Table Of ContentsNext Page

Introgression and biochemical manifestation of the gene(s) for white rust resistance in Indian mustard (Brassica juncea (L.) Coss.)

S.S. Banga, Kulwinder Kaur, K.L. Ahuja and S.K. Banga

Department of Plant Breeding, Genetics and Biotechnology, Punjab Agricultural University, Ludhiana, India, E-mail : surin11@rediffmail.com

Abstract

Genes for white rust resistance were introgressed into B. juncea cv. RL 1359 from B. napus, B. carinata and B. tournefortii following interspecific hybridization. Selection for white rust (Albugo candida) incidence was carried out under aided natural epiphytotic conditions in the selfed progenies of BC3 derivatives of the respective interspecific crosses. This led to the development of seven genotypes having a significantly higher degree of resistance than the susceptible check genotype, RL 1359. These were RL-1-3, RL 1-22, RL 1-25, RL 2-30, RL 2-41, RL 3-48 and RL 3-69. Further backcrossing B. juncea cv. RL 1359 as recurrent parent with these newly identified resistant stocks for five generations helped in synthesis of near isogenic lines of B. juncea cv. RL 1359, differing only for gene(s) for white rust resistance. Biochemical analysis of the isogenic lines and the susceptible recurrent parent RL 1359 revealed association of white rust resistance with peroxidase and polyphenol oxidase activities, especially under diseased conditions compared to disease free conditions. Total phenols, sugars, flavonoids and waxes were higher in leaves of the resistant genotype. In contrast, cellulase activity was higher in the susceptible check, especially under diseased conditions.

Media summary

Gene(s) for white rust resistance were introgressed into susceptible B. juncea cv. RL 1359 from resistant species B. napus / B. carinata / B. tournefortii. Analysis of isogenic lines suggested association of waxes, flavonoids, and peroxidase with resistance.

Key words

White rust, Albugo candida, introgression, resistance, flavonoids, waxes, peroxidase

Introduction

Indian mustard (Brassica juncea (L.) Coss.) is attacked by many pathogenic diseases which are the major impediment to improving crop productivity. Yield losses vary depending upon severity of pathogenic attack, tolerance level of cultivars and prevailing environmental conditions. Among the various diseases white rust, caused by Albugo candida, is widespread and damaging, especially when stag head formation occurs alone or in combination with downy mildew. Upto 23-60 percent yield losses have been reported (Saharan 1993). Based on host specific differentials, at least ten physiological races of A. candida have been identified (Tewari and Mithen, 1999), with host range extending to all the species sharing a common ‘A’ genome. A number of genetic stocks conferring resistance against one or some races have been bred and genetically characterized in B. juncea. Deployment of such gene sources, however, has not helped significantly in evolving cultivars with durable resistance due to continuous emergence of new races. Occurrence of a high level of resistance in Brassica species carrying the C genome (Delwiche and Williams, 1974; Saharan, 1993) and in B. tournefortii Gouan prompted us to explore the possibility of genetically enriching B. juncea by introgressing genes for white rust resistance from these genomically related Brassica species.

Methods

i) Introgression of gene(s) for white rust resistance

Introgression of gene(s) for white rust resistance was attempted from related Brassica species namely B. carinata (2n=34; BBCC), B. napus (2n=38; AACC) and B. tournefortii (2n=20; TT). All these species as a group remain free from white rust, and show close genomic affinity with B. juncea (2n=36; AABB). Introgression of genes from B. carinata and B. napus to B. juncea was accomplished by means of simple hybridization and backcrossing for three generations using B. juncea as the initial female parent for the synthesis of interspecific hybrids. For subsequent backcross generations, B. juncea cv RL 1359 was the recurrent male parent. Backcrossing was followed by selfing and selection for B. juncea (2n=36) type plants for four generations. A large number of segregants having excellent pollen fertility and euploid chromosome number (2n=36) were identified in BC3S4 generation. In each generation of backcrossing / selfing the segregants were exposed to aided epiphytosis of white rust. Only the resistant plants, devoid of any staghead formation were used for the next cycle of backcrossing / selfing. To facilitate the desired introgression from B. tournefortii, an amphiploid (2n=40; AATT) between B. rapa and B. tournefortii was first synthesized. This amphiploid was backcrossed thrice, with B. juncea as the recurrent male parent followed by three cycles of selfing and selection for fully fertile, B. juncea type segregants. Plants with euploid chromosome number (2n=36) were available by BC3S3 generation.

ii) Synthesis of isogenic lines

The isogenic lines of B. juncea cv. RL 1359 were developed by recurrent backcrossing (five times) of identified resistant stocks (RL 1-3, RL 1-22, RL 1-25, RL 2-30, RL 2-41, RL 3-48 and RL 3-69) as donor parents and cv RL 1359 as a common recurrent parent. BC5 lines were selfed for two generations to obtain lines homozygous for resistant gene(s).

iii) Screening for white rust resistance

Experimental material was sown unreplicated in a white rust sick plot. The susceptible check, RL 1359 was interplanted as repeat infector rows. High humidity was maintained by applying frequent irrigations. Disease incidence on the foliage was recorded by using 0-5 scale (Sokhi and Khangura 1992) with slight modifications. Incidence of floral malformation (staghead) was recorded by counting the percentage of plants having stagheads.

iv) Biochemical studies

Isogenic lines differing for resistance to white rust were analysed for their biochemical constituents such as total phenols, polyphenol oxidase, cellulase, waxes and flavonoids etc. using standard laboratory protocols (Swain and Hillis 1959; Balbaa et al. 1974; Mondols et al. 1994; Bastin and Unluer 1972). The levels of various such constituents were then correlated with disease incidence.

Results and Discussion

Based on lower percent disease index (PDI) and absence of stagheads, the isogenic lines were categorized as resistant. Genotype RL 1359 (3-48) had the least PDI followed by RL 1359 (1-25) and RL 1359 (2-28). None of the resistant genotypes showed any staghead formation whereas in the susceptible check, 66.8% plants had staghead formation. The identified resistant stocks, RL 1-(3,22,25), RL2-(25,30,41) and RL3-(48,69) had introgressions carrying gene(s) for white rust resistance from B. napus, B. carinata and B. tournefortii respectively. Such intergenomic introgressions were facilitated by the homoeologous pairing and subsequently the genetic exchange during meiosis in the trigenomic hybrids [AACB (B. juncea x B. napus), BBAC (B. juncea x B. carinata) and AABT [B. juncea x (B. rapa x B. tournefortii)]. A high degree of pairing between ‘A’ and ‘C’ (Attia and Rbbelen, 1986), A-T (Banga et al., 1987) and B-T (Narain and Prakash 1972) genome chromosomes has been reported in the past. Successive backcrossing of the trigenomic hybrids with B. juncea and selection of rust resistant mustard type segregants in each backcross / self generation helped to recover plants with euploid B. juncea chromosome number (2n=36) having desired introgressed gene(s). Genetic studies have previously revealed monogenic dominant inheritance for resistance in RL-1-3, RL-1-25, RL-2-28, RL-2- 41, RL-3-48 whereas duplicate recessive epistasis occurred in RL 2-30 and RL 3-69 (Singh, 1994).

Biochemical Studies

Biochemical constituents of the plant parts act as physiological barriers and / or are involved in the suppression of pathogen growth and establishment on plant surface and inner plant parts. In the present investigation, seven white rust resistant isogenic lines were analysed for various biochemical parameters reportedly having association with the resistance mechanisms in different crop plants. Analysis of the susceptible check and the isogenic resistant lines revealed higher values or activity for phenols, flavonoids, peroxidase and polyphenol oxidase in genotypes showing resistance to white rust as compared to the susceptible check RL 1359 (Tables 1 and 2).

Table 1. Biochemical analysis of isogenic lines in B. juncea.

 

Total phenols (mg/g)

Sugar (mg/g)

Total free amino acids (mg/g)

Flavonoids (g/g)

Waxes (mg/g dry wt.)

Glucosi-nolates
(mg/g)

Chl
a / b

PDI
(%)

RL 1359 (1-3)

7.7

14.7

1.4

46.0

5.9

1.7

1.0

19.3

RL 1359 (1-22)

8.9

14.4

1.8

46.0

5.5

1.6

1.0

6.5

RL 1359 (1-25)

8.8

16.1

1.7

36.9

5.5

1.6

1.0

7.2

RL 1359 (2-30)

7.1

15.7

1.4

33.3

4.0

1.8

0.8

5.4

RL 1359 (2-41)

9.0

15.9

1.7

44.5

5.5

1.3

1.0

17.3

RL 1359 (3-48)

8.9

14.8

1.4

34.9

5.1

1.9

0.9

3.1

RL 1359 (3-69)

6.0

13.8

1.3

34.0

4.0

1.2

0.7

19.5

RL 1359

6.1

11.5

1.1

26.2

3.5

1.0

0.8

73.8

Correlation PDI vs. phenols (-0.46) / sugars (-0.86) / free amino acid (-0.66) / flavonoids (-0.57) / waxes (-0.57) / glucosinolates (-0.79) / chl a/b (-0.35) (P0.05=0.63).

Table 2. Activity of key enzymes under diseased and disease free condition in isogenic lines of B. juncea.

 

Peroxide

 

Polyphenol oxidase

 

Cellulase
(g / mg protein/min)

PDI
(%)

DFC

DC

DFC

DC

DFC

DC

RL 1359 (1-3)

2.3

3.4

7.9

8.6

2.3

3.1

19.3

RL 1359 (1-22)

4.7

5.3

9.7

10.4

3.1

4.1

6.5

RL 1359 (1-25)

5.5

6.0

7.6

8.3

2.6

3.5

7.2

RL 1359 (2-30)

2.2

3.9

9.9

10.3

4.4

5.3

5.4

RL 1359 (2-41)

6.0

6.2

9.9

10.4

4.1

5.0

17.3

RL 1359 (3-48)

5.3

7.7

10.5

10.1

3.0

3.6

3.1

RL 1359 (3-69)

2.3

3.7

10.2

11.2

2.8

3.3

19.5

RL 1359

2.2

2.3

2.2

2.2

6.0

7.8

73.8

DFC : Disease free conditions ; DC : Diseased conditions

Correlation : PDI vs. Peroxidase (DFC=-0.46 / DC=-0.67) / Polyphenol oxidase (DFC=-0.90 / DC=-0.90) / Cellulase (DFC=0.74 / DC=0.79) (P0.05=0.63).

Association studies of plant disease index with all these four constituents confirmed their involvement with resistance. Further the demonstration of increased activity of peroxidase and polyphenol oxidase enzymes in resistant lines in conditions of disease epidemic as compared to disease free conditions was indicative of their role in defensive responses of the plants. The activity of both these enzymes remained the same under both the conditions in the susceptible check RL 1359 (Table 2). Epicuticular wax is one of the most important resistance mechanism available with the plants. The leaf surface waxes are water repellent and thus significantly reduce the conidial retention. Besides they may also have a role in suppressing or stimulating the infection by pathogens (Lampard and Carter, 1973). The flavonoids have a protective function in plants in relation to disease resistance as some of them are highly toxic to microorganisms. Interestingly, the susceptible recurrent parent RL 1359 showed significantly higher cellulase activity as compared to its resistant isogenic versions. Cellulase activity was also higher under disease epiphytotic conditions.

Based on the analysis of the genotypes included in the present study, the waxes having direct association with resistance, may be ideal to fit in the role of developing initial phenotypic screens for the purpose of evaluating segregating populations. Biochemically, the resistant genotypes, especially, RL-1-25, RL-3-48 appeared to have better fortification against white rust infection as these carry phenols, waxes and flavonoids in higher proportions. This was confirmed by lower PDI in these accessions. It will be very interesting to observe the cosegregation pattern of these components in F2 along with the gene for white rust resistance.

Conclusions

The present study constituted the first organized attempt to biochemically characterize introgressed variability for white rust resistance in B. juncea using isogenic lines differing only for the gene(s) for resistance.

References

Attia T and Rbbelen G (1986). Cytogenetic relationship within cultivated Brassica analyzed in amphihaploids from the three diploid ancestors. Can J Genet Cytol 28, 323-329.

Balbaa SI, Ashgan YZ and Ali MES (1974). Total flavonoid and rutin content of different organs of Sophora japonica L. J Assoc oft Anlyt Chemists 57, 752-755.

Banga SS, Labana KS and Singh K (1987). Wide hybridization in the genus Brassica. I. B. tournefortii x B. campestris and B. nigra x B. alboglabra. Proc 7th Int Rapeseed Cong, 11-14 May, 1987. Poland. pp 404-409.

Bastin M and Unluer O (1972). Effect of actinomycin-D on the formation of enzymes in Jerusalem artichoke tuber slices. Planta 102, 357-361.

Delwiche PA and Williams PH (1974). Resistance to Albugo candida race 2 in Brassica sp. Proc Phytopathol Soc 1, 66 (Abstr).

Lampard JF and Carter GA (1973). Chemical investigations on resistance to coffee berry disease in Coffea arabica. An antifungal compound in coffee cuticular wax. Annal of Applied Biology 73, 31-37.

Mondols M, Hontz L and Nyslrom J (1994). Enzymatic hydrolysis of waste cellulase. Biotechnol Bioengg 16, 1471-1493.

Narain A and Prakash S (1972). Investigations on the artificial synthesis of amphidiploids of Brassica tournefortii Gouan with the other elementary species of Brassica. I. Genomic relationships. Genetica 43, 90-97.

Saharan GS (1993). Disease resistance in: Labana KS, Banga SS, Banga SK (eds.) Breeding Oilseed Brassicas Springer Verlag, Germany.

Singh G (1994). Evaluation of germplasm and inheritance studies resistance to white rust in Indian mustard (Brassica juncea (L.) Coss). Unpubd. M.Sc. thesis, PAU, Ludhiana.

Sokhi SS and Khangura RK (1992). White rust of rapeseed and mustard. Punjab Agric Univ Ldh. DR / Publication pp. 11.

Swain T and Hillis WE (1959). The phenolic constituents of Prunus domestica-I. The quantitative analysis of phenolic constituents. J Sci Fd Agric 10, 63-68.

Tewari JP and Mithen RF (1999). Diseases. In : Gmez-Campo C (ed) Biology of Brassica Coeonspecies. Elsevier, Amsterdam. pp 375-412.

Top Of PageNext Page