand. 2005. Symbiosis-induced 466

adaptation to oxidative stress. J. Exp. Biol. 208: 277–285. 467

49. Saikkonen, K., S.H. Faeth, M.L. Helander, and T.J. Sullivan. 1998. Fungal endophytes: a 468

continuum of interactions with host plants. Annu. Rev. Ecol. Syst. 29: 319–343. 469

50. Santamariá, J., and P. Bayman. 2005. Fungal epiphytes and endophytes of coffee leaves 470

(Coffea arabica). Microb. Ecol. 50: 1–8. 471

51. Schadt, C.W., R.B. Mullen and S.K. Schmidt. 2001. Isolation and phylogenetic identification 472

of a dark septate fungus associated with the alpine plant Ranunculus adoneus. New Phytol. 150: 473

747–755. 474

52. Schmidt, S.K., L.C. Sobieniak-Wiseman, S.A. Kageyama, S.R.P. Halloy, and C.W. Schadt. 475

2008. Mycorrhizal and dark septate fungi in plant roots above 4270 meters elevation in the 476

Andes and Rocky Mountains. Arct. Antarct. Alp. Res. 40: 576–583. 477

53. Senthilkumar, M., M. Madhaiyan, S.P. Sundaram, H. Sangeetha, and S. Kannaiyan. 2008. 478

Induction of endophytic colonization in rice (Oryza sativa L.) tissue culture plants by 479

Azorhizobium caulinodans. Biotechnol. Lett. 30: 1477–1487. 480

54. Sieber, T.N. 2002. Fungal root endophytes. In: Plant roots: the hidden half. (Wasel, Y., Eshel, 481

A., and Kafkafi, U., eds). Marcel Dekker, New York, pp. 887–917. 482


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

22

55. Sieber, T.N., T.K. Riesen, E. Muller, and P.M. Fried. 1988. Endophytic fungi on four winter 483

wheat cultivars (Triticum aestivum L.) differing in resistance against Stagonospora nodorum 484

(Berk.) Cast. & Germ. = Septoria nodorum (Berk.) Berk. J. Phytopathology 122: 289–306. 485

56. Solaiman, M.Z., and H. Hirata. 1997. Effect of arbuscular mycorrhizal fungi inoculation of 486

rice seedlings at the nursery stage upon performance in the paddy field and greenhouse. Plant 487

Soil. 191: 1–12. 488

57. Suryanarayanan, T.S., and V. Kumaresan. 2000. Endophytic fungi of some halophytes from 489

an estuarine mangrove forest. Mycol. Res. 104: 1465–1467. 490

58. Tao, G., Z.Y. Liu, K.D. Hyde, X.Z. Liu, and Z.N. Yu. 2008. Whole rDNA analysis reveals 491

novel and endophytic fungi in Bletilla ochracea (Orchidaceae). Fungal Divers. 33: 101–122. 492

59. Thomas, S.E., J. Crozier, M.C. Aime, H.C. Evans, and K.A. Holmes. 2008. Molecular 493

characterisation of fungal endophytic morphospecies associated with the indigenous forest tree, 494

Theobroma gileri in Ecuador. Mycol. Res. 112: 852–860. 495

60. Tian, X.L., L.X. Cao, H.M. Tan, W.Q. Han, M. Chen, Y.H. Liu, and S.N. Zhou. 2007. 496

Diversity of cultivated and uncultivated actinobacterial endophytes in the stems and roots of rice. 497

Microb. Ecol. 53: 700–707. 498

61. Tian, X.L., L.X. Cao, H.M. Tan, Q.G. Zeng, Y.Y. Jia, W.Q. Han, and S.N. Zhou. 2004. 499

Study on the communities of endophytic fungi and endophytic actinomycetes from rice and 500

their antipathogenic activities in vitro. World J. Microbiol. Biotechnol. 20: 303–309. 501

62. Unterseher, M., and M. Schnittler. 2009. Dilution-to-extinction cultivation of leaf-inhabiting 502

endophytic fungi in beech (Fagus sylvatica L.)-different cultivation techniques influence fungal 503

biodiversity assessment. Mycol. Res. 113: 645–654. 504

63. Vallino, M., D. Greppi, M. Novero, P. Bonfante, and E. Lupotto. 2008. Rice root 505

colonisation by mycorrhizal and endophytic fungi in aerobic soil. Ann. Appl. Biol. 154: 506


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

23

195–204. 507

64. Vandenkoornhuyse P., SL. Baldauf, C. Leyval, J. Straczek, and J.P. Young. 2002. 508

Extensive fungal diversity in plant roots. Science 295: 2051. 509

65. Wang, C.J.K., and H.E. Wilcox. 1985. New species of ectendomycorrhizal and 510

pseudomycorrhizal fungi: Phialophora finlandia, Chloridium paucisporum, and Phialocephala 511

fortinii. Mycologia 77: 951–958. 512

66. White, T. J., T. Bruns, S. Lee, J.W. Taylor. 1990. Amplification and direct sequencing of 513

fungal genes for phylogenetics. In: PCR protocols: A guide to methods and applications. 514

Academic Press, San Diego. pp. 315–322. 515

67. Wicklow, D.T., S. Roth, S.T. Deyrup, and J.B. Gloer. 2005. A protective endophyte of maize: 516

Acremonium zeae antibiotics inhibitory to Aspergillus flavus and Fusarium verticillioides. 517

Mycol. Res. 109: 610–618. 518

68. Wilberforce, E.M., G.W. Griffith, L. Boddy, and R. Griffiths. 2002. The widespread 519

occurrence of dark septate endophyte fungi in grassland communities, abstr. P. 319. The 7th 520

International Mycological Congress, Oslo, Norway. 521

69. Yamato, M., S. Ikeda, and K. Iwase. 2008. Community of arbuscular mycorrhizal fungi in a 522

coastal vegetation on Okinawa island and effect of the isolated fungi on growth of sorghum 523

under salt-treated conditions. Mycorrhiza 18: 241–249. 524

70. Yuan, Z.L., C.C. Dai, X. Li, L.S. Tian, and X.X. Wang. 2007. Extensive host range of an 525

endophytic fungus affects the growth and physiological functions in rice (Oryza sativa L.). 526

Symbiosis 43: 21–28. 527

71. Zhang, Y., Y. Zhang, M. Liu, X. Shi, and Z. Zhao. 2008. Dark septate endophyte (DSE) 528

fungi isolated from metal polluted soils: their taxonomic position, tolerance, and accumulation 529

of heavy metals in vitro. J. Microbiol. 46: 624–632. 530


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

24

LEGENDS TO FIGURES 531

Figure 1 A longitudinal section of O. granulate roots. Mature melanized (A) and blue stained (B) 532

microsclerotia occupy intracellularly within root cortex cells. (Bar = 20µm) 533

Figure 2 The extensive colonization of dark septate endophytes and other root endophytic fungi 534

in wild rice roots (shown in longitudinal section, Bar = 20µm). A: heavy colonization of 535

blue-stained hyphae in epidermal cells with some chlamydospore-like structures; B: 536

Co-occurrence of melanised hyphae and developing sclerotium-like structures in or on the root 537

cortex layer; C: Colonization of melanised hyphae in epidermis and cortex; hyphae growing 538

along the epidermis or cortex parallel to the longitudinal axis of the roots; D: Other trypan 539

blue-stained endophytic fungi colonize intracellularly within root cortex; E: Co-occurrence of 540

melanised hyphae and other blue-stained endophytic fungi; F: Initiation, development and 541

formation of microsclerotia. 542

Figure 3 The frequency of 31 different ITS-based genotypes determined from total cultured fungi. 543

∗ denotes the undescribed fungal species. Genus and/or species names of identified fungi are 544

indicated above the corresponding column. 545

Figure 4 Taxon accumulation curves illustrating observed genotypic or phylotypic richness and 546

estimated total richness (based on bootstrap estimates) of endophyte communities in wild rice 547

roots. A: culturing method; B: direct clone library sequencing method. The 95% confidence 548

intervals for each curve were also shown. 549

Figure 5 Position of two isolates of culturable DSEs (Harpophora sp. and Periconia 550

macrospinosa) on the phylogenetic trees as inferred based on ITS1-5.8S-ITS2 sequence. MP 551

(Maximum-parsimony) bootstrap values > 50% are indicated above branch nodes. Number of 552

bootstrap replicates = 1,000. Each tree is rooted with corresponding outgroup. Macro and 553


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

25

microscopic features of each DSE are also indicated. ∗ denotes the described DSE species. 554

Figure 6 Phylogeny of Rhizoctonia-related species using maximum parsimony analysis based on 555

ITS1-5.8S-ITS2 sequence (CI = 0.7828, RC = 0.6697, RI = 0.8556, HI = 0.2172). Tree 556

length=907. Bootstrap values > 50% are shown above branch nodes. Agaricus bisporus 557

(AF465404) is designated as outgroup. 558

Figure 7 Neigbor-joining phylogenetic tree showing the placement of all the phylotypes based on 559

the sequences of 5.8S of rDNA. The Kimura two-parameter model is used for pairwise distance 560

measurement. The tree is rooted with Rhizopus microsporus (a zygomycete, EU798703). Only 561

bootstrap values > 50% (1000 replicates) are shown at the branches.562


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

26

TABLE 1 Putative taxonomic affinities of sequence types (phylotypes) inferred from BALST searches of ITS sequences and 563

frequency of occurrence of different phylotypes. 564

565

Representative clone

(phylotype)

Putative taxonomic affinity (GenBank number)

Similarity

Score

(expected value)

No. of clones Proportion to

total (%)

R3-67 Aspergillus vitricola (EF652046) 99 1114(0.0) 2 1

R3-21* Mycena rubromarginata (EF530939) 87 745 (0.0) 6 3.2

R3-54* Wallemia sp. (FJ755832) 97 1044 (0.0) 15 8

R1-29* Uncultured soil fungus clone (DQ421246) 100 654 (3e-146) 1 0.5

R3-18* Marasmius oreades (EF 187911) 98 1146 (0.0) 11 5.9

R3-50 Aspergillus sp. (FJ755829) 99 1085 (0.0) 8 4.3

R3-16 Fusarium sp. (EU750682) 98 998 (0.0) 4 2.2

R3-13 Uncultured fungus (AJ875343) 89 676 (0.0) 15 8

R3-11 Penicillium sp.( AJ279476) 99 1116 (0.0) 1 0.5

R3-8* Uncultured fungus clone (FJ553308) 84 604 (1e-169) 11 5.9

R2-67* Uncultured soil fungus (FM866339) 87 723 (0.0) 3 1.6

nR3-4* Uncultured soil fungus clone (DQ421269) 91 915 (0.0) 1 0.5

R2-52 Endophytic ascomycete sp. (AM922199) 94 765 (0.0) 5 2.7

R2-5 Cylindrocarpon sp. (DQ682573) 98 955 (0.0) 2 1

R2-48 Diaporthe sp.( EF488448) 98 1000 (0.0) 3 1.6

R2-19 Xylaria venosula (AB462754) 95 944 (0.0) 7 3.8

R2-51 Exophiala pisciphila (DQ826739) 98 1105 (0.0) 3 1.6

R2-1 Uncultured ascomycete clone (EU003012) 95 1011 (0.0) 1 0.5


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

27

R2-3 Uncultured soil fungus clone (EU826909) 93 845 (0.0) 3 1.6

R1-35 Cladophialophora chaetospira (EU035406) 92 929 (0.0) 1 0.5

R1-23* Uncultured fungus clone (EF434028) 95 1055 (0.0) 7 3.8

R3-70 Kernia pachypleura (DQ318208) 93 966 (0.0) 2 1

R3-58 Phaeomoniella capensis (FJ372391) 87 704 (0.0) 4 2.2

R1-1 Berkleasmium sp. (EU543255) 93 795 (0.0) 1 0.5

R3-62 Arthrinium phaeospermum (EU326200) 99 1129 (0.0) 1 0.5

R2-62 Rhizopycnis sp. (DQ682600) 99 1177 (0.0) 1 0.5

R3-29 Podosphaera fusca (FJ625796) 99 1098 (0.0) 1 0.5

R1-34* Uncultured basidiomycete clone (EU489884) 98 487 (9e-102) 13 7

nR2-4* Uncultured basidiomycete fungus (FN296244) 97 905 (0.0) 47 25.3

nR1-10* Uncultured soil fungus clone (DQ421246) 87 691 (0.0) 1 0.5

nR1-11 Uncultured soil fungus clone (EU480266) 85 577 (3e-161) 1 0.5

R2-66 Uncultured fungus (AJ875342) 81 460 (3e-126) 1 0.5

R3-10 Uncultured Helotiales clone (FJ553766) 90 754 (0.0) 1 0.5

R3-40 * Trichosporon mucoides (AF455482) 99 996(0.0) 1 0.5

R3-68 Uncultured Xylariales clone (EF619915) 96 854 (0.0) 1 0.5

Note: * denotes the sequences of clones within basidiomycota. The highlighted text indicated the frequently detected clones. 566


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

1 title: identity, diversity and molecular phylogeny of ...(n 22°04 -22°17 ; e 100°32 -100°44 )...

Documents