Direct Effects of Physcion, Chrysophanol, Emodin and Pachybasin on

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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Direct Effects of Physcion, Chrysophanol, Emodin, and Pachybasin on Germination and Appressorium Formation of the Barley (Hordeum vulgare L.) Powdery Mildew Fungus Blumeria graminis f. sp. hordei (DC.) Speer Ulrich Hildebrandt,* Alexander Marsell, and Markus Riederer Universität Würzburg, Julius-von-Sachs-Institute for Biosciences, Chair of Botany II, Julius-von-Sachs-Platz 3, 97082 Würzburg, Germany S Supporting Information *

ABSTRACT: Several anthraquinone derivatives are active components of fungicidal formulations particularly effective against powdery mildew fungi. The antimildew effect of compounds such as physcion and chrysophanol is largely attributed to host plant defense induction. However, so far a direct fungistatic/fungicidal effect of anthraquinone derivatives on powdery mildew fungi has not been unequivocally demonstrated. By applying a Formvar-based in vitro system we demonstrate a direct, dose-dependent effect of physcion, chrysophanol, emodin, and pachybasin on conidial germination and appressorium formation of Blumeria graminis f. sp. hordei (DC.) Speer, the causative agent of barley (Hordeum vulgare L.) powdery mildew. Physcion was the most effective among the tested compounds. At higher doses, physcion mainly inhibited conidial germination. At lower rates, however, a distinct interference with appressorium formation became discernible. Physcion and others may act by modulating both the infection capacity of the powdery mildew pathogen and host plant defense. Our results suggest a specific arrangement of substituents at the anthraquinone backbone structure being crucial for the direct antimildew effect. KEYWORDS: anthraquinones, barley, fungicide, physcion, powdery mildew, germination, appressorium

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INTRODUCTION Many anthraquinones are known to exhibit antimicrobial properties. Therefore, it is not surprising that anthraquinones are found among the active components of plant extracts commercialized as botanical pesticides.1 The efficacy of anthraquinone containing plant extracts such as from giant knotweed (Reynoutria sachalinensis), rhubarb (Rheum sp.), dock (Rumex sp.), or senna (Cassia sp.) against fungal plant pathogens has been demonstrated by several studies.2−6 An ethanolic extract of giant knotweed leaves proved to be extremely effective in controlling powdery mildew disease on wheat, cucumber, tomato, and other crop plants. Its major mode of action was attributed to triggering induced local resistance, resulting in phytoalexin accumulation, inhibition of fungal development, and consequently disease reduction.2,7,8 The major resistance eliciting constituents of giant knotweed extract were identified as the anthraquinone compounds physcion (1,8-dihydroxy-3-methoxy-6-methylanthracene-9,10dione, 1) and emodin (1,3,8-trihydroxy-6-methylanthracene9,10-dione, 3) (Figure 1).9,10 Consequently, the Fungicide Resistance Action Committee (FRAC) currently lists the formulations based on giant knotweed extract by their mode of action as host plant defense inducing compounds.11 However, until now direct fungistatic or fungitoxic effects of these anthraquinone compounds on powdery mildew fungi were not unambiguously demonstrated. In vivo, on cucumber leaves, the germination of conidia of Podosphaera f uliginea (cucumber powdery mildew) was only slightly inhibited by treatment with Milsana, a formulated giant knotweed extract.1 On the contrary, the majority of conidia of © XXXX American Chemical Society

the wheat powdery mildew (Blumeria graminis f. sp. tritici) and of the tomato powdery mildew (Leveillula taurica) remained nongerminated in vitro on agar plates supplemented with Milsana as well as in vivo on Milsana sprayed leaves, indicating a direct fungistatic effect resulting from knotweed extract treatment.5,6 Spraying wheat leaves with a physcion (1) solution gave similar in vivo results with respect to germination inhibition, again indicating a direct effect of this anthraquinone derivative on B. graminis germination, whereas a germination suppressing effect due to a physcion-induced chemical defense reaction of the host plant was not excluded.12,13 Induced antifungal compounds present in or on the plant cuticle, such as lipophilic surface flavonoids or antimicrobial lipid transfer proteins, could affect the prepenetration behavior of B. graminis conidia.14−16 However, despite comparable results in vivo, a subsequent study with B. graminis f. sp. hordei even showed increased conidial germination rates in vitro on physcion (1) treated agar plates.17 The authors concluded that physcion (1) does not directly affect conidial germination of B. graminis but instead is effective against barley powdery mildew by inducing a localized defense response. In support of that mode of action, physcion (1) was recently demonstrated to strongly regulate expression of plant defense related genes in cucumber.18 Nevertheless, studies excluding the host plant as a source of potentially antifungal compounds are frequently hampered by Received: Revised: Accepted: Published: A

December March 14, March 20, March 20,

21, 2017 2018 2018 2018 DOI: 10.1021/acs.jafc.7b05977 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

essential prepenetration processes of the world’s sixth most important fungal plant pathogen.28

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MATERIALS AND METHODS

Plant and Fungal Material. Barley (Hordeum vulgare cv. ‘Stendal’, Poaceae, IG Pflanzenzucht, Munich, Germany) seeds were sown in plastic pots (diameter 9 cm) filled with Typ ED73 standard potting soil (SteuderComp, Schermbeck, Germany) and kept in growth chambers with a light intensity of 300 μmol photons/m2/s in a 16 h:8 h (L:D) photoperiod at 20 °C:18 °C and 70% relative humidity. Blumeria graminis f. sp. hordei (race A6) was propagated on its hosts until distinct white powdery pustules appeared. One day before conidia were required for experimentation, spore-bearing leaves were shaken to remove older conidia, so that freshly emerged conidia were available for subsequent assays. Preparation of Artificial and Natural Surfaces. For obtaining total cuticular leaf wax extracts, primary and secondary entire leaves (apart from cut edges) from 14 day old plants were dipped for 2 min into chloroform (50 mL; >99%) (Roth, Karlsruhe, Germany). The solvent was removed under a gentle flow of nitrogen, and the dry residue was dissolved in chloroform. The solution was supplemented with Formvar resin (polyvinyl formal) (Applichem, Darmstadt, Germany) and acetone to final concentrations of 0.5% (w/v) Formvar and 3% (v/v) acetone adjusted to 250 μg/mL cuticular leaf wax.22 Microscopy cover glasses (21 × 26 mm) were immersed in chloroform for 1 h, dried, and then carefully cleansed with a few drops of Deconex 11 UNIVERSAL detergent (Applichem, Darmstadt, Germany), subsequently rinsed with distilled water, immersed in isopropanol for 10 s, again in distilled water for 1 min, and finally dried at room temperature. The cover glasses were coated by dipping for 30 s into the wax/Formvar solution and drying at room temperature for at least 24 h to ensure complete solvent evaporation. For analyzing B. graminis in vitro prepenetration processes in the presence of anthraquinones, Formvar/wax dipping solutions were supplemented with stock solutions of either physcion, 1 (≥98%), chrysophanol, 2 (1,8dihydroxy-3-methylanthracene-9,10-dione, ≥98%), emodin, 3 (≥97%), pachybasin, 4 (1-hydroxy-3-methylanthracene-9,10-dione), danthron, 5 (1,8-dihydroxyanthracene-9,10-dione, 96%), anthraquinone, 6 (anthracene-9,10-dione, 97%), rhein, 7 (4,5-dihydroxy-9,10dioxoanthracene-2-carboxylic acid, ≥95%), aloe-emodin, 8 (1,8dihydroxy-3-(hydroxymethyl)anthracene-9,10-dione, ≥97%), alizarin, 9 (1,2-dihydroxyanthracene-9,10-dione, 97%), quinizarin, 10 (1,4dihydroxyanthracene-9,10-dione, 96%), 2,3-dimethylanthraquinone, 11 (2,3-dimethylanthracene-9,10-dione, 98%), or 2-methylanthraquinone, 12 (2-methylanthracene-9,10-dione, ≥95%), in acetone to final concentrations ranging from 10−4 to 10−1 mM (all from SigmaAldrich, Munich, Germany). All of the chemicals were applied as received without further purification. The dipping solutions were adjusted to contain equal amounts of cuticular leaf waxes, 0.5% Formvar (w/v), and 3% acetone (v/v). Exemplifying anthraquinone coating, cover glasses that had been dipped into Formvar/wax solutions with different concentrations of physcion (1) (0.1 mM, 1 mM and 2.5 mM) were re-extracted in acetone and the amount of eluted physcion/cm2 was determined spectrophotometrically using the molar extinction coefficient (ε) of 12,133 at 434 nm (λmax).29 The coated cover glasses were fixed on microscopy glass slides that were positioned at the base of a settling tower. Conidia from infected barley leaves were blown into the tower using pressurized air to ensure their even distribution at a density of approximately 2 × 103 conidia/cm2. In order to study effects of physcion (1) treatment on fungal germination and appressorium formation in vivo we analyzed fungal development on detached primary barley leaves that were sprayed with 10−4−10−1 mM physcion (1) 6 h before inoculation with B. graminis conidia. By using a standard glass chromatography sprayer the leaves were sprayed until runoff with aqueous spray mixtures containing 0.025% Tween 20, 10% acetone, and the respective concentrations of physcion (1). Control leaves were mock treated with a spray mixture without physcion (1). The leaf surface was completely dry 6 h after spraying. Subsequently, the sprayed barley leaves with their adaxial surface up

Figure 1. Structures of the assayed anthraquinones physcion (1), chrysophanol (2), emodin (3), pachybasin (4), danthron (5), anthraquinone (6), rhein (7), aloe-emodin (8), alizarin (9), quinizarin (10), 2,3-dimethylanthraquinone (11), and 2-methylanthraquinone (12). Carbon numbering is indicated for anthraquinone (6). Common structural moieties of physcion (1), chrysophanol (2), emodin (3), and pachybasin (4) are indicated by a rectangle.

the obligately biotrophic nature of powdery mildew fungi, since these are fully dependent on the presence of living host tissue for the completion of their life cycle. For an efficient induction of germination and formation of infection structures, summarized as prepenetration processes, the grass and cereal powdery mildew fungus B. graminis requires a hydrophobic surface in combination with specific host cuticular wax constituents.19−22 By applying a Formvar-based in vitro system, even-numbered very long chain aldehydes (C22−C30) present in the host cuticular wax were demonstrated to be mainly responsible for promoting the prepenetration processes of B. graminis spores in a dose- and chain-length-dependent manner.23−25 This in vitro system essentially consists of a Formvar membrane containing host leaf wax or host wax constituents that induce B. graminis prepenetration processes. The Formvar/wax membrane can be easily supplemented with lipophilic substances of interest, such as anthraquinones, in order to analyze their effects on B. graminis prepenetration processes. It avoids possible interference(s) with enzymatic activities and/or a chemical property of the otherwise underlying plant tissue and largely complies with the proposed requirements for powdery mildew in vitro germination tests.26 Hence, analyzing the germination behavior of B. graminis simply on hydrophilic agar or glass surfaces in the absence of the naturally germination and differentiation inducing very long chain aldehydes results in abnormal germination and differentiation and consequently in misleading or inconsistent data.27 By applying a Formvar resin based in vitro system and following a pharmacological approach, the present study aimed at analyzing the direct dose-dependent effects of several commercially available anthraquinone derivatives on the B

DOI: 10.1021/acs.jafc.7b05977 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Table 1. Prepenetration Development of Blumeria graminis f. sp. hordei Conidia on Cover Glasses Coated with Formvar/Barley Wax Supplemented with Equimolar Amounts of Anthraquinonesa concentration in the dipping solution [mM] −4

0 physcion (1) chrysophanol (2) emodin (3) pachybasin (4) danthron (5) anthraquinone (6) rhein (7) aloe-emodin (8) alizarin (9) quinizarin (10) 2,3-dimethylanthraquinone (11) 2-methylanthraquinone (12) physcion (1) chrysophanol (2) emodin (3) pachybasin (4) danthron (5) anthraquinone (6) rhein (7) aloe-emodin (8) alizarin (9) quinizarin (10) 2,3-dimethylanthraquinone (11) 2-methylanthraquinone (12)

physcion (1) chrysophanol (2) emodin (3) pachybasin (4)

10

10−3

10−2

10−1

Germinated Conidia ± SD 91 ± 9 b 58 ± 9 c 23 ± 6 d 23 ± 6 d 96 ± 8 ab 87 ± 10 b 58 ± 4 c 18 ± 6 c 100 ± 6 a 98 ± 8 a 95 ± 9 a 68 ± 11 b 100 ± 11 a 100 ± 11 a 97 ± 12 a 65 ± 11 b 96 ± 6 ab 97 ± 7 ab 92 ± 8 b 79 ± 8 c 97 ± 10 a 101 ± 10 a 100 ± 12 a 103 ± 12 a 102 ± 6 a 97 ± 8 a 98 ± 9 a 95 ± 9 a 99 ± 12 a 99 ± 12 a 104 ± 10 a 107 ± 9 a 102 ± 11 a 100 ± 9 a 94 ± 12 a 92 ± 6 a 94 ± 7 ab 93 ± 9 b 96 ± 6 ab 104 ± 7 a 92 ± 7 b 95 ± 4 b 99 ± 5 a 103 ± 4 a 100 ± 5 a 100 ± 5 a 101 ± 5 a 103 ± 4 a Conidia with Normal Appressorial Germ Tubes/Appressoria ± SD 100 ± 9 a 64 ± 11 ab 14 ± 5 b 4±2c 4±2c 100 ± 13 a 92 ± 8 a 64 ± 9 b 9±6c 2±3c 100 ± 7 a 104 ± 7 a 91 ± 5 ab 60 ± 13 b 18 ± 5 b 100 ± 9 a 102 ± 12 a 100 ± 9 a 95 ± 9 a 9±5b 100 ± 8 a 97 ± 8 ab 100 ± 8 a 89 ± 10 b 75 ± 8 c 100 ± 14 a 100 ± 10 a 103 ± 12 a 98 ± 10 a 102 ± 14 a 100 ± 11 a 98 ± 5 a 98 ± 9 a 98 ± 9 a 79 ± 11 b 100 ± 12 a 98 ± 14 a 100 ± 15 a 103 ± 15 a 97 ± 17 a 100 ± 10 a 98 ± 7 a 100 ± 10 a 95 ± 10 ab 90 ± 5 b 100 ± 11 a 97 ± 8 a 93 ± 10 a 98 ± 7 a 100 ± 8 a 100 ± 6 a 93 ± 7 a 97 ± 4 a 99 ± 6 a 97 ± 7 a 100 ± 6 a 97 ± 6 a 100 ± 6 a 96 ± 6 a 96 ± 6 a Conidia with Normal Appressorial Germ Tubes/Appressoria Plus Malformed Appressorial Germ Tubes (= Generally Differentiated) ± SD 100 ± 9 a 86 ± 9 a 34 ± 9 b 18 ± 4 c 18 ± 6 c 100 ± 12 a 94 ± 8 a 82 ± 13 b 48 ± 8 c 12 ± 6 d 100 ± 7 a 105 ± 6 a 98 ± 7 ab 90 ± 11 b 49 ± 13 c 100 ± 9 a 102 ± 12 a 101 ± 8 a 95 ± 9 a 47 ± 10 b 100 100 100 100 100 100 100 100 100 100 100 100

± ± ± ± ± ± ± ± ± ± ± ±

8a 11 a 6a 9a 9a 10 a 11 a 10 a 9a 12 a 5a 4a

Data represent the mean values of two independent experiments each recording the developmental categories of 8 × 100 conidia. Means were normalized to the results from the respective mock/control treatment set as 100% ± standard deviation (SD). Different letters within a line indicate significant differences (P < 0.05) according to a nonparametric Kruskal−Wallis test followed by a Dunn−Bonferroni post hoc test.

a

graminis prepenetration processes, the contact angles of 2 μL droplets of distilled water deposited on the coated glass surfaces were determined with an OCA 15 contact angle system and SCA20 software system (Dataphysics Instruments, Filderstadt, Germany). Four measurements were performed on each of four independent surface samples. Microscopic Analysis. For microscopic analysis, leaves were initially destained on filter paper moistened with ethanol/acetic acid (1:1, v/v) until they appeared transparent and were then transferred to filter paper soaked with fixation solution (lactic acid/glyercol/water (1:1:1, v/v/v)) for 5 h.30 To visualize the fungal structures leaf surfaces were stained with droplets of 0.05% (w/v) Trypan Blue (Merck, Darmstadt, Germany) in acetic acid/glycerol/water (1:1:1, v/v/v) for 30 min. The conidia inoculated onto the coated cover glasses were observed directly without staining. It was determined whether conidia subjected to different treatments had remained nongerminated or had formed a primary germ tube only, a secondary nonswollen germ tube, an elongated swollen secondary germ tube (= appressorial germ tube), or a fully differentiated hooked appressorium with septum. Conidia bearing either an appressorium or an appressorial germ tube were defined as being differentiated. On surfaces supplemented with some of the anthraquinone molecules distinctly malformed appressorial germ tubes were recorded. In addition, the loss rate of conidia that were apparently damaged, burst, or desiccated before germination was recorded. Only single, well-separated conidia were counted at each

were subjected to the inoculation procedure. The barley leaves or the microscopy slides bearing the coated cover glasses were kept in a humid atmosphere with wet filter paper applied underneath to achieve a relative humidity of at least 90%. The samples were incubated for 16 h in darkness at 20 °C. In order to study effects of physcion (1) treatment on haustorium formation and colony establishment in vivo we analyzed fungal development on primary barley leaves that were sprayed with 10−2 mM physcion (1) 24 h before inoculation with B. graminis conidia. In brief, 10 d old barley germlings were cut at their coleoptile and the secondary leaves were removed. The germlings, still bearing their primary leaves, were then each inserted into a water filled tube that provided a continuous water supply during the experiment. The germlings were sprayed as described above. Accordingly, control leaves were mock treated with a spray mixture without physcion (1). As light is known to play an important role in haustorium formation, the leaves were then incubated for 24 h in a growth chamber with a light intensity of 200 μmol photons/m2/s in a 16 h:8 h (L:D) photoperiod at 20 °C and over 90% relative humidity. 24 h after spray treatment the adaxial leaf surfaces were inoculated as described above. Subsequently, the barley germlings were kept for 24, 48, or 120 h in the growth cabinet under the same culture conditions. Leaves were destructively sampled at 24, 48, and 120 h after inoculation (hai) with B. graminis. Determination of Surface Contact Angles. As surface hydrophobicity is known to play an important role for the initiation of B. C


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Journal of Agricultural and Food Chemistry observation to eliminate the possibility of inhibition caused by crowding. Two independent experiments were performed per in vitro anthraquinone treatment. A total of 8 × 100 conidia were counted per experiment (4 coated cover glasses with 2 × 100 conidia each). 15 × 100 conidia were counted for the analysis of fungal development on physcion-pretreated leaves. In order to facilitate the comparison of data sets, all data were normalized to the results from the respective mock/control treatment. On leaf surfaces incubated for 48 h after inoculation B. graminis colony frequency/cm2 was determined. The average lengths of malformed and normally differentiated appressoria were determined in vitro on cover glasses coated with Formvar/wax or coated with Formvar/wax supplemented with physcion (1) (10−1 mM in the dipping solution) from 20 individual conidia each. Likewise, appressorial lengths were recorded on mocktreated and 10−1 mM physcion-sprayed leaves. Statistical Analysis. The basis for statistical analyses of in vitro data was n = 2 independent experiments, where n = 1 represents 8 × 100 examined conidia. For in vivo data it was n = 1 with 15 × 100 examined conidia. Significant differences (P < 0.05) between multiple data sets were analyzed by a nonparametric Kruskal−Wallis test followed by Dunn−Bonferroni post hoc test. The analyses were performed with IBM SPSS statistics (version 23).

Conidial Germination. After landing on a suitable surface and extrusion of the extraconidial matrix, the formation of the primary germ tube marks an important step at the very beginning of pathogenesis. Since the average proportion of germinated conidia in the control treatments varied from 63 to 75%, data were normalized to the control values of the respective treatment in order to facilitate the comparability of the prepenetration effects of the different anthraquinone compounds. Physcion (1) significantly inhibited germination by 42% at 10−3 mM, while similar values for chrysophanol (2) were obtained at 10−2 mM (Table 1). At 10−1 mM these two substances finally reduced the proportion of germinated conidia to 23 ± 6 and 18 ± 6% of the respective control values. At 10−4−10−2 mM emodin (3) and pachybasin (4) were ineffective in preventing primary germ tube formation. Even at 10−1 mM in the dipping solution, germination rates were decreased only by 32 and 35%, respectively. On cover glasses dipped into 10−1 mM danthron (5) germination was inhibited by roughly 21%. Anthraquinone (anthracene-9,10-dione, 6), rhein (7), aloe-emodin (8), alizarin (9), quinizarin (10), 2,3 dimethylanthraquinone (11), and 2-methylanthraquinone (12) did not substantially affect conidial germination within the given concentration range. Although germination was largely inhibited at higher concentration, the conidia were not immediately killed after contact with physcion (1), chrysophanol (2), emodin (3), or pachybasin (4). Even 24 h after inoculation on the cover glasses bearing the respective anthraquinone derivatives, the nongerminated conidia had sustained their cellular integrity with characteristic vacuoles still visible under the microscope. Conidial Differentiation. After primary germ tube emergence B. graminis conidia start with the formation of a secondary germ tube. Normally, this second germ tube elongates, swells, and finally differentiates a septate appressorium. The terminal appressorial cell then starts penetrating the cuticle and the cell wall of the underlying host epidermal cell. However, in the case of treatment with different concentrations of physcion (1), chrysophanol (2), emodin (3), and pachybasin (4), a substantial proportion of conidia elongated their secondary germ tubes without a distinct terminal swelling, failing to form a hooked, mature appressorium (Figure 2). The up to three septae present in these abnormally elongated, malformed appressorial germ tubes suggest that up to three mitoses have taken place. In vitro, normally differentiated appressoria on average exhibited a length of 33 ± 6 μm while their elongated malformed counterparts showed a significantly increased length of 62 ± 10 μm (Student’s t test, P < 0.001, n = 20 conidia) . At 10−4 mM only the physcion (1) treatment resulted in a distinct decrease in the proportion of normally differentiated conidia (with normal appressorial germ tube or appressorium) to 64 ± 11% of the control value (Table 1). Physcion (1) was therefore the most effective of the tested anthraquinone derivatives followed by chrysophanol (2) > emodin (3) > pachybasin (4) > danthron (5). With average values below 5% of the control treatment, physcion (1) at 10−1 and 10−2 mM and chrysophanol (2) at 10−1 mM resulted in substantially decreased proportions of mature appressoria competent for penetration. Emodin (3) at increasing concentrations from 10−3 to 10−1 mM showed a much steeper decrease of normal appressorium formation than seen for its effect on germination. Likewise, pachybasin (4) treatment resulted in a sharp decrease from 10−2 to 10−1 mM, while treatments with danthron (5) or

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RESULTS AND DISCUSSION Linearity of Anthraquinone Deposition. Dipping cover glasses in a mixture of Formvar and barley cuticular leaf wax supplemented with different amounts of anthraquinone derivatives is expected to result in coated glass surfaces reflecting the respective anthraquinone concentrations of the dipping solution. In order to demonstrate the linearity of anthraquinone derivative deposition we performed an exemplary assay re-extracting Formvar/wax/physcion (1) coated cover glasses and subjected the eluates to spectrophotometric quantification. The resulting data confirmed that cover glass deposition of physcion (1) is a linear function of physcion (1) concentration in the dipping solution (y = 0.2736x − 0.0017) demonstrating the suitability of our Formvar in vitro system. Dipping a cover glass into a mixture of Formvar and barley leaf wax in chloroform supplemented with 1 mM physcion (1) resulted in a physcion (1) deposition of 0.59 ± 0.05 μg/cm2. Surface Contact Angles of Cover Glasses Coated with Anthraquinone Derivatives. Besides the presence of very long chain aldehydes, a sufficiently high surface hydrophobicity, with surface contact angles >80°, is required for efficiently promoting B. graminis prepenetration processes.19 Applying the Formvar in vitro system resulted in cover glass surfaces with contact angles of at least 98° on the average. The surface contact angles of the Formvar/barley wax coated control slides (0 mM anthraquinone in the dipping solution) varied from 102° to 115° on average. In most cases supplementing the Formvar wax coating with different amounts of the anthraquinone derivatives did not significantly affect surface contact angles with respect to the barley wax control slides. However, slides supplemented with danthron consistently exhibited significantly decreased surface contact angles. Anthraquinone Derivatives and Blumeria graminis in Vitro Prepenetration. Among the selected anthraquinone derivatives only physcion (1), chrysophanol (2), emodin (3), pachybasin (4), and danthron (5) markedly affected germination and differentiation of B. graminis conidia within the given concentration range (Table 1). The other molecules did not result in a substantial decrease of B. graminis germination and appressorium differentiation. D


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(Table 1). The portion of malformed appressorial germ tubes within this group was dependent on the concentration of the applied anthraquinone derivatives (Table 2). In the absence of physcion (1), chrysophanol (2), emodin (3), and pachybasin (4), however, conidia differentiated normally and did not show the formation of malformed appressorial germ tubes as exemplified by danthron (5) and anthraquinone (6). On slides dipped in solutions with 10−1 mM physcion (1) and chrysophanol (2), conidia with malformed appressorial germ tubes made up 76 ± 16% and 80 ± 22% of the differentiated conidia, respectively. The proportion of conidia with malformed germ tubes in the group of differentiated conidia also largely prevailed at 10−1 mM, with 82 ± 13% for pachybasin (4) and 62 ± 11% for emodin (3). However, for pachybasin (4) a steep increase in this proportion became evident from 10−2 to 10−1 mM. Conidial Differentiation on Physcion-Sprayed Leaves. Our in vitro experiments suggested that physcion (1) significantly affected fungal prepenetration even at comparably low concentrations (10−4 mM). In order to find out whether this also applied to the in vivo situation, detached barley leaves were sprayed with different concentrations of physcion (1). The effect on conidial germination became discernible even at 10−4 mM with a decrease of 29% (Figure 3). However, at 10−1 mM primary germ tube formation decreased to at best 32% of the control values. As in the in vitro experiments, the appressorial differentiation at 10−1 mM was decreased to 5% of the initial control values. Under physcion (1) treatment the proportion of conidia bearing malformed appressorial germ tubes ranged from 4 ± 2% at 10−4 mM to 81 ± 10% at 10−1 mM. Their average length of 65 ± 11 μm did not significantly differ from the malformed appressorial germ tubes formed in vitro (Student’s t test, n = 20 conidia). Haustorium Formation and Colony Establishment. In order to study potential effects of physcion (1) treatment on haustorium formation and colony establishment we analyzed fungal development at 24, 48, and 120 hpi on host leaves that had been sprayed with 10−2 mM physcion (1) or a mock spray mixture 24 h before inoculation. At 24 hpi the proportion of conidia with normally developed appressoria on mock treated leaves was 82 ± 5%, with 9 ± 5% exhibiting the formation of a haustorium. Spraying leaves with the 10−2 mM physcion (1) spray mixture resulted in 24 ± 5% of conidia with appressoria that did not show the typical appressorial hook in contrast to the mock treatment. None of the counted 5 × 100 conidia had formed a haustorium. At 48 hpi young colonies had formed on

Figure 2. Normal and malformed appressorial germ tubes of Blumeria graminis f. sp. hordei. (A) A conidium with a normal appressorial germ tube in vitro on a cover glass coated with Formvar/barley wax and (B) a conidium with a malformed appressorial germ tube in vitro on a cover glass coated with Formvar/barley wax supplemented with 10−1 mM physcion (1) in the dipping solution. (C) A conidium with a normal appressorial germ tube in vivo on a barley primary leaf and (D) a conidium with a malformed appressorial germ tube in vivo on a barley primary leaf sprayed with 10−1 mM physcion (1) in the spray mixture. Fungal structures are stained with Trypan Blue. Septae are indicated with an arrow. The bar indicates 30 μm.

rhein (7) at 10−1 mM resulted in less than 30% reduction of control treatment appressorium formation. The presence of anthraquinone (anthracene-9,10-dione, 6), aloe-emodin (8), alizarin (9), quinizarin (10), 2,3-dimethylanthraquinone (11), and 2-methylanthraquinone (12) did not substantially affect conidial differentiation within the given concentration range. Since mitosis is an essential component of fungal differentiation, the sum of conidia with malformed appressorial germ tubes and of conidia with normally formed appressorial germ tubes/appressoria represents generally differentiated conidia

Table 2. Percentage of Malformed Appressorial Germ Tubes among the Generally Differentiated Appressorial Germ Tubes ± Standard Deviation (SD)a concentration in the dipping solution [mM] −4

0 physcion (1) chrysophanol (2) emodin (3) pachybasin (4) danthron (5) anthraquinone (6)

0 0 0 0 0 0

± ± ± ± ± ±

10−3

10 0 0 0 0 0 0

a a a a a a

10−2

10−1

% Malformed Germ Tubes among All Differentiated Germ Tubes ± SD 25 ± 12 b 56 ± 14 c 81 ± 15 d 2±2a 21 ± 11 b 80 ± 13 c 1±2a 7±3b 35 ± 9 c 1±1a 0±1a 1±1a 0±0a 0±0a 0±0a 0±0a 0±0a 0±0a

76 ± 16 80 ± 22 62 ± 11 82 ± 13 0±0a 0±0a

cd c c b

Data represent the mean values of two independent experiments each recording the developmental categories of 8 × 100 conidia. Different letters within a line indicate significant differences (P < 0.05) according to a nonparametric Kruskal−Wallis test followed by a Dunn−Bonferroni post hoc test. a

E


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severely affect the prepenetration processes of the barley powdery mildew fungus B. graminis f. sp. hordei, thereby excluding a potential contribution of host defense reactions. By applying the Formvar-based in vitro system we provide unambiguous evidence for a direct effect of these anthraquinone derivatives on germination and formation of infection structures. Treating agar plates before inoculation with Milsana formulation based on R. sachalinensis extract resulted in a significant decrease of conidial germination.6 In contrast, another study showed increased conidial germination rates in vitro on physcion (1) treated agar plates.17 However, due to the limitations of their experimental conditions, the authors of these studies could not analyze the in vitro effects of physcion (1) and Milsana on the appressorial differentiation of B. graminis conidia. Nevertheless, our in vitro data support the strong inhibition of conidial germination in response to treatment with physcion (1) or chrysophanol (2) under in vivo conditions reported previously.12,17,31−33 In addition, conidia were shown to respond to host leaf treatment with Reynoutria extract or physcion (1) with the formation of abnormally elongated, malformed and penetration incompetent appressorial germ tubes/appressoria.12,31,33 Our quantitative in vitro data suggest that this specific morphological response is also a direct and concentration dependent effect caused by the intimate contact with anthraquinone derivatives such as physcion (1), chrysophanol (2), emodin (3), or pachybasin (4). The majority of abnormally elongated appressorial germ tubes exhibited the presence of two septae. Therefore, in these germ tubes at least two rounds of mitosis must have taken place, which is at least one more than in normally developed appressoria. Consequently, one may conclude that hyphal growth and the cell cycle per se are not affected by treatment with anthraquinone derivatives. Interestingly, treatment with the commercially relevant mildewcides, proquinazid and quinoxyfen, known to interfere with Blumeria surface signal perception/transduction also results in the formation of abnormally elongated secondary germ tubes.34−36 However, there is currently no information available, whether these elongated germ tubes also exhibited septae. Due to the striking similarities with respect to appressorial germ tube elongation/ malformation it is tempting to speculate that the anthraquinone derivatives might also exert their antimildew activity by perturbing fungal surface signal perception/transduction processes. Interestingly, the two fungicides proquinazid and quinoxyfen also activated host defense gene expression.37 A recent study demonstrated in vitro a direct effect of physcion (1) on conidial germination and appressorium formation of the rice blast fungus Magnaporthe oryzae.38 Physcion (1) led to the reduction of M. oryzae appressorium diameters and to the deformation of germ tubes and appressoria when applied at a concentration of 10 mg/L. This might hint at similar modes of action in M. oryzae and B. graminis. Recently, physcion (1) was shown to exert an antibacterial effect through the efficient photosensitized generation of singlet oxygen and superoxide radical anion species.39 In our in vitro experiments, however, B. graminis conidia were incubated in darkness. Therefore, it seems unlikely that irradiation triggered effects might have influenced the outcome of our in vitro studies. While the applied Formvar in vitro system facilitates a more or less homogeneous and reproducible coating of glass surfaces with lipophilic compounds, the efficient and even deposition of

Figure 3. Prepenetration development of Blumeria graminis f. sp. hordei conidia on barley primary leaves sprayed with mixtures containing different concentrations of physcion (1). Data were normalized to the results from the respective mock/control treatment. Relative proportions of germinated conidia (●), of generally differentiated conidia (formation of normal appressorial germ tubes/ appressoria plus malformed appressorial germ tubes) (○), and of normally differentiated conidia (▼), and relative proportions of malformed appressorial germ tubes within the group of generally differentiated conidia (△). Data represent the mean values of 15 × 100 conidia per treatment ± standard deviation. Different letters within a line indicate significant differences (P < 0.05) according to a Kruskal−Wallis test followed by a Dunn−Bonferroni post hoc test.

the mock sprayed leaves with a density of 132.5 ± 13.3/cm2, while the physcion (1) treated leaves exhibited a colony density of only 0.8 ± 1.3/cm2. This dramatic difference became clearly visible by the naked eye at 120 hpi (Figure 4).

Figure 4. Colony development of Blumeria graminis f. sp. hordei at 5 days after inoculation on barley primary leaves sprayed 24 h before inoculation. (A) Mock treated leaf surface. (B) Leaf surface sprayed with a mixture containing 10−2 mM physcion (1). The bar indicates 5 mm.

Direct Effect of Anthraquinone Derivatives on Blumeria graminis Prepenetration. The applied Formvar in vitro system facilitates the largely homogeneous and reproducible coating of glass surfaces with lipophilic compounds, such as plant wax constituents.23 However, the cover glass surfaces coated with Formvar/barley wax were not fully homogeneous as indicated by the surface contact angle data. Nevertheless, all surfaces exhibited contact angles above 90°, hence generally facilitating germination and differentiation of B. graminis conidia.20 Our results unequivocally demonstrate that, in vitro, physcion (1) in particular and three other anthraquinone derivatives F


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sustained their cellular integrity and appeared vital, which hints more at a fungistatic than at a fungitoxic mode of action of these molecules. However, it remains to be determined whether the conidia would retain their ability to germinate after transfer onto a Formvar/wax-coated glass surface without the presence of anthraquinone derivatives. Structure−Function Relationships. All tested anthraquinones and anthraquinone derivatives are lipophilic and at best poorly soluble in water. Therefore, it is likely that the contact of these compounds with the conidial cell is facilitated by the hydrophobic conidial cell wall mainly impregnated with long chain fatty acids and long chain fatty acid methyl esters rendering the conidial walls largely impermeable to water and aqueous solutes.42,43 Anthraquinones exhibit a basal structure composed of three benzene rings, including two ketone groups on the central ring. The diversity of the anthraquinone derivatives relies on the nature and position of the substituents on the characteristic anthraquinone backbone structure (Figure 1). Interestingly, physcion (1), chrysophanol (2), emodin (3), and pachybasin (4), the anthraquinone derivatives affecting conidial differentiation, have a specific structural motif in common. Physcion (1) and emodin (3) bear a hydroxyl substituent at position 8 in combination with a methyl group at position 6, while chrysophanol (2) and pachybasin (4) bear a hydroxyl group at position 1 and a methyl substituent at position 3 (Figure 1). Although the number of tested anthraquinone derivatives is by far not sufficient for drawing clear-cut structure−function relationships, it is tempting to speculate that this specific motif may be associated with the antimildew activity of the tested anthraquinone derivatives. Likewise, the distinct differences in antimildew effectiveness of the four compounds may be attributable to the presence/absence of specific substituents at the opposing part of the molecule. At the lowest concentration, physcion (1) with a methoxy group at position 6 and a hydroxyl group at position 8 exhibited the highest activity of all molecules tested. Chrysophanol (2), however, which shares all substituents with physcion (1) except the methoxy group, turned out to be distinctly less effective than physcion (1). Nevertheless, it remains to be elucidated whether other anthraquinone derivatives bearing this specific substituent motif at least on one side of the molecule, such as helminthosporin (1,5,8-trihydroxy-3-methylanthraquinone) or fragilin (2-chloro-1,8-dihydroxy-3-methoxy-6-methylanthraquinone), also exhibit an antimildew activity. As yet, there is also no information available whether water-soluble monoglucosides of active anthraquinone derivatives (such as physcion-8-O-β-Dmonoglucoside) still exert an inhibitory activity on powdery mildew conidia. Consequently, a more systematic study is required to address the detailed structural and functional relationships. Effect of Physcion in Vivo on Prepenetration and Penetration. Despite the substantial inhibition of conidial germination on physcion-sprayed barley leaves a comparably small proportion of conidia still formed appressoria of normal length exhibiting only one septum, however without the typical hook frequently seen on mock-treated leaves. Since none of the assayed 500 conidia successfully penetrated the epidermal cell wall forming a haustorium, either the induction of host defense or a direct effect on the penetration capacity of the formed appressoria could be responsible for the observed effect.18 The fact that neither the malformed nor the normally developed appressoria formed the typical hook might again point more at

active ingredients by leaf spraying is highly dependent on the physicochemical properties of the plant cuticle, the efficacy of spray mixture adjuvants, and environmental conditions, which clearly affects the general comparability of in vitro and in vivo data sets.23 Within the tested concentration ranges physcion (1), chrysophanol (2), emodin (3), and pachybasin (4) turned out to be highly efficient in inhibiting germination and causing an aberrant prepenetration development. Our in vitro results basically corroborate data from experiments where barley or wheat leaves had been sprayed with physcion (1) or chrysophanol (2) before inoculation.3,40 The spray mixture concentrations required for 50% disease control were 4.7 μg/ mL for chrysophanol (2) and 0.48 μg/mL for physcion (1) on barley leaves and 5.63 μg/mL for chrysophanol (2) and 0.14 μg/mL for physcion (1) on wheat leaves.3,40 Likewise, at concentrations of 10−3 mM and 10−2 mM in the dipping solution our in vitro data suggest that chrysophanol (2) is in fact less effective than physcion (1) in eliminating normal differentiation. Hence, physcion (1) is the most effective anthraquinone derivative used in the present assay. Our in vitro data suggest that physcion (1) at a surface coverage of only 59 ng/cm2 can reduce conidial germination by roughly 75%. Treatment of barley with the anthraquinone derivatives rhein (7) and aloe-emodin (8) at spray mixture concentrations of 0.0625 to 1 g/L did not exhibit any antifungal effect against B. graminis.4 Likewise, rhein (7) and aloe-emodin (8) applied via the Formvar in vitro system did at best slightly affect the prepenetration processes of the barley powdery mildew fungus, thus largely corroborating previous in vivo results.4 However, in the same study physcion (1) and emodin (3) showed 50% disease control at comparable concentrations of 73 μg/mL and 46 μg/mL, respectively. In contrast, our in vitro results suggest that emodin (3) might be even less effective than chrysophanol (2). Another study analyzed the antifungal effects of danthron (5), alizarin (9), and quinizarin (10) on the barley powdery mildew disease and determined the spray mixture concentrations required for 50% disease control: 64 μg/mL for danthron (5), 94 μg/mL for alizarin (9), and 101 μg/mL for quinizarin (10).41 Taken together these data suggest that physcion (1), emodin (3), danthron (5), alizarin (9), and quinizarin (10) are almost equally effective against B. graminis, which is contradictory to our in vitro data. At the tested concentrations neither alizarin (9) nor quinizarin (10) exhibited a significant effect on B. graminis prepenetration development in vitro. Only danthron (5) at a concentration of 10−1 mM in the dipping solution resulted in slightly reduced germination values. With respect to the proportion of conidia with appressoria, danthron (5), alizarin (9), and rhein (7) at the highest concentration in the dipping solution resulted in a slight though significant decrease. From these results one might infer that with increasing concentrations even some of the so far largely inactive compounds such as danthron (5), rhein (7), and alizarin (9) might exert significant effects on fungal prepenetration. Interestingly, with the strongly effective derivatives physcion (1), chrysophanol (2), emodin (3), and pachybasin (4), conidial germination and total differentiation (including the malformed appressorial germ tubes) were reduced almost to the same extent, while the portion of normally differentiated conidia showed a more distinct decrease. One day after inoculation and thus after contact with anthraquinone derivatives, most of the nongerminated conidia G


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(3) Choi, G. J.; Lee, S.-W.; Jang, K. S.; Kim, J.-S.; Cho, K. Y.; Kim, J.C. Effects of chrysophanol, parietin, and nepodin of Rumex crispus on barley and cucumber powdery mildews. Crop Prot. 2004, 23, 1215− 1221. (4) Kim, Y.-M.; Lee, C.-H.; Kim, H.-G.; Lee, H.-S. Anthraquinones isolated from Cassia tora (Leguminosae) seed show an antifungal property against phytopathogenic fungi. J. Agric. Food Chem. 2004, 52, 6096−6100. (5) Konstantinidou-Doltsinis, S.; Markellou, E.; Kasselaki, A.-M.; Fanouraki, M. N.; Koumaki, C. M.; Schmitt, A.; Liopa-Tsakalidis, A.; Malathrakis, N. E. Efficacy of Milsana®, a formulated extract from Reynoutria sachalinensis, against powdery mildew of tomato (Leveillula taurica). BioControl 2006, 51, 375−392. (6) Randoux, B.; Renard, D.; Nowak, E.; Sanssené, J.; Courtois, J.; Durand, R.; Reignault, P. Inhibition of Blumeria graminis f.sp. tritici germination and partial enhancement of wheat defenses by Milsana. Phytopathology 2006, 96, 1278−1286. (7) Daayf, F.; Schmitt, A.; Bélanger, R. R. Evidence of phytoalexins in cucumber leaves infected with powdery mildew following treatment with leaf extracts of Reynoutria sachalinensis. Plant Physiol. 1997, 113, 719−727. (8) Fofana, B.; McNally, D. J.; Labbé, C.; Boulanger, R.; Benhamou, N.; Séguin, A.; Bélanger, R. R. Milsana-induced resistance in powdery mildew-infected cucumber plants correlates with the induction of chalcone synthase and chalcone isomerase. Physiol. Mol. Plant Pathol. 2002, 61, 121−132. (9) Schmitt, A.; Ibarra, F.; Francke, W. Resistance inducing constituents in extracts of Reynoutria sachalinensis. In Modern fungicides and antifungal compounds IV: Proceedings of the 14th international Reinhardsbrunn symposium, Friedrichroda, Germany, April 25−29, 2004; Dehne, H. W., Gisi, U., Kuck, K. H., Russell, P. E., Lyr, H., Eds.; British Crop Production Council: Alton, 2005; pp 259−262. (10) Su, H.; Blai, R.; Johnson, T.; Marrone, P. Regalia® bioprotectant in plant disease management. Outlooks Pest Manage. 2012, 23, 30−34. (11) FRAC Code List© 2018. http://www.frac.info/docs/defaultsource/publications/frac-code-list/frac_code_list_2018-final. pdf?sfvrsn=6144b9a_2. Accessed: 20.02.2018. (12) Yang, X.; Yang, L.; Ni, H.; Yu, D. Effects of physcion, a natural anthraquinone derivative, on the infection process of Blumeria graminis on wheat. Can. J. Plant Pathol. 2008, 30, 391−396. (13) Yang, L.; Gong, S.; Yang, X.; Yu, D. Activities of botanical fungicide physcion on several plants pathogenic fungi. Nongyao 2010, 49, 133−141. (14) Onyilagha, J. C.; Grotewold, E. The biology and structural distribution of surface flavonoids. Recent Res. Dev. Plant Sci. 2004, 2, 1−18. (15) Fofana, B.; Benhamou, N.; McNally, D. J.; Labbé, C.; Séguin, A.; Bélanger, R. R. Suppression of induced resistance in cucumber through disruption of the flavonoid pathway. Phytopathology 2005, 95, 114− 123. (16) Yeats, T. H.; Rose, J. K. C. The biochemistry and biology of extracellular plant lipid-transfer proteins (LTPs). Protein Sci. 2008, 17, 191−198. (17) Ma, X.; Yang, X.; Zeng, F.; Yang, L.; Yu, D.; Ni, H. Physcion, a natural anthraquinone derivative enhances gene expression of leafspecific thionin of barley against Blumeria graminis. Pest Manage. Sci. 2010, 66, 718−724. (18) Li, Y.; Tian, S.; Yang, X.; Wang, X.; Guo, Y.; Ni, H. Transcriptomic analysis reveals distinct resistant response by physcion and chrysophanol against cucumber powdery mildew. PeerJ 2016, 4, e1991. (19) Tsuba, M.; Katagiri, C.; Takeuchi, Y.; Takada, Y.; Yamaoka, N. Chemical factors of the leaf surface involved in the morphogenesis of Blumeria graminis. Physiol. Mol. Plant Pathol. 2002, 60, 51−57. (20) Zabka, V.; Stangl, M.; Bringmann, G.; Vogg, G.; Riederer, M.; Hildebrandt, U. Host surface properties affect prepenetration processes in the barley powdery mildew fungus. New Phytol. 2008, 177, 251−263.

a direct effect of physcion (1) on the fungal penetration efficiency. The present study demonstrates a direct dose-dependent antifungal effect of the anthraquinone derivatives physcion (1), chrysophanol (2), emodin (3), and pachybasin (4) on the prepenetration processes of the barley powdery mildew fungus B. graminis f. sp. hordei. Due to the striking inhibition of conidial germination and the impairment of appressorial functioning, the repeated preventive application of these anthraquinone derivatives effectively reduces the major part of the fungal infection potential. Hence, the contribution of host defense induction might play a more or less subordinate role in affecting fungal infestation. However, plant defense induction could become more relevant affecting secondary haustorium formation and colony growth. Nevertheless, without forming a haustorium even a germinated and fully differentiated B. graminis conidium will finally starve to death after residing on the plant surface for a couple of days. The present study shows that the Formvar-based in vitro system is ideally suited for screening potentially antifungal compounds in order to assess their effects on the prepenetration processes of the obligate biotroph B. graminis. Future investigations should aim at identifying fungal and host plant molecular targets of anthraquinone derivatives and address the fate of these compounds in the host cuticle with respect to accumulation, absorption, permeation, and metabolic modification and/or degradation within the host tissue.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b05977. Absorbance of physcion in acetone re-extracted from coated cover glasses and surface contact angles of coated cover glasses (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: ++49(0)931-3186206. Fax: ++49(0)931-3186235. Email: [email protected]. ORCID

Ulrich Hildebrandt: 0000-0002-5142-9240 Markus Riederer: 0000-0001-7081-1456 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Andrea Knorz for excellent and dedicated technical assistance and R. Hückelhoven (Chair of Phytopathology, Technical University, Munich, Germany) for providing barley powdery mildew race A6.

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ABBREVIATIONS USED hpi, hours post inoculation REFERENCES

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Direct Effects of Physcion, Chrysophanol, Emodin and Pachybasin on

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