Resistance to heterodera carotae and methods for use

Abstract

The present application relates to the use of Daucus carota plants which do not belong to the Daucus carota subsp. sativus subspecies of cultured carrots to disinfect a culture medium infested with Heterodera carotae nematodes, using the nematicidal power of the plants. The present application also relates to Daucus carota plants which are resistant to the Heterodera carotae nematode, and to methods for culturing such plants which make it possible to reduce the population of Heterodera carotae nematodes present in a culture medium.

Claims

1. A method for reducing the population of Heterodera carotae nematodes in a cultivation medium infested with such nematodes, comprising cultivating Daucus carota plants which do not belong to the sub-species of cultivated Daucus carota subsp. sativus carrots in such medium so that said plants have a nematicidal effect on said nematodes, wherein said plants are grown from seeds deposited under accession number NCIMB 42351.

2. The method according to claim 1, wherein said reduction in the population of nematodes involves a reduction in the number of cysts and/or J2 juveniles.

3. The method according to claim 1, wherein said cultivating is for at least 4 months.

4. The method according to claim 1, wherein the infested cultivation medium is earth or sand in an open field.

5. The method according to claim 1, wherein the level of infestation of the cultivation medium is reduced to less than one J2 female Heterodera carotae larva per gram of medium.

6. The method according to claim 1, wherein the population of Heterodera carotae nematodes in the cultivation medium is reduced by at least 40%.

7. The method according to claim 1, wherein the number of cysts or female nematodes in the cultivation medium is reduced by at least 50%.

8. The method according to claim 1, wherein said cultivating is carried out at a density of 0.8 to 1.4 million seeds per hectare.

9. A population of Daucus carota plants obtained from seeds deposited under the accession number NCIMB 42351, said plants having a nematicidal effect on a population of Heterodera carotae nematodes.

10. The population of carrot plants according to claim 9, wherein the nematicidal effect involves (a) a reduction in the number of J2 juveniles, (b) a reduction in the number of cysts, (c) a masculinization of more than 50% of the larvae of the Heterodera carotae nematode, or (d) combinations of (a), (b) and (c), after a cultivation period is of at least 3 months.

11. The method of claim 3 wherein said cultivation period is at least 5 months.

12. The method of claim 3 wherein said cultivation period is at least 6 months.

13. The method of claim 6, wherein the population of Heterodera carotae nematodes in the cultivation medium is reduced by at least 50%.

14. The method of claim 6, wherein the population of Heterodera carotae nematodes in the cultivation medium is reduced by at least 60%.

15. The method of claim 6, wherein the population of Heterodera carotae nematodes in the cultivation medium is reduced by at least 75%.

Description

KEY TO FIGURES

(1) FIG. 1: The graph illustrates, for various hybrid combinations obtained from genotypes identified in Example 1, as well as for the susceptible variety Nanco, the distribution of the number of Heterodera carotae females per plant, the mean number of females per plant and the percentage of plants with less than 10 Heterodera carotae females.

(2) FIG. 2: The table indicates, for the two F2 lines obtained from the HCR10 genotype, the distribution of the number of females as an average per plant over two years of cultivation.

(3) FIGS. 3 and 4: These figures describe the mean number of females present in the roots, approximately 80 to 90 days after planting in infested cultivation media taken from 5 distinct geographic zones in crop fields infested with Heterodera carotae in Crance in the Manche department, Plouhinec in the Morbihan department, Machecoul in the Vende department, Carpentras in the Vaucluse department and Lambesc in the Bouches-du-Rhone department. Their level of contamination varied from 4.46 to 13.86 J2 larvae per gram of soil. FIG. 3 concerns plantings on 3.sup.rd May with readings on 19.sup.th July, while FIG. 4 concerns plantings on 28.sup.th May with readings on 24.sup.th August.

(4) FIG. 5: FIG. 5 illustrates the results shown in Table 2. Along the abscissa are the various genotypes A to I which were tested. Up the ordinate is the percentage of plants in a genotype belonging to each of the following 9 classes: 0; 1 to 5 females; 5 to 10 females; 10 to 20 females; 20 to 30 females; 30 to 40 females; 40 to 50 females; 50 to 100 females and 100 to 1000 females.

(5) FIG. 6: FIG. 6 illustrates the degree of multiplication of cysts in the soil of planters in which various batches of seeds, susceptible and resistant, were cultivated, after 4 months and 6 months of cultivation. The level of infestation at the time of planting is identical in each planter. % Tm indicates the level of multiplication of the cysts in the soil.

EXPERIMENTAL SECTION

Example 1: Identification of Plants Resistant to Heterodera carotae

(6) The inventors proceeded in three successive steps.

(7) Firstly, they tested more than 3700 different plants of various genetic and geographical origins using a root inoculation test. Only plants presenting no female Heterodera carotae nematode, particular no cysts, were retained for the second analysis (test for confirmation of resistance in vivo).

(8) A. Obtaining Heterodera carotae Juveniles (J2)

(9) The cysts were harvested in infected plots in lower Normandy and multiplied in greenhouses before being stored at 4 C. prior to use. The cysts were then moistened in water over 24 h before being transferred into carrot root exudate and deposited in an air-conditioned chamber at 20 C. The larvae, a maximum of one week old, were recovered and stored at 4 C. in water.

(10) B. Decontamination of Carrot Seeds and Inoculation of Heterodera carotae Larvae (Step 1)

(11) The carrot seed decontamination was carried out at 52 C. in a water bath for 18 minutes in sterile water supplemented with 10% SDS. The seeds were then rinsed in sterile water and deposited onto 1.5% gelose medium.

(12) Germinated seeds were transferred individually into a Petri dish on gelose medium supplemented with micro-elements, MS medium (Murashige T and Skoog F (1962)). The J2 stage juvenile Heterodera carotae were inoculated onto the plantlets which had a root length of 1 to 2 cm.

(13) Seven J2 were deposited on the apex of the root using a fine brush. The dishes were left in the laboratory for 48 h before being deposited in an air-conditioned chamber at 20 C. for an irradiation period of 16 h.

(14) 60 seeds were germinated for each genotype (plant) tested. The number of plants actually evaluated (inoculated) for their resistance then depended on the degree of germination obtained in the Petri dishes.

(15) A commercial hybrid Daucus carota sativus susceptible to the carrot cyst nematode was used as the susceptible control. The Nanco variety was selected as the susceptible control for this purpose.

(16) The dishes were observed between 15 and 20 days after inoculation with the second stage (J2) juveniles. All of the plants which had not allowed the development of nematodes or which had only allowed the development of males were retained. Plants with females were eliminated.

(17) C. Resistance Confirmation Tests (Steps 2 and 3)

(18) Step 2:

(19) The plants retained at the end of the in vitro test were re-potted into an earth-sand mixture before being tested a second time in vivo in a natural soil contaminated with Heterodera carotae cysts.

(20) The device was constituted by a PVC tube 20 cm long and 4.5 cm in diameter containing a sandy soil (70% sand and 30% earth). The assembly was placed on a bed of Fontainebleau sand, kept moist to keep the humidity constant. The plants were pricked out in contact with a pack of 10 cysts of Heterodera carotae obtained from the same population as during the in vitro test. The pack could advantageously be replaced by adding larvae in solution directly to the base of each plant. The test was carried out in a greenhouse under controlled temperature conditions of 20 C.5 C. and with an irradiation period of 16 hours.

(21) Two months after planting, the plants were checked one by one for their resistance to Heterodera carotae by observing the roots, which meant that the presence or absence of females could be detected, particularly the presence or absence of cysts. The plants with developed females, particularly cysts, were automatically eliminated because they were susceptible; the others were retained as they were potentially resistant.

(22) Of 3720 different plants which were tested, only 32 were retained as this step.

(23) Step 3:

(24) Thirdly, the 32 retained plants were initially multiplied in vitro using somatic embrogenesis protocols which are well known to the person skilled in the art; see, for example, Steward, F. C. et al (1958), then four clones of each genotype were re-potted into an earth-sand mixture containing cysts of Heterodera carotae in accordance with the test of step 2. Only the genotypes which did not present a female Heterodera carotae nematode, in particular no cyst, on none of the 4 cloned plants was retained: 11 genotypes were finally retained at the end of these three successive steps. They are presented in Table 1 below.

(25) TABLE-US-00001 TABLE 1 Tested genotypes with a resistance to Heterodera carotae (HCR = Heterodera carotae Resistance) Number of genotype Sub-species HCR1 Daucus carota gummifer HCR2 Daucus carota carota HCR3 Daucus carota carota HCR4 Daucus carota carota HCR5 Daucus carota gummifer HCR6 Daucus carota carota HCR7 Daucus carota gummifer HCR8 Daucus carota commutatus HCR9 Daucus carota commutatus HCR10 Daucus carota dentatus HCR11 Daucus carota gummifer

(26) The inventors retained and multiplied these 11 genotypes in vitro.

Example 2: Validation and Identification of Resistance to Heterodera carotae

(27) The 11 plants retained in the preceding step were crossed with plants of the cultivated carrot Daucus carota sativus susceptible to Heterodera carotae in order to study the heritability of the resistance.

(28) The first generation hybrid plants obtained from crossing resistant genotypes with susceptible Daucus carota sativus plants were sown into pots in earth removed from fields naturally infested with Heterodera carotae. The mean level of Heterodera carotae infestation for this earth was 16 larvae per gram of soil. Before planting, the seeds were decontaminated in water at 51 C. for 12 minutes. The pots were placed in a greenhouse, at a temperature of 20 C. 100 seeds per hybrid combination that was produced were employed.

(29) The plants were analysed 70 to 90 days after planting; the root system of each plant was carefully washed then followed with a jet of water applied to the roots in order to detach the adult and stage J4 females.

(30) It was observed that 70% of the hybrid plants with the HCR10 genotype as a parent plant had fewer than 10 females in the roots. The results are shown in FIG. 1 for a few combinations of hybrids as well as for Nanco, the susceptible variety selected as the control: the table shows, for each hybrid combination, the distribution of the number of Heterodera carotae females per plant, the mean number of Heterodera carotae females per plant and the percentage of plants with fewer than 10 Heterodera carotae females.

(31) Only plants with a reduced number of 0 to 5 female Heterodera carotae larvae were retained and self-pollinated to produce second generation plants.

(32) The second generation was tested under identical conditions to those of the tests for the first generation, sown into pots, into earth removed from fields naturally infested with Heterodera carotae. The mean level of Heterodera carotae infestation of this earth was 55 larvae per gram of soil. The control Nanco behaved as expected, with more than 97% of the tested control plants having more than 200 Heterodera carotae females. The analysis of second generation plants obtained from the HCR10 genotype showed high levels of resistance, more than 80% of the plants having fewer than 100 Heterodera carotae females. The analysis was repeated over two years, the results being substantially identical and being presented in FIG. 2. This figure illustrates the distribution of the number of females over two years for two F2 lines obtained from the HCR10 genotype.

(33) Again, only the plants with a reduced number of larvae but this time 0 to 10 Heterodera carotae females were retained and self-pollinated to produce third generation plants.

(34) Similarly, the third generation plants were sown into pots in earth removed from fields naturally infested with Heterodera carotae. Only plants with a reduced number of 0 to 10 females per plant of female Heterodera carotae larvae were retained.

(35) During the tests and generations, the inventors identified the HCR10 genotype as being a potential source of resistance to Heterodera carotae. The multiplication of nematodes was very restricted in this genotype: not only are larvae blocked in the J2 stage, but also they are highly masculinized, thereby demonstrating a nematicidal action for said plants. The third generation plants with the IICR10 genotype were again phenotypically very close to the wild type; they had white roots which were very slightly tuberized with waxy and glossy foliage and a spreading growth habit.

Example 3: Stability of Resistance Compared with Various Heterodera carotae Populations

(36) The second generation plants obtained from the HCR10 genotype (F2.HCR10) were used for this analysis: they were sown into pots into earth removed from naturally infested fields but of multiple geographical origins representing various French cultivation zones where Heterodera carotae is present. The susceptible variety Nanco was used as the susceptible control.

(37) The 5 populations of Heterodera carotae were taken from crop fields infested with Heterodera carotae from Crance in the Manche department, Plouhinec in the Morbihan department, Machecoul in the Vende department, Carpentras in the Vaucluse department and Lambesc in the Bouches-du-Rhne department. Their level of contamination varied from 4.46 to 13.86 larvae J2 per gram of soil.

(38) FIGS. 3 and 4 describe the mean number of females present in the roots. The plants were analysed approximately 80 to 90 days after planting. The plants were carefully pulled up then the root system of each plant was carefully washed. Finally, the cysts were counted using a binocular magnifying glass.

(39) This experiment shows that the second generation plants obtained from the HCR10 genotype had a resistance against the various populations of Heterodera carotae which were tested.

Example 4: Identification of Resistance Mechanism

(40) Clones of resistant plants were pricked out into a soil contaminated with Heterodera carotae cysts. The clones of a susceptible control served to monitor the dynamics of penetration of the nematode by means of an inspection, under a microscope, of the roots at different times after the start of the experiment. The inventors observed that the larvae were blocked at the J2 stage and also that they were strongly masculinized.

Example 5: Greenhouse Tests and Validation

(41) The aim of this experiment was to test the level of resistance of various plants: second (F2.HCR10), third (F3.HCR10) and fourth (F4.HCR10) generation plants described or obtained as described in Examples 1 to 3 (genotypes A, B and E, respectively), third generation plants obtained from brother-sister crosses obtained by crossing a second generation plant with the HCR10 genotype, selected for its high level of resistance, with another second generation plant with the HCR10 genotype selected for its high level of resistance (genotypes C and D, corresponding to AxA), hybrid plants obtained by crossing a second generation plant with the HCR10 genotype selected for its high level of resistance with fertile male lines of the carrot Daucus carota sativus susceptible to the nematode Heterodera carotae (genotypes F, G and H); genotype I, corresponding to the susceptible control Nanco.

(42) The plants were sown in three distinct series (with the exception of genotypes D and E which were sown in two series of the 3) in an amount of approximately 20 plants per series for each genotype, into plastic (8*8*8) pots containing about 380 g of soil which had previously been homogenized in a cement mixer. The soil was naturally infested and came from Crance, in the Manche department. The mean measurement of the level of infestation was J2 larvae per gram of soil. Watering was carried out pot by pot, from above.

(43) The test was inspected between 70 and 90 days after planting using a rapid reading method: counting all of the females (including cysts) for plants with fewer than 100 females. Counting was stopped above this value of 100 females and the value more than 100 attributed to those plants.

(44) Plants obtaining a score of fewer than 5 females were retained and pricked out into a mixture of earth and sand free from contamination.

(45) The series were sown at specific times between September and November.

(46) The results are shown in Table 2 which, for each series, summarizes the plants as a function of their level of infestation (classes of plants with 0 females, 1 to 5 females, 5 to 10 females, etc.). Thus, for genotype A, 4 plants out of 63 tested plants did not present any females, 17 plants had 1 to 5 females, etc.

(47) The genotype A corresponded to the resistant genotype (resistant second generation plant obtained from HCR10 genotype), while the genotype I corresponded to the susceptible control. For the three series, the level of control was sufficiently high for the test to be considered to be discriminating.

(48) The mean M represents the total number of cysts counted for each plant of each series, divided by the number of plants analysed per genotype.

(49) The mean distribution between the various classes is illustrated in FIG. 5 for each of the tested genotypes.

(50) TABLE-US-00002 TABLE 2 Number of cysts per plant for various genotypes. Genotypes Class A B C D E F G H I 0 4 5 4 13 5 0 1 0 0 1 to 5 17 16 8 13 13 0 0 0 0 5 to 10 11 14 4 6 6 1 0 2 0 10 to 20 13 8 9 2 8 2 0 0 0 20 to 30 8 0 5 3 4 3 2 1 1 30 to 40 2 3 1 1 1 1 4 0 1 40 to 50 1 3 2 0 2 7 3 1 0 50 to 100 3 1 4 0 1 21 10 8 2 100 to 1000 4 1 3 1 0 22 42 47 61 S 63 51 40 39 40 57 62 59 65 M 21.397 13.3529 28.275 12.538 12.075 85.421 86.371 92.4375 323.615

(51) The results were as follows, for each genotype tested:

(52) A: (F2.HCR10) resistant control genotype. The results were in agreement with those obtained in the preceding tests, with a high level of resistance (general mean over the 3 series of 21.4) and the presence of a small percentage of multiplicative plants (about 10%) at the tail of the distribution.

(53) B: (F3.HCR10, F3 derived by self-fertilization of the F2.HCR10 population) genotype of interest for the 3 series with approximately 50% of plants having fewer than 10 females. However, the presence of a small percentage of multiplicative precursors (approximately 10%) at the tail of the distribution should be noted.

(54) E: (F4.HCR10, F4 derived by self-fertilization of the F3.HCR10 population) highly resistant genotype (general mean over the two series of 12.5). Note the total absence of multiplicative plants at the tail of the distribution.

(55) From one generation to another (F2, F3 then F4), an increase in resistance and a reduction in the number of multiplicative plants was observed.

(56) C: Atypical profile with a practically equivalent percentage of plants in all classes. This type of profile tends to indicate an oligogenic character for the resistance.

(57) D: Highly resistant genotype (general mean over two series of 12.5). note the almost complete absence of multiplicative plants at the tail of the distribution.

(58) F, G and H: Identical profile to that of susceptible control I.

(59) These three genotypes (F, G, H) were obtained from a cross with a commercial elite line susceptible to Heterodera carotae; the results show that the resistance is recessive. In consequence, the self-crossing of individuals obtained from the HCR10 genotype meant that individuals could be obtained which were homozygous for the resistance characteristic in the case of a monogenic resistance. Since the resistance is more likely to be oligogenic, though, the inventors carried out crosses in order to stabilize a resistant descendancy.

Example 6: Container Tests

(60) The aim of this experiment was to test the nematicidal capability of third generation plants with the HCR10 genotype.

(61) F3 plants were planted in 5 lines of planting in containers (32*28*20 cm) comprising 18 cm of naturally infested soil (and originating from Crance). The infestation was measured at the start and at the end of the experiment after pulling, for each container. The experiment was carried out twice.

(62) TABLE-US-00003 Rep 1 Rep 1 Rep 2 Rep 2 Genotype Genotype I Genotype Genotype I Genotype susceptible B susceptible B Pop initial 10.92 18.80 10.6 22.07 (J2/g) Pop final (J2/g) 14.46 9.86 3 February Pop final (J2/g) 27.51 9.33 28 March Tm (Pf/Pi) 1.32 0.52 2.59 0.42 % +32.5 47.53 +159 56

(63) Susceptible genotype I (Nanco): in agreement with expectations, an increase in the soil population of Heterodera carotae was observed after pulling up the carrots. This increase was even higher when the cultivation period was longer (+32% and +159%), which corresponded to producing an additional generation in the case of the second harvesting date. Genotype B: In both cases, the level of the population reduced by approximately 50%. The reduction was all the more marked with a longer period. The results could have been even better if the genotype used had been more genetically homogeneous: the test of Example 5 shows that approximately 10% of the plants were multiplicative for this genotype. However, these results are extremely encouraging and are on the level of those obtained in the case of a successful trap cultivation carried out with a conventional variety. With a conventional variety however, destroying the crop too late causes the opposite effect to that desired, i.e. maintains or increases the population, as confirmed with the susceptible genotype I.

(64) With other genotypes (in particular the genotypes E or D, free from multiplicative plants), values close to those obtained in the case of chemical disinfection (80-90%) should be able to be obtained in this test.

Example 7: Validation of Nematicidal Capability

(65) An artificial greenhouse test was carried out in order to measure the level of the nematicidal capability of a carrot crop resistant to Heterodera carotae identified in the preceding examples.

(66) Protocol:

(67) This test was carried out in planters containing naturally contaminated soil from the Crance region (Normandy). The criterion measured was the level of infestation of the soil (number of cysts) at planting then at various dates after planting, which meant that a level of multiplication of the Heterodera carotae nematode could be measured during cultivation. The method used was of the complete block randomization type with 2 repeats per genotype.

(68) Plant Material Tested:

(69) Susceptible control: Nanco

(70) Resistant Plants: 2 different batches of F3.HCR10 (genotype B in Example 5): F3.HCR10a and F3.HCR10b; 2 different batches of plants obtained from F3.HCR10 by multiplication: F3.HCR10.Ma and F3.HCR10.Mb

(71) Results:

(72) The inventors observed very poor germination when this test was being carried out: 33-34% for Nanco and 0 to 14% for the 4 resistant F3 lines, including 0% for the F3.HCR10b line. This poor level of germination observed for all of the test plants was probably linked to the poor conditions during germination.

(73) The inventors observed that with Nanco, the susceptible control, the increase in the nematode population was 430% after 6 months of cultivation, despite the poor germination conditions. For the lines F3.HCR10a and F3.HCR10.Ma, a reduction in the level of infestation of the soil by approximately 20% (F3.HCR10.Ma) to approximately 40% (F3.HCR10a) was observed after 6 months of cultivation despite the poor germination conditions.

(74) Conclusion:

(75) The nematicidal capability is confirmed by this test.

(76) However, given the very poor germination during this test, it was not possible to draw any accurate conclusions regarding the level of the nematicidal capability of this Heterodera carotae-resistant material.

Example 8: Creation of Batches of Seeds with the Most Resistant Genotypes

(77) Multiplication of the best F3s which were identified was carried out in large cages and in a plastic tunnel (fertilization carried out randomly by insects), with a view to depositing seeds, in order to obtain: a synthetic population created by intercrossing the most resistant F3 plants; a population F4.HCR10.M, corresponding to a multiplication of the F4.HCR10 line described in Example 5 for which the nematicidal capability had been ascertained.

(78) The seeds deposited on 20 Jan. 2015 at the NCIMB, with accession number NCIMB 42351 and reference Daucus carota DCHR1, corresponded to a multiplication of the F4.HCR10.M population, baptised F4.HCR10.MM.

Example 9: New Validation of Nematicidal Capability in Planters

(79) This test was carried out in order to test the nematicidal effect of a carrot population on Heterodera carotae. It was carried out in planters, each genotype being tested in a planter over two rows. In order to guarantee the robustness of the test, it was carried out twice.

(80) The nematicidal effect was monitored by counting the number of cysts in the earth infected by means of sampling before planting, half way through the test 4 months after planting, and at the end of the test 6 months after planting, which meant that the degree of multiplication of the nematode Heterodera carotae during cultivation could be measured.

(81) Inoculum:

(82) Natural inoculum: earth from a field infested with Heterodera carotae nematodes. This inoculum was homogenized well in a cement mixer.

(83) Preparation of Planters:

(84) 29 litre apertured planters, H: 20 cm, L: 98 cm, W: 25 cm (1 planter per genotype and per repeat) were filled with strictly the same quantity of earth.

(85) Plant Material Tested:

(86) Batch 1. Nanco: susceptible control

(87) Batch 2. Resistant F4 line (F4.HCR10.M, see Example 8)

(88) Batch 3. A synthetic population as described in Example 8

(89) Batch 4. Pseudo resistant F3 line

(90) Batch 5. Pseudo resistant F3 line

(91) Planting: the seeds were planted leaving a 5 cm space between the seeds and the edge in order to avoid a border effect. Planting was carried out in two parallel 70 cm rows with 60 seeds per row distributed homogeneously over the entire length.

(92) Samples: during the test, 3 samples with 3 specimens in each planter were taken. Specimen number 1 corresponded to sampling before planting, No. 2 corresponded to sampling 4 months after planting and No. 3 corresponded to sampling 6 months after planting.

(93) Results: The inventors observed good germination for all of the batches tested except for batch 4 (25%), but very poor development of the plants for the susceptible control Nanco (very significant growth retardation). Because of the lateness of the growth of the susceptible control, the results for multiplication of nematodes do not agree with the results obtained in preceding years in this type of test. In fact, the retarded growth results in the introduction of a bias into the results: while in all of the preceding tests a multiplication of the nematode in the soil was observed (from 159% to 430% for the susceptible controls), here a reduction of 52% in the nematodes was observed. The poor development of the roots probably had an impact on the cycle of the nematode. 3 of the genotypes tested had a reduction in the level of infestation in the soil of 62% to 87% after 6 months of cultivation; for one of the genotypes (batch 5, pseudo resistant F3), there was an increase in the nematode population of 60% after 6 months of cultivation, which was unexpected.

(94) The results are illustrated in FIG. 6. The tables below record the analysis of the variance and the results of a Newman-Keuls test with a 5% threshold.

(95) Variance Analysis:

(96) TABLE-US-00004 S.C.E DDL C.M. TEST F PROBA VarTOTAL 28367.288735 9 3151.920971 Var. FACTOR 1 26750.325501 4 6687.581375 25.584440 0.006094 Var. BLOCKS 571.393143 1 571.393143 2.185958 0.212694 VAR. RESIDUAL 1 1045.570091 4 261.392523

(97) Newman-Keuls Test, 5% Threshold

(98) TABLE-US-00005 Modality Mean Homogeneous groups Batch 5 59.842520 A Batch 1: NANCO 51.968504 B Batch 4 62.204724 B Batch 3 64.566929 B Batch 2 86.614173 B

(99) Conclusions:

(100) The nematicidal capability linked to this resistance is again confirmed by the test which has been carried out.

(101) The genotype F4.HCR10.M which is resistant to Heterodera carotae, with a nematicidal capability of the order of 80% after 4 to 6 months of cultivation, means that an effect comparable to a chemical treatment can be obtained (with an efficiency of 80-90%).

(102) This genotype had already demonstrated the best nematicidal capability in the preceding test; seeds obtained by multiplication of this genotype have been deposited with the NCIMB under the number NCIMB 42351.

LIST OF REFERENCES

(103) Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15(3): 473-497. Steward, F. C., Mapes, M. O., and Smith, J. (1958). Growth and organized development of cultured cells. I, II & III. Growth and division of freely suspended cells. Am. J. Bot. 45