Identity, Diversity, and Molecular Phylogeny of the Endophytic ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2010, p. 1642–1652 0099-2240/10/$12.00 doi:10.1128/AEM.01911-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 76, No. 5

Identity, Diversity, and Molecular Phylogeny of the Endophytic Mycobiota in the Roots of Rare Wild Rice (Oryza granulate) from a Nature Reserve in Yunnan, China䌤† Zhi-lin Yuan,1,2 Chu-long Zhang,1* Fu-cheng Lin,1* and Christian P. Kubicek3 State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China1; Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, Zhejiang 311400, China2; and Institute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, Vienna University of Technology, 1060 Vienna, Austria3 Received 9 August 2009/Accepted 16 December 2009

Rice (Oryza sativa L.) is, on a global scale, one of the most important food crops. Although endophytic fungi and bacteria associated with rice have been investigated, little is known about the endophytic fungi of wild rice (Oryza granulate) in China. Here we studied the root endophytic mycobiota residing in roots of O. granulate by the use of an integrated approach consisting of microscopy, cultivation, ecological indices, and direct PCR. Microscopy confirmed the ubiquitousness of dark septate endophytes (DSEs) and sclerotium-like structures in root tissues. Isolations from 204 root segments from 15 wild rice plants yielded 58 isolates, for which 31 internal transcribed spacer (ITS)-based genotypes were recorded. The best BLAST match indicated that 34.5% of all taxa encountered may represent hitherto undescribed species. Most of the fungi were isolated with a very low frequency. Calculation of ecological indices and estimation of taxon accumulation curves indicated a high diversity of fungal species. A culture-independent approach was also performed to analyze the endophytic fungal community. Three individual clone libraries were constructed. Using a threshold of 90% similarity, 35 potentially different sequences (phylotypes) were found among 186 positive clones. Phylogenetic analysis showed that frequently detected clones were classified as Basidiomycota, and 60.2% of total analyzed clones were affiliated with unknown taxa. Exophiala, Cladophialophora, Harpophora, Periconia macrospinosa, and the Ceratobasidium/Rhizoctonia complex may act as potential DSE groups. A comparison of the fungal communities characterized by the two approaches demonstrated distinctive fungal groups, and only a few taxa overlapped. Our findings indicate a complex and rich endophytic fungal consortium in wild rice roots, thus offering a potential bioresource for establishing a novel model of plant-fungal mutualistic interactions. 14,000 years ago, rice is today the main staple for more than 3 billion people (i.e., half of the world’s population). Its consumption exceeds 100 kg per capita annually in many Asian countries, and it is the principal food for most of the world’s poorest people, particularly in Asia. The association of arbuscular mycorrhizal fungi and endophytic bacteria with rice plants has been well documented (15, 32, 35, 44, 53, 56, 60). Less, however, is known about its fungal endophytes. Fungal endophytes have been detected in cultivated rice (Oryza sativa L.) (12, 14, 37, 61, 70), and antagonistic or plant growthstimulating properties have been claimed for some of these isolates. For example, endophytic Fusarium spp. from cultivated rice roots proved to be effective in biocontrol of a rootknot nematode (28). The occurrence of mycorrhizal and endophytic fungi in a variety of rice cultivars has also recently been reported (63). Nondomesticated, wild plant species may live in symbiosis with a unique and rich mycoflora that may have been lost during breeding of the cultivars used in agriculture (20, 59). The purpose of this research was to characterize the endophytic fungal community of the roots of rare (nearly extinct) wild rice (Oryza granulate) from a nature reserve in Yunnan, China. Our results showed that arbuscular mycorrhizal fungi were apparently absent from wild rice roots. This finding was confirmed by standard root staining techniques and molecular detection using the arbuscular mycorrhizal (AM)-specific primer pairs (69). The characterization of root endophytes in

The majority of terrestrial plant roots are intimately associated with mycorrhizal fungi, and many aspects of the ecological roles played by these mycorrhizal fungi are well understood. In recent years, however, endophytic fungi have been gaining increasing interest. There is accumulating evidence that plant roots usually harbor mycorrhizal as well as endophytic fungi (29, 30, 34, 39, 52, 63). Dark septate endophytes (DSEs), which are characterized by dark pigmented hyphae and sclerotiumlike structures, are believed to represent primary nonmycorrhizal root-inhabiting fungi (23). In some cases, DSEs are even more frequent than mycorrhizal fungi (68). Endophytic fungi have frequently been reported to be associated with crop plants, including wheat (Triticum aestivum), wild barley (Hordeum brevisubulatum and Hordeum bogdanii), soya bean (Glycine max), and maize (Zea mays) (6, 9, 11, 13, 21, 26, 27, 33, 36, 67). Some of the endophytic fungi in these crops conferred resistance of the plant to insect or fungal pathogens (55). Domesticated from the wild grass Oryza rufipogon 10,000 to

* Corresponding author. Mailing address: State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China. Phone for Chu-long Zhang: 86-571-86971185. Fax: 86-571-86971516. E-mail: [email protected]. Phone and fax for Fu-cheng Lin: 86-571-86971516. E-mail: [email protected]. † Supplemental material for this article may be found at http://aem .asm.org/. 䌤 Published ahead of print on 28 December 2009. 1642

VOL. 76, 2010

ENDOPHYTIC FUNGI ASSOCIATED WITH WILD RICE

1643

FIG. 1. A longitudinal section of O. granulate roots. Mature melanized (A) and blue-stained (B) microsclerotia occupy intracellularly root cortex cells. Bar ⫽ 20 ␮m.

wild rice as reported in this study will improve our knowledge concerning the ecology and evolution of mutualistic plantfungus interactions. MATERIALS AND METHODS Site of study. The site of this study was located in Xishuangbanna Nature Reserve, Yunnan Province, southwest of China (22°04⬘–22°17⬘N; 100°32⬘–100°44⬘E). Sampling. We sampled a total of 15 O. granulate plants within an approximate 50-m radius of the georeference point in September 2008. Due to the endangered species status of wild rice, additional sampling in other sites was not permitted. The O. granulate plants grew in latosol (acidic red soil, pH ⬍6.0) at an altitude of 650 m within a bamboo forest. Usually, they grew with two bamboo species, including Pleioblastus amarus and Oxytenantheca nigrociliata. Upon collection, healthy and intact wild rice plants with bulk soil were carefully packed into a box and transported to a laboratory within 48 h. Microscopic analysis of roots. To detect the presence of fungal developmental structures, such as arbuscular mycorrhizal fungi or root endophytes, roots were stained using a modification of a previously described protocol (42). Specifically, roots were cleaned with sterile deionized water and fixed in 50% (vol/vol) ethanol for 24 h. They were then rinsed three times with deionized water and placed in 5% (wt/vol) KOH for 2 h at 90°C. After they were further rinsed with deionized water, the roots were submerged in 2% (wt/vol) lactic acid for 2 min, stained with 0.05% (wt/vol) trypan blue at 50°C for 5 h, and destained in 50% (vol/vol) glycerin for 24 h. Squash preparations of the root segments in 50% (vol/vol) glycerol were examined by light microscopy (Olympus BX51, Japan). Isolation and identification of endophytic fungal cultures. Visual and microscopic inspection was first performed to ensure that roots were free of obvious lesions. Healthy roots were rinsed with tap water immersed in ethanol (75% [vol/vol]) for 40 s and then in sodium hypochlorite 1% (vol/vol) for 4 min and finally rinsed three times in sterile distilled water. Equal numbers of old and young roots from the 15 sampled plants were cut into 0.5-cm lengths for a total of 204 segments (root tips were not included because of the low frequency of fungal colonization with previous microscopic staining examination) and transferred to plates with 2% (wt/vol) malt extract agar (MEA; 20 g malt extract plus 20 g agar/liter) medium supplemented with chloromycetin (50 mg/liter) to prevent bacterial growth. Six segments per plate were distributed to MEA. A total of 34 plates were sealed with Parafilm to avoid desiccation and cultured at 25°C in darkness. Hyphae emerging from segments were subcultured onto fresh PDA

(potato [200 g] plus glucose [20 g/liter]) for purification of isolates. The remaining root samples were used for extraction of total DNA. To ensure that the surface sterilization had removed all hyphae and chlamydospores externally adhering to the roots, they were placed in MEA agar plates and incubated. Only roots that were negative in this test were used for isolation of endophytes. All fungal isolates were initially identified to the genus and/or species level using cultural and morphological characteristics, which included colony appearance, conidia morphology, and conidiophore/conidial structures. Some isolates sporulated readily on PDA media after 1 week of inoculation in darkness at 25°C. The microscopic characteristics of strains were based on light microscopy (Olympus BX51, Japan) and/or cryo-scanning electron microscopy (cryo-SEM; HITACHI S-3000N, Japan). Specimens for light microscopy were mounted in 3% KOH or sterile distilled water for observation. Remaining sterile fungal isolates were subjected to a molecular method of identification. Fungal ITS amplification and sequencing. Fungal DNA was extracted from 58 pure culture isolates using the multisource genomic DNA miniprep kit (Axygen Incorporation, China) according to the manufacturer’s instructions. Primers ITS1 (5⬘-TCCGTAGGTGAACCTGCGG-3⬘) and ITS4 (5⬘-TCCTCCGCTTAT TGATATGC-3⬘) (66) were used for amplification of the fungal ribosomal DNA (rDNA) internal transcribed spacer (ITS) regions 1 and 2 of all isolates. The PCR mixture (50 ␮l, total volume) contained 5 ␮l 10⫻ PCR buffer, 4 ␮l 25 mM Mg2⫹, 2 ␮l 10 mM deoxynucleoside triphosphates (dNTPs), 1 ␮l of each primer (10 ␮M), 2 ␮l original template, 1 ␮l Taq polymerase, and double-distilled water (ddH2O) (34 ␮l). Thirty-five cycles were run, each cycle consisting of a denaturation step at 94°C (40 s), an annealing step at 54°C (60 s), and an extension step at 72°C (60 s). After the 35th cycle, a final 10-min extension step at 72°C was performed. The reaction products were separated in 1.0% (wt/vol) agarose gel, and the amplicons were purified using a gel band purification kit (Axygen Incorporation, China) and sequenced in an ABI 3730 sequencer (Applied Biosystems, United States) using the ITS1 and ITS4 primers. Direct amplification of fungal ITS sequences from roots. All root samples were stored at ⫺70°C until used in DNA extraction. For extraction of DNA, all root slices were pooled together and grouped into three batches which were treated independently. One hundred mg of root material was cut into sections approximately 1 cm in length and ground to a fine powder in liquid nitrogen. Total DNA was subsequently extracted using the multisource genomic DNA miniprep kit (Axygen Incorporation, China) according to the manufacturer’s instructions. The fungal specific primers ITS1-F (5⬘-CTTGG TCATTTAGAGGAAGTAA-3⬘) and ITS4 (5⬘-TCCTCCGCTTATTGATAT

1644

APPL. ENVIRON. MICROBIOL.

YUAN ET AL.

FIG. 2. The extensive colonization of dark septate endophytes and other root endophytic fungi in wild rice roots (shown in longitudinal section, bar ⫽ 20 ␮m). (A) Heavy colonization of blue-stained hyphae in epidermal cells with some chlamydospore-like structures; (B) cooccurrence of melanized hyphae and developing sclerotium-like structures in or on the root cortex layer; (C) colonization of melanized hyphae in epidermis and cortex, and hyphae growing along the epidermis or cortex parallel to the longitudinal axis of the roots; (D) other trypan blue-stained endophytic fungi colonize intracellularly within root cortex; (E) cooccurrence of melanized hyphae and other blue-stained endophytic fungi; (F) initiation, development, and formation of microsclerotia.

GC-3⬘) were used for amplification of the ITS 1 and 2 region of ascomycetes and basidiomycetes (30). The PCR mixture (50 ␮l, total volume) contained 5 ␮l 10⫻ PCR buffer, 7 ␮l 25 mM Mg2⫹, 2 ␮l 10 mM dNTPs, 2 ␮l of each primer (10 ␮M), 4 ␮l original template, 1 ␮l Taq polymerase, and 27 ␮l ddH2O. Amplification conditions were 35 cycles of 94°C for 40 s, an annealing step at 55°C for 50 s, and an extension step at 72°C for 60 s. After the 35th cycle, a final extension step at 72°C for 10 min was performed. The reaction products were then separated and purified as above. The products from three individual PCRs (R1, R2, and R3) were ligated into pGEM-T Easy (Promega, United States), respectively, and transformed into Escherichia coli JM109 (Promega) according to the manufacturer’s instructions, resulting in three individual clone libraries. The transformants were plated on LB agar plates containing 50 ␮g/ml ampicillin and X-Gal/IPTG (5-bromo-4-chloro-3-indolyl␤-D-galactopyranoside/isopropyl-␤-D-thiogalactopyranoside). A total of 214 positive clones were randomly selected and subjected to sequencing. Primer M13F was used for sequencing. A flow diagram of the detailed procedure is presented in Fig. S1 in the supplemental material. Phylogenetic analysis. Vector sequences of sequenced fungal clones were removed using VecScreen (http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen .html). To remove potential chimeric sequences, the sequences were first manually inspected for the presence of signature shifts and then subjected to analysis by Bellerophon (http://foo.maths.uq.edu.au/⬃huber/bellerophon.pl) (19). Only 28 of 214 positive clones were chimeric and thus removed. The frequency of unique phylotypes was determined by assembling sequences with 90% similarity threshold (2) using Sequencher version 4.1.4 (http://www .genecodes.com/). For the culture-based method, the sequences were then

aligned, and those with ⱖ99% sequence identity over the whole amplicon length were defined as one genotype. The final sequence sets were then submitted to BLAST analysis, and identities ⱖ99% were considered conspecific. To verify the phylogenetic position of DSE genotypes, they and corresponding best BLAST hits were aligned by Clustal X and manually corrected in GENEDOC. Maximum parsimony (MP) analysis was performed with PAUP* 4.0b10, using the heuristic search option with tree bisection-reconnection (TBR) branch swapping; the stability of clades was tested using 1,000 bootstrap replications. For inferring the phylogeny of all detected phylotypes in the clone libraries, the 5.8 S rDNA analysis was performed with sequences retrieved in BLAST searches. Neighbor-joining (NJ) trees were built using a Kimura two-parameter (K2P) model in PAUP* 4.0b10. The robustness of the internal branches was also assessed with 1,000 bootstrap replications. Calculation of ecological indices and estimation of taxon accumulation curves for quantifying fungal biodiversity. The rates of colonization and isolation were calculated by the following formula:

Colonization rate ⫽

Isolation rate ⫽

Total number yielding ⱖ 1 isolate Total number of samples in that trial

Total number of isolates yielded in a given trial Total number of samples in that trial

To quantify fungal diversity of rice roots, Fisher’s alpha (␣), the Shannon diversity index (H), Simpson’s diversity index (1-D), and Margalef’s richness

VOL. 76, 2010


1645

index (R1) were calculated (18, 57, 58) by the following equations, respectively:

冘 N

S ⫽ ␣ ⫻ In共1 ⫹ n/␣兲

冘

D⫽

P i2

i⫽l

N

H⫽ ⫺

Pi InPi

R1 ⫽ 共S ⫺ 1兲/In共N兲

i⫽l

Therein, S is the number of taxa (ITS genotype or phylotype), N is the number of individuals (defined by numbers of isolates or sequenced clones), and i is the proportion of species relative to the total number of species (Pi). Taxon accumulation curves and bootstrap estimates of total species richness based on recovered fungal isolates and sequenced clones were generated in EstimateS version 7.5 (8) using 50 randomizations of sample order (http://viceroy.eeb.uconn .edu/EstimateS). Nucleotide sequence accession numbers. Sequences were obtained from cultures, and clones were deposited in GenBank (accession numbers FJ752597 to FJ752627, FJ524295 to FJ524304, FJ524306, FJ524308 to FJ524311, FJ524313 to FJ524316, FJ524318, FJ524320 to FJ524321, FJ524324 to FJ524329 and FJ882005 to FJ882011).

RESULTS Microscopic detection of endophytic fungi in wild rice roots. To assess the presence of fungal endophytes in rice roots, we first examined the roots microscopically. The presence of DSEs in root tissues can be seen by the presence of intraradical microsclerotia (Fig. 1). Large sclerotium-like structures were detected inside of the root cortex, some occupying the whole cortical cell volume. Other small sclerotium-like structures also coexisted with dark septate hyphae, suggesting that they represent different stages of development of DSEs or different DSE species (Fig. 2B). In addition, we found extensive colonization by other fungal endophytes whose hyphae stained blue (Fig. 2A, D, and E). Further, some chlamydospore-like structures similar to those found with Piriformospora indica-infected maize roots (24) were also observed. In most cases, the dark and the blue hyphae grew along the epidermis or cortex parallel to the longitudinal axis of the roots (Fig. 2C, D, and E). Also, the root epidermis contained a dense hyphal network which was infrequent in the cortex layers (Fig. 2C). In the early stage of development, the formation of microsclerotia was apparent by the detection of dark hyphal fragments (Fig. 2F). Isolation and identification of endophytic fungi. A total of 58 fungal isolates were recovered and purified from 204 root tissue samples. A proportion of isolates were identified to genus and/or species level based on the morphology of conidia and conidiophores and unique phenotypic characters (Fig. 3). The identified fungi belonged to the phylum Ascomycota, with the exception of one basidiomyceteous Rhizoctonia-like isolate. To confirm the reliability of morphological identification, all 58 isolates were subjected to molecular identification based on rDNA ITS sequence analysis. In total, 31 distinctive genotypes were detected at a 99% sequence similarity threshold (Fig. 3), which corresponded well with morphological differences between these fungal cultures. This allowed the placement of these isolates into several ascomycete lineages representing at least seven orders (Hypocreales, Diaporthales, Eurotiales, Xylariales, Microascales, Capnodiales, and Magnaporthales). In addition, the Rhizoctonia-like fungus was identified as an anamorphic species of Ceratobasidium (anastomosis group AG-G) (see Fig. 7). Nine genotypes, however, could not be

FIG. 3. The frequency of 31 different ITS-based genotypes determined from total cultured fungi. * denotes undescribed fungal species. Genus and/or species names of identified fungi are indicated above the corresponding column.

assigned to any genus or species, as their ITS sequences did not resemble any described species in the GenBank database and thus likely represent novel fungal lineages (Fig. 3). Molecular phylogeny assigned all nine to the class Dothideomycetes (Ascomycota) group (data not shown). To characterize the biodiversity of our samples, we calculated Fisher’s alpha, the Shannon diversity index, Simpson’s diversity index, and Margalef’s richness index. The values ob-

1646

YUAN ET AL.


FIG. 4. Taxon accumulation curves illustrating observed genotypic or phylotypic richness and estimated total richness (based on bootstrap estimates) of endophyte communities in wild rice roots. (A) culturing method; (B) direct clone library sequencing method. The 95% confidence intervals for each curve are also shown.

tained by these tests (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 sequenced clones, respectively) indicate that the biodiversity of fungal endophytes in wild rice roots is very high. On the other hand, unlike for other perennial herbaceous and woody plants, the colonization rate and isolation rate in root tissues of O. granulate were low (23% and 28.4%, respectively). Nineteen fungal genotypes were recovered only once (61.3%) (Fig. 3). With the exception of the fungi exhibiting the ITS type 13, for which 9 conspecific isolates were found, no other genus or species dominated. This suggests that the ITS type 13 fungi are potentially important root symbionts in wild rice. These findings would be in accordance with the hypothesis that horizontally transmitted endophytes reinfect annual grasses yearly and accumulate seasonally, while, in contrast, they accumulate in older tissues for evergreen plants and perennial grasses (5, 49). Estimated species and phylotype richness accumulation curves were generated using EstimateS (8). Two “taxon-based” accumulation curves showed a declining rate of accumulation of ITS genotypes or phylotypes (Fig. 4). The relative steepness of the curves and the high number of estimated total richness implied that more endophytes are waiting to be discovered. Bootstrap estimates of species richness exceeded the observed species richness. The observed species richness fell within the 95% confidence intervals of the estimated richness, indicating that the sampling method used was effective in recovering the fungal species of the endophyte community. It was also shown that the estimated richness curve based on sequenced clones (SC) increased more quickly with fewer clone numbers (Fig. 4B) than the curve based on cultured isolates (CI). However, with increasing numbers of analyzed isolates or clones, the SC curve gradually flattened and the CI curve did not reach saturation level. The final values of the abundance-based estimator (ACE) for CI and SC were 71.4 and 54.5, respectively. Endophytic fungal community evaluation via environmental PCR. In order to learn whether the culturable endophytes would represent the actual biodiversity of fungi in rice roots, we analyzed which fungal sequences would be retrieved by an analysis of direct PCR from the total root DNA. A total of 186 clones were obtained from the three individual clone libraries, sequenced, and subjected to phylogenetic analysis. BLAST and

NJ analysis placed the cloned sequences into various groups of Ascomycota and Basidiomycota (Fig. 5). In contrast to the results obtained by the cultivation-based approach, the endophytic fungal community in roots was dominated by basidiomycetes (63%) (Table 1). A total of 60.2% of the clones had sequences that were close but not identical to currently known taxa (Table 1). The most frequently detected phylotype was genetically close to an uncultured basidiomycetes fungus (FN296244). Four phylotypes were closely related to the basidiomycetes Trichosporon, Wallemia, Marasmius, and Mycena (Table 1). All these data provide strong evidence that roots act as large reservoirs for the colonization of unexplored endophytic fungi. DISCUSSION Fungal symbionts, comprising mainly mycorrhizal fungi and fungal endophytes, are ubiquitously distributed in terrestrial plant roots. They act beneficially on the plant by modulating host nutrition, metabolites, and stress response (4, 45–47). Based on previous investigations, it can be concluded that the colonization rate of these two types of fungi is highly variable and dependent on habitat, host, and seasonal fluctuations in the climate (31). In this study, we show that roots of wild rice exhibit a very high biodiversity of endophytic but not mycorrhizal fungi. Recently, 16 fungal genera were recovered from the roots of O. sativa from the Bhadra River Project area, Karnataka (India), and only 6 genera were found with rice varieties cultivated in Italy and in Guangdong province, China (37, 61, 63). Results of these studies involved culture-based approaches, not environmental PCR methodology. Moreover, they did not look for the occurrence of dark septate endophytes. The difference in the results from those and the present study may be due to the fact that those authors investigated cultivated rice. It appears plausible to assume that wild rice roots host more and novel endophytes relative to cultivated plants. Among root-associated fungi, dark septate endophytes (DSEs) are ubiquitous and cosmopolitan and found in a wide range of plant species. While the abundance of DSEs in arctic, alpine, and temperate habitats has been investigated widely

VOL. 76, 2010


1647

FIG. 5. Neighbor-joining phylogenetic tree showing the placement of all the phylotypes based on the sequences of 5.8S of rDNA. The Kimura two-parameter model is used for pairwise distance measurement. The tree is rooted with Rhizopus microsporus (a zygomycete, EU798703). Only bootstrap values of ⬎50% (1,000 replicates) are shown at the branches.

1648

YUAN ET AL.


TABLE 1. Putative taxonomic affinities of sequence types (phylotypes) inferred from BLAST searches of ITS sequences and frequency of occurrence of different phylotypesa Representative clone (phylotype)

R3-67 R3-21* R3-54* R1-29* R3-18* R3-50 R3-16 R3-13 R3-11 R3-8* R2-67* nR3-4* R2-52 R2-5 R2-48 R2-19 R2-51 R2-1 R2-3 R1-35 R1-23* R3-70 R3-58 R1-1 R3-62 R2-62 R3-29 R1-34* nR2-4* nR1-10* nR1-11 R2-66 R3-10 R3-40* R3-68 a

Putative taxonomic affinity (GenBank no.)

Aspergillus vitricola (EF652046) Mycena rubromarginata (EF530939) Wallemia sp. (FJ755832) Uncultured soil fungus clone (DQ421246) Marasmius oreades (EF 187911) Aspergillus sp. (FJ755829) Fusarium sp. (EU750682) Uncultured fungus (AJ875343) Penicillium sp.(AJ279476) Uncultured fungus clone (FJ553308) Uncultured soil fungus (FM866339) Uncultured soil fungus clone (DQ421269) Endophytic ascomycete sp. (AM922199) Cylindrocarpon sp. (DQ682573) Diaporthe sp.(EF488448) Xylaria venosula (AB462754) Exophiala pisciphila (DQ826739) Uncultured ascomycete clone (EU003012) Uncultured soil fungus clone (EU826909) Cladophialophora chaetospira (EU035406) Uncultured fungus clone (EF434028) Kernia pachypleura (DQ318208) Phaeomoniella capensis (FJ372391) Berkleasmium sp. (EU543255) Arthrinium phaeospermum (EU326200) Rhizopycnis sp. (DQ682600) Podosphaera fusca (FJ625796) Uncultured basidiomycete clone (EU489884) Uncultured basidiomycete fungus (FN296244) Uncultured soil fungus clone (DQ421246) Uncultured soil fungus clone (EU480266) Uncultured fungus (AJ875342) Uncultured Helotiales clone (FJ553766) Trichosporon mucoides (AF455482) Uncultured Xylariales clone (EF619915)

Similarity (%)

Score (expected value)

No. of clones

Proportion to total (%)

99 87 97 100 98 99 98 89 99 84 87 91 94 98 98 95 98 95

1,114 (0.0) 745 (0.0) 1,044 (0.0) 654 (3e-146) 1,146 (0.0) 1,085 (0.0) 998 (0.0) 676 (0.0) 1,116 (0.0) 604 (1e-169) 723 (0.0) 915 (0.0) 765 (0.0) 955 (0.0) 1,000 (0.0) 944 (0.0) 1,105 (0.0) 1,011 (0.0)

2 6 15 1 11 8 4 15 1 11 3 1 5 2 3 7 3 1

1 3.2 8 0.5 5.9 4.3 2.2 8 0.5 5.9 1.6 0.5 2.7 1 1.6 3.8 1.6 0.5

93 92 95 93 87 93 99 99 99 98

845 (0.0) 929 (0.0) 1,055 (0.0) 966 (0.0) 704 (0.0) 795 (0.0) 1,129 (0.0) 1,177 (0.0) 1098 (0.0) 487 (9e-102)

3 1 7 2 4 1 1 1 1 13

1.6 0.5 3.8 1 2.2 0.5 0.5 0.5 0.5 7

47

25.3

1 1 1 1 1 1

0.5 0.5 0.5 0.5 0.5 0.5

97

905 (0.0)

87 85 81 90 99 96

691 (0.0) 577 (3e-161) 460 (3e-126) 754 (0.0) 996 (0.0) 854 (0.0)

* denotes the sequences of clones within Basidiomycota. Boldface text indicates the frequently detected clones.

(31, 39, 51, 52), the role of DSEs in tropical ecosystems is still poorly understood. The melanized hyphae, typical for these fungi, are considered to be of importance for the host to survive stress conditions because cell wall melanin can trap and eliminate oxygen radicals generated during abiotic stress (48). Therefore, the dominant colonization of wild rice by DSEs may confer tolerance to a variety of environmental stress factors. Surprisingly, even though light microscopy revealed that DSE fungi were ubiquitous in rice roots, sequence analysis indicated that only 4 of 186 sequenced clones matched potential DSE fungi, i.e., 3 clones of Exophiala pisciphila and one clone of Cladophialophora chaetospira. Likely, our total DNA extraction protocol was not well suited for efficient recovery of genomic DNA from dark hyphae and/or sclerotia. The genus Exophiala is phylogenetically close to Phialophora (23), and recent experimental data confirm that Exophiala sp. is responsible for DSE appearance (22, 71). Also, some species of Cladophialophora are morphologically and phylogenetically similar to Heteroconium chaetospira (38), a recognized DSE, which is, however, phylogenetically different from most DSE taxa. Interestingly, none of the endophytes isolated by cultiva-

tion could be identified as Exophiala or Cladophialophora, indicating that the methods for disinfection and/or cultivation media may be inappropriate for some DSE species. Alternatively, some unculturable DSE fungi may also exist within the roots. We propose that the application of high-throughput cultivation methods should be applied in the future to complement the drawback of traditional isolation methods. The so called “dilution-to-extinction” culture method has recently been successfully used to recover more diverse endophytic fungi species from leaf tissues than the segment plating method (7, 62). The members of the genera Phialocephala and Phialophora are typical DSEs and frequently observed with roots (54, 65). Phialocephala fortinii preferentially colonizes roots of woody plant species, while Phialophora spp. usually live in herbaceous plant roots as hosts, especially in Gramineae (54). The ITS type 10 sequence was 96% identical to Phialophora sp. (anamorph of Gaeumannomyces, now widely named Harpophora sp.), although only one strain was isolated in culture (Fig. 3). In vitro inoculation testing has verified that this isolate is capable of living endophytically in cultivated rice (Oryza sativa L.) roots

VOL. 76, 2010


1649

FIG. 6. Position of two isolates of culturable DSEs (Harpophora sp. and Periconia macrospinosa) on the phylogenetic trees, as inferred based on ITS1-5.8S-ITS2 sequence. Maximum-parsimony bootstrap values of ⬎50% are indicated above branch nodes. Number of bootstrap replicates ⫽ 1,000. Each tree is rooted with corresponding outgroup. Macro- and microscopic features of each DSE are also indicated. * denotes the described DSE species.

and formed dark brown hyphae in the tissues. After 30 days of coculture under controlled aseptic conditions, Harpophora sp. significantly promoted the growth and biomass of rice seedlings (data not shown). Further characterization of colony and conidial morphology confirmed the isolate to be a member of the genus Harpophora, but its ITS sequence was not conspecific with any of the species of Harpophora described so far (Fig. 6). Harpophora graminicola (previously referred to as Phialophora graminicola) was also shown to be a beneficial dark endophyte in grass roots (40). In addition, both BLAST and phylogenetic analysis revealed

that the ITS genotype 22 was closely related to Periconia macrospinosa (98% sequence identity; Fig. 6). P. macrospinosa was recently reported as a unique DSE that inhabits various plant roots (31). Furthermore, one isolate (ITS type 31) was morphological and phylogenetically identified as a member of the Ceratobasidium/Rhizoctonia complex (Fig. 7). This genus comprises a diverse group of soil inhabitant fungi that include important crop pathogens, and orchid mycorrhizal symbionts. Most Rhizoctonia orchid mycorrhizae are anamorph members of Tulasnella, Ceratobasidium, and Thanatephorus (41). Members of the Ceratobasidium/Rhizoctonia complex may also act

1650

YUAN ET AL.


FIG. 7. Phylogeny of Rhizoctonia-related species using maximum parsimony analysis based on ITS1-5.8S-ITS2 sequence (consistency index [CI] ⫽ 0.7828, rescaled consistency index [RC] ⫽ 0.6697, retention index [RI] ⫽ 0.8556, homoplasy index [HI] ⫽ 0.2172). Tree length ⫽ 907. Bootstrap values of ⬎50% are shown above branch nodes. Agaricus bisporus (AF465404) is designated the outgroup.

as fungal symbionts in nonorchid hosts, as recently suggested for Fagopyrum esculentum and Fagopyrum tataricum (30). Only five ascomycete genera (Cylindrocarpon, Fusarium, Xylaria, Phomopsis, and Penicillum) were detected both by direct isolation from the roots and by direct PCR analysis. This observation could indicate a potential technical bias in examining fungal diversity (1). The direct PCR method effectively recognized endophytic fungi belonging to the phylum Basidiomycota, while pure-culture isolation preferentially detected those fungi within the phylum Ascomycota (25). Thus, these results highlight the importance of integrating multiple approaches for analyzing endophytic microbial biodiversity in plants (25). Some of our isolates were identified as being members of commonly observed genera of soil fungi, e.g., Fusarium, Penicillium, Trichoderma, and Paecilomyces. Representatives of these genera have been identified as endophytes in cultivated rice roots (37, 61, 63). These fungi are characteristically freeliving saprophytes that can also be opportunistic root symbionts (3, 17). Our data demonstrate that roots of wild rice are associated with a surprisingly rich endophyte community. The combination of microscopy, isolation of pure cultures, ecological analysis, and clone sequencing yielded comprehensive information about the identity, diversity, and phylogeny of fungal endophytes. Our results also provide additional evidence that endophyte diversity in gramineous grass roots may be as rich as other perennial grasses and woody plants (49). In comparison to previous studies, 49 and 51 fungal phylotypes and operational taxonomic units (OTUs) were found in the grasses

Arrhenatherum elatius and Bouteloua gracilis, respectively (43, 64). It must be noted that the authors of those studies (43, 64) actually targeted root-associated fungi (endophytes, epiphytes, and some rhizosphere soil fungi) and therefore probably have also detected Chytridiomycota and Zygomycota. Most of the endophytic lineages belong to the Ascomycota clade, and some belong to the phylum Basidiomycota. Currently, direct clone library sequencing (also called “environmental PCR”) has been applied in studying foliar and root endophytic fungi diversity (2, 10, 16, 58). It must be admitted that the sterilization of roots as described in this paper as a technique will certainly kill all the microorganisms in the root surface, but the dead cells may still contain DNA that becomes extracted and thus amplified. However, the extraction of total endophytic fungal DNA is still considered to be reliable because some epiphytic fungi may also penetrate the cortex tissue and live endophytically (50). Furthermore, none of our clones matched any sequences of “lower fungi,” which indicates that the sterilization procedure in this study was effective in degrading the DNA of most epiphytic and rhizosphere soil fungi. There was no indication of overestimation of endophytic fungal diversity. In summary, we obtained consistent results (microscopy, culturing, and molecular detection) that indicated that nonmycorrhizal fungi, including several lineages of ascomycetes and basidiomycetes, may constitute the dominant fungal consortium in wild rice roots. This highlights the similar contributions of mycorrhizal fungi and endophytic fungi to modulating host growth and development. It should also be conceded that the limited sample size (15 wild rice roots collected in one site) in this study may bias conclusions. Analyzing additional samples

VOL. 76, 2010


from multiple locations will be necessary to determine whether the endophytic fungal lineages found in samples from the Yunnan site reflect a pattern common to wild rice from other regions. The specific DSE fungi also represent a novel system for exploring mutualistic plant-fungus interactions. Further work is needed to elucidate the roles that these cohabiting colonizers play in plant performance and stress response. Considering the global importance of the rice plant for food production, examination of endophyte-mediated plant growth promoting and/or disease resistance will aid in the production of this crop. ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China grant no. 30600002 and 30970097 to Chu-long Zhang. We would also like to express our great appreciation to Yang Yun for collecting samples. REFERENCES 1. Allen, T. R., T. Millar, S. M. Berch, and M. L. Berbee. 2003. Culturing and direct DNA extraction find different fungi from the same ericoid mycorrhizal roots. New Phytol. 160:255–272. 2. Arnold, A. E., D. A. Henk, R. L. Eells, F. Lutzoni, and R. Vilgalys. 2007. Diversity and phylogenetic affinities of foliar fungal endophytes in loblolly pine inferred by culturing and environmental PCR. Mycologia 99:185–206. 3. Bacon, C. W., and I. E. Yates. 2006. Endophytic root colonization by Fusarium species: histology, plant interactions, and toxicity, p. 133–152. In B. J. E. Schulz, C. J. C. Boyle, and T. N. Sieber (ed.), Microbial root endophytes. Springer-Verlag, Berlin, Germany. 4. Baldi, A., A. Jain, N. Gupta, A. K. Srivastava, and V. S. Bisaria. 2008. Co-culture of arbuscular mycorrhiza-like fungi (Piriformospora indica and Sebacina vermifera) with plant cells of Linum album for enhanced production of podophyllotoxins: a first report. Biotechnol. Lett. 30:1671–1677. 5. Carroll, G. C. 1986. The biology of endophytism in plants with particular reference to woody perennials, p. 205–222. In N. Fokkema and J. van den Heuval (ed.), Microbiology of the phyllosphere. Cambridge University Press, Cambridge, United Kingdom. 6. Clement, S. L., A. D. Wilson, D. G. Lester, and C. M. Davitt. 1997. Fungal endophytes of wild barley and their effects on Diuraphis noxia population development. Entomol. Exp. Appl. 82:275–281. 7. Collado, J., G. Platas, B. Paulus, and G. F. Bills. 2007. High-throughput culturing of fungi from plant litter by a dilution-to-extinction technique. FEMS Microbiol. Ecol. 60:521–533. 8. Colwell, R. K. 2005. EstimateS: statistical estimation of species richness and shared species from samples, version 7.5. User’s guide and application. http://purl.oclc.org/estimates. 9. Crous, P. W., O. Petrini, G. F. Marais, Z. A. Pretorius, and F. Rehder. 1995. Occurrence of fungal endophytes in cultivars of Triticum aestivum in South Africa. Mycoscience 36:105–111. 10. Dearnaley, J. D. W., and A. F. Le Brocque. 2006. Molecular identification of the primary root fungal endophytes of Dipodium hamiltonianum. Aust. J. Bot. 54:487–491. 11. Dingle, J., and P. A. McGee. 2003. Some endophytic fungi reduce the density of pustules of Puccinia recondita f. sp. tritici in wheat. Mycol. Res. 107:310– 316. 12. Fisher, P. J., and O. Petrini. 1992. Fungal saprobes and pathogens as endophytes of rice (Oryza sativa L.). New Phytol. 122:137–143. 13. Fisher, P. J., O. Petrini, and H. M. Lappin-Scott. 1992. The distribution of some fungal and bacterial endophytes in maize (Zea mays L.). New Phytol. 122:299–305. 14. Fisher, P. J., and J. Webster. 1992. A Trematosphaeria endophyte from rice roots and its Zalerion anamorph. Nova Hedwigia 54:77–81. 15. Glassop, D., R. M. Godwin, S. E. Smith, and S. W. Smith. 2007. Rice phosphate transporters associated with phosphate uptake in rice roots colonized with arbuscular mycorrhizal fungi. Can. J. Bot. 85:644–651. 16. Gotz, M., H. Nirenberg, S. Krause, H. Wolters, S. Draeger, A. Buchner, J. Lottmann, G. Berg, and K. Smalla. 2006. Fungal endophytes in potato roots studied by traditional isolation and cultivation-independent DNA-based methods. FEMS Microbiol. Ecol. 58:404–413. 17. Harman, G. E., C. R. Howell, A. Viterbo, I. Chet, and M. Lorito. 2004. Trichoderma species-opportunistic, avirulent plant symbionts. Nat. Rev. Microbiol. 2:43–56. 18. Hoffman, M., M. Gunatilaka, J. Ong, M. Shimabukuro, and A. E. Arnold. 2008. Molecular analysis reveals a distinctive fungal endophyte community associated with foliage of Montane oaks in southeastern Arizona. J. ArizonaNevada Acad. Sci. 40:91–100.

1651

19. Huber, T., G. Faukner, and P. Hugenholtz. 2004. Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2319. 20. Hung, P. Q., S. M. Kumar, V. Govindsamy, and K. Annapurna. 2007. Isolation and characterization of endophytic bacteria from wild and cultivated soybean varieties. Biol. Fertil. Soils 44:155–162. 21. Istifadah, N., and P. A. McGee. 2006. Endophytic Chaetomium globosum reduces development of tan spot in wheat caused by Pyrenophora triticirepentis. Australas. Plant Pathol. 35:411–418. 22. Julou, T., B. Burghardt, G. Gebauer, D. Berveiller, C. Damesin, and M. A. Selosse. 2005. Mixotrophy in orchids: insights from a comparative study of green individuals and nonphotosynthetic individuals of Cephalanthera damasonium. New Phytol. 166:639–653. 23. Jumpponen, A., and J. M. Trappe. 1998. Dark septate endophytes: a review of facultative biotrophic root-colonizing fungi. New Phytol. 140:295–310. 24. Kumar, M., V. Yadav, N. Tuteja, and A. K. Johri. 2009. Antioxidant enzyme activities in maize plants colonized with Piriformospora indica. Microbiology 155:780–790. 25. Kwasna, H., G. L. Bateman, and E. Ward. 2008. Determining species diversity of microfungal communities in forest tree roots by pure-culture isolation and DNA sequencing. Appl. Soil Ecol. 40:44–56. 26. Larran, S., A. Perello ´, M. R. Simo ´n, and V. Moreno. 2006. The endophytic fungi from wheat (Triticum aestivum L.). World J. Microbiol. Biotechnol. 23:565–572. 27. Larran, S., C. Rollan, H. A. Bruno, H. E. Alippi, and M. I. Urrutia. 2002. Nota corta: endophytic fungi in healthy soybean leaves. Investig. Arg. Prod. Prot. Veg. 17:173–178. 28. Le, H. T. T., J. L. Padgham, and R. A. Sikora. 2009. Biological control of the rice root-knot nematode Meloidogyne graminicola on rice using endophytic and rhizosphere fungi. Int. J. Pest Manage. 55:31–36. 29. Li, L. F., A. N. Yang, and Z. W. Zhao. 2005. Seasonality of arbuscular mycorrhizal symbiosis and dark septate endophytes in a grassland site in southwest China. FEMS Microbiol. Ecol. 54:367–373. 30. Likar, M., U. Bukovnik, I. Kreft, N. K. Chrungoo, and M. Regvar. 2008. Mycorrhizal status and diversity of fungal endophytes in roots of common buckwheat (Fagopyrum esculentum) and tartary buckwheat (F. tataricum). Mycorrhiza 18:309–315. 31. Mandyam, K. 2008. Dark septate fungal endophytes from a tallgrass prairie and their continuum of interactions with host plants. Ph. D. thesis. Kansas State University, Manhattan, KS. 32. Mano, H., and H. Morisaki. 2008. Endophytic bacteria in rice plant. Microbes Environ. 23:109–117. 33. Marshall, D., B. Tunali, and L. R. Nelson. 1999. Occurrence of fungal endophytes in species of wild Triticum. Crop Sci. 39:1507–1512. 34. Mathew, A., and R. M. Malathy. 2008. The evidence of mycorrhizal fungi and dark septate endophytes in roots of Chlorophytum borivillianum. Acta Bot. Croat. 67:91–96. 35. Mattos, K. A., V. L. Pa ´dua, A. Romeiro, L. F. Hallack, B. C. Neves, T. M. Ulisses, C. F. Barros, A. R. Todeschini, J. O. Previato, and L. Mendonc¸aPreviato. 2008. Endophytic colonization of rice (Oryza sativa L.) by the diazotrophic bacterium Burkholderia kururiensis and its ability to enhance plant growth. An. Acad. Bras. Cienc. 80:477–493. 36. Monfort, E., L. V. Lopez-Llorca, H. B. Jansson, J. Salinas, J. O. Park, and K. Sivasithamparam. 2005. Colonisation of seminal roots of wheat and barley by egg-parasitic nematophagous fungi and their effects on Gaeumannomyces graminis var. tritici and development of root-rot. Soil Biol. Biochem. 37:1229–1235. 37. Naik, B. S., J. Shashikala, and Y. L. Krishnamurthy. 2009. Study on the diversity of endophytic communities from rice (Oryza sativa L.) and their antagonistic activities in vitro. Microbiol. Res. 164:290–296. 38. Narisawa, K., S. Hambleton, and R. S. Currah. 2007. Heteroconium Chaetospira, a dark septate root endophyte allied to the Herpotrichiellaceae (Chaetothyriales) obtained from some forest soil samples in Canada using bait plants. Mycoscience 48:274–281. 39. Newsham, K. K., R. Upson, and D. J. Read. 2009. Mycorrhizas and dark septate root endophytes in polar regions. Fungal Ecol. 2:10–20. 40. Newsham, K. K. 1999. Phialophora graminicola, a dark septate fungus, is a beneficial associate of the grass Vulpia ciliata ssp. ambigua. New Phytol. 144:517–524. 41. Otero, J. T., J. D. Ackerman, and P. Bayman. 2002. Diversity and host specificity of endophytic Rhizoctonia-like fungi from tropical orchids. Am. J. Bot. 89:1852–1858. 42. Peterson, R. L., H. B. Massicotte, and L. H. Melville. 2004. Mycorrhizas: anatomy and cell biology. NRC Research Press, Ottawa, Ontario, Canada. 43. Porras-Alfaro, A., J. Herrera, R. L. Sinsabaugh, K. J. Odenbach, T. Lowrey, and D. O. Natvig. 2008. Novel root fungal consortium associated with a dominant desert grass. Appl. Environ. Microbiol. 74:2805–2813. 44. Prayitno, J., J. Stefaniak, J. McIver, J. J. Weinman, F. B. Dazzo, J. K. Ladha, W. Barraquio, Y. G. Yanni, and B. G. Rolfe. 1999. Interactions of rice seedlings with bacteria isolated from rice roots. Aust. J. Plant Physiol. 26: 521–535. 45. Rai, M., D. Acharya, and A. Singh. 2001. Positive growth responses of the

1652

46.

47.

48. 49.

50. 51.

52.

53.

54.

55.

56.

57. 58.

59.

YUAN ET AL.

medicinal plants Spilanthes calva and Withania somnifera to inoculation by Piriformospora indica in a field trial. Mycorrhiza 11:123–128. Rai, M. K., A. Varma, and A. K. Pandey. 2004. Antifungal potential of Spilanthes calva after inoculation of Piriformospora indica. Mycoses 47:479– 481. Redman, R. S., K. B. Sheehan, R. G. Stout, R. J. Rodriguez, and J. M. Henson. 2002. Thermotolerance generated by plant/fungal symbiosis. Science 298:1581. Richier, S., P. Furla, A. Plantivaux, P. L. Merle, and D. Allemand. 2005. Symbiosis-induced adaptation to oxidative stress. J. Exp. Biol. 208:277–285. Saikkonen, K., S. H. Faeth, M. L. Helander, and T. J. Sullivan. 1998. Fungal endophytes: a continuum of interactions with host plants. Annu. Rev. Ecol. Syst. 29:319–343. Santamaría, J., and P. Bayman. 2005. Fungal epiphytes and endophytes of coffee leaves (Coffea arabica). Microb. Ecol. 50:1–8. Schadt, C. W., R. B. Mullen, and S. K. Schmidt. 2001. Isolation and phylogenetic identification of a dark septate fungus associated with the alpine plant Ranunculus adoneus. New Phytol. 150:747–755. Schmidt, S. K., L. C. Sobieniak-Wiseman, S. A. Kageyama, S. R. P. Halloy, and C. W. Schadt. 2008. Mycorrhizal and dark septate fungi in plant roots above 4270 meters elevation in the Andes and Rocky Mountains. Arctic Antarct. Alp. Res. 40:576–583. Senthilkumar, M., M. Madhaiyan, S. P. Sundaram, H. Sangeetha, and S. Kannaiyan. 2008. Induction of endophytic colonization in rice (Oryza sativa L.) tissue culture plants by Azorhizobium caulinodans. Biotechnol. Lett. 30: 1477–1487. Sieber, T. N. 2002. Fungal root endophytes, p. 887–917. In Y. Wasel, A, Eshel, and U. Kafkafi (ed.), Plant roots: the hidden half. Marcel Dekker, New York, NY. Sieber, T. N., T. K. Riesen, E. Muller, and P. M. Fried. 1988. Endophytic fungi in four winter wheat cultivars (Triticum aestivum L.) differing in resistance against Stagonospora nodorum (Berk.) Cast. & Germ. ⫽ Septoria nodorum (Berk.) Berk. J. Phytopathology 122:289–306. Solaiman, M. Z., and H. Hirata. 1997. Effect of arbuscular mycorrhizal fungi inoculation of rice seedlings at the nursery stage upon performance in the paddy field and greenhouse. Plant Soil 191:1–12. Suryanarayanan, T. S., and V. Kumaresan. 2000. Endophytic fungi of some halophytes from an estuarine mangrove forest. Mycol. Res. 104:1465–1467. Tao, G., Z. Y. Liu, K. D. Hyde, X. Z. Liu, and Z. N. Yu. 2008. Whole rDNA analysis reveals novel and endophytic fungi in Bletilla ochracea (Orchidaceae). Fungal Divers. 33:101–122. Thomas, S. E., J. Crozier, M. C. Aime, H. C. Evans, and K. A. Holmes. 2008. Molecular characterisation of fungal endophytic morphospecies associated


60.

61.

62.

63.

64. 65.

66.

67.

68.

69.

70.

71.

with the indigenous forest tree, Theobroma gileri in Ecuador. Mycol. Res. 112:852–860. Tian, X. L., L. X. Cao, H. M. Tan, W. Q. Han, M. Chen, Y. H. Liu, and S. N. Zhou. 2007. Diversity of cultivated and uncultivated actinobacterial endophytes in the stems and roots of rice. Microb. Ecol. 53:700–707. Tian, X. L., L. X. Cao, H. M. Tan, Q. G. Zeng, Y. Y. Jia, W. Q. Han, and S. N. Zhou. 2004. Study on the communities of endophytic fungi and endophytic actinomycetes from rice and their antipathogenic activities in vitro. World J. Microbiol. Biotechnol. 20:303–309. Unterseher, M., and M. Schnittler. 2009. Dilution-to-extinction cultivation of leaf-inhabiting endophytic fungi in beech (Fagus sylvatica L.)—different cultivation techniques influence fungal biodiversity assessment. Mycol. Res. 113:645–654. Vallino, M., D. Greppi, M. Novero, P. Bonfante, and E. Lupotto. 2008. Rice root colonisation by mycorrhizal and endophytic fungi in aerobic soil. Ann. Appl. Biol. 154:195–204. Vandenkoornhuyse, P., S. L. Baldauf, C. Leyval, J. Straczek, and J. P. Young. 2002. Extensive fungal diversity in plant roots. Science 295:2051. Wang, C. J. K., and H. E. Wilcox. 1985. New species of ectendomycorrhizal and pseudomycorrhizal fungi: Phialophora finlandia, Chloridium paucisporum, and Phialocephala fortinii. Mycologia 77:951–958. White, T. J., T. Bruns, S. Lee, and J. W. Taylor. 1990. Amplification and direct sequencing of fungal genes for phylogenetics, p. 315–322. In M. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols: a guide to methods and applications. Academic Press, San Diego, CA. Wicklow, D. T., S. Roth, S. T. Deyrup, and J. B. Gloer. 2005. A protective endophyte of maize: Acremonium zeae antibiotics inhibitory to Aspergillus flavus and Fusarium verticillioides. Mycol. Res. 109:610–618. Wilberforce, E. M., G. W. Griffith, L. Boddy, and R. Griffiths. 2002. The widespread occurrence of dark septate endophyte fungi in grassland communities, abstr. P. 319. Abstr. 7th Int. Mycol. Cong., Oslo, Norway. Yamato, M., S. Ikeda, and K. Iwase. 2008. Community of arbuscular mycorrhizal fungi in a coastal vegetation on Okinawa island and effect of the isolated fungi on growth of sorghum under salt-treated conditions. Mycorrhiza 18:241–249. Yuan, Z. L., C. C. Dai, X. Li, L. S. Tian, and X. X. Wang. 2007. Extensive host range of an endophytic fungus affects the growth and physiological functions in rice (Oryza sativa L.). Symbiosis 43:21–28. Zhang, Y., Y. Zhang, M. Liu, X. Shi, and Z. Zhao. 2008. Dark septate endophyte (DSE) fungi isolated from metal polluted soils: their taxonomic position, tolerance, and accumulation of heavy metals in vitro. J. Microbiol. 46:624–632.