Antifungal activity of three spermidine conjugates

Dale Walters a;*, Barbara Meurer-Grimes b;1, Ivelisse Rovira b

a Department of Plant Biology, The Scottish Agricultural College, Auchincruive, Nr Ayr, KA6 5HW, UKb Department of Biological Sciences, Lehman College, The City University of New York, 250 Bedford Park Boulevard West, Bronx, NY 10468, USA

Received 9 May 2001; accepted 5 June 2001

First published online 28 June 2001

Abstract

Three tri-substituted spermidines, di-p-coumaroyl-caffeoylspermidine, tri-caffeoylspermidine and tri-p-coumaroylspermidine, isolatedfrom pollen of Quercus alba, were examined for antifungal activity. Both di-p-coumaroyl-caffeoylspermidine and tri-p-coumaroylspermidinereduced mycelial growth of the oat leaf stripe pathogen, Pyrenophora avenae and reduced powdery mildew (Blumeria graminis f. sp. hordei)infection of barley seedlings when applied as a post-inoculation treatment. When used as a pre-inoculation treatment, only di-p-coumaroyl-caffeoylspermidine reduced powdery mildew infection significantly. Growth of P. avenae in the presence of 100 WM di-p-coumaroyl-caffeoylspermidine reduced activity of S-adenosylmethionine decarboxylase (AdoMetDC), and led to a reduction in the incorporation oflabelled ornithine into spermidine. The other two spermidine conjugates increased AdoMetDC activity and the flux label from ornithineinto spermine in P. avenae significantly. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V.All rights reserved.

Keywords: Spermidine conjugate; Polyamine biosynthesis; Antifungal ; Fungicide

1. Introduction

Hydroxycinnamic acid amides (HCAs) of the di- andpolyamines are widely distributed in higher plants [1].They are particularly associated with reproductive struc-tures and a number of workers have isolated them frompollen. Thus, di-substituted hydroxycinnamoylspermidineswere described from the pollen of Quercus dentata [2], butHCAs are also found in other plant tissues and have forsome time been associated with plant-pathogen interac-tions [3]. Most of this work relates to the possible involve-ment of HCAs in the hypersensitive response of the hostto virus infection [4,5], but some reports suggest an in-volvement of HCAs in interactions between plants andfungal pathogens [6,7]. As part of a programme of workstudying the possible involvement of HCAs in plant resis-

tance to fungal infection, it was decided to examine theantifungal activity of HCAs. Here we report the e¡ects ofthree tri-substituted hydroxycinnamic acid spermidines, di-p-coumaroyl-ca¡eoylspermidine, tri-ca¡eoylspermidineand tri-p-coumaroylspermidine, on mycelial growth ofthe oat leaf strip pathogen Pyrenophora avenae and oninfection of barley with the powdery mildew fungus, Blu-meria graminis f. sp. hordei.

2. Materials and methods

2.1. Isolation of spermidine conjugates from oak pollen

The three spermidine conjugates were isolated from pol-len of the oak Q. dentata and their structures elucidated asdescribed previously [2]. The chemical structure of theconjugates is shown in Fig. 1. These compounds havevery limited solubility in water and so, for experimentalpurposes, were dissolved in methanol. In the experimentsdescribed below, methanol was added to the controls togive the appropriate concentrations. In preliminary experi-ments, it was con¢rmed that the concentration of metha-nol (0.1%) had no e¡ect on fungal growth, fungal infectionor polyamine biosynthesis.

0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 2 7 8 - 6

* Corresponding author. Tel. : +44 (1292) 525307;Fax: +44 (1292) 525314.

E-mail address: d.walters@au.sac.ac.uk (D. Walters).

1 Present address: Cerylid Pty Ltd, Natural Products Chemistry,576 Swan Street, Richmond, Vic. 3121, Australia.

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2.2. Determination of the fungicidal activity of thespermidine conjugates

The e¡ects of the three compounds on infection of bar-ley seedlings with the powdery mildew fungus B. graminisf. sp. hordei Marchal were determined as described previ-ously [8].

2.3. E¡ect of the spermidine conjugates on growth andpolyamine biosynthesis of P. avenae

2.3.1. Growth of P. avenae on solid mediumP. avenae was maintained on potato dextrose agar

(PDA). Filter-sterilised solutions (10 ml) containing theconjugates were added to 70 ml of sterile PDA at 45^47³C to obtain a ¢nal concentration of 100 WM. Sterilemedium (20 ml) was added aseptically to a 90 mm single-vent sterile plastic Petri dish, and a sterile 8 mm diametercork borer used to remove plugs of mycelium from theedges of stock cultures, which were inverted and placedin the centre of each Petri dish. Control plates containedculture medium plus 0.1% methanol, and inoculated plateswere incubated in the dark at 24³C. Radial mycelialgrowth, excluding the mycelial plug, was measured 3, 6and 8 days after inoculation.

2.3.2. Growth of P. avenae in liquid medium anddetermination of the formation of polyamines fromradio-labelled ornithine in the fungus

Filter-sterilised solutions (10 ml) containing the com-pounds were added to 140 ml of sterile potato dextrosebroth in 250 ml conical £asks to obtain the desired ¢nalinhibitor concentration. Control £asks contained medium

only. Flasks were inoculated with a 10 mm disc of myce-lium and placed in a Gallenkamp orbital shaker (140 rpm)at 24³C. After 4 days, the fungus was washed with distilledwater through a ¢ne-mesh sieve and centrifuged at16 000Ug at 0³C for 10 min. The pellet obtained wasweighed and used to determine the formation of poly-amines from radio-labelled ornithine as described previ-ously [9].

2.4. Statistical analysis

Results were calculated as the means of eight replicates.For fungal growth on solid medium and in liquid culture,results were calculated as the means of 12 and ¢ve repli-cates respectively, while results for the formation of poly-amines from radio-labelled ornithine were calculated asthe means of four replicates. All experiments were re-

Fig. 1. Structural formulas for the tri-substituted hydroxycinnamic acid spermidines.

Fig. 2. E¡ects of three spermidine conjugates on mycelial growth of P.avenae. A: Di-p-coumaroyl-ca¡eoylspermidine; B: tri-ca¡eoylspermi-dine; C: tri-p-coumaroylspermidine ; all compounds used at 100 WM. Aand B signi¢cantly di¡erent from the control at P6 0.001.

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peated with similar results and statistical signi¢cance wasassessed using Student's t-test.

3. Results and discussion

The three spermidine conjugates reduced mycelialgrowth of P. avenae signi¢cantly when used at 100 WM(Fig. 2). The greatest reduction was obtained with di-p-coumaroyl-ca¡eoylspermidine, which reduced mycelialgrowth by 56%, while tri-p-coumaroylspermidine reducedgrowth by 50% (Fig. 2). Interestingly, although di-p-cou-maroyl-ca¡eoylspermidine reduced powdery mildew infec-

tion on barley seedlings when applied both pre- and post-inoculation, tri-p-coumaroylspermidine only reduced mil-dew infection when applied as a post-inoculation treat-ment. When applied in this way, it reduced mildew infec-tion on barley seedlings by 92% (Fig. 3). Tri-ca¡eoylspermidine had no signi¢cant e¡ect on mildew in-fection. This could indicate that the p-coumaroyl residuein either or both, the N1 and N10 position of the spermi-dine, is required for the activity of the compound. There isa paucity of data on the involvement of HCAs in inter-actions between plants and pathogenic fungi. However, ithas been shown that feruloylputrescine accumulates in po-tato tubers in response to infection by Phoma exigua [13]and p-coumaroyl-2-hydroxyputrescine accumulated inwheat challenged with rust [9]. In more recent work, anovel HCA, p-coumaroyl-hydroxyagmatine (pCHA) wasfound to accumulate in barley expressing resistance topowdery mildew [10]. Indeed, pCHA was also shown toexhibit signi¢cant activity against the powdery mildewfungus both in vivo and in vitro [10].

All of the conjugates a¡ected activity of the polyaminebiosynthetic enzyme AdoMetDC and the incorporation oflabelled ornithine into polyamines. Thus, di-p-coumaroyl-ca¡eoylspermidine reduced AdoMetDC activity (Fig. 4)and the £ux of label into spermine, while the other twocompounds increased AdoMetDC activity and the incor-poration of labelled ornithine into spermine (Table 1).Whether these alterations are related to the antifungalactivity of the compounds is not known. Although sper-mine concentrations were not determined in this work, thedata presented indicate an increased biosynthesis of sper-mine. Spermine is known to exert a direct toxic e¡ect onanimal and plant cells [11,12], and work on cell-free mod-els suggests that spermine accumulation triggers the acti-vation of caspase-3, leading to the induction of apoptosis[13]. It would be useful to determine whether spermineaccumulation is responsible, at least in part, for the anti-fungal activity of tri-ca¡eoylspermidine and tri-p-coumar-oylspermidine. However, spermine accumulation seemsunlikely to be responsible for the antifungal activity ofdi-p-coumaroyl-ca¡eoylspermidine, because spermine bio-

Fig. 3. E¡ects of three spermidine conjugates on powdery mildew infec-tion of barley seedlings. a: treatments applied pre-inoculation, b: treat-ments applied post-inoculation. A: Di-p-coumaroyl-ca¡eoylspermidine;B: tri-ca¡eoylspermidine; C: tri-p-coumaroylspermidine; all compoundsused at 100 WM. Signi¢cant di¡erences are shown at *P6 0.001.

Fig. 4. E¡ects of three spermidine conjugates on AdoMetDC activity inP. avenae. A: Di-p-coumaroyl-ca¡eoylspermidine; B: tri-ca¡eoylspermi-dine; C: tri-p-coumaroylspermidine ; all compounds used at 100 WM.Signi¢cant di¡erences are shown at *P6 0.001.

Table 1E¡ect of three spermidine conjugates on the incorporation of radio-la-belled ornithine into polyamine in P. avenae

Compound Radioactivity in polyamine (dpm mg protein31)

putrescine spermidine spermine

Control 14 þ 2.9 17 þ 2.4 28 þ 7.2A 21 þ 4.2 15 þ 1.7 18 þ 1.0*B 25 þ 3.9* 17 þ 1.8 56 þ 4.1**C 34 þ 3.7** 29 þ 1.7** 80 þ 5.4**

A: Di-p-coumaroyl-ca¡eoylspermidine, 100 WM.B: Tri-ca¡eoylspermidine, 100 WM.C: Tri-p-coumaroylspermidine, 100 WM.Signi¢cant di¡erences as shown at *P6 0.01 and **P6 0.001.

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synthesis was reduced in P. avenae treated with this com-pound. Indeed, it is unlikely that reduced intracellularpolyamine levels are responsible for the activity of thisconjugate since the incorporation of labelled ornithineinto putrescine and spermidine was una¡ected by growthof P. avenae in the presence of di-p-coumaroyl-ca¡eoyl-spermidine. Further work will be required to pinpointthe mode of action of these spermidine conjugates.

Acknowledgements

The Scottish Agricultural College receives grant-in-aidfrom SERAD. I.R. was supported by a NIH/MARC fel-lowship (5T34GMO882).

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