Cremenolide, a new antifungal, 10-member lactone from Trichoderma cremeum with plant growth promotion activity

Abstract Trichoderma based products are considered an alternative to synthetic pesticides and fertilizers. These Trichoderma spp. are among the most studied and applied fungal BCAs in industry and agriculture and are known to secrete several secondary metabolites with different biological activities. The analysis of metabolic profiles (the ‘metabolome’) of Trichoderma species is complex because of the wide range of compounds produced and the molecular activities identified, including the recently determined role in the activation of plant resistance to biotic and abiotic stresses and growth promotion. A new 10-member lactone, but-2-enoic acid 7-acetoxy-6-hydroxy-2-methyl-10-oxo-5,6,7,8,9,10-hexahydro-2H-oxecin-5-yl ester, named cremenolide (1), has been isolated from culture filtrates of Trichoderma cremeum. The structure of cremenolide was determined by spectroscopic methods, including UV, MS, and 1D and 2D NMR analyses. In vitro tests showed that the purified compound inhibited the radial mycelium growth of Fusarium oxysporum, Botrytis cinerea and Rhizoctonia solani, and exerted a significant promotion of growth of tomato seedlings.


Introduction
Fungi of the genus Trichoderma have been extensively studied, and are used as bioactive components of biopesticides and biofertilisers . They have been ABSTRACT Trichoderma based products are considered an alternative to synthetic pesticides and fertilizers. These Trichoderma spp. are among the most studied and applied fungal BCAs in industry and agriculture and are known to secrete several secondary metabolites with different biological activities. The analysis of metabolic profiles (the 'metabolome') of Trichoderma species is complex because of the wide range of compounds produced and the molecular activities identified, including the recently determined role in the activation of plant resistance to biotic and abiotic stresses and growth promotion. A new 10-member lactone, but-2-enoic acid 7-acetoxy-6-hydroxy-2-methyl-10-oxo-5,6,7,8,9,10-hexahydro-2H-oxecin-5-yl ester, named cremenolide (1), has been isolated from culture filtrates of Trichoderma cremeum. The structure of cremenolide was determined by spectroscopic methods, including UV, MS, and 1D and 2D NMR analyses. In vitro tests showed that the purified compound inhibited the radial mycelium growth of Fusarium oxysporum, Botrytis cinerea and Rhizoctonia solani, and exerted a significant promotion of growth of tomato seedlings. applied for their beneficial effects on treated plant that include: antagonism of plant pathogens by using a variety of mechanisms (mycoparasitism or hyperparasitism; antibiosis; competition; cell wall-lytic enzyme activity) in addition to enhancement of plant growth, acquisition of soil nutrients and induction of plant defense responses (Harman 2000;Howell 2003;Harman et al. 2004;Marra et al. 2006;Vinale et al. 2008). The genus Trichoderma are well known producers of secondary metabolites that are biologically active on plants and fungal pathogens (Sivasithamparam & Ghisalberti 1998;Reino et al. 2008;Vinale et al. 2014;Ahluwalia et al. 2015). The production of Trichoderma secondary metabolites is strain dependent and varies in relation to the equilibrium between elicited biosynthesis and biotransformation rates ). in the search for new 'effectors' with antibiotic and plant growth promotion activities among Trichoderma, we have studied the metabolites of Trichoderma cremeum. From the ethyl acetate extracts of the culture filtrate, we have isolated a new 10-member lactone, but-2enoic acid 7-acetoxy-6-hydroxy-2-methyl- 10-oxo-5,6,7,8,9,10-hexahydro-2H-oxecin-5-yl ester, named cremenolide (1). This compound inhibited the mycelium radial growth of Fusarium oxysporum, Botrytis cinerea and Rhizoctonia solani, and promoted growth of tomato seedlings.

Results and discussion
The culture filtrate of T. cremeum was extracted with ethyl acetate and the residue recovered was fractionated by silica gel column chromatography (CC). Fraction 2 obtained 312 mg of a new metabolite named herein cremenolide (1), that represents the major compound of the crude extract. Cremenolide (1) (32 mg/L) was a colourless solid with a molecular ion [M] + at 327.1416 and 16 carbons ( 13 C NMR coupled with DePT spectra). With these data, the molecular formula was estimated to be C 16 70.53, 71.63, 77.17 and 78.50 and that one of the protons in the molecule is bound to oxygen (3CH 3 , 2CH 2 , 8CH, and 3 fully substituted C atoms). 1 H-1 H COSY, 1 H-13 C HSQC spectra and the chemical shift evaluation allowed identification of the cerinolactone fragments ( Figure 1).
The connectivity of the fragments was deduced by a long-range 1 H-13 C heterocorrelated experiment which was obtained with the HMBC. The correlation of C-1 with 9-H, 2-Ha, 2-Hb, 3-Ha, 3-Hb and of C-4 with 2-Ha, 2-Hb, 3-Ha, 3-Hb and 5-H indicated the presence of a 10-member lactone ring. The key cross peaks of C-4 with 2″-H, C-1″ with 4-H and C-1′ with 6-H allowed us to correlate the other two remaining spin systems to the lactone ring. Due to the chemical shift and to complete the number of hydrogen the C-5 is bounded with an -OH.
The configuration of cremenolide was evident from the NOeSY experiments on 1. The NOe correlation observed from the protons of the lactone ring and the methyl C-10 led to the relative stereochemistry of (1) that was established unambiguously, as shown in Figure  1. NOeSY also permitted to confirm the assignment performed for 5-H peak (4.5 ppm). Despite the apparent leaking of multiplicity, due to the minimised 1 H-1 H coupling resulting from the unfavourable dihedral angle formed with protons H-4 and H6, this peak was indeed vicinally coupled with these protons, as proved by TOCSY correlations and its relatively broadened shape. Moreover, the MM-2 energy calculation of cremenolide (41.253 kcal/ mol) presented the most stable conformational model that was completely in agreement with the NOe data.
Cremenolide (1) significantly inhibited, in a concentration-dependent manner, the mycelial growth of F. oxysporum, B. cinerea and R. solani. in particular, the most significant effects were registered on B. cinerea at 10 μg plug −1 (15% of inhibition - Figure 2). Tomato seedlings assays showed that (1) promotes plant growth in terms of root length and fresh weight at 10 and 1 ppm (no effect at 0.1 and 0.01 ppm). Moreover, no significant effect was registered on seedling height at all concentrations used (Figure 3).
10-Member lactones belonging to the polyketides class, have been recently isolated by Malmierca and coauthors (2015) from a tri5 gene-disrupted mutant of Trichoderma arundinaceum that lacks Harzianum A production. This microbe overproduced aspinolides B and C, which were not detected in the wild-type strain, and produced four new aspinolides (D-G). Aspinolide C had antibiotic effect against B. cinerea and Fusarium sporotrichioides, while  aspinolide B did not show any activity against these two plant pathogens (Malmierca et al. 2015). Aspinolides A-C were obtained also from the cultures of Aspergillus ochraceus but the antifungal activity of aspinolide A was not tested (Sun et al. 2012).
The treatment of tomatoes seedlings with 50 μg ml −1 of aspinolide B resulted in the proliferation of lateral roots, while aspinolide C caused a significant reduction in the number of lateral roots and seedling height as compared to control (Malmierca et al. 2015). Furthermore, higher concentration of aspinolide C (250 μg ml −1 ) caused a strong reduction in development of the plant. Finally, Aspinolide C induced expression of tomato SA-related genes, which indicates that it may play a role in activation of tomato defence-related genes (Malmierca et al. 2015).

General experimental procedures
CC was performed on silica gel (Merck silica gel 60 GF 254 ), and TLC with glass pre-coated silica gel GF 254 plates (Merck Kieselgel 60 GF 254 , 0.25 mm). The compounds were detected on TLC plates using UV light (254 or 366 nm) and/or by spraying the plates with 5% (v/v) H 2 SO 4 solution in etOH followed by heating at 110 °C for 10 min. electrospray mass spectra were recorded on a 6540 accurate-mass Q-TOF LC-MS instrument with a 1200 SL series HPLC (Agilent Technologies, Santa Clara, USA). The QTOF-MS instrument was operated with an electrospray ion source in positive ionisation mode.
isolated molecules were solubilised in 1000 μL of deuterated chloroform (99.8% CDCl 3 -Sigma Chemical Co) and transferred into a stoppered NMR tube (5 mm, 7″, 507-HP-7 -NORELL) where remaining void volume was gently degassed by a N 2 flux. Peak of chloroform was used as reference to calibrate both 1 H and 13 C axes.
A 400 MHz Bruker Avance spectrometer, equipped with a 5 mm Bruker Broad Band inverse probe, working at the 1 H and 13 C frequencies of 400.13 and 100.61 MHz, respectively, was employed to conduct all liquid state NMR measurements at a temperature of 298 ± 1 K.
1D 1 H and 13 C acquisitions were conducted as follows: proton spectrum was acquired with 2 s of thermal equilibrium delay, a 90° pulse length ranging between of 8.65 μs, 64 transients and 16 ppm (6410.26 kHz) as spectral width, whereas proton-decoupled carbon acquisitions were executed by both inverse gated and DePT 135° pulse sequences, adopting 7 and 4 s of equilibrium delay, respectively, as well as 12000 transients and a spectral width of 250 ppm (25.152 kHz). A time domain of 32768 points was adopted for all cited mono-dimensional experiments.
Homo-nuclear and hetero-nuclear 2D experiments were acquired with 48 scans, 16 dummy scans, a time domain of 2 k points (F2) and 256 experiments (F1). Moreover, TOCSY and NOeSY experiments were conducted with a mixing time of 80 and 800 ms, respectively, while HSQC and HMBC experiments were optimised accounting for a short and long-range J CH coupling of 145 and 10 Hz, respectively. All of 2D experiments were gradient enhanced, except for TOCSY and NOeSY experiments.
The free induction decay of mono-dimensional spectra was multiplied by an exponential factor corresponding to 0.1 and 1 Hz for 1 H and 13 C 1D experiments, respectively. each spectrum was baseline corrected and processed by using both Bruker Topspin Software (v.1.3) and MestReC NMR Processing Software (v.4.9.9.9).

Fungal strains
T. cremeum strain 506 was isolated from decaying wood in Central Poland (Warsaw). This isolate was identified by molecular identification with primers for the iTS1, iTS2 rRNA (internal transcribed region 1 and 2 of the rRNA gene cluster) and tef1 (translation elongation factor 1-alpha) sequences and kindly provided by Prof. Jerzy Chelkowski and Dr Lidia Blaszczyk of the institute of Plant Genetics of the Polish Academy of Sciences, Poznań, Poland. The pathogens F. oxysporum, B. cinerea and R. solani were isolated from field crops in italy. T. cremeum, F. oxysporum and R. solani were maintained on potato dextrose agar (PDA, High Media Pvt. Ltd., Mumbai-india) slants at room temperature and sub-cultured bimonthly. B. cinerea was maintained on malt extract peptone (MeP, SiGMA, St. Louis, MO., USA) slants at room temperature and subcultured bimonthly.

Liquid culture, extraction and metabolite isolation
Ten 7 mm diameter plugs of T. cremeum, from actively growing margins of PDA cultures, were inoculated in a 5 L conical flasks containing 1 L of sterile potato dextrose broth (PDB -High Media). The stationary cultures were incubated for 25 days at 25 °C, then the biomass was removed by filtration and the culture filtrate was collected.
The culture filtrate (10 L) was acidified to pH 4 with 5 M HCl and extracted exhaustively three times with etOAc to obtain the crude extract (dried with Na 2 SO 4 ; solvent evaporated under reduced pressure at 35 °C). The brown residue recovered (1.007 g) was fractionated by CC (silica gel; 300 g) eluted with etOAc. Twelve fractions were collected and pooled on the basis of similar TLC profiles. Fraction 2, yielded 1 (301,7 mg) a colourless solid (Rf 0.6 by silica gel, eluent system CH 2 Cl 2 /MeOH 98:2 v:v).

Antifungal assays
The purified compound 1 was tested against the fungal phytopathogenic agents F. oxysporum, B. cinerea and R. solani. The method described by Dunlop et al. (1989) was used with some modifications. Briefly, pathogen plugs (5 mm diameter) were placed at the centre of 1/5 PDA Petri dishes. Ten microliters of the purified compound was applied to the surface of each plug at concentrations ranging from 0.1 to 100 μg plug −1 . The controls were treated only with 10 μl of etOAc. The pathogen growth was evaluated daily by measuring the colony diameter (mm). each treatment consisted of three replicates and each experiment was repeated three times.

Plant growth promotion assay
To test the plant growth promotion activity, tomato seeds (Lycopersicum esculentum cv. San Marzano 823) were treated (coating) with different amounts of cremenolide (10-0.01 ppm corresponding to 100-0.1 ng per seed). The method described by Vinale, Flematti, et al. (2009) was used to determine the effect on seed germination and plant growth.

Conclusion
These results indicated that cremenolide can act as an antifungal compounds produced in the Trichoderma-pathogen interaction and as a plant growth promoters. Such compounds that affect plant metabolism or that can play a role in the environmental interactions established by beneficial microbes with other organisms present in the rhizosphere, may help the plant host to overcome the challenges from deleterious pathogens in agricultural production and help development of new biopesticides and biofertilisers formulations based on the bioactive metabolites.

Disclosure statement
No potential conflict of interest was reported by the authors.