Heterosmilax japonica Kunth is well recognized for its diuretic effects in China. However, little is known about its endophytic fungi. In this study, microbial communities inhabiting the stems of H. japonica in spring and summer were investigated by light microscopy and cultivation-independent approaches, such as RFLP analysis and sequencing of rDNA ITS library. Molecular phylogenetic analysis showed that a broad spectrum of fungi, including Mycosphaerella, Phomopsis, Aureobasidium, Cladosporium, Glomerella, Botryosphaeria, Guignardia, is able to colonize the plants internally. Particularly, several rDNA sequences determined in this study like YJ4-61 are not specifically affiliated with any currently documented fungal sequences in the public database. Several sequence types, such as YJ4-9 and YJ4–70, are significantly similar to some uncultured environmental samples. Furthermore, our result also showed that the samples collected in spring harbored more abundant endophytic populations than that in summer, implying a seasonal fluctuation for the endophytes in H. japonica.
Endophytes are microorganisms that live asymptomatically within plant tissues . They are presumably ubiquitous in the plant kingdom [2,3]. The colonization and propagation of endophytes may in some ways offer significant benefits to their host plants by producing a plethora of substances that provide protection and survival value to the plants, such as enhancement of stress-, insect- and disease-resistance, productivity improvement, and herbicide activities . These facts indicate that endophytes play an important role in ecological community.
Endophytes have also been recognized as a repository of novel metabolites of pharmaceutical importance . The discovery that an endophytic fungus (Taxomyces andreanae) colonizing Taxus brevifolia also produced the anticancer drug Taxol was a huge surprise . Thus, traditional Chinese herbs may provide us with a profuse potential source of endophytic fungi for finding new therapeutic drugs. Endophytic fungi produce antimicrobial agents that may have applications outside the host plant in which they normally reside, whose production will not use the herbal resources. Heterosmilax japonica Kunth (Family Smilacaceae) is a well known pharmaceutical plant used in Chinese traditional medicine. Dried rhizomes or the whole plant of H. japonica, called as “white-tufuling”, has diuretic, antipyretic, detoxifying, and aphrodisiac effects . It is likely that H. japonica host certain endophytic fungi species producing bioactive secondary metabolites. To our knowledge, endophytic fungi directly obtained from plant tissues have rarely been studied and there has been no comparable investigation of H. japonica.
Until recently, most studies on endophyte have been conducted by cultivation. However, as known from environmental microbiology in general, it can be expected that the actual diversity of endophyte colonizing plants will be significantly higher than that suggested by cultivation based approaches . Therefore, in this study, endophytic fungi associated with H. japonica were characterized using culture-independent method that relies on the rRNA genes (rDNA) obtained from the plant sample. We chose to study the diversity of the internal transcribed spacer (ITS) regions of fungal rDNA, which have been identified as discriminative targets for molecular analysis of fungal communities [9,10]. Their high sequence variability relative to the flanking sequences makes them valuable for genus- and species-level identification.
Meanwhile, the effect of biotic or abiotic factors on endophytic communities has been investigated [11–13]. These intensive works had shown that the variations could be generated by parameters such as different organs of the host, physiopathologic status of host, and seasonal changes (temperature, relative air humidity, water content in the soil, etc.). In order to obtain a better overview of the endophytic fungi populations in H. japonica and the seasonal fluctuation that they may experience, we sampled apparently symptomless stems of H. japonica at the same site in spring and summer of 2004, respectively. By using the methods of molecular biology such as RFLP analysis and sequencing of rDNA ITS library, our analysis showed for the first time the high diversity of endophytic fungi that colonized in the stems of the pharmaceutical plant H. japonica.
2Materials and methods
2.1Plant materials and surface sterilization
Healthy stems of Heterosmilax japonica Kunth (Family Smilacaceae) were collected from South China Institute of Botany, PR China, on 12 April 2004 (the samples named YJ) and 28 June 2004 (the samples named XJ), respectively. The samples were cross-sectioned at various locations with knives to obtain pieces of 3–5 cm. Parallel samples were processed immediately for light microscopy and within 24 h for total DNA extraction following collection.
To amplify fungal rDNA fragments within plant tissues, all organisms attached on the surface of the stems were removed. The pieces were surface sterilized following a procedure modified from Rodrigues . Initially, the pieces were rigorously scrubbed with detergent and tap water and dried with sterile filter paper. They were submerged subsequent in 70% ethanol (v/v) for 1 min to wet the surface, surface sterilized for 1 min in a solution of 15% hydrogen peroxide (v/v), and then were submerged again for 1 min in 70% ethanol. The segments were then dried with sterile filter paper for DNA extraction.
2.2DNA extraction, PCR amplification and cloning
Total genomic DNA was extracted from fresh surface-sterilized stem tissue of H. japonica with a CTAB method  with modifications. Approximately 50 mg fresh stem tissues were ground with mortar and pestle under liquid nitrogen. The frozen powdered tissues were transferred into a 1.5-ml Eppendorf microcentrifuge tube with 1 ml of pre-warmed (65°C) 2 × CTAB extraction buffer (2% w/v CTAB, 100 mM Tris–HCl, 1.4 M NaCl, 20 mM EDTA, 1%β-mercaptoethanol, pH 8.0), and then incubated in a 65°C water bath for 60–90 min with occasional gentle swirling. After centrifugation, the aqueous phase of the mixture containing the total DNA was reextracted with an equal volume phenol:chloroform:isoamyl alcohol (25:24:1). The residual phenol was removed with chloroform:isoamyl alcohol (24:1) twice. DNA in the aqueous phase was precipitated by adding 2 vol ethanol and 0.1 vol 3 M NaAc (pH 5.2), and then incubated at −20°C overnight. The DNA pellet was washed with 70% ethanol twice, and suspended in 15 ml of TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0).
A pair of eukaryote universal primers, LH2 described by Bachellerie and Qu  and Sm73 designed by authors, were used to amplify the 5.8S gene and flanking ITS1 and ITS2 regions from total DNA. Primer LH2, 5′-CGTAGGTGAACCTGCGGAAGGATCA-3′, corresponds to 18 rDNA 3′ terminal region of eukaryotic organisms. Primer Sm73, 5′-TTCGCTCGCCGTTACTAGGGGAATC-3′, corresponds to positions 70–100 bp from the 5′ end of eukaryotic 26S rDNA. Another ITS primer pair ITS1/ITS4  were used to amplify the rDNA ITS fragments from total DNA in our pre-experiment.
The PCR mixtures (20 μl) each contained 2.0 mM MgCl2, 12.5 mM dNTPs, 2 μl 10 × buffer, 5% (V/V) dimethyl sulfoxide and 0.5 U ExTaq DNA polymerase (TaKaRa Biotechnology). PCRs were performed under the following conditions: 30 cycles (denaturation at 94°C for 1 min, annealing at 50°C for 1 min, extension at 72°C for 2 min) a 4-min denaturation at 94°C and followed by a final elongation step of 72°C for 10 min. The PCR products were separated on a 2.0% agarose gel and the band of interest was excised from the gel and purified with the QIA quick Gel Extraction Kits (Qiagen). The purified products were ligated into the pMD 18-T vector (TaKaRa Biotechnology), and then recombinants were transformed into Escherichia coli TG1 competent cells with ampicillin and blue/white screening.
The positive transformants of rDNA libraries were screened by PCR amplification of inserts using the vector primer pair P47/P48. PCR amplification products containing the correct size of insert were digested with 1 U of restriction enzyme Msp I μl−1 for 6–12 h at 37°C. The digested DNA fragments were separated in 2.0% agarose gels and the analyses of RFLP patterns were performed manually. Plasmid DNA from recombination clones was extracted and purified for sequencing .
2.4rDNA sequencing and phylogenetic analysis
Sequencing reactions were performed with an automatic ABI 377 sequencer (Dye-Terminator Cycle Sequencing Ready Reaction FS Kit; PE Applied Biosystem) by using M13/pUC universal sequencing primers P47/P48.
For each sequence, the entire ITS region (ITS1–5.8S–ITS2) was used as query sequences in GenBank and EMBL searches by using the FASTA  and BLAST  programs. The rDNA ITS sequences were aligned using ClustalX version 1.8 . Pre-aligned sequences were checked manually for correct alignment and edited by using the BioEdit 5.0.9 program . Phylogenetic analyses were conducted by using maximum parsimony (MP) and neighbor-joining (NJ) methods in PAUP* versions 4.0 b8 . NJ tree was constructed by using the Kimura two-parameter distances. Heuristic MP analyses were conducted with TBR (tree bisection-reconnection) branch swapping with 100 random addition sequence replicates. All characters were equally weighted and unordered. Alignment gaps were treated as missing data. Support for specific nodes on the MP and NJ trees was estimated by bootstrapping 1000 replications. In addition to the MP and NJ analyses, Bayesian method of phylogenetic inference using a Metropolis-coupled Markov chain Monte Carlo (MCMC) approach was carried out as implemented in the computer program MRBAYES 3.0 . The Bayesian analysis was run for 3,000,000 generations and sampled every 500 generations, resulting in an overall sampling of 6000 trees. To obtain estimates for the a posteriori probabilities, a majority rule consensus tree was computed from those trees that were sampled after the process had reached stationarity (burnin = 2000).
Stems were collected as above and immediately fixed in 4% paraformaldehyde (Sigma, United States) in 0.1 M phosphate buffer (pH 7.2) overnight at 4°C. The fixed specimens were rinsed with the same buffer 3 times and then dehydrated through a series of ethanol (30%, 50%, 70%, 83%, 95% and 100%), embedded in Paraplast-plus?. Paraffin sections 8–10 μm thick were cut serially, mounted on microscope slides, and then stained with 2,3,5-triphenyltetraaolium chloride  and 1% aniline blue  for light microscopy.
2.6Nucleotide sequence accession numbers
The ITS sequences have been deposited in the EMBL nucleotide sequence database under Accession Nos.: AJ877092–AJ877107, AJ877175–AJ877207.
3.1Abundant endophytic hyphae proliferating within H. japonica
For a preliminary screening to determine the location of fungal endophytes, stem tissues of H. japonica were first examined by light microscopy. Transverse sections of stem showed aniline blue-stained intercellular hyphae of endophytic fungi located below the epidermis. The area of stem tissue colonized was highly variable, ranging from 2 to 3 layers of cells by scattered hyphae in some sections of July (Fig. 1D), to 5 to 6 layers of cells by dense hyphae as indicated in the sample of April (Fig. 1A–C).
Remarkably, no blue-stained hyphae appeared outside the epidermis in all the sections we observed, which indicated that the surface contamination had been completely removed in our sterile process.
3.2High diversity of endophytic fungi in H. japonica revealed by rDNA ITS libraries analysis
Initially, using the primer pair ITS1/ITS4, two fragments were amplified from DNA of samples YJ (Fig. 3B). The expected DNA amplicons of 600 bp were eluted and rDNA ITS library were constructed. Fifty positive clones were analyzed by RFLP analysis, and sequencing of the representatives resulted in four different sequence types. An appropriate primer pair should be as broad-spectrum as possible for detecting endophytic fungi within plant tissues. Sequence data from the large ribosomal subunit (LSU) genes of different taxa in the eukaryota and bacteria groups were aligned. Fig. 2 gives a representative selection from these alignments. The primer Sm73 was designed according to the conserved regions to amplify toward the 5′ sense. Its 3′ end is located 34 bp (tested with Smilax bona-nox AF293852) downstream of the 3′ end of the ITS4 primer site. With the eukaryotic universal primers LH2/Sm73, PCR amplifications of the rDNA ITS region successfully resulted in two fragments (Fig. 3A): one is approximately 700 bp, a typical rDNA ITS size for angiosperms ; the other is slightly shorter than the angiosperm band, ca. 600 bp, corresponding a typical rDNA ITS size for fungi [28,29]. The products of both sizes were gel purified, with which three rDNA ITS libraries (YJ5, YJ4 and XJB) were constructed.
The rDNA ITS sequences from the YJ5 library that contain the larger DNA fragments were homogenous in RFLP pattern (Fig. 4A, Lanes 12–16) and then identified as the rDNA ITS of Smilacaceae (data not shown). However, the RFLP patterns of the sequences from the YJ4 and XJB libraries that contain the smaller PCR fragments revealed a high degree of genetic variability (Fig. 4A–D). Interestingly, 14 RFLP types detected in XJB library were completely recovered by 44 RFLP types from the YJ4 library (Table 1), implying more complexity of endophytes in YJ4 library.
Table 1. Comparison of RFLP patterns detected in the two rDNA ITS libraries
YJ4 library (spring)
XJB library (summer)
No. of total clones (% representation)
No. of RFLP patterns
% Sequence similarity to its closest relativesa
No. of sequence types that exhibit <75% similarities to its closest relativea
No. of total clones (% representation)
No. of RFLP patterns
aClosest relatives were determined by BLAST analysis of sequence database.
3.3Molecular identification and phylogenetic analysis of endophyte in H. japonica
At least two clones representing each RFLP type from the YJ4 library were selected for DNA sequencing. A total of 49 rDNA ITS representative sequences were determined after four chimeric sequences were identified and excluded from subsequent analyses. BLAST searches in GenBank showed that all the rDNA ITS sequences from the clones could be grouped within the Fungi domain. Only 12 sequence types had significant identities (>99% similarity) to recorded species in GenBank, such as clone YJ4-1, YJ4-26, and YJ4–46 showed greater 100% similarity to their closest relatives (Table 1). The remaining 37 sequences had similarity with known ITS rDNA sequences, ranging from 47.0% to 98.3%. Initially, a NJ tree, containing all the sequences we found, shown that our samples contained diverse fungi (Fig. 5). Of the 49 sequences, 48 sequences related to Ascomycota and the remaining one belonging to Basidiomycota formed paraphyletic clades. Further phylogenetic analysis was performed based on Maximum parsimony and Bayesian methods. Maximum parsimony and Bayesian analysis generated similar tree topologies, thus only the Bayesian inference tree were shown in this study.
The 15 sequences derived from clade I, together with Acremonium strictum, Dictyochaeta simplex, Phialemonium dimorphosporum, three Glomerella species, four Phomopsis species, and three representative species of the Xylariales, were conducted for phylogenetic analysis (Fig. 6). In this tree, 13 fungal clones, represented by six sequences and accounting for 14.1% of the YJ4 library, were phylogenetically associated with Phomopsis species. Ten fungal clones, represented by six sequence types and accounting for 10.8% of the YJ4 library, clustered with the Glomerella species. YJ4–72 and YJ4-II14 were grouped with Dictyochaeta simplex. YJ4-49 and Acremonium strictum formed a terminal cluster with 100% bootstrap percentages and 0.95 posterior probabilities (PP).
The 12 sequences derived from clade II, together with 15 Mycosphaerellaceae species and seven representative species of different families belonging to the Dothideomycetidae, were conducted for phylogenetic analysis (Fig. 7). Eleven sequence types, representing 21 fungal clones and accounting for 22.8% of the rDNA library, phylogenetically associated with Mycosphaerella species. In this tree, six sequences formed a separate clade with 100% bootstrap percentages and 1.00 PP. They were related to each other with sequence similarity ranging from 74.9% to 99.4%, but the group was not affiliated with any currently documented sequences. Remarkably, YJ4-70, YJ4-73, and YJ4-II12, were not closely related to any known cultivated fungi members, and they were only closely related (84.0–99.6% similarities) to their closest environmental sequences in the GenBank. Sequence type YJ4–56 and an anamorph genera, Cladosporium oxysporum, formed a terminal cluster with 60% bootstrap percentages and 0.97 PP.
Six sequences derived from clade III, representing 14 fungal clones and accounting for 15.2% of the YJ4 library, had less than 65% sequence identity to any previously sequenced clone or organism in GenBank (Fig. 5 and Table 1). The remaining two sequence types YJ4-9 and YJ4-III9, accounting for 4.3% of the rDNA library, were allocated to the cluster of Herpotrichiellaceae (data not shown). YJ4-III9 was closely related (99.7% similarity) to p4–4, an uncultured fungal clone obtained from Livistona chinensis. YJ4-9 was closely related (98.3% similarity) to its 288, a leaf litter ascomycete strain residing in host Picea abies (GenBank description).
The seven sequences derived from clade IV, together with Helminthosporium asterinum, four species of Dothioraceae, five species of Botryosphaeriaceae, and three representative species of different families belonging to the Pleosporales, were conducted for phylogenetic analysis (Fig. 8). In this tree, sequence type YJ4-8 and YJ4-78, representing three clones and accounting for 3.3% of the gene library, were clustered with the Botryosphaeriaceae. YJ4–8 was remotely related (89.8% similarity) to Botryosphaeria dothidea, a cultivated member obtained from specific host Malus sp. (GenBank description). YJ4-78 was closely related (99.1% similarity) to Guignardia mangiferae, a cultivated member isolated from tropical plants. Seven clones, represented by four sequence types and accounting for 7.6% of the rDNA library, were grouped with the Dothioraceae. YJ4-26, YJ4-30, and YJ4-II35, were closely related (99.4–100% similarities) to Aureobasidium pullulans, a cosmopolitan yeast-like fungus. Sequence type YJ4-6, representing 3 clones and accounting for 3.3% in the rDNA library, was related (95.1% similarity) to H. asterinum, a cultivated species of Ascomycota.
Within clade V, YJ4–61 was distantly related (54.4%) to its closest relatives in the GenBank. Its exact phylogenetic position was not clear, although it was distributed in Ascomycota clade. The other five sequence types within clade, representing seven fungal clones and accounting for 7.6% of the YJ4 library, were closely related (89.7–96.8% similarities) to WMS30, a white morphotype strains of endophytic fungi from Pinus tabulaeformis.
YJ4-III4 was the only one sequence belonging to Basidimycota, accounting for 1.1% in rDNA library. Fifteen Erythrobasidium clade genera were obtained from GenBank for the phylogenetic analysis (Fig. 9). In this tree, YJ4-III4 and Rhodotorula lactosa formed a terminal cluster with 50% bootstrap percentages and 0.86 PP.
Culture-independent molecular techniques has been widely used to study bacterial communities in soil ecosystems and other extreme environments [32–35], which has also been proved useful for evaluating endophyties communities of plants . Compared with the traditional culture techniques, the DNA-based methods may have advantage to identify microorganisms that are difficult to culture in vitro. It has been found that cultivation techniques captured only 48% of the bacterial diversity retrieved by cultivation-independent clonal assessment . Moreover, when compared endophytic fungi assemblages within Livistona chinensis, which obtained from traditional  and culture-independent molecular techniques , it reported that some species of basidiomycetous and Herpotrichiellaceae could only been identified with the latter techniques. In our study, two Herpotrichiellaceae species were obtained and identified closely related to the uncultivated species derived from L. chinensis. Noticeably, the use of different primer pairs would influence to some extent the identification of endophytic fungal conmunity. Several fungal ITS primers for PCR, such as ITS1, ITS4, and ITS5 had been designed to amplify fungal rDNA from a wide range of samples . In our pre-experiment, merely four fungal ITS sequences were detected by using primer pair ITS1/ITS4. However, the new primer pair LH2/Sm73 developed in this study for PCR, succeeded in amplifying both fungal and host plant rDNAs fragments. Remarkably, a wide variety of fungi were found in H. japonica stems including Mycosphaerella, Phomopsis, Aureobasidium, Cladosporium, Glomerella, Botryosphaeria, Guignardia. Another precaution concerns the treatment of the host tissue. We preformed the surface sterilization techniques (with ethanol, detergents, and reducing agents), as there could be phylloplane fungi and other organisms on the host surface. To confirm the removal of surface DNA contamination (or dead cells), we collected the sterilized distilled water (3dH2O) in which the surface sterilize tissue was rinsed. With the water as DNA template, PCR result was negative. Combined with the light microscopy results, we considered that the surface contamination had been effectively removed.
Among the fungal community living within H. japonica, Mycosphaerella species was the most abundant and largest assemblages, represented by 12 sequences and accounting for 24.5% of the gene library. Cladosporium is the anamorph of some Mycosphaerella species and is an extremely large and important genus of plant pathogens which has been isolated as endophytic fungi from plant host [30,38]. In addition, Diaporthe and its anamorph Phomopsis species are frequent colonizers in various hosts in different geographic areas, including tropical regions [39–42] and temperate regions [38,43]. A. pullulans, a cosmopolitan yeast-like fungus, has already been cultivated from a number of plants and reported as potential biocontrol agent of pathogens . Glomerella and its anamorph Colletotrichum species (Phyllachoraceae family), pathogens on over 100 species of plants, distribute worldwide and have also frequently been isolated as endophytic fungi [14,30,35,37].
Fluctuation of endophytic assemblages during spring and summer season was detected in this study, demonstrating there may be more species within the spring samples (active growth stages). In detail, RFLP analysis revealed that there was only a small pattern in the summer samples and those patterns were 100% recorded in the spring samples. Clay and Holah  reported the numbers of fungal endophyte species were higher in spring samples compared with fall within tall fescue, the most abundant perennial grass in the eastern United States. One explanation of this situation might be that the endophyte population depends on the physiological status of the host plant, which, in turn, is partly related to the seasonal weather variation, though only two seasonal samples were collected in this experiment.
We are grateful to Dr. Jian Shu-Guang at South China Institute of Botany, Prof. Liao Wen-Bo and Fan Qiang at School of Life Sciences of Zhongshan University for identifying the plant samples, Prof. Ou-Yang Xue-Zhi at Key Laboratory of Gene Engineering of the Ministry of Education for microscopy helps. This work was supported by the National Science Foundation of China (No. 30270009), and by the Fund for Distinguished Young Scholars from the Ministry of Education of China.