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Published online 31 May 2007
Published in Crop Sci 47:1119-1124 (2007)
© 2007 Crop Science Society of America
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CROP PHYSIOLOGY & METABOLISM-NOTES

Constitutive Coumarin Accumulation on Sunflower Leaf Surface Prevents Rust Germ Tube Growth and Appressorium Differentiation

E. Pratsa,*, M. J. Llamasa, J. Jorrinb and D. Rubialesa

a Instituto de Agricultura Sostenible, CSIC, Apdo. 4084, E-14080, Córdoba, Spain
b Dpto. Bioquímica y Biología Molecular, Universidad de Córdoba, Campus Rabanales, Edificio C-6, Córdoba, Spain

* Corresponding author (bb2prpee{at}uco.es).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Resistance against sunflower rust incited by Puccinia helianthi Schwein was characterized in three sunflower (Helianthus annuus L.) genotypes: AMES 18925 that was highly resistant; AMES 3442, partially resistant; and Hysun 33, susceptible. Microscopic studies showed a differential rust development on the genotypes. Impairment of rust spore germination and of appressorium formation was observed only in the resistant genotype. A differential excretion of coumarins to the leaf surface was also observed among genotypes, with the highest levels corresponding to the most resistant genotype and the lowest to the most susceptible. Impairment of rust germ tube growth and of appressorium formation was associated with the excreted level of ayapin. Reduction in germination frequency was not associated with coumarins but probably to other phenolic compounds present at higher concentrations in the resistant than in the susceptible genotype.

Abbreviations: ASM, acibenzolar-S-methyl • CS, colony size • DPI, days post inoculation • IF, infection frequency • LP, latency period • TLC, thin layer chromatography.


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
SUNFLOWER RUST caused by Puccinia helianthi Schwein is a widespread disease of importance in Argentina, Australia, Canada, Egypt, Israel, Turkey, USA, and the former USSR (Gulya et al., 1997). Yield losses as high as 50% due to a reduction in the capitulum and seed size and in oil content have been reported in Canada and Argentina (Siddiqui and Brown, 1977; Zimmer and Zimmerman, 1972). The rust infection process starts with the germination of the urediospores on the leaf surface. When the germ tube contacts a stoma an appressorium develops over the guard cells and then a penetration hypha penetrates through the stoma (Hoch and Staples, 1987; Prats et al., 2002). The penetration hypha develops a substomatal vesicle from which the haustorium mother forms. Then, an infection peg penetrates the mesophyll cells and forms a feeding structure, the haustorium, that takes up nutrients for fungal growth (Mendgen et al., 1996). To date, several sunflower rust races have been described: the North American Races 1, 2, 3, 4, 5, and 6; the Argentine Race ARG 340; and the Australian Races 0 and 1 (reviewed by Miller and Fick, 1997). Resistance genes to different rust races, in most cases deriving from wild annual sunflower, have also been described (Putt and Sackston, 1963; review by Miller and Fick, 1997). However, recent surveys indicated that new rust races are evolving and overcoming resistance genes, resulting in a continuous threat for the crop (Gulya et al., 1997). A better understanding of the plant–pathogen interaction is necessary for a durable control of rust disease.

A battery of resistance mechanisms, acting either before or after stomatal penetration, might prevent rust development. Prestomatal penetration mechanisms include poor germling adhesion to the leaf surface (Mendgen, 1978; Wynn and Staples, 1981), deviating micromorphology of the epidermal surface that serves as cues in guiding the thigmosensing germ tube toward stoma (Wynn and Staples, 1981), stomatal guard cell morphology (Wynn, 1976), and leaf pubescence (Mmbaga and Steadman, 1992). However, the limited differences among genotypes in germination and germtube directional growth reported within host species suggested that reduction of urediospore germination and/or fungal development on the leaf surface were of marginal importance, at best, in reducing infection levels and offered little opportunity to be exploited for resistance breeding (Niks and Rubiales, 2002; Rubiales and Moral, 2004). Recently, Prats et al. (2002) found a reduced P. helianthi spore germination and appressorium formation in sunflower leaves after cotyledon treatment with acibenzolar-S-methyl (ASM; 1,2,3-benzothiadiazole-7-carbothiotic acid S-methyl ester), an inducer of systemic acquired resistance. The treatment did not interfere with rust postpenetration events; however, it led to excretion of the sunflower coumarins scopolin, scopoletin, and ayapin to the leaf surface, which inhibited the rust appressorium formation and therefore conferred resistance (Prats et al., 2002). These results bring to the mind the question of whether natural excretion of coumarins to the leaf surface might be an early defense mechanism of certain sunflower genotypes. As far as we know, there is no information on whether constitutive concentration of coumarins in different sunflower cultivars might be correlated with resistance.

This work sought to further characterize sunflower cultivars with resistance mechanisms acting in the early stages of rust infection for their subsequent use in sunflower breeding. Furthermore we aimed to determine whether constitutive excreted coumarins accumulate on the sunflower leaf surface of resistant cultivars, and whether this could be involved in the prestomatal penetration resistance to P. helianthi by inhibition of germination and/or appressorium formation.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Fifteen sunflower genotypes with reported resistance against P. helianthi and containing different resistance genes were kindly supplied by the United States Department of Agriculture (USDA). The genotypes AMES 18925 containing the resistance gene R4, AMES 3442 with R1, and Hysun 33 with phRR3 were selected in a preliminary experiment based on visible symptoms as highly resistant, moderately resistant, and susceptible against a Spanish isolate of P. helianthi (CO97). In this preliminary experiment our isolate was found to be avirulent to genes R4, R5, R10, Pu6, Ph2, and Ph3 and virulent to genes R1, R2, R6, R7, R8, R9, phRR3, Ph1, and Ph2.

Seeds were surface sterilized, pregerminated in petri dishes, and sown in 1-L pots filled with perlite. Plants were grown under a 14-h photoperiod, in a growth chamber (Model Climatec01, Climatec, Córdoba, Spain) provided with white fluorescent lamp tubes with a photosynthetic photon flux density of 350 µmol m–2 s–1 and at 20°C temperature and 75/85% relative humidity. The P. helianthi isolate was obtained from a field of infected sunflower plants in Córdoba, Spain, in 1997. Urediospores were maintained in liquid N and multiplied on plants of the susceptible cultivar Sun-Gro 380 before use. When plants had two expanded pairs of leaves (approx. 20 d old) they were inoculated with 20 mg of freshly collected urediospores mixed with pure talcum (1:10, w/w) by dusting them over the plants. Homogeneous inoculation was ensured by placing leaves horizontally with the help of metallic clips. After inoculation, plants were incubated for 24 h in the dark at 20°C, covered with a polyethylene bag to ensure high relative humidity, and then transferred to a growth chamber under the conditions indicated above. Controls were inoculated with pure talcum only.

Latency period (LP) and infection frequency (IF) were determined as macroscopic parameters of the disease as previously described (Prats et al., 2002). Latency period, the time elapsing between inoculation and appearance of 50% of the pustules, was determined by daily counting of the number of pustules visible in a 4-cm2 marked area on the leaves. This was done by using a pocket lens (magnification 7x) until the number of pustules in the marked areas ceased to increase. The time at which 50% of the final number of pustules had appeared was estimated by interpolation. Infection frequency, given as the number of pustules per unit area, was calculated from the same leaf area in which LP was estimated. Two leaf segments were studied per plant, using five plants per genotype, and data were subjected to standard analysis of variance. For microscopic assessment of fungal development, middle segments of 1 to 3 cm2 from each fully expanded second pair of leaves (five leaves per treatment) were excised at 2 and 6 d post inoculation (DPI). Samples collected at 2 DPI were processed according to Rubiales and Niks (1992). They were bleached on pads moistened with fixative (absolute ethanol/glacial acetic acid, 3:1 v/v) and then stained on pads moistened in trypan blue in lactophenol ethanol at 0.5% (w/v). This procedure prevents the displacement of ungerminated spores and loosely attached germlings. Observations were made under a Leica DM LS phase contrast microscope (Leica Microsystems, Wetzlar, Germany) at 400x magnification. Percentages of germinated urediospores were determined from 100 random urediospores per leaf segment. Percentages of germtubes forming an appressorium over a stoma were determined from 100 germinated urediospores per leaf segment. Samples collected at 6 DPI were processed as described by Rubiales and Niks (1995) and stained with Uvitex (Ciba, Barcelona, Spain). Fifty colonies per leaf segment were studied. The presence of host cell necrosis associated with infection structures was recorded. The length (L) and width (W) of 25 random colonies per leaf were measured with an eyepiece micrometer. Colony size (CS) was calculated according to Niks (1986) using the formula CS = (1/4{pi}LW)1/2.

To evaluate excreted coumarins, leaves from 22-d-old control plants were washed with 5 mL of methanol by dripping the volume along the leaf surface. The methanol was recovered with a pipette and dripped again along the leaf. Then the same procedure was repeated using 5 mL of chloroform instead of methanol. The methanol and chloroform washes were combined, concentrated under vacuum, and then redissolved in 0.1 mL of methanol. Coumarins were identified and quantified by high performance liquid chromatography as reported by Gutiérrez-Mellado et al. (1996). Methanolic samples were applied to a Lichrocart column (125 by 4 mm, Merck, Darmstadt, Germany) and compounds eluted with water/acetonitrile 9:5 (v/v) at a flow rate of 1 mL min–1. Eluant was monitored by a fluorescence detector (Model Waters 474, Beckman Coulter, Madrid, Spain) with excitation and emission wavelengths of 340 and 430 nm, respectively. Coumarins were identified and quantified by scopoletin, scopolin, and ayapin reference standards. Total phenolics were determined by using the Folin–Ciocalteu reagent. Thin layer chromatography (TLC) analysis of the methanolic extract was performed as previously described (Prats et al., 2003). Coumarins were visualized by illuminating the plate with UV light (254 nm) and identified by scopoletin and ayapin reference standards.

Data were calculated from 100 observations made on each of the five replicate leaves. For ease of understanding, means of raw percentage data are presented in tables. However, for statistical analysis, replicate percentages were transformed to arcsine square roots (transformed value = 180/{pi}[arcsine (%/100)1/2] to normalize data and stabilize variances throughout the data range. Transformed data were subjected to analysis of variance using Statistix 8 (Analytical Software, 2003), after which residual plots were inspected to confirm data conformity to normality. Significance of differences between means was determined by calculating least significant difference (LSD).


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Macroscopic assessment of inoculated leaves showed the genotype AMES 18925 as most resistant with no pustules observed (Table 1), AMES 3442 as moderately resistant with a low IF and a long LP, and Hysun 33 as the most susceptible with the highest IF and shortest LP (P < 0.05; Table 1). The detailed microscopic study showed that the resistance of the AMES 18925 genotype was mainly due to a low spore germination frequency together with a lack of success in appressorium formation. Indeed, in this genotype none of the germinated spores assessed were able to form an appressorium over a stoma. This was due to a poor growth of the germ tube that often did not reach the stoma together with the inability of the germ tube to form an appressorium when finding a stoma. No disoriented germ tube growth or continued germ tube growth across stomata were observed. Due to the lack of success in appressorium formation, neither cell death nor colony size could be assessed for this genotype (Table 1). No significant differences in appressorium formation were found between AMES 3442 and Hysun 33. However, the moderate resistance of AMES 3442 was largely explained by the high proportion of mesophyll cell death, significantly higher than in Hysun 33 (P < 0.05). This high percentage of cell death in AMES 3442 probably led to the smaller colony size compared to Hysun 33 (P < 0.05; Table 1).


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  		Table 1. Macroscopic and microscopic assessment of P. helianthi development in three sunflower genotypes. Data are based on five replicate leaves in all cases.{dagger}

 
The observed dramatic reduction in spore germination and appressorium formation in a particular sunflower genotype against its appropriate rust species found in this work is an important observation. To our knowledge, this is the first report of such strong genotypic variation for rust resistance mechanisms acting before stoma penetration and it offers opportunities for breeding for these traits. To date, breeding programs against rusts have been mainly based on postpenetration resistance mechanisms such as the hypersensitive response. The great disadvantage of this type of resistance is that it is often ephemeral (Niks and Rubiales, 2002). There are alternative resistance mechanisms that might prove more durable, for instance those arresting the pathogen before haustorium formation. Prehaustorial resistance has usually been named "partial resistance" and has been identified in a range of crop–rust interactions (Niks and Rubiales, 2002). It includes different resistance mechanisms that may act at the cell penetration stage or even earlier, before stomatal penetration. In most cases, prehaustorial resistance is polygenetically inherited and can be selected by recurrent selection or by marker assisted selection. However, there are examples of prehaustorial resistance conferred by single genes, such as Lr34 and Lr46, against P. triticina in wheat (Triticum aestivum L.) that are associated with reduced haustorium formation, but not with hypersensitivity (Martínez et al., 2001; Rubiales and Niks, 1995). Prestomatal penetration mechanisms are frequent in nonhosts, but have seldom, if ever been reported in hosts against their appropriate rusts. They are even rare in nonhost species if these are not morphologically too distinct from their host plant (Heath, 1974; Niks, 1987). A notable exception is the low appressorium formation by various leaf rusts of cereals (P. triticina Ericks., P. hordei Otth., P. recondita Rob. ex Desm.) in some genotypes of Hordeum chilense Roem. et Schult and of other wild barleys (up to 10 fold reduction) but unfortunately not in accessions of the cultivated barley (H. vulgare L.) or any cultivated cereal (Rubiales and Niks, 1992; Rubiales and Niks, 1996). In these, only a little reduction in appressorium formation by P. hordei in some cer-mutants (Rubiales et al., 2001) and in stomatal recognition by P. striiformis in some resistant wheat cultivars (Broers and Lopez-Atilano, 1996) were found.

Interestingly, there are reports of variation within crops for pre-appressorium differentiation resistance against other fungi. For instance, Rhynchosporium secalis (Oud.) Davis spore germination is inhibited in certain barley cultivars, although the responsible factor or factors are unknown (Lehnackers and Knogge, 1990). It is known that topographical signals mediate the processes leading to appressorium formation (Hoch et al., 1987). However, physical features are not the only important determinant of rust appressorium formation. Specific plant chemical signals may also influence appressorium differentiation. Thus, hexenols can induce appressorium formation by the wheat stem rust on artificial substratum (Collins et al., 2001). On the other hand, antifungal plant metabolites extracted from the diffusates of resistant rice (Oryza sativa L.) genotypes have been shown to strongly inhibit Magnaporthe grisea (T.T. Hebert) M.E. Barr germination and appressorium formation (Pasechnik et al., 1997). Among these metabolites, polyamines have great effect, impairing appressorium formation in M. grisea (Choi et al., 1998). It has also been found that phenolic compounds excreted from dead cells of outer scales to the surface in resistant onion (Allium cepa L.) varieties prevent urediospore germination of Colletotrichum circinans (Berk.) Voglino (Walker and Stahmann, 1955). Phenolics among other substances may inhibit fungal hydrolytic enzymes restricting their activity (Godman et al., 1986).

In the present study we found no significant differences in total soluble phenolics excreted to the leaf surface among genotypes with mean values approximately 130 µg equivalents chlorogenic acid g–1 fresh weight (data not shown). However, quantification of coumarins excreted to the leaf surface showed that the most resistant genotype, AMES 18925, and the moderately resistant AMES 3442, had a higher content of total excreted coumarins than the susceptible genotype Hysun 33 (Table 2). Among the sunflower phenolic compounds, the coumarins, scopolin, scopoletin, and ayapin, have been extensively described as phytoalexins since synthesis of these antifungal compounds is induced in response to pathogen attack (Tal and Robeson, 1986a, 1986b). Furthermore, recent work has described that constitutive coumarin accumulation in sunflower corollas and bracts may lead to resistance against fungal pathogens such as Sclerotinia sclerotiorum (Lib.) de Bary (Prats et al., 2006). A detailed analysis of each coumarin in the present work showed that the most resistant genotype, AMES 18925, excreted higher amount of ayapin than AMES 3442 or Hysun 33 (Table 2). Ayapin is considered the most potent antifungal sunflower coumarin to pathogens such as Sclerotinia and has been shown to inhibit wheat stem rust (Puccinia graminis f. sp. tritici [Puccinia graminis Pers.:Pers. f. sp. tritici Eriks. E. Henn.]) rediospore germination (Prats et al., 2006; Urdangarin et al., 1999). Indeed, it is known that ayapin concentration in a range similar to that observed on the leaf surface in the present work reduces up to 84% the percentage of P. helianthi appressorium formation (Prats et al., 2002). Therefore, our results suggest that ayapin is, at least partly, responsible for the lack of success in appressorium formation observed in AMES 18925. Ayapin is highly fungitoxic, but also highly phytotoxic so when cells accumulate high ayapin concentration it is excreted to the leaf surface to avoid phytotoxicity (Jorrín and Prats, 1999). There, this compound might exert its inhibitory effect to pathogens like rust impeding germtube development and appressorium differentiation. Scopoletin concentration at the level found on genotype AMES 3442, has been shown to reduce appressorium formation, though less effectively than ayapin (Prats et al., 2002). This could explain the tendency of AMES 3442 to reduce appressorium formation although differences with susceptible Hysun 33 were not significant (Table 1).


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  		Table 2. Coumarin content excreted to the leaf surface in three sunflower genotypes. Values are the means of five independent replicates.{dagger}

 
In previous in vitro bioassays, the sunflower coumarins did not affect the germination of sunflower rust urediospores in the range of concentrations observed in the present work (Prats et al., 2002). Therefore, excreted coumarins by themselves may not explain the reduction in percentage of germination observed in AMES 18925. Prats et al. (2002) observed in sunflower leaves an inhibition of sunflower rust urediospore germination following cotyledon treatment with the systemic acquired resistance inducer, ASM. However, this inhibition could not be associated with the excretion of coumarins that followed the treatment. It might be possible that other excreted compounds different from coumarins interfered with rust germination. Indeed, TLC analysis revealed that AMES 18925 contained higher concentrations than AMES 3442 or Hysun 33, of at least three other phenolic compounds different from the sunflower coumarins (data not shown). Excretion of other phenolic compounds was also observed after ASM treatment. These other compounds are also constitutive in sunflower genotypes with Sclerotinia resistance (Prats et al., 2003), and they are currently under further characterization. Altogether, data from the present work highlight the importance of constitutive sunflower coumarins in hampering sunflower rust germ tube growth and appressorium formation and suggest the involvement of other related phenolic compounds inhibiting rust germination. The present study also opens the possibility of breeding for high coumarin content to improve resistance at these early stages of fungal development.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Received for publication July 21, 2006.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 





This Article
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