Generation of oilseed rape with resistance to turnip yellows luteovirus
Klaus Graichen1 and Herbert Peterka2
Federal Centre for Breeding Research on Cultivated Plants, 1Institute for Epidemiology and Resistance, Theodor-Roemer-Weg 4, D-06449 Aschersleben, 2Institute for Breeding Methods in Vegetables, Neuer Weg 22/23, D-06484 Quedlinburg
Abstract
In recent years, high levels of infection of winter oilseed rape fields by turnip yellows luteovirus (TuYV) have been detected in Germany. Resistance to TuYV was found only in a resynthesized rapeseed and in Brassica rapa ssp. pekinensis, whereas most Brassica species and all rape varieties and breeding lines tested were highly susceptible.
Experiments were carried out to transfer TuYV resistance in current winter oilseed rape cultivars and breeding lines by crossing with resistant plants of the resynthesized rapeseed. In field experiments, the progenies of the crosses of three susceptible oilseed rape cultivars with the TuYV-resistant resynthesized rapeseed were screened on virus resistance. The investigations resulted in F2, F3, F4, F5 and backcross progenies with different ratios of TuYV resistant plants. Using the DAS-ELISA and a IC-RT-PCR technique it was possible to show that some of the resistant plants were absolutely virusfree. Furthermore, the virus concentrations in the infected plants were distinctly decreased and most TuYV infected plants showed no virus symptoms.
The successful transfer of TuYV resistance to oilseed rape suggests that it will be possible in future to prevent virus induced yield losses by breeding and cultivating virus resistant cultivars.
Keywords: Virus resistance, progenies, field experiments, ELISA
Introduction
Luteoviruses infect world-wide many important crops and cause severe economic losses (Casper, 1988). In recent time, very high levels of infection of winter oilseed rape by turnip yellows luteovirus (TuYV, syn. beet western yellows luteovirus, BWYV) were detected in Germany, Great Britain, France, the Czech Republic, and the USA (Smith and Hinckes, 1985; Kerlan, 1991; Schröder, 1994; Polak and Majkowa, 1992; Thomas et al., 1993; Hardwick et al., 1994; Graichen et al., 1997). In a three-year experiment at Aschersleben, Germany, virus-free experimental plots of the oilseed rape cvs. Falcon and Zeus yielded 12% to 34% more seed than plots with 90% to 100% TuYV infection (Graichen, 1997).
The detected high infection degree of oilseed rape fields in different growing regions and yield losses caused by TuYV infections showed that measures are necessary to control virus infection of oilseed rape. The best way to prevent yield losses caused by virus infection in oilseed rape is the breeding and growing of cultivars resistant to TuYV.
In glasshouse and field experiments a total of 652 genotypes of summer and winter oilseed rape cultivars, actual breeding lines and resynthesized rape forms, were screened for resistance to a highly virulent TuYV isolate from oilseed rape (Graichen, 1994; Graichen and Peterka, 1995). All of the rapeseed genotypes were susceptible to TuYV. The resynthesized rapeseed R 54 from the University of Göttingen (Gland, 1980), from which single plants were selected resistant to TuYV, was an exception. The resynthesized rapeseed R 54 was used for cross experiments to transfer the TuYV resistance in oilseed rape cultivars. An effective method based on glasshouse and field experiments was developed for screening the progenies to evaluate their reaction to TuYV.
This paper reviews the results of field experiments from the 1997/98 growing season.
Materials and methods
Plant material
In 1994, plants of the susceptible cvs. Express, Falcon and Wotan were crossed with two selected virus resistant S1 plants of the resynthesized rapeseed R 54 (line R 54-15). After selfing of the F1 plants, the F3, F4 and F5 progenies were produced by selfing of the selected resistant plants of the previous generation. Several resistant F2 plants were used for crossing with the cvs. Express and Falcon to produce the first backcross generation.
Field experiments design
At the end of August 1997 in field experiments, 128 single plant progenies of the crosses of the three cvs. mentioned above, furthermore the cultivars Falcon, Express, Sollux and Wotan and the resynthesized rape line R 54-15 were sowed each in two until to five rows. The cv. Sollux which showed very high degree of susceptibility to TuYV in former experiments was used as susceptible control. At the end of September/ beginning of October, all rows of each genotype containing 25 plants were inoculated with TuYV by colonisation with about five viruliferous Myzus persicae per plant.
Virus symptoms
At the end of February, beginning of April and end of May, the virus symptoms were visually assessed as reddening and/or anthocyanous discoloration and growth reduction.
Virus detection
At first in April 1998, virus infections of 46 progenies and two cultivars were assessed by means of direct tissue print immunoassay (TP; Hsu et al., 1995). However, this method has low sensitivity and hence is only suitable for preliminary tests. Furthermore, in April and May 1998 the percentage of infected plants and the relative virus concentration (absorbance value at 405 nm) were assessed of 57 progenies by means of DAS-ELISA using a polyclonal TuYV antiserum produced by Dr. F. Rabenstein, Aschersleben. Samples of three leaves per plant were homogenised by use of a roller press and tested as mixture in DAS-ELISA. As a negative threshold, the mean absorbance value for four healthy control wells was used. The results of DAS-ELISA from the progenies were compared with the results of the highly susceptible cv. Sollux and the three cvs. used in the cross experiments.
We have divided the plants according their absorbance values (OD) into three groups:
1. E405 <0,1: Plants with OD <0,1 can be considered as virus free and, consequently, as extremely resistant.
2. E405 = 0,1 to 0,6: Plants that contain reduced amounts of virus. They can be considered as resistant.
3. E405 > 06: Individuals of cultivars, used as susceptible controls, all belonging to group 3, never showed reduced absorbance values as found for resistant progenies.
No serological tests were carried out with plants of 63 progenies which showed distinct virus symptoms or strong phenotype of the R 54. Using a RT-IC-PCR technique developed by Dr. J. Schubert, Aschersleben, 106 apparently virus free plants were tested to confirm that they were really free of virus.
Results
Virus symptoms
Susceptible cultivars
First symptoms of TuYV infection were visible as anthocyanous and/or red discolorations at the margin and tips of leaves of the plants of susceptible cvs. at the end of October. During late autumn until early spring infected plants showed conspicuous anthocyanous discoloration of the whole leave. The virus symptoms are most obvious before stem extension. However, virus induced growth reductions were not observable on infected plants before spring. After stem extension, the new developed leaves showed no discoloration symptoms. But, since this time the TuYV infected plants showed distinct growth reductions and a reduced number of lateral shoots. Later in spring and during early summer, strong reddening was visible on many plants, especially after a period with temperatures over 25 °C.
Resynthesized rapeseed R 54 and cross progenies
In contrast to the plants of the four cvs., the resynthesized rapeseed R 54 did not show any symptoms in the field after repeated virus inoculation.
The crosses of susceptible oilseed rape cultivars with the two resistant R 54-15 plants resulted in selected progenies with different levels of symptom expression. In the 1997/98 field experiment, in F2 and BC1F2 plants appeared both with and without virus symptoms. No virus symptoms were observed on plants of the most F3, F4 and F5.
Virus detection
Susceptible cultivars
The testing of the plants of the three cvs. by DAS-ELISA resulted in total infection and average absorbance value in the range of 1.65 to 1.94, indicating that the use of infectious aphids for virus inoculation is a very effective method for virus transmission.
Resynthesized rapeseed R 54
Among the pool of progenies of both selected R 54-15 plants, which were used for crosses with all three cultivars, 29% of the plants were free of virus and 13% of the plants belonged to group 2.
F2 progenies
The percentage of virus resistant plants in the F2 varied depending from the oilseed rape parents.
Express: Four F2 populations were tested. The part of virus free individuals varied from 2% to 15%. OD values characteristically for group 2 showed of 8% to 26%, so 27% of the individuals were scored as resistant.
Falcon: For virus testing TP and DAS-ELISA were used. All five F2 populations showed a high proportion of virus free plants with both detection techniques, 45% to 75% for TP and 36% to 50% for ELISA. 7% to 16% of the plants belonged to resistance group two. Consequently, 60% of the progenies were scored as resistant.
Wotan: Thirteen F2 populations were tested. 2% to 16% of the plants proved to be virus free. Proportion of plants belonging to group 2 varied from 3% to 38%. Hence, 25% of F2 plants in this cross can be considered as virusresistant.
F3 progenies
Among the nine populations, differing proportions of virus resistant plants were found. It was shown by means of TP that 30% to 90% of the Falcon progenies were virus free. Five of the tree pedigrees revealed 24% to 67% of virus free plants as shown by ELISA. An uniform virus resistance could not yet be found in this generation.
F4 progenies
Eighteen F4 populations were tested. Among nine of them all or almost all of the plants were virus infected. Nine F4 populations showed a high proportion of virus free plants ranging between 42% to 95%. Including plants of resistance group 2 80% to 100% of the progenies can be scored as resistant.
F5 progenies
Six populations, three each from Express and Falcon pedigrees were tested. Striking differences between both pedigrees were found. Among pedigrees of Express most were virus-free in ELISA, e.g. for 60% to 70% of the plants no virus infection could be detected. 35% to 45% of the plants revealed a reduced virus concentration. Consequently, 88% to 100% of the plants can be considered as resistant. Among pedigrees of Falcon only 3% to 7% were virus-free.
Table 1. Comparison of the results of ELISA (OD E405) of oilseed rape cvs., resynthesized rapeseed R 54 and Falcon progenies (assortment of genotypes, OD graded)
|
|
genotype |
||||||
|
plant |
Sollux |
Falcon |
R 54-15 |
F2 |
F3 |
F4 |
BC1F2 |
|
1 |
0,821 |
0,644 |
0,004 |
0,000 |
0,002 |
0,001 |
0,000 |
|
|
|
|
|
|
|
|
|
|
10 |
1,523 |
1,067 |
0,014 |
0,009 |
0,008 |
0,002 |
0,009 |
|
|
|
|
|
|
|
|
|
|
20 |
1,811 |
1,326 |
0,388 |
0,017 |
0,012 |
0,005 |
0,034 |
|
|
|
|
|
|
|
|
|
|
30 |
2,034 |
1,502 |
0,797 |
0,037 |
0,020 |
0,007 |
0,355 |
|
|
|
|
|
|
|
|
|
|
40 |
2,500 |
1,642 |
1,405 |
0,732 |
0,041 |
0,010 |
1,887 |
|
|
|
|
|
|
|
|
|
|
50 |
2,500 |
1,994 |
1,888 |
1,323 |
0,247 |
0,114 |
2,500 |
|
|
|
|
|
|
|
|
|
|
60 |
|
|
|
2,500 |
0,785 |
0,818 |
|
|
|
|
|
|
|
|
|
|
|
|
1,940 |
1,383 |
0,845 |
0,626 |
0,227 |
0,072 |
0,754 |
BC1F2 progenies
Among ten of the 16 tested BC1F2 all or nearly all of the plants were infected. Only for four of the Falcon-BC1F2 no virus infection could be detected for 40% to 72% of them by means of TP. By means of ELISA it was demonstrated that 56% of the plants of one of them was really virus free.
DISCUSSION
The investigations resulted TuYV-resistant plants in different proportion in the progenies.
Whereas in field experiments clear virus symptoms were visible on the four susceptible cvs. from autumn until early spring and newly appeared in early summer, plants of the resistance source R 54 and 62 progenies from crosses with it remained without symptoms of virus infection over the whole period. But viruslike symptoms can be induced by environmental conditions, by a lack of nutritive elements or by soil compression, too. Furthermore, in some years in spring TuYV is symptomless in oilseed rape. Therefore, tests by serological methods are necessary for assessment of the infestation level of crossing progenies.
By means of direct tissue print immunoassay (TP) and/or DAS-ELISA no virus infection were detectable in the most plants of 15 populations. Furthermore, the virus concentration in several progenies were distinctly decreased and virus symptoms were not visible, indicating that these progenies were virus resistant, too.
Using a IC-RT-PCR technique it was possible to show that 78 plants selected from different generations of selfing to generate extreme virus resistant basic material were absolute resistant.
The genetic interpretation of obtained results remains unclear because segregation of resistance vs. susceptibility in the selfing progenies of selected resistant plants follows not a consistent theoretical ratio. Inhomogeneity, heterozygosity and cytogenetic instability of resynthesized rape donor line R54-15 are probable causes of this. The varying proportions of virus resistant plants in progenies with different cvs. of rape could be due to the different presence of genes modifying the expression of resistance.
Although it seems that the resistance donor line R 54-15 is inhomogen experiments were successful to transfer the TuYV resistance into current breeding material. In the growing season 1998/1999 in field experiments 253 progenies of them were examined for resistance to TuYV. Several of them proved to be free of virus after artificial colonisation with viruliferous aphids.
The successful transfer of TuYV resistance in susceptible oilseed rape cultivars and breeding lines showed that it will be possible to prevent virus induced yield losses by virus resistant cultivars in future.
Acknowledgement
The authors thank Dr. F. Rabenstein, Institute for Resistance Research and Pathogen Diagnostics Aschersleben for supplying polyclonal TuYV antiserum used in the experiments for virus detection. Furthermore, we wish to thank Dr. J. Schubert and Dr. Kerstin Richter for the virus testing of selected single plants using the IC-RT-PCR. We thank M. Knauft, U. Fuhrmann and S. Gropp for excellent technical assistance.
The work was supported by grant from the Ministry of Food, Agriculture and Forestry Germany.
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