1. Introduction
The use of microbes for pest management in agriculture is one of the most effective biological control strategies. The beneficial effects are strain dependent and the advantages for the associated plant include the suppression of pathogens by using a variety of mechanisms (
i.e., antibiosis, parasitism, competition for nutrients,
etc.), the promotion of plant growth and the improvement of host resistance to both biotic and abiotic stresses [
1,
2,
3].
Secondary metabolites are chemically different natural compounds of relatively low molecular weight (in most cases < 3 kDa), that are mainly produced by microorganisms and plants, and typically associated to individual genera, species or strains. They are biosynthesized along specialized pathways from primary metabolites, exhibit a wide range of biological activities and play an important role in regulating interactions between organisms [
4]. Included in this group are antibiotics, which are natural products capable of inhibiting or killing microbial competitors [
5,
6].
In fungi, the production of secondary metabolites has been often correlated to specific stages of morphological differentiation, and associated to the phase of active growth [
7]. Interestingly, some fungal secondary metabolites can modify the growth and the metabolism of plants, while others seem to target specific fungal processes such as sporulation and hyphal elongation [
7]. Thus, the expression of secondary metabolites may occur at a predictable point during the normal life cycle of some fungi, including those used for agriculture applications [
7].
Some fungi of the genus
Trichoderma may act as symbionts of plants, and are presently marketed as biopesticides and biofertilizers due to their ability to protect crops and promote vegetative growth [
1,
2,
3]. These microbes are well known producers of secondary metabolites with different biological activities [
8,
9,
10]. The production of such compounds varies according to the strain and in relation to the equilibrium between elicited biosynthesis and biotransformation rates (or degradation by other microbes) [
11].
In this work we report the isolation and the characterization of a new metabolite named isoharzianic acid (iso-HA), a stereoisomer of harzianic acid, from the culture filtrate of a T. harzianum strain isolated from decomposing hardwood bark. The biological activity of this metabolite was investigated both in vitro against the fungal pathogens Sclerotinia sclerotiorum and Rhizoctonia solani and in vivo in terms of plant growth promotion and induction of disease resistance. Moreover, the influence of plant tissue on the production of HA and iso-HA has been also examined.
2. Results and Discussion
T. harzianum culture filtrate was extracted exhaustively with ethyl acetate to give a red-brown residue from which HA (
1) and iso-HA (
2) (
Figure 1) were isolated after RP-18 vacuum chromatography or semi-preparative HPLC (
Figure 2). The structure of
1 was determined by comparison of its NMR spectroscopic data with those of an authentic standard [
12,
13]. The absolute configuration of
1, determined by X-ray diffraction studies, its antibiotic activity and plant growth promotion effect have been reported in a previous study [
13]. Recently, we demonstrated the ability of this tetramic acid to bind with a good affinity essential metals such as Fe
3+, thus representing a previously unrecognized siderophore [
14].
Figure 1.
Chemical structures of (1) HA; (2) iso-HA.
Figure 1.
Chemical structures of (1) HA; (2) iso-HA.
Figure 2.
Chromatogram of T. harzianum extract, as monitored by HPLC-DAD at 360 nm (10 to 20 min).
Figure 2.
Chromatogram of T. harzianum extract, as monitored by HPLC-DAD at 360 nm (10 to 20 min).
iso-HA was obtained as a yellow solid and its ESI-MS/MS spectrum showed a molecular ion peak at 366.1909
m/z corresponding to C
19H
27NO
6. HA (
1) and iso-HA (
2) had similar mass spectra with molecular ions at
m/z 366 ([M+H]
+) and main fragments at
m/z 320 ([M+H-HCO
2H]
+), 224 and 138. Interestingly, HA showed an extra peak at 348
m/z ([M+H-H
2O]
+). The optical rotation of iso-HA is [α]
D −15 (c 1.1, MeOH),while is [α]
D +16 (c 1.06, MeOH) for HA [
12].
The
1H- and
13C-NMR spectra of these two compounds (
Table 1) showed high similarities. The analyses of mono- and bidimensional NMR spectra showed that these two metabolites have the same signals except H-5' and H-6', suggesting a different stereochemistry of C5 and C6. NOESY experiments were performed to confirm this hypothesis and allowed to determine the configuration of the two stereoisomers. In particular, NOESY experiments revealed, in case of HA, a through-space correlation between the H-5' and H-6' b protons, which, conversely, was not detected in case of iso-HA. However, no other different correlations between HA and iso-HA were observed, thus suggesting the different orientation of H-5'.
Table 1.
1H- and 13C-NMR spectral data of HA and iso-HA (in CD3OD).
Table 1.
1H- and 13C-NMR spectral data of HA and iso-HA (in CD3OD).
Position | HA (1) | iso-HA (2) |
---|
δ 13C | δ 1H | Multi | J (Hz) | δ 13C | δ 1H | Multi | J (Hz) |
---|
1 | 174.0 | _ | | _ | 174.0 | _ | | _ |
2 | 119.3 | 7.0 | d | 15.6 | 119.4 | 7.05 | d | 15.25 |
3 | 146.2 | 7.57 | m | _ | 146.2 | 7.47 | dd | 10.17; 5.4 |
4 | 129.7 | 6.35 | m | _ | 129.7 | 6.35 | m | _ |
5 | 148.5 | 6.30 | m | _ | 148.5 | 6.30 | m | _ |
6 | 35.4 | 2.19 | m | _ | 35.4 | 2.19 | m | _ |
7 | 21.8 | 1.50 | m | _ | 21.8 | 1.50 | m | _ |
8 | 13.7 | 0.93 | m | _ | 13.7 | 0.93 | m | _ |
2' | 174.0 | _ | | _ | 174.0 | _ | | _ |
3' | 99.6 | _ | q | _ | 99.5 | _ | q | _ |
4' | 195.0 | _ | | _ | 195.1 | _ | | _ |
5' | 63.7 | 3.62 | dd | 1.17, 9.3 | 63.6 | 3.80 | dd | 2.7, 4.6 |
6'a | 34.9 | 2.20 | dd | | 34.8 | 2.2 | c | |
6'b | | 2.51 | | | | 2.2 | | |
7' | 78.1 | _ | q | _ | 78.0 | _ | q | _ |
8' | 35.9 | 2.02 | m | _ | 35.8 | 2.02 | m | _ |
9' | 17.2 | 0.98 | m | _ | 17.2 | 0.98 | m | _ |
10' | 16.4 | 0.99 | m | _ | 16.4 | 0.99 | m | _ |
11' | 27.4 | 2.99 | s | _ | 27.3 | 2.98 | s | _ |
12' | 176.8 | _ | | _ | 176.7 | _ | | _ |
All these data implies that the metabolite 2-hydroxy-2-[4-(1-hydroxyocta-2,4-dienylidene)-1-methyl-3,5-dioxopyrrolidin-2-ylmethyl]-3-methylbutyric acid named iso-HA is a diastereoisomer of HA with the stereochemistry reported in
Figure 1. The difference between these two tetramic acids determines their different chemical and physical characteristics (UV λ
max nm (log ε) HA = 344 (3.11), iso HA = 340 (4.04), different solubility in organic solvents).
In vitro assays were performed to assess the iso-HA antibiotic activity. This compound, at concentration of 10
−3 M, inhibited the growth of the phytopathogenic agents
R. solani and
S. sclerotiorum of about 40% and 20%, respectively, while at concentrations of 10
−4 M and 10
−5 M it showed lower effects compared to untreated control (
Figure 3). No significant growth inhibitions were observed with other two fungal pathogens,
Botrytis cinerea and
Phytium ultimum (data not shown).
Figure 3.
Antibiotic activity of iso-HA at different concentrations on Rhizoctonia solani (Rhizoctonia -■-) and Sclerotinia sclerotiorum (Scletotinia -♦-). % Inhibition of radial growth.
Figure 3.
Antibiotic activity of iso-HA at different concentrations on Rhizoctonia solani (Rhizoctonia -■-) and Sclerotinia sclerotiorum (Scletotinia -♦-). % Inhibition of radial growth.
The
in vitro effect of iso-HA and HA on tomato growth was evaluated in terms of seed germination, stem and root lengths (
Figure 4). Both metabolites promoted seed germination (
Table 2) and plant growth, with an increase of 35% in stem length (iso-HA and HA 10
−7 M) and of 65% in root length (iso-HA and HA 10
−7 M). Interestingly, iso-HA enhanced the plant fresh weight at 10
−6 and 10
−7 M, while small differences were observed with HA treatments.
Figure 4.
In vitro plant growth promotion effects of iso-HA (10−5, 10−6, 10−7, 10−8 and 10−9 M) on tomato seedlings. (a) Stem and (b) root lengths. Values indicates the % increase of growth as compared to untreated control plants. Each bar is the mean ± the standard deviation.
Figure 4.
In vitro plant growth promotion effects of iso-HA (10−5, 10−6, 10−7, 10−8 and 10−9 M) on tomato seedlings. (a) Stem and (b) root lengths. Values indicates the % increase of growth as compared to untreated control plants. Each bar is the mean ± the standard deviation.
Table 2.
In vitro effect of iso-HA and HA on tomato seed germination (12, 24, 36, 48 h after sowing). DS = standard deviation.
Table 2.
In vitro effect of iso-HA and HA on tomato seed germination (12, 24, 36, 48 h after sowing). DS = standard deviation.
% of Germination |
---|
Treatment | 12 h | DS | 24 h | DS | 36 h | DS | 48 h | DS |
---|
Control | 0% | 0% | 0% | 0% | 55% | 3.6% | 100% | 0% |
HA 10−5 M | 0% | 0% | 50% | 3.9% | 100% | 0% | 100% | 0% |
HA 10−6 M | 0% | 0% | 72% | 4.1% | 88% | 3.4% | 100% | 0% |
HA 10−7 M | 0% | 0% | 55% | 3.1% | 100% | 0% | 100% | 0% |
HA 10−8 M | 0% | 0% | 61% | 7.3% | 76% | 12.3% | 100% | 0% |
HA 10−9 M | 0% | 0% | 56% | 7.9% | 63% | 11.1% | 100% | 0% |
iso-HA 10−5 M | 0% | 0% | 72% | 3.9% | 100% | 0% | 100% | 0% |
iso-HA 10−6 M | 0% | 0% | 66% | 5.2% | 100% | 0% | 100% | 0% |
iso-HA 10−7 M | 0% | 0% | 88% | 5.6% | 100% | 0% | 100% | 0% |
iso-HA 10−8 M | 0% | 0% | 62% | 2.6% | 83% | 11.8% | 100% | 0% |
iso-HA 10−9 M | 0% | 0% | 56% | 2.8% | 67% | 3.8% | 100% | 0% |
In vivo treatment of tomato plants with iso-HA increased stem length by 22%, 35% and 19% at concentrations of 10
−5, 10
−6, 10
−7 M, respectively, compared to untreated control (
Figure 5). Moreover, the ability of iso-HA to induce systemic resistance against
B. cinerea was evaluated
. A reduction of the necrotic area (90%) caused by the pathogen was observed 48 h after a drench application of iso-HA at 10
−5 M (
Figure 6).
Some
Trichoderma strains produce compounds that can cause substantial changes in the metabolism of the host plant [
10,
15]. The involvement of secondary metabolites in the ability of
Trichoderma spp. to activate plant defence mechanisms and regulate plant growth has been investigated [
16,
17]. HA is a natural product that demonstrates antifungal and plant growth promoting activities [
13]. In this paper we indicate that iso-HA is an antifungal compound and also an inducer of plant disease resistance.
Figure 5.
In vivo plant growth promotion effects of iso-HA on tomato. (Left) untreated control; (Right) plant treated with iso-HA 10−6 M.
Figure 5.
In vivo plant growth promotion effects of iso-HA on tomato. (Left) untreated control; (Right) plant treated with iso-HA 10−6 M.
Figure 6.
Induction of disease resistance against B. cinerea. Plants were drenched with iso-HA at different concentrations (10−5, 10−6, 10−7 M). Each bar is the mean ± the standard deviation. Treatments with the same letter are not significantly different (p < 0.05).
Figure 6.
Induction of disease resistance against B. cinerea. Plants were drenched with iso-HA at different concentrations (10−5, 10−6, 10−7 M). Each bar is the mean ± the standard deviation. Treatments with the same letter are not significantly different (p < 0.05).
In order to test if the presence of plant may influence the production of HA and iso-HA in
T. harzianum, tomato tissue was added to the cultivation media (PDB, PDB 1/5 strength and SM) in order to mimic the composition of a natural substrate or a naturally occurring plant-microbe interaction. The presence of tomato plant modulated the production of the tetramic acid derivatives as reported in
Figure 7. For both metabolites, the production was significantly higher in potato dextrose broth (PDB—full and 1/5 strength) compared to the salt medium. The biosynthesis of HA was elicited by tomato tissue in PDB (both full and 1/5 strength). On the contrary, this was not observed for iso HA, whose accumulation was reduced by the presence of plant tissue added in the cultivation substrate. However no significant differences were observed in salt medium amended or not with tomato tissue (data not shown). Moreover, HA and iso-HA were not detected in the mycelium extracts (data not shown).
Interestingly, the dual culture of
T. harzianum and calli of
Catharathus roseus produced another tetramic acid compound named trichosedin (
3 in
Figure 8), that was not produced in the single culture of
T. harzianum or
C. roseus callus [
18]. This fungal metabolite affects the root and shoot growth of several plant species [
19]. Dual cultures of a fungus and a plant provide a simple method of establishing plant-fungus interaction and allow isolation of metabolites induced by one of the system components. Previous studies also demonstrated that the production of secondary metabolites was induced by fungal cell wall material or by the presence of pathogens [
11].
Figure 7.
Production of HA (a) and iso-HA (b) in different media amended with tomato plant tissue. PDB = full PDB (-♦-); PDB 1/5 = 1/5 strength PDB (-▲-); PDB + plant tissue = full PDB amended with tomato plant tissue (-■-); PDB 1/5 + plant tissue = 1/5 strength PDB emended with tomato plant tissue (-x-); SM = salt medium with 1% glucose (-●-). Each point on the line is the mean ± the standard deviation of four independent biological replicates. Point on the line with the same letter are not significantly different; point on the line without the letter are significantly different (p < 0.05).
Figure 7.
Production of HA (a) and iso-HA (b) in different media amended with tomato plant tissue. PDB = full PDB (-♦-); PDB 1/5 = 1/5 strength PDB (-▲-); PDB + plant tissue = full PDB amended with tomato plant tissue (-■-); PDB 1/5 + plant tissue = 1/5 strength PDB emended with tomato plant tissue (-x-); SM = salt medium with 1% glucose (-●-). Each point on the line is the mean ± the standard deviation of four independent biological replicates. Point on the line with the same letter are not significantly different; point on the line without the letter are significantly different (p < 0.05).
Figure 8.
Chemical structure of trichosedin (3).
Figure 8.
Chemical structure of trichosedin (3).
3. Experimental Section
3.2. Fungal Strains
The phytopathogens R. solani, S. sclerotiorum, P. ultimum and Botrytis cinerea, as well as the antagonistic fungus, T. harzianum strain M10 were maintained on potato dextrose agar (PDA, Sigma, St Louis, MO, USA) at room temperature and sub-cultured bimonthly. Two 7-mm diameter plugs of T. harzianum, obtained from actively growing margins of PDA cultures, were inoculated into conical flasks containing 1.5 L of sterile potato dextrose broth (PDB). The stationary cultures were incubated for 30 days at 25 °C.
3.4. Antifungal Assay
Iso HA was tested against R. solani, S. sclerotiorum, P. ultimum and B. cinerea to evaluate its antifungal properties. Pathogen plugs (5-mm diameter) from growing edges of colonies were placed at the centre of Petri dishes containing PDA. Iso HA was assayed starting from a 10−2 M water solution. The pathogen growth was measured daily as colony diameter for ten days. Each treatment consisted of three replicates and the experiment was repeated twice.
3.7. Production of HA and Iso HA in Presence of Plant Tissue
The production of HA and iso HA by T. harzianum in liquid cultures amended with plant tissue was evaluated. Tomato seedlings (Solanum lycopersicum cv. San Marzano), 15 day-old, were harvested, surface sterilised with sodium hypochlorite 1% solution, homogenized and added to the substrate (PDB, 1/5 strenght PDB or salt medium +1% glucose) at a concentration of 10 g L−1. Four 7 mm diameter plugs of T. harzianum, obtained from actively growing margins of PDA cultures, were inoculated to 500 mL conical flasks containing 100 mL of medium. Culture broths of T. harzianum without amendment with plant were used as controls. The stationary cultures were incubated for 10, 20, 30 and 40 days at 25 °C and then filtered under vacuum. The filtered broths (0.2 μm) were subjected to HPLC/DAD analysis for the quantification of metabolites. Each treatment consisted of four replicates and the experiment was repeated twice. Data from the experiments were combined since statistical analysis determined homogeneity of variance (p ≤ 0.05).