Composition of the endophytic filamentous fungi isolated from the tea plant Camellia sinensis
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It has been found by ribosomal DNA analysis that the endophytic filamentous fungi isolated from the tea plant Camellia sinensis (Theaceae) are composed of six groups; one Fusarium sp., one Penicillium sp., two Schizophyllum sp., and two Diaporthe sp..
KeywordsEndophyte Filamentous fungi Camellia sinensis rDNA Fusarium sp. Penicillium sp. Schizophyllum sp. Diaporthe sp.
It is known that there are several symbiotic relationships in nature. We are, among them, paying attention to that between plants and microorganisms, which are divided into endophytes  living inside of a plant and epiphytes living at surface of a plant. To know the composition of endophytes is useful since they create the distinctiveness of each plant.
We previously reported that 35 filamentous endophytic fungi were obtained from the young stems of Camellia sinensis (L.) O.K. collected in West Java, Indonesia [2,3]. In this paper, we deal with classification in a genus level of the endophytic filamentous fungi by ribosomal DNA analysis.
Materials and methods
Each endophytic fungus was ground down in lysis buffer (400 μl, 50 mM Tris–HCl, 50 mM EDTA-2Na, 3% SDS, 1% mercaptoethanol; pH 7.2) containing alumina powder. The suspended liquid was heated at 60°C for 1 h. After cooling, chloroform (200 μl) and TE-saturated phenol (200 μl) were added to the mixture and then submitted to centrifugation at 15,000 rpm for 5 min. The upper phase was collected and treated with 3 M aqueous sodium acetate (10 μl) and isopropanol (200 μl). The whole mixture was submitted to centrifugation at 15,000 rpm for 15 min. The precipitates were collected and treated with 70% EtOH. The whole mixture was again submitted to centrifugation, and the precipitates were collected and dried under reduced pressure, then treated with RNase (0.5 μg, Sigma, MO, USA) in TE buffer solution (10 μl, TE buffer: 10 mM Tris–HCl, 1 mM EDTA 2Na; pH 8.0) at room temperature for 10 min. Finally, the removal of RNA was confirmed by electrophoresis.
PCR amplification of 18S rDNA and ITS1-5.8S rDNA-ITS2 regions was performed in a reaction mixture of 50 mM Tris-HCl buffer (pH 8.0), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 1.0% Triton X-100, 2.4 mM MgCl2, 100 mM of each dNTP (Takara), 0.15 μM of primers described in our previous paper  with about 1 ng of genomic DNA and 1 unit of the Taq DNA Polymerase (Promega, UK). The reaction mixture was placed in a thermal cycliing process programmed for 0.5 min at 95°C, followed by 30 cycles of 1 min at 95°C, 0.5 min at 55°C, 2 min at 72°C, and a final extension at 72°C for 10 min. The PCR product was checked by electrophoresis in 1% agarose gel with ethidium bromide 0.5 mg/ml. The presence of a single bright band in each lane confirmed successful amplification. The PCR product was suspended in 95% EtOH and placed at –80°C for 27 min, and then the whole was submitted to centrifugation at 15,000 rpm for 15 min. After removal of the supernatant, the residue was resuspended in 250 μl of 70% EtOH and centrifuged at 15,000 rpm for 5 min, and the precipitates were collected and dried under reduced pressure to obtain a DNA template.
DNA sequencing and phylogenetic analyses
Amplification was performed in a reaction mixture of 1 unit of BigDye Terminator Cycle Sequencing Ready Reaction Kit V2.0 (ABI), 0.3 μM of primers described in our previous paper  and 15 ng of the DNA template. The reaction mixture was placed in a thermal cycling process programmed for 1 min at 96°C, followed by 25 cycles of 0.5 min at 96°C, 0.25 min at 55°C, 4 min at 60°C, and a final extension at 60°C for 10 min. One microliter of 6 M aqueous ammonium acetate and 30 μl of 99.5% EtOH were added to the reaction mixture and the whole was shaken briefly and then allowed to stand in an ice-bath for 15 min. The mixture was centrifuged at 14,000 rpm for 15 min. The supernatant was removed and the residue was suspended in 200 μl of 70% EtOH and centrifuged at 14,000 rpm for 10 min. The resulting precipitates were collected and dried under reduced pressure to afford the cloned DNA fragments. Those fragments were analyzed using a DNA sequencer. Sequence alignment, a similarity matrix, and a neighbor-joining phylogenetic tree were created on a Macintosh personal computer using the DNASTAR program (DNASTAR Inc, Madison, WI, USA) and the Clustal V software package [5,6].
Results and discussion
The endophytic filamentous fungi isolated from the tea plant C. sinensis were classified into six species (fungi A–F) by RAPD analysis (Fig. 1).
As to fungus A, the generic name was supposed to be Gibberella or Fusarium, since the base sequence of the 18S rDNA region showed 99.7% and 99.6% similarities with those of Gibberella fujikuroi (DDBJ/EMBL/GenBank, accession No. AR168094) and Fusarium oxysporum (AB110910). The genus Fusarium is an anamorph stage of the genus Gibberella [7,8].
From these findings, it has been clarified that the endophytic fungus A (registered as AB245442) belongs to the genus Fusarium.
The RAPD fragment patterns of funguses C and D were mutually different as shown in Fig. 1; however, the base sequences for the 18S rDNA, ITS1, 5.8S rDNA and ITS2 regions were completely identical. The length was 2145, 153, 152 and 243 bp for the 18S rDNA, ITS1, 5.8S rDNA and ITS2 regions respectively.
These findings indicated that the endophytic fungi C (registered as AB245444) and D (registered as AB245445) might belong to the genus Schizophyllum and be new species.
With regard to fungi E (registered as AB245446) and F (registered as AB245447), we already reported these to be quite closely-related species to Diaporthe phaseolorum var. sojae (IFO6709) and D. phaseolorum strain sw-93-13 (AF001018) respectively [3, 9]
As a conclusion, it has been clarified that the endophytic filamentous fungi isolated from the tea plant C. sinensis consist of one Fusarium sp., one Penicillium sp., two Schizopyllum sp. and two Diaporthe sp.
We thank Dr. Partomuan Simanjuntak for collection of the plant material, Mrs. Admarita Agusta for technical assistance, and the National Collection of Industrial, Food and Marine Bacteria (NCIMB) Japan for morphological observation of the endophytic filamentous fungus B Penicillium sp. This work was supported by the “High-Tech Research Center” Project for Private Universities: matching fund subsidy from the Minister of Education, Culture, Sports, Science and Technology, 2004–2008.
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