Crinipellins a and I, Two Diterpenoids from the Basidiomycete Fungus Crinipellis Rhizomaticola, As Potential Natural Fungicides
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molecules Article Crinipellins A and I, Two Diterpenoids from the Basidiomycete Fungus Crinipellis rhizomaticola, as Potential Natural Fungicides Jae Woo Han 1,†, Mira Oh 1,2,†, Yu Jeong Lee 1,2, Jaehyuk Choi 3 , Gyung Ja Choi 1,2 and Hun Kim 1,2,* 1 Center for Eco-friendly New Materials, Korea Research Institute of Chemical Technology, Daejeon 34114, Korea; [email protected] (J.W.H.); [email protected] (M.O.); [email protected] (Y.J.L.); [email protected] (G.J.C.) 2 Department of Green Chemistry and Environmental Biotechnology, University of Science and Technology, Daejeon 34113, Korea 3 Division of Life Sciences, Incheon National University, Incheon 22012, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-42-860-7436 † These authors contributed equally to this work. Received: 27 August 2018; Accepted: 14 September 2018; Published: 17 September 2018 Abstract: In the course of screening for microbes with antifungal activity, we found that the culture filtrate of the IUM00035 isolate exhibited strong antifungal activity against Magnaporthe oryzae and Colletotrichum coccodes in planta. Based on the phylogenetic analysis with the ITS region, the IUM00035 isolate was identified as Crinipellis rhizomaticola. To identify antifungal compounds from the C. rhizomaticola IUM00035 isolate, the culture filtrate of the isolate was partitioned with ethyl acetate and n-butanol and, consequently, two active compounds were isolated from the ethyl acetate extract. The chemical structures of the isolated compounds were determined as crinipellin A (1) and a new crinipellin derivative, crinipellin I (2), by NMR spectral analyses and a comparison of their NMR and MS data with those reported in the literature. Crinipellin A (1) exhibited a wide range of antifungal activity in vitro against C. coccodes, M. oryzae, Botrytis cinerea, and Phytophthora infestans (MICs = 1, 8, 31, and 31 µg/mL, respectively). Furthermore, when plants were treated with crinipellin A (1) (500 µg/mL) prior to inoculation with fungal pathogens, crinipellin A (1) exhibited disease control values of 88%, 65%, and 60% compared with non-treatment control against tomato late blight, pepper anthracnose, and wheat leaf rust, respectively. In contrast to crinipellin A (1), crinipellin I (2) showed weak or no activity (MICs > 250 µg/mL). Taken together, our results show that the C. rhizomaticola IUM00035 isolate suppresses the development of plant fungal diseases, in part through the production of crinipellin A (1). Keywords: antifungal; Crinipellis rhizomaticola; crinipellin; biological control; mushroom 1. Introduction Plant-pathogenic fungi cause severe plant diseases, resulting in a loss of crop yields and economic damage to the agricultural industry [1]. Synthetic fungicides have been recognized as a primary method to control plant diseases caused by fungal pathogens, although the overuse of chemical pesticides has caused severe problems, such as toxicity for human health and environmental pollution. Therefore, the development of safer and more effective alternatives is necessary to replace chemical pesticides [2]. Among the various natural resources, antagonistic microbes (or microbial metabolites) have been recognized as an attractive source for new antifungal agents [3]. Molecules 2018, 23, 2377; doi:10.3390/molecules23092377 www.mdpi.com/journal/molecules Molecules 2018, 23, 2377 2 of 11 Mushrooms have been used as a functional food or as active ingredients of folk medicine due to their various bioactive compounds [4]. Currently, over 140,000 species of mushrooms have been reported,Molecules and 2018, more 23, x than 200 species have been known to possess antimicrobial properties2 of 11 [5 ,6]. The fruiting bodies, mycelial biomass, or culture filtrates of mushrooms contain bioactive compounds reported, and more than 200 species have been known to possess antimicrobial properties [5,6]. The with a wide range of antimicrobial activity, including antifungal and antibacterial activities [7–9]. fruiting bodies, mycelial biomass, or culture filtrates of mushrooms contain bioactive compounds Strobilurus tenacellus In particular,with a wide strobilurins, range of antimicrobial produced byactivity, wood-inhabiting including antifungal forest mushroomand antibacterial activities [7–9]. Inand Oudemansiellaparticular, mucidastrobilurins,, are oneproduced of the by greatest wood-inhabiting findings in forest the history mushroom of fungicides Strobilurus [10tenacellus]. Strobilurins and act asOudemansiella inhibitors ofmucida the, respiratoryare one of the chain greatest at findings the quinone in the outerhistory binding of fungicides site of[10]. the Strobilurins cytochrome act bc1 complexas inhibitors [11]. Due of the to the respiratory unique andchain specific at the quinone modes ofouter action, binding a number site of the of strobilurincytochrome analogues bc1 complex have been[11]. synthesized Due to the with unique improved and specific antifungal modes activity, of action, stability, a number and dissemination,of strobilurin analogues of which have several been have beensynthesized developed with as highly improved successful antifungal commercial activity, stability, products and such dissemination, as kresoxim-methyl, of which several pyraclostrobin, have and trifloxystrobinbeen developed [as12 highly]. successful commercial products such as kresoxim-methyl, pyraclostrobin, andIn the trifloxystrobin search for antifungal[12]. agents from mushrooms, we recently found that a culture filtrate of CrinipellisIn the rhizomaticola search for antifungalIUM00035 agents isolate from hasmushroom the potentials, we recently to control found plant that a diseasesculture filtrate caused of by MagnaportheCrinipellis oryzaerhizomaticolaand Colletotrichum IUM00035 isolate coccodes has. Furthermore,the potential to we control isolated plant and diseases identified caused the activeby Magnaporthe oryzae and Colletotrichum coccodes. Furthermore, we isolated and identified the active small molecules from the culture filtrate of C. rhizomaticola IUM00035 and investigated their in vitro small molecules from the culture filtrate of C. rhizomaticola IUM00035 and investigated their in vitro and in vivo antifungal activity. Taken together, our results provide a useful information for the and in vivo antifungal activity. Taken together, our results provide a useful information for the developmentdevelopment of plant-protectingof plant-protecting agents agents using using microbial-derivedmicrobial-derived materials materials to tocontrol control plant plant diseases diseases causedcaused by fungal by fungal pathogens. pathogens. 2. Results2. Results and and Discussion Discussion 2.1. In2.1. Vivo In Vivo Antifungal Antifungal Activity Activity of of the the Culture Culture Filtrate Filtrate ofof thethe IUM00035 Isolate Isolate To findTo find a mushroom a mushroom isolate isolate showing showing a plant a plant disease disease control control activity, activity, we we have have examined examined the thein in vivo antifungalvivo antifungal activity ofactivity the culture of the filtratesculture filtrates of over of 100 over mushroom 100 mushroom isolates isolates against against seven seven plant plant diseases: rice bdiseases:last (RCB, rice caused blast (RCB, by M. caused oryzae ),byr iceM. soryzaeheath), bricelight sheath (RSB, blight caused (RSB, by causedRhizoctonia by Rhizoctonia solani), tomato solani),g ray moldtomato (TGM, gray caused mold by (TGM,Botrytis caused cinerea by), Botrytistomato cinerealate blight), tomato (TLB, late caused blight by (TLB,Phytophthora caused by infestansPhytophthora), barley powderyinfestansmildew), barley (BPM, powdery caused mildew by Blumeria (BPM, caused graminis by Blumeriaf. sp. hordei graminis), wheat f. sp.leaf hordeirust), wheat (WLR, leaf caused rust by Puccinia(WLR, triticina caused), by and Pucciniapepper triticinaanthrac), andnose pepper (PAN, anthracn causedose by (PAN,C. coccodes caused). by Among C. coccodes the). culture Among filtrates the of overculture 100 filtrates mushroom of over isolates, 100 mush theroom treatment isolates, of the undiluted treatment IUM00035 of undiluted culture IUM00035 filtrate culture suppressed filtrate the suppressed the development of RCB, TGM, TLB, WLR, BPM, and PAN; however, there was no effect development of RCB, TGM, TLB, WLR, BPM, and PAN; however, there was no effect on RSB (Figure1). on RSB (Figure 1). In particular, this culture filtrate had strong effects against RCB and PAN with In particular, this culture filtrate had strong effects against RCB and PAN with disease control values of disease control values of 70% and 65% compared with the non-treatment control (distilled water 70%containing and 65% compared 0.025% Tween with 20), the respectively. non-treatment Three-fold control diluted (distilled culture water filtrate containing of the IUM00035 0.025% Tween isolate 20), respectively.further showed Three-fold disease diluted suppression culture filtrate of RCB, of the TLB, IUM00035 WLR, and isolate PAN further (Figure showed 1). Thus, disease our suppression results of RCB,suggest TLB, that WLR, the andIUM00035 PAN (Figure culture1 ).filtrate Thus, may our contai resultsn suggestantifungal that substances the IUM00035 for the culture control filtrate of plant may containdiseases antifungal caused substances by some plant-pathogenic for the control offungi. plant diseases caused by some plant-pathogenic fungi. FigureFigure 1. Antifungal 1. Antifungal activity activity of