Inheritance of partial resistance to race 2 of Albugocandida in canola-quality mustard (Brassica juncea) andits role in resistance breeding

V. K. Bansal, M. R. Thiagarajah, G. R. Stringam* and J. P. TewariDepartment of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5

The inheritance of partial resistance to race 2 of Albugo candida was studied in a canola-quality line of Brassicajuncea. This partially resistant line was crossed with the susceptible B. juncea cultivar Commercial Brown. F1,F1(reciprocal), F2, BC and doubled haploid generations from the cross were inoculated with a zoospore suspension ofrace 2 to study segregation of partial resistance. The partially resistant phenotype appeared to be controlled by a singledominant gene that has variable expression. This partial resistance can have implications in breeding for diseaseresistance against white rust, as adult plants did not develop hypertrophic growth or stagheads under greenhouse andfield conditions.

Keywords: Albugo candida, Brassica juncea, genetics, resistance

Introduction

The oomycete Albugo candida is the cause of white rustand staghead diseases of cruciferous plants. White rust isknown to be present wherever rapeseed and mustard aregrown (Nyvall, 1989). When the infection is confined toleaves it may not result in any appreciable yield loss.However, in the staghead phase, racemes develop intomalformed structures and substantial yield losses mayoccur (Kolte, 1985). Harper & Pittman (1974) devel-oped an equation to estimate the yield loss caused by thestaghead phase of the disease.

The staghead disease is widespread mainly in Brassicajuncea and Brassica rapa cultivars, and less so in those ofBrassica napus grown extensively in Canada. Total yieldlosses attributed to this disease are not high in Canada.However, the presence of conspicuous white pustules atthe vegetative stage and stagheads at the reproductivestage can be of serious concern to farmers. A number ofchemicals have been suggested to control this disease(Verma & Petrie, 1979), but genetic resistance is thepreferred choice to reduce yield losses. Breeding fordisease resistance in this host–pathogen system has beenquite successful because the biological races of thispathogen are well established (Pound & Williams,1963), and a single dominant gene can confer completeresistance in oilseed B. juncea and B. rapa crops. The

greenhouse inoculation and screening techniques againstwhite rust disease are simple and reliable.

Tiwari et al. (1988) reported that resistance in B.juncea cv. R-908 against race 2 is controlled by a singlegene and, under artificial inoculation conditions,expressed as complete absence of pustules on leaves.Paladhi et al. (1993) reported that field resistance of B.juncea strain PI-15 to natural infection by A. candidawas also controlled by a single gene. Pound & Williams(1963) studied variability in the genus Brassica againstraces 1 and 2, and concluded that several genes governthe resistance and that host populations are highlyheterogeneous. Variations in the amount of sporulationand location of sori or pustules were also reported onBrassica oleracea and B. rapa. In this study, they alsoreported that the progeny of one resistant plant of B.oleracea italica cv. Green Mountain segregated in a 3 : 1ratio for resistance, indicating resistance controlled by asingle dominant gene. The genetics of resistance in B.rapa to a white rust sample collected from B. juncea wasalso studied by Edwards & Williams (1987) and Kole etal. (1996). Both reported a partially resistant phenotypecharacterized by small pinhead-size pustules, mostly onthe adaxial side of cotyledonary leaves.

Williams & Pound (1963) studied the inheritance ofrust resistance in radish (Raphanus sativus) to race 1 ofA. candida, and reported three phenotypes of host–pathogen interaction on cotyledons. Resistance wasindicated by no visible expression of the disease orminute hypersensitive flecking, tolerance by discretepustules on the adaxial surface of leaves, and the

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*To whom correspondence should be addressed (e-mail:gstringam@afns.ualberta.ca)

Accepted 19 July 1999.

susceptible reaction consisted of diffuse relatively largesori on the abaxial surface surrounded by secondarypustules. In studies where B. juncea cultivars were used,the resistant phenotype was characterized by thecomplete absence of pustules and was reported to bedetermined by a single dominant gene. In B. rapa, B.napus and R. sativus cultivars, in addition to completeresistance controlled by a single gene, partial resistancecharacterized by small, noncoalescing lesions was alsodescribed. The lesions appeared mostly on the adaxialsurface and were accompanied by reduced sporulation.This partial resistance was suggested to be underpolygenic control and affected by environmental con-ditions (Pound & Williams, 1963; Williams & Pound,1963; Ferreira et al., 1995; Kole et al., 1996).

In the canola breeding programme at the University ofAlberta a research project was initiated to transfer whiterust resistance from the B. juncea cv. Cutlass toagronomically superior canola-quality mustard lines.In the doubled haploid (DH) progenies of this cross,several lines carried partial resistance characterized byinteraction phenotype 1–3 (Anonymous, 1985). Thispartial resistance in B. juncea appears to be similar tothat reported earlier in B. napus, B. rapa and R. sativuscultivars. The genetic control, mode of inheritance andpractical implications of this partial resistance forbreeding are reported here.

Materials and methods

Pathogen race

The sample of race 2 used in this study was initiallyinoculated on Brassica juncea cultivars Cutlass andCommercial Brown, and on B. rapa cv. Torch (Table 1).Although Commercial Brown and Cutlass had similardisease incidence, the level of disease severity wascomparatively much lower in Cutlass. Similar resultsfor mean white rust incidence were reported forCommercial Brown and Cutlass when pathotype 2vwas used in the evaluation of brown and orientalmustards (Rakow & Gugel, 1999). A mean incidenceof 13 and 38% was also reported for pathotyype 2a onCutlass in different screening experiments. Because ofthis variation reported for pathotype 2a, race 2 waschosen for the present study and was not confirmed aseither pathotype 2a or 2v. The infection rating on B.

juncea cv. Commercial Brown and B. rapa cv. Torch ineach screening experiment confirmed that the sampleused was race 2 of A. candida and not race 7.

Plant materials

The parents used in this study were a DH B. juncea linewith partial resistance to white rust and a susceptible B.juncea cultivar, Commercial Brown. The parent DH linewith partial resistance was initially selected from a crossof a canola-quality susceptible B. juncea line and aresistant B. juncea cv. Cutlass plant. The expression ofpartial resistance in the parent DH line was variable,some of the plants expressing complete resistance. Forinitial crossing to produce F1 and F1(reciprocal), plantswith the partially resistant phenotype were used. Allcrosses were performed in the greenhouse. From the F1

and F1(reciprocal), F2, BC and DH generations werederived (Table 2). DH lines were produced as describedby Thiagarajah & Stringam (1993). For inoculation andscreening, plants (nine per pot) from various generationswere grown in 10-cm pots filled with soil-free mix(Stringam, 1971). Plants from various generations werescreened as they were produced over the period fromNovember 1994 to April 1996. Both parents and B. rapacv. Torch were included as controls in each run of thescreening experiments.

Inoculum preparation

Race 2 of A. candida was maintained and increased oncotyledons of B. juncea cv. Commercial Brown. Frominfected cotyledons, fresh zoosporangia were collectedby scraping the fungus off with a spatula into Petri platescontaining sterile distilled water. The zoosporangialsuspension was incubated in a refrigerator at 48C untilmotile zoospores were visible. The zoospore concentra-tion was adjusted to approximately 4–5 ×104 spores permL. A 25-mL drop of the zoospore suspension was theninoculated on cotyledons of 6–7-day-old seedlings. Thedrop inoculation method was used to avoid thepossibility of any escapes in the screening procedure.

Incubation conditions and plant growth

Before inoculation, plants were maintained in a green-house at approximately 208C with a 16-h photoperiod.Inoculated plants were incubated for approximately 15 hin a dark humidity chamber maintained at 17–188C and100% relative humidity. From the humidity chamber,plants were moved to a growth cabinet maintained atapproximately 178C under a 16-h photoperiod. Theinoculated plants were incubated at this lower tempera-ture as higher temperatures are reported to suppressasexual sporulation (Williams & Pound, 1963).

Disease evaluation at the seedling stage

Plants were scored for disease symptoms 9–10 days after

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Table 1 Mean percentage white rust disease incidence on Brassicajuncea and B. rapa control cultivars challenged with Albugo candidarace 2

Mean percentageGenotype white rust incidence

Brassica juncea cv. 100Commercial Brown

B. juncea cv. Cutlass 100

B. rapa cv. Torch 5

inoculation. Resistance, partial resistance and suscept-ibility were estimated from the lesion size and location ofpustules on cotyledon surfaces. The host was consideredresistant when there was no visible expression of thedisease, partially resistant when a few pinhead-sizepustules were observed mainly on the upper surface ofthe cotyledonary leaf, and susceptible when largecoalescing pustules were present, mainly on the lowerside of the cotyledonary leaf.

Adult plant resistance in the greenhouse

Eight seedlings having the highest levels of diseaseseverity based on cotyledon symptoms were saved fromeach of the four genotypes for adult plant evaluations(Table 3). Eight plants from each genotype weretransplanted in pairs to four 12-cm pots. Growingconditions for the adult plants were the same asdescribed above for seedlings. Plants were observedregularly for any symptoms of hypertrophic growth and

staghead formation. Finally, the numbers of plantsattaining full maturity were also counted.

Adult plant resistance in the field

Two of the DH lines used in the above greenhouse adultplant evaluation, along with partially resistant andsusceptible parents, and Torch, were grown as fourrow plots in the summer of 1996 for field observations.In the field, all lines were inoculated at the rosette stage.Plants in each line were evaluated at flowering for whiterust symptoms on leaves, and at maturity for stagheadformation.

Statistical analysis

Chi-square analyses was used to test the goodness-of-fitof genetic ratios in F2, backcross and DH generationsderived from F1 and F1(reciprocal) crosses of partiallyresistant and susceptible parents. Plants in the resistant

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Table 2 Segregation for resistance and susceptibility to race 2 of Albugo candida in parents, F2, backcross and doubled-haploid populationsin Brassica juncea

Generationa Observed reactionb

and putative Expected x2

Pedigree genotype R PR S Total R : S ratio calculated P-value

Parent (R) P1 Wpr Wprc 30 30 0 60 — — —Parent (S) P2 wpr wpr 3 0 56 59 — — —S ×R F1 Wpr wpr 1 12 0 13 — — —R ×S F1(rec) Wpr wpr 3 20 0 23 — — —From F1 F2 11 34 16 61 3 : 1 0·088 0·766From F1(rec) F2 16 47 24 87 3 : 1 0·243 0·622F1 ×P2 BCF1 2 25 36 63 1 : 1 1·286 0·257F1(rec) ×P2 BCF1 8 25 56 89 1 : 1 5·944 0·014*

F1 ×P1 BCF1 26 27 0 53 — — —F1(rec) ×P1 BCF1 35 50 0 85 — — —From F1 DH 5 0 6 11 1 : 1 0·091 0·763

aRec, reciprocal; BC, backcross; DH, doubled haploid.bR, resistant; PR, partially resistant; S, susceptible.cWpr, dominant partial resistance; wpr, recessive susceptible.*Significant deviation at the 0·05 level of probability.

Table 3 Adult plant evaluation for white rust resistance against race 2 of Albugo candida under greenhouse and field conditions

Greenhouse————————————————————————————Hypertrophic Plant

Genotype growth survival Field

Partially resistant DH line 0a 8(100)b Tracec WR on leaves, no staghead formationSusceptible DH line 6(75) 4(50) Moderate WR on leaves, few stagheadsPartially resistant parent 0 8(100) No WR on leaves, and no staghead formationSusceptible parent 8(100) 1(13) Severe WR on leaves and several stagheadsTorch (host for race 7) – – No WR on leaves, and no staghead formation

aThe number of plants showing hypertrophic growth.bThe values in parentheses are percentage plant survivals.cWhite rust (WR) on leaves was rated according to the scale of P. R. Verma (Saharan & Verma, 1992), lesion size in partially resistant line hadpinhead-size lesions.

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and partially resistant classes were grouped together foranalysis.

Results

The disease reactions of partially resistant and suscep-tible parents, and of DH lines produced from this cross,are given in Fig. 1. The parent DH line with partialresistance had some plants with complete resistance(Table 2). The expression of complete resistance in anumber of the plants was carried over in subsequentgenerations. The disease reactions of F1 and F1(recipro-cal) progenies of the cross between partially resistantand susceptible parents were similar to those of thepartially resistant parent. Segregation among F2 seed-lings from both types of F1 generations fitted a 3-resistant : 1-susceptible genetic ratio (Table 2), suggest-ing that the partially resistant phenotype is controlledby a single gene. Its expression in the F1 andF1(reciprocal) generations confirmed that the gene isdominant in its expression (Table 2). The BCF1

generations from F1 ×P2 and F1(reciprocal) ×P2 did notsegregate in a 1-resistant : 1-susceptible ratio (Table 2).The lower probability for x2 for F1(reciprocal) ×P2 mayhave resulted from misclassification of some partiallyresistant seedlings as susceptible, as there was con-siderable variation in the partially resistant class. Thisobservation does not negate the hypotheses that thepartial resistance of the line studied is conferred by asingle gene, but indicates that its final expression isprobably affected by the environment and/or back-ground genotype. The BCF1 generations from F1 ×P1

and F1(reciprocal) ×P1 did not segregate, supportingthe hypothesis of a dominant gene.

DH lines derived from F1 plants were rated as resistantor susceptible based on various numbers of plantsscreened in each line. The segregation of DH linesfitted a 1 : 1 ratio, consistent with its control by a singlegene (Table 2). In resistant DH lines only a few plantshad a susceptible phenotype, whereas in susceptible DHlines most of the plants were of this phenotype (data notpresented).

In the greenhouse, where a partially resistant parentand partially resistant DH line were screened and grownto maturity, none of the plants showed any symptoms ofhypertrophy. However, in the susceptible parent andsusceptible DH line, 100 and 75% of the plants,respectively, showed hypertrophic growth and only 13and 50%, respectively, grew to maturity (Table 3). In thefield experiment, a parent and a DH line with partialresistance had either no white rust or only trace levels onthe leaves, and stagheads were not formed at maturity.

The susceptible parent and a susceptible DH line hadsevere and moderate leaf infection, and stagheads wereformed on both lines (Table 3). The absence of leafsymptoms and stagheads on cv. Torch grown in the fieldsuggested that natural inoculum in the field was also race2 (Table 3).

It is proposed that the partially resistant dominantgene reported here be designated Wpr with thecorresponding recessive allele designated as wpr.

Discussion

The segregation data presented in this paper suggest thatpartial resistance for white rust disease in the line testedis simply inherited, being controlled by a single gene thatcould be easily exploited in resistance breeding. In earlierstudies (Pound & Williams, 1963; Williams & Pound,1963; Ferreira et al., 1995; Kole et al., 1996) where thistype of resistance was reported, no attempt was made tostudy its genetics and inheritance. The segregation ofDH lines for the partial resistance showed no distortionfrom the expected 1 : 1 Mendelian ratio but only 11 lineswere tested. Foisset & Delourme (1996) reviewed theoccurrence of segregation distortion in androgenicprogenies of several species studied for different typesof markers. Despite these distortions they favour the useof DH lines as long as the user is aware of thisphenomenon. Pauls (1996) outlined several advantagesof using DH populations for genetic studies comparedwith commonly used F2 populations. The lack ofheterozygotes in DH lines reduces the number ofphenotypic classes and, consequently, simplifies segrega-tion ratios.

The partial resistance reported here has the advantagethat the pathogen may have limited reproduction andmay therefore be under reduced selection pressure forthe emergence of new virulent strains. Host andpathogen can coexist and resistance can be effective fora longer period of time. The dominant, single geneinheritance of this partial resistance in B. juncea makes itmore desirable for resistance breeding in mustard, sinceresistance can be transferred easily by backcross and DHbreeding methods. A similar partially resistant pheno-type in B. napus lines genotypes against race 7 was alsofound (unpublished data). The mode of inheritance ofthis resistance needs further study.

Initial evaluation of adult plants in the greenhouse andfield experiments suggests that plants in breeding lineswith partial resistance do not develop hypertrophicgrowth or stagheads, indicating that partial resistancemay protect the plants from yield losses that result fromthese malformations.

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Figure 1 Disease reaction of Albugo candida race 2 on Brassica juncea. (a) Reaction of partially resistant parent (P1) at cotyledon stage onupper and lower surfaces. (b) Reaction of susceptible parent (P2) Commercial Brown at cotyledon stage on upper and lower surfaces. (c)Reaction of partially resistant parent, partially resistant (96–15016), susceptible (96–15020) DH lines, and susceptible parent CommercialBrown, respectively. (d–g) Growth and reaction of partially resistant parent, partially resistant and susceptible DH lines; and susceptible parentat adult plant stage, respectively.

Acknowledgements

We thank S. W. Wu for technical assistance in thegreenhouse, R. Gadoua for the field studies, andS. Brezden for photography. This research was sup-ported by funding from the Alberta AgriculturalResearch Institute.

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