Endophytic Fungi of Salt-Tolerant Plants: Diversity and Ability to Promote Plant Growth

Suaeda australis, Phragmites australis, Suaeda maritima, Suaeda glauca Bunge, and Limonium tetragonum in the Seocheon salt marsh on the west coast of the Korean Penincula were sampled in order to identify the endophytes inhabiting the roots. A total of 128 endophytic fungal isolates belonging to 31 different genera were identified using the fungal internal transcribed spacer (ITS) regions and the 5.8S ribosomal RNA gene. Fusarium, Paraconiothyrium and Alternaria were the most commonly isolated genera in the plant root samples. Various diversity indicators were used to assess the diversity of the isolated fungi. Pure cultures containing each of the 128 endophytic fungi, respectively, were tested for the plant growth-promoting abilities of the fungus on Waito-C rice germinals. The culture filtrate of the isolate Lt-1-3-3 significantly increased the growth of shoots compared to the shoots treated with the control. Lt-1-3-3 culture filtrate was analyzed and showed the presence of gibberellins (GA1 2.487 ng/ml, GA3 2.592 ng/ml, GA9 3.998, and GA24 6.191 ng/ml). The culture filtrate from the Lt-1-3-3 fungal isolate produced greater amounts of GA9 and GA24 than the wild-type Gibberella fujikuroi, a fungus known to produce large amounts of gibberellins. By the molecular analysis, fungal isolate Lt-1-3-3 was identified as Gibberella intermedia, with 100% similarity.


Collection of Plant Samples
Five plant species were collected from the Seocheon salt marsh, located in the middle of the west coast of Korea. Within each species, two to four samples were collected at different locations at each site. In Table 1, lists of the scientific name, codes of the plants, and global positioning system information were organized. The samples were placed into zipped bags during collection, transported to the laboratory, and stored at 4°C until further processing for isolation of endophytes. All sample preparation was done within 36 h of collection.

Surface Sterilization and Isolation of Endophytic Fungi from the Roots
Samples were washed with distilled water to remove the soil particles and residues from the plant roots. Washed samples were sterilized with Tween 80 detergent solution for 5 min and surface of samples was sterilized by perchloric acid (1%) solution. The samples were then washed twice with sterile distilled water. The prepared root samples were then cut into 2.0-2.5 cm pieces by using scissors. The pieces were cultured on Hagem minimal medium (glucose 5.0 g/l, malt extract 5.0 g/l, NH 4 Cl 0.5 g/l, KH 2 PO 4 0.5 g/l, MgSO 4°7 H 2 O 0.5 g/l, and Fe-EDTA 11.3 mg/l) containing 80 ppm streptomycin [13]. The plates were incubated at 25°C until single colonies of fungi were emerged. As fungi emerged, they were transferred to potato dextrose agar (PDA; potato extract 4 g/l and dextrose 20 g/l) plates for pure cultures.

DNA Extraction, PCR Amplification, and Identification of Fungal Strains
The fungal isolates were inoculated into Erlenmeyer flasks containing 50 ml of the potato dextrose broth medium and were incubated at 25°C ± 2°C for 7 days on a rotary shaker at 120 rpm. Lyophilized fungal isolates were collected using vacuum. Fungal genomic DNA extracted using the DNeasy Plant Mini Kit (Qiagen, USA) was identified by amplification and sequencing of the internal transcribed spacer (ITS) region. Among the various ITS, ITS primers named ITS-1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS-4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) were used in this study. The reaction cycles comprised an initial denaturation step (95°C for 2 min); 30 cycles of denaturation (95°C for 30 sec), annealing (55°C for 1 min), and extension (72°C for 1 min); and a final extension (72°C for 7 min). Detection of PCR production was processed using agarose gel electrophoresis with ethidium bromide staining. The products were directly purified by the QIAquick PCR Purification Kit (Qiagen). Then, purified PCR products were sequenced by the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems, USA) and an ABI 310 Genetic Analyzer (Perkin Elmer, USA). The obtained sequences were identified using the National Center for Biotechnology Information GenBank database BLASTN tool.

Statistical Analysis
Diversity of the fungal endophytes isolated from the plant samples was assessed. Diversity at the genus level was assessed using the Shannon diversity index (H′), Fisher's alpha index (α), and Simpson's index of diversity (1 -D). Richness was evaluated using Menhinick's richness index (D mn ) and Margalef 's index (D mg ) [14][15][16].

Effect of Fungal Filtrates on Plant Growth Promotion on Waito-C Rice Seedlings
To test the ability of the fungal isolates to promote plant growth, Waito-C rice germinals were exposed to culture filtrates from the fungal isolates. The fungal isolates were cultured in Czapek Dox broth medium on a shaking incubator for 7 days at 25°C and 180 rpm and were harvested using 20~25 μm/15 cm Whatman filter papers (Brentford, UK). The harvested fungal culture filtrates were immediately frozen at −70°C and then lyophilized for concentration. The lyophilized culture filtrates were concentrated 50 times by mixing with 1 ml of distilled water. In order to minimize the activity of gibberellins in the seed coat, Waito-C rice grains were treated with uniconazole for 24 h. The treated seeds were washed and soaked in distilled water until radical emergence. The young seedlings were then transplanted to glass tubes with 0.6% water agar medium and the seedlings were grown in a growth chamber under conditions of 12 h at 25°C and 12 h at 30°C for a total of 5 days. After rice seedlings reached the two-leaf stage, 10 microliters of concentrated lyophilized culture filtrate from each fungal isolate was applied to the apical meristem. In order to compare with G. fujikuroi, whose performance was previously confirmed, the same amount with same concentrate as in other experimental groups was inoculated into the same site and the experiment was conducted. In this study, plant and shoot lengths of rice became apparent 1 week after application compared to rice seedlings treated with wild-type G. fujikuroi, which served as positive controls.

Extraction and Quantification of Gibberellins from Fungal Culture Filtrates
Fungal isolates were incubated for 7 days in Czapek Dox broth medium with 1% (w/v) glucose and peptone. Gibberellins were extracted from culture filtrates and were analyzed using reverse-phase C 18 high-performance

Molecular Identification of Endophytic Fungi
The nucleotide sequences of endophytic fungal isolates from the Seocheon salt marsh were grouped by host plant species and registered in GenBank with the following accession numbers: S. australis (KP017930-KP017952), P. australis (KP017953-KP017967), S. maritima (KP017968-KP017981), S. glauca Bunge (KP017982-KP018018), and L. tetragonum (KP018019-KP018057). In table 1, the accession numbers of fungal isolates were listed. Total 128 fungal isolates, representing 31 genera and 37 species, were isolated from plants on the west coast of Korea.
Endophytic fungi were also analyzed at the class and genus levels ( Fig. 1) and Dothideomycetes was found to account for the highest percentage of isolates at the class level. Except for the isolates from the plant L. tetragonum, Dothideomycetes accounted for more than a half of all of the fungal isolates in every plant sample. Fusarium was the most prevalent (18%) among all of the fungal strains, followed by Paraconiothyrium (11.7%), and Alternaria (10.9%). The remaining genera of the fungal isolates constituted only 0.7-7% of the strains. A study by Nalini et al. (2014) showed that fungi from the Fusarium genus are the most salient fungi discovered in stem and root fragments of medicinal plants that are native to the western coast of the Indian peninsula [17]. Fungal species abundant in the host, including fungi from the genera Fusarium, Alternaria, and Penicillium can associate with plants [18,19].

Diversity of Endophytic Fungi Isolated from Halophytes
In Table 2, more details of fungal components were organized. Depending on the plants, fungal isolates from S. australis were classified as 12 genera and 11 species. Isolates from P. australis were classified as 11 genera and 8 species. Isolates from S. maritima were classified as 13 genera and 8 species. Isolates from S. glauca Bunge were classified as 16 genera and 16 species. Fungal isolates from L. tetragonum were classified as 13 genera and 17 species.
The richness and diversity of the fungal isolates were analyzed at the genus level and more detail of fungal  information was arranged on the Table 3. For general abundance calculated using Menhinick and Margalef indices, S. maritima scored the highest at 3.47 and 4.55, respectively. In terms of genetic diversity, which was calculated using Shannon's, Fisher's α, and Simpson's indices, S. maritima had the highest scores of 2.54, 88.78, and 0.99, respectively. The diversity scores vary greatly because the diversity indices are based on the proportional abundance of genera in a sample, and are more sensitive toward genus evenness [20]. The S. maritima specimen had the highest diversity indices, indicating the most diverse fungal community isolated from this plant.

Bioassay for Plant Growth-Promoting Effects of Fungal Culture Filtrates on Waito-C Rice Seedlings
The culture filtrate from the Lt-1-3-3 fungal isolate was superior to that of G. fujikuroi in promoting plant growth, as the average shoot length of plants exposed to Lt-1-3-3 was 9.3 cm and the plant length was 20.1 cm in Waito-C seedlings (Fig. 2). These results are consistent with those of another study in which the endophytic fungus P. citrinum, isolated from the roots of Ixeris repens, was shown to promote the growth of Waito-C rice and Atriplex gemelinii seedlings [21].
To confirm the presence of gibberellins, microbial culture filtrates were screened to identify biologically active molecules. Waito-C rice seedlings were used for the detection of plant growth-promoting hormones in the culture filtrates of the fungal endophytes isolated from the roots of halophytes. Waito-C rice is a known dwarf rice cultivar with reduced biosynthesis of gibberellins. Waito-C seeds were treated with uniconazole as a gibberellin biosynthesis inhibitor. The Lt-1-3-3 fungal isolate, which has plant growth-promoting capacity (Fig. 2), was analyzed using Waito-C rice seedlings.
Including gibberellins, various secondary metabolites are known to be produced from fungal endophytes. The GC/MS with the SIM method makes it possible to analyze the complex mixtures and detect the compounds of different classes. Therefore, we used this technique for the cultural analysis of the Lt-1-3-3 fungal isolate. GC/MS SIM is helpful for the analysis of a number of compounds and is often used in plant experimentation. Due to its reliability, GC/MS SIM has been previously used in quantitative analyses of various plant hormones [22,23].
In summary, 128 fungal isolates are isolated from 5 species of coastal plants. These fungal isolates were classified into 2 phyla, 6 classes, 12 orders, 17 families, and 31 genera. The dominant genus was Fusarium (class Sordariomycetes), followed by Paraconiothyrium and Alternaria. Based on the diversity analysis, the most diverse fungi were the endophytic fungi isolated from S. maritima. Culture filtrates from the 128 fungal isolates were assessed on Waito-C rice seedlings and the filtrate from fungal isolate Lt-1-3-3 exhibited excellent plant growthpromoting capacity. This isolate was confirmed to produce gibberellins GA 1 , GA 3 , GA 9 , and GA 24 . Using sequence homology, the Lt-1-3-3 isolate was identified as G. intermedia. This study provides basic data on the microbial resources present in plants from coastal salt marshes.