Diversity and Plant Growth Promotion of Fungal Endophytes in Five Halophytes from the Buan Salt Marsh

J. Microbiol. Biotechnol. 2021. 31(3): 408–418 https://doi.org/10.4014/jmb.2012.12041 Review Diversity and Plant Growth Promotion of Fungal Endophytes in Five Halophytes from the Buan Salt Marsh Irina Khalmuratova1†, Doo-Ho Choi1†, Hyeok-Jun Yoon1, Tae-Myung Yoon2*, and Jong-Guk Kim1* 1School of Life Science and Biotechnology, Kyungpook National University, Daegu 701-701, Republic of Korea 2Department of Horticultural Science, Kyungpook National University, Daegu 41566, Republic of Korea The diversity and plant growth-promoting ability of fungal endophytes that are associated with five halophytic plant species (Phragmites australis, Suaeda australis, Limonium tetragonum, Suaeda glauca Bunge, and Suaeda maritima) growing in the Buan salt marsh on the west coast of South Korea have been explored. About 188 fungal strains were isolated from these plant samples’ roots and were then studied with the use of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2). The endophytic fungal strains belonged to 33 genera. Alternaria (18%) and Fusarium (12.8%), of the classes Dothideomycetes and Sordariomycetes, were most rampant in the coastal salt marsh plants. There was a higher diversity in fungal endophytes that are isolated from S. glauca Bunge than in isolates from other coastal salt marsh plants. Plant growth-promoting experiments with the use of Waito-C rice seedlings show that some of the fungal strains could encourage a more efficient growth than others. Furthermore, gibberellins (GAs) GA1, GA3, and GA9 were seen in the Sa-1-4-3 isolate (Acrostalagmus luteoalbus) culture filtrate with a gas chromatography/mass spectrometry. Keywords: Coastal salt marsh plants, fungal endophytes, Buan salt marsh, growth promotion, gibberellin Introduction The world’s population is estimated to rise to 9 billion by 2050 [1]. There is a need to increase food production under less than optimal conditions due to population growth and climate change. In the mid- to late twentieth century, the cultivation efforts’ purpose is to improve crop varieties, introduce hybrids, and increase agricultural productions in terms of fertilizer, crop management practices, herbicides, water delivery systems, and pesticides resulted in the “Green Revolution” [2, 3]. Worldwide, there is a similar global challenge, leading to a need of breed- improved varieties as well as agricultural production practices [4]. The expansion of agricultural production to Received: December 22, 2020 marginal lands and the effects of global climate change also need an increased biotic and abiotic stress tolerance Accepted: December 31, 2020 and efficient nutrient utilization in crop plants for the future global food requirements to be met. Endophytic fungi are organisms that are found anywhere (intercellularly or intracellularly in plants from nearly First published online: all genera of Kingdom Plantae). These fungi live in host plants for at least a portion of their lives without January 04, 2021 generating any immediate overt disease symptoms. These associations can encourage tissue differentiation and plant growth and can help in managing abiotic and biotic stresses to which the host plants are subjected [5, 6]. In *Corresponding authors addition, endophytic fungi may prevent pathogenic organisms and provide nutrients, benefiting the plant host J.-G. Kim [7]. These well-distributed fungi form diverse plant associations and thus constitute outstanding sources of new Phone: +82-53-950-5379 Fax: +82-53-955-5379 bioactive secondary metabolites [8]. Accordingly, there are several new bioactive compounds with insecticidal, E-mail: [email protected] antimicrobial, cytotoxic, and anticancer activities that have been separated from endophytic fungi recently [9, 10]. T.-M. Yoon Plant signaling compounds, also called phytohormones, regulate plant responses to environmental change as E-mail : [email protected] well as control plant growth and development [11]. Notably, recent studies have reported that certain endophytes encourage host plant growth through the synthesis of phytohormones, for example, gibberellins (GAs), †These authors contributed cytokinins, and indole-3-acetic acid (IAA) [12-14]. Indeed, endophytic fungi promote plant growth by secreting equally to this work. gibberellins in the rhizosphere of their hosts, which leads to an increase in plant biomass production as well as disease resistance. Apart from this, some endophytic fungi secrete both IAA and GAs into culture media [15]. pISSN 1017-7825 This research surveys both distribution and diversity of fungal endophytes in a certain coastal region of Korea. eISSN 1738-8872 We have investigated fungal isolates’ capacity to encourage growth of Waito-C rice seedlings and have identified Copyright© 2021 by whether secondary metabolites like gibberellins were seen in fungal culture filtrates. The Korean Society for Microbiology and Biotechnology J. Microbiol. Biotechnol. Diversity and Plant Growth-Promoting Effects of the Fungal Endophytes 409 Table 1. Geographic coordinates and scientific names of plants native to the Buan salt marsh. No Scientific name Code Site of collection Habitat 1 Phragmites australis Pa 35°35'34.69"N/126°36'2.42"E Halophytic 2 Suaeda australis Sa 35°35'10.99"N/126°32'44.70"E Halophytic 3 Limonium tetragonum Lt 35°35'10.73"N/126°32'51.40"E Halophytic 4 Suaeda glauca Bunge Su 35°35'10.80"N/126°32'51.90"E Halophytic 5 Suaeda maritima Sm 35°35'34.92"N/126°36'3.95"E Halophytic Materials and Methods Plant Materials and Sampling Sites Healthy plants and roots of Phragmites australis, Suaeda australis, Limonium tetragonum, Suaeda glauca Bunge, and Suaeda maritima were gathered from different places in the Buan salt marsh in South Korea. The samples were carefully sealed in sterile plastic bags and were then processed in the laboratory within 24 hours of collection. The scientific names, codes, and local sites of the five plant species are listed in Table 1. Sterilization and Isolation of Endophytes from the Roots of Halophytes Fungal endophytes were isolated from healthy roots of halophytic plants that were gathered from the salt marsh. Each plant’s root samples were washed with tap water, cut into 2–2.5-cm-long segments, and treated with Tween 80 solution for 10 min on a shaker with 160 rotations per minute (rpm). Afterward, root segments were incubated in a solution of 1% (w/v) perchloric acid for 10 min and then rinsed with double-distilled water [16, 17]. Then, they were dehydrated for 5–6 min at a temperature of 22°C on a clean bench, and two to three root segments were placed in a 90-mm Petri plate with Hagem minimal media that contain 80 ppm streptomycin. Samples were incubated at 25°C in dark conditions until there is growth of fungi from the root segments seen [18, 19]. Lastly, pure fungal strains were separated from the root segments and were kept on potato dextrose agar at 25°C [20]. DNA Extraction, PCR Amplification, and the Identification of Fungal Strains Fungal endophyte cultures were grown in an Erlenmeyer flask that contains 50 ml potato dextrose broth for 7– 10 days at a temperature of 26°C on a shaker at 120 rpm. All the 188 lyophilized endophyte samples were known. There was a fungal genomic DNA extraction using a DNeasy Plant Mini Kit (Qiagen, USA). There was identification of fungi performed by sequencing the internal transcribed spacer (ITS) region with the universal primers ITS-1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS-4 (5′-TCCTCCGCTTATTGATATGC-3′). Reaction cycling comprised of an initial denaturation step at 95°C for 2 min, which is then 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. The final extension was done at 72°C for 7 min. PCR products were electrophoresed on agarose gels with an ethidium bromide stain and purified using the QIAquick PCR purification kit (Qiagen). The products were then sequenced with the use of the ABI PRISM BigDye terminator cycle sequencing kit (PE Biosystems, USA) on an ABI 310 DNA automated sequencer (Perkin, USA). The sequences were identified with the use of the BLAST (Basic Local Alignment Search Tool) tool of the National Center for Biotechnology Information (NCBI). Statistical Analyses The fungal endophytes’ diversity at the genus level was indicated by the Shannon diversity index (H’), Fisher’s alpha index (α), and Simpson’s index of diversity. Richness was assessed by Menhinick’s richness index (Dmn) and Margalef’s index (Dmg) [21, 22]. The Menhinick’s index was calculated through the following formula: Dmm= S⁄ N ; 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 [23]. Fisher’s alpha index (α) was calculated as follow; S=α·ln(1+N/α), where S is the number of genera, and N is the total number of individuals. The formula for Shannon’s diversity index is ' = –ΣR ⋅ H i = 1piln pi, where pi is the proportion of individuals found in genera i in a sample. The values of the Shannon diversity index generally range from 1.5 to 3.5. Simpson’s index of diversity (1-D) was calculated as = ΣR ()– ⁄ ()– follow; D i = 1ni ni 1 NN 1 , 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 from 0 to 1; the greater the magnitude, the greater the sample diversity. Screening of Fungal Cultures on Waito-C rice Seedlings To test whether the fungi have growth-promoting capacities, Waito-C rice sprouts were exposed to fungal culture filtrates.

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