Physiological and Molecular Plant Pathology 72 (2008) 8086

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Physiological and Molecular Plant Pathology

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A novel role for Trichoderma secondary metabolites in the interactions with plants
F. Vinale a, *, K. Sivasithamparam b, E.L. Ghisalberti c, R. Marra a, M.J. Barbetti d, H. Li b, S.L. Woo a, M. Lorito a
a

` degli Studi di Napoli Federico II, Via Universita ` 100, 80055 Portici, Naples, Italy Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Universita School of Earth and Geographical Sciences, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Nedlands, WA 6009, Australia c School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Nedlands, WA 6009, Australia d School of Biological Sciences, The University of Western Australia, Nedlands, WA 6009, Australia
b

a r t i c l e i n f o
Article history: Accepted 28 May 2008 Keywords: Trichoderma Induced systemic resistance Plant growth promotion Secondary metabolites T22azaphilone T39butenolide 1-Hydroxy-3-methyl-anthraquinone 1,8-Dihydroxy-3-methyl-anthraquinone Harzianolide 6PP Harzianopyridone

a b s t r a c t
Secondary metabolites play a pivotal role in the antagonistic activities of some biocontrol species of Trichoderma resulting in the suppression of plant pathogens, but their involvement in complex interactions with plants has not been specically studied. In this work the major secondary metabolites produced by biocontrol strains of Trichoderma (T. harzianum strains T22, T39 and A6, and T. atroviride strain P1) have been investigated for their effect on plant growth promotion. An auxin-like activity was observed on etiolated pea (Pisum sativum) stems treated with harzianolide and 6-n-pentyl-6H-pyran-2-one (6PP), which also affected the growth of tomato (Lycopersicum esculentum) and canola (Brassica napus) seedlings. The ability of these molecules to induce systemic defence responses in planta was also investigated. Tomato and oilseed rape seedlings were treated with the metabolites and then inoculated with a spore suspension of Botrytis cinerea or Leptosphaeria maculans, respectively. In both cases, a reduction of disease symptoms was observed, particularly on 6PP-treated plants. Moreover an over-expression of pathogenesis-related (PR) proteins was also detected in treated plants. These results clearly indicate that secondary metabolites of Trichoderma spp. may have a role in both plant growth regulation and activation of plant defence responses. 2008 Elsevier Ltd. All rights reserved.

1. Introduction Secondary metabolites are an heterogeneous group of natural compounds that are considered to aid the producing organism in survival and basic functions, such as competition, symbiosis, metal transport, differentiation, etc. [1]. Do to chemical and biological properties of such compounds, they are widely used for medical, pharmaceutical, or agricultural purposes [2]. This group includes antibiotics, which are natural products that are capable of inhibit microbial growth, and are produced by various microbes during processes of development and sporulation [35]. Fungi of the genus Trichoderma are biocontrol agents (BCAs) that are successfully used as biopesticides worldwide, and many species are well known producers of secondary metabolites with antibiotic activity [68]. Antibiotic production is often combined with other mechanisms of biocontrol [such as mycoparasitism and the production of cell wall-degrading enzymes (CWDEs), competition for

* Corresponding author. Tel.: 39 081 253 9338; fax: 39 081 253 9339. E-mail address: frvinale@unina.it (F. Vinale). 0885-5765/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2008.05.005

nutrients/space, and induced resistance in the plant] and thus is considered involved in Trichodermas interactions with the host plant and the resulting benecial effects [816]. The production of secondary metabolites in Trichoderma spp. is strain-dependent and includes volatile and non-volatile antifungal substances, such as 6-n-pentyl-6H-pyran-2-one (6PP), gliotoxin, viridin, harzianopyridone, harziandione and peptaibols [7,8,17]. Synergistic effects between CWDEs and different classes of antibiotics on fungal pathogen growth have been well documented [1821]. The overall biocontrol effect of Trichoderma spp. was signicantly enhanced by the stimulation of plant defence responses to pathogen attack. In addition, the inoculation of the living fungal antagonist produced a growth promotion effect in planta [13,15,22]. However, the role that secondary metabolites produced by BCAs such as Trichoderma play in the complex three-way interaction between plant, pathogens and antagonistic fungi has received little attention. In this work, we isolated the major secondary metabolites produced in liquid culture by different Trichoderma biocontrol strains (T. harzianum commercial strains T22 and T39, T. atroviride P1 and T. harzianum A6). Seven known compounds were extracted,

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isolated and characterized from fungal culture ltrates: (1) T22azaphilone; (2) T39butenolide; (3) harzianopyridone; (4) harzianolide; (5) 1-hydroxy-3-methyl-anthraquinone; (6) 1,8-dihydroxy-3-methyl-anthraquinone; and (7) 6PP (Fig. 1). The effects on plant growth and the induction of defence mechanisms of the puried metabolites were determined in vivo. 2. Materials and methods 2.1. Microbial strains and culture conditions The phytopathogens Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici, as well as the antagonistic fungi T. harzianum strains T22, T39 and A6 and T. atroviride strain P1 were maintained on potato dextrose agar (PDA, SIGMA, St Louis, MO) slants at room temperature and sub-cultured bimonthly. Similarly, Leptosphaeria maculans isolate UWA P11 was grown on V8 agar plates and cultured as above. The pathogens were isolated from eld crops in Western Australia. 2.2. Production and isolation of Trichoderma secondary metabolites Two 7 mm diameter plugs of each Trichoderma strain were obtained from actively growing margins of PDA cultures and inoculated to 5 L conical asks containing 1 L of sterile 1/5 strength potato dextrose broth (PDB, SIGMA). The stationary cultures were incubated for 31 days at 25  C. The cultures were ltered under vacuum through lter paper (Whatman, Brentford, UK). The ltered culture broth of T. harzianum T22 and T39 were extracted and treated as previously described [23]. The ltered culture broth (2 L) of T. harzianum A6 was extracted exhaustively with ethyl acetate (EtOAc). The redbrown residue obtained was subjected to ash column chromatography (Si gel; 50 g), by eluting with a gradient of EtOAc:petroleum ether (1:1 to 10:0). Fractions showing similar thin-layer chromatography (TLC) proles were combined and further puried by using preparative TLC separation (Si gel; EtOAc:petroleum ether; 6:4). The culture ltrate of T. atroviride P1 was

extracted with EtOAc as above and the extract was separated by using ash column chromatography (Si gel; 50 g), with gradient mixtures of petroleum ether and EtOAc (0100% EtOAc) used as the eluent. Secondary metabolites were isolated from Trichoderma spp. culture ltrates as previously described [23]. Vacuum liquid (VLC) and column chromatography were carried out using silica gel 60 GF254 and GF60 3570 mesh (Merck, Darmstadt, Germany). For the characterization of the metabolites, 1H and 13C NMR spectra were recorded with a Bruker AM 500 spectrometer operating at 500 (1H) and 125 (13C) MHz using residual and deuterated solvent peaks as reference standard. Low and high resolution mass spectra were obtained by using a VG Autospec mass spectrometer (EI mode). Analytical and preparative TLC were performed on silica gel (Kieselgel 60, GF254, 0.25 and 0.5 mm, respectively, Merck); compounds were detected with UV radiation (254 or 366 nm) and/or by spraying the plates with CeSO4 (10% w/v in water) or H2SO4 (5% v/v in ethanol) and heating at 110  C for 10 min. 2.3. Antifungal assays The antifungal activity of the puried Trichoderma compounds were tested against the plant pathogens R. solani, P. ultimum and G. graminis var. tritici as described by Vinale et al. [23]. Briey, pathogen plugs were placed at the center of 1/5 PDA Petri dishes. Ten ml of the puried compounds were applied to the surface of each plug at concentrations ranging from 0.01 to 200 mg plug1. The controls were treated with 10 ml of only EtOAc [24]. The pathogen growth was determined daily by measuring the colony diameter (mm). Each treatment consisted of three replicates and each experiment was repeated at least twice. 2.4. Effect of Trichoderma metabolites on plants inoculated with fungal pathogens B. cinerea conidial suspension was obtained from 10 day-old sporulating cultures on PDA in 0.1% Tween-20 solution, ltered through glass wool and diluted to a nal concentration of 5 105 conidia ml1. A spore suspension of L. maculans isolate UWA P11 was obtained as previously reported by Li et al. [25]. Fifteen seeds of tomato (Lycopersicum esculentum cv. San Marzano 823) and canola (Brassica napus cv. Westar) were sown in 30 60 cm polisterol supports (consisting of 60 inserts 4 4 4 cm) containing sterile soil. Plants were grown in a growth chamber (22  C, 75% RU; 16 h photoperiod) and watered daily to eld capacity. The inoculations of the puried Trichoderma secondary metabolites and pathogens were performed singly on each cotyledon of 7 day old canola and tomato seedlings, previously wounded with a 26 G sterile needle. In particular, each Trichoderma metabolite was applied to a single cotyledon at concentrations ranging from 1 to 10 mg Ll. Three hours later the opposite cotyledon of each canola and tomato plant was inoculated with a 10 ml droplet of a 1 106 spore ml1 suspension (containing a single drop of Tween-20 as wetting agent) of L. maculans and B. cinerea, respectively. A solvent (1 ml of methanol in 15 ml of sterilized water) was used as the control treatment on the plants. The inoculated plants were covered with a plastic sheet in order to maintain high humidity for 72 h. Disease symptoms were evaluated 14 days after inoculation using a disease severity scale 09 for L. maculans [0 no visible symptoms, 1 necrotic hypersensitive, 2 tissue collapse (1 mm diameter) with distinct margin, 3 collapsed spots (2 mm diameter) with diffuse margin, 4 collapsed spot (3 mm diameter) with diffuse margin, 5 collapsed spot (4 mm diameter) with diffuse margin, 6 collapsed spot (5 mm diameter) with diffuse margin, 7 collapsed spot (>6 mm diameter) with diffuse

O HO H

O O O O OH O O O O

2
OH

MeO MeO N H O O

3
OH O CH3

5R=H 6 R = OH

Isolated compound
T22azaphilone (1) T39 butenolide (2) Harzianopyridone (3) Harzianolide (4) 1-hydroxy-3-methyl-anthraquinone (5) 1,8-dihydroxy-3-methyl-anthraquinone (6) 6-n-pentyl-6H-pyran-2-one (6PP) (7)

T22 T39 A6 P1 ++ ++ + + + ++ - ++ + + - +++

Fig. 1. Secondary metabolites produced by Trichoderma spp. Level of production not produced; produced (05 mg L1); produced (525 mg L1); produced (2550 mg L1).

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A
6PP (7) T39butenolide (2) Harzianopyridone (3) Anthraquinone (5, 6) Harzianolide (4) T22azaphilone (1) Control 5.0 5.5

Treatments at 10 mg L-1
a a b b b a a 6.0 6.5 7.0 7.5 8.0 8.5

Treatments at 1 mg L-1
b a b a b b a

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

Disease severity (0-9)

Disease severity (0-9)

Fig. 2. Lesion severity on B. napus cotyledons treated with Trichoderma secondary metabolites and inoculated 3 h later with L. maculans. Disease severity was assessed after 14 days. A: Treatments at 10 mg Ll; B: treatments at 1 mg Ll. Bars indicates standard deviation. Means followed by the same letter are not signicantly different (P < 0.05). In parenthesis the compound number as in Fig. 1.

margin and a few pycnidia, 8 collapsing of cotyledon tissue with a few pycnidia, 9 collapsing of cotyledon tissue with masses of pycnidia] and 05 for B. cinerea (lesion size: 0 no symptoms, 1 < 2 mm, 2 25 mm, 3 510 mm, 4 1020 mm, 5 > 20 mm). Cotyledons from infected and control plants were harvested, ground in liquid nitrogen, and stored at 80  C until used for RNA extraction. Each experiment was duplicated. Data from two independent experiments (20 replicates each) were combined since statistical analysis determined homogeneity of variance (P 0.05). 2.5. Extraction of total RNA Total RNA was isolated from approximately 150 10 mg of frozen tomato and canola cotyledons, homogenized in TRI REAGENT (SIGMA) (1 ml per 50 mg of tissue). The samples were centrifuged at 12,000 g for 10 min at 4  C and incubated 5 min at room temperature. Chloroform (0.6 ml) was added; the mixture was shaken for 15 s, left at room temperature for 10 min then centrifuged (12,000 g for 15 min at 4  C). The supernatant was removed and 1.5 ml of isopropanol was added. The samples were left 10 min at room temperature and centrifuged. The precipitated total RNA was washed twice with 70% ethanol, and dissolved in diethylpyrocarbonate (DEPC) treated water. 2.6. Reverse transcriptase-polymerase chain reaction (RT-PCR) RT-PCR was used to detect transcripts of several genes in total RNA extracted from treated and control plants. The experiments on chitinase class IV of canola and endochitinase of tomato were carried out by using SuperScript One-Step RT-PCR system with

platinum Taq DNA polymerase (INVITROGEN, Carlsbad, CA), that permits the production of both complementary DNA (cDNA) and PCR products in a single procedure. Two different primer sets for the detection of plant transcripts involved in induced resistance were used: CHITINASE CLASS IV (50 -CTGCGGTTGTGCCCCAAACC-30 and 50 -AACCACAGACCCGTCCTG-30 [26,27]) and YLED (50 -AAAACAGCAACTACAACT-30 and 50 -TACCTCCTGTAAAATCCA-30 [28]). The primers were predicted to amplify from B. napus cDNA a 600 bp region encoding chitinase class IV, and, in the second case, a region of 300 bp fragment from L. esculentum cDNA encoding an endochitinase protein. For one step RT-PCR each reaction mixture (50 ml) contained: 1 ml of RNA (500 ng ml1), 1 ml of sense and anti-sense primers (10 mM), 25 ml of 2 Reaction Mix (a buffer containing 0.4 mM of dNTPs, 2.4 mM MgSO4), 1 ml of RT/Platinum Taq Mix (1.5 U). The thermal cycler was programmed to have cDNA synthesis followed immediately by PCR amplication: 1 cycle of 45 min at 30  C; 1 cycle at 94  C for 2 min; 40 cycles at 94  C for 15 s, 60  C for 30 s (B. napus) or 40  C for 30 s (L. esculentum), 72  C for 1 min; 1 cycle of 10 min at 72  C. The experiments on PR-1 of canola were carried out by using a double step RT-PCR (INVITROGEN). cDNA was synthesized by adding reverse transcriptase (INVITROGEN), 5 ml reverse transcriptase buffer, 5 ml of deoxynucleotides (containing 2.5 mM each of dATP, dCTP, dGTP, dTTP), and 6 ml oligo (dT)15 primer (500 mg/ml) to the RNA solution (500 ng; nal volume 50 ml) and incubating at 37  C for 1 h, then at 95  C for 5 min. The rst-strand cDNA was used as a template for PCR with primers PR1 (50 -CCACAAGACTAT GTCAACGC-30 and 50 -CATAATTGCCCCGAGGATC-30 [26]; P. Zhang and B. Fristensky, GenBank accession no. U70666). The primers were predicted to amplify a region of 370 bp from B. napus cDNA encoding a PR-1 protein. Actin was used as control (B. napus:

A
6PP (7) T39butenolide (2) Harzianopyridone (3) Anthraquinone (5, 6) Harzianolide (4) T22azaphilone (1) Control 3.5 3.7 3.9

Treatments at 10 mg L-1
a a b b b a a 4.1 4.3 4.5 4.7 4.9 5.1 5.3

Treatments at 1 mg L-1
b a b a b a a

3.5

3.7

3.9

4.1

4.3

4.5

4.7

4.9

5.1

5.3

Disease severity (1-6)

Disease severity (1-6)

Fig. 3. Effect of Trichoderma secondary metabolites treatments on disease severity of tomato cotyledons inoculated with B. cinerea. The disease was assessed after 14 days. A: Treatments at 10 mg Ll; B: treatments at 1 mg Ll. Bars indicates standard deviation. Means followed by the same letter are not signicantly different (P < 0.05). In parenthesis the compound number as in Fig. 1.

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ACTBN-R 50 -CATCACCAGAGTCGAGCAC-30 ; ACTBN-F 50 -AGGCTGG GTTTGCTGGTG-30 ). For PR-1 gene amplication, PCR conditions were as follows: 4 min at 94  C, 28 cycles of 94  C for 15 s, 56  C for 50 s, 72  C for 1 min. The reaction (50 ml) contained 2.5 ml of cDNA template, 10 pmol of each primer, 1 ml of 10 mM dNTP, 4 ml of 25 mM MgCl2, 5 ml of 10 PCR buffer without MgCl2 (INVITROGEN) and 1.5 U Taq DNA polymerase (INVITROGEN). 2.7. Plant growth promotion assay The method of Gillespie-Sasse et al. [29] was used, with some modications, to screen the plant growth promotion effect induced by the Trichoderma isolated compounds. Sterile 25 1 cm glass tubes were lled with 4 ml of test solution (puried Trichoderma secondary metabolites at concentrations ranging from 1 to 10 mg Ll). Six day old wheat (Triticum aestivum) seedlings were supported in sterile cotton wool and placed singly in the glass tube, with the roots in the test solutions. The plants were grown under indirect natural light at 25  C. Growth of stem height, rst and second leaves, were measured daily for 14 days. A solvent (1 ml of methanol in 15 ml of sterilized water) was used as control. Each treatment consisted of ten replicates and the experiment was repeated twice. 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). The activity of Trichoderma secondary metabolites as plant hormones were tested on pea (Pisum sativum) as determined by the methods of Findlay and Dougherty [30], with some modications. Surface sterilized pea seeds were planted in humid perlite in closed boxes until the seedlings had reached a height of 1215 cm. Etiolated pea stems were longitudinally split and washed with sterilized water. The stems were immersed in Petri dishes (6 cm diameter), containing 5 ml of the test solutions (ve stems per plate with four replications). After 48 h the split curvature on stems was evaluated as positive response. The activity of the test solutions were determined at a concentration of 106 M and compared to the indoleacetic acid- and water-treated samples used as controls. In order to analyze the effects of 6PP and harzianolide on plant growth, a 106 M solution of each compound was sprayed (10 ml) daily directly on tomato seedling for 21 days. Moreover, surface sterilized canola and tomato seeds were planted in water agar amended with 6PP or harzianolide (nal concentration: 106 M). The plant responses were measured as plant height, area of green leaves and root density. 3. Results 3.1. Characterization of Trichoderma secondary metabolites Major secondary metabolites obtained from culture ltrates of T22 and T39 T. harzianum strains (1: T22azaphilone; 2:

Size (Kb) - 0.36

PR-1 - 0.41 Actin

Fig. 5. RT-PCR analysis of B. napus defence genes of treated with Trichoderma metabolites using primers specic for a gene encoding a PR-1 protein (370 bp). Lanes 1: harzianolide 1 mg Ll; 2: anthraquinone 1 mg Ll; 3: harzianopyridone 1 mg Ll; 4: T22azaphilone 1 mg Ll; 5: T39butenolide 1 mg Ll; 6: 6PP 1 mg Ll; 7: water control; 8: solvent control.

T39butenolide; 3: harzianopyridone; 4: harzianolide; 5: 1-hydroxy-3-methyl-anthraquinone; 6: 1,8-dihydroxy-3-methyl-anthraquinone) (Fig. 1, 16) were isolated and characterized as previously reported [23]. The separation of T. harzianum A6 extract (520 mg) yielded 11 homogeneous fractions. Preparative TLC of fraction no. 2 gave 12 mg of the known anthraquinones (Figs. 1, 5 and 6). Fraction no. 5 was further fractionated using silica gel ash chromatography, giving 15 mg of harzianolide (Figs. 1 and 4). Eight chromatographic fractions were obtained from the oil residue (280 mg) of T. atroviride P1 liquid culture. Fraction no. 3 gave 72 mg of the known compound 6PP (Figs. 1 and 7). The metabolites isolated from P1 and A6 strains showed chromatographic and spectroscopic properties similar to those reported in literature [24,31,32]. Fig. 1 reports the compounds isolated from the culture ltrates of the Trichoderma strains used in this study. In vitro antibiotic activity of the secondary metabolites produced by T22 and T39 strains against G. graminis var. tritici, R. solani and P. ultimum were reported in Vinale et al. [23]. The anthraquinones (Figs. 1, 5 and 6), isolated from T. harzianum A6, showed little inhibition of G. graminis var. tritici and no activity towards R. solani or P. ultimum. Conversely, harzianolide (Figs. 1 and 4) inhibited G. graminis var. tritici at concentration of 200 mg plug1, but was less effective against R. solani and P. ultimum. 6PP (Figs. 1 and 7) isolated from T. atroviride P1 did not exhibit a specic antibiotic activity, since it completely inhibited the growth of the test pathogens when applied at concentration of 100 mg plug1. 3.2. Effect of Trichoderma secondary metabolites on plant disease severity The application of Trichoderma metabolites 3 h before inoculation with L. maculans signicantly reduced blackleg severity on B. napus seedling cotyledons (Fig. 2A,B). In particular, a 16% reduction of disease symptoms was obtained using 6PP (7) at 1 mg Ll; 12% and 9.5% reduction with harzianopyridone (3) at 10 and 1 mg Ll, respectively; about 13% reduction using harzianolide (4) at both concentrations, and 9.5% with T22azaphilone (1) at 1 mg Ll. A lower effect (8%) was registered with anthraquinones at 10 mg Ll.

Cs C 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Size (Kb) - 0.60

1
Fig. 4. RT-PCR analysis of B. napus defence genes of using primers specic for a gene encoding a chitinase class IV (600 bp) on: (i) seedlings treated with different puried Trichoderma secondary metabolites (lanes 112); (ii) seeds coated with T. harzianum T22 (1 107 spore ml1 for 1 g of seeds) 14 days post-treatment (lane 13); (iii) seedling infected with L. maculans 14 days post-inoculation (lane 14). Lanes Cs: control solvent; C: control water; 1: T22azaphilone 10 mg Ll; 2: T22azaphilone 1 mg Ll; 3: harzianolide 10 mg Ll; 4: harzianolide 1 mg Ll; 5: anthraquinone 10 mg Ll; 6: anthraquinone 1 mg Ll; 7: harzianopyridone 10 mg Ll; 8: harzianopyridone 1 mg Ll; 9: T39butenolide 10 mg Ll; 10: T39butenolide 1 mg Ll; 11: 6PP 10 mg Ll; 12: 6PP 1 mg Ll; 13: T. harzianum T22 seed coating; 14: L. maculans infection; 15: Molecular Weight (MW) Marker.

9 10 11 12 Cs C

Size (Kb) - 0.30

Fig. 6. RT-PCR analysis of L. esculentum defence genes of treated with Trichoderma secondary metabolites. The amplications were performed using specic primers for a gene encoding an endochitinase (300 bp) in L. esculentum seedlings. Lanes 1: T22azaphilone 10 mg Ll; 2: T22azaphilone 1 mg Ll; 3: harzianolide 10 mg Ll; 4: harzianolide 1 mg Ll; 5: anthraquinone 1 mg Ll; 6: anthraquinone 10 mg Ll; 7: harzianopyridone 10 mg Ll; 8: harzianopyridone 1 mg Ll; 9: T39butenolide 10 mg Ll; 10: T39butenolide 1 mg Ll; 11: 6PP 10 mg Ll; 12: 6PP 1 mg Ll; Cs: solvent control; C: water control; MW: Molecular Weight Marker.

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CONTROL

HAR 10-6 M

6PP 10-6 M

Fig. 7. Tomato plants foliar sprayed with 106 M 6PP [0.166 mg L1] and 106 M harzianolide [0.222 mg L1] solutions 20 days after treatments. (A) From left: control, harzianolideand 6PP-treated plants. (B) Particular of the root mass increase in the 6PP-treated plant (right) compared to the control (left).

Trichoderma metabolites treatments reduced the gray mold severity caused by B. cinerea on tomato cotyledons (Fig. 3A,B). An 18% reduction in disease symptoms was obtained by applying 6PP at 1 mg Ll; 17% and 12% with harzianopyridone (3) at 10 and 1 mg Ll, respectively; about 15% by using harzianolide (4) at both concentrations; 10% with anthraquinones at 10 mg Ll.

3.3. Induction of PR-protein expression in plants treated with Trichoderma secondary metabolites The expression of plant defence genes (chitinase class IV and PR1) was investigated by RT-PCR 36 h after treatment of B. napus cotyledons with Trichoderma metabolites (Figs. 4 and 5). The expected size of the amplied bands were 600 bp for the chitinase class IV and 360 bp for the PR-1 genes. Chitinase RNAs were detected when T22azaphilone (1 mg Ll), anthraquinones (10 mg Ll) or harzianopyridone (10 and 1 mg Ll) were applied (Fig. 4, lines 2, 5, 7 and 8). PR-1 genes were induced by anthraquinone, harzianopyridone and 6PP at 1 mg Ll (Fig. 5, lines 2, 3 and 6). No induction was detected at 10 mg Ll for any metabolites (data not shown). The transcription of both classes of genes was detectable in plants 14 d after inoculation with L. maculans and in plants emerging from seeds coated with T. harzianum T22 (Fig. 4, lines 13 and 14; for PR-1 data not shown). A 300 bp fragment was amplied from L. esculentum plants treated with Trichoderma metabolites by using YLED primers (Fig. 6). In particular, endochitinase gene expression was detected when harzianolide (10 and 1 mg Ll), anthraquinones (10 mg Ll), harzianopyridone (10 and 1 mg Ll), T39butenolide (10 mg Ll) and 6PP (10 and 1 mg Ll) were applied (Fig. 6, lines 3, 4, 6, 7, 8, 9, 11 and 12).

After immersion in 6PP and harzianolide solutions (106 M), etiolated pea stems showed a positive response (split curvature) compared to the controls (water- and indoleacetic acid-treated stems). The effect of 6PP and harzianolide on tomato plants was further investigated by foliar spray treatments to growing plants. Signicant increases in plant height and leaf area were observed on 106 M (0.166 mg L1) 6PP-treated plants, which appeared after 20 days much more developed and vigorous then the controls (Fig. 7). Moreover, the root system of plants treated with 6PP was more extensive and developed than those of untreated plants (Fig. 7B). Increased growth was not detected with 106 M harzianolide (0.222 mg L1) (Fig. 7A). Finally, seeds of tomato and oilseed rape, germinated in Water Agar (WA) containing 6PP (106 M), showed a signicant increase of seedling height (Fig. 8). 4. Discussion In the rst part of this work we isolated the major secondary metabolites produced by three T. harzianum strains (T22, T39 and A6) and one T. atroviride strain (P1). Seven known compounds obtained from fungal culture ltrates were extracted and characterized (Fig. 1). These compounds, with the exception of 6PP, showed different levels of antibiotic activity against the pathogens belonging to taxonomically unrelated groups, G. graminis var. tritici (Ascomycete), R. solani (Basidiomycete), P. ultimum (Oomycete), thus suggesting that each compound exhibits specic antibiotic activity and may play different roles in the direct antagonism of Trichoderma against diverse microbial pathogens. Although the production of some secondary metabolites is considered to be directly involved in the antagonism by some
Table 1 Growth promotion of wheat seedlings treated with Trichoderma secondary metabolites measured as stem leaf lengths (cm) and average of dry weight (mg) 12 days after treatment Stem leaf length (cm) Control T22azaphilone 10 mg Ll T22azaphilone 1 mg Ll 6PP 10 mg Ll 6PP 1 mg Ll T39butenolide 10 mg Ll T39butenolide 1 mg Ll Anthraquinone 10 mg Ll Anthraquinone 1 mg Ll Harzianolide 10 mg Ll Harzianolide 1 mg Ll Harzianopyridone 10 mg Ll Harzianopyridone 1 mg Ll 18.67 a 18.63 a 20.20 a 15.88 b 20.60 a 20.90 a 24.18 c 23.30 c 20.27 a 20.75 a 24.85 c 17.89 a 19.33 a SD 1.30 5.88 4.14 0.85 1.80 1.70 1.56 2.30 2.72 2.80 1.80 2.72 2.75 Dry weight (mg) 34 a 27 a 36 a 25 b 28 a 36 a 38 a 33 a 32 a 29 a 43 c 30 a 31 a SD 5.16 2.83 5.20 3.50 9.54 4.92 2.16 9.15 0.50 6.68 1.53 6.85 7.83

3.4. Effect of Trichoderma secondary metabolites on plant growth The effect of Trichoderma metabolites on plant growth promotion was initially evaluated by measuring shoot lengths and dry weights of wheat seedlings grown in contact with metabolite solutions (Table 1). Plant growth was improved by treatments with harzianolide (1 mg Ll), anthraquinones (10 mg Ll) and T39butenolide (1 mg Ll). On the other hand, T22azaphilone and harzianopyridone did not signicantly promote seedling growth in comparison to control. An effect on plant dry weight was only observed with 6PP at 10 mg Ll which reduced, and with harzianolide at 1 mg Ll which increased plant growth in comparison with other treatments, which were not signicantly different from the controls. Our results suggest a role of these metabolites in plant growth regulation. Therefore 6PP and harzianolide were further tested as plant growth regulators, eventually with an auxin-like activity.

Values are means of ten replicates. SD: standard deviation. Values with the same letter do not differ signicantly (P < 0.05).

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Fig. 8. Tomato (A) and canola (B) seedlings grown in WA containing 6PP (106 M) after 10 days from sowing.

biocontrol agents such as Trichoderma spp., the activity of these compounds in the soil environment and particularly towards the plant has not yet been fully elucidated [15,33]. Recently in the past 10 years, it is being well documented that the interaction of some Trichoderma strains with the plant may result in promotion of growth, increased nutrient availability, improved crop yield and enhance disease resistance [13]. In addition to direct activity against phytopathogens, some Trichoderma spp. are able to colonize root surfaces, interact with the plant, and exchange compounds that can cause substantial changes in plant metabolism [13,34,35]. The induction of plant resistance mechanisms by different Trichoderma strains has been shown to enhance the production of defence-related metabolites in the plant such as enzymes involved in the biosynthesis of phytoalexins or in the response to oxidative stress, pathogenesis-related proteins (PR-proteins), etc. [3436]. The induced systemic resistance (ISR) has been related to the activation of plant defence genes by pathogens and/or elicitors [9]. Trichoderma metabolites may act as elicitors of the plant defence system by enhancing plant resistance against biotic stress, such as invading fungi and bacteria [37], as well as towards abiotic stresses [13]. Different classes of compounds may act as elicitors or resistance inducers during the interaction of Trichoderma with plants [13,16]. These molecules may include: (i) proteins with enzymatic activity, such as endoxylanase; (ii) avirulence (avr) gene products able to induce defence reactions in plants having the correspondent resistance gene; and (iii) low-molecular-weight compounds released from fungal or plant cell walls by the activity of Trichoderma enzymes [13]. However, the role that secondary metabolites may have in the interaction of Trichoderma spp. with plants has not been determined to date. In order to investigate the potential involvement of secondary metabolites in the induction of systemic resistance during the Trichoderma plant interaction, we evaluated the ISR-inducing ability of the compounds isolated from different Trichoderma culture ltrates. A reduction of disease symptoms on tomato and canola seedlings treated with the puried metabolites and inoculated, respectively, with the pathogens B. cinerea or L. maculans was observed. A positive effect was obtained by using 6PP (7) at 1 mg Ll on both plants, or harzianopyridone (3) at 10 mg Ll on canola and at both concentrations on tomato (Figs. 2 and 3). Interestingly, harzianolide (4) showed similar results both at 1 and 10 mg Ll for canola and tomato plants. 6PP (7) at 10 mg Ll did not reduce disease symptoms on either plants, probably because of a phytotoxic effect at high concentrations (Table 1). RT-PCR analysis carried out by using primers for chitinase IV, PR-1 and endochitinase, indicated that treatments with the puried fungal compounds induced an over-expression of the tested defence genes in planta. Thus, the results of the in vivo experiments indicated that the isolated Trichoderma metabolites are directly involved in the activation of plant defence genes, as previously reported for peptaibols [38]. In particular, peptaibols induced an over-expression of defence related genes involved in the systemic responses of the plant.

Previous studies carried out in greenhouse reported a considerable yield increase when plant seeds were pre-treated with a Trichoderma spore suspension [10]. A similar result was observed when seeds were separated from Trichoderma by a cellophane membrane, indicating that Trichoderma produces diffusible metabolites promoting plant growth [39]. The dose-effect responses of Trichoderma secondary metabolites on plant growth regulation are yet to be determined [15]. Trichoderma secondary metabolites may possibly act as auxin-like compounds [15], which typically have an optimum activity at low concentrations (105 and 106 M) while having an inhibitory effect at higher doses [4042]. In particular our data conrm a possible involvement of Trichoderma secondary metabolites in plant growth regulation. In fact, both 6PP and harzianolide affected the growth of wheat seedlings and showed an auxin-like activity on etiolated pea stems, thus supporting the hypothesis that these natural compounds actively inuenced the growth of Trichoderma-colonized plant. Moreover, the experiments carried out in our laboratories showed that 6PP-treated plants were much better developed and vigorous than controls. In agreement with our results, Parker et al. [43] showed that 6PP has a concentration-dependent activity when acting as plant growth regulator in a wheat coleoptile assay (phytotoxic activity detected at 103 M, but not at 104 M). The results reported in this work clearly indicated that some Trichoderma secondary metabolites are directly involved in the Trichoderma-plant interactions, and particularly that the compound 6PP may be considered to act as an auxin-like compound and/or may act as an auxin inducer. The identication of new molecular effectors may support the application of new biopesticides and biofertilizers based on Trichoderma metabolites to be used instead of the living microbes as elicitors of plant defence mechanisms and plant growth stimulants.

Acknowledgments The authors thank the graduate student Antonio Mauro for his assistance. This work was supported by the following projects: FIRB 2002 prot. RBNE01K2E7; MIUR-PON numbers: DD12935, DD5266, 5911, 10418; EU TRICHOEST QLK3-2002-02032; EU 2E-BCAs; Uni` degli Studi di Napoli Federico II Programma di Scambi versita `. Internazionali per la Breve Mobilita

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