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Keywords:

  • Hypovirulence;
  • Mycovirus;
  • Double-stranded RNA;
  • Botrytis cinerea

Abstract

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

Botrytis cinerea CCg425 contains a 33-nm isometric mycovirus whose genome is a 6.8-kb double-stranded RNA (dsRNA) molecule. Virulence bioassays, performed by direct plug mycelial inoculation on bean plant leaves, showed that B. cinerea CCg425 displays less fungal aggressivity than B. cinerea CKg54, a virulent fungal strain that is not infected by dsRNA mycoviruses. B. cinerea CCg425 also showed lower laccase activity and conidiation rate than B. cinerea CKg54. Furthermore, infection of B. cinerea CKg54 with viral particles purified from B. cinerea CCg425 resulted in diminished virulence of the infected fungus. Collectively, our results indicate that mycovirus infection confers hypovirulence to the fungal host.


1Introduction

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

Although viral infection generally has no effect on the phenotype of phytopathogenic fungi [3,16], the presence of some mycoviruses has been associated with particular phenotypic traits, including killer toxin production in Ustilago maydis[15,18], debilitant diseases in Helminthosporium victoriae[10] and virulence reduction in both Sclerotinia sclerotiorum[1] and Leucostoma persoonii[12]. The presence of multiple double-stranded RNAs (dsRNAs) has also been shown to confer reduced growth rates and virulence to Chalara elegans[2,19], and the presence of a 4.3-kb dsRNA molecule in Diaphorte ambigua has been reported to confer hypovirulence-associated traits, such as reduced laccase activity, diminished oxalic acid accumulation, and suppressed sporulation and pathogenicity [20]. The best-studied example of virus-mediated modulation of fungal virulence is that of infection of the chestnut blight fungus Cryphonectria parasitica by the dsRNA mycovirus CHV1-EP713. This virus has been shown to dramatically decrease fungal virulence and the application of hypovirulent fungal strains onto chestnut resulted in the conversion of resident virulent strains to the hypovirulent phenotype [17]. Furthermore, expression of viral sequences in several species of the genus Cryphonectria confirmed that the cytoplasmic-replicating L dsRNA is the hypovirulence-conferring agent [5–7].

Botrytis cinerea is a fungus that infects vegetables, ornamental plants and economically important fruits such as grapes, strawberries, raspberries, kiwi and pears [8]. The disease associated with B. cinerea infection is called gray mould and causes great economic losses world-wide. Although some strains of wild-type B. cinerea have been shown to contain dsRNA mycoviruses [4,13,22], the effect that these agents exert on the virulence of this important phytopathogenic fungus has not been investigated.

In this study we investigated the phenotypic effects that are conferred to the fungus by a mycovirus that infects B. cinerea CCg425. Our results strongly suggest that the mycovirus confers hypovirulence-associated traits to the fungus.

2Materials and methods

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

2.1Fungal strains and culture conditions

B. cinerea CKg54 and B. cinerea CCg425 were isolated from rotten grapes that were collected near the city of Rancagua (VI Región, Chile). Fungi were grown at 20°C in culture medium containing 1.5% (w/v) malt extract and 0.75% (w/v) yeast extract (Merck, Darmstadt, Germany).

2.2Nucleic acid purification and analysis

Viral dsRNA was purified by CF11 cellulose chromatography as described by Castro et al. [4]. Purified dsRNA was incubated for 15 min at 37°C with 2 μg ml−1 of RNase A in high (2×SSC) or low (0.1×SSC) ionic strength buffers (1×SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). Digestion with S1 nuclease has been described [22].

For electrophoretic analysis, nucleic acids were resuspended in triple-distilled water and resolved in 0.8% (w/v) agarose or 5% (w/v) polyacrylamide gels. The gels were subsequently stained by incubation in 0.5 μg ml−1 ethidium bromide.

2.3Virus purification and analysis

Mycovirus was purified by sedimentation on linear 10–40% (w/v) sucrose gradients, as described by Castro et al. [4]. Negative staining of viral particles was performed as previously described [22]. Virus images were obtained with a Philips Tecnai 12 Bio Twin electron microscope, at 80 kV. The polypeptide composition of viral particles was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and protein bands were visualized by staining with Coomassie blue R-250. BenchMark Protein Ladder 10–220 kDa (Invitrogen Life Technologies) was used as a molecular mass standard.

2.4Preparation and infection of spheroplasts from B. cinerea CKg54

Mycelium obtained from a 5-day culture was resuspended in a buffer containing 50 mM sodium phosphate, pH 5.8, and 0.7 M KCl. The mixture was then supplemented with one volume of Novozyme 234 (1 mg ml−1), prepared in the same buffer, shaken gently for 2.5 h at 25°C, and residual mycelium was removed by filtration through sterile gauze. Spheroplasts, whose integrity was checked by optical microscopy, were washed three times with 0.7 M KCl, twice with 1 M sorbitol and then diluted to 107 spheroplasts ml−1. A 100-μl aliquot of this spheroplast suspension was gently mixed with 20 μl of purified viral particles in 100 μl of PEG solution (40% (w/v) PEG 8000 in 50 mM CaCl2 and 100 mM Tris–HCl, pH 7.5). The resulting mixture was incubated on ice for 30 min, supplemented with 500 μl of PEG solution and incubated for 30 min at room temperature. The volume of the mixture was adjusted to 1 ml with 0.7 M KCl and 50 mM CaCl2. Infected spheroplasts were then plated and incubated in regeneration medium (1.5% (w/v) agar-agar, 1.5% (w/v) malt extract, 0.75% (w/v) yeast extract and 0.7 M KCl) for 5 days at 20°C to allow mycelium development [11].

2.5Sporulation rate and laccase activity determinations

B. cinerea strains were grown in 250-ml Erlenmeyer flasks containing 50 ml of solid medium (1.5% (w/v) agar-agar and 0.75% (w/v) malt extract) in the dark for 15 days at 20°C. Then, 20 ml of 0.005% (v/v) Triton X-100 was added and the spores were removed by incubation in a horizontal shaker for 15 min. The spore-containing liquid was filtered through sterile gauze and subsequently centrifuged at 8000×g for 10 min. The supernatant was discarded and the spores were resuspended in 1 ml of distilled water and counted in a Neubauer chamber.

Laccase activity was determined using 2,6-dimethoxyphenol as substrate, as previously described [20].

2.6Virulence bioassays

Virulence tests were performed by placing mycelial plugs of 7 mm in diameter (extracted from a 3-day-old nutrient agar plate) on intact bean leaves that had been surface-sterilized by washing first with 0.5% (v/v) sodium hypochlorite for 5 min and then three times with abundant sterile distilled water. Leaves were incubated in a dark wet chamber for 4 days at room temperature, and then the diameters of the injuries on the surface of the vegetal tissue were measured. These experiments were done in triplicate.

3Results

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

3.1Identification of a dsRNA in B. cinerea CCg425

Agarose gel electrophoresis of the nucleic acids extracted from B. cinerea CCg425 revealed the presence of an extrachromosomal nucleic acid with higher electrophoretic mobility than the fungal genomic DNA (not shown). Our finding that this nucleic acid is resistant to digestion with both RNase A in high ionic strength buffer and with S1 nuclease indicates that it is a dsRNA (not shown). Purification of the dsRNA molecule by CF11 cellulose chromatography and subsequent analysis by PAGE revealed that the molecular size of this dsRNA was 6.8 kb (Fig. 1).

image

Figure 1. PAGE of dsRNA from B. cinerea CCg425. Lane 1, λ DNA/HindIII fragments; lane 2, dsRNA from B. cinerea CCg425. The numbers on the left side indicate molecular sizes expressed in kb. The arrow on the right indicates the dsRNA position.

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3.2Mycovirus purification and analysis

To assess whether this dsRNA molecule was associated with viral particles, particulate material obtained from B. cinerea CCg425 was resolved by ultracentrifugation on a 10–40% linear sucrose gradient. The gradient profile at 280 nm revealed a sharp peak of absorbance centered on fractions 6–8. The material present in these fractions was subsequently analyzed by electron microscopy to visualize viral particles and by electrophoresis to detect the presence of both proteins and nucleic acids. Analysis by electron microscopy, after negative staining with potassium phosphotungstate, revealed that these fractions contained isometric viral particles, apparently icosahedral, of about 33 nm in diameter (Fig. 2A). Electrophoresis on agarose gels, after phenol extraction, revealed the presence of a nucleic acid band with the same electrophoretic mobility as the dsRNA isolated from B. cinerea CCg425 by CF11 cellulose chromatography (Fig. 2B). Finally, analysis of these fractions by SDS–PAGE revealed the presence of a major polypeptide of about 70 kDa that very likely represents the major viral capsid polypeptide (Fig. 2C).

image

Figure 2. A: Electron micrograph of mycovirus from B. cinerea CCg425. The sample was negatively stained with 1% (w/v) potassium phosphotungstate at pH 7.0. The bar marker represents 100 nm. B: Agarose gel electrophoresis analysis of dsRNAs extracted from mycoviral particles present in the gradient fractions shown on top. C: SDS–PAGE analysis of polypeptides present in the gradient fractions shown on top.

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3.3Analysis of virulence-associated traits in wild-type B. cinerea strains

Bioassays performed on bean leaves showed that the dsRNA-lacking CKg54 fungal strain is much more aggressive than the mycovirus-containing CCg425 strain (Fig. 3). As a first approach to establish whether there is a cause–effect relationship between mycovirus infection and hypovirulence, we performed a comparative analysis of two hypovirulence-associated traits, sporulation rate and laccase activity, on the two fungal strains. The results shown in Fig. 4 revealed that strain CCg425 displayed lower sporulation rates and laccase activity than strain CKg54, suggesting that mycovirus infection is associated with reduced fungal virulence.

image

Figure 3. Bioassays on bean plant leaves. Leaves were inoculated with mycelial plugs of either (A) B. cinerea CKg54 (B) B. cinerea CCg425 or (C) B. cinerea CKg54vi425.

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image

Figure 4. A: Comparison of sporulation rates in different B. cinerea strains. Strain CKg54vi425 resulted from infection of B. cinerea CKg54 with purified mycovirus particles isolated from B. cinerea CCg425. Results are expressed as means±S.E.M. (n=5). B: Laccase activity of different B. cinerea strains. Results are expressed as means±S.E.M. (n=3).

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3.4Mycovirus infection confers hypovirulence-associated traits to the fungus

To confirm whether mycovirus infection confers hypovirulence to B. cinerea, spheroplasts of the B. cinerea CKg54 virulent strain were infected with mycovirus particles isolated from B. cinerea CCg425, to generate the infected B. cinerea strain CKg54vi425. The resulting mycelium was shown to contain a 6.8-kb dsRNA molecule and isometric viral particles of 33 nm in diameter, indicating that the regenerated spheroplasts have acquired the mycovirus in a stable way. Interestingly, strain CKg54vi425 also acquired hypovirulence-associated traits, including diminished sporulation rates and laccase activity (Fig. 4A,B). Furthermore, the invasivity degree of CKg54vi425 on the vegetable tissue was reduced to levels similar to those of CCg425 hypovirulent strain (Fig. 3). These results demonstrate that mycovirus infection confers reduced virulence to B. cinerea.

4Discussion

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

In this study we show for the first time that a dsRNA mycovirus is able to confer hypovirulence-associated traits to B. cinerea. Observation by electron microscope revealed that the mycoviral particles were isometric and very similar to other mycoviral particles previously detected in our laboratory [4,22], but different from the rod-shaped mycoviral particles of 25×63 nm, whose genome is a single-stranded (ss) RNA molecule of 6827 nucleotides [14]. The genome of the mycovirus described in this study was characterized as dsRNA by its resistance to digestion with S1 nuclease and with RNase A in high ionic strength buffer, and by its chromatographic properties on CF11 cellulose, a resin specifically used to separate dsRNAs from ssRNAs and DNA [9]. Copurification of the 6.8-kb dsRNA with viral particles and with a specific protein strongly suggests that this nucleic acid is indeed the mycovirus genome. This suggestion was further supported by our finding that the migration of this dsRNA on agarose gels was highly retarded when phenol–chloroform extraction of sucrose gradient fractions was omitted, indicating a strong association between genomic dsRNA and viral polypeptides.

Our finding that the degrees of several virulence-associated parameters of B. cinerea, including sporulation rate, laccase activity and invasivity, were much higher in the mycovirus-lacking strain CKg54 than in the mycovirus-containing strain CCg425 suggests an association between mycovirus infection and fungal virulence. To confirm this suggestion we infected spheroplasts of B. cinerea CKg54 with purified viral particles obtained from B. cinerea CCg425, an infection approach that has been used successfully by other investigators [21]. After infection, the virulent B. cinerea CKg54 strain not only acquired and stably maintained the mycovirus, but also showed hypovirulence-associated traits characteristic of the donor strain. This is clear evidence that mycovirus is the agent responsible for diminished virulence of B. cinerea CCg425. The molecular mechanisms by which this mycovirus reduces B. cinerea virulence are still unknown.

Acknowledgements

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

This work was supported by Grants from FONDECYT 1000077, Universidad Mayor and DICYT-USACH.

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