Institute of Applied Genetics, Freie Universitaet Berlin, A.-Thaer-Weg 6,
14195 Berlin, Germany, e-mail: email@example.com
Blackleg caused by Leptosphaeria maculans (Phoma lingam) is one of the most important global diseases of oilseed rape. To enlarge the narrow genetic basis of resistance in Brassica napus different crucifers were used for sexual interspecific and intergeneric blackleg resistance transfer into winter and spring cultivars of the crop. Current data for dihaploid B. napus-B. juncea lines and several backcross progenies with resistance from Sinapis arvensis and Coincya monensis, respectively, are shown.
Comparative phytopathological studies including two aggressive (Tox+) isolates from Germany (W4) and Australia (M1), respectively, and the three hybrid groups were performed. First results indicate that M1, although able to damage severely some plants of the B. juncea population tested, causes less lesions on the resistent B. napus-B. juncea hybrids than W4. Special attention is focused on the mode of inheritance and the question of correlation (in some B. napus-S. arvensis lines) or non-correlation (B. napus-B. juncea lines) of cotyledon and adult plant resistance. The influence of different inoculation methods is discussed as well as environmental effects. Stability of resistance is studied by cytological analysis.
In the Coincya monensis derived lines, even on the BC3 level, wide sterility due to postzygotic incompatibility requires continual large-scale use of embryo rescue techniques.
First attempts to localise the introgressions by GISH and DNA markers are presented.
Keywords Leptosphaeria maculans, interspecific hybrids, Brassica juncea, Sinapis arvensis, Coincya monensis, genomic in situ hybridisation (GISH)
Leptosphaeria maculans (Desm.) Ces. et De Not. [anamorph Phoma lingam (Tode ex Fr.) Desm.], the causal agent of blackleg, induces worldwide severe damages on basal parts (stem canker) of oilseed rape (Brassica napus L., genome AACC, 2n=38) and other susceptible plants of the Brassicaceae family. Moreover it can also provoke lesions and necroses on leaves, pods and seeds.
The narrow genetic basis of recent rapeseed resistance in Europe mainly originates from the French cultivar „Jet Neuf“, which shows only a partial, polygenically controlled adult plant resistance. In contrast to this, like all Brassica species containing the B genome, B. juncea (L.) Czern. (genome AABB, 2n=36) possesses an absolute and stable resistance to most of the agressive isolates (Tox+) of the pathogen studied so far, including W4 from Germany. It is mono- or oligogenically controlled and efficient already in the seedling stage (Roy 1978, Rimmer and van den Berg 1992). Several attempts of interspecific resistance transfer have been reported (Roy 1978, Sacristán and Gerdemann 1986, Sjödin and Glimelius 1989, Chèvre et al. 1996, Struss et al. 1996). Blackleg resistance genes have been mapped in B genome chromosomes (Chèvre et al. 1996 and 1997) and B. juncea introgressions in the rapeseed genome have been localised (Plieske et al. 1998).
Some Australian Tox+ isolates of the fungus have been recently shown to overcome the resistance of B genome Brassica species. Especially Brassica juncea is severely damaged by such isolates like M1 (Howlett, University of Melbourne, personal communication). For that reason interspecific and intergeneric resistance transfer from wild crucifers, such as Sinapis arvensis L. (2n=18) and Coincya monensis (L.) Greuter & Burdet (2n=24), becomes an important alternative.
In this study dihaploid (DH) B. napus-B. juncea lines as well as backcross progeny from hybrids B. napus x S. arvensis and B. napus x C. monensis, respectively, were characterised according to chromosome numbers and resistance behavior to two different aggressive isolates, W4 and M1. First data about genomic in situ hybridisation (GISH) and RAPD analysis are reported.
Seventeen DH lines derived from microspores from selfed plants of the second and third backcross generation (BC2 and BC3) from hybrids B. napus x B. juncea were examined in previous studies (Winter and Sacristán 1998, Winter et al. 1998a and b). Data for sublines derived from them by twice or three times selfing are presented.
BC3, BC3S and BC4 plants of B. napus cv. „Madora“ (winter oilseed rape) x Sinapis arvensis (backcrossed with winter oilseed rape cultivar „Ceres“) were examined.
One BC2 plant with high adult plant resistance derived from the hybrid B. napus cv. „Loras“ (spring oilseed rape) x Coincya monensis, BC3 individuals and a progeny from open pollinations of the BC2 plant, all obtained through embryo rescue, have been studied.
For cytological examinations, resistance tests, GISH and PCR analysis plants were grown in the greenhouse or in a growth chamber (Winter and Sacristán 1998). Chromosome numbers of selected plants were determined in root tips or styles from young flower buds (Wu et al. 1997). A freezed pycnidiospore suspension of aggressive isolates W4 and M1 of L. maculans was inoculated on plates with modified V8-media. Fungal culture and isolation of spores were made as described by Sacristán (1982). The standard spore concentration in the resistance tests was 107 spores/ml (exception field tests). The level of resistance to the pathogen in all three hybrid groups was studied by various inoculation methods. Cotyledon tests (not possible for most of the embryo rescue derived B. napus-C. monensis hybrids), adult tests on cotyledon inoculated plants, hypocotyl tests (Sacristán 1982, Winter and Sacristán 1998) and adult tests with double inoculation (combined hypocotyl and cotyledon inoculation; Winter et al. 1998a and b) were performed. For selected lines resistance to W4 was also studied in field tests by spraying spores (Winter and Sacristán 1998) or by using infected plant debris. DNA was extracted from fresh leaves modified according to Edwards et al. (1991; for RAPD-PCR) and Rogers and Bendich (1985; CTAB method, for GISH and RAPD-PCR). GISH analysis was performed as described by Snowdon et al. (1997), with slight modifications. Especially for the B. napus-C. monensis hybrids styles from young flower buds were used for in situ hybridisation experiments instead of root tips. The procedure of RAPD-PCR follows the method of Williams et al. (1990).
Preliminary results concerning resistance tests, chromosome numbers, isozyme patterns, presence of erucic acid and fluorescence microscopic analysis of control pollinations to exclude prezygotic incompatibility have been presented elsewhere (Winter and Sacristán 1998, Winter et al. 1998a and b).
Among the B. napus-B. juncea hybrids the DH genotypes 7/10, 8/1 and 8/10 revealed an increased level of resistance not reaching the B. juncea-like resistance (Roy 1978). Resistance on the cotyledon level could not be detected, but the lines 8/1 and 8/10 are usually characterised by smaller lesions. DH sublines derived from the same resistant DH genotype behaved similar but not identical. Included in various resistance tests BC3S DH S2 7/10-6, BC3S DH S3 8/1-7 and BC3S DH S3 8/10-28 (all 2n=38) were selected because of the best expression and inheritance of the resistance phenotype. With these plants and the susceptible BC2S DH S3 4/9-1-32 (2n=38) crosses for studying the inheritance of resistance were made. In an adult test with double inoculation M1 induced lighter symptoms than W4, while susceptible genotypes, among them most plants of B. juncea, were severely damaged. The resistance level of this group seems to be similar to that of the B. napus-B. juncea lines described by Plieske et al. (1998).
A number of B. napus-S. arvensis BC3 plants showed a high seedling (cotyledon) and adult plant resistance to L. maculans. Most of them had a chromosome number near to that of B. napus (2n=38-43). GISH analyses of higly resistant BC3S plants confirmed the hybrid character by identifying addition lines. For example plant BC3S 13.6-11 contains one acrocentric chromosome from S. arvensis (Fig. 1d) while BC3S 16.3-1 has two additional (one acrocentric and one metacentric) chromosomes. Data on segregation of BC3S derived from resistant BC3 plants suggest that resistance (Fig. 1a and b) and susceptibility (Fig. 1a and c) in this progeny is correlated with the presence and absence, respectively, of „resistance chromosomes or introgressions“. For other hybrid plants cotyledon and adult plant resistance was not correlated, confirming findings for the B. napus-B. juncea group and results from literature (Cargeeg and Thurling 1979, Sacristán 1982, Pang and Halloran 1996, Wiechel et al. 1998). Until now in this group differences in plant reaction after inoculation with isolate W4 or M1 were not obtained. The large amount of resistant genotypes available makes it probable to identify resistant plants with 2n=38 and to detect the donor chromatin (translocation or substitution) carrying resistance by GISH.
One BC2 genotype (16/1) from the cross B. napus x C. monensis showed a high level of adult plant resistance. Plant 16/1 (2n=53) was highly sterile, but it did not show prezygotic incompatibility mechanisms (Winter et al. 1998b). It contains twelve C. monensis chromosomes as revealed by GISH. From more than 1000 attempts of embryo rescue only two vital BC3 plants were obtained. One was highly susceptible, the other one showed moderate resistance and two additional chromo-somes from the resistant parent with a total chromosome number of about 40. Some plants originated from open pollinations of plant 16/1 showed adult plant resistance. In one case cotyledon resistance, which was not to study for most of the plants because of the in vitro derived individuals, was also detected. For each of the two hybrids derived from open pollinations (probably BC2S) involved in GISH analysis four additional C. monensis chromosomes (Fig. 1e) were found. Several resistant and susceptible genotypes could be distinguished by RAPD polymorphisms.
Current results confirm suppositions previously made that deviating results in adult plant tests according to different inoculation methods indicate the possibility of resistance mechanisms in the lamina of the leaves (cotyledons) or petioles (Hammond and Lewis 1987, Pang and Halloran 1996). Symptom expression varies according to environmental conditions, the test locations (field, greenhouse, growth chamber) and the way of inoculation. We suggest adult tests with double inoculation as the method of choice for accurate resistance evaluation. It is the severest attack to the plant (more or less additive effect in comparison with adult tests on cotyledon inoculated plants and hypocotyl tests), it widely prevents escapes and levels differences in the reaction of one genotype although it is possible to overlook specific resistances with this test.
Fig. 1: Resistance behavior to L. maculans and results of genomic in situ hybridisation for selected hybrids. (a) Coty-ledon resistance (left plant) and susceptibility (right plant) in B. napus-S. arvensis line BC3S 13.6. (b) Resistant adult plants of BC3S 13.6, right individual is BC3S 13.6-11. (c) Susceptible adult plants of BC3S 13.6. (d) Monosomic addition chromosome in root tip metaphases of resistant B. napus-S. arvensis hybrid plant BC3S 13.6-11 detected by GISH analysis. (e) GISH in styles reveals four addition chromosomes of C. monensis in a resistant B. napus-C. monensis hybrid derived from open pollinated resistant BC2 plant 16/1
The interspecific and intergeneric hybrids of these three groups presented are included in further studies, which are focused on the identification of additional and substitutional chromosomes and the localisation of introgressions conferring resistance to L. maculans using GISH and DNA markers. The stability of the lines will be observed by classical cytology including meiosis analysis.
We thank Dr. Ulrike Bellin, Ernst-August University Goettingen, Germany, and Dr. Rod Snowdon, Justus-Liebig University Giessen, Germany, for their valuable and kind help in learning the GISH technique and for useful discussions. The authors express the hope that these useful cooperations will lead to further common success in future. This work was financed by a grant from the Deutsche Forschungsgemeinschaft (DFG: Sa 488/8-1).
1. Cargeeg, L.A. and N. Thurling 1979: Seedling and adult plant resistance to blackleg [Leptosphaeria maculans (Desm.) Ces. et de Not.] in spring rape (Brassica napus L.). Australian Journal of Agricultural Research 30, 37-46.
2. Chèvre, A.M., F. Eber, P. This, P. Barret, , X. Tanguy, H. Brun, M. Delseny and M. Renard 1996: Characterization of Brassica nigra chromosomes and of blackleg resistance in B.napus-B.nigra addition lines. Plant Breeding 115, 113-118.
3. Chèvre, A.M., P. Barret, F. Eber, P. Dupuy, H. Brun, X. Tanguy and M.Renard 1997: Selection of stable Brassica napus-B.juncea recombinant lines resistant to blackleg (Leptosphaeria maculans). 1: Identification of molecular markers, chromosomal and genomic origin of the introgression. Theoretical and Applied Genetics 95/ 7, 1104-1111.
4. Edwards, K., C. Johnstone and C. Thompson 1991: A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research 19 (6), 1349.
5. Hammond, K.E. and B.G. Lewis 1987: Variation in stem infections caused by aggressive and non-aggressive isolates of Leptosphaeria maculans on Brassica napus var. oleifera. Plant Pathology 36, 53-65.
6. Pang, E.C.K. and G.M. Halloran 1996: The genetics of blackleg [Leptosphaeria maculans (Desm.) Ces. et De Not.] resistance in rapeseed (Brassica napus L.). II. Seedling and adult-plant resistance as quantitative traits. Theoretical and Applied Genetics 93, 941-949.
7. Plieske, J., D. Struss and G. Röbbelen 1998: Inheritance of resistance derived from the B-genome of Brassica against Phoma lingam in rapeseed and the development of molecular markers. Theoretical and Applied Genetics 97, 929-936.
8. Rimmer, S.R. and C.G.J. van den Berg 1992: Resistance of oilseed Brassica spp. to blackleg caused by Leptosphaeria maculans. Canadian Journal of Plant Pathology 14, 56-66.
9. Rogers, S.O. and A.J. Bendich 1985. Detection of small amounts of human DNA in human-rodent hybrids. Journal of Cell Science 38, 391-403.
10. Roy, N.N. 1978: A study on disease variation in the populations of an interspecific cross of Brassica juncea L. x B. napus L. Euphytica 27, 145-149.
11. Sacristán, M.D. 1982: Resistance responses to Phoma lingam of plants regenerated from selected cell and embryogenic cultures of haploid Brassica napus. Theoretical and Applied Genetics 61, 193-200.
12. Sacristán, M.D. and M. Gerdemann 1986: Different behavior of Brassica juncea and B. carinata as sources of Phoma lingam resistance in experiments of interspecific transfer to B. napus. Plant Breeding 97, 304-314.
13. Sjödin, C. and K. Glimelius 1989: Transfer of resistance against Phoma lingam to Brassica napus by asymmetric somatic hybridization combined with toxin selection. Theoretical and Applied Genetics 78, 513-520.
14. Snowdon, R.J., W. Köhler, W. Friedt and A. Köhler 1997: Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids. Theoretical and Applied Genetics 95, 1320-1324.
15. Struss, D., C.F. Quiros, J. Plieske and G. Röbbelen 1996: Construction of Brassica B genome synteny groups based on chromosomes extracted from three different sources by phenotypic, isozyme and molecular markers. Theoretical and Applied Genetics 93, 1026-1032.
16. Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and S.V. Tingey 1990: DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18, 6531-6535.
17. Winter, H. and M.D. Sacristán 1998: Cytological and isozyme analysis of dihaploid Brassica napus-B. juncea backcross lines and their resistance behavior against Leptosphaeria maculans. Cruciferae Newsletter 20, 29-30.
18. Winter, H., A. Diestel and M.D. Sacristán 1998a: Resistance to Leptosphaeria maculans and Alternaria brassicicola transferred into Brassica napus by interspecific and intergeneric hybridization. Abstract International Congress of Plant Pathology, August 1998, Edinburgh/Scotland.
19. Winter, H., A. Diestel, S. Gärtig and M.D. Sacristán 1998b: The use of wild crucifers for blackleg resistance transfer into Brassica napus. Abstract 11th International Crucifer Genetics Workshop, October 1998, Montreal/ Canada.
20. Wiechel, T.J., G.J. Ash, N. Wratten and C.E. May 1998: The Leptosphaeria maculans: Brassica napus interaction in Australia. Abstract International Congress of Plant Pathology, August 1998, Edinburgh/Scotland.
21. Wu, J.G., Z. Li, Y. Liu, H.L. Liu and T.D. Fu 1997: Cytogenetics and morphology of the pentaploid hybrid between Brassica napus and Orychophragmus violaceus and its progeny. Plant Breeding 116, 251-257.