Ó Springer 2006
Mycopathologia (2006) 162: 65 68 DOI 10.1007/s11046-006-0030-0
Direct evidence of plant-pathogenic activity of fungal metabolites of Trichothecium roseum on apple Martin Zˇabka1, Kamila Drastichova´1, Alexandr Jegorov2, Julie Soukupova´3 & Ladislav Nedbal3 Faculty of Agriculture, University of South Bohemia, Cˇeske´ Budeˇjovice, Czech Republic; 2IVAX Pharmaceuticals, Research and Development Unit, Cˇeske´ Budeˇjovice, Czech Republic; 3Institute of Systems Biology and Ecology CAS and Institute of Physical Biology JU, Nove´ Hrady, Czech Republic
1
Received 22 January 2004; accepted in revised form 13 April 2006
Abstract Apples were exposed to various concentrations of roseotoxins metabolites of Trichothecium roseum and kinetic fluorescence imaging was used to detect the area influenced by the phytotoxin. Contrast was quantified within these images between the areas exposed to roseotoxins and the untreated areas. It was proved that roseotoxin B is able to penetrate apple peel and produce chlorotic lesions. Activity of roseotoxin B is similar as the activity of destruxins, host specific phytotoxins of Alternaria brassicae parasitic on canola. Key words: apples, chlorophyll fluorescence imaging, Trichothecium roseum
Introduction The vast majority of fungal secondary metabolites have no known biological function, their production, however, is most probably not accidental, but has developed as a phylogenetic adaptation providing some distinct advantage in the hostile or competitive environment. Production of some secondary metabolites has been shown to correlate with the virulence or pathogenity of the fungi. These metabolites are usually denominated as host selective toxins (HST’s) and a fungus that makes them causes more disease on its host than one that is otherwise identical, but does not make HST’s [1]. Cyclic peptides have been described as HST’s from a number of phytopathogenic fungi, e.g., Alternaria brassicae destruxins, Alternaria alternata tentoxin, Phoma lingam phomalide and Cochliobolus carbonum HC-toxin [2 7]. Trichothecium roseum, a typical saprobe reported also as
phytopatogen on apples, tomatoes, nectarines, plums, and prunes, produces several potentially toxic secondary metabolites, particularly trichothecenes and roseotoxins [8 11]. Roseotoxins represent a group of lipophilic and neutral cyclohexadepsipeptides containing linear or branched C5- or C6-a-hydroxy acid residues (Figure 1) [12, 13]. The fluorescence emission of chlorophyll is widely used as a non-invasive tool to determine the photosynthetic activity including applications where plant response to fungal infection was investigated [14, 15]. The fungal infection is usually well localised on the leaf surface and the signals from infected and healthy tissues can be separated only by acquiring fluorescence images [16, 17]. The aim of the present study was to test the capacity of fluorescence imaging technique to detect the impact of roseotoxins on apples. In addition, the objective of the study was to prove if the changes observed on apples are related to
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Roseotoxin B 1
Figure 1. Structures of destruxin A, cyclo(D-2-hydroxypent-4-enoic acid -Pro2-Ile3-MeVal4-MeAla5-b-Ala6), and roseotoxin B, cyclo(D-2-hydroxypent-4-enoic acid1 3-Me-Pro2-Ile3-MeVal4-MeAla5-b-Ala6).
those observed with destruxins on Alternaria brassicae.
Material and methods Cultivation of the fungus, isolation and characterisation of roseotoxins are described elsewhere [12, 13]. Solution of roseotoxin B in acetone was applied on the surface of apple (Golden delicious) and the same volume of acetone was used for control spots. Chlorophyll fluorescence imaging on apples was performed using a method, which we have previously developed, for measurement of effect of destruxins, metabolites of phytopathogenic fungus Alternaria brassicae, on leaves of Brassica napus [18]. Briefly, a commercial imaging fluorometer (FluorCam, P.S. Instruments, Brno, Czech Republic), as described in [19], was used. The fluorescence emission of the apples was measured 24 h following the roseotoxin treatment. The images of several fluorescence parameters (F0, FM, FP, and FS) were captured. The F0 fluorescence emission yield was measured in dark-adapted apple when the primary quinone acceptor QA was oxidized and the non-photochemical quenching was inactive. Strong light pulses (1.6 s, 2000 lmol (photon) m)2 s)1) were used to close transiently all the reaction centres of Photosystem II that led to zero photochemical efficiency and, consequently, to maximum fluorescence emission yield FM. The actinic light
(60 lmol (photon) m)2 s)1) was used to generate photosynthetic activity reflected in the Kautsky effect of fluorescence induction. The FP parameter was measured as the maximum fluorescence yield reached after ca. 1 s in the actinic light (FP < FM). The FS is the steady-state fluorescence emission yield attained after ca. 100 s of the actinic light exposure.
Results Green apple was used to monitor exposure to various levels of roseotoxins phytotoxins of Trichothecium roseum. Images of fluorescence signals F0, FM, FP, and of FS were captured in a single kinetic experiment. First, the dark-adapted apple was exposed to dim flashes measuring F0. FM was measured during the bright flash of light that transiently reduced the plastoquinone pool effectively blocking the photochemistry. After ca. 10 s of dark relaxation the apple was exposed to a moderate actinic light that induced a fluorescence transient reaching a maximum FP before declining to a steady-state level of FS. The recently developed fluorescence imaging technique was applied here to identify a reporter parameter that offers the highest image contrast between the healthy areas and the phytoxoxin-affected tissue [19]. The highest contrast was found in the image constructed by pixel-to-pixel division of images F0 by FP and of F0
67 by FM. The symptoms appear to be light-dependent [3]. The roseotoxin-treated segments exhibited elevated FS fluorescence and were detected as the bright lesions on the surface (Figure 2). The roseotoxins-free control solution had no effect on the fluorescence emission. Visual symptoms of the roseotoxin intoxication were observed always significantly later than in fluorescence imaging. The fluorescence emission kinetics within the affected and non-affected areas was used for further statistical evaluation. The fluorescence kinetics measured in the roseotoxin-treated areas and control areas were averaged and the standard deviations of roseotoxin-treated and control segments were calculated. Discussion The fluorescence imaging technique was proved to provide sensitive detection of phytotoxic activity of roseotoxin B prior to any visual detection. Thus, this technique makes it possible to localise phytotoxin-affected and healthy tissues. The increase in F0/FM ratio was identified as the fluorescence parameter yielding the highest contrast between the roseotoxin-treated and control areas. It was proved that roseotoxin B on apples exhibits essentially the same characteristic as we obtained previously with destruxins on canola leaves (Brassica napus) [18]. It is interest-
Figure 2. The F0/FM image of the apple treated with 20 ll droplet of 10 mg ml)1 of roseotoxin B (light blot), control droplet of roseotoxin-free acetone and droplets containing 1 mg ml)1 and 0.1 mg ml)1 (not visible, symmetrical along the apple C4-axis).
ing to note that roseotoxin B was applied on the intact apple surface without any pre-treatment. Even in such case, some amount of roseotoxin B was able to penetrate through apple peel and produce chlorotic lesions. In the case of any puncture, the effect of roseotoxin B was detectable at much lower dose. However, because of non-homogeneous surface of apples, flat canola leaves is more suitable model for testing. Although roseotoxins were originally described as a distinct group of fungal toxins, no wonder from both the chemical point of view and biological effect, that they actually belong to the only one group including destruxins, roseotoxins, and bursaphelocides. Despite a number of studies, molecular basis of selective phytotoxicity is poorly understood. The phytotoxic effect manifested by formation of chlorotic and necrotic lesions facilitated by various cyclic peptides can have several possible explanations and it may differ for sensitive and resistant plants. On one hand, destruxin B, the major phytotoxin of A. brassicae, appears to be the virulence factor, contributing mostly to the aggressiveness phytotoxicity of A. brassicae by conditioning the host tissue [3]. Similar effects exhibit also, e.g., tentoxin and phomalide [4 7]. On the other hand, cyclic peptides can act as elicitors facilitating a hypersensitive reaction of a host plant, defensive production of phytoalexins and leaf remove effect [6, 7, 20, 21]. Alternatively, the observed effect can be connected with other activities of roseotoxins (destruxins), which are not related to direct toxicity, for example with the membrane activity of cyclic peptides, which might help the fungus to affect osmotic regulation of host cells and to obtain water or minerals [22]. Another question is connected with the definition of host-specificity. Destruxins have been described as host specific toxins of Alternaria brassicae. The production of destruxins and roseotoxins by the fungus Trichothecium roseum on apples in vivo has been also described [13]. It is a striking fact that the production of the same group of compounds has been developed in entomopathogenic fungi Metarhizium anisopliae, Aschersonia aleyrodis, and some imperfect nematicidal fungus, strain D1084 [23, 24]. We contributed in this work to the description of one novel activity of roseotoxins. Although numerous activities described so far for the group of ‘‘destruxins’’, their
68 exact role in either the insect or phytopathogenesis still remains obscure. Acknowledgement This work was supported by grant No. 203/04/0799 from the Grant Agency of the Czech Republic. JS and LN were supported in part by the grants LN00A141, LC545, and MSM6007665808 of the Czech Ministry of Education and by the AV0Z60870520 project of Institute of Syst. Biology & Ecology CAS. References 1.
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Address for correspondence: Alexandr Jegorov, IVAX Pharmaceuticals, Research and Development Unit, Branisovska 31, 370 05 Cˇeske´ Budeˇjovice, Czech Republic E-mail:
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