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Keywords:

  • Capsicum annuum;
  • CASC1;
  • Fusarium oxysporum Fo47;
  • induced resistance;
  • Phytophthora capsici;
  • Verticillium dahliae

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The application of the nonpathogenic isolate Fusarium oxysporum 47 (Fo47) reduced the symptoms of verticillium wilt, phytophthora root rot and phytophthora blight in pepper plants. Botrytis cinerea was also tested on the leaves of plants treated with Fo47, but no protection was observed. Verticillium dahliae colonies cultured in the presence of Fo47 grew slower than control cultures, but Phytophthora capsici growth was unaffected by Fo47. At least part of the protection effect observed against V. dahliae could therefore be due to antagonism or competition. In order to search for induced resistance mechanisms, three defence genes previously related to pepper resistance were monitored over time. These genes encode a basic PR-1 protein (CABPR1), a class II chitinase (CACHI2) and a sesquiterpene cyclase (CASC1) involved in the synthesis of capsidiol, a phytoalexin. These three genes were transiently up-regulated in the roots by Fo47 in the absence of inoculation with the pathogen, but in the stem only CABPR1 was up-regulated. In plants that were inoculated with V. dahliae after the Fo47 treatment, the three genes had a higher relative expression level than the control in both the roots and the stem.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Pepper is an economically important crop in the region of Galicia (Spain). However, two soilborne diseases, verticillium wilt and phytophthora root rot, attack pepper plants in this region (Pomar et al., 2001). Phytophthora root rot management is based on phenylamide fungicides, but fungicide-tolerant strains have been detected (Silvar et al., 2006). Verticillium wilt is difficult to control, both in pepper and other crops (Goicoechea, 2009). Alternative methods of control are therefore increasingly being investigated, with biological control one of the most promising.

Nonpathogenic Fusarium oxysporum strain Fo47 is a biological control agent isolated from a soil that was suppressive to fusarium wilt at Châteaurenard (France), and its efficiency against pathogenic Fusarium has been demonstrated in tomato and other plants (Fravel et al., 2003; Alabouvette et al., 2009). Other strains of Fusarium have proven to effectively control fusarium wilts (Larkin & Fravel, 1998; Fravel et al., 2003), but there are reported cases of protection against other pathogens. In this laboratory, F. oxysporum f. sp. lycopersici was able to protect pepper against Verticillium dahliae, Phytophthora capsici and Botrytis cinerea, suggesting that the induced response is both local and systemic, as well as nonspecific (Diaz et al., 2005). This protection conferred by the pathogenic strain F. oxysporum f. sp. lycopersici involved the activation of different defence related genes (Silvar et al., 2009). These genes reached the maximum expression level in the leaves, where the pathogen P. capsici was not detected in the induced plants (Silvar et al., 2009). Three of the pepper defence genes induced by F. oxysporum f. sp. lycopersici (CABPR1, CACHI2 and CASC1) have also been reported to be induced by some Trichoderma isolates assayed for their biocontrol activity against Phytophthora capsici (Bae et al., 2011), so they can be used as markers for pepper induced resistance.

In the two studies mentioned above, the assayed Fusarium strain was not pathogenic for the pepper plants, but this strain belongs to a forma specialis able to cause disease in tomato plants. Therefore, its use in agricultural practice is not possible. The alternative is to use putative nonpathogenic strains, such as Fo47. Indeed, nonpathogenic strains of F. oxysporum have been successfully assayed to protect several crops (Fravel et al., 2003; Olivain et al., 2006). The mode of action of these strains covers a wide range of mechanisms. Each strain seems to act differently from each other; the nonpathogenic strain F. oxysporum F2 has been demonstrated to protect aubergine against V. dahliae by having an antagonistic interaction with the pathogen (Pantelides et al., 2009), whereas strain CS-20 acts against pathogenic Fusarium mainly by inducing the defence response (Fravel & Larkin, 2002). In the case of Fo47, results in tomato point to an antagonist effect against pathogenic Fusarium based on competition for nutrients and a minor effect on induced resistance (Larkin & Fravel, 1999). As far as is known, Fo47 has not been tested in pepper as a biocontrol strain against fungal diseases.

The aim of the present study was to test whether Fo47 is effective in protecting pepper plants against the same range of pathogens that are controlled by F. oxysporum f. sp. lycopersici according to a previous report (Diaz et al., 2005), namely Verticillium dahliae, Phytophthora capsici and Botrytis cinerea. Moreover, the in vitro antagonism of Fo47 against two of these pepper pathogens was tested, and the enhancement by Fo47 of the expression of three defence genes (CABPR1, CACHI2 and CASC1) previously related to F. oxysporum f. sp. lycopersici induced resistance is reported.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant material and Fo47 treatment method

Capsicum annuum seeds were disinfected in 10% (v/v) commercial bleach for 30 min. Seeds were then washed and soaked overnight in distilled water before being sown in sterile vermiculite. Plants were grown in a growth chamber at 25°C and a photoperiod of 16 h light and 8 h darkness. Plants were used for treatment with Fo47 15 days after plant emergence.

Fusarium oxysporum Fo47 was kindly provided by C. Alabouvette and C. Steinberg (UMR INRA, Dijon). The Fusarium treatment of the plants was performed according to Diaz et al. (2005) with some modifications. Fo47 inoculum was obtained from cultures growing in potato dextrose broth medium for 7 days at 25°C in a shaker at 150 rpm. The culture was filtered and the filtrate was centrifuged at 2500 g for 5 min. The pellet was resuspended in sterile distilled water and the concentration was adjusted to 106 conidia mL−1. The plant roots were dipped into the conidial suspension of Fo47 for 3 h. Control plants were treated with sterile distilled water instead of Fo47 conidia.

Pathogen material and inoculum preparation

The V. dahliae isolate UDC53Vd was previously obtained by this research group in Galicia (northwest of Spain) from a diseased pepper plant collected on a farm. The isolate was grown on potato dextrose agar (PDA) for 28 days at 25°C. The inoculum was obtained by gently flooding the dishes with sterile distilled water (Diaz et al., 2005). The concentration was adjusted to 106 conidia mL−1.

The B. cinerea isolate B0510 was kindly provided by Dr J. van Kan (Wageningen University), and grown in tomato-PDA for 10 days at 25°C. The inoculum was prepared by flooding the dishes with sterile distilled water and filtering the suspension. The concentration was adjusted to 106 conidia mL−1 as described by Diaz et al. (2005).

The Phytophthora capsici isolate PC450 was kindly provided by F. Panabieres (UMR INRA, Sophia-Antipolis), and grown in V8 agar for 7 days at 25°C. Plugs of 5 mm diameter from 1-week-old cultures in V8 agar were used as leaf inoculum. In the case of root inoculation, zoospores were obtained as indicated in Diaz et al. (2005), and the concentration of the inoculum was adjusted to 1000 zoospores mL−1.

Inoculation with Verticillium dahliae

After the treatment with Fo47 or water, the plants were placed in sterile flasks with nutrient solution (for composition, see Chmielowska et al., 2010) and incubated for 48 h in a growth chamber at 25°C. Then, the plant roots were placed into the V. dahliae inoculum for 45 min. A challenge control group treated with sterile water instead of V. dahliae was also prepared. Afterwards, the plants were placed into pots containing a sterile 4:1 mixture of soil and perlite and grown in a culture chamber under controlled conditions, 16 h light and 25°C. The first measurements were taken 7 days after inoculation. The stem length and the number of wilted leaves were recorded every 7 days for 4 weeks. At the end of the fourth week the fresh and dry weights were also measured. Three independent experiments were carried out with eight plants per treatment and per experiment. In additional experiments, plants were kept for 6 weeks after inoculation and photographed.

Soil inoculation with Phytophthora capsici

After treatment with Fo47 or water, the plants were placed in sterile flasks with nutrient solution and incubated for 24 h in a growth chamber. The plants were then transferred into pots containing a sterile 4:1 mixture of soil and perlite and grown for another 24 h. The plants were then inoculated with P. capsici: 5 mL of a zoospore suspension (1000 zoospores mL−1) were poured uniformly over the surface of the soil in each pot (pot size 150 mL). Symptom severity was rated periodically on a scale from 0 (no symptoms) to 7 (dead plant), both 48 and 72 h post-inoculation. Two independent experiments were carried out with eight plants per treatment and per experiment.

Leaf inoculation with Phytophthora capsici

After the induction treatment, the plants were transferred into pots containing a sterile 4:1 mixture of soil and perlite and grown for 48 h in a culture chamber under controlled conditions, 16 h of light and 25°C. The plants were then inoculated by putting a plug of mycelia on the two first true leaves and the cotyledons. After inoculation the plants were placed in a wet chamber at room temperature to preserve high humidity conditions. The severity of phytophthora blight was scored for each cotyledon and leaf according to a scale from 0 (no lesion) to 4 (>75% of area with symptoms), both 48 and 72 h post-inoculation. An average value of severity was calculated for every plant. Two independent experiments were carried out with eight plants per treatment and per experiment.

Inoculation with Botrytis cinerea

After the induction treatment, the plants were transferred into pots containing a sterile 4:1 mixture of soil and perlite and grown for 48 h in a culture chamber under controlled conditions, 16 h light and 25°C. Plants were then inoculated.

Inoculation with B. cinerea was carried out by putting a 3 μL drop of the inoculum on the two first true leaves and the cotyledons. After inoculation the plants were placed into a wet chamber at room temperature to preserve high humidity conditions. The diameters of the lesions caused by B. cinerea were measured 48 and 72 h post-inoculation. Three independent experiments were carried out with eight plants per treatment and per experiment.

Pairing assay

Dual cultures were used according to the method of Diaz et al. (2005), with some modifications. The Fo47–pathogen combinations were assayed on PDA in 9-cm Petri dishes. Mycelial plugs, taken from actively growing colonies of Fo47 or the pathogens (V. dahliae or P. capsici), were placed 5 cm apart on the PDA. Control plates consisted of two plugs of the same isolate placed 5 cm apart on the PDA. The colonies were incubated together for several days and the diameter of the colony was recorded. The colonies were also observed with an optical microscope. The experiments were performed twice.

Gene expression study

In a first set of experiments, the roots and the stem were sampled 48 and 120 h after Fo47 treatment without pathogen inoculation. A second set of experiments was performed and samples were collected 8, 24 and 72 h after inoculation with V. dahliae. In all cases, the samples (five plants per sample) were frozen with liquid nitrogen and stored at −80°C. The extraction and retrotranscription was carried out following the protocol of the Bio-Rad Aurum™ Total RNA Mini kit and iScript™ cDNA Synthesis Kit, respectively.

The cDNA samples were analysed with the Bio-Rad iCycler™ iQ System following the protocol described by Silvar et al. (2008, 2009). The assay was performed for three defence related genes: a sesquiterpene cyclase (CASC1,AF061285), a basic PR-1 protein (CABPR1, AF053343) and a chitinase (CACHI2, AF091235) (for primer and amplicon details, see Silvar et al., 2009). The constitutively expressed actin gene (AY572427) was used as reference gene (Silvar et al., 2008, 2009).

The PCR reaction mixes contained Bio-Rad 1× iQ SYBR Green Supermix, 0·3 μm of each primer and 2·5 μL of cDNA for a 50 μL end volume reaction. The PCR program started with a 2 min denaturation step at 95°C followed for 40 cycles of amplification (95°C for 20 s, 58°C for 25 s and 72°C for 50 s) and finished with an elongation step of 5 min at 72°C. The data analysis was carried out with the Bio-Rad Optical System Software 3·0. The efficiency was calculated and the resulting Ct values were processed by the Pfaffl method (Pfaffl, 2001) to obtain the relative expression values. In this method the relative expression is defined as follows:

  • image

where E is the efficiency and Ct the number of cycles to detect the amplicon signal.

Statistical analysis

All statistical analyses were performed using statgraphics 5·1 for Windows. Percentage of wilted leaves was analysed by logistic regression (α = 0·05). In the rest of the Verticillium inoculation experiments a one-way anova was performed (α = 0·05) followed by Duncan tests for multiple comparisons. Comparison of two treatments in pairing assays and in Botrytis and Phytophthora inoculation experiments was performed by Student’s t-test (α = 0·05). Statistically significant differences (< 0·05) are reported in the text and shown in the figures.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Treatment of Fo47 against Verticillium dahliae

Treatment with Fo47 before inoculation with V. dahliae protected the plants against this pathogen. The plants treated with Fo47 and challenged with V. dahliae showed a reduction of symptoms from the third week after inoculation. At this time, differences became significant as the stems of the Fo47 treated plants grew longer than those from plants which were nontreated but inoculated with V. dahliae (Fig. 1a). The leaves started to wilt at the third week but the plants induced with Fo47 showed reduced wilt symptoms compared with the noninduced (Fig. 1c). Differences increased at the fourth week. As expected, Fo47 did not change the normal development of the plant, as the induced plants did not differ from the control (Fig. 1a). The fresh weight (Fig. 1b) was higher in the plants treated with Fo47, and was in fact the same as in the control noninoculated plants. This was also observed for the dry weight (Fig. 1d), indicating that the differences in fresh weight were not due to an imbalance in the water content. The overall reduction in the symptoms of verticillium wilt indicates that Fo47 was effective in the control of V. dahliae in pepper. In experiments where plants were grown for 6 weeks after inoculation, the differences in symptoms were even more evident (Fig. 2).

image

Figure 1.  Effect of Fo47 on verticillium wilt symptoms affecting growth parameters and the wilting of leaves: length of stem (a), fresh weight (b), wilted leaves (c) and dry weight (d). There were four groups: plants treated with Fo47 (Fo47), plants inoculated with V. dahliae (Vd), plants treated with Fo47 and subsequently inoculated with V. dahliae (Fo47 & Vd) and control plants. Data are means ± SE of three independent experiments with eight plants per treatment and per experiment. Asterisks indicate statistical differences (α = 0·05) in anova and Duncan test (a, b, d) or a logistic regression test (c).

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image

Figure 2.  Nontreated plants (a) and Fo47-induced plants (b) 6 weeks after inoculation with Verticillium dahliae. Note that plants that were not treated with Fo47 showed more severe symptoms of verticillium wilt. The experiment was repeated twice with similar results.

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Treatment of Fo47 against Botrytis cinerea and Phytophthora capsici

Plants treated with Fo47 were not protected against B. cinerea. The incidence (percentage of expanding lesions per plant) and the severity (area of expanding lesions per plant) of the symptoms showed by the Botrytis infection were not significantly alleviated by the treatment either at 48 or 72 h post-inoculation.

However, Fo47 protected pepper from P. capsici when inoculated in the soil (Fig. 3a). Severity index was significantly lower in Fo47-treated plants than in control plants. When P. capsici was inoculated on the leaves, Fo47 protected the plants 48 h after inoculation, but 72 h after inoculation, protection was surpassed (Fig. 3b).

image

Figure 3.  Effect of Fo47 on Phytophthora symptoms when inoculated in the soil (a) or on the leaves (b). Data are means ± SE of two independent experiments with eight plants per treatment and per experiment. Asterisks indicate statistical differences (α = 0·05) in Student’s t-test.

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Pairing assay

Verticillium dahliae grew slower in the presence of Fo47, with a reduction of approximately 25% compared to the control. Phytophthora capsici growth was not affected by Fo47. In both cases Fo47 did not change its growth rate. For both pathogens no apparent interactions with Fo47 were observed under the microscope.

Relative gene expression

The three studied genes were all up-regulated in the roots 48 h after Fo47 treatment (Fig. 4a,c,e), but only CABPR1 in the stems (Fig. 4d). If no further inoculation with a pathogen was done, CASC1 and CABPR1 decreased their expression 120 h after Fo47 treatment (Fig. 4a,c,d). However, if plants were inoculated with V. dahliae 48 h after Fo47 treatment, the three genes increased their expression compared to the inoculated control both in the roots (Fig. 5a,c,e) and in the stem (5b,d,f). The up-regulation of the genes was maintained from 8 to 72 h after Verticillium inoculation. A peak maximum (approximately 14-fold increase in expression) of CASC1 was observed 24 h after inoculation. It is also interesting to note that CASC1 and CACHI2 were not up-regulated in the stem with just Fo47 treatment (Fig. 4b,f), but they were induced in Fo47 treated plants after a subsequent inoculation with V. dahliae (Fig. 5b,f). This fact means that these two genes were primed by Fo47 in the stem.

image

Figure 4.  Effect of Fo47 on relative expression levels of three defence related genes. (a) CASC1 in the roots, (b) CASC1 in the stem, (c) CABPR1 in the roots, (d) CABPR1 in the stem, (e) CACHI2 in the roots and (f) CACHI2 in the stem. Data are means ± SE of two independent experiments.

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image

Figure 5.  Effect of Fo47 on relative expression levels of three defence related genes after inoculation with Verticillium dahliae. (a) CASC1 in the roots, (b) CASC1 in the stem, (c) CABPR1 in the roots, (d) CABPR1 in the stem, (e) CACHI2 in the roots and (f) CACHI2 in the stem. Data are means ± SE of two independent experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fusarium oxysporum Fo47 has been demonstrated to be an effective biocontrol agent in several crops against pathogenic Fusarium spp. In this study protection conferred by Fo47 was observed against V. dahliae and P. capsici. In both cases, antagonism by competition cannot be excluded. The same concentration of conidia was applied for Fo47 and the pathogen V. dahliae, but a lower concentration of spores for P. capsici. In both cases competition is possible based on the ratio between Fo47 inoculum and pathogen inoculum. Moreover, a dual culture of V. dahliae and Fo47 showed a decrease on the growth rate of the pathogen. Therefore, Fo47 could be acting at least partially through antagonistic interactions in the case of V. dahliae. Many nonpathogenic strains of Fusarium seem to act through competition for carbon source, and nonpathogenic strains consume it more efficiently than pathogenic strains (Couteaudier & Alabouvette, 1990). Moreover, a nonpathogenic F. oxysporum strain (F2) has been reported to reduce verticillium wilt in aubergine by a competition mechanism (Pantelides et al., 2009).

Another possibility to explain the Fo47 effect on the disease development is mycoparasitism. Indeed, mycoparasitism has been reported for Fo47, when paired with different Pythium species (Benhamou et al., 2002; Floch et al., 2009). In dual culture or in the rhizosphere of tomato, Pythium growth in the presence of Fo47 was associated with ultrastructural disorganizations such as cytoplasm aggregations or loss of protoplasm. Nevertheless, in the pairing assays Fo47 did not destroy the mycelium of V. dahliae or P. capsici, and no interactions were observed under the microscope.

Fo47 is also able to induce defence responses in several crops (Fuchs et al., 1997; Benhamou & Garand, 2001). Fo47 remains in the outermost layers of the root where several induced responses are triggered and prevent colonization (Benhamou & Garand, 2001; Benhamou et al., 2002; Alabouvette et al., 2009). The contact of Fo47 with the root triggers the elaboration of newly formed barriers, papillae and the occlusion of the intercellular spaces with a dense material. These responses can be locally induced by the cell death caused by Fo47, triggering Ca2+ and H2O2 release that are physiological responses related to induced response (Olivain et al., 2006). Other strains of Fusarium are able to confer a protective effect by inducing defence responses in other crops such as chickpea, tomato, pea or basil (Larkin & Fravel, 1998; Benhamou et al., 2002; Kaur & Singh, 2007; Aiméet al., 2008). In the present study, induced resistance was achieved by Fo47 application against P. capsici both by inoculation in the soil or in the leaves (Fig. 3). Silvar et al. (2009) found that plants pre-inoculated with F. oxysporum f. sp. lycopersici were more resistant to P. capsici root rot and such resistance was accompanied by the up-regulation of several resistance related genes. The response triggered in pepper by different strains of Fusarium could have some similar points of action, but the protective effect induced was not exactly the same. Diaz et al. (2005) found a protective effect against B. cinerea when pre-inoculating with F. oxysporum f. sp. lycopersici, but in the present study Fo47 did not confer protection against B. cinerea. Resistance to necrotrophic fungi, such as B. cinerea, depends on ethylene and jasmonate signalling pathways (Diaz et al., 2002), so it is tempting to speculate that ethylene is involved in response to Fo47. Another nonpathogenic Fusarium strain (Fs-K) protects tomato against F. oxysporum f. sp. radicis-lycopersici, but the effect is lost in mutants impaired in ethylene response (Kavroulakis et al., 2007). However, the Fs-K strain attenuates PR gene expression whereas Fo47 induces PR gene expression. Indeed, the two pepper PR genes induced by Fo47 (CACHI2 and CABPR1) are also induced by the ethylene-releasing compound ethephon (2-chloroethylphosphonic acid) (Hong et al., 2000; Kim & Hwang, 2000; Hong & Hwang, 2006). Moreover, the promoters of these genes are activated both by ethylene and salicylate (Hong et al., 2005; Hong & Hwang, 2006). All these facts point to a role for ethylene in the pepper signalling mechanism induced by Fo47.

In the present work, one hallmark of Fo47 treatment was the enhancement of the expression of CASC1, a gene encoding a sesquiterpene cyclase. Sesquiterpene cyclases are a group of enzymes that form a branch point in the general isoprenoid pathway involved in the conversion of the acyclic isoprenoid intermediate farnesyl pyrophosphate into a wide variety of cyclic sesquiterpene skeletons. Those enzymes comprise the early steps of the synthesis of the antimicrobial sesquiterpene phytoalexin capsidiol, which inhibits fungal growth (Taller, 2006). Capsidiol accumulates more in the resistant pepper cultivars against P. capsici (Garcia-Perez et al., 1998), confirming the important role of this gene in the resistance in pepper. Capsidiol has been described in Nicotiana and Capsicum as a pathogen- and elicitor-inducible defence metabolite in intact plants and cell cultures (Bohlmann et al., 2002). The enhancement of CASC1 expression was observed in the resistance induced by F. oxysporum f. sp. lycopersici in pepper (Silvar et al., 2009) and after pepper inoculation with some Trichoderma isolates that confer protection against P. capsici (Bae et al., 2011). Fo47 primed CASC1 and CACHI2 in the stem because its expression level did not increase until pathogen inoculation. However, in the roots Fo47 induced the expression of CASC1 even in the absence of the pathogen, but transiently: induction was only maintained if the pathogen was applied to the plant. Therefore, a sort of priming of this gene was caused by the Fo47 treatment. Priming is a recognized mechanism in ISR (Induced Systemic Resistance) triggered by plant growth promoting rhizobacteria and some beneficial fungi such as Trichoderma (Van Wees et al., 2008). Indeed, a strain of Bacillus cereus causes priming of CABPR1 in pepper (Yang et al., 2009).

Furthermore, CASC1 has been previously related to pepper resistance against P. capsici (Silvar et al., 2008, 2009), the response to copper stress associated to V. dahliae resistance (Chmielowska et al., 2010) and the response to UV light (Back et al., 1998). As far as is known, there are no clues regarding CASC1 regulation by plant hormones, so the connection of this gene to the defence signalling and the necrotroph/biotroph resistance scheme is still unexplored. In any case, the three defence genes (CABPR1, CACHI2 and CASC1) were induced by Fo47 and they take part in the protective effect against several pathogens (Hong et al., 2000; Kim & Hwang, 2000; Silvar et al., 2009).

In summary, the data demonstrate that Fo47 confers protection in pepper against V. dahliae and P. capsici. In the case of V. dahliae the results point to antagonism and induced defence response by Fo47 as possible reasons for the protection.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Dr Claude Alabouvette and Dr Christian Steinberg from the INRA Dijon for kindly providing us with the Fo47 strain. This research was funded by the Xunta de Galicia (PGDIT01AGR10301PR) and INIA (RTA2007-00062-C02-02). JV is in receipt of a Maria Barbeito program scholarship from the Xunta de Galicia.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References