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Received Date : 21-Jun-2013 Revised Date : 16-Jul-2013 Accepted Date : 28-Jul-2013 Article type : Research Letter Editor : Paolina Garbeva Title
Harzianic acid: a novel siderophore from Trichoderma harzianum Francesco Vinale*a, Marco Nigroa,b, Krishnapillai Sivasithamparamc, Gavin Flemattid, Emilio L. Ghisalbertid, Michelina Ruoccoa, Rosaria Varleseb, Roberta Marrab, Stefania Lanzuiseb, Ahmed Eidb, Sheridan L. Wooa,b and Matteo Loritoa,b. a
CNR – Istituto per la Protezione delle Piante (IPP-CNR) 80055 Portici, Italy, Dipartimento di Arboricoltura Botanica e Patologia Vegetale, Università degli Studi di Napoli ‘Federico II’, Portici, 80055 Naples, Italy c School of Earth and Environment - Faculty of Natural and Agricultural Sciences d School of Chemistry and Biochemistry - Faculty of Life and Physical Sciences, The University of Western Australia, W.A. 6009, Australia. b
* Corresponding author: Francesco Vinale Phone number: +39 081 2539338 Fax number: +39 081 2539339 e-mail address: [email protected] Via Università 133 – 80055 Portici (Naples) – Italy Running title: Harzianic acid, a T. harzianum siderophore Keywords: - Secondary metabolites - Harzianic acid - Iron (III) - Trichoderma - Siderophores
Abstract Agriculture-relevant microbes are considered to produce secondary metabolites during processes of competition with other micro- and macro-organisms, symbiosis, parasitism or This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6968.12231 This article is protected by copyright. All rights reserved.
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pathogenesis. Many different strains of the genus Trichoderma, in addition to a direct activity against phytopathogens, are well known producers of secondary metabolites and compounds that substantially affect the metabolism of the host plant. Harzianic acid is a Trichoderma secondary metabolite showing antifungal and plant growth promotion activities. This report demonstrates the ability of this tetramic acid to bind with a good affinity essential metals such as Fe3+, which may represent a mechanism of iron solubilisation that significantly alters nutrient availability in the soil environment for other microorganisms and the host plant.
Introduction Trichoderma spp. are well studied filamentous fungi commonly found in the soil community that are widely marketed as biopesticides, biofertilizers and soil amendments, due to their ability to protect plants, enhance vegetative growth and contain pathogen populations (Vinale et al., 2008a). To date, hundreds of different Trichoderma-based preparations are commercially used to protect and/or increase the productivity of various crops (Lorito et al., 2010). One factor that contributes to the beneficial biological activities exerted by Trichoderma strains is the wide variety of secondary metabolites that they are able to produce (Sivasithamparam & Ghisalberti, 1998; Reino et al., 2008). Fungal secondary metabolites may be considered a large and heterogeneous group of small molecules not directly essential for growth, but having an important role in signalling, development and interaction with other organisms (Song et al., 2006; Mukherjee et al., 2012). Trichoderma metabolites may act by directly inhibiting the growth of pathogens, or by indirectly triggering the defence system in the host plant, thus increasing disease resistance, and by promoting plant growth (Vinale et al., 2012). The accumulation of Trichoderma
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metabolites varies according to the species or the strain used and is related to the biosynthesis and biotransformation rates (Vinale et al., 2009a). Siderophores are low-molecular-weight metabolites produced for scavenging iron from the environment and have an high affinity for iron(III) (Hider & Kong, 2010). Fe3+-chelating molecules can be beneficial to plants because they can solubilise the iron which is otherwise unavailable and can suppress the growth of pathogenic microorganisms by depriving the pathogens of this necessary micronutrient (Leong, 1986). We have recently purified from the culture filtrate of a strain of T. harzianum a nitrogen heterocyclic compound named harzianic acid (HA) and demonstrated its growth promotion effect (Vinale et al., 2009b). Growth of different fungal pathogens and canola seedlings was shown to be affected by treatments with HA in a concentration dependent manner, suggesting a role of this metabolite in plant growth regulation, as well as in the antagonism against phytopatogenic agents. In this work we demonstrate that HA has a Fe3+ chelating activity. This secondary metabolite strongly binds essential metals such as iron, thus representing a previously unrecognized siderophore.
Materials and methods Microbial strain and culture conditions T. harzianum strain M10 was maintained on potato dextrose agar (PDA, SIGMA, St Louis, Mo., USA) slants at room temperature and sub-cultured bimonthly. Genomic DNA was isolated in order to analyse ribosomal DNA. Using PCR approach, were amplified fragments containing the internal transcribed spacer 1 (ITS1-partial sequence), the 5.8S ribosomal RNA gene and the internal transcribed spacer 2 (ITS2-complete sequence), by using primers SR6R (5’-AAGTAGAAGTCGTAACAAGG-3’) and LR1 (5’-GGTTGGTTTCTTTTCCT-3’).
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The PCR products were gel electrophoresed for quantification and assessment of PCR specificity and finally sequenced. Sequence analysis of the internal transcribed spacers (ITS) of the rDNA indicated 99% similarity with GenBank sequences of T. harzianum confirming the identity of this specie. Ten 7 mm diameter plugs of M10 strain obtained from actively growing margins of PDA cultures were inoculated to 5 L conical flasks containing 1 L of sterile potato dextrose broth (PDB, SIGMA). The stationary cultures were incubated for 21 days at 25° C. The cultures were filtered under vacuum through filter paper (Whatman No. 4, Brentford, UK).
Extraction and isolation of HA The filtered culture broth (2 L) was acidified to pH 4 with 5 M HCl and extracted exhaustively with ethyl acetate (EtOAc). The combined organic fraction was dried (Na2SO4) and evaporated in vacuo at 35 °C. The red residue recovered was dissolved in CHCl3 and extracted three times with 2 M NaOH. HA was then precipitated with 2 M HCl. The solid was recovered (135 mg), solubilised and subjected to RP-18 vacuum chromatography (20 g), eluting with a gradient of methanol (MeOH):H2O:CH3CN (1:8:1 to 10:0:0). After the separation, 45 mg of pure HA were collected. The homogeneity of pure pooled products was verified by analytical reverse-phase TLC (glass pre-coated Silica gel 60 RP-18 plates - Merck Kieselgel 60 TLC Silica gel 60 RP-18 F254s, 0.25 mm) using 3:4:3 CH3CN - MeOH -H2O as eluent (Rf of HA: 0.3). The compounds were detected on TLC plates using UV light (254 or 366 nm) and/or by spraying the plates with 5% (v/v) H2SO4 in EtOH followed by heating at 110 °C for 10 min. UV, IR, 1H NMR, 13C NMR and HR-FABMS were identical to those reported by Sawa et al. (1994). ESMS (+) m/z 753.3 [M2 + Na]+ , 404.2 [M + K]+, 388.2 [M + Na]+, 366.2 [M + H]+; 264.2 [M +H – C7H18]+. 1H and 13C NMR spectra were recorded with a Varian 400
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instrument operating at 400 (1H) and 125 (13C) MHz, using residual and deuterated solvent peaks as reference standards.
CAS agar plate assays The method to detect siderophore production was previously described by Schwyn & Neilands (1987). Orange halos around the colonies, growth on Chrome Azurol S (CAS) plates, are indicative of siderophore activity. CAS solution was also used for detection of siderophore production in culture filtrate (50 μl of culture was added to 950 μl of CAS solution, after reaching equilibrium the absorbance was measured at 630 nm). The CAS assay was also used to test the chelating properties of a 10-3 M HA solution in methanol. The CAS assay was modified according to Milagres et al. (1999) to test the ability of strain M10 to produce iron-binding compounds eventually avoiding the growth inhibition caused by the toxicity of the CAS-blue agar medium. Petri dishes (10 cm in diameter) were prepared with the Malt Extract Agar (MEA) medium. After solidification, the medium was cut into halves, one of which was replaced by CAS-blue agar. The halves containing culture medium (MEA) were inoculated with M10 plugs. The plates were incubated at 25°C for 6 days. P. fluorescens strain CHA0 (bacterial strain collection of prof A. Zoina - University of Naples) was used as a positive control (Maurhofer et al., 1994; Youard et al., 2007).
Iron binding affinity of HA In order to measure the iron binding affinity of HA, the method of Kaufmann et al. (2005) was used with some modifications. Stock solutions of ferric chloride (10 mM) and HA (10 mM) were prepared with 4:1 MeOH / 0.1 M NaOAc buffer solution (pH 7.4). Aliquots of both stock solutions were diluted and the absorbances of the formed complexes were
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measured at 290 nm in triplicate in the presence and absence of EDTA (10 mM and saturated solution).
LC/MS of HA–Fe(III) complex The Fe(III)-binding properties of the HA were investigated by adding 100 μl of a Fe(III) chloride solution (10 mM) to 100 μL of 10 mM HA in MeOH. The solution turned red and was directly infused into the LC/MS system at 5 μL/min using a syringe pump. Full-scans in the range m/z 100–1,200 were performed on a Bruker 6340 ion trap mass spectrometer equipped with an electrospray ionization source and operating in the positive ion mode. High resolution spectra were recorded using a Waters Alliance e2695 HPLC connected to a Waters LCT Premier XE mass spectrometer with an electrospray ionisation source (ESI). Samples were injected using the onboard injector in 10 µL injection volumes and eluted with 20% acetonitrile/water at a flow rate of 0.3 mL/min to the time-of-flight mass spectrometer. For the HA-Fe(III) complex, positive ESI-HRMS found m/z 491.0574 ([C19H27NO6FeCl2 ]+ requires 491.0565).
Plant growth promotion assay Tomato (Solanum lycopersicum cv. Roma) seeds were surface sterilized using 70% EtOH for 2 min, followed by 2% NaClO for 2 min, thoroughly washed with sterile distilled water, then placed on magenta box containing half-strength Murashige and Skoog salt (MS) medium (ICN Biomedicals, Irvine, California, USA) containing 1 % agar and 1.5% sucrose, adjusted to pH 5.7, and vernalized for 2 days at 4°C in the dark. Media with iron concentrations different from the standard 50 μM were prepared according to Murashige & Skoog (1962), except that defined amounts of Fe III were added (diluted 1/10 and 1/100 corresponding to 5 and 0.5 μM, respectively). Sterile solutions of HA were added to the substrate before the agar
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solidification to have final concentrations of 100 μM, 10 μM, 1 μM. Untreated substrates were used as controls. Seedlings were grown in a growth chamber (16 h photoperiod); the temperature was maintained at 25 ±1°C with a relative humidity of 65–75 %. Growth of stem height was measured daily. Each treatment consisted of twenty replicates and the experiment was repeated four times. At the end of each experiment, the whole plants were dried and weighed. Data from the experiments were combined since statistical analysis determined homogeneity of variance (P ≤ 0.05). Plant tissues (50–100 mg) were ground with liquid N2, mineralized according to Beinert (1978) and the iron contents were determined using Atomic Absorption analysis.
Results Purification and identification of HA T. harzianum M10 was grown in PDB and the culture filtrate was extracted with ethyl acetate, from which HA was purified as described above. The molecular mass of this compound, as determined by electrospray ionization-mass spectrometry, was 365 Da, and its pattern corresponded to that of 2-hydroxy-2-[4-(1-hydroxy-octa-2,4-dienylidene)-1-methyl3,5-dioxo-pyrrolidin-2-ylmethyl]-3-methyl-butyric acid (Figure 1) described by Sawa et al. (1994) and Vinale et al. (2009b). The HA structure was confirmed by nuclear magnetic resonance.
Iron(III) binding activity of M10 and HA The assay described by Schwyn & Neilands (1997) and Millagres et al. (1999) was used for the detection of siderophores released in the substrate by M10 strain. The fungus grew on CAS blue agar and the iron(III) chelating compounds, excreted by the microorganism, diffused through the medium producing a colour change from blue to orange. Purified HA
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decolorizes CAS blue agar, indicating that it could form a complex with Fe(III) (Figure 2). While the free compound in aqueous solution is pale orange, addition of Fe(III) results in the appearance of a red colour, indicating that an iron complex is formed.
Characterization of Fe(III)-HA complex To investigate the formed Fe(III)-HA complexes, experiments were carried out adding FeCl3 to a solution of HA. The uncomplexed HA molecule is detected as [M+H]+ (m/z 366.2), [M+Na]+ (m/z 388.2), [M+K]+ (m/z 404.2) and [M2 + Na]+ (m/z 753.3) (Figure 1-A). After adding Fe(III), the spectrum showed additional signals at m/z 455.1 and m/z 491.1 corresponding to two 1:1 Fe(III) containing complexes which were assigned the following signals based on the chloride isotope pattern, [M-H+Fe(III)+Cl2+H]+ (m/z 491.1) or [M2H+Fe(III)+Cl+H]+ (m/z 455.1), as determined by isotopic distribution. The resulting mass spectrum is depicted in Figure 1-B. High resolution spectrum of the Fe(III)-HAcomplex showed signals at m/z 491.0574 ([C19H27NO6FeCl2 ]+ requires 491.0565), confirming the 1:1 HA–Fe complex.
The apparent binding constant (Kd,app) was determined in order to measure the affinity of HA for iron (III). These experiments were performed by using a previously described protocol based on the competition between HA and EDTA for iron and the detection of the complexes by measuring the characteristic absorption (Wang et al., 2002). The loss of Fe(III)-HA absorbance at 340 nm upon addition of EDTA was used to calculate the equilibrium constant, presuming the formation of a 1:1 Fe-HAcomplex, according to the following equation: EDTA-Fe + HA = EDTA + Fe-HA Keq = [EDTA][Fe-HA] / [HA][EDTA-Fe] = Kd;EDTA / Kd;HA
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By using the known affinity of EDTA for Fe(III) (5.00 x 10-23 M), we were able to determine the relative affinity (Kd,app) of HA for Fe3+ to be 1.79 x 10-25 M.
Plant growth promotion The activity of HA as plant growth promoter was evaluated by analyzing the effect on seed germination and measuring lengths, fresh or dry weights of tomato seedlings treated with metabolite solutions as compared to controls.
Seed germination was significantly affected by HA applications. In particular, when 100 or 10 μM HA solutions were used, the percentages of germinated seeds were 4-5 times higher than in control (Table 1). Shoot and root growth of tomato seedlings was enhanced by treatments with HA used at different concentrations. The highest increase of shoot and root length (+76% and +66%, respectively) was registered using 10 μM of HA compared to control (Figure 3). A promotion effect on both fresh and dry plant weights was observed using HA at 10 and 1 μM. Conversely, a higher concentration of HA (100 μM) did not significantly affect plant weights or seedling lengths compared to the untreated control (Table 1, Figure 3).
To further characterize the role of HA in iron availability, plants were supplemented with low concentrations of iron (5 and 0.5 μM). Root length of tomato seedlings grown in media containing the lowest iron concentration was improved by treatments with both 10 and 1 μM HA; conversely, no significant effect was detected in shoot growth (Figure 4-A) or in plant weight in comparison to control (data not shown). However, in these growing conditions, iron levels increased in plants treated with HA, particularly when 1μM HA was used (Figure 4-B).
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Discussion The interaction of Trichoderma spp. with plants confers several benefits for the associated host that include: i) the suppression of phytopathogens by using direct antagonistic mechanisms (i.e. antibiosis, mycoparasitism, competition for nutrient and space); ii) plant growth promotion; iii) enhanced nutrient availability and uptake, and iv) induction of planthost resistance (Harman et al., 2004).
In addition, some Trichoderma strains produce compounds that can cause substantial changes in the metabolism of the host plant. The involvement of secondary metabolites in the ability of Trichoderma spp. to activate plant defence mechanisms and regulate plant growth has been investigated (Vinale et al., 2008b). HA is one of these natural products that shows antifungal and plant growth promoting activities (Vinale et al., 2009b). In the present study we investigated the Fe+3 chelating properties of HA.
HA, isolated by RP-18 vacuum chromatography from a 2M NaOH 2 M extract, showed the same 1H and 13C parameters as those previously reported for 2-hydroxy-2-[4-(1-hydroxyocta-2,4-dienylidene)-1-methyl-3,5-dioxo-pyrrolidin-2-ylmethyl]-3-methyl-butyric acid, a compound belonging to the chemical class of tetramic acids. The naturally occurring tetramic acids derivatives have attracted significant attention because these metabolites have a wide distribution and have been found to display a remarkable diversity of biological activities playing a significant role in ecological interactions. These activities may be enhanced significantly through the chelation of the tetramic acid nucleus with metal ions (important for transport across membranes in biological tissues). It was found that in some cases the metal complexes obtained revealed higher biological activity than their ligands (Ghisalberti, 2003; Schobert & Schlenk, 2008; Athanasellis et al., 2010).
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Both the living microorganism and the isolated metabolite HA when tested in the CAS blue agar plates caused a colour change of the substrate from blue to orange indicating that the iron(III) binding properties of the fungus is related also to the production of the tetramic acid derivative. Moreover, the LC-MS analysis of a HA – Fe+3 solution showed additional signals at 455.1 m/z and 491.1 m/z corresponding to a 1:1 chloride containing complex, [MH+Fe(III)+Cl2+H]+ (m/z 491.1) or [M-2H+Fe(III)+Cl+H]+ (m/z 455.1). Since chloride is a coordinating ligand for iron, it is possible that the chloride anion is directly bound to the metal (Caudle et al., 1994).
The value of Kd of Fe – HA complex (1.79 x 10-25 M) may be directly compared with that of other chelators showing a 1:1 Fe:ligand stoichiometry, such as desferrioxamine (DFO), EDTA, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (HPD), pyoverdin and pyochelin (Kaufmann et al., 2005). As shown in Table 2, HA has lower affinity to Fe(III) than DFO, pyoverdin and HPD, while it has a stronger affinity for iron(III) than EDTA and pyochelin. This data suggest that HA could compete for available iron in solution and provide a method for iron solubilization.
Microbial siderophores are used as iron chelating agents that can regulate the availability of iron in the plant rhizosphere. It has been assumed that competition for iron in the rhizosphere is controlled by the affinity of the siderophore for iron. The important factors, which participate, are concentration of various types of siderophores, kinetics of exchange, and availability of Fe-complexes to microbes as well as plants. Siderophores produced by beneficial agents may have important effects on both microbial and plant nutrition. Fe3+ siderophore complexes can be recognized and taken up by several plant species and this
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process is considered crucial for plant iron uptake, particularly in calcareous soils (Sharma et al., 2003). Trichoderma metabolites may help the plant to withstand pathogens by both promoting the growth and development of root and shoot systems, and stimulating the defence mechanisms (Vinale et al., 2008b). In the present work we confirm a possible involvement of HA in plant growth regulation probably because of its Fe(III) binding activity. In fact, HA affected the germination of tomato seeds and improved the growth of the seedlings even in iron deficient conditions (increment of iron concentration into the plants was also registered). This finding supports the hypothesis that HA actively influences the growth of Trichoderma-colonized plants.
The isolation and application of bioactive compounds produced by beneficial microbes responsible for the desired positive effects on plants may be a promising alternative to the use of living antagonists.
Acknowledgments The authors thank Davide Stellitano, Gelsomina Maganiello and Alberto Pascale for their technical assistance. The authors thank Antonio De Martino for the iron concentration measurements. Work by the authors FV and MR was supported by a dedicated grant from the Italian Ministry of Economy and Finance to the National Research Council for the project "Innovazione e Sviluppo del Mezzogiorno - Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy Agroalimentare - Legge n. 191/2009". Work by the authors MN, RS, RM, SL, AE, SW and ML has been supported by the following projects: “S.Re.Va.Pr.O.”, “CA.SVI.PR.OLI.” “MI.P.RE.VEGE.”; PRIN MIUR 2008 - prot. 2008SNPNC2 - prot. 2008WKPAWW. The authors also acknowledge the facilities, scientific and technical assistance of the Centre for Microscopy, Characterisation & Analysis at the University of Western Australia.
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References
Athanasellis G, Igglessi-Markopoulou O & Markopoulos J (2010) Tetramic and tetronic acids as scaffolds in bioinorganic and bioorganic chemistry. Bioinorg Chem Appl 2010: 1-11. Beinert H (1978) Micro methods for the quantitative determination of iron and copper in biological material. Methods Enzymol 54: 435–445. Caudle MT, Stevens RD & Crumbliss AL (1994) Electrospray mass spectrometry study of 1:1 ferric dihydroxamates. Inorg Chem 33: 843-844. Chiang Y, Lee K, Sanchez JF, Keller NP & Wang CCC (2009) Unlocking fungal cryptic natural products. Nat Prod Commun 4: 1505–1510. Ghisalberti EL (2003) Bioactive tetramic acid metabolites. In: Atta-ur Rahman, Editor(s), Studies in Natural Products Chemistry, Elsevier, Vol 28/1, pp 109-163. Harman GE, Howell CR, Viterbo A, Chet I & Lorito M (2004) Trichoderma species opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2: 43-56. Hider RC & Kong X (2010) Chemistry and biology of siderophores. Nat Prod Rep 27: 637657. Kaufmann GF, Sartorio R, Lee SH, Rogers CJ, Meijler MM, Moyss JA, Clapham B, Brogan AP, Dickerson TJ & Janda KD (2005) Revisiting quorum sensing: Discovery of additional chemical and biological functions for 3-oxo-N-acylhomoserine lactones. Proc Natl Acad Sci USA 102: 309 - 314. Youard ZA, Mislin GL, Majcherczyk PA, Schalk IJ & Reimmann C (2007) Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin. J Biol Chem 282: 35546-35553. Leong J (1986) Siderophores : their biochemistry and possible role in the biocontrol of plant pathogens. Annu Rev Phytopathol 24: 187–209.
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Lorito M, Woo SL, Harman GE & Monte E (2010) Translation research on Trichoderma: from ‘omics to the field. Annu Rev Phytopathol 48: 395-417. Maurhofer M, Hase C, Meuwly P, Metraux JP & Defago G (1994) Induction of systemic resistance of tobacco to tobacco necrosis virus by the root-colonizing Pseudomonas fluorescens strain CHA0: influence of the gacA gene and of pyoverdine production. Phytopathology 84: 139-146. Milagres AMF, Machuca A & Napoleão D (1999) Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay. J Microbiol Methods 37: 1–6 Mukherjee PK, Horwitz BA & Kenerley CM (2012) Secondary metabolism in Trichoderma – a genomic perspective. Microbiology 158: 35–45. Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497. Reino JL, Guerriero RF, Hernàndez-Galà R & Collado IG (2008) Secondary metabolites from species of the biocontrol agent Trichoderma. Phytochem Rev 7: 89-123. Sawa R, Mori Y, Iinuma H, Naganawa H, Hamada M, Yoshida S, Furutani H, Kajimura Y, Fuwa, T & Takeuchi T (1994) Harzianic acid, a new antimicrobial antibiotic from a fungus. J Antibiot 47: 731–732. Schobert R & Schlenk A (2008) Tetramic and tetronic acids: an update on new derivatives and biological aspects. Bioorg Med Chem 16: 4203–4221. Schwyn B & Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160: 47-56. Sharma A, Johri BN, Sharma AK & Glick BR (2003) Plant growth promoting bacterium Pseudomonas sp., strain GFP(3) influences iron acquisition in mung bean (Vigna radiata L. Wilzeck). Soil Biol Biochem 35: 887–894.
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Sivasithamparam K & Ghisalberti EL In Trichoderma and Gliocladium; Harman, G. E., Kubicek, C. P., Eds.; Taylor and Francis Ltd: London, 1998; Vol. 1, pp 139-191. Song XY, Shen QTXT, Chen XL, Sun CY & Zhang YZ (2006) Broad-spectrum antimicrobial activity and high stability of trichokonins from Trichoderma koningii SMF2 against plant pathogens. FEMS Microbiol Lett 260: 119–125. Vinale F, Sivasithamparam K, Ghisalberti EL, Marra R, Woo SL & Lorito M (2008a) Trichoderma – plant – pathogen interactions. Soil Biol Biochem 40: 1–10. Vinale F, Sivasithamparam K, Ghisalberti EL, Marra R, Barbetti MJ, Li H, Woo SL & Lorito M (2008b) A novel role for Trichoderma secondary metabolites in the interactions with plants. Physiol Mol Plant Pathol 72: 80–86. Vinale F, Ghisalberti EL, Sivasithamparam K, Marra R, Ritieni A, Ferracane R, Woo SL & Lorito M (2009a) Factors affecting the production of Trichoderma harzianum secondary metabolites during the interaction with different plant pathogens. Lett Appl Microbiol 48: 705–711. Vinale F, Flematti G, Sivasithamparam K, Lorito M, Marra R, Skelton BW & Ghisalberti EL (2009b) Harzianic acid, an antifungal and plant growth promoting metabolite from Trichoderma harzianum. J Nat Prod 72: 2032–2035. Vinale F, Sivasithamparam K, Ghisalberti EL, Ruocco M, Woo S & Lorito M (2012) Trichoderma secondary metabolites that affect plant metabolism. Nat Prod Commun 7: 1545 – 1550. Wang J, Buss JL, Chen G, Ponka P & Pantopoulos K (2002) The prolyl 4-hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate generates effective iron deficiency in cultured cells. FEBS Lett 529: 309–312.
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Table 1: Growth promotion of tomato seedlings treated with HA at different concentrations fresh and dry weights 12 days after treatment; germination 2 days after treatment). Values are means of ten replicates. SD: ±Standard Deviation. Values with the same letter do not differ significantly (P < 0.05).
Treatment
Fresh weight (mg) SD Dry weight (mg) SD % of Germination SD
Control
46.9 a
8
5.4 a
0.4
16.5 a
4.2
HA 100 μM
59.8 a
7
6.3 a, b
0.6
88.0 b
3.5
HA 10 μM
73.7 b
6
6.6 b
0.5
71.5 b
4.1
HA 1 μM
72.5 b
8
6.5 b
0.7
27.5 c
1.9
Table 2. Affinity constants of chelators for Fe(III). Compound EDTA DFO Pyoverdin Pyochelin HPD HA
Kd 5.00 x 10-23 M 2.51 x 10-26 M 10-32 M 10-5 M 1.6 x 10-29 M 1.79 x 10-25 M
Figure legends Figure 1: Characterization of HA-Fe(III) complex by LC/MS. (A) Harzianic Acid (HA) and its mass spectrum ;(B) mass spectrum of HA-Fe(III).
Figure 2: CAS-blue agar plate assays: (A – Left) plate amended with 10 μl of HA 10-3 M; (B – Middle) plate inoculated with T. harzianum M10; (C- Right) plate inoculated with Pseudomonas fluorescens strain CHA0 (used as positive control).
Figure 3. Growth promotion of tomato seedlings treated with 100, 10 or 1 μM HA, measured as shoot and root lengths 12 days after treatment. Values are means of twenty replicates. Bars: ± Standard Deviation. Values with the same letter do not differ significantly (P < 0.05).
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Figure 4. Plant growth promotion activity of HA in iron deficient conditions. A) Growth of tomato seedlings supplemented with low concentration of iron (0.5 μM) and treated with 10 or 1 μM HA, measured as shoot and root lengths, 12 days after treatments. B) Analysis of iron content in tomato seedlings supplemented with low concentration of iron (0.5 μM) and treated with 10 or 1 μM HA, 12 days after treatments. Values are means of twenty replicates. Bars: ± Standard Deviation. Values with the same letter do not differ significantly (P < 0.05). Figure 1 HOOC O
A
B
H O
H N
O H O
Figure 2
A
B
C
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12,0
Shoot
Length (cm)
b
8,0
b
b
b
Root
10,0
a,b
b
a a
6,0 4,0 2,0 0,0
HA 100 μM
Control
HA 10 μM
HA 1 μM
Treatments
Figure 4
A
B
Root length Shoot length
0,016
b
d
b
–1
b c
4
a 2
0 Control
HA 10 uM
Iron in plant (100 μg Fe g DW)
6
Length (cm)
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Figure 3
c 0,012
0,008
a
b
0,004
HA 1 uM
Treatments
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0 Control
HA 10 uM Treatments
HA 1 uM
Received Date : 21-Jun-2013 Revised Date : 16-Jul-2013 Accepted Date : 28-Jul-2013 Article type : Research Letter Editor : Paolina Garbeva Title
Harzianic acid: a novel siderophore from Trichoderma harzianum Francesco Vinale*a, Marco Nigroa,b, Krishnapillai Sivasithamparamc, Gavin Flemattid, Emilio L. Ghisalbertid, Michelina Ruoccoa, Rosaria Varleseb, Roberta Marrab, Stefania Lanzuiseb, Ahmed Eidb, Sheridan L. Wooa,b and Matteo Loritoa,b. a
CNR – Istituto per la Protezione delle Piante (IPP-CNR) 80055 Portici, Italy, Dipartimento di Arboricoltura Botanica e Patologia Vegetale, Università degli Studi di Napoli ‘Federico II’, Portici, 80055 Naples, Italy c School of Earth and Environment - Faculty of Natural and Agricultural Sciences d School of Chemistry and Biochemistry - Faculty of Life and Physical Sciences, The University of Western Australia, W.A. 6009, Australia. b
* Corresponding author: Francesco Vinale Phone number: +39 081 2539338 Fax number: +39 081 2539339 e-mail address: [email protected] Via Università 133 – 80055 Portici (Naples) – Italy Running title: Harzianic acid, a T. harzianum siderophore Keywords: - Secondary metabolites - Harzianic acid - Iron (III) - Trichoderma - Siderophores
Abstract Agriculture-relevant microbes are considered to produce secondary metabolites during processes of competition with other micro- and macro-organisms, symbiosis, parasitism or This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6968.12231 This article is protected by copyright. All rights reserved.
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pathogenesis. Many different strains of the genus Trichoderma, in addition to a direct activity against phytopathogens, are well known producers of secondary metabolites and compounds that substantially affect the metabolism of the host plant. Harzianic acid is a Trichoderma secondary metabolite showing antifungal and plant growth promotion activities. This report demonstrates the ability of this tetramic acid to bind with a good affinity essential metals such as Fe3+, which may represent a mechanism of iron solubilisation that significantly alters nutrient availability in the soil environment for other microorganisms and the host plant.
Introduction Trichoderma spp. are well studied filamentous fungi commonly found in the soil community that are widely marketed as biopesticides, biofertilizers and soil amendments, due to their ability to protect plants, enhance vegetative growth and contain pathogen populations (Vinale et al., 2008a). To date, hundreds of different Trichoderma-based preparations are commercially used to protect and/or increase the productivity of various crops (Lorito et al., 2010). One factor that contributes to the beneficial biological activities exerted by Trichoderma strains is the wide variety of secondary metabolites that they are able to produce (Sivasithamparam & Ghisalberti, 1998; Reino et al., 2008). Fungal secondary metabolites may be considered a large and heterogeneous group of small molecules not directly essential for growth, but having an important role in signalling, development and interaction with other organisms (Song et al., 2006; Mukherjee et al., 2012). Trichoderma metabolites may act by directly inhibiting the growth of pathogens, or by indirectly triggering the defence system in the host plant, thus increasing disease resistance, and by promoting plant growth (Vinale et al., 2012). The accumulation of Trichoderma
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metabolites varies according to the species or the strain used and is related to the biosynthesis and biotransformation rates (Vinale et al., 2009a). Siderophores are low-molecular-weight metabolites produced for scavenging iron from the environment and have an high affinity for iron(III) (Hider & Kong, 2010). Fe3+-chelating molecules can be beneficial to plants because they can solubilise the iron which is otherwise unavailable and can suppress the growth of pathogenic microorganisms by depriving the pathogens of this necessary micronutrient (Leong, 1986). We have recently purified from the culture filtrate of a strain of T. harzianum a nitrogen heterocyclic compound named harzianic acid (HA) and demonstrated its growth promotion effect (Vinale et al., 2009b). Growth of different fungal pathogens and canola seedlings was shown to be affected by treatments with HA in a concentration dependent manner, suggesting a role of this metabolite in plant growth regulation, as well as in the antagonism against phytopatogenic agents. In this work we demonstrate that HA has a Fe3+ chelating activity. This secondary metabolite strongly binds essential metals such as iron, thus representing a previously unrecognized siderophore.
Materials and methods Microbial strain and culture conditions T. harzianum strain M10 was maintained on potato dextrose agar (PDA, SIGMA, St Louis, Mo., USA) slants at room temperature and sub-cultured bimonthly. Genomic DNA was isolated in order to analyse ribosomal DNA. Using PCR approach, were amplified fragments containing the internal transcribed spacer 1 (ITS1-partial sequence), the 5.8S ribosomal RNA gene and the internal transcribed spacer 2 (ITS2-complete sequence), by using primers SR6R (5’-AAGTAGAAGTCGTAACAAGG-3’) and LR1 (5’-GGTTGGTTTCTTTTCCT-3’).
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The PCR products were gel electrophoresed for quantification and assessment of PCR specificity and finally sequenced. Sequence analysis of the internal transcribed spacers (ITS) of the rDNA indicated 99% similarity with GenBank sequences of T. harzianum confirming the identity of this specie. Ten 7 mm diameter plugs of M10 strain obtained from actively growing margins of PDA cultures were inoculated to 5 L conical flasks containing 1 L of sterile potato dextrose broth (PDB, SIGMA). The stationary cultures were incubated for 21 days at 25° C. The cultures were filtered under vacuum through filter paper (Whatman No. 4, Brentford, UK).
Extraction and isolation of HA The filtered culture broth (2 L) was acidified to pH 4 with 5 M HCl and extracted exhaustively with ethyl acetate (EtOAc). The combined organic fraction was dried (Na2SO4) and evaporated in vacuo at 35 °C. The red residue recovered was dissolved in CHCl3 and extracted three times with 2 M NaOH. HA was then precipitated with 2 M HCl. The solid was recovered (135 mg), solubilised and subjected to RP-18 vacuum chromatography (20 g), eluting with a gradient of methanol (MeOH):H2O:CH3CN (1:8:1 to 10:0:0). After the separation, 45 mg of pure HA were collected. The homogeneity of pure pooled products was verified by analytical reverse-phase TLC (glass pre-coated Silica gel 60 RP-18 plates - Merck Kieselgel 60 TLC Silica gel 60 RP-18 F254s, 0.25 mm) using 3:4:3 CH3CN - MeOH -H2O as eluent (Rf of HA: 0.3). The compounds were detected on TLC plates using UV light (254 or 366 nm) and/or by spraying the plates with 5% (v/v) H2SO4 in EtOH followed by heating at 110 °C for 10 min. UV, IR, 1H NMR, 13C NMR and HR-FABMS were identical to those reported by Sawa et al. (1994). ESMS (+) m/z 753.3 [M2 + Na]+ , 404.2 [M + K]+, 388.2 [M + Na]+, 366.2 [M + H]+; 264.2 [M +H – C7H18]+. 1H and 13C NMR spectra were recorded with a Varian 400
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instrument operating at 400 (1H) and 125 (13C) MHz, using residual and deuterated solvent peaks as reference standards.
CAS agar plate assays The method to detect siderophore production was previously described by Schwyn & Neilands (1987). Orange halos around the colonies, growth on Chrome Azurol S (CAS) plates, are indicative of siderophore activity. CAS solution was also used for detection of siderophore production in culture filtrate (50 μl of culture was added to 950 μl of CAS solution, after reaching equilibrium the absorbance was measured at 630 nm). The CAS assay was also used to test the chelating properties of a 10-3 M HA solution in methanol. The CAS assay was modified according to Milagres et al. (1999) to test the ability of strain M10 to produce iron-binding compounds eventually avoiding the growth inhibition caused by the toxicity of the CAS-blue agar medium. Petri dishes (10 cm in diameter) were prepared with the Malt Extract Agar (MEA) medium. After solidification, the medium was cut into halves, one of which was replaced by CAS-blue agar. The halves containing culture medium (MEA) were inoculated with M10 plugs. The plates were incubated at 25°C for 6 days. P. fluorescens strain CHA0 (bacterial strain collection of prof A. Zoina - University of Naples) was used as a positive control (Maurhofer et al., 1994; Youard et al., 2007).
Iron binding affinity of HA In order to measure the iron binding affinity of HA, the method of Kaufmann et al. (2005) was used with some modifications. Stock solutions of ferric chloride (10 mM) and HA (10 mM) were prepared with 4:1 MeOH / 0.1 M NaOAc buffer solution (pH 7.4). Aliquots of both stock solutions were diluted and the absorbances of the formed complexes were
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measured at 290 nm in triplicate in the presence and absence of EDTA (10 mM and saturated solution).
LC/MS of HA–Fe(III) complex The Fe(III)-binding properties of the HA were investigated by adding 100 μl of a Fe(III) chloride solution (10 mM) to 100 μL of 10 mM HA in MeOH. The solution turned red and was directly infused into the LC/MS system at 5 μL/min using a syringe pump. Full-scans in the range m/z 100–1,200 were performed on a Bruker 6340 ion trap mass spectrometer equipped with an electrospray ionization source and operating in the positive ion mode. High resolution spectra were recorded using a Waters Alliance e2695 HPLC connected to a Waters LCT Premier XE mass spectrometer with an electrospray ionisation source (ESI). Samples were injected using the onboard injector in 10 µL injection volumes and eluted with 20% acetonitrile/water at a flow rate of 0.3 mL/min to the time-of-flight mass spectrometer. For the HA-Fe(III) complex, positive ESI-HRMS found m/z 491.0574 ([C19H27NO6FeCl2 ]+ requires 491.0565).
Plant growth promotion assay Tomato (Solanum lycopersicum cv. Roma) seeds were surface sterilized using 70% EtOH for 2 min, followed by 2% NaClO for 2 min, thoroughly washed with sterile distilled water, then placed on magenta box containing half-strength Murashige and Skoog salt (MS) medium (ICN Biomedicals, Irvine, California, USA) containing 1 % agar and 1.5% sucrose, adjusted to pH 5.7, and vernalized for 2 days at 4°C in the dark. Media with iron concentrations different from the standard 50 μM were prepared according to Murashige & Skoog (1962), except that defined amounts of Fe III were added (diluted 1/10 and 1/100 corresponding to 5 and 0.5 μM, respectively). Sterile solutions of HA were added to the substrate before the agar
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solidification to have final concentrations of 100 μM, 10 μM, 1 μM. Untreated substrates were used as controls. Seedlings were grown in a growth chamber (16 h photoperiod); the temperature was maintained at 25 ±1°C with a relative humidity of 65–75 %. Growth of stem height was measured daily. Each treatment consisted of twenty replicates and the experiment was repeated four times. At the end of each experiment, the whole plants were dried and weighed. Data from the experiments were combined since statistical analysis determined homogeneity of variance (P ≤ 0.05). Plant tissues (50–100 mg) were ground with liquid N2, mineralized according to Beinert (1978) and the iron contents were determined using Atomic Absorption analysis.
Results Purification and identification of HA T. harzianum M10 was grown in PDB and the culture filtrate was extracted with ethyl acetate, from which HA was purified as described above. The molecular mass of this compound, as determined by electrospray ionization-mass spectrometry, was 365 Da, and its pattern corresponded to that of 2-hydroxy-2-[4-(1-hydroxy-octa-2,4-dienylidene)-1-methyl3,5-dioxo-pyrrolidin-2-ylmethyl]-3-methyl-butyric acid (Figure 1) described by Sawa et al. (1994) and Vinale et al. (2009b). The HA structure was confirmed by nuclear magnetic resonance.
Iron(III) binding activity of M10 and HA The assay described by Schwyn & Neilands (1997) and Millagres et al. (1999) was used for the detection of siderophores released in the substrate by M10 strain. The fungus grew on CAS blue agar and the iron(III) chelating compounds, excreted by the microorganism, diffused through the medium producing a colour change from blue to orange. Purified HA
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decolorizes CAS blue agar, indicating that it could form a complex with Fe(III) (Figure 2). While the free compound in aqueous solution is pale orange, addition of Fe(III) results in the appearance of a red colour, indicating that an iron complex is formed.
Characterization of Fe(III)-HA complex To investigate the formed Fe(III)-HA complexes, experiments were carried out adding FeCl3 to a solution of HA. The uncomplexed HA molecule is detected as [M+H]+ (m/z 366.2), [M+Na]+ (m/z 388.2), [M+K]+ (m/z 404.2) and [M2 + Na]+ (m/z 753.3) (Figure 1-A). After adding Fe(III), the spectrum showed additional signals at m/z 455.1 and m/z 491.1 corresponding to two 1:1 Fe(III) containing complexes which were assigned the following signals based on the chloride isotope pattern, [M-H+Fe(III)+Cl2+H]+ (m/z 491.1) or [M2H+Fe(III)+Cl+H]+ (m/z 455.1), as determined by isotopic distribution. The resulting mass spectrum is depicted in Figure 1-B. High resolution spectrum of the Fe(III)-HAcomplex showed signals at m/z 491.0574 ([C19H27NO6FeCl2 ]+ requires 491.0565), confirming the 1:1 HA–Fe complex.
The apparent binding constant (Kd,app) was determined in order to measure the affinity of HA for iron (III). These experiments were performed by using a previously described protocol based on the competition between HA and EDTA for iron and the detection of the complexes by measuring the characteristic absorption (Wang et al., 2002). The loss of Fe(III)-HA absorbance at 340 nm upon addition of EDTA was used to calculate the equilibrium constant, presuming the formation of a 1:1 Fe-HAcomplex, according to the following equation: EDTA-Fe + HA = EDTA + Fe-HA Keq = [EDTA][Fe-HA] / [HA][EDTA-Fe] = Kd;EDTA / Kd;HA
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By using the known affinity of EDTA for Fe(III) (5.00 x 10-23 M), we were able to determine the relative affinity (Kd,app) of HA for Fe3+ to be 1.79 x 10-25 M.
Plant growth promotion The activity of HA as plant growth promoter was evaluated by analyzing the effect on seed germination and measuring lengths, fresh or dry weights of tomato seedlings treated with metabolite solutions as compared to controls.
Seed germination was significantly affected by HA applications. In particular, when 100 or 10 μM HA solutions were used, the percentages of germinated seeds were 4-5 times higher than in control (Table 1). Shoot and root growth of tomato seedlings was enhanced by treatments with HA used at different concentrations. The highest increase of shoot and root length (+76% and +66%, respectively) was registered using 10 μM of HA compared to control (Figure 3). A promotion effect on both fresh and dry plant weights was observed using HA at 10 and 1 μM. Conversely, a higher concentration of HA (100 μM) did not significantly affect plant weights or seedling lengths compared to the untreated control (Table 1, Figure 3).
To further characterize the role of HA in iron availability, plants were supplemented with low concentrations of iron (5 and 0.5 μM). Root length of tomato seedlings grown in media containing the lowest iron concentration was improved by treatments with both 10 and 1 μM HA; conversely, no significant effect was detected in shoot growth (Figure 4-A) or in plant weight in comparison to control (data not shown). However, in these growing conditions, iron levels increased in plants treated with HA, particularly when 1μM HA was used (Figure 4-B).
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Discussion The interaction of Trichoderma spp. with plants confers several benefits for the associated host that include: i) the suppression of phytopathogens by using direct antagonistic mechanisms (i.e. antibiosis, mycoparasitism, competition for nutrient and space); ii) plant growth promotion; iii) enhanced nutrient availability and uptake, and iv) induction of planthost resistance (Harman et al., 2004).
In addition, some Trichoderma strains produce compounds that can cause substantial changes in the metabolism of the host plant. The involvement of secondary metabolites in the ability of Trichoderma spp. to activate plant defence mechanisms and regulate plant growth has been investigated (Vinale et al., 2008b). HA is one of these natural products that shows antifungal and plant growth promoting activities (Vinale et al., 2009b). In the present study we investigated the Fe+3 chelating properties of HA.
HA, isolated by RP-18 vacuum chromatography from a 2M NaOH 2 M extract, showed the same 1H and 13C parameters as those previously reported for 2-hydroxy-2-[4-(1-hydroxyocta-2,4-dienylidene)-1-methyl-3,5-dioxo-pyrrolidin-2-ylmethyl]-3-methyl-butyric acid, a compound belonging to the chemical class of tetramic acids. The naturally occurring tetramic acids derivatives have attracted significant attention because these metabolites have a wide distribution and have been found to display a remarkable diversity of biological activities playing a significant role in ecological interactions. These activities may be enhanced significantly through the chelation of the tetramic acid nucleus with metal ions (important for transport across membranes in biological tissues). It was found that in some cases the metal complexes obtained revealed higher biological activity than their ligands (Ghisalberti, 2003; Schobert & Schlenk, 2008; Athanasellis et al., 2010).
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Both the living microorganism and the isolated metabolite HA when tested in the CAS blue agar plates caused a colour change of the substrate from blue to orange indicating that the iron(III) binding properties of the fungus is related also to the production of the tetramic acid derivative. Moreover, the LC-MS analysis of a HA – Fe+3 solution showed additional signals at 455.1 m/z and 491.1 m/z corresponding to a 1:1 chloride containing complex, [MH+Fe(III)+Cl2+H]+ (m/z 491.1) or [M-2H+Fe(III)+Cl+H]+ (m/z 455.1). Since chloride is a coordinating ligand for iron, it is possible that the chloride anion is directly bound to the metal (Caudle et al., 1994).
The value of Kd of Fe – HA complex (1.79 x 10-25 M) may be directly compared with that of other chelators showing a 1:1 Fe:ligand stoichiometry, such as desferrioxamine (DFO), EDTA, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (HPD), pyoverdin and pyochelin (Kaufmann et al., 2005). As shown in Table 2, HA has lower affinity to Fe(III) than DFO, pyoverdin and HPD, while it has a stronger affinity for iron(III) than EDTA and pyochelin. This data suggest that HA could compete for available iron in solution and provide a method for iron solubilization.
Microbial siderophores are used as iron chelating agents that can regulate the availability of iron in the plant rhizosphere. It has been assumed that competition for iron in the rhizosphere is controlled by the affinity of the siderophore for iron. The important factors, which participate, are concentration of various types of siderophores, kinetics of exchange, and availability of Fe-complexes to microbes as well as plants. Siderophores produced by beneficial agents may have important effects on both microbial and plant nutrition. Fe3+ siderophore complexes can be recognized and taken up by several plant species and this
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process is considered crucial for plant iron uptake, particularly in calcareous soils (Sharma et al., 2003). Trichoderma metabolites may help the plant to withstand pathogens by both promoting the growth and development of root and shoot systems, and stimulating the defence mechanisms (Vinale et al., 2008b). In the present work we confirm a possible involvement of HA in plant growth regulation probably because of its Fe(III) binding activity. In fact, HA affected the germination of tomato seeds and improved the growth of the seedlings even in iron deficient conditions (increment of iron concentration into the plants was also registered). This finding supports the hypothesis that HA actively influences the growth of Trichoderma-colonized plants.
The isolation and application of bioactive compounds produced by beneficial microbes responsible for the desired positive effects on plants may be a promising alternative to the use of living antagonists.
Acknowledgments The authors thank Davide Stellitano, Gelsomina Maganiello and Alberto Pascale for their technical assistance. The authors thank Antonio De Martino for the iron concentration measurements. Work by the authors FV and MR was supported by a dedicated grant from the Italian Ministry of Economy and Finance to the National Research Council for the project "Innovazione e Sviluppo del Mezzogiorno - Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy Agroalimentare - Legge n. 191/2009". Work by the authors MN, RS, RM, SL, AE, SW and ML has been supported by the following projects: “S.Re.Va.Pr.O.”, “CA.SVI.PR.OLI.” “MI.P.RE.VEGE.”; PRIN MIUR 2008 - prot. 2008SNPNC2 - prot. 2008WKPAWW. The authors also acknowledge the facilities, scientific and technical assistance of the Centre for Microscopy, Characterisation & Analysis at the University of Western Australia.
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References
Athanasellis G, Igglessi-Markopoulou O & Markopoulos J (2010) Tetramic and tetronic acids as scaffolds in bioinorganic and bioorganic chemistry. Bioinorg Chem Appl 2010: 1-11. Beinert H (1978) Micro methods for the quantitative determination of iron and copper in biological material. Methods Enzymol 54: 435–445. Caudle MT, Stevens RD & Crumbliss AL (1994) Electrospray mass spectrometry study of 1:1 ferric dihydroxamates. Inorg Chem 33: 843-844. Chiang Y, Lee K, Sanchez JF, Keller NP & Wang CCC (2009) Unlocking fungal cryptic natural products. Nat Prod Commun 4: 1505–1510. Ghisalberti EL (2003) Bioactive tetramic acid metabolites. In: Atta-ur Rahman, Editor(s), Studies in Natural Products Chemistry, Elsevier, Vol 28/1, pp 109-163. Harman GE, Howell CR, Viterbo A, Chet I & Lorito M (2004) Trichoderma species opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2: 43-56. Hider RC & Kong X (2010) Chemistry and biology of siderophores. Nat Prod Rep 27: 637657. Kaufmann GF, Sartorio R, Lee SH, Rogers CJ, Meijler MM, Moyss JA, Clapham B, Brogan AP, Dickerson TJ & Janda KD (2005) Revisiting quorum sensing: Discovery of additional chemical and biological functions for 3-oxo-N-acylhomoserine lactones. Proc Natl Acad Sci USA 102: 309 - 314. Youard ZA, Mislin GL, Majcherczyk PA, Schalk IJ & Reimmann C (2007) Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin. J Biol Chem 282: 35546-35553. Leong J (1986) Siderophores : their biochemistry and possible role in the biocontrol of plant pathogens. Annu Rev Phytopathol 24: 187–209.
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Lorito M, Woo SL, Harman GE & Monte E (2010) Translation research on Trichoderma: from ‘omics to the field. Annu Rev Phytopathol 48: 395-417. Maurhofer M, Hase C, Meuwly P, Metraux JP & Defago G (1994) Induction of systemic resistance of tobacco to tobacco necrosis virus by the root-colonizing Pseudomonas fluorescens strain CHA0: influence of the gacA gene and of pyoverdine production. Phytopathology 84: 139-146. Milagres AMF, Machuca A & Napoleão D (1999) Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay. J Microbiol Methods 37: 1–6 Mukherjee PK, Horwitz BA & Kenerley CM (2012) Secondary metabolism in Trichoderma – a genomic perspective. Microbiology 158: 35–45. Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497. Reino JL, Guerriero RF, Hernàndez-Galà R & Collado IG (2008) Secondary metabolites from species of the biocontrol agent Trichoderma. Phytochem Rev 7: 89-123. Sawa R, Mori Y, Iinuma H, Naganawa H, Hamada M, Yoshida S, Furutani H, Kajimura Y, Fuwa, T & Takeuchi T (1994) Harzianic acid, a new antimicrobial antibiotic from a fungus. J Antibiot 47: 731–732. Schobert R & Schlenk A (2008) Tetramic and tetronic acids: an update on new derivatives and biological aspects. Bioorg Med Chem 16: 4203–4221. Schwyn B & Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160: 47-56. Sharma A, Johri BN, Sharma AK & Glick BR (2003) Plant growth promoting bacterium Pseudomonas sp., strain GFP(3) influences iron acquisition in mung bean (Vigna radiata L. Wilzeck). Soil Biol Biochem 35: 887–894.
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Sivasithamparam K & Ghisalberti EL In Trichoderma and Gliocladium; Harman, G. E., Kubicek, C. P., Eds.; Taylor and Francis Ltd: London, 1998; Vol. 1, pp 139-191. Song XY, Shen QTXT, Chen XL, Sun CY & Zhang YZ (2006) Broad-spectrum antimicrobial activity and high stability of trichokonins from Trichoderma koningii SMF2 against plant pathogens. FEMS Microbiol Lett 260: 119–125. Vinale F, Sivasithamparam K, Ghisalberti EL, Marra R, Woo SL & Lorito M (2008a) Trichoderma – plant – pathogen interactions. Soil Biol Biochem 40: 1–10. Vinale F, Sivasithamparam K, Ghisalberti EL, Marra R, Barbetti MJ, Li H, Woo SL & Lorito M (2008b) A novel role for Trichoderma secondary metabolites in the interactions with plants. Physiol Mol Plant Pathol 72: 80–86. Vinale F, Ghisalberti EL, Sivasithamparam K, Marra R, Ritieni A, Ferracane R, Woo SL & Lorito M (2009a) Factors affecting the production of Trichoderma harzianum secondary metabolites during the interaction with different plant pathogens. Lett Appl Microbiol 48: 705–711. Vinale F, Flematti G, Sivasithamparam K, Lorito M, Marra R, Skelton BW & Ghisalberti EL (2009b) Harzianic acid, an antifungal and plant growth promoting metabolite from Trichoderma harzianum. J Nat Prod 72: 2032–2035. Vinale F, Sivasithamparam K, Ghisalberti EL, Ruocco M, Woo S & Lorito M (2012) Trichoderma secondary metabolites that affect plant metabolism. Nat Prod Commun 7: 1545 – 1550. Wang J, Buss JL, Chen G, Ponka P & Pantopoulos K (2002) The prolyl 4-hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate generates effective iron deficiency in cultured cells. FEBS Lett 529: 309–312.
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Table 1: Growth promotion of tomato seedlings treated with HA at different concentrations fresh and dry weights 12 days after treatment; germination 2 days after treatment). Values are means of ten replicates. SD: ±Standard Deviation. Values with the same letter do not differ significantly (P < 0.05).
Treatment
Fresh weight (mg) SD Dry weight (mg) SD % of Germination SD
Control
46.9 a
8
5.4 a
0.4
16.5 a
4.2
HA 100 μM
59.8 a
7
6.3 a, b
0.6
88.0 b
3.5
HA 10 μM
73.7 b
6
6.6 b
0.5
71.5 b
4.1
HA 1 μM
72.5 b
8
6.5 b
0.7
27.5 c
1.9
Table 2. Affinity constants of chelators for Fe(III). Compound EDTA DFO Pyoverdin Pyochelin HPD HA
Kd 5.00 x 10-23 M 2.51 x 10-26 M 10-32 M 10-5 M 1.6 x 10-29 M 1.79 x 10-25 M
Figure legends Figure 1: Characterization of HA-Fe(III) complex by LC/MS. (A) Harzianic Acid (HA) and its mass spectrum ;(B) mass spectrum of HA-Fe(III).
Figure 2: CAS-blue agar plate assays: (A – Left) plate amended with 10 μl of HA 10-3 M; (B – Middle) plate inoculated with T. harzianum M10; (C- Right) plate inoculated with Pseudomonas fluorescens strain CHA0 (used as positive control).
Figure 3. Growth promotion of tomato seedlings treated with 100, 10 or 1 μM HA, measured as shoot and root lengths 12 days after treatment. Values are means of twenty replicates. Bars: ± Standard Deviation. Values with the same letter do not differ significantly (P < 0.05).
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Figure 4. Plant growth promotion activity of HA in iron deficient conditions. A) Growth of tomato seedlings supplemented with low concentration of iron (0.5 μM) and treated with 10 or 1 μM HA, measured as shoot and root lengths, 12 days after treatments. B) Analysis of iron content in tomato seedlings supplemented with low concentration of iron (0.5 μM) and treated with 10 or 1 μM HA, 12 days after treatments. Values are means of twenty replicates. Bars: ± Standard Deviation. Values with the same letter do not differ significantly (P < 0.05). Figure 1 HOOC O
A
B
H O
H N
O H O
Figure 2
A
B
C
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12,0
Shoot
Length (cm)
b
8,0
b
b
b
Root
10,0
a,b
b
a a
6,0 4,0 2,0 0,0
HA 100 μM
Control
HA 10 μM
HA 1 μM
Treatments
Figure 4
A
B
Root length Shoot length
0,016
b
d
b
–1
b c
4
a 2
0 Control
HA 10 uM
Iron in plant (100 μg Fe g DW)
6
Length (cm)
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Figure 3
c 0,012
0,008
a
b
0,004
HA 1 uM
Treatments
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0 Control
HA 10 uM Treatments
HA 1 uM
