Production of Ergot Alkaloids by the Japanese Isolate Claviceps purpurea var. agropyri on Rice Medium

Ergot alkaloids (EAs) are secondary metabolites produced by ergot fungi (e.g., Claviceps purpurea), which are parasites of Gramineae grasses. EAs and their analogs are used to treat migraine, postpartum uterine bleeding, and Parkinson's syndrome. Recent studies have reported additional new bioactive activities of EAs and their analogs, making them essential compounds for drug development, drug repositioning, and clinical applications. EAs are produced industrially by field cultivation of ergot or liquid fermentation in the mycelial phase, but there are few published studies of the production of EAs by cereal culture and thus this approach is poorly understood. This study searched for Claviceps strains that produce EAs cultured artificially in the mycelial phase, then the selected strains were cultured on cereal media (white rice, brown rice, and rye) to examine their ability to produce EAs on each medium. C. purpurea var. agropyri produced the Clavine-type EAs pyroclavine (1), festuclavine (2), and agroclavine (3) in the mycelial phase. When cultured with white rice, brown rice, or rye, C. purpurea var. agropyri produced 1 - 3 on all cereal media. The total amount of 1 - 3 in each cereal medium (150 g of cereal per Roux flask) was 2220.5 ± 564.1 μg for white rice, 920.0 ± 463.6 μg for brown rice, and 595.4 ± 52.1 μg for rye. The white rice medium supported the highest production of 1 - 3, with the total amount of EAs (150 g of white rice per Roux flask) being about 34 times higher than that in the T25 liquid medium (190 mL per 1 L Erlenmeyer flask) (equivalent amount per flask).


Introduction
Ergot alkaloids (EAs) are secondary metabolites produced by a wide range of fungi, predominantly Claviceps spp. (e.g., C. purpurea), a parasite of wheat, rye, and other Gramineae grasses. EAs are synthesized within the sclerotia (ergot) that form in the ears of host rice plants. EAs were discovered as the causative compound of wheatgrass poisoning, the main symptom of which is cramps and miscarriages [1] [2].
EAs and their analogs are classified into three types: Clavine-type, peptide-type, and simple amides of the lysergic acid-type, and more than 70 compounds are currently known [3]. EAs and their analogs are compounds with many pharmacological activities; for example, ergotamine is used to treat migraine, ergometrine is used to prevent postpartum hemorrhage after childbirth, and bromocriptine and pergolide are used to treat Parkinson's syndrome [4]. In particular, the market size of ergot-derived dopamine agonists in Japan was approximately 1.5 billion yen in FY2017 and a stable supply is required [5]. EAs and their analogs are thus essential compounds for the development of new drugs and clinical applications, including antibiotics, and treating cancer, psychiatric disorders, and coronavirus infection [6] [7] [8] [9].
EAs have a sterically complex chemical structure and industrial chemical synthesis has not been achieved due to economic disadvantages [10]. The industrial method for producing EAs is to extract them from ergot on rye artificially cultivated with C. purpurea in the field, or by the liquid culture of the mycelial phase of Claviceps spp. [11] [12]. The field production is greatly influenced by weather conditions, but liquid culture production covered these shortcomings. In addition, solid media changed the EAs composition ratio and increased the amount of EAs production [13]. The production of EAs by cultivating Claviceps spp. on solid media has been reported using wheat, rye, or sugarcane dregs but few studies on cereal culture have been published and the process is poorly understood [13] [14]. Furthermore, the ability to produce EAs in the mycelial phase varies significantly between species and strains of Claviceps [15].
This study searched for Claviceps strains cultured artificially in the mycelial phase and the produced EAs were identified. We cultured selected strains on cereal media (white rice, brown rice, and rye) to examine their ability to produce EAs on each medium.
Twenty-two strains of Claviceps spp. isolated in Japan were examined to produce EAs in the mycelial phase of liquid media. The results showed that C. purpurea var. agropyri produced the Clavine-type EAs pyroclavine (1), festuclavine (2), and agroclavine (3) in the mycelial phase. We report the types and total amounts of EAs produced by the mycelial phase of C. purpurea var. agropyri cultured on cereal media made from white rice, brown rice, or rye.

Experimental Instruments
Analytical TLC was performed on silica gel 60 F 254 (Merck Ltd., Darmstadt,

Materials
Twenty-two strains of the genus Claviceps were isolated from parasites of 18 grass hosts from 1990 to 2019 in 12 prefectures in Japan (Table 1). Ergotamine tartrate standard was purchased from Tokyo Chemical Industry Co., Ltd.

Fermentation and Extraction of Claviceps Spp. in T25 Culture
The culture conditions using a liquid medium were based on the method of Amici et al. [16]. Claviceps species were pre-cultured on T2 agar (sucrose 100 g, l-asparagine 10 g, yeast extract 0.1 g, Ca(NO 3 ) 2
First, C. purpurea var. agropyri was pre-cultured on PDA and grown for 21 days at 25˚C, then two pieces of cultured agar (1 × 2 cm 2 ) were transferred into 500 mL Erlenmeyer flasks containing 200 mL of M102 medium and grown at 25˚C and 150 rpm for seven days. Next, 10 mL of cultured M102 was transferred

Isolation of Pyroclavine (1), Festuclavine (2) and Agroclavine (3)
C. purpurea var. agropyri was incubated using 10 Roux flasks containing rice medium for 28 days at 25˚C, then the cultures were extracted with chloroform. The crude chloroform extract (7.6 g) was obtained by evaporation under vacuum, suspended in 100 mL of 4% tartaric acid aqueous solution, and parti-

TLC Analysis
Chloroform extracts of T25 culture were dissolved in chloroform at 50 mg/mL and ergotamine tartrate was dissolved at 1 mg/mL, then 2 μL aliquots were spotted on a TLC plate and developed with chloroform/methanol/acetic acid (90/15/0.1). Chromatographed ergot alkaloids were observed by spraying the plates with van Urk's reagent.

Culture of Claviceps Spp. in T25 Liquid Medium
Twenty-two strains were cultured in T25, which has already been reported to produce EAs during culture. All strains except C. purpurea var. sasae and C. microspora grew in T25 (Table 1).

Detection of EA-Producing Strains by TLC Analysis and Identification of Compounds
The EAs detected in chloroform extracts of the T25 culture media were confirmed by TLC analysis with van Urk's color reagent, using ergotamine tartrate as a color control. C. purpurea var. agropyri produced compounds showing blue-violet spots 1 (Rf 0.07), 2 (Rf 0.11), and 3 (Rf 0.24) with the same color as ergotamine tartrate (Rf 0.54) (Figure 1(A)).

Effects of Moisture Content of White Rice Medium on the Production of 1 -3 by C. purpurea var. agropyri
C. purpurea var. agropyri efficiently produced 1 -3 in white rice medium culture. Osmotic pressure (water activity) of the liquid medium affects the production of EAs in the mycelial phase of Claviceps spp. [21] [22]. Therefore, we examined the relationship between the water content of white rice medium and the     production of 1 -3 by C. purpurea var. agropyri. The production of EAs 1 -3 was maximal at 90 mL of water per 150 g of white rice (Figure 3).

Discussion
The purpose of our study was to identify Claviceps sp. that can be cultured in the mycelial phase, determine their ability to produce EAs, and cultivate the strains on cereal media (white rice, brown rice, and rye), and examine their ability to produce EAs on each medium.
Twenty-two strains of Claviceps spp. isolated in Japan were examined for their production of EAs in the mycelial phase using a liquid medium. The results showed that C. purpurea var. agropyri, isolated from Elymus tsukusiensis var. transiensis as a host plant, produced the Clavine-type EAs pyroclavine (1), festuclavine (2), and agroclavine (3)  Clavine-type EAs 1 -3 were first isolated by Abe et al. from Claviceps sp. parasitic on Elymus tsukusiensis var. transiensis [23] [24] [25]. Previous studies reported various physiological activities of agroclavine (3), including partial agonist or antagonist effects on adrenergic receptors, dopamine receptors, and serotonin receptors [26]. Furthermore, the carcinocidal effect of agroclavine (3) was enhanced by derivatization [7].  However, 1 -3 are difficult to obtain and there are no commercially available standards. C. fusiformis, isolated in South Asia and Africa, is a representative Clavine-type EA-producing fungus whose host plant is Pennisetum typhoideum Rich [27] [28] [29]. C. fusiformis belongs to section Pusillae within the genus Claviceps, and C. purpurea belongs to section Purpurea in the taxonomic study by Píchová et al. [30]. In that study, Clavine-type alkaloids were isolated from a phylogenetically distinct species of fungi from which Clavine-type alkaloids had previously been isolated, suggesting that C. purpurea var. agropyri could be a useful new Clavine-type EA-producing fungus distinct from C. fusiformis. When cultured with white rice, brown rice, or rye, C. purpurea var. agropyri Osmotic pressure (water activity) of the liquid medium affects the production of EAs in the mycelial phase of Claviceps spp. [21] [22]. The production of EAs by C. purpurea var. agropyri was affected by the moisture content of the white rice medium. C. purpurea var. agropyri produced the highest amount of 1 -3 when 90 mL of water was added to 150 g of white rice. These data suggest that water content is a major factor affecting the production of EAs in white rice me- dium. An osmosensor of filamentous fungi is the group III histidine kinase up-  [31]. The production of aurofusarin of Fusarium graminearum is regulated by the group III histidine kinase FgOs1 [32]. On the other hand, the type III histidine kinase CpHK1 in Claviceps purpurea does not directly regulate EAs production, and the relationship between osmotic pressure and EAs production remains unclear [22].

Conclusion
This study showed that white rice medium is effective for the production of the Clavine-type EAs pyroclavine (1), festuclavine (2), and agroclavine (3)