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Diversity and Plant Growth Promoting Capacity of Endophytic Fungi Associated with Halophytic Plants from the West Coast of Korea

Five halophytic plant species, Suaeda maritima, Limonium tetragonum, Suaeda australis, Phragmites australis, and Suaeda glauca Bunge, which are native to the Muan salt marsh of South Korea, were examined for fungal endophytes by sequencing the internal transcribed spacer (ITS) region containing ITS1, 5.8S rRNA, and ITS2. In total, 160 endophytic fungal strains were isolated and identified from the roots of the 5 plant species. Taxonomically, all 160 strains belonged to the phyla Ascomycota, Basidiomycota, and Zygomycota. The most dominant genus was Fusarium, followed by the genera Penicillium and Alternaria. Subsequently, using 5 statistical methods, the diversity indices of the endophytes were determined at genus level. Among these halophytic plants, P. australis was found to host the greatest diversity of endophytic fungi. Culture filtrates of endophytic fungi were treated to Waito-C rice seedlings for plant growth-promoting effects. The fungal strain Su-3-4-3 isolated from S. glauca Bunge provide the maximum plant length (20.1 cm) in comparison with wild-type Gibberella fujikuroi (19.6 cm). Consequently, chromatographic analysis of the culture filtrate of Su-3-4-3 showed the presence of physiologically active gibberellins, GA1 (0.465 ng/mL), GA3 (1.808 ng/mL) along with other physiologically inactive GA9 (0.054 ng/mL) and GA24 (0.044 ng/mL). The fungal isolate Su-3-4-3 was identified as Talaromyces pinophilus.

Marshes are transitional areas between terrestrial and aquatic ecosystems and are dominated by various living species that provide numerous ecological services such as coastal protection, carbon sequestration, and buffering of coastal waters from terrestrial pollutants, which helps to improve water quality. Coastal salt marshes also reduce storm damage by absorbing high wind and wave energy [1]. Salt marshes contain highly diverse hydrophytes, salttolerant plants, and microorganisms [2]. Soil microbes are directly connected to the productivity and diversity of plants [3]. Symbiosis between plants and microbes is important for the settlement of coastal plants.
Endophytes are microorganisms (fungi, actinomycetes, and other bacteria) that live within host plant tissues without causing any detectable symptoms of disease to the host. Endophytic microorganisms have been isolated from nearly all plant families, including species growing in many different climatic regions. Fungal endophytes live in symbiotic association with all plants in natural ecosystems, play an important role in the resistance of plants to various diseases and abiotic and biotic stresses, and also promote plant growth [4,5]. Such symbiotic fungal endophytes produce a number of important plant hormones including gibberellins (GAs), indole acetic acid, and abscisic acid [6,7]. The purpose of the present study was to investigate the distribution of fungal endophytes in the roots of halophytic plants and analyze their diversity. Additionally, isolated strains were screened on Waito-C rice seedlings to investigate 374 Khalmuratova et al. their plant growth promoting activity. The fungal culture filtrates were subjected to chromatographic techniques to isolate and detect secondary metabolites.

MATERIALS AND METHODS
Collection of plant materials. For the isolation of fungal endophytes, plant samples were collected from a salt marsh located in Muan County in South Korea. Healthy and fresh roots of the plants Suaeda maritima, Limonium tetragonum, Suaeda australis, Phragmites australis, and Suaeda glauca Bunge were collected in separate sterile plastic bags, labeled, transported to the laboratory, and stored at 4 o C until processed. The local sites, scientific names, and codes of the 5 plant species from which samples were taken are listed in Table 1.
Isolation of endophytic fungi from roots. Root samples of the halophytes were washed with tap water to remove sand particles and treated with Tween 80 solution (200 µL in 100 mL distilled water) for 10 min. Samples were surface sterilized twice with 1% (w/v) perchloric acid solution for 10 min, followed by washing with distilled water. The sterilized roots were cut into 3~4 cm pieces, cultured on Hagem minimal media containing streptomycin, and incubated at 25 o C. After the emergence of fungi from inside the root pieces, the fungi were then transferred onto potato dextrose agar. The isolated pure cultures of root fungi were stored on potato dextrose agar plates and slants [8,9]. DNA extraction, PCR, and identification. The fungal strains were subcultured and incubated in potato dextrose broth for 6~8 days. For DNA extraction, mycelia of fungi were transferred into 100 mL Erlenmeyer's flasks containing 50 mL potato dextrose broth medium in a shaking incubator for 7~9 days at 28 ± 2 o C and 110 rpm. The lyophilized samples were used for identification. Fungal genomic DNA was isolated using a DNeasy Plant Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer's instructions. PCR was performed using the primers ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT  TAT TGA TAT GC-3 Statistical analysis of fungi. The richness and diversity of fungi were analyzed at the genus level in the plant samples. Fungal genus diversity was evaluated using the Simpson's, Fisher's alpha (α), and Shannon (H') indices of diversity [10][11][12]. Menhinick's (Dmn) and Margalef 's (Dmg) indices were used to determine the richness of each genus among the fungal community [13,14]. The formulas used for calculating diversity indices are listed in Table 2.
Bioassay on Waito-C rice seedlings. The filtered culture media of the isolated fungal strains were screened on Waito-C rice seedlings for their plant growth promoting activity. The fungal isolates were grown on a shaking incubator for 7 days at 25 o C and 180 rpm on Czapek broth medium. Forty-five milliliters of culture fluid was harvested pellet and the supernatant were stored at −70 o C and then lyophilized. The lyophilized supernatants were mixed with 1 mL of autoclaved distilled water. The Waito-C rice seeds were surface sterilized in spotac solution for 1 day and treated with growth inhibitor uniconazol (20 ppm). After treatment Waito-C rice seeds were washed clearly and soaked in autoclaved distilled water until sprouts emerged. These Waito-C rice seedlings were transplanted in glass tubes containing 0.6% water-agar medium and grown in a growth chamber. When Waito-C rice seedlings had reached the two leaf stage, the meristems were treated with 10 µL of supernatant solution of each fungal culture filtrates. One week after treatment, the shoot and plant length was observed and compared with culture filtrate of Gibberella fujikuroi, lyophilized Czapek broth medium, and distilled water. Culture filtrates of G. fujikuroi, Czapek broth medium and distilled water were applied as controls to determine the shoot and plant length of Waito-C rice seedlings.
Quantification of endogenous GAs. The culture filtrates of fungal isolate was analyzed for the presence of GAs by gas chromatography/mass spectrometry (GC/MS). For these experiments, fungal strains were cultured in 250 mL Czapek broth medium for 7 days at 25 o C in a shaking incubator at 180 rpm. The extracted GAs was analyzed with reversephase C 18 high-performance liquid chromatography (HPLC). The fractions were collected and were prepared for GC/MS with a selected ion monitoring (SIM). After the GC/MS data were analyzed, the three major ions of the supplemented [ 2 H 2 ] GAs internal standards and the fungal GAs were monitored. The retention time was determined using hydrocarbon standards to calculate the Kovats Retention Index value, while the GAs quantification was based on peak area rations of nondeuterated (extracted) GAs to deuterated GAs.

RESULTS AND DISCUSSION
Endophyte identification. The endophytic fungal strains were identified by obtaining the nucleotide sequences of Table 3. Identification of endophytic fungal isolates from the roots of coastal plants

Fungal isolates
Closest relative based on sequence homology Similarity (%) Accession No.
Previously, molecular methods have been successfully used for the identification of the strains comprising endophytic fungal communities [15,16]. In this study, we followed a similar molecular strategy to those previously reported in order to identify these endophytic fungi by means of sequencing internal transcribed spacer rRNA genes and employing a phylogenetic classification system.
Previous studies have reported that these endophytes could play a role in plant development. Fusarium oxysporum, which was isolated from all Suaeda species, reportedly produces GAs and indole acetic acids that stimulate plant growth and development and may reduce the hazardous effect of salinity on the host plant [17]. GAs are known to influence stem elongation, seed germination, pollen maturation, leaf expansion, and the induction of flowering [18], while indole acetic acid modulates cell division and enlargement, tissue differentiation, and responses to gravity and light [19]. Endophytic fungi of the species Alternaria alternata were isolated from all plant samples, and were previously isolated from the leaves of Solanum nigrum, where they were shown to produce indole acetic acid [20]. Members of the genus Penicillium were represented in all of the studied plant samples. Previous studies have revealed that some species of Penicillium can promote plant growth by several different mechanisms, such as the production of plant growth promoting secondary metabolites (auxin and GAs), antagonism to plant pathogens, and solubilization of    minerals [21][22][23][24][25].
Diversity of endophytic fungi at the genus level in the sampled plant species. The identified endophytic fungal strains were characterized into 11 genera and 21 species from S. maritima, 13 genera and 10 species from L. tetragonum, 12 genera and 13 species from S. australis, 14 genera and 11 species from P. australis, and 12 genera and 18 species from S. glauca Bunge (Table 4). Generic richness and diversity were calculated based on counting of fungal genera by plant samples ( Table 5). The results showed that P. australis had the highest score in Margalef 's (4.15) and Menhinick's (2.92) indices of richness and in Fisher's α (15.18), Shannon's (1.95) and Simpson's (0.94) indices of diversity. The Shannon diversity index (H') ranged from 1.24~1.95 (this index is usually between 1.5~3.5; 3.5 representing the highest diversity and 1.5 the lowest). Based on this result, the Shannon index is less sensitive to evenness, compared with other diversity indices [12]. The plant P. australis had the highest diversity indices, showing that the endophytic fungal community isolated from this plant had the most diversity among those from our plant specimens. P. australis is often found to form homogenous belts in temperate zone freshwater lakes, and it is ecologically important because it filters pollutants, stabilizes shores, and houses rich wildlife. P. australis is adapted to its aquatic environment, most importantly, in its ability to form aerenchyma to supply the underground plant parts with oxygen, allowing the plant to survive in anoxic and waterlogged sediments [26,27]. Thus, this creation of an oxygen rich environment may facilitate the establishment of an especially rich and diverse endophytic community in its roots. Environmental conditions may also play an important role in the assemblages and diversity of endophytic fungi.
Bioassay of culture filtrates for plant growth promotion Waito-C rice seedlings. The bioassay on Waito-C rice seedlings was carried out to check plant growth promotion capacity of fungal culture filtrates. All fungi were checked, of which Su-3-4-3 fungal strain indicated 20.1 cm of plant length and 9.2 cm of shoot length and was found as growth promoter. The fungal isolate Su-3-4-3 significantly promoted whole plant length as compared with Gibberella fujikuroi (Fig. 2).
The use of Waito-C rice seedlings is profitable as they can easily grow under controlled and sterilized conditions, hydroponically, using autoclaved water-agar media. Since this media is free of any nutrient, the sole effect of culture filtrate can easily be evaluated. Waito-C rice is a known dwarf rice mutant with reduced GA biosynthesis. Treatment of its seeds with uniconazol, as a GA biosynthesis retardant, further suppresses the endogenous GAs production by blocking its biosynthesis pathway in the plant. Shoot elongation of these seedlings can thus efficiently be related to activity of plant growth promoting secondary metabolites from fungal culture filtrates applied [28,29]. Similarly, it has been reported the biotechnological application of Piriformospora indica, a culturable mycelium possessing growth promoting effects in a vast range of plant hosts. The Su-3-4-3 fungal strain, which has strain plant growth promoting effects, was analyzed using Waito-C rice seedlings.
Analysis of culture filtrates of Su-3-4-3 for the presence of GAs. GA, the plant hormone produced by fungal endophytes isolated from salt tolerant plants, was analyzed with HPLC and GC/MS. Therefore, a variety of GAs were confirmed from the culture filtrate of the Su-3-4-3 fungal strain; the result of the GC/MS SIM analysis showed that Su-3-4-3 produced GA 1 (0.465 ng/mL), GA 3 (1.808 ng/mL), other inactive GA 9 (0.054 ng/mL) and GA 24 (0.044 ng/mL) (Fig. 3). It was confirmed that Su-3-4-3 produced as much GA 1 , GA 3 , GA 9 and GA 24 as G. fujikuroi.
The GC/MS with SIM technique has the ability to analyze highly complex mixtures and to detect compounds of different classes [30], and so was used for culture filtrate analysis of the Sm-3-7-5 fungal strain. GC/MS SIM is useful to investigate a number of compounds and is often used in plant experimentation [31,32]. By reason of its reliability, GC/MS SIM was used in quantitative analysis of various plant hormones.
In summary, a total of 160 fungal strains were isolated from 5 plants inhabiting the Muan salt marsh and were classified into 3 phyla, 5 classes, 10 orders, 18 families, and 28 genera. Fusarium (class Sordariomycetes) was the most dominant genus followed by Penicillium. The group of endophytic fungi isolated from Phragmites australis was the Fig. 3. Gibberellins (GAs) content of fungal culture filtrates of the Su-3-4-3 strain and wild type Gibberella fujikuroi. Gas chromatograph/mass spectrometer selected ion monitoring analysis of culture filtrate extracts from the Su-3-4-3 fungal strain detected two bioactive GAs. Su-3-4-3 showed the presence of bioactivity of GA 1 , GA 3 , and other inactive GA. The standard deviation from means was calculated using Microsoft Excel. most diverse according to the diversity analysis. Plant growth promotion activity of Waito-C rice seedlings was confirmed by culture filtrate of Talaromyces pinophilus Su-3-4-3. Our recent study reports the information on the capacity of Talaromyces pinophilus Su-3-4-3 producing GAs. Therefore, the present study was performed to provide basic data on the symbiosis of halophytic plants and fungi. Understanding such endophytic interactions may significantly improve the quality and productivity of agricultural crops.