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Atypical scab-like symptoms were reported for the first time in 2007 in the south of France on fruits of apple cultivars carrying the Rvi6 (=Vf) major resistance gene to Venturia inaequalis. With microscopic observations, nucleotide sequence data and pathological tests, it was shown that the causal agent was Venturia asperata. Scanning electron microscopy was used to compare its infection process and conidiogenesis to those of Venturia inaequalis on apple and Venturia pirina on pear. Venturia asperata produced fewer hyphae and fewer spores than the two other Venturia species, and resulted in weaker symptoms. This fungal species was previously described as a saprotroph on apple leaf litter. This is the first report of damage on apple fruits caused by V. asperata. Changes in host and cultural practices may have created a new context favourable for the emergence of this pathogen. It was also detected on symptomless leaves and on overwintered leaves on the ground. Pseudothecia developed on overwintered leaves and released ascospores over a 2-month period from the end of March until the end of May, suggesting that the fungus is able to survive from season to season. However, it is not yet known if this new disease will establish over coming years and become an emergent disease.
New diseases may appear on a crop as a result of changes concerning the pathogen, the host or the environment. The pathogen may acquire new virulences overcoming resistance genes of its habitual host or switch from one host to another, through mutation, hybridization or lateral transfer (Friesen et al., 2006; Desprez-Loustau et al., 2007; Oliver & Solomon, 2008). The use of new crops or cultivars, or changes in environment conditions or cultural practices may create a new ecological niche favourable for new pathogens (Anderson et al., 2004). In particular, the use of cultivars resistant to a disease may lead to the emergence of new pathogen species that were absent or present at only a low frequency previously (Vincenot et al., 2008).
On apple, the main source of resistance deployed in cultivars resistant to Venturia inaequalis, the causal agent of apple scab, is based on the major gene Rvi6 (=Vf) (Laurens, 1999). These cultivars have been deployed in Europe since the 1970s and have made it possible to reduce the number of fungicide sprayings. Albeit scarcely deployed, this resistance has been overcome by virulent isolates of V. inaequalis since 1985 in northern Europe (Parisi et al., 1993, 2004) and since 1995 in northwest France (Parisi et al., 2000; Gladieux et al., 2011). Since 2002, marketing in France of the new Rvi6 cultivar Ariane (Laurens et al., 2005) has appreciably increased the area of orchards growing a cultivar carrying this gene up to 500 ha in 2007. In order to delay the development of virulent isolates of V. inaequalis and delay the breakdown of Rvi6 resistance in orchards, it was recommended that growers destroy the leaf litter in winter and apply a light fungicide spraying schedule. The Rvi6 resistance was still efficient in most orchards in the south of France in 2007 (Didelot et al., 2009). However, atypical scab-like symptoms (Fig. 1) were reported on fruits of cv. Ariane in at least three orchards in the south of France in 2007 and at least six orchards in 2008 (Fig. 2). These orchards were separated by distances ranging from a few kms (<10 km) to more than 300 km. The atypical symptoms were first observed at the end of July and beginning of August, and became more pronounced at harvest time. The percentage of damaged fruits varied from one orchard to another, from <10% up to 60%.
The aims of this work were (i) to identify the causal agent of the atypical scab-like symptoms observed on fruits of the Rvi6 cultivar Ariane, based on morphological and molecular characteristics; (ii) to test its pathogenicity in controlled conditions on leaves and fruits of the Rvi6 cultivar Ariane and the non-Rvi6 cultivar Gala; (iii) to analyse its infection process and conidiogenesis by scanning electron microscopy, in comparison to V. inaequalis on apple and V. pirina on pear; and (iv) to follow its ascospore release in spring in comparison to V. inaequalis.
Materials and methods
Origin of fruits with atypical scab-like symptoms
Fruits of apple cv. Ariane with atypical scab-like symptoms were collected during August–September in one orchard (Lot-et-Garonne, France) in 2007 and 2008, and in three orchards (Bouches-du-Rhone, Dordogne and Lot-et-Garonne, France) in 2008. The distance between these orchards varied from 6 to 360 km.
Observation of conidia from atypical scab-like symptoms by light microscopy
Spores were collected from atypical scab-like symptoms by scratching or by adhesive tape, and mounted on a glass slide for observation under a light microscope. They were compared with conidia prepared from cultures of reference isolates of V. inaequalis, V. pirina and V. asperata (Table 1).
Table 1. Description of seven reference isolates of Venturia sp. used in the present study
Twenty-nine monoconidial isolates were obtained from atypical scab-like symptoms on fruits from the four orchards, according to techniques used for V. inaequalis: conidia were recovered in 100 μL sterile water and spread over a medium with 15 gL−1 agar, 10 gL−1 malt and antibiotics (25 mg streptomycin, 5 mg chlortetracycline and 12·5 mg penicillin L−1). After 24–48 h incubation in darkness at 18°C, a small agar piece with a single germinated conidium was removed with a needle under a stereomicroscope and laid in one Petri dish with malt agar medium. These isolates were cultivated at 18°C. After 1–2 weeks in darkness, cultures were incubated with 16 h light per day, and then stored at 4°C. The isolates were multiplied on cellophane laid on malt agar medium incubated at 18°C. For molecular characterization, mycelium was scratched from the cellophane after 2 or 3 weeks of incubation in darkness. For tests of pathogenicity, spores were obtained after 5 days of incubation with 16 h light. The cellophane was air-dried in a sterile air flow for 48 h and frozen at −20°C until use.
DNA was extracted following methods described in previous studies (Le Cam et al., 2001) from mycelium of the 29 isolates obtained from atypical scab-like symptoms and seven reference isolates of V. asperata, V. carpophila, V. pirina and V. inaequalis (Table 1). To distinguish these isolates, the following species-specific primers, defined previously (Schnabel et al., 1999; Stehmann et al., 2001), were used: Vina specific for V. inaequalis (5′-GTCTGAGAACAAGTTAAATAA-3′), Vpir specific for V. pirina (5′-GTCTGAGAACGAGTTAAAT-3′), Vasp specific for V. asperata (5′-GTCTGAGAAACAAGTAATAG-3′) and Vcar specific for V. carpophila (5′- GTCTGAGGAGAAAGCCAANCG-3′). These forward primers were used in combination with the primer ITS4 (White et al., 1990). PCR conditions to amplify the ITS locus were the same as described by Stehmann et al. (2001). The nucleotide sequence of the rDNA was obtained for one of the 29 isolates (isolate 2345) after amplification by PCR using the ITS1 primer from the 18S (5′-TCCGTAGGTGAACCTGCGG-3′) and the ITS4 primer from the 28S rDNA. Sequencing reactions of this PCR product were carried out using these two primers.
Tests of pathogenicity
The pathogenicity of one of the 29 isolates (isolate 2203) was evaluated on two apple cultivars, Ariane and Gala, on grafted plants (eight plants per cultivar) and on detached fruits (10 fruits per cultivar). Two experiments were carried out on grafted plants and one experiment on detached fruits. An additional experiment was carried out with the reference isolate of V. asperata (isolate ATCC34052) on grafted plants of Ariane and Gala. Plants were grafted on rootstock MM106 and used 4 weeks after pruning. The youngest expanded leaf at inoculation time was labelled F0 (the leaves below F0 being named F1, F2 and so on). Fruits were sampled at the end of May, 1 month after the beginning of flowering, in an orchard in Val de Loire (France). This orchard was not infected by V. asperata, had a low level of V. inaequalis infection and had not been sprayed with fungicides against scab during the previous month. Ten fruits were sprayed with water as a control for natural infection.
Plants or fruits were inoculated until runoff with a manual sprayer with suspensions of conidia adjusted to a concentration of 2·5 × 105 spores mL−1 after counting on a Mallassez cell. Three droplets from the inoculum suspension were laid on a Petri dish with malt agar to evaluate the germination rate, which varied from 69 to 85%. After inoculation, leaves and fruits were maintained wet with an atomizer for 3–4 days in darkness at 18°C. Plants were then incubated at 17°C with 16 h light and relative humidity fluctuating between 70 and 90% (mean = 83%). Fruits were wrapped with moistened paper and incubated in closed boxes to maintain high humidity.
Twenty-eight days after inoculation, symptoms were assessed on leaves with an estimation of the percentage of sporulating leaf area on a 0–7 scale (Parisi et al., 1993), and on fruits by counting the number of scab lesions.
Scanning electron microscopy
Leaf samples of cv. Ariane were collected from the tests of pathogenicity described above at 72 h, 6 and 18 days after inoculation. Samples were fixed in 4% glutaraldehyde in phosphate buffer (0·1 m; pH 7·2) for 2 h at 4°C under vacuum. Subsequently, samples were rinsed in three changes of buffer and then post-fixed in 2% osmium tetroxide for 2 h. Samples were washed three times in distilled water and dehydrated in a graded ethanol series and acetone, and dried by the critical-point method (Boyde, 1978). Samples were sputter-coated with gold–palladium and examined with a JEOL JSM-6301 F scanning electron microscope. These observations were compared with leaf samples of apple cv. Gala inoculated by the V. inaequalis isolate 1680 and with leaf samples of pear cv. Angelys inoculated with a mixture of V. pirina isolates multiplied together on different pear cultivars. The sampling dates were similar to those for isolate 2203, except for V. inaequalis, for which the last sampling date was 12 days after inoculation instead of 18 days.
Detection of the causal agent of atypical scab-like symptoms in symptomless leaves
In orchards where atypical symptoms were observed on fruits, no symptoms were observed on leaves. Evidence was sought as to the capacity of the fungus to colonize and to overwinter on leaves. Molecular detection of the causal agent of fruit atypical scab-like symptoms was carried out on leaves collected from apple trees in September 2007 at the end of the growing season and on leaves collected from the ground in March 2008 after overwintering. These leaves, which presented no apparent disease symptoms, were collected in one orchard from apple trees of cv. Ariane that presented atypical scab-like symptoms on fruits. For each sampling date, 18 discs of 1 cm2 were cut randomly in these leaves. DNA was extracted from these discs as described in Guérin et al. (2007) and then amplified with species-specific primers. Three discs from leaves collected from cv. Ariane in a production area where no atypical scab-like symptoms have ever been observed (Val de Loire, France), were used as a control.
Release of ascospores in spring
In autumn 2008, a sample of 200 leaves was collected at leaf fall on the ground in an apple orchard of cv. Ariane presenting 60% of fruits with atypical scab-like symptoms at harvest. For comparison, another sample of 200 leaves was collected in an untreated orchard of cv. Tentation® Delblush presenting more than 90% of fruits infected by V. inaequalis at harvest in the same location (Dordogne, France). The two leaf samples were placed in mesh bags on ground covered by grass for overwintering. In February 2009, the leaf samples were removed from the mesh bags and leaf beds were prepared for monitoring ascospore release, using a Marchi spore trap. This trap is derived from the tower aspirator technique (MacHardy, 1996), adapted by the Experimental Station of Laimburg (South Tyrol) for outside use (M. Varner, Mezzocorona, Italy, personal communication) and then modified and described by Giraud (2009). It consisted of a pump with a manometer sucking a constant air volume from a chamber. This chamber was connected to the leaf beds and contained a rotating drum with a 7-day movement. A plastic sheet, with 2-hourly graduated bands corresponding to each leaf bed, was fixed on the drum. The trap was placed outside. Ascospores released from each leaf bed were sucked and projected on the plastic band through a pipette tip. After removing the plastic bands from the drum, the trapped ascospores were counted under a light microscope.
Identification of the fungus
Spores isolated either from fruits of cv. Ariane in the four orchards, from in vitro cultures, or from leaves after inoculation in controlled conditions, had lengths varying from 9 to 12 μm (Fig. 3a). Conidia presented a rounded tip and were very slightly truncated at the base. These characteristics differed from conidia of V. inaequalis, which were piriform and had a length of 15–20 μm (Fig. 3b). Conidia of V. pirina had a characteristic spindle shape, with a length of 18–22 μm (Fig. 3c). The characteristics of the spores isolated from atypical scab-like symptoms were compatible with the species V. asperata (Samuels & Sivanesan, 1975). DNA amplification was obtained by primers specific for V. asperata (Fig. 4), while no amplification was observed using primers specific for V. inaequalis, V. pirina or V. carpophila. ITS1-5.8S-ITS2 sequences from isolate 2345 showed 100% identity with published sequences of V. asperata. These results confirmed microscopic observations, indicating that the fungus isolated from the atypical scab-like symptoms was V. asperata. The ITS1-5.8S-ITS2 sequences of isolate 2345 were assigned GenBank accession no. JN695772.
Pathogenicity of V. asperata
Two weeks after inoculation with isolate 2203 of V. asperata, sporulating symptoms appeared on leaves of both apple cvs Gala and Ariane. Sporulation covered a large leaf area (41% on Ariane and 49% on Gala) but was weak and difficult to see (Fig. 5a). The reference isolate of V. asperata ATCC34052 was also pathogenic on leaves of cvs Ariane and Gala. The most infected leaf was generally F1 or F2. In comparison, after inoculation with V. inaequalis isolates in the same conditions, symptoms generally appeared 1 week after inoculation, with the most infected leaves being generally leaves F0 and F1. There was no significant difference between the two cultivars Ariane and Gala for the percentage of sporulating leaf area. On one fruit of Ariane, one symptom similar to an atypical scab-like symptom was observed (Fig. 5b), but spores could not be recovered from this symptom.
Infection process and conidiogenesis of V. asperata
The germination of V. asperata conidia gave very long germ tubes. The appressorium was of small size and was applied preferentially at the junction between two host cells. A limited host cellular reaction was observed below the appressorium attack point. No network of mucopolysaccharides was observed (Fig. 6a-Va). In the case of V. inaequalis and V. pirina, the germ tube was short; the appressorium was clearly differentiated and a fine network of mucopolysaccharides was observed (Fig. 6a-Vi and -Vp).
After cuticle penetration, hyphae were subcuticular for the three Venturia species. Venturia asperata made a wide network of thin hyphae (Fig. 6b-Va), whereas V. inaequalis and V. pirina established dense networks of thick mycelium strings with large stromatic areas in some places (Fig. 6b-Vi and -Vp).
The V. asperata conidiophores arose at the wider ends of the hyphae and gave at their top one to five conidia with smooth scars; the conidiophore state was a Fusicladium. Numerous conidiophores were sterile, and grew extensively without differentiating conidia (Fig. 6c-Va). The V. inaequalis and V. pirina conidiophores arose from thick stromatic mycelium or from large mycelium strings. The conidia succeeded one another at the top of the V. inaequalis conidiophores; after their release they left an annular scar; their conidiophore state was a Spilocea (Fig. 6c-Vi). The conidia of V. pirina arose side by side at the extremity of the conidiophore; their conidiophore state was a Fusicladium. They left a circular scar with a smooth surface after release (Fig. 6c-Vp). The conidiophores of V. inaequalis and V. pirina gave numerous conidia (i.e. numerous scars were visible).
The conidium surface of V. asperata was covered by a thin network (Fig. 6d-Va). Conidia of V. inaequalis had a rough surface (Fig. 6d-Vi) and those of V. pirina had a slightly rough surface (Fig. 6d-Vp).
Detection of V. asperata on symptomless leaves in orchards
With specific primers, V. asperata was detected on 10 out of 18 symptomless leaf discs collected at the end of the growing season in September and on 17 out of 18 leaf discs collected in March on the leaf litter. As a control, no V. asperata DNA was detected on leaves sampled in an area where no atypical scab-like symptoms were reported.
Release of ascospores
Pseudothecia were observed on the overwintered leaves, and at the end of March they contained ascospores of shape and size in accordance with a previous description of V. asperata (Samuels & Sivanesan, 1975). The first ascospores were trapped from leaf beds on 4 March for V. inaequalis and on 25 March for V. asperata. From this time, release of V. asperata ascospores was abundant and the main peaks corresponded to those of V. inaequalis (Fig. 7). The last ascospores were trapped on 6 June for both V. inaequalis and V. asperata. The total amount of trapped spores reached 84 657 and 58 492 for V. asperata and V. inaequalis, respectively.
The present study showed that V. asperata, previously known as a saprotrophic organism on overwintered leaves of apple (Samuels & Sivanesan, 1975; Corlett, 1985), was also pathogenic on apple, giving atypical scab-like symptoms on fruits. The identification of V. asperata was based on microscopic observations and nucleotidic sequence data. Scanning electron microscopy showed that V. asperata produced fewer hyphae and fewer spores than V. pirina or V. inaequalis, with conidiophores that were sometimes sterile. These characteristics may explain why the symptoms produced by V. asperata on apple were less intense than those produced by V. inaequalis on apple or V. pirina on pear.
Venturia asperata was previously reported in New Zealand (Samuels & Sivanesan, 1975) and in Canada (Corlett, 1985) on overwintered leaves of apple. Symptoms on living leaves or fruits had never been observed. This species has not been reported previously in France. It was observed for the first time in southwest France in 2007, on fruits of cv. Ariane which carries Rvi6, a major resistance gene to V. inaequalis. The disease was observed again in 2008 and 2009 on the same cultivar. Within the same period, symptoms were also observed on the Rvi6 cultivars Goldrush and Prima in the same geographic area as cv. Ariane. As V. asperata was observed in a short period of time on several orchards (separated by distances ranging from a few kilometres to over 300 km), it is likely that the fungus was already present in the environment as a previously undetected native species, either already in apple orchards but without causing damage, or on other unknown host species in the proximity of apple orchards.
Changes concerning the host and cultural practices may be responsible for the emergence of V. asperata. In orchards planted with scab-susceptible cultivars, V. asperata may suffer from high competition with V. inaequalis, which intensively develops on apple and causes severe damage on fruits. As a consequence, V. asperata may be masked by this more damaging pathogen, as observed on cucurbits for melon severe mosaic virus in the presence of a potyvirus (Ciuffo et al., 2009). In these orchards, V. asperata may also be controlled by the repeated fungicide treatments applied on apple against V. inaequalis. Symptoms of V. asperata appeared on Rvi6-resistant cultivars in a geographic area where Rvi6 was not overcome by isolates of V. inaequalis (Didelot et al., 2009). The new Rvi6 cultivars offered a new and empty ecological niche for V. asperata, where competition with V. inaequalis disappeared and where fungicide sprayings were reduced. This might be a typical situation where control of one disease creates the conditions for the emergence of a new disease (Oliver & Solomon, 2008). A similar situation occurred on crucifers, with the occurrence of a new subclade of Leptosphaeria biglobosa on cultivars carrying efficient major genes of resistance to L. maculans (Vincenot et al., 2008). Venturia asperata has been reported in conventional farming (cv. Ariane) as well as in organic farming (cv. Goldrush), showing that the increase of V. asperata populations is independent of the farming system. It can be argued that the increase of V. asperata populations has been caused by the deployment of major resistance combined with the setting up of new practices based on low-inputs against scab.
The appearance of damage caused by V. asperata may also have been enhanced by favourable climatic conditions in the period 2007–2009. Literature on optimal climatic conditions for epidemics of V. inaequalis, V. pirina or V. nashicola is abundant (MacHardy & Gadoury, 1989; Villalta et al., 2000; Li et al., 2003), but nothing is known about these conditions for V. asperata, or concerning the stage of receptivity of leaves or fruits to the disease. In controlled environmental conditions that were optimal for V. inaequalis, weak development of V. asperata was obtained on leaves, and attempts to produce symptoms on fruits almost all failed. This could indicate that optimal conditions for V. asperata infection are different from those required for V. inaequalis.
Changes concerning the pathogen cannot be excluded, such as host-range expansion or an increase in aggressiveness. Such an increase in aggressiveness may indeed be responsible for the emergence of a new disease or worsening of an existing disease, as observed for L. biglobosa on crucifers (Vincenot et al., 2008) or for yellow rust on wheat (Hovmøller et al., 2008). As atypical scab-like disease caused by V. asperata was reported only on Rvi6 cultivars, the hypothesis that the Rvi6 cultivars might be more susceptible to V. asperata than scab susceptible cultivars, either by chance or because of particular linkages between resistance genes to different pathogens, was tested. Pathogenicity tests on leaves did not show any difference of susceptibility to V. asperata between the Rvi6 cultivar Ariane and the non-Rvi6 cultivar Gala, and therefore did not confirm the hypothesis of hypersusceptibility of cv. Ariane to V. asperata. It is likely that V. asperata was reported on Rvi6 cultivars because Rvi6 is the most frequently used scab resistance gene. It does not mean that V. asperata is unable to develop on cultivars with other resistance genes. However, difficulty in managing inoculation of fruits with V. asperata did not allow any conclusions on fruit susceptibility to be drawn. Further experiments on the susceptibility of fruits over a large range of cultivars should be performed to determine if there is any variation of susceptibility to this new disease.
In conclusion, V. asperata has been identified as a new agent of apple scab disease, whereas up to now only V. inaequalis was known to cause apple scab. The most likely reason explaining its emergence is the use of a new cultivar associated with modification of agricultural practices. Indeed, it is argued that the changes in host and cultural practices may have created a new context favourable for the emergence of this pathogen that had been described as a saprotroph until now. It is important to breed cultivars adapted to low-input practices and to set up adapted disease management to avoid the emergence of new diseases, or re-emergence of pathogens considered as secondary in high-input systems (Trapman & Jansonius, 2008). To attain the status of ‘emergent disease’, the new disease caused by V. asperata requires a series of conditions and steps, including enhanced fecundity of the new pathogen, enhanced survival from season to season, and increase in incidence and geographic area (Rachowicz et al., 2005; Oliver & Solomon, 2008). Its capacity to overwinter in orchards on leaf litter and to release ascospores in spring means that one of the conditions is already fulfilled. However, it is not yet known if this new disease will establish over future years and become an emergent disease.
We thank J. Champeix and C. Pitiot (Pomalia, France) for information on the atypical scab-like disease, G. Assié, F. Aubert, M. Charmasson, S. Limousin (Pomalia, France) and E. Koké (CEFEL, France) for providing samples of Ariane fruits with atypical scab-like symptoms. We also thank G. Samuel (USDA, USA) for helpful discussion during the diagnosis phase, and P. Loevenbruck and J. Hubert (LNPV, France) for confirmation of the diagnosis.