Diversity of endophytic fungi from roots of Panax ginseng and their saponin yield capacities

Endophytes of medicinal plants have the capacity to synthesis same or similar active substances with their hosts. To investigate the diversity and capacity to produce saponins of endophytic fungi of Panax ginseng, thirty-eight strains of were isolated. Polymerase chain reaction (PCR) and sequencing were used to identify the isolates, and saponins concentrations in the cultures were measured. Agar diffusion method was used to test antimicrobial activity. High-performance liquid chromatography (HPLC) was used to analyze ginsenosides produced by representative strains. Nectria, Aspergillus, Fusarium, Verticillium, Engyodontium, Plectosphaerella, Penicillium, Cladosporium, and Ascomycete species were isolated. Overall, 18.4% of the isolates belonged to Nectria (Nectria haematococca), 13.2% belonged to Aspergillus, and 10.5% belonged to Penicillium. The highest concentration of triterpenoid saponin was 0.181 mg/ml (Pg27), followed by 0.144 mg/ml (Pg30 and Pg42-1). According to the results of the phylogenetic results, these isolates were species of Fusarium, Aspergillus and Verticillium, respectively. The culture filtrate of Pg30 exhibited its antibacterial activity Staphylococcus aureus. Pg 27 and Pg30 could excrete the substances to inhibit the growth of Rhizoctonia solani. Pg42-1 showed strong inhibition against Klebsiella pneumoniae. From HPLC results, the ginsenoside Rb2 was detected in both Pg27 and Pg30 cultures. The ginsenoside Rc was found in Pg42-1 cultures. In conclusion, thirty-eight endophytic fungal strains were isolated and Pg27 (Fusarium sp.) has a potential application value in saponins production.


Introduction
Ginseng is one of the most famous medicinal plants in the Araliaceae, which occupies an important position in traditional Chinese medicine in China. With the excessive and predatory exploitation, wild ginseng resources become scarce. Cultivated ginseng has gradually become the mainstream of the market. To develop the ginseng farming, deforestation reclaimed to new participants is necessary, because the humus in forest area is essential for ginseng cultivation. However, this deforestation has greatly broken the ecological balance and biodiversity in forest area. How to obtain medicinal ingredients from ginseng without damage to the environment has become a very important issue.
Endophytic fungi, which are fungi that colonize a plant without causing visible disease symptoms (Schulza and Boyle 2005), are common in plants (Lin et al. 2010;De Siqueira et al. 2011;Suto et al.2002), and have been found to be ubiquitous within all examined plants Tadych et al. 2012;Li et al. 2012). In addition, endophytic fungi have been isolated from different plant tissues, including flowers, seeds, roots, stems and leaves (Lupo et al. 2001;Bayman et al. 1997). Previous studies have found that some endophytic fungi have roles within the plant in relation to growth (Doty 2011), enhanced stress resistance (Ownley et al. 2010), degradation of pollutants , and the production of bioactive substances in the host (Guimarães et al. 2008).
In medicinal plants, some endophytic fungi have been found to produce secondary metabolites that have medicinal value. Indeed, since the discovery that the endophytic fungi isolated form Taxus brevifolia, T. celebica, T. mairei, T. chinensis var. mairei, and T. wallachiana produced the anti-cancer drug taxol, many researchers have studied the endophytic fungi of medicinal plants to identify potential sources of novel medicine (Lin et al. 2007;De Siqueira et al. 2011;Kumaran et al. 2010). Saponin is the main medicinal product of Panax ginseng and has multiple therapeutic values, including anti-tumor and anti-aging properties and blood vessels softening. Studying the saponin yield capacity of Panax ginseng endophytes could provide new sources for producing saponins and protect wild ginseng resources indirectly. Antimicrobial activity of endophytes is also one research direction. Endophytes and their metabolites are generally not harmful to their host. Therefore, endophytes which are resistant to pathogens may become the natural sources for pesticides (Yang et al. 2006).
Researches on ginseng endophytes mainly focused on the diversity and the biological activity of metabolites. Xu et al. isolated Paecilomyces sp. from the ginseng and studied its antifungal and antitumor properties. The results showed that the extracts derived from Paecilomyce sp. and ginseng samples contained the same compound falcarinol, an atural pesticide and anti-cancer agent (Xu et al. 2009). Park et al. isolated 38 fungal isolates from three cultivars of Panax ginseng in Korea. They were classified into Phoma radicina, Fusarium oxysporum, Setophoma terrestris and Ascomycota sp. 2-RNK. The most dominant fungal endophyte was P. radicina in 3 cultivars (Park et al. 2012). In the present study, to select the endophytes with the capacity of producing saponins, we investigated the diversity of the endophytic fungi in the roots of Panax ginseng cultivated in the forest of Northeast China. The saponin concentrations of typical strains were measured. The antimicrobial activity of representative strains was tested and ginsenosides produced by typical strains were analyzed.

Sampling and isolation
Panax ginseng (PG) specimens were respectively sampled from Fu-yuan City and Ji-an City (Jinlin, China) and had been grown for 15 years in the forest. The PG samples were immediately put into sterile plastic bags and stored at 4°C. The endophytes were isolated within 48 hours.
Before disinfection, the plant samples were thoroughly washed under running tap water for 10 h. The PG root samples were surface-disinfected with 70% (v/v) ethanol for 1 min, 5% NaOCl 10 min, 70% (v/v) ethanol for 1 min and burning for 30 sec. The samples were subsequently rinsed with sterile water, and the outer tissue was removed with a sterile scalpel. Small pieces (0.5×0.5 cm) of PG were placed in Petri dishes containing malt extract agar (MEA, Difco, USA), Czapeck agar (CZA, Difco, USA), or potato dextrose agar (PDA, Difco, USA), and incubated at 28°C for five days. Following the incubation, single colonies of distinctive morphotypes were separated on the basis of their morphological characteristics and appearance. The colonies were subsequently re-isolated by plating on PDA and incubated at 28°C for 24-48 h to obtain pure cultures. All of the isolates were vacuum freeze-dried and deposited in the College of Life Sciences, Northeast Forestry University.

DNA extraction and PCR amplification of the 28S rRNA gene
The genomic DNA was extracted using the EZNA Fungal DNA Mini Kit (OMEGA, USA) according to the manufacturer' instructions. The 50 μl PCR mixtures contained 15 ng of template DNA, 1× PCR buffer (Mg 2+ free), 0.16 mM of each dNTP, 1.5 mM MgCl 2 , 0.45 μM of each primer, and 1 U of Takara rTaq DNA polymerase (Takara, Japan). The primers for the amplification of the D1/D2 region of the fungal 28S rRNA gene were NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3') (Redecke 2000). The thermocycler program consisted of initial an DNA denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 52°C for 45 s, and elongation at 72°C for 1 min 30 s, and ending with a final elongation step at 72°C for 6 min (Yang et al. 2007).
The PCR amplification products were separated by electrophoresis through 1% (W/V) agarose gels and stained with ethidium bromide for visual examination. The PCR products were purified using the Agarose Gel DNA Extraction Kit (Takara, Japan) and sequenced at Sangon Bioteck (Shanghai, China).

Phylogenetic analysis and nucleotide sequence accession numbers
The sequences generated in this study were compared with those in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and the sequences with a similarity ≥99% to the partial 28S rDNA regions (a. 600 bp) were considered to belong to identical genera. A neighbor-joining tree (Thompson et al. 1997) was constructed using MEGA 5.0 software (Tamura et al. 2011). The number of bootstrap replications was 1000. The sequences were deposited in GenBank under the accession numbers shown in Table 1.

Determination of triterpenoid saponins
Each isolate was cultured in 100 ml PDA liquid medium (250 ml flask), and stirred at 150rpm at 28°C for two weeks. After ultrasonication, the supernatant was separated from the cell debris by centrifugation at 4,000 × g for 20 min. A 20 ml aliquot of the supernatant was poured into a 50 ml centrifuge tube (Corning, USA), and 20 ml ethyl acetate was added into the same tube. After mixing, ultrasonication and incubation for 5 min, 5 ml of the supernatant was evaporated to dryness under a vacuum at 50°C. The residue was dissolved in 2 ml methanol. The methanol solutions were centrifuged at 4,000 × g for 10 min, and the supernatants were used for the subsequent analysis of the total saponins and ginsenoside. Cladosporium cladosporioides (AY213695) 100 Passalora fulva (AB100653) The measurement of the total extracted saponins was based on a color reaction of the acid-hydrolysis products of the saponins (i.e. sapogenins) with vanillin. In total, 5 ml of the supernatant was added to a test tube and evaporated at 60°C in a water bath. The residue was dissolved in 0.2 ml 5% vanillin, mixed with 0.8 ml perchloric acid, incubated at 60°C in a water bath for 15 min and quickly cooled in ice water. The concentration of saponins (mg/ml) in the reaction sample was detected using a spectrophotometer at 560 nm against a calibration curve established with an oleanolic acid standard (National Institutes for Food and Drug Control, Bei-jing, China) (Liu et al. 2011).

Antimicrobial activity of the representative strains
The 14-day culture filtrates were assessed for antimicrobial activity by the agar diffusion method (Hormazabal and Piontelli 2009) against the test microorganism strains showed as Table 3. Three 6-mm wells were made in each disk. Culture filtrates (0.2 ml) was added in each well. Except for Fusarium sporotrichioides (isolated in our lab), the other strains were purchased from Agricultural Culture Collection of China (ACCC). As a reference, the Streptomycin Sulfate (5 mg/well), the Amoxicillin (5 mg/ well) and the Itraconazole Hydrochloride (4.4 mg/well) were used as antibacterial standards. The activity of the extracts was estimated from growth inhibition (in mm).

Similarity of the sequences
Thirty-eight strains were identified on the basis of their morphological characteristics. The sequences were compared with those in the GenBank database, and the results are shown in Table 1.

Analysis of triterpenoid saponins
The concentration of triterpenoid saponins of typical isolates are showed in Table 2 The highest concentration of saponins was 0.181 mg/ml in Pg27, which was significantly higher than Pg30 and Pg42-1 (0.144 mg/ml) (P<0.05). According to the results of phylogenetic results, Pg27 was identified as a Fusarium sp., Pg30 was identified as an Aspergillus sp., and Pg42-1 was identified as a Verticillium sp. .The saponin concentrations among the strains of the same genus were different significantly (P<0.05), for example Pg14 (0.023 mg/ml), Pg34 (0.133 mg/ml) and Pg12-1 (0.042 mg/ml); these isolates were also identified as Fusarium spp.

Antimicrobial activity
To test the Pg27, Pg30 and Pg42-1 potential use, the antimicrobial activity was analyzed. From Table 3, the culture filtrate of Pg30 exhibited its antibacterial activity against Gram-positive bacteria Staphylococcus aureus ACCC10499. Pg 27 and Pg30 could excrete the substances to inhibit the growth of Rhizoctonia solani ACCC36233, which was a pathogenic fungi of Panax notoginseng. The culture filtrate of Pg42-1 showed strong inhibition against Klebsiella pneumoniae ACCC10498. This result indicated that Pg42-1 might be a potential medical source.

Ginsenosides analyses
Accoridng the result of total saponins, Pg27, Pg30 and Pg42-1 produced higher concentrations of saponins. To further analyze the composition of saponins, the standards of eight ginsenosides were injected into HPLC. The spectrums showed as Figure 2. Rb2 was detected in both Pg27 and Pg30 cultures. The concentration of Pg30 was especially high. Rc was found in Pg42-1 cultures.

Discussion
Thirty-eight endophytic fungi were isolated, and they were classified into nine genera according to the morphological types and 28S rDNA sequencing results. Three isolates (Pg45, Pg47, and Pg60) were not identified because of lack of comparative sequences: their sequences were significantly similar to unknown fungal sequences in the GenBank database. Nectria, Aspergillus and Penicillium were the predominant genera. The host materials were all healthy in this study. Park et al. (2012) reported that Phoma radicina, Fusarium oxysporum, Setophoma terrestris and Ascomycota were the predominant endophytic fungi in Korean Ginseng, and Xing et al. (2010) reported that Cladosporium sp. was the dominant species in the root of Panax quinquefolium. These previous results were different from this present study, indicating the specificity of the endophytic fungi from different areas and plants.
Nectria was reported as endophytic fungi in European beech (Danti et al. 2002) and red alder (Dorworth et al. 1996) as the endophytic fungi. Nectria has also been associated with the canker diseases of tree species. However, the Nectria isolates in this study didn't show their pathogenicity to the host plant. Therefore, the pathogenicity of Nectria had their specificity. Aspergillus species could be sources of new medicines. For example, Kusari and Zhao had reported that an Aspergillus sp. was a source of anticancer medicines (Kusari et al. 2009;Zhao et al. 2009). Therefore, further research on the Aspergillus sp. isolated in this study may be interesting. Penicillium is the source of penicillin, and recent results showed that endophytic Penicillium sp. had the capacity to secrete anti-tumor substances (Aly et al. 2010) or hypocrellin (Meng et al. 2011). We propose that the endophytic fungi isolated in this study from a medical plant are potential sources of medicines.
The growth-promotion factors and metabolites produced by endophytic fungi have been widely applied in medicine and agriculture. The most famous substance is taxol, a mitotic inhibitor used in cancer chemotherapy, which was originally produced by the yew tree and can be produced by endophytic fungi of yew trees (Rivera-Orduña et al. 2011). Similarly, a filterd liquid culture of endophytic fungi was analyzed to identify endophytes that could produce triterpenoid saponins. Overall, 19 of the isolated fungi showed a color reaction, which indicated that they could produce triterpenoid saponins.  Culture filtrate (0.2 ml) was added in each well (6 mm); (−) no inhibiton, (+) inhibition zone, +++ width of growth inhibition zone > 10 mm, ++ 5-10 mm, + 1-5 mm; Figure 2 HPLC spectrums of culture filtrates from the representative strains. 1, Rg1; 2, Re; 3, Rf; 4, Rb1; 5, Rc; 6, Rb2; 7, Rb3; 8, Rd.