Diversity and Plant Growth-Promoting Effects of Fungal Endophytes Isolated from Salt-Tolerant Plants

Fungal endophytes are symbiotic microorganisms that are often found in asymptomatic plants. This study describes the genetic diversity of the fungal endophytes isolated from the roots of plants sampled from the west coast of Korea. Five halophytic plant species, Limonium tetragonum, Suaeda australis, Suaeda maritima, Suaeda glauca Bunge, and Phragmites australis, were collected from a salt marsh in Gochang and used to isolate and identify culturable, root-associated endophytic fungi. The fungal internal transcribed spacer (ITS) region ITS1-5.8S-ITS2 was used as the DNA barcode for the classification of these specimens. In total, 156 isolates of the fungal strains were identified and categorized into 23 genera and two phyla (Ascomycota and Basidiomycota), with Dothideomycetes and Sordariomycetes as the predominant classes. The genus Alternaria accounted for the largest number of strains, followed by Cladosporium and Fusarium. The highest diversity index was obtained from the endophytic fungal group associated with the plant P. australis. Waito-C rice seedlings were treated with the fungal culture filtrates to analyze their plant growth-promoting capacity. A bioassay of the Sm-3-7-5 fungal strain isolated from S. maritima confirmed that it had the highest plant growth-promoting capacity. Molecular identification of the Sm-3-7-5 strain revealed that it belongs to Alternaria alternata and is a producer of gibberellins. These findings provided a fundamental basis for understanding the symbiotic interactions between plants and fungi.


DNA Extraction, PCR Amplification, and Identification of Fungal Strains
The mycelia of the fungal strains were cultivated in potato dextrose broth for 7-10 days at 120 rpm and 25°C. The lyophilized fungal strains were used for identification. The fungal genomic DNA was extracted with a DNeasy Plant Mini Kit (Qiagen, USA) and the internal transcribed spacer (ITS) region of the DNA was amplified using universal primers ITS-1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS-4 (5′-TCC GCT TAT TGA TAT GC-3′). The reaction conditions consisted of an initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 1 min, and extension at 72°C for 1 min, and a final extension at 72°C for 7 min. The PCR products were observed by agarose gel electrophoresis with ethidium bromide staining. The products were purified with a QIAGEN QIAquick PCR Purification Kit and sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) on an ABI 310 DNA sequencer (Perkin-Elmer). The resulting DNA sequence was identified using the Basic Local Alignment Search Tool (BLAST) search program (http://www.ncbi.nlm.gov/BLAST/) from the National Center for Biotechnology Information (NCBI).

Statistical Analysis
The generic richness and diversity of the fungal endophytes were analyzed at the genus level in the plant samples. Menhinick's index (Dmn) and Margalef 's index (Dmg) were used to determine the richness of each genus in the community [28][29]. The Menhinick's index was calculated via the following equation: ; Margalef 's index was calculated as follows: Dmg = (S−1)/ln(N), where S is the number of genera in a sample, and N is the total number of individuals in a community. Both indices ranged from 0 to . The genus diversity was evaluated using the Shannon diversity index (H'), Fisher's alpha index ( α), and Simpson's index of diversity [30][31]. Fisher's alpha index (α) was calculated as , where S is the number of genera, and N is the total number of individuals. The equation for Shannon's diversity index is , where pi is the proportion of individuals found in genera i in a sample. The values of the Shannon diversity index generally fall between 1.5 and 3.5. Simpson's index of diversity (1-D) was calculated as , where N is the total number of individuals in a sample, and ni is the number of individuals found in genera i in a sample. The magnitude of this index ranges between 0 and 1; the greater the magnitude, the greater the sample diversity.

Effect of Fungal Filtrates for Plant Growth Promotion in Waito-C Rice Seedlings
The culture filtrates of the isolated fungal strains were bioassayed in Waito-C rice seedlings to determine their plant growth-promoting activity. The fungal isolates were grown in a shaking incubator for 7 days at 25°C and 180 rpm in Czapek broth medium (1% glucose, 1% peptone, 0.1% K 2 HPO 4 , 0.05% KCl, 0.05% MgSO 4 ·7H 2 O, and 0.001% FeSO 4 ·7H 2 O; /L pH 7.3 ± 0.2). Forty-five milliliters of culture fluid was harvested; the pellet and the supernatant were stored at −70°C and then lyophilized. The lyophilized supernatants were mixed with 1 ml of autoclaved distilled water. The Waito-C rice seeds were treated overnight with uniconazole 20 ppm to minimize the activity of the seed coat gibberellins. The treated Waito-C rice seeds were washed and soaked in autoclaved distilled water until the sprouts emerged. Then the Waito-C rice seedlings were transplanted into glass tubes containing a 0.6% water-agar medium and grown in a growth chamber. Ten microliters of the supernatant solution for each fungal culture filtrate was applied to the apical meristem of the Waito-C rice seedlings at the twoleaf stage. One week after treatment, the plant and shoot length were observed and compared with the controls. The controls included culture filtrate of Gibberella fujikuroi, Czapek broth medium, and distilled water [30,32].

Extraction and Quantification of Gibberellins (GAs) from the Fungal Culture Filtrates
The culture filtrate of each endophytic fungal isolate was analyzed for the presence of GAs by gas chromatography/mass spectrometry (GC/MS). Endophytic fungal isolates were cultured in 250 ml of Czapek broth medium (containing 1% (w/v) glucose and peptone) for 7 days at 25°C in a shaking incubator at 180 rpm. The extracted GAs were analyzed by reverse-phase C18 high-performance liquid chromatography (HPLC). The fractions were collected and prepared for GC/MS via selected ion monitoring (SIM) (6890N Network GC System, 5973 Network Mass Selective Detector, Agilent Technologies, USA). Following the GC/MS, all data were collected and analyzed. The three major ions of the supplemented [ 2 H 2 ] GAs' internal standards and the fungal GAs were monitored simultaneously. The retention time was determined using hydrocarbon standards to calculate the Kovats retention index (KRI) value, while quantification of the GAs was based on peak area ratios of the nondeuterated (extracted) GAs to deuterated GAs [31,33].

Molecular Identification of the Endophytic Fungi
In total, 156 fungal endophytes, belonging to 26 species and 23 genera, were isolated from the roots of five halophytic plant species native to the Gochang salt marsh. The nucleotide sequence for each strain was submitted to the GenBank database of the National Center for Biotechnology Information (Accession Nos. KP018058-KP018213). The fungal similarity scores obtained were at or close to 100%.
Taxonomic placement of endophytic fungi in each plant sample was analyzed at the class and genus levels ( Fig. 1). Dothideomycetes from the Ascomycota phylum accounted for the highest percentage at the class level, except in the species L. tetragonum. Dothideomycetes accounted for more than half of the fungi in each plant sample. At the genus level, Alternaria (33.3%) was the most prominent, followed by Cladosporium (25%) in all of the plant samples except S. glauca Bunge, where Cladosporium was the most prominent genus followed by Fusarium (10.2%). In the plant samples of L. tetragonum, Fusarium was the most prevalent genus. Alternaria was found in all of the plant samples. The proportion of remaining fungal genera was approximately 0.5~2.5%. A previous study [26,27] reported that the genus Alternaria was the most prominent fungus found in the roots of halophytic plants native to the Buan salt marsh. Fungal species, including those of the genera Fusarium, Penicillium, Alternaria, and Cladosporium, which are abundant in the host, occasionally establish endophytic associations with plants [34][35][36][37][38].
The current study isolated 156 fungal endophytes from salt-tolerant plants that were identified at the genus level and included Alternaria, Aspergillus, Cladosporium, Cochliobolus, Colletotrichum, Epicoccum, Exophiala, Fusarium, Kabatiella, Khuskia, Lecanicillium, Macrophoma, Meira, Paraconiothyrium, Paraphaeosphaeria, Penicillium, Pestalotiopsis, Phomopsis, Pleospora, Pseudozyma, Stemphylium, Talaromyces, and Trichoderma. Among the endophytic fungi isolated, the genera Alternaria, Cladosporium, and Fusarium were the most abundantly distributed in the plant samples. The phyla of various endophytic fungi were confirmed to be Ascomycota and Basidiomycota. These findings revealed that the roots of the plants growing in the coastal salt marsh are inhabited by diverse endophytic mycobiota.
Molecular techniques and sequencing of the genomic DNA are used widely and successfully for fungal identification [39]. DNA sequence analysis methods provided important information for this study. Most of the ribosomal DNA genes were highly conserved taxonomically for identification. The internal transcribed spacer (ITS) region has been commonly used as a DNA barcode for the molecular identification of fungi. This study was conducted with the 5.8S gene and ITS1/2 regions for the identification of the fungal strains [35].

Diversity of the Endophytic Fungi Isolated from Halophytes
According to the host plant specimen results, the fungal isolates comprised 10 genera and 12 species isolated from L. tetragonum, 7 genera and 9 species from S. australis, 7 genera and 7 species from S. maritima, 8 genera and 6 species from S. glauca Bunge, and 9 genera and 6 species from P. australis (Table 2).
Generic richness and diversity were determined by counting the genera present in the fungal communities among the plant samples (Table 3). P. australis exhibited high scores compared to the other plant species in terms of both genus richness and diversity. Statistical analysis of richness demonstrated that P. australis had a Margalef 's index of 2.95 and a Menhinick's index of 2.32. The analysis of genetic diversity revealed a Fisher's α index of 9.50, a Simpson's index of diversity of 0.88, and Shannon's index of 1.95. Thus, the Shannon index was less sensitive to evenness than the Fisher's α and Simpson's indices [40]. P. australis also exhibited the highest diversity index, as the fungi isolated from this plant were the most diverse compared to the other plant specimens. Statistical analysis of the endophytic fungi via counting the genera from the plant samples revealed that P. australis possessed the most diverse type of endophytic fungi. [15].
Fungal symbionts associated with plants in natural ecosystems help plants overcome abiotic stress, such as soil salinity, drought, and heat [41]. Shoreline habitats are frequently exposed to high salt stress, as they remain rhythmically submerged in saltwater. The capacity to resist high salinity stress is necessary for survival in coastal environments. Thus, fungal strains such us Penicillium citrinum and Fusarium oxysporum could improve the  survival and growth of their hosts by enhancing tolerance to environmental stress [10,11,42,43]. Many species of plant symbiotic fungi are known to produce a number of phytohormones [21,22,37,38]. Aspergillus terreus and P. citrinum GAs that confer biotic stress resistance to pathogenic attack [44]. Plant hormones like GAs are essential for many developmental processes in plants, including seed germination, stem elongation, leaf expansion, ripening, and the induction of flowering [45]. Many researchers have found that fungal endophytes could limit the damage caused by pathogenic microorganisms and protect the host from diseases [12,46,47]. Moreover, it has been reported that Penicillium species increased salt stress resistance in the host plant [48,49]. Alternaria alternata was the most abundant fungus among the collected samples and was isolated from plants S. australis, S. maritima, S. glauca Bunge, and P. australis. Previous reports have confirmed that A. alternata produces the plant growth regulator indole-acetic acid for the host plant [44] Indole-acetic acids are considered essential for important physiological processes such as cell division or cell elongation, tissue differentiation, phototrophic or geotropic responses, and all subsequent effects on plant growth and development [50]. Moreover, Trichoderma harzianum and F. oxysporum could produce indole-acetic acid [4,51,52]. Thus, these fungal species helped their host plants procure nutrients and promoted growth.

Screening for Plant Growth-Promoting Effects of Fungal Culture Filtrates on Waito-C Rice Seedlings
Waito-C seeds were treated with uniconazole as a GA biosynthesis inhibitor. The Sm-3-7-5 fungal strain, which has plant growth-promoting capacity, was analyzed using the Waito-C rice seedlings. Screening of the microbial culture filtrates was used to identify the biologically active molecules to confirm the presence of gibberellins. In this study, plant growth-promoting hormones were detected in the culture filtrates of the fungal endophytes isolated from the roots of the halophytes using the Waito-C rice seedlings. Waito-C rice is a known dwarf rice cultivar with reduced GA biosynthesis [7,18].
The culture filtrates of all of the fungal endophytes were applied to the Waito-C rice seedlings to analyze the plant growth promoting capacity (Fig. 2). The culture filtrate treatments using the Sm-3-7-5 fungal strain resulted in a 9.3 cm shoot length and 19.6 cm plant length as plant growth-promoting effects. Treatments with Sm-3-7-5 fungal strain or the G. fujikuroi strain revealed marked similarity in plant growth promotion between the two strains. These results were consistent with those of a previous study in which the endophytic fungus Aspergillus clavatus Y2H0002, isolated from the roots of Nymphoides peltata, was shown to promote the growth of various rice plants [53].

Quantitative Analysis of Culture Filtrate of Sm-3-7-5 for the Presence of Gibberellins
In an analysis of growth promoting effect, Sm-3-7-5 produced the most effective growth promotion results. Comparing with G. fujikuroi, the two strains showed quite similar results in Waito-C growth. Because of that, additional comparison between the two strains was necessary. As GC/MS with selected ion monitoring technique has the ability to analyze highly-complex mixtures and detect compounds of different classes [54], it was used to analyze the culture filtrate of Sm-3-7-5 fungal strain. GC/MS SIM was important for the investigation of a number of compounds and was often used in plant experimentation [55,56]. We also employed GC-MS SIM in the quantitative analysis of various plant hormones.
In summary, 156 strains of endophytic fungi were isolated from the roots of five halophytic plant species native to the Gochang salt marsh. These halophytes included L. tetragonum, S. australis, S. maritima, S. glauca Bunge, and P. australis. A study of the plant growth-promoting effects of A. alternata Sm-3-7-5 and GA production was conducted. All fungal strains were identified by molecular methods and were classified into 2 phyla, 5 classes, 11 orders, 15 families, and 23 genera. Alternaria and Cladosporium were found to be the dominant genera among the collected isolates. The most diverse group of fungi determined by the diversity analysis was isolated from the roots Fig. 2. Waito-C rice seedlings with fungal culture filtrates of fungal endophytes plant growth promotion results (A, B, C, D, E). Ten microliters of lyophilized culture filtrates were applied to the Waito-C rice seedlings. The shoot length and plant length of the Waito-C rice seedlings were measured following 7 days of treatment. The standard deviation from the means was calculated using Microsoft Excel. of P. australis. In conclusion, this study provides key information to the literature toward understanding the interactions between coastal plants and fungi. Sm-3-7-5 showed the presence of bioactivity of GA1, GA3, and other inactive GAs. The standard deviation from the means was calculated using Microsoft Excel.