Introduction

Root-knot nematodes (RKNs), Meloidogyne spp., are extremely polyphagous plant parasites, responsible for estimated losses of more than 80 billion Euros/year (Blok et al. 2008). These yield losses are a problem throughout the EU and are exacerbated by the sophisticated and intensive character of EU agriculture. In addition, new regulations have withdrawn the use of most chemical nematicides since 2006, and nematode problems have been increasing in horticultural crops consequently. In this context, cultivation of grafted vegetables is expanding in Europe, and has been adopted as a non-chemical alternative to methyl bromide in several countries (Methyl Bromide Technical Options Committee 2006). Because of this situation, breeding programs for the selection of resistant cultivars and/or rootstocks have recently become a major challenge for breeders. However, the diversity of Meloidogyne species and isolates infecting vegetables in most major production areas worldwide, is a threat to the use of single major resistance genes (R-genes) (Castagnone-Sereno 2002). Since resistance sources against these nematodes are rare, management of the R-genes currently available is of crucial importance to preserve their durability.

In Solanaceous species (e.g., tomato, pepper), several R-genes have been identified, that control the three main RKN species, M. arenaria, M. incognita and M. javanica (Williamson and Kumar 2006), and are currently used in breeding programs. However, the diversity and availability of such R-genes is limited. In tomato, all the modern fresh-market and processing resistant cultivars carry the single dominant gene Mi-1, introgressed from a single interspecific hybrid (Williamson and Roberts 2009). For more than 60 years, this gene has been the only source of resistance to RKNs in all the available tomato cultivars worldwide, and may be considered as a very stable R-gene in terms of durability. However, although this gene has been an excellent example of the use of host resistance to effectively reduce the need for pesticide application (Sorribas et al. 2005), the lack of diversity in the resistance sources exploited explains in part the emergence and spread of Mi-1-virulent nematode populations able to overcome it (Castagnone-Sereno 2002). Indeed, natural virulent isolates have been reported in many areas of the world where tomato is a major crop, and this trend has been increasing recently (e.g., Tzortzakakis et al. 2005, 2008; Verdejo-Lucas et al. 2009; Devran and Söğüt 2010), which could significantly reduce the duration of the commercial exploitation of the Mi-1 gene in tomato. Moreover, the artificial selection of virulent lineages from progenies of avirulent Meloidogyne isolates, was demonstrated by repeated inoculations onto Mi-1-resistant tomato genotypes under laboratory and greenhouses conditions (Jarquin-Barberena et al. 1991; Castagnone-Sereno et al. 1994b; Meher et al. 2009).

In pepper, resistance to RKNs is controlled by several linked dominant genes, i.e., the Me genes. Three of them (Me1, Me3, Me7), in inbred lines originating from three genetically distant accessions, are effective against M. incognita, M. arenaria and M. javanica (Hendy et al. 1985; Djian-Caporalino et al. 1999; 2001; 2007). However, in laboratory experiments, the selection of virulent variants toward the Me3 gene was achieved by using drastic selection pressure of avirulent M. incognita populations, whereas the resistance conferred by the Me1 gene could not be overcome (Castagnone-Sereno et al. 1996; 2001). Although Mi-1 and Me- virulent populations were obtained in experimental assays, the results suggested that these R-genes may differ in their durability if repeatedly used under field conditions.

Few data are available in the literature about the reproductive potential of RKNs virulent against R-genes from Solanaceous vegetables, and reports showed differences that might be revealed at different developmental stages during the nematode-host interaction. Experimental evaluation of the fecundity (i.e., production of egg-masses by mature females) of Meloidogyne field populations virulent against the tomato Mi-1 gene did not show any significant differences when the nematodes were inoculated onto susceptible versus resistant plants (Castagnone-Sereno et al. 1994a; Tzortzakakis et al. 1998). Another study indicated that Mi-1-virulent nematodes produced fewer eggs per gram of root on resistant tomatoes compared to susceptible ones (Huang et al. 2004). More recently, a lower reproduction of M. incognita was found associated to virulence against Mi-1 when inoculated onto susceptible tomatoes, using a combination of female fecundity, fertility and egg hatching as an overall measure of reproduction (Castagnone-Sereno et al. 2007). In addition, as far as we know, no such information is currently available concerning RKN populations exhibiting virulence against the pepper Me R-genes.

The first objective of the present study was to compare the reproduction of several laboratory-selected and wild Mi-1- and Me-virulent isolates of M. incognita on both susceptible and resistant tomatoes and peppers. For that purpose, we evaluated the ability of the juveniles to mature into adult females and produce eggs. A further objective was to determine whether reproductive fitness costs may be associated with the ability of the nematodes to overcome the tomato and pepper R-genes, by comparing reproductive parameters of M. incognita isogenic lines that differ only for their virulence on a set of resistant and susceptible host genotypes. Specificity of virulence and fitness costs of virulence could have important consequences for the management of plant resistance in the field.

Materials and methods

Plant material

Tomato (Solanum lycopersicon) cultivars Saint Pierre and Marmande, susceptible to M. arenaria, M. incognita and M. javanica, and cultivars Piersol and VFN8, homozygous for the Mi-1 gene and resistant to the same three Meloidogyne species, were used in the experiments. Saint Pierre and Piersol are near-isogenic lines (Laterrot 1975).

Pepper (Capsicum annuum) genotypes used in this work were inbred lines with differential resistances to the three RKN species. Doux Long des Landes is a susceptible cultivar; Yolo Wonder exhibits a partial (i.e., a low level of quantitative) resistance to some RKN populations; the two resistant haplo-diploid lines, HD149 and HD330, produced through in vitro androgenesis (Dumas de Vaulx et al. 1981) are homozygous for the Me3 and Me1 genes, respectively (Hendy et al. 1985); the inbred pepper line Criollo de Morelos 334 (CM334) was selfed in insect-proof cages to eliminate outcrossing and was assumed to be homozygous for the major dominant resistance gene previously named Me7 (Djian-Caporalino et al. 2007).

Nematode isolates

Twelve M. incognita isolates were used. Their geographical origin and (a)virulence status against the tomato Mi-1 or pepper Me3 and Me7 R-genes are reported in Table 1. All the nematode isolates originated from single egg mass cultures, except the greenhouse naturally-selected isolates CREAT, pop A, pop B and pop C that consisted of mixed cultures. Prior to multiplication, each isolate was specifically identified according to its isoesterase electrophoretic pattern (Dalmasso and Bergé 1978) and/or by SCAR-PCR (Zijlstra et al. 2000). All nematode isolates have been maintained for at least 2 years (i.e., 10 successive generations) in the greenhouse on susceptible or resistant cultivars for the avirulent or virulent isolates, respectively. Hatched second-stage juveniles (J2s) were obtained in a mist chamber from previously inoculated roots. Nematodes were collected in water every 48 h and used immediately for inoculating the plants.

Table 1 Meloidogyne incognita isolates used in this study
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The Mi-1 and Me-avirulent nematode isolates used in this study were collected originally from heavily infested fields or greenhouses, in areas where resistant tomatoes and peppers have not been cultivated. They are avirulent for both the Mi-1-gene in tomato and Me-genes in pepper.

From the Morelos M. incognita avirulent isolate, virulent lines were laboratory-selected and reared by successive re-inoculation on the Mi-1-tomato cultivar Piersol, or the Me3-pepper HD149 line, or the Me7-pepper CM334 line for more than 25 generations, starting from the progeny of one single female, according to the procedure of Jarquin-Barberena et al. (1991). The Mi-1 virulent SM1 and SM2V isolates have been laboratory-selected under controlled greenhouse conditions from the avirulent wild-type isolates avir Mifield1 and avir MifieldV, respectively, by repeated inoculations on Mi-1-resistant tomato for about 40 generations. The Me3-virulent isolates CREAT from La Baronne, France, and pop A, pop B, and pop C from Hungary have been naturally selected on resistant peppers carrying Me3 in experimental field (greenhouses). Vir CREAT was issued from a single year of selection in field (approximately 3 generations). Pop A, pop B, pop C from Hungary were well-established virulent populations arising from several years (not precisely determined) of selection in field.

Experimental procedures and infestation parameters

Tomato and pepper seedlings of cultivars susceptible and resistant to RKNs, were grown individually in 100 ml pots containing steam-sterilized sandy soil covered by a 1 cm layer of loam. Six to twenty replicates were performed for each cultivar and each nematode isolate tested. Experiments were conducted in climatic chambers maintained at 24°C (±2°C) with a 12-h light cycle and a relative humidity of 60–70%. For each nematode isolate, six to seven-week-old plants (4–6 true leaves) were inoculated with a water suspension of 300 to 500 J2s.

Six to seven weeks after inoculation (i.e., a duration that allowed completion of the nematode life cycle), plants were harvested, carefully washed individually with tap-water, and stained for 10 min in a cold aqueous solution of eosin yellow (0.1 g/l water), to specifically stain egg masses (EMs) in red (Roberts et al. 1990).

The roots were then rinsed and examined under a magnifying glass and the number of EMs counted for each plant.

For each plant, up to 10 EMs were removed at random from the root system and the eggs were separated from the gelatinous matrix in 0.9% NaOCl, mounted in water between a glass side and a cover slip and counted under a stereomicroscope. Three repeats per plant were assessed.

Since two different tomato cvs (for both nematode susceptible and resistant), a variable inoculum rate (300–500 J2s) and a variable number of replicates (6–20) were described, three parameters of infection were calculated :

  • Infection Frequency (IF) was calculated according to the following ratio: IF = number of EMs/number of J2s inoculated. IF theoretically ranged from 0 (no reproduction at all) to 1 (each juvenile inoculated could develop into a gravid female and generate one EM);

  • Female Fertility (FF) was evaluated as the average number of eggs per EM (i.e., the number of eggs produced by each female);

  • Reproduction Potential (RP) of the nematode population defined as the number of eggs/number of J2s inoculated, following formula: RP = IF x FF.

The virulence costs (Ch) of the laboratory-selected virulent lines on the susceptible host were estimated as follows: Ch (%) = 1—RP(virulent line)/RP(avirulent line) (Castagnone-Sereno et al. 2007).

Statistical analysis

In order to investigate a possible cost of virulence, the RP on the susceptible tomato or pepper of a restricted set of nematode genotypes (avir F, avir Mifield1, avir MifieldV, vir SM1, vir SM2V, vir Me3, and vir Me7) were compared. A Kruskal-Wallis test was first carried out. Wilcoxon-Mann-Whithney unilateral tests were then used for post hoc comparisons in order to check if, for a similar nematode genetic background, avirulent strains have higher RP than virulent ones. This led us to perform four pairwise comparisons (avir F versus vir Me3 or vir Me7; avir Mifield1 versus vir SM1; avir MifieldV versus vir SM2V). Bonferroni correction was consequently applied (significance level at ε = 0.05/4 = 0.0125 instead of α = 0.05). Analyses were performed using the free software R (http://www.r-project.org/).

Non-parametric tests were also applied to compare the RP of the nematode isolates on each plant genotype except “R Me1-pepper” (see Fig. 2 and associated Results). After a significant Kruskal-Wallis test, Wilcoxon-Mann-Whithney bilateral tests were carried-using Bonferonni correction (significance level at ε = 0.05/28 = 0.0018). These post hoc comparisons should nevertheless be interpreted with caution since they are not only sensitive to differences of RP between populations but also to differences in sample sizes.

Results

All the M. incognita isolates tested were able to infect both the susceptible tomatoes and peppers, with IF ranging from 0.10 to 0.87 (IF not determined for avir Mifield1 and vir Mi-1) (Table 2). The Mi-1 and Me-avirulent isolates avir MifieldV and avir F produced fewer egg masses on the partially resistant pepper cultivar Yolo Wonder compared to the susceptible pepper Doux Long des Landes and tomato St Pierre or Marmande plants. As expected, i) avirulent isolates (avir MifieldV and avir F) were unable to infect the resistant tomatoes or peppers (IF values ranging from 0 to 0.002); ii) selected isolates virulent on Mi-1-tomato exhibited a high infection frequency on resistant tomatoes (0.35 ≤ IF ≤ 0.82); and iii) both selected and natural isolates virulent on Me3-pepper and selected isolates virulent on Me7-pepper exhibited a high infection frequency on the Me3-pepper (0.23 ≤ IF ≤ 0.81). Nevertheless, none of the Mi-1, Me3 and Me7-virulent isolates (overcoming either tomato or pepper R genes) were able to infect Me1-pepper, and both selected and natural isolates virulent on resistant peppers were unable to reproduce on Mi-1-tomato. One or two egg-masses were obtained on a few Me1-peppers, but these egg masses contained very few eggs (i.e., < 65 eggs; data not shown) compared to those on Me3-peppers (i.e., > 800 eggs; data not shown) and the nematodes obtained from these eggs did not survive to a successive inoculation, so it was not possible to obtain a Me1-virulent isolate.

Table 2 Infection frequency (IF) of greenhouse and laboratory-selected Meloidogyne incognita isolates on susceptible vs. resistant tomato and pepper genotypes. Values are the mean (±standard error) of 6–20 replicates
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Considering the RP of the nematodes, we first observed that the RP of avirulent isolates was higher on susceptible tomato than on susceptible pepper (496.78 ± 46.30 and 371.21 ± 71.77 for the avir F isolate on tomato and pepper, respectively; and 238.78 ± 35.13 and 157.73 ± 17.93 for the avir MifieldV isolate on tomato and pepper, respectively) (data not shown). When comparing the RP of a restricted set of the avirulent and virulent nematode populations on susceptible plants, we firstly demonstrated significant differences between nematode populations (χ²6df = 40.8, p < 10−3) (Fig. 1). More interestingly, the hypothesis of a virulence cost was strongly supported in three of the four pairwise comparisons: avir F versus vir Me3 (W = 200; p < 10−3); avir F versus vir Me7 (W = 279; p < 10−3); avir MifieldV versus vir SM2V (W = 34; p = 0.004). In these three cases, the estimated fitness cost ranged from 54 to 71%. In the last comparison (avir Mifield1 versus vir SM1), the same tendency was observed but the difference was not significant (W = 23; p = 0.24); it is noteworthy that the sample size was nevertheless low (6 replicates).

Fig. 1
figure 1

Comparison of the reproduction potential (mean RP of 6 to 20 replicates ± standard error) of avirulent and virulent laboratory-selected M. incognita isolates on tomato and pepper susceptible genotypes. NS = no significant difference; * = significant difference; ** = highly significant difference (significance level at ε = 0.05/4 = 0.0125 instead of α = 0.05)

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Except for the resistant Me1-pepper, significant differences of RP were found between virulent nematode lines on a same host plant (Fig. 2). The number of eggs produced ranged from 106 to 496 times the number of J2s inoculated on susceptible cultivars (RP = 106 ± 9 to 496 ± 19) and from 0 to 436 times the number of J2s inoculated on resistant cultivars (RP = 0 to 436 ± 32). The Me3-virulent isolates A, B and C from Hungary reproduced much more on all susceptible and Me3-resistant pepper genotypes (RP ranging from 200 ± 3 to 496 ± 19) than the Me3-virulent isolate arising from a single year (approximately 3 generations) of selection in field (vir CREAT) (RP ranging 117 ± 11 from to 209 ± 21), and than the laboratory selected isolates (vir Me3 and vir Me7) (RP ranging from 63 ± 10 to 322 ± 18).

Fig. 2
figure 2

Comparison of the reproduction potential (mean RP of 6 to 20 replicates ± standard error) of laboratory-selected and natural M. incognita virulent isolates on both susceptible and resistant tomatoes and peppers. The combinations missing a datum bar (for tests on R Mi-1-tomato, R Me3-pepper and R Me1-pepper) have values of 0. The results of Kruskal-Wallis tests are given under each line

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Discussion

Selection for RKN resistance in crops, including tomato and pepper, is a major challenge for plant breeders. Therefore, because the introgression of R-genes into cultivars of agronomic interest by successive back-crosses generally takes several years, a long-term goal of breeders is to select the genotypes (robust R-genes, their combination, and associated genetic backgrounds) that are expected to exhibit the best durability under field conditions (late emergence and low fitness of nematode virulent populations). In pepper, the Me1 R-gene appears to be a good candidate for such a purpose. Previous studies had shown that none of the 22 RKN avirulent isolates tested, belonging to the three major species (M. incognita, M. arenaria and M. javanica), were able to overcome this R-gene under laboratory artificial conditions (Castagnone-Sereno et al. 1996; 2001). In the present study, we further showed that neither natural nor selected virulent RKN isolates (overcoming either Mi-1 from tomato or Me3 or Me7 from pepper) were able to reproduce on Me1-peppers. Such a result is in good agreement with previous data indicating that these R-genes are not equally overcome by RKNs, and that Me1 behaves as a robust R-gene. Conversely to Mi-1, Me3 and Me7, Me1 induces a late hypersensitive reaction in the vascular cylinder of infected roots, thus inhibiting the development of egg-laying females (Bleve-Zacheo et al. 1998; Pegard et al. 2005). Our observations here support previous data suggesting that late expression of the hypersensitive reaction by Me1 prevents (or at least strongly reduces) the frequency of emergence of Me1-virulent nematode genotypes.

Additionally, this study demonstrates that both natural and selected RKN isolates virulent on Me3 or Me7 resistant peppers are unable to reproduce on Mi-1 tomato, and confirmed that selected isolates virulent on Mi-1 tomato are unable to reproduce on Me3 and Me1 resistant peppers. The virulent isolate overcoming Me7 was able to reproduce on Me3-pepper, which may indicate that Me3 and Me7 may be one single allele at the same gene. Indeed, two independent allelism tests performed with 385 test-cross lines [F1(CM334xHD149) x DLL] strongly suggested that Me3 and Me7 are allelic, since no susceptible plant was obtained on these 385 lines inoculated with 500 avirulent M. incognita juveniles (Djian-Caporalino & Castagnone-Sereno, unpublished). Moreover, the fact that RKN selected for virulence over Me7 also broke down Me3 (and reciprocally, data not shown) indicates these are a single allele. So, RKN isolates virulent on one resistant crop are definitively not virulent on a different resistant crop. These findings indicate that virulence is highly specific to the R-gene on which selection has occurred. As a result of such strict specificity of virulence, once virulent isolates are selected on a determined R-gene, it is very likely that alternation in the rotation with a different gene will reduce the number of nematodes in the soil under their damage threshold, except if multiple virulence can be progressively selected, which remains to be demonstrated.

The reproduction potential of Mi-1-, Me3- or Me7-virulent laboratory-selected and wild M. incognita isolates exhibited significant high levels of variability on a same host, either susceptible or resistant. As nematode reproduction on resistant genotypes is generally explained by the interaction between the plant genotype and nematode isolate, but not by either factor alone (Ornat et al. 2001; Jacquet et al. 2005; Lopez-Perez et al. 2006; Cortada et al. 2008), we can hypothesize that the variability observed here in the infection and reproduction of virulent lines on a same host can be attributed to the nematode genetic background, because they did not originate from the same nematode population and they were not selected on the same resistant plants in laboratory and in fields, even if the R-gene in these plants was the same. The natural Me3-virulent isolates selected on resistant peppers seemed to reproduce better on susceptible and resistant Me3-peppers than laboratory-selected isolates. This observation may be correlated to the way these isolates acquired their virulence, and suggests that the genetic changes induced under managed pressure in artificial conditions are different from those occurring in natural Me3 resistance-breaking biotypes, as previously reported for laboratory-selected or natural isolates virulent against the tomato Mi-1 R-gene (Castagnone-Sereno et al. 1994a; Roberts 1995). It may also result from the fact that laboratory-selected Me3-virulent isolates are very recent and have not yet been submitted to alternative hosts, conversely to the case with natural virulent isolates that may be older and whose genetic background may have co-evolved with the virulence, thus restoring the nematode fitness.

Comparing the RP of the avirulent and laboratory-selected virulent nematodes revealed that both Me3- (or Me7-) and Mi-1-virulent isolates showed significantly lower RPs on susceptible plants compared to the avirulent isolates, mainly due to a decrease in female fertility. This result indicates that some fitness cost reduced the nematode reproduction on the susceptible plants because of unnecessary virulence. This fitness cost may be associated to the virulence trait (pleiotropic effect), but may also result in part from the reduction of genetic variability operated by the selection pressure of the R-genes. Experimental evidence of such virulence fitness costs penalizing the aggressiveness of virulent pathogens on susceptible plants has been frequently reported for bacteria, fungi and viruses (Vera Cruz et al. 2000; Parlevliet 2002; Desbiez et al. 2003; Ayme et al. 2007; Janzac et al. 2009). However, similar data on RKNs remain scarce (Petrillo and Roberts 2005; Castagnone-Sereno et al. 2007). The present study reinforces these previous observations and suggests that such a fitness cost of virulent lines on susceptible crops is a general trend in plant pathogens, including nematodes. In our study, the virulence costs were estimated at 33 to 56% on susceptible tomatoes and at 54 to 71% on susceptible peppers. Thus, Me3 (or Me7) seemed to exert a higher selection pressure than Mi-1 on RKN populations, resulting in an increased fitness cost of virulence. Moreover, RP of avirulent isolates was higher on susceptible tomatoes than on susceptible peppers, suggesting that the so-called ‘susceptible’ peppers had some undetermined and partial resistance factors, conferring a quantitative resistance to some Me3- (or Me7-) virulent RKN isolates that are not present in susceptible tomatoes. In that respect, it should be noted that resistance to some isolates of the RKN species M. chitwoodi was previously observed in Doux Long des Landes (Berthou et al. 2003; Djian-Caporalino et al. 2007). Further investigations are currently underway in the laboratory to confirm this hypothesis, which may have major practical implications in terms of durability of the resistance (Palloix et al. 2009).

Host resistance is considered as an important component of integrated management of RKNs. Because few R-genes acting against these pests are currently available, it is an urgent need to protect them and promote their durability. Since resistance deployment is influenced by the variation in (a)virulence and host range, two primary attributes of host resistance for nematode resistance breeding and management are relevant: i) the value of resistance in crop self-protection, based on the level of resistance to injury caused by initial infection, and ii) the rotational value of resistance in cropping systems for protecting subsequent crops, based on the ability to decrease the nematode population densities in soil by restricting nematode reproduction (Roberts 1995). In pepper, the Me1 R-gene confers a high level of resistance without affecting the plant growth, is still active at high temperature conversely to the tomato Mi-1 resistance gene, and is not overcome by both selected and natural virulent isolates overcoming Mi-1 in tomato nor Me3 nor Me7 in pepper. This R-gene therefore appears to be suitable for either combination with Me3 in pepper cultivars (‘pyramiding’) or alternating with other R-genes (Me3 or Me7, Mi-1) in crop rotations to strengthen and increase the durability of resistance. Experiments are now necessary to validate the obtained results under field agronomic conditions comparing: i) the alternation of single R-genes in rotation; ii) the mixture of genotypes bearing single R-genes sown in the same plot; and iii) the pyramiding of two R-genes in one genotype. Results will allow the identification of conditions lowering the emergence of virulent biotypes of RKN in the field, and the time required for the reduction of parasites under their damage threshold using the R-plants as RKN ‘traps’. This approach will help to promote the durability of resistance against RKN, and making breeders and farmers (resistance users) sensitive to the ‘directions for use’ necessary to maintain their durability.