Secondary metabolites from an endophytic fungus Trichoderma erinaceum with antimicrobial activity towards Pythium ultimum

Abstract Chemical investigation of the endophytic fungus, Trichoderma erinaceum, isolated from healthy and asymptomatic common bean field crop, resulted in the isolation of a new alkene, (Z)-5-amino-5-(1,1,2-trihydroxybuta-1,3-dienyloxy)pentane-6,7,8,9-tetraol (1), together with five known compounds (2–6). The structures of the compounds were elucidated by analysis of their spectroscopic data including 1 D, 2 D NMR, ESI–HRMS and literature data. The organic crude extract and the compound isolated from T. erinaceum significantly (p ≤ 0.05) inhibited the mycelial growth of Pythium ultimum. Graphical Abstract


Introduction
Natural products from endophytes such as Trichoderma strains are an important source of biomolecules that can be used successfully to control many plant diseases (Hermosa et al. 2012). Trichoderma spp. is a saprophytic filamentous fungus that is effectively used as a biological control agent and bio-pesticide against phytopathogens (Harman et al. 2004). The modes of action of Trichoderma as a biological control agent include many mechanisms such as competition for nutrients, production of lytic cell wall enzymes, antibiosis, mycoparasitism and induction of defenses plants (Shoresh et al. 2010;Tchameni et al. 2020). Among these, the production of antibiotics is considered to play an important role during biological control events. From the bioguide isolation, different secondary metabolites were isolated from liquid extracts of Trichoderma. These volatile and non-volatile metabolites could possess antibiotic properties and thus play a major role during antagonism. A literature survey on the genus Trichoderma has shown that different kinds of secondary metabolites were isolated earlier from the genus. These include flavonoids, triterpenoids, phenols such as 1hydroxy-3-methyl-anthraquinone; 1,8-dihydroxy-3-methyl-anthraquinone; harzianolide; butenolide; harzianopyridone; viridepyrone; massoilactone; koninginins; viridin; viridiofungines; trichostromaticines; harzianic acid (Keswani et al. 2014;Vinale et al. 2014). Recently, other classes of compounds such as sorbicillinoids (Ma et al. 2021), dithiodiketopiperazine derivatives (Harwoko et al. 2021), cyclopentenone derivative (Yin et al. 2021;Zhu et al. 2021), octahydronaphthalene derivatives (Sofian et al. 2021), a-pyrone and decalin derivatives (Nuansri et al. 2021) have been reported from Trichoderma.
In our laboratory, preliminary research has shown that many species of Trichoderma are antagonists of Pythium ultimum, one of the most responsible agents of common beans root rot (Bedine et al. 2020). Among them, Trichoderma erinaceum was the most effective. In addition, the organic extract of this antagonist fungus significantly inhibited mycelial growth and conidial germination of P. ultimum. However, to the best of our knowledge, there is no report on the chemical composition of its organic extract. Here we report the isolation and structural elucidation of a new compound (Z)-5amino-5-(1,1,2-trihydroxybuta-1,3-dienyloxy)pentane-6,7,8,9-tetraol (1) together with five known compounds ( Figure 1) from the isolated endophytic fungus T. erinaceum (IT-58). The isolated compounds as well as the endophytic fungal extract were tested for their inhibitory efficacies against P. ultimum.

Results and discussion
Compound 1 was isolated as a white paste and its molecular formula was determined to be C 9 H 17 O 8 N by ESI-HRMS data (m/z 268.1041 [M þ H] þ , calcd. for C 9 H 18 O 8 N þ , 268.1027) indicating two degrees of unsaturation. The 1 D NMR data in combination with the HSQC spectrum revealed a total of nine carbon atoms: one methylene, six methines, and two quaternary carbons (Experimental, Figures S3ÀS6, supplementary material). The 1 H NMR spectrum of compound 1 (Experimental, Table S1, supplementary material) exhibited two cis-coupled protons at d H 5.64 (1H, d, J ¼ 8.0 Hz, H-3) and 7.89 (1H, d, J ¼ 8.0 Hz, H-4). Six protons at d H 5.78 (1H, d, J ¼ 5.5 Hz, H-5), 4.02 (1H, br s, H-6), 3.96 (1H, br s, H-7), 3.84 (1H, q, J ¼ 3.4 Hz, H-8), 3.62 (1H, m, H-9), and 3.54 (1H, m, H-9), linked to a carbon bonded to a high electronegative atom are also observed in the middle field of the 1 H NMR spectrum. The 1 HÀ 1 H COSY ( Figure S5, supplementary material) correlations of H-3 to H-4, H-5 to H-6, H-6 to H-7, H-7 to H-8 and H-8 to H 2 -9 identified a connectivity sequence in the chain. The presence of signals ranging from d C 61.3 to 88.1 indicated polyhydroxy carbons in the molecule. According to HMBC spectrum, strong HMBC correlations from cis-coupled proton at 7.89 (H-4) to C-1, C-2, C-5 and from H-3 (d H 5.64) to C-1 and C-4 were observed, determined the position of the double bonds at C-1 and C-3. The down field nature of C-1 (d C 163.6) indicated the presence of an additional hydroxyl group at this position. Similarly, the polyhydroxy saturated chain was established using COSY and HMBC correlations. Thus, the doublet proton at d H 5.78 (H-5) had COSY correlations with d H 4.02 (H-6) and HMBC correlations with C-3, C-4, C-6 and C-7. COSY correlation of H-8 with H-7 and H 2 -9 and HMBC correlation of H-8 with C-6, C-7, and C-9 also supporting the structure. Moreover, the amino group has to be placed at C-5 due to its characteristic up-field shift compared with analogous hydroxy carbon signal. Thus, the structure of compound 1 was elucidated as (Z)-5-amino-5-(1,1,2-trihydroxybuta-1,3-dienyloxy)pentane-6,7,8,9-tetraol.

Fungal material
Trichoderma erinaceum used in this study came from the core collection of the Laboratory of Biochemistry of the University of Douala. Molecular identification was done by analysis of the ITS region of its rDNA and the sequence data have been deposited at Gene Bank under the accession number KF040478 (Bedine et al. 2020).

Plant pathogen
Pythium ultimum used in this study was isolated from common bean showing the typical symptoms of root rot in Cameroon. The pathogen was purified according to the method describe by Agrios (2005) with some modifications. It was maintained on Potato Dextrose Agar (PDA) slants at room temperature and sub-cultured bimonthly. The details concerning the identification were previously reported by Bedine et al (2020) 3.2. General experimental procedures NMR experiments were performed with a Bruker Avance III spectrometer operating at 600 MHz ( 1 H) and 150 MHz ( 13 C) using standard pulse sequences and referenced to residual solvent signals. High-resolution mass spectra (ESI-HRMS) were carried out on a LTQ Orbitrap spectrometer (Thermo Scientific, USA) equipped with a HESI-II source. The spectrometer was operated in positive mode with a nominal mass resolving power of 60,000 at m/z 400 with a scan rate of 1 Hz under following parameters: spray voltage 6 kV, capillary temperature 300 C, tube lens 100 V. Ar served as collision gas and N2 was used as sheath gas (66 arbitrary units) and auxiliary gas (8 arbitrary units).
Semi-preparative HPLC was performed on Shimadzu LC-20AP pump equipped with DGU-20A5R degassing unit, a Shimadzu SPD-M20A detector, a Shimadzu SIL-20ACHT auto-sampler and a Phenomenex Gemini C18 column (10 Â 250 mm, 10 lm particle size) using a LabSolutions software system. Separation was achieved using MeOH (B) -H 2 O (A) (0.1% formic acid) gradient program. All the solvents used were of analytical grade.

Extraction and isolation
Five millimeter diameter plugs of T. erinaceum obtained from 3-days growing margins of PDA cultures were inoculated into 1000 mL conical flasks containing 500 mL of sterile potato dextrose broth (PDB). The cultures were incubated for 7 days at 30 C under shaking at 180 rpm, filtered under vacuum through Whatman paper (No. 4). The filtrates were stored at À20 C for 24 h and lyophilised for 7 days. The powder obtained was extracted exhaustively with ethyl acetate and organic fraction was concentrated by evaporating the solvent under reduce pressure at 40 C. The residue (4-5 mL of crude extract or pure component) was concentrated by using vacuum apparatus and diluted with 1 mL of methanol. The extract was subjected to semi-preparative HPLC with gradient of MeOH-H 2 O, from 5% to 70% methanol to afford compound 1-6.

Antimicrobial assay
The organic crude extract and the pure fractions were tested in vitro against Pythium ultimum. The inhibition of mycelia growth was performed using the food poisoning method. Five millimeters diameter plug from the margin of a 3-day-old pure culture of the pathogen was inoculated in the center of a 90-mm-diameter PDA plate. Ten microliters of the crude extracts and chromatographic pure fractions at concentrations ranging from 0.01 mg/mL to 200 mg/mL were applied on the top of each plug. The controls were made by applying 10 ml of ethyl acetate and the plates were incubated at 30 C for 5 days. The diameter of mycelia growth was measured daily, and the growth inhibition was evaluated according to the following formula: %I ¼ ((Do-d) -(D-d)/(Dod)) x 100 where %I is inhibiting percentage, Do diameter of control growth and D pathogen growth diameter, d the diameter of the pathogen plug (5 mm). Each treatment was consisted of 3 plates with three replicates and the experiment was repeated twice.

Statistical analysis
SPSS, 22.0 version was used to analyse all the data. The results were expressed as means. The comparison of the mean values was done by using one-way parametric ANOVA (Analysis of Variance) at p 0.05.