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Keywords:

  • Alternaria dauci;
  • Daucus carota;
  • disease resistance screening;
  • progeny testing

Abstract

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

The objective of this study was to find a technique for plant resistance screening to alternaria leaf blight (ALB), caused by the fungus Alternaria dauci, in controlled environments. Glasshouse and laboratory screening methods were compared using three cultivars and F2 genotypes segregating for ALB resistance evaluated against self-pollinated F3 field-grown plants. Plant disease was assessed through a disease index obtained from the size and number of symptoms on carrot leaves. The results indicated the value of glasshouse evaluation and the inadequacy of detached leaf and hypocotyl assays for carrot screening for ALB resistance. Spearman's rank correlation, applied to results obtained with both F2 plants and their progeny, indicated that the optimal evaluation stage for ALB resistance in carrot is 20 days after inoculation. This test was powerful enough to be used as a prescreening test in breeding programmes.


Introduction

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

Cultivated carrot (Daucus carota ssp. sativus) is grown worldwide. In 2003, c. 21 million tonnes of carrots were produced from 1 million ha (FAO, 2004). Carrot ranks in the top 10 economically important vegetables in France. Alternaria leaf blight (ALB), caused by Alternaria dauci, is recognized as one of the most common and destructive foliar diseases of carrot (Souza et al., 2001). Symptoms are characterized by necrotic spots on leaves and petioles (Champion, 1997). In the worst situation, leaves are entirely destroyed and harvesting becomes difficult. As much as 40–60% of the yield may be lost (Vintal et al., 1999).

Rainy, hot weather and overhead irrigation favour this disease, with conidial spores, which are responsible for pathogen spread, being dispersed by air and water splash. The optimum temperature for growth and infection is 27°C, with some infections occurring at temperatures as low as 14°C and as high as 35°C (Rubatzky et al., 1999).

Development of ALB can be suppressed by the use of fungicides. This approach to disease management is, however, costly and not always effective. Moreover, it may result in pesticide residues accumulating in roots and soil (Pryor et al., 2002; Gaube et al., 2004). It is desirable to control this disease using resistant cultivars (Rotem, 1998). Carrot cultivars partially resistant to ALB have been developed and provide various levels of disease control, but none of these cultivars is completely resistant (Simon & Strandberg, 1998). Thus breeders are still searching for cultivars showing higher levels of resistance to ALB. One of the main difficulties of such breeding is the identification of resistant genotypes. Field screening is the routine procedure (Boiteux et al., 1993; Vintal et al., 1999; Santos et al., 2000; Ben-Noon et al., 2001; Pryor et al., 2002), but is time-consuming, expensive and highly affected by uncontrollable environmental conditions and the simultaneous presence of other pathogens. Moreover, field conditions unfavourable to A. dauci development often result in substantial material waste.

In the present study, several techniques were tested in order to improve the cost efficiency of traditional breeding. The aims were: (i) to find a rapid and reliable technique for screening carrot plants showing resistance to ALB; and (ii) to validate the technique by evaluation of individual F2 plants and their F3 progeny.

Materials and methods

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

Plant and fungal material

Three cultivars showing different levels of resistance to A. dauci were used: Presto, a highly susceptible cultivar (Ben-Noon et al., 2001; Villeneuve et al., 2001); and Bolero and B5280, which are partially resistant to ALB (Corbaz & Perko, 1995; Simon & Strandberg, 1998). Presto and Bolero are two hybrids widely grown in France, whereas the inbred line B5280 was evaluated in the USA (Simon & Strandberg, 1998). For whole-plant assays, F2 plants were used. These were obtained from a cross between a susceptible line and a resistant line related to Bolero. These F2 progeny were previously shown to segregate for resistance to A. dauci.

A single-conidial isolate of A. dauci taken from naturally infected carrots grown in Cestas, Gironde, France, and reported as aggressive by Gaube et al. (2004), was used for all experiments.

Inoculum preparation

Alternaria dauci was grown on V8 agar medium (200 mL V8 vegetable juice, 3 g CaCO3, and 15 g agar L−1) autoclaved for 20 min at 105°C and dispensed into 9-cm Petri dishes. Cultures were incubated in the dark at 20 ± 2°C for 10 days. Petri dishes were then exposed to 12 h near-ultraviolet light (36 W) alternating with 12 h darkness at 20 ± 2°C for 10–15 days to induce sporulation.

A suspension of conidia was prepared by flooding the culture with sterile water and gently dislodging conidia with a glass plate. Mycelial and conidial suspensions were filtered through two layers of cheesecloth. Spore density was counted using a haemocytometer and adjusted to the required concentration.

Disease screening

Detached leaf assay

This assay was carried out using cvs Presto, Bolero and B5280. Tests were performed on leaves of glasshouse-grown young plants (two or three true leaves). For each cultivar, six replications were used. After excision, leaves were placed in plastic containers on two layers of filter paper soaked with sterile distilled water. The leaves rested on a plastic grid to avoid direct contact with the humid paper. Leaves were sprayed with 5 mL conidial suspension per container at 7 × 103 conidia mL−1. The containers were covered with a glass plate and relative humidity was kept at 90–100%. This was followed by incubation in a greenhouse at 20 ± 2°C, with protection from direct light to prevent the leaves from withering. Scores were taken 8 days after incubation.

Hypocotyl assays

This assay was performed with cvs Presto and Bolero only. Seeds were surface-sterilized in sodium hypochlorite solution (30 g L−1) for 10 min and then rinsed three times in sterile deionized water. Four seeds were placed in a 9-cm-diameter Petri dish containing a single layer of filter paper moistened with 3 mL sterile water. Petri dishes were sealed with Parafilm and incubated at 24°C with a 14-h photoperiod. Ten-day-old seedlings were tested twice. In the first test, 4- to 6-day-old mycelial explants were placed on hypocotyls. In the second test, hypocotyls were inoculated with one drop of a conidial suspension at a concentration of 1 × 103 conidia mL−1. Both experiments were repeated three times. Disease development was assessed 7 and 14 days after inoculation.

Glasshouse trials on whole plants

Experiment 1

Seeds of cvs Presto, Bolero and B5280 were sown in a peat mixture (35% black peat, 25% white peat, 10% clay, 30% sand) in trays. Seedlings were then transplanted into 8-cm3 pots containing this peat mixture (three seedlings per pot). Pots were maintained at 20 ± 2°C in a glasshouse. Inoculation was performed when plants showed three true leaves. A conidial suspension of 1·2 × 104 conidia mL−1 was sprayed to runoff with an atomizer over both sides of leaves. Two replicates were used. Plants were incubated under plastic covers with day/night temperatures of 25/18°C and a 12-h photoperiod. Pots and plastic covers were sprayed regularly with sterile water to provide a water source for the plants and to maintain high relative humidity. Disease development was assessed 8 days after inoculation.

Experiment 2

Seeds of the F2 population segregating for ALB resistance were sown in 20-cm-diameter pots (one plant per pot). Pots were placed in a glasshouse at 20 ± 2°C. After inoculation, they were covered with a small polyethylene tunnel to maintain high relative humidity. Inoculation was carried out 1 month after sowing, when all plants had developed three true leaves. Fifty plants were individually sprayed to runoff with a conidial suspension of 1·2 × 104 conidia mL−1. Disease development was assessed 7, 10, 15 and 20 days after inoculation. Soon after the final disease assessment, plants were treated with the fungicide iprodione. They were then transferred to a chamber at 3 ± 2°C for 10 weeks to initiate vernalization. Once vernalized, the plants were transferred into an insect-proof glasshouse at 20 ± 2°C to encourage flowering. Subsequently, seeds were harvested from individual self-pollinated plants to provide F3 progenies. To check for the presence of contaminating conidia of A. dauci, 40 seeds of each F3 progeny were incubated in Petri dishes on filter paper moistened with water. After 7 days’ incubation at 20 ± 2°C, the level of infestation in each seed was determined microscopically.

The following spring, F3 progenies were scored for ALB resistance using standard conditions. Small field plots 1 m long were arranged in two replicates in a randomized block design and sown with 80 seeds m−1; the distance between rows was c. 30 cm. At the three-true-leaves stage, plants were sprayed to runoff with the same isolate used to inoculate F2 plants. Plants were inoculated twice at 2-week intervals with a suspension of 5 × 103 conidia mL−1. To maintain continuous wetness, plants were watered using a robotic system 1 day prior to inoculation and for another 72 h. Leaf symptoms were recorded 35 and 75 days after the second inoculation.

Disease assessment

Disease severity was rated as previously described (Gaube et al., 2004) by visual assessment based on a 0–9 scale (Table 1). A disease index (DI), obtained from the average of each leaf value, was used to assess plant resistance. For the field trial, one single average score was given to the 80 plants of each replicate. Differences between cultivars were tested using the Newman–Keuls method. The F2 and F3 plants were compared using Spearman's rank correlation coefficients.

Table 1.  Disease rating scale for alternaria leaf blight of carrot
ScaleSymptoms
0No visible disease damage
1Few scattered lesions, < 5% leaf area damaged
3Few scattered lesions on petioles, lesions on c. 30% of foliage, 5–20% leaf area damaged
5Lesions on c. 60% of foliage, 20–40% leaf area damaged
7Lesions on c. 90% of foliage, 40–60% leaf area damaged, few leaves wilted
9Severe defoliation, only new leaves remaining

Results

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

Disease screening

Detached leaf assay

Comparison of the level of resistance of cvs Presto, Bolero and B5280 to A. dauci showed no significant differences among cultivars. The DI scores did not exceed 3·5 (on cv. Presto; Table 2).

Table 2.  Resistance of carrot cvs Presto, Bolero and B5280 to Alternaria dauci, using a 0–9 scale disease index and three types of assay
Evaluation techniqueCultivarDisease indexa
  • a

    Numbers (for each assay) followed by the same letter are not significantly different at P = 0·05 according to the Newman-Keuls method. For disease index scale see Table 1.

Detached-leaf assayPresto3·5 A
Bolero1·9 A
B52802·4 A
Hypocotyl assays
 conidial suspensionPresto5·5 A
Bolero7·0 A
 explant of mycelial culturePresto7·5 A
Bolero8·0 A
Whole-plant assay (experiment 1)Presto6·0 A
Bolero2·0 B
B52802·5 B
Hypocotyl assays

The first symptoms were observed on hypocotyls 7 days after inoculation. On cv. Bolero, the DI scores were 7 and 8 for inoculation with conidial suspension and mycelial culture, respectively (Table 2). Irrespective of the inoculation method used, no significant differences in susceptibility were observed between cultivars, regardless of previous ratings for susceptibility or resistance.

Glasshouse trials on whole plants

Experiment 1

Cultivars Bolero and B5280 showed clear resistance to A. dauci. No significant differences in resistance were identified between both cultivars, but cv. Presto showed clear susceptibility to A. dauci (Table 2).

Experiment 2

Irrespective of cultivar, all inoculated plants showed disease symptoms on the leaves. The DI score ranged from 0 (7 days after inoculation) to 8·5 (20 days after inoculation). From a total of 50 F2 plants assessed, nine did not flower, 18 failed to produce seeds and 12 showed low levels of germination, leaving 11 producing viable seeds being shown to be free of A. dauci contamination. Spearman's rank correlation coefficients between glasshouse-grown F2 and field-grown F3 disease scores showed significant nonzero correlations (P ≤ 0·1) between the DI values of F2 and F3 plants (Table 3). The evaluation of F2 plants 20 days after inoculation was most strongly correlated to disease measurements of F3 plants. Table 4 showed that discrimination between genotypes was maximized 20 days after inoculation, with DI scores ranging from 1 to 8·5, compared with ranges of 0–2·5, 1–4 and 1–5 obtained 7, 10 and 15 days after inoculation, respectively.

Table 3.  Spearman's rank correlations between scores for alternaria leaf blight resistance of F2 carrot plants (7, 10, 15 and 20 days after inoculation) and scores of their self-fertilized F3 progeny (35 and 75 days after inoculation)
 F2 (7 days)F2 (10 days)F2 (15 days)F2 (20 days)
  1. P values < 0·1 indicate statistically significant nonzero correlation at 90% confidence level.

F3 (35 days)0·460·690·530·66
(P = 0·15)(P = 0·03)(P = 0·09)(P = 0·04)
F3 (75 days)0·620·540·610·69
(P = 0·05)(P = 0·09)(P = 0·06)(P = 0·03)
Table 4.  Disease index of F2 carrot plants 7, 10, 15 and 20 days after inoculation with Alternaria dauci under glasshouse conditions
F2 genotypeDisease index
7 days10 days15 days20 days
  1. For disease index scale see Table 1.

11(z)112·53·5
15/50(z)1133·5
5(z)0·51·533
6/41(z)223·54·5
30(z)01·52·53·5
20(z)0·5122
10/45(z)1·51·52·53·5
12(z)1·53·53·58·5
31(z)2·53·557
33(z)0111
8/43(z)2444·5

To classify levels of resistance of F3 genotypes, comparisons were made with cv. Bolero, the standard reference genotype for partial resistance to A. dauci used by breeders or producers in evaluation trials. Genotypes with F3 DI values lower than those of cv. Bolero (6) showed a maximum F2 DI of 3·5 (Table 5). Two genotypes, 12(z) and 31(z), were highly susceptible in the F2 evaluation, and the symptoms shown by their F3 progeny indicated susceptibility, with DI values of 4 and 7 at 35 and 75 days after inoculation, respectively. Depending on the genotype, a score of 3·5 in an F2 evaluation could correspond to a DI score of 5, 6 or 7 for F3 progeny 75 days after inoculation.

Table 5.  Disease index of 11 glasshouse-grown F2 plants (20 days after inoculation) and their field-grown self-fertilized F3 progeny 35 and 75 days after inoculation with Alternaria dauci
F2 genotypeF2 (20 days)F3 (35 days)F3 (75 days)
  1. –, No evaluation in F2 test. For disease index scale see Table 1.

11(z)3·537
15/50(z)3·526
5(z)335
6/41(z)4·537
30(z)3·536
20(z)226
10/45(z)3·535
12(z)8·547
31(z)747
33(z)136
8/43(z)4·538
Bolero2·56

Discussion

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

The main goal of the present study was to develop a miniaturized screening method for carrots for resistance to ALB, which could be reliable when used under controlled environments. The aim was to provide optimal conditions for disease development and to establish a screening programme that could be performed at any time during the year at a location distant from the main production area.

Breeding for disease resistance requires efficient, low-cost and rapid screening techniques (Foolad et al., 2000). For ALB in carrot, field screening is a routine procedure. It is, however, time-consuming and can be done only once a year. Dugdale et al. (2000) proposed a disease bioassay based on detached-leaf inoculation, but correlation between detached-leaf evaluation and field evaluation was not calculated.

Among the three screening procedures described here, the highest discrimination between carrot genotypes was obtained using whole plants grown in a glasshouse. This test correctly classified cultivars with known susceptibility to A. dauci. The two other tests (detached-leaf and hypocotyl tests) failed to show such discrimination. In detached-leaf assays, the DI scores of resistant and susceptible cultivars were not significantly different, indicating that cvs Bolero, B5280 and Presto had similar levels of resistance. However, these three cultivars show well established and different levels of resistance to ALB. Similarly, the hypocotyl test failed to screen effectively for ALB resistance. Indeed, there was no correlation between hypocotyl evaluation and the known susceptibility of the cultivars. Therefore it appears that neither hypocotyl assays nor detached-leaf assays can be used with confidence to evaluate carrot susceptibility/resistance to ALB.

In contrast, in the whole-plant assay (experiment 1) a highly significant difference in DI scores between cultivars was recorded, indicating that the experimental procedure discriminated between genotypes. The ranking of the three cultivars, Bolero, B5280 and Presto, agreed well with previous studies on their resistance to ALB (Corbaz & Perko, 1995; Simon & Strandberg, 1998; Villeneuve et al., 2001). Therefore the whole-plant assay offers a possible method for assessing carrot resistance to A. dauci, although the results are based on work with only three cultivars and a single fungal isolate.

The possibility of using this test in individual plant evaluation was examined using a larger number of plants (experiment 2; F2 plants segregating for A. dauci resistance) and comparing results with those of a traditional field test on the self-fertilized F3 progenies. Statistical correlation between F2 and F3 tests confirmed the potential of the glasshouse test, particularly when F2 plants were assessed 20 days after inoculation. The highest level of discrimination, based on disease severity (1–8·5), was also observed at this time of evaluation. Therefore, in the selection process of F2 plants, the optimal stage of observation for susceptibility or resistance was 20 days after inoculation (Table 4). Use of later evaluation stages (e.g. 30–35 days after inoculation) may lead to underevaluation of plant susceptibility. Indeed, after a few weeks some of the most severely attacked leaves may have rotted or abscised from the diseased plants and may or may not be replaced by healthy leaves (data not shown). Genotypes showing high levels of susceptibility in the glasshouse test [e.g. 6/41(z), 12(z), 31(z), 8/43(z)] were also recorded as highly susceptible in the F3 field test. This concordance between F2 and F3 results confirmed that the variability in disease susceptibility of F2 progeny in the pot evaluations was caused more by differences between genotypes than by experimental errors. Therefore the former test may be used by breeders as a miniaturized procedure to screen potentially interesting material. In this situation, a DI ≤ 3·5 for F2 plants 20 days after inoculation could be proposed as the threshold with which to select potentially interesting genotypes (with a resistance level similar to or higher than the resistance level of cv. Bolero). According to the present results, such a threshold would carry only a minor risk for breeders in selecting genotypes that show limited resistance, such as genotype 11(z), as opposed to eliminating resistant genotypes. Several mother plants with similar DI values in the F2 evaluation subsequently generated F3 progeny with different scores. This means that the test is not powerful enough for use in single-plant evaluation. For example, in a research programme where phenotypic evaluation of each segregating individual plant is required, an approximate result is of no value.

The results obtained on individual plants may be used as a good indicator of resistance to A. dauci infection in subsequent progeny. As the test was performed in a controlled environment, it is not subject to environmental fluctuations (temperature, pathogen activity and inoculum) and can be performed year round. Use of this selection process can substantially reduce both the cost and duration of breeding programmes.

Acknowledgements

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

We thank J. Delettre and A. Suel for their technical assistance and D. Dumet for reviewing the English language. This research was supported by a grant from Vilmorin and Mairie d’Angers.

References

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