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Keywords:

  • environmental conditions;
  • epidemiology;
  • Nicotiana tabacum;
  • pathotype;
  • Potyvirus;
  • serotype

Abstract

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

To investigate the role of environmental conditions on the selection of virulent Potato virus Y (PVY) isolates subject to pressure from the recessive resistance gene va in tobacco, a field survey was performed in Brazil where va-derived genotypes have been recently introduced and now represent less than one-third of cultivated tobacco genotypes. A serological analysis of 397 leaves collected from different Brazilian tobacco-growing areas and mainly from plants with symptoms indicated that 52·4% of samples were infected by at least one of the viral species tested. PVY was present in 72·1% of infected samples. The probability of a plant being infected with PVY was reduced in va hosts. However, the biological characterization of PVY isolates on indicator hosts showed that 20 of the 29 tested isolates were able to overcome the alleles of the va gene. Moreover, the observed biological diversity of isolates was higher in susceptible tobacco genotypes than in va-resistant ones. Comparison of these data with the PVY diversity in French tobacco fields shows that the use of va-derived genotypes in two environments with contrasting climatic conditions, local hosts and cultural contexts, leads to a similar outcome: the prevalence of virulent isolates. These results strongly suggest an important role of the va gene in the modification of PVY populations.


Introduction

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

RNA viruses are described as dynamic populations (Domingo, 2002). Indeed, the characteristics of these viral populations can be modified by several processes, including the selection pressure imposed by the host. Potato virus Y (PVY, Potyvirus genus) is one of the most variable plant RNA viral species (Shukla et al., 1994). The filamentous and flexuous PVY particle contains a viral genome which consists of a single-stranded positive-sense RNA molecule of about 10 kb in length. A VPg protein is covalently attached at the 5′ end of the RNA molecule and a polyadenylated tail at the 3′ end. The viral genome includes one large open reading frame (ORF), which codes for a polyprotein cleaved into nine products by three viral proteases, and a second short ORF (PIPO; Chung et al., 2008) embedded within the previously described large ORF. PVY, transmitted in a non-persistent manner by more than 40 aphid species, has a wide host range including cultivated (e.g. potato, tomato, tobacco and pepper) and wild species of the Solanaceae family (Singh et al., 2008). PVY is both one of the most economically important plant viruses and one of the most damaging viruses affecting tobacco and potato crops (Blancard, 1998; Valkonen, 2007). PVY infections can induce necrotic symptoms on infected tissues (e.g. tobacco leaves) and organs (e.g. potato tubers) that can reduce yield and product quality (Le Romancer et al., 1994; Verrier et al., 2001). To limit the impact of PVY in tobacco fields, breeders since the early 1980s have produced cultivars resistant to PVY infection. Three allelic forms (0, 1 and 2) of the recessive resistance gene va, conferring different degrees of resistance to the necrotic symptoms induced by PVY infection, have been identified (Yamamoto, 1992; Blancard et al., 1995) and introduced into cultivars of Nicotiana tabacum (Ano et al., 1995). The va gene does not prevent viral infection of plants but (i) limits cell-to-cell movement of viral particles during the systemic infection of the host (Acosta-Leal & Xiong, 2008) and (ii) reduces the development of vein necrosis symptoms induced by PVY on tobacco leaves (Verrier & Doroszewska, 2004). Even though tobacco cultivars with the recessive resistance va gene represent the most reported genetic resistance source against PVY in this host species (Blancard, 1998), few data are available on the impact of the deployment of the va alleles on the structure of PVY populations. Field surveys have described the presence of virulent PVY isolates on va-derived tobacco genotypes (Latorre & Flores, 1985; Blancard et al., 1995; Verrier & Doroszewska, 2004). Virulence refers here to the genetic ability of a pathogen to overcome a genetically determined host resistance and cause a compatible interaction (Shaner et al., 1992). A recently published French field survey reported both the prevalence of PVY isolates able to overcome the resistance mediated by each of the three alleles va0, va1 and va2 and a reduction in the biological diversity of PVY isolates collected in va tobacco plants (Lacroix et al., 2010). For the last three decades, the culture of French tobacco crops has been based on intensive deployment of the alleles 0 and 2 of the va gene. Indeed, 77·5% of the tobacco cultivated by farmers in 2006 corresponded to va-derived tobacco genotypes (J.-L. Verrier, unpublished data). Such an intensive use of a resistance gene can lead to the emergence of variants putatively more virulent and/or aggressive than the parental viral entities (Pelham et al., 1970; Fargette et al., 2002; Garcia-Arenal et al., 2003; Chain et al., 2007). However, both abiotic and biotic factors (e.g. climatic parameters, the composition of the local host community) can also influence the strength and direction of evolution that drives host–parasite interactions (Thompson & Cunningham, 2002; Laine, 2009). Consequently, a 30-year period of massive deployment of the va gene and/or environmental characteristics of French tobacco-growing areas could have both influenced the modification of PVY population structure. To test the impact of these parameters on the virulence of PVY, biological properties of isolates collected in a contrasted environment need to be studied and compared to previously published results associated with PVY isolates collected in France. Brazil, the second main tobacco leaf producer in the world (http://www.fao.org), is an appropriate environment for such a study because the deployment of va-resistant cultivars, which constitutes the only resistance source used to limit PVY expansion in tobacco fields in Brazil, is both more recent (one decade) and limited (33% of the tobacco crop in 2009, C. Lorencetti, unpublished data) than in the French tobacco context. The main tobacco production in Brazil is situated in the south of the country (376 000 ha in 2009; C. Lorencetti, unpublished data). In addition, PVY has been frequently reported in this region on the basis of samples with symptoms collected from either tobacco or potato plants (Verrier & Doroszewska, 2004; de Avila et al., 2009). Thus, biological properties of PVY isolates present in tobacco samples collected in Brazil were studied and the resulting data compared to previously published results associated with PVY isolates collected in France.

Materials and methods

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

Sampling of tobacco plants

Tobacco leaves were collected in the three southern states of Brazil: Parana (PAR), western Santa Catarina (SCW); and Rio Grande do Sul (in the mountains [RGM] and in the centre [RGC] of the state). Samples were collected during a 2-week period (1–13 November 2009) from individual susceptible and PVY-resistant plants that belong to flue-cured (F) or burley (B) types. The collected plants were distributed in 55 fields from PAR (N = 15), SCW (N = 19), RGM (N = 12) and RGC (N = 9). For each field, leaves showing characteristic PVY symptoms, unconventional symptoms and no symptoms were sampled. Sampled leaves were individually stored at −20°C until use.

Serological identification of viral species in tobacco leaves

The presence of the four main viruses affecting tobacco crops in Brazil was determined for each of the collected leaves using enzyme-linked immunosorbent assays (ELISA) (Clark & Adams, 1977) and appropriate specific antisera. Polyclonal antisera raised against PVY (INRA Rennes/FNPPPT), Cucumber mosaic virus (CMV; INRA Avignon), Tobacco mosaic virus (TMV; LCA France) and Tobacco vein mottling virus (TVMV; Sediag France) and monoclonal antibodies raised against either PVYO (Neogen) or PVYN (INRA Rennes/FNPPPT) isolates were used in double antibody sandwich ELISA procedures according to previously published protocols [for PVY and CMV (Lacroix et al., 2010)] or to manufacturer’s instructions (for TMV, LCA France and TVMV, Sediag France).

Tobacco cultivars used in the experiments and biological characterization of PVY isolates

Tested PVY isolates were first inoculated to susceptible N. tabacum cv. Xanthi plants according to the previously published protocol (Lacroix et al., 2010) to produce viral inoculum for the biological characterization experiments. Their virulence was then analysed using the tobacco genotype MN944 as a susceptible host and three indicator resistant tobacco genotypes: VAM (Koelle, 1958), Wislica (Verrier & Doroszewska, 2004) and PBD6 (Schiltz, 1967), carrying the alleles 0, 1 and 2 of the va gene, respectively. For each PVY isolate, four plants from each tobacco genotype were mechanically inoculated according to Lacroix et al. (2010). This biological typing procedure was repeated twice. Systemic infection of each inoculated plant was tested 25 days post-inoculation using noninoculated leaves and polyclonal antibodies raised against PVY.

Statistical analyses

Statistical analyses of infection data were performed using the software R (R Development Core Team, 2005) by means of generalized linear models (McCullagh & Nelder, 1989) assuming a binomial distribution and a logit link function. Pairwise comparisons (contrasts analysis) were carried out with the esticon function in the doBy package (Author: S. Højsgaard) of the R software. A principal component analysis (PCA) was performed, using the Pearson correlation coefficient (Joliffe & Morgan, 1992), on arcsine-root transformed proportions of infected plants per tobacco cultivar for each of the tested PVY isolates. Then, an hierarchical ascendant classification (HAC) was carried out using the matrix of Euclidian distances calculated from the coordinates of isolates along the PCA F1 axis and using the Ward aggregative method (Arabie et al., 1998). Additional statistical analyses were performed by means of chi-squared tests (xlstat software, 2009, Addinsoft, France) to compare the geographical and host origins of PVY isolates included in each HAC group.

Results

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

Characterization of collected tobacco leaves

A total of 397 leaf samples were collected from cultivated tobacco hosts in Brazil. Taking into account variations in the number of plants sampled per field (from three to nine), 286 leaves with and 111 leaves without symptoms were collected (Table 1). The number of collected samples for each area and susceptible/resistant host ranged from 36 (RGM, resistant) to 84 (SCW, susceptible). Each collected sample was tested by ELISA for the specific detection of CMV, TMV, TVMV and PVY. Overall, none of the tested viruses were detected in 47·6% (189/397) of the collected samples, whereas one or a combination of several viruses was detected in 52·4% (208/397) of leaves (Table 1). The percentage of infected samples in the different sampled areas ranged from 27·4% in RGM to 80·9% in SCW. The majority of the collected leaves with symptoms (62·6%, 179/286) were infected. In addition, 26·1% (29/111) of symptomless tobacco leaves were infected. PVY, TVMV, CMV and TMV particles were detected, alone or in mixed infection, in 72·1% (150/208), 51·4% (107/208), 27·4% (57/208) and 7·7% (16/208) of infected samples, respectively (Table 2). The percentage of infected samples that contained PVY particles, alone or in mixed infection, for each area and host ranged from 45·5% (5/11) in resistant tobacco sampled in RGM to 81·3% (26/32) in susceptible leaves sampled in PAR. Mixed infections represented 46·2% (96/208) of infected samples. Each type of mixed infection was limited to a few samples (from three to 14), except for PVY/TVMV, which represented 28·4% (59/208) of infected samples (Table 2).

Table 1.   Percentages of infected Nicotiana tabacum leaves according to area and host origins of samples
OriginInfected samplesa
AreabHostcSym+SymTotal
  1. Sym+ and Sym indicate the presence and absence of symptoms on tested materials, respectively.

  2. aInfected samples according to results of Potato virus Y, Cucumber mosaic virus, Tobacco mosic virus and Tobacco vein mottling virus detection by ELISA.

  3. bPAR: Parana; SCW: Santa Catarina west; RGM: Rio Grande do Sul mountains; RGC: Rio Grande do Sul centre.

  4. cS: susceptible tobacco genotypes; R: resistant tobacco genotypes.

  5. dNumber of infected plants/number of collected plants.

PARS62·2 (28/45)d21·1 (4/19)50·0 (32/64)
R32·4 (12/37)8·3 (1/12)26·5 (13/49)
SCWS93·4 (57/61)47·8 (11/23)81·0 (68/84)
R89·7 (35/39)53·8 (7/13)80·8 (42/52)
RGMS34·4 (11/32)6·3 (1/16)25·0 (12/48)
R42·3 (11/26)0·0 (0/10)30·6 (11/36)
RGCS54·3 (25/46)27·8 (5/18)46·9 (30/64)
TotalS65·8 (121/184)27·6 (21/76)54·6 (142/260)
R56·9 (58/102)22·9 (8/35)48·2 (66/137)
Total 62·6 (179/286)26·1 (29/111)52·4 (208/397)
Table 2.   Percentages of Nicotiana tabacum leaves infected by Potato virus Y (PVY) in single and/or mixed infections with other targeted viral species according to the geographical and host origins of samples
Origin of samplesType of PVY infection
AreaaHostbSingleMixedTotal 
CMVTVMVCMV+TVMVTMV+TVMVCMV+TMV+TVMV
  1. aPAR: Parana; SCW: Santa Catarina west; RGM: Rio Grande do Sul mountains; RGC: Rio Grande do Sul centre.

  2. bInfected samples according to results of PVY, Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV) and Tobacco vein mottling virus (TVMV) detection by ELISA.

PARS3·10·068·83·10·06·381·3
R7·70·046·20·00·00·053·8
SCWS32·48·820·68·84·45·980·9
R31·019·011·92·42·40·066·7
RGMS58·30·00·00·00·00·058·3
R45·50·00·00·00·00·045·5
RGCS26·70·040·00·06·70·073·3
TotalS26·84·233·84·93·54·277·4
R28·812·116·71·51·50·060·6
Total 27·46·728·43·82·92·972·1

PVY-infected tobacco leaves were analysed in ELISA using two monoclonal antibodies raised against either PVYO or PVYN isolates. This serological analysis allowed the classification of 11·3% (17/150) and 18·7% (28/150) of isolates as belonging to the YO and YN serogroups, respectively. In addition, 4·7% (7/150) of infected samples were efficiently detected by the two monoclonal antibodies (YON serogroup). However, the majority of the PVY samples (65·3%, 98/150), were not detected by these two antisera and were assigned to an ‘unconventional’ PVY serogroup (YU). In addition, the percentage of infected samples that contained YU isolates ranged from 23·1% in resistant cultivars sampled in PAR to 64·3% in resistant cultivars sampled in SCW (Fig. 1).

image

Figure 1.  Percentages of Potato virus Y (PVY)-infected Nicotiana tabacum leaves according to geographical and host origins of samples. S: susceptible tobacco genotypes; R: resistant (va-derived) tobacco genotypes. PAR: Parana; SCW: Santa Catarina west; RGM: Rio Grande do Sul mountains; RGC: Rio Grande do Sul centre. For each area and host, the percentage of PVYO (grey), PVYN (black), PVYNO (white) and PVYU (hatched) is illustrated according to ELISA results obtained with monoclonal antibodies raised against PVYO (Neogen) and PVYN (INRA Rennes/FNPPPT).

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Analysis of infected samples

The impact of the factors associated with the collected tobacco leaves (e.g. sampling areas, the presence of the va gene in the host) on the probability of a plant being infected by PVY and more precisely by isolates belonging to the different PVY serogroups (YO, YN, YON and YU) was analysed by means of a generalized linear model (see above). The probability of being infected by PVY isolates was significantly smaller in va-resistant tobacco hosts (< 0·01). Moreover, the distribution of samples infected by PVY, YO, YN and YU isolates was significantly affected by the sampling area (< 0·001). The percentages of tobacco leaves infected with PVY and YU isolates were both statistically higher in SCW when compared with the other sampled areas (< 0·01). The percentage of samples infected with YN isolates was significantly higher in both PAR and SCW (< 0·02), whereas YO were more frequent in RGC (= 0·01).

Biological characterization of PVY isolates

From the 150 identified PVY-infected leaves, 29 isolates, selected among samples not described in these assays as mixed infected materials, were selected according to their geographical (17, 10 and two from SCW, RGM and RGC, respectively) and host (18 and 11 from PVY-susceptible and va-resistant plants, respectively) origins (Table 3). The virulence of these selected PVY isolates was analysed by mechanical inoculation of four tobacco cultivars (four plants per cultivar; two replicates) bearing none or one of the alleles (0, 1 or 2) of the va gene. According to the ELISA results obtained for each isolate/host combination, the 29 tested isolates were assigned to a PVY pathotype (Table 3). The pathotype VA, which included four out of the 29 tested PVY, describes isolates with an infection pattern restricted to the ability to infect plants of the susceptible tobacco MN944 genotype. The other tested PVY isolates were able to infect, in addition to the MN944 genotype, one (pathotypes 1 and 2), two (pathotypes 0-2 and 1-2) or all three (pathotype 0-1-2) resistant hosts used in the experiment. The majority of the isolates (20/29) belonged to the pathotype 0-1-2. In addition, members of the PVY pathotype 0-1-2 included all isolates (N = 11) collected on va-derived tobacco genotypes and all pathotype 0-1-2 members were described as PVYU isolates. The 18 isolates collected on PVY-susceptible hosts were assigned to six different pathotypes (Table 3).

Table 3.   Characteristics and biological properties of a selection of Potato virus Y (PVY) isolates collected in different hosts and tobacco-growing areas in Brazil
OriginIsolates Mean infected plants per tobacco genotyped  
HostaAreabSerogroupcNameMN944VAMWislicaPBD6PathotypeHAC groupe
  1. aB va0: resistant burley (B) tobacco genotypes possessing allele 0 of the va gene; S: susceptible tobacco genotypes.

  2. bPAR: Parana; SCW: Santa Catarina west; RGM: Rio Grande do Sul mountains; RGC: Rio Grande do Sul centre.

  3. cPVY serogroups were defined according to detection assays performed using monoclonal antibodies raised against PVYN (INRA Rennes/FNPPPT) and PVYO (Neogen). YO, YN, YON and YU serogroups were defined according to specific detection of isolates by YO, YN, both YO/C and YN and neither of these two monoclonal antibodies, respectively.

  4. dMN944 is a PVY-susceptible host; VAM, Wislica and PBD6 each present a PVY-resistant host phenotype because of the presence in the genotype of allele 0, 1 and 2, respectively.

  5. eTested PVY isolates were assigned to three groups by a hierarchical ascendant classification (HAC) analysis performed on the basis of the coordinates along the principal component F1 axis.

B va0SCWYU76, 121, 122 125, 131, 132B6·78·07·87·80-1-23
B va0RGMYU139, 144, 173, 179, 2096·87·88·08·00-1-23
SSCWYU51, 52, 64, 97, 1017·07·87·87·80-1-23
SRGMYU167, 172, 186, 1876·87·88·07·80-1-23
SSCWYO3873010-22
SSCWYN10360211-22
SSCWYON36702011
SSCWYN39701011
SSCWYON31700121
SSCWYN1006000VA1
SRGMYN1987000VA1
SRGCYO370, 3896·5000VA1

Analysis of the virulence of PVY isolates

To test the possible impact of the allelic forms of the recessive resistance gene va on the virulence of PVY isolates, a PCA was performed. This calculated three axes that illustrated 96·7%, 2·3% and 1% of the variability contained in the dataset, respectively. Each of the va0, va1 and va2 initial variables was associated with the F1 axis (cos2 = 0·969, cos2 = 0·955 and cos2 = 0·977, respectively). Moreover, the absolute contributions of va0, va1 and va2 to the F1 axis were 33·4%, 32·9% and 33·7%, respectively. To group PVY isolates according to their biological properties, an HAC was carried out, which led to the definition of three groups of isolates (Table 3). PVY isolates in the three HAC groups were not structured according to the sampling area. For example, the numbers of tested PVY isolates from SCW and RGW that belonged to HAC group 3 (11 and nine, respectively) were not statistically different (= 0·56). On the contrary, the distribution of PVY isolates in HAC groups 1 and 3 was statistically different (= 0·036). These two HAC groups were comprised of isolates collected only on susceptible hosts (HAC group 1) or collected either on susceptible or resistant hosts (HAC group 3).

Discussion

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

The objectives of this study were (i) to estimate the prevalence of PVY on cultivars with symptoms in tobacco crops in Brazil, (ii) to characterize the serological and biological diversity of ‘Brazilian’ PVY isolates and (iii) to compare the virulence of PVY isolates collected in two contrasted cultural contexts (Brazil and France).

The serological characterization of 397 leaf samples collected in 2009 in Brazil showed that PVY is prevalent (72·1%, 150 samples) within the 208 leaves infected by at least one of the four tested viral species (PVY, TMV, CMV and TVMV). As the sampling procedure was biased towards the sampling of leaves with symptoms (74% of sampled leaves), the presented detection frequencies for each virus do not represent the global epidemiological situation for viruses in tobacco fields in Brazil. Indeed, 26·1% (29/111) of symptomless tobacco leaves were infected. These samples may have been collected during the latency period between inoculation and symptom expression, which can last up to several weeks, or could correspond to asymptomatic infections. In addition, symptom expression varies according to both genetic characteristics of the virus and environmental conditions (Kerlan & Moury, 2008). However, the percentage of tobacco samples infected with PVY (72·1%) was similar to the infection results obtained from an analysis of potato leaves collected randomly (79·5%, 190/239) and on plants with symptoms (80·2%, 840/1047) in 2005/2006 from fields located in seven states of Brazil (de Avila et al., 2009). Moreover, most of the PVY samples (65·3%, 98/150) were not detected by the two monoclonal antisera raised against PVYO (Neogen) or PVYN (INRA Rennes/FNPPPT) isolates. Original PVY isolates with nonconventional biological, serological and/or molecular characteristics have already been sporadically described in various studies (Chikh Ali et al., 2007; Lorenzen et al., 2008; Karasev et al., 2010; Lacroix et al., 2010). This study reveals for the first time, in a specific growing area, that the most prevalent viral serotype cannot be assigned to the classical PVYO/PVYN serogroups with standard monoclonal antibodies. It would be interesting to characterize these isolates further with serological reagents produced with non-European PVY isolates and to investigate if they share, in addition to serological properties, other characteristics with the 49 PVYU isolates collected in France in 2007 (Lacroix et al., 2010).

To describe factors involved in the prevalence of PVY isolates in tobacco plants, and of the isolates belonging to the different PVY serogroups (YO, YN, YON and YU), the susceptible/resistant status of the sampled host and the sampled areas were used as variables for statistical analyses. Percentage of PVY-infected samples was significantly smaller in resistant than in susceptible tobacco hosts (< 0·01), which is consistent with the role of the va gene in the reduction of PVY spread in tobacco fields (Verrier & Doroszewska, 2004; Acosta-Leal & Xiong, 2008). The distribution of samples infected by PVY, YO, YN and YU isolates was significantly affected by the sampling area (< 0·001). The variation of environmental conditions, including biotic factors and cultural practices, could explain the different percentages of PVY according to the sampling area. Indeed, the transplanting dates of tobacco seedlings in the field differed between the different sampled areas. During the sampling period, the tobacco plants in SCW and RGC were taller (older) than those in PAR and RGM. Thus, the development stage of tobacco plants in SCW and RGC (young plants), together with a possible heterogeneous pattern (e.g. frequency, abundance and distribution) of aphid flights (Schuber et al., 2009; Cividanes & dos Santos-Cividanes, 2010) could have favoured viral infections in these areas. Moreover, as previously reported (Lacroix et al., 2010), the other plant species cultivated in the vicinity of tobacco crops can constitute host reservoirs for PVY and can influence the distribution of PVY isolates from the different serogroups. Potato plants, which are mainly cultivated in the centre and south-east of Brazil, including the states of Parana and Santa Catarina, allow PVY multiplication and could thus contribute to the increase of PVY incidence in those areas. Analysis of samples of potato plants collected from fields located in seven states of Brazil indicated that more than 90% of the tested isolates belonged to the PVYN group (de Avila et al., 2009). Even if the link between serological characterization and biological properties of PVY isolates is known to be weak (Chrzanowska, 1991; Lorenzen et al., 2008), most PVYN serogroup members are able to induce necrosis on tobacco. Thus, the high percentage of YN isolates in the northern sampled areas (i.e. PAR and SCW) could be the result of the connection between the potato and the tobacco compartments. Besides potato production, the main plant species cultivated in the south of Brazil, e.g. rice, bean, maize, wheat and sugarcane, are not host plants for PVY. In contrast, South America is the origin of many Solanaceae species (Olmstead et al., 2008) and wild hosts from this family are likely to be often found around tobacco fields in Brazil. The possible role of these wild Solanaceae hosts in the emergence and spread of PVY epidemics in Brazil, especially in the southern part of the country, remains to be investigated.

Although the probability of infection by PVY is less for a va-resistant than for a susceptible host, the characterization of biological properties of PVY isolates using four Nicotiana hosts with either none or one allele (0, 1 or 2) of the va gene showed that most of the tested isolates (N = 20/29 isolates) were assigned to pathotype 0-1-2. In addition, PVY isolates collected on susceptible and resistant hosts were distributed in six (VA, 1, 2, 1-2, 0-2 and 0-1-2) and only one pathotype (0-1-2), respectively. Thus, the selection pressure imposed by the va alleles seems to lead to the reduction of the biological diversity of PVY isolates affecting resistant tobacco crops. Moreover, all the tested YU isolates were included in the pathotype 0-1-2. Similar biological characterization performed with isolates collected in tobacco fields in France also resulted in the classification of a YU isolate in the 0-1-2 pathotype. The quantitative analysis of the number of infected plants for each tested host genotype and viral isolate by PCA and HAC led to the definition of three groups of isolates. The distribution of isolates in these groups was not determined by the sample area (= 0·56) but was dependent on the susceptible and resistant status of the host origin. The low-virulence populations, included in the HAC group 1, were collected in susceptible tobacco, while highly virulent ones (HAC group 3) were collected from resistant tobacco plants. Thus, the selection of virulent PVY isolates in the different sampled areas, which are characterized by contrasted landscapes, seems not to be linked to the geographical parameters (i.e. flat land versus mountains). This contrasted distribution of PVY isolates in HAC groups suggests that the emergence of virulent PVY isolates is associated with a va-resistant host selection pressure.

In conclusion, these results showed that the selection pressure imposed by the va gene deployed in tobacco crops in Brazil is associated with both the reduction of biological diversity of PVY isolates affecting resistant tobacco hosts and a high frequency of pathotype 0-1-2 isolates. It is important to note that these results are similar to those obtained for PVY isolates collected in tobacco fields in France, indicating that the contrasted climatic conditions, local host communities and cultural contexts of these two tobacco-growing countries did not differentially impact the virulence characteristics of PVY populations present in tobacco fields. Thus, in these two tobacco-growing systems, va-selection pressure led to the emergence and selection of virulent PVY isolates. Variation of both the frequency of these virulent isolates and the PVY incidence in tobacco crops should be monitored during several successive growing periods to acquire data on the dynamics of PVY populations under these growing/environmental conditions. Resulting data will be important for estimating the need for development of new strategies based on the deployment of va-derived tobacco genotypes.

Acknowledgements

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

We are grateful to Dr Yannick Outreman and Dr Luc Madec for statistical analyses, to Dr Thomas Baldwin for critical reading of the manuscript, and to Agnès Delaunay and Laize Espindula for technical support. The work presented was supported by the Institut National de la Recherche Agronomique (France), Imperial Tobacco Group, Alliance One and the Association for Research for Nicotianae.

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