Ⅰ. INTRODUCTION

Epichloë is a fungal endophyte classified into 43 species (Leuchtmann et al., 2014), and often naturally infects crops of the genus Festuca-Lolium, including tall fescue used as forage (Schardl, 1996). The majority of Epichloë endophytes are parasitic on plants throughout their lifetime and maintain a symbiosis by propagating through seeds without injuring or damaging the host (Schardl et al., 2004, Moon et al., 2000). Some endophytes of Epichloë such as E. coenophialum, also produce alkaloids that are toxic to predatory insects or mammals, thereby benefiting the host by increasing their survival and dominance in grasslands through increased environmental stress resistance, predator avoidance, and growth promotion (Decunta et al., 2021, Wang et al., 2020). Such a symbiotic relationship can be advantageous for the cultivation of grasses, but caution is required, as toxic alkaloids such as ergovaline and lolitrem B can accumulate in infected plants (Chujo et al., 2019). This can cause problems, such as convulsions, paralysis, high fever, decreased milk production, reproductive problems, and even death, in livestock that feed on them (Riet-Correa et al., 2013, Guerre, 2015). The infection rate depends on the region; for example it was 58–100% in the United States, 64% in the UK, and 98% on average in Finland (Clay et al., 2005). However, not all endophytes are toxic to mammals; certain strains are only toxic to herbivorous insects, while also improving drying stress resistance in infected plants. Furthermore, there was a case of commercializing these endophyte spores that are harmless to mammals in New Zealand (Hume et al., 2020). Since the quantitative evaluation of endophyte toxicity remains scarce, despite the endophytes being widespread in nature, recent studies are beginning to focus on cross-evaluating their genetic diversity, geographical characteristics, and alkaloid levels (Cagnano et al., 2019). Toxic alkaloids, such as ergovaline and lolitrem B, are produced by gene products expressed in approximately 11 biosynthetic gene clusters located in the genome of Epichloë (Chujo et al., 2019, Hettiarachchige et al., 2021). Lolitrem B is synthesized by ltmE, ltmJ, ltmP, ltmQ, ltmF, ltmC, ltmB, ltmG, ltmS, ltmM, and ltmK, while ergovaline is synthesized by lpsB, easE, easG, easA, easH, easC, easD, dmaW, cloA, and lpsA (Schardl et al., 2013). These alkaloids provide the host with increased resistance to droughts and herbivorous insects. Currently, two methods for endophyte testing are available: one using microscopy, and the other using a commercial detection kit (Agrinostics Phytoscreen kit) based on an immunoblot assay. Skilled research personnel are required for the microscopy method, with diagnosis being time-consuming and inconsistent. On the other hand, the immunoblot assay is quicker, simpler to perform, and has greater consistency than the microscopy method (Trento et al., 2007). However, this method still has limitations, including long incubation times (overnight) and variations in the results depending on the skill-level and subjectivity of the tester. Therefore, an attempt was made to develop a polymerase chain reaction (PCR)-based diagnostic method using a primer specific to Epichloë beta-tubulin (Dombrowski et al., 2006). However, only the presence or absence of Epichloë could be identified; whether the samples could generate toxic alkaloids could not be determined. Moreover, the sensitivity was relatively low due to the adoption of a single conventional PCR method. Since the synthesis of toxic alkaloids can be prevented by damaging the corresponding biosynthetic gene clusters (Panaccione et al., 2001), a technique was developed for monitoring the endophyte infection rate and toxic alkaloids by identifying not only the house-keeping genes of Epichloë, but also the genes involved in the biosynthesis of three alkaloids (peramine, lolines, and ergovaline, which are indole diterpenes) using multiplex PCR (Vikuk et al., 2019). However, the conventional multiplex PCR method involving electrophoresis has difficulties in interpretation due to false positives, background PCR bands from excessive DNA addition or sample contamination, and non-specific reactions. This makes its application difficult in monitoring the status of endophytes on large grasslands and monitoring endophyte contamination during seed breeding, production, and distribution as well as analysis of whether imported and exported grasses are infected with endophytes. Therefore, in this study, we aimed to develop a real-time PCR-based diagnostic method that extracts DNA from the seeds and tissues of tall fescue and detects the presence of Epichloë infections and alkaloid synthesis genes.

Ⅱ. MATERIALS AND METHODS

1. Designing of real-time PCR primer and collection of template nucleic acid sequence information

For the development of a real-time PCR method to specifically detect Epichloë spp., the sequences of the beta-actin gene (a house-keeping gene of Epichloë spp.), and the LtmC and easD genes (key genes from the gene cluster involved in the biosynthesis of alkaloids toxic to mammals), registered in NCBI GenBank, were obtained (Table 1). These sequences were then aligned and visualized using MAFFT (Katoh and Standley, 2013) to search for conserved sites (Fig. 1, Table 1). Thereafter, the quantitative PCR (qPCR) primer was designed according to a previous study (Bustin and Huggett, 2017) after analyzing the melting temperature (Tm) value and hairpin structure (Table 2).

2. Genomic DNA extraction

DNA was extracted from seeds and plant tissues of five cultivars of tall fescue (Festuca arundinacea) (Greenmaster, Purmi, Greenmaster2ho, Greenmaster3ho, and Greenmaster4ho) developed by the Grassland and Forages Division of the National Institute of Animal Science. Ten grains or 50 mg of tissue per sample were placed in a 3.0 mm pre-filled ceramic bead tube (Invirustech, Prep-HD30, Gwang-ju, Korea) for crushing. For DNA extraction, the lysis buffer included in the Clear-S™ Quick DNA extraction kit (Invirustech, IVT3002) was added according to the product manual before the sample was crushed twice using a homogenizer (Bead Ruptor Elite; Omni International, GA, USA) for 30 s at 4.5 ms⁻¹. The extracted DNA was then measured using an epoch microplate reader (BioTek, VT, USA) and stored at -65 ℃ until PCR amplification.

3. Real-time and conventional PCRs

The extracted DNA (50 ng) was amplified using EzAmp™ qPCR 2X Master Mix (Elpisbiotech, EBT-1805, Daejeon, Korea). The detection mix was prepared by mixing the tub2, easD, and ltmC-specific primers and probes (Table 1), with 10 nM of primer and 2.5 nM of probe. For PCR amplification, heat activation was performed at 95 °C for 5 min using qTower3 (Analytik Jena, Jena, Germany), and the denaturation was repeated 40 times in total by heating at 95 °C for 15 s and at 60 °C for 60 s for primer annealing and extension, respectively. The fluorescence signal was read at each cycle. To compare sensitivity and confirm infection, conventional PCR was used to amplify the tub2 gene by nested PCR, which was then electrophoresed on a 1.5% agarose TBE gel at 130 V for 40 min to confirm the PCR band (Dombrowski et al., 2006).

Ⅲ. RESULTS AND DISCUSSION

A total of 227 Epichloë genes, including Tub2, easD, and LtmC, were obtained from NCBI GenBank to design an Epichloë-specific PCR primer. All sequences entered into the reverse complement were modified to fit the ORF. Thereafter, multiple sequence alignment was performed with MAFFT v7.487 and was visualized using UGENE before the consensus sequence was secured. After checking the Tm value, amplicon size, and primer secondary structure from the obtained consensus, primers and internal oligos were selected for production. Subsequently, the genomic DNA was extracted from seeds and plant tissue samples for each tall fescue cultivar, which was measured using a spectrophotometer. The extracted DNA was quantified and diluted to not exceed 500 ng per PCR, and following PCR analysis, Epichloë spp. were detected in some samples. In the Epichloë Tub2-specific nested PCR, a product of the expected size, at approximately 364 bp, was confirmed (Fig. 2). Multiplex PCR confirmed the presence of Epichloë infection and detected the toxic alkaloid genes LtmC and easD, as detected in the nested PCR (Fig. 3). Based on the sequences of known Epichloë spp. genes, real-time PCR primers and probes were designed, and PCR conditions were established. Based on the references, infection was compared and analyzed using nested PCR primer sets, and the sensitivity and specificity were confirmed to be consistent. We also noted that the new diagnostic method developed through this research does not require skilled research technicians, unlike microscopy- or immune blot-based diagnostic methods. In addition, the limitations of the nested PCR method, such as high cross-contamination despite increased sensitivity and accuracy, were improved. Importantly, the existing diagnostic method could only confirm the presence of endophytes, whereas our multiplex real-time PCR method was able to detect endophyte infection and the toxic alkaloid genes. As this multiplex real-time PCR method indirectly confirms whether the infected endophyte is toxic to livestock, it has the potential to be utilized as a basic monitoring tool for more precise and safe grass production.

Ⅳ. CONCLUSION

Epichloë, a type of fungus, is well known as an endophyte of major plants and herbs, such as tall fescue. Some Epichloë spp. have positive functions, such as increasing the drying stress resistance of host plants through alkaloid synthesis and accumulation, or improving grass survival owing to increased avoidance by herbivorous insects. However, the absence of endophytes is more advantageous in the use of high-quality grasslands for livestock production as alkaloids that are toxic to mammals, such as lolitrem B or ergovaline, may accumulate in the plants. In conclusion, a multiplex real-time PCR method was developed in this study that could detect easD and LtmC, which are toxic alkaloid synthesis genes, as well as Tub2, which is a house-keeping gene. The test results were consistent with those obtained by the nested PCR method, with a detection accuracy of up to 0.1 ng or less. Epichloë spp.-specific gene amplification through real-time PCR could not only diagnose endophyte infection in grasses but also determine whether the imported seeds were infected. The findings of this study may aid in the risk assessment of endophytes.