Inheritance and Allelic Relationship among Gene(s) for White Rust Resistance in Indian Mustard [ Brassica juncea (L.) Czern & Coss]

: White rust [ Albugo candida (Pers. Ex. Lev) Kuntze] is an important oomycetes disease of Indian mustard [ Brassica juncea (L.) Czern & Coss] that causes a drastic reduction in seed yield and quality when the conditions are favorable. A set of 25 Indian mustard genotypes were screened against A. candida Delhi isolate (Ac-Dli) under both controlled and natural epiphytotic conditions. Out of 25, only six genotypes (Pusa Karishma, PDZ-3, Heera, BEC-144, BIO YSR, and Donskaja) were found highly resistant in both environments. To study the inheritance of resistance, four resistant genotypes (BEC-144, BIO YSR, Pusa Karishma, and Donskaja) were crossed with three susceptible genotypes (PM-24, Pusa Vijay, and MSTWR-17-15) in a deﬁnite design. The parents, F 1 , F 2, and BC 1 F 1 (F 1 backcrossed with susceptible parent) generations were screened under both artiﬁcially controlled and natural epiphytotic conditions and the results indicated that the resistance in BIO YSR, BEC-144, and Pusa Karishma is governed by a single dominant gene, but more than one dominant gene is responsible for resistance in Donskaja. A test of an allelism conducted on the F 2 population derived by crossing resistant (BEC-144) × resistant (BIO YSR) genotypes indicated that the gene imparting resistance to Ac-Dli isolate in the resistant parents BEC-144 and BIO YSR is the same and they are allelic to each other. Therefore, in broader terms, the information generated in the present study will be useful in Indian mustard breeding programs for the development of durable resistant cultivars.


Introduction
Brassica is an economically important genus, being home to many species that are widely used as edible oilseed crops, leafy vegetables, green fodder crops, and condiments [1][2][3].Globally, rapeseed mustard plays a significant role in terms of edible oilseed production and ranks third after soybean and oil palm.In India, rapeseed mustard is the second major oilseed crop in terms of area (≈22.2% of the total oilseed cultivated area) and production (≈32% of the country's oilseed production) after soybean [4]; however, as edible oil production is concerned, it ranks first [5].Among different species of rapeseed mustard crops, Indian mustard [Brassica juncea (L.) Czern & Coss] is the most important and widely cultivated species in India, occupying about 90% of the area (9.168 million ha) and production (11.75 MT) of the rapeseed mustard group of crops, with a productivity of 1178 kg/ha during 2021-2022 [6].The production of rapeseed mustard is highly affected by biotic and abiotic factors.Among biotic factors, diseases such as stem rot (Sclerotinia sclerotiorum), white rust (Albugo candida), Alternaria blight (Alternaria brassicae), powdery mildew (Erysiphe cruciferarum), downy mildew (Hyaloperonospora parasitica), and blackleg (Leptoshaeria maculans) are more devastating and significantly reduce the seed yield and oil quality of rapeseed mustard depending on the prevailing climatic and agro-ecological conditions [7].
White rust [Albugo candida (Pers.Ex.Lev) Kuntze] is an obligate oomycetes pathogen of the oilseed Brassicas worldwide, including India, which causes localized and systemic infections in plants [8][9][10].With the increase in area under mustard cultivation, the intensity and severity of white rust have increased gradually throughout the mustard-growing areas of tropical and subtropical India [11].White rust disease in Indian mustard is favored when low temperature (15-20 • C) and high humidity (>65%) with intermittent rainfalls occur from the cotyledonary to the complete flowering stage [12].Yield losses have been reported to the range of 23-89.9% in B. juncea [13].The infected plants show localized white to pale-colored pustules on the abaxial surface of leaves, stems, and inflorescence, along with chlorosis on the adaxial surface of leaves; however, systemic infection leads to the formation of staghead and accounted for complete loss of seed formation which causes up to 90% yield losses [14].The yield loss depends upon disease severity, which is affected by prevailing environmental conditions (temperature and humidity), planting geometry, the date of sowing [15], etc.In India, many isolates of white rust have been reported but until today systematic information is lacking about the "dominant race(s)" present in different mustard-growing regions of India [7,16].
The pathogen survives through oospores lying in the soil (Figure 1) that are formed in the hypertrophied plant tissues fallen from diseased plants or seeds, and the oospores can act as a source of inoculum after germination [14].The oospore can survive for more than 21 years in diseased host tissue even under dry storage conditions.In natural conditions, oospore germinates once favorable environmental factors (temperature 10-20 • C and RH > 70%) occur and cause primary infection in the host leaves directly or indirectly (by entering via stomata or natural openings).However, the secondary infection takes place through sporangia and/or zoospores, which develop symptoms in the form of pustules [17].The sporangia move away by air current from one place to another after being released from the matured and dehiscence pustules.The germination and infection process of zoospores and sporangia essentially require moisture on the host leaf surface.Later, oospores are formed in the hypertrophied tissues such as roots, stems, leaves, inflorescence, and even in the siliquae of diseased plants [12].Disease resistance plays a major role in crop production, quality assurance, environmental safety and ultimately crop yield.Disease resistance can be controlled by a single gene, viz., R-gene, or by many genes with small effects [18,19].A wide range of genotypic variability exists among Brassica species for white rust resistance.The genotypes of B. juncea and B. rapa are comparatively less tolerant to white rust than B. napus, but few susceptible genotypes in B. napus were also reported [20,21].B. juncea germplasm belonging Disease resistance plays a major role in crop production, quality assurance, environmental safety and ultimately crop yield.Disease resistance can be controlled by a single gene, viz., R-gene, or by many genes with small effects [18,19].A wide range of genotypic variability exists among Brassica species for white rust resistance.The genotypes of B. juncea and B. rapa are comparatively less tolerant to white rust than B. napus, but few susceptible genotypes in B. napus were also reported [20,21].B. juncea germplasm belonging to the Indian gene pool is highly susceptible to white rust, whereas the east European germplasm is highly resistant [22,23].In addition, genotypes of B. juncea accessed from Australia and China were more resistant than Indian genotypes at the leaf and/or inflorescence stage [24,25].Earlier, many donor sources such as Donskaja IV, Heera, BIO YSR, BEC-144, and NRCDR 515 were reported and registered as resistant genetic stocks for different isolates of white rust occurring in various states of India.Wild Brassicaceae members such as B. fruticulosa and Thlaspi arvense have also been found to be resistant to white rust, whereas few species of genus Diplotaxis and Sinapis are reported as moderately resistant [26,27].Although several Brassica species have been reported to carry white rust resistance genes, rapid evolutionary pathways of pathogen overcome the prevailing host resistance due to the occurrence of a high level of selection pressure in the present day mustard based cropping system, therefore searching for new resistance gene(s) is always a necessary basic and continuous process [16,18].In the unpredictable climatic change and global warming, inbuilt resistance became imperative to stabilize and sustain the yield potential of Indian mustard cultivars under different growing conditions in India.With this background, in the present study, a set of 25 genotypes of B. juncea were screened under both artificial epiphytotic and field conditions, and four stable resistant and three susceptible genotypes were studied to understand the inheritance pattern of white rust resistance, and the allelic relationship among resistance conferring genes present in resistant genotypes.

Plant Materials
The plant material used in the present investigation includes 25 genotypes of Indian mustard of both indigenous and exotic origin.The details of genotypes, their pedigree, and disease reaction against A. candida Delhi isolate (Ac-Dli) under both controlled and natural field conditions were presented in Table 1.The genotypes were maintained as pure lines by continuous selfing at ICAR-Indian Agricultural Research Institute (IARI), New Delhi, India.

Inoculum Collection, Purification, and Multiplication
The inoculum of white rust zoosporangia (primary inoculum) was collected from highly infected fresh leaves of the decidedly susceptible B. juncea cv.Varuna grown at the experimental farm of IARI, New Delhi.Further, the single pustule method was used for inoculum purification [27] and multiplied on the genetically pure highly susceptible plants of variety Varuna.

Pathogen Inoculation and Disease Development
White rust/blisters from fresh leaves were scrapped by sterile scalpel into petriplates containing sterile double distilled water and allowed to germinate for 3-4 h at 4 • C. Hemocytometer mounted on a simple microscope was used to count the density of zoospores and a population of 7-8 × 10 4 zoospores were maintained for inoculation [28].In artificial epiphytotic conditions, 8-10 days old seedlings at two cotyledon stage were used and 5 µL zoospore suspension was drop inoculated on the adaxial surface of each lobe of cotyledon manually employing a micropipette.The inoculated trays were kept in a humid chamber covered tightly by a thin polythene sheet (700-gauge thickness) for maintenance of relative humidity.The dark condition was maintained inside the plastic chamber for the first 24 h after inoculation by covering the polythene sheet with a light-blocking thick cloth made sheet to enable the onset of disease.Low temperature and high humidity are prerequisites for congenial white rust disease development in Indian mustard cultivars.Therefore, low temperature and high humidity inside the polythene chamber were maintained by supplying water (up to 2-3 cm height) at regular intervals once in 2-3 days and a hand automizer was used to wet the surrounding area of the chamber during the entire period of disease development.
For screening under the natural field conditions, the seeds of parents and three generations, (F 1 , F 2 , BC 1 F 1 ) of four crosses (PM-24 × BEC-144; Pusa Vijay × Donskaja; PM-24 × Pusa Karishma; MSTWR-17-15 × BIO YSR) were raised at the experimental farm area of IARI, New Delhi, where the white rust occurs regularly.All the cultural practices were followed to raise a healthy crop and plants were irrigated manually to maintain soil moisture as and when required.

Experimental Design and Crop Maintenance
The experiments on disease screening were conducted under both artificially controlled epiphytotic and natural field conditions at IARI, New Delhi, India.Initially, 100 plants from each of 25 genotypes were screened at cotyledonary stage in a complete randomized design (CRD) with three replications for two consecutive years (2016-2018) against Ac-Dli isolate of A. candida under controlled conditions.In each year, two sets of genotypes were screened after 10-15 days intervals and scored, and mean values of percent disease index (PDI) were used for classification of disease reaction (Table 1).Additionally, the same set of 25 genotypes was evaluated under field conditions in a randomized complete block design (RCBD) with three replications during 2020-2021.Resistant and susceptible genotypes were identified and selected for further study.In addition, two rows of each parent (PM-24, MSTWR-17-15, Pusa Vijay, Pusa Karishma, BEC-144, BIO YSR, and Donskaja) and F 1 s, three rows of BC 1 F 1 s and fifteen rows of F 2 s were sown and evaluated at true leaf stage under field conditions during rabi 2019-2020 and all the cultural practices were followed to raise a healthy crop stand.
Under artificial epiphytotic conditions, 2-3 untreated seeds each of 25 genotypes were sown in plastic trays (34 × 22 × 9 cm) each having 40 pores of 4 cm diameter.The parents and three generations, (F 1 , F 2 and BC 1 F 1 ) of four crosses (PM-24 × BEC-144; Pusa Vijay × Donskaja; PM-24 × Pusa Karishma; MSTWR-17-15 × BIO YSR); parents and two generations (F 1 and F 2 ) of the single cross (BEC-144 × BIO YSR) were raised and screened in two separate experiments conducted at 20-day intervals with two replications in each experiment.Different population size was maintained for each of parent, F 1 , BC 1 F 1 and F 2 generations depending on the optimum criteria required for the segregation of mendelian genes.Soil mixed with FYM (3:1) was autoclaved (at 121 • C temperature and at a pressure of 106 kPa for 30-60 min) before it was filled in plastic trays for raising the seedlings.In each pore, seeds were sown at the depth of 2-3 cm and lightly covered by sand.The trays were kept in the glasshouse and watered on a regular basis for uniform and vigorous germination.

Disease Scoring
Under field conditions, 60 plants each from P 1 , P 2 , F 1 , BC 1 F 1 and 350 plants in F 2 population were selected and tagged at the seedling stage; however, scoring was initiated at true leaf stage during flowering.In artificial epiphytotic conditions, disease scoring was performed 12-15 days after inoculation.The disease scoring was conducted using 0-9 scale at both cotyledonary and true leaf stages modified from Fox and Williams [29] and the detailed procedure is highlighted in Table 2 and Figure 3.After scoring, the percent disease index (PDI) was calculated, which indicates the percentage of host tissue or plant part covered by lesions or symptoms or damaged by the disease.Disease severity is the result of the number and size of the lesions and expresses the extent of damage caused by the disease.

Statistical Analysis
Disease scores obtained from the artificial epiphytotic and field conditions at the cotyledonary and true-leaf stages were analyzed using Microsoft Excel.The mean values of selected and tagged plants at each replication were used for statistical analysis.Genotypic mean values were also compared using critical differences at a 5% level of significance.The Chi-square test was used for the analysis of goodness of fit in different breeding populations by comparing observed and expected frequencies [29].The PDI can be calculated using the following formula: PDI = sum of all numerical ratings (number of cot yledons or leaves scored × maximum grade of scale) × 100

Statistical Analysis
Disease scores obtained from the artificial epiphytotic and field conditions at the cotyledonary and true-leaf stages were analyzed using Microsoft Excel.The mean values of selected and tagged plants at each replication were used for statistical analysis.Genotypic mean values were also compared using critical differences at a 5% level of significance.The Chi-square test was used for the analysis of goodness of fit in different breeding populations by comparing observed and expected frequencies [29].

Inheritance of White Rust Disease Resistance
Among the six genotypes which were highly resistant under both natural and artificial screening, only the four genotypes (BEC-144, Donskaja, Pusa Karishma and BIO YSR) were taken for inheritance studies.The results of disease reaction in the different parents and breeding generations (F 1 , F 2 and BC 1 F 1 ) of four crosses (PM-24 × BEC-144, Pusa Vijay × Donskaja, PM-24 × Pusa Karishma and MSTWR-17-15 × BIO YSR) against A. candida Ac-Dli isolate in natural and artificial epiphytotic conditions are illustrated in Table 3.In these crosses, PM-24, Pusa Vijay and MSTWR-17-15 were used as susceptible parents.Under the field conditions, 60 plants used for recording disease reaction of each parent viz., PM-24 and BEC-144 were found susceptible and resistant, respectively and 60 F 1 plants of this cross exhibited resistance to the disease (Table 3).The complete susceptibility of PM-24 against Ac-Dli isolate suggested the presence of susceptible allele in this genotype.All the F 1 plants were resistant to Ac-Dli isolate, indicating the dominant nature of resistance over susceptibility.Among 350 F 2 plants, 277 were resistant and 73 were susceptible with the best fit for a 3:1 R/S ratio (χ 2 = 3.204; p = 0.073), indicating that resistant donor BEC-144 carries a single dominant gene.Further, this monogenic dominant nature of resistance gene was also confirmed from the result of the backcross population as well.The 60 BC 1 F 1 plants were segregated into 35 resistant and 25 susceptible plants, which was a good fit for 1R:1S ratio (χ 2 = 1.667; p = 0.197), suggesting that trait is controlled by monogenic dominance gene.In two independent experiments under artificial epiphytotic conditions also both the parents and F 1 plants responded similarly to Ac-Dli isolate as under the field condition.In experiment-I, 434 plants were resistant, and 158 plants were susceptible in F 2 generation, while 530 and 195 plants were resistant and susceptible, respectively in experiment-2.In the BC 1 F 1 generation, 137 and 128 plants in experiment-1, 64 and 45 plants in experiment-2 were resistant and susceptible, respectively.Therefore, the segregation in F 2 and BC 1 F 1 was best fit to 3:1 R/S and 1:1 R/S ratio, respectively in experiment-1 (χ 2 F2 = 0.901; χ 2 BC1F1 = 0.306) and experiment-2 (χ 2 F2 = 1.391; χ 2 BC1F1 = 3.352) imparting that the resistance is governed by single dominant gene in the resistant genotype BEC-144.

Cross-II: Pusa Vijay × Donskaja
Under the field conditions, 60 plants each of Pusa Vijay and Donskaja were found susceptible and resistant, respectively; 60 F 1 plants were resistant at true leaf stage, imparting that resistance is governed by dominant gene(s) in Donskaja (Table 3).In the F 2 generation, 280 plants were resistant, and 70 plants were susceptible to Ac-Dli isolate and do not fit to 3:1 R/S ratio (χ 2 = 4.667; p = 0.031), inferring that resistance is governed by more than one dominant gene.In BC 1 F 1 , 38 plants were resistant, and 22 plants were susceptible to Ac-Dli isolate and do not fit to 1:1 R/S ratio (χ 2 = 4.267; p = 0.039), confirming the earlier finding based on segregation in F 2 .Under artificially controlled epiphytotic conditions, in the experiments 1 and 2, both parents and F 1 plants showed similar response to Ac-Dli isolate as it was found under the field condition.In F 2 population, 658 and 129 plants were resistant and susceptible in experiment-1 and 550 and 110 plants were resistant and susceptible in experiment-2, respectively; however, the segregating F 2 population does not fit the 3:1 R/S ratio (χ 2  Exp-1 = 31.106;χ 2 Exp-2 = 24.444).In BC 1 F 1 generation also, 70 plants were resistant, and 40 plants were susceptible in experiment-1; and 62 plants were resistant and 33 plants were susceptible and do not fit to1:1 R/S ratio (χ 2  Exp-1 = 9.713; χ 2 Exp-2 = 8.853).Based on the segregation pattern of resistance in F 2 and BC 1 F 1 it can be inferred that the resistance is governed by more than one dominant gene in the resistant genotype Donskaja.

Cross-III: PM-24 × Pusa Karishma
Under the field conditions, 60 plants of PM-24 were susceptible, and 60 plants of Pusa Karishma were resistant to Ac-Dli isolate of A. candida at true leaf stage (Table 3).The 60 F 1 plants were also found resistant signifying the dominant nature of resistance over susceptibility.In F 2 population, 250 plants were resistant, and 100 plants were susceptible, which were best fitted into 3:1 R/S ratio (χ 2 = 2.381; p = 0.123), highlighting that the resistance is governed by a single dominant gene.Further, 28 plants were resistant, and 32 plants were susceptible in BC 1 F 1 and well-matched to 1:1 R/S ratio (χ 2 = 0.267; p = 0.606), confirming the findings of F 2 generation.Under artificially controlled epiphytotic conditions, both parents and F 1 plants exhibited similar disease reaction as under field conditions.In addition, 264 and 84 plants in F 2 generation and 55 and 44 plants in BC 1 F 1 population were reported as resistant and susceptible to the isolate and were best fitted to 3:1 R/S (χ 2 F2 = 0.138; p = 0.710) and 1:1 R/S (χ 2 BC1F1 = 1.222; p = 0.269) ratio in experiment-1, respectively.Likewise, the F 2 and BC 1 F 1 generations of the cross followed the monogenic mendelian segregation and were best fitted to 3:1 R/S (χ 2 F2 = 1.410; p = 0.235) and 1:1 R/S (χ 2 BC1F1 = 3.139; p = 0.076) ratio, respectively, in experiment-2, confirming that resistance is governed by a single dominant gene in the resistant genotype-Pusa Karishma.
(score = 0) against Ac-Dli isolate at the cotyledonary stage under controlled epiphytotic conditions.Likewise, five genotypes, including BEC-144, Heera and BIO YSR of B. juncea were found highly resistant at true leaf stage under field conditions [32].Similarly, Yadav et al. [33] reported 27 Indian mustard germplasm accessions with resistance reaction at Hisar, Ludhiana and Pantnagar under field conditions and eight of them were identified as highly resistant to "Delhi isolate" of A. candida at both cotyledonary and true leaf stages under artificial conditions.In the present study, four genotypes (PDZ-1, PDZ-4, PDZ-5, EC-399299) were resistant at true leaf stage under field conditions but were susceptible at cotyledonary stage under controlled conditions, highlighting that there is no chance for the seedlings to escape from the disease infection in controlled conditions where proper temperature and humidity were maintained, which is highly congenial for disease development.Similarly, it was testified at Pantnagar that the advanced breeding lines of B. juncea were completely free from disease (score = 0) under field conditions, whereas under glass house the same lines were found to show variable disease response (score = 0-5) at cotyledonary and true leaf stages [34].In the present study, 19 genotypes were categorized from moderately susceptible to highly susceptible groups based on disease severity under both natural and artificial screening conditions (Table 1), highlighting that these genotypes do not possess any resistance gene(s), therefore the pathogen establishes very well due to compatible pathogen-host interaction leading to heavy incidence of disease.Similarly, Singh et al. [16] reported that 13 out of 30 genotypes of B. juncea studied by them have been reported to be highly susceptible (score = 6) to Ac-Dli isolate at cotyledonary stage under controlled conditions.The wide variation in susceptible disease reaction of different genotypes against pathogens might be due to the differential expression of resistance gene(s) and genetic background of genotypes that affects genotype-pathogen interaction [16].The overall disease on the susceptible genotypes was higher in the experimental year 2017-2018 than in 2016-2017 at the cotyledonary stage in controlled conditions.Environment, both micro and macro, plays a vital role in changing the dynamics of host-pathogen interaction and ultimately affects disease severity [35].As the inoculum from the fresh symptomatic leaves of infected plants from the first year was used to multiply the pathogen in the next year, the disease severity in the second year might be high due to adaption and acclimatization of the pathogen on the host.In addition, day and night temperature fluctuations (16-24 • C) at different growth stages of host plants might affect disease severity of A. candida [36,37].Therefore, genotypes must be tested at both cotyledonary and true leaf stages in the controlled conditions before evaluating them in the field conditions to identify reliable and stable resistance source/s against white rust disease.
In the present inheritance study, all the F 1 s developed from S × R crosses displayed complete resistance against the Ac-Dli isolate, inferring the dominant nature of resistance over susceptibility in all the resistant genotypes.In earlier studies also, F 1 s derived from S × R crosses were found resistant to Ac-Dli isolate of A. candida under epiphytotic field conditions and reported dominance type of resistance in Indian mustard genotypes [32,38].In addition, previous studies revealed that susceptible genotypes lack the resistant alleles and are thus more vulnerable to white rust disease [39][40][41][42].Likewise, resistance alleles were described in different B. juncea genotypes such as Vniimik-405 [39], J90-2733 [40], BEC-144 [43], BEC-286 [44], NPC-12 [45], Heera and Donskaja IV [22], BIO YSR, EC-399301, JM-1 and JM-2 [46], JMY11 [42] and EC-399299 [32] for different isolates of A. candida in different environmental conditions across India.In the present study, the F 2 generations of all S × R crosses (PM-24 × BEC-144, PM-24 × Pusa Karishma, MSTWR-17-15 × BIO YSR), except Pusa Vijay × Donskaja, were best fitted to 3:1 R/S ratio (p > 0.05), signifying that the resistance is governed by a single dominant gene in all three resistant genotypes (BEC-144, Pusa Karishma and BIO YSR).Further, it was reconfirmed by employing the corresponding BC 1 F 1 generation where resistant and susceptible plants segregated and fitted into 1:1 R/S ratio (p > 0.05).Likewise, Singh et al. [47] reported a single dominant gene by analyzing the different breeding generations derived by crossing susceptible and resistant genotypes against AcB1 isolate of A. candida.In the present study, the segregation of resistant and susceptible plants in F 2 generation of S × R cross (Pusa Vijay × Donskaja) did not fit to 3:1 R/S ratio (p < 0.05), concluding that resistance is controlled by more than one dominant gene in resistant parent Donskaja.It was also reconfirmed using their BC 1 F 1 generation in which resistant and susceptible plants did not segregate into 1:1 R/S (p < 0.05) ratio against the Ac-Dli isolate.Likewise, two duplicate dominant resistance genes for Ac-Dli isolate were reported in B. napus by screening the breeding populations developed by crossing S × R (BN-38 Sel.× BN-Sel.)genotypes [48].Similarly, digenic inheritance was proposed in segregating generations that were developed by S × R cross (Varuna of B. juncea × ISN 706 of B. napus) against Ac-Dli isolate of A. candida [49].However, several earlier studies have also reported that resistance to white rust has been governed by a single dominant gene in different genotypes of B. juncea [22,30,[39][40][41]43,44,47].
In the present study with four resistance sources (BEC-144, Pusa Karishma, BIO YSR and Donskaja) it was revealed that the resistance against the Ac-Dli isolate was governed by a single dominant gene in BEC-144, Pusa Karishma, and BIO YSR, whereas more than one dominant gene is controlling the resistance in Donskaja, a widely used donor source.These resistance sources could be more rewarding if they carry different resistance genes and could be employed for achieving durable resistance against multiple isolates of A. candida by gene pyramiding through backcrossing.By keeping this view, test of allelism was conducted by crossing the two stable resistant genotypes BEC-144 and BIO YSR, identified in this study, under artificial epiphytotic conditions against Ac-Dli isolate.Interestingly, no segregation (all plants were resistant) in F 2 generation of cross R × R (BEC-144 × BIO YSR) was observed, indicating that the same gene is governing resistance (i.e., allelic to each other) in both the resistant parents, BEC-144 and BIO YSR.In similar studies, allelic relationship between resistance genes was reported in F 2 generations of direct and reciprocal crosses of R × R genotypes (BEC-144, BEC-286, EC399299, Heera and BIO YSR) which were screened against Ac-Dli isolate [32].In contrast, two dominant genes showing duplicate gene interaction were reported in two resistant B. juncea genotypes, BIO YSR and NPC-12, against Ac-Dli isolate [45].Further, two independent loci governing resistance to A. candida race 2V (AcB1) were also tagged in two east European B. juncea resistant genotypes viz., Heera and Donskaja-IV [22].
It was reported earlier that only the European genotypes of B. juncea showed resistance to white rust whereas genotypes from the Indian gene pool were all susceptible [22,50].Therefore, two resistant genotypes, BEC-144 and Donskaja, exotic collection from Europe, were widely used in Indian resistant breeding programs with two other resistant Indian genotypes Pusa Karishma and BIO YSR (developed in India which may or may not have a European background).The pedigree of two resistant genotypes BEC-144 and BIO YSR highlighted that they have diverse genetic background, however, they were found to have the same gene for white rust resistance against the Ac-Dli isolate.Nevertheless, these two genotypes may not be rejected as they might have additional genes for different isolates of A. candida, which has to be revealed in further studies.The Donskaja (score = 0) and Pusa Karishma (score = 0) were found to be resistant for most of the isolates of A. candida collected from the different parts of India when screened under controlled conditions at ICAR-IARI, New Delhi, India [16], whereas, Pusa Vijay (score = 6) and PM-24 (score = 6) were found to be susceptible to Ac-Dli isolate under artificially controlled conditions [16].
It is very much essential to identify diverse resistance genes for white rust and pyramid them in elite breeding lines through breeding.In the fast-changing climatic conditions, it is imperative to identify diverse resistance genes in any crop species to battle the ever-evolving pathogens.In India, in recent times, extensive efforts are underway to horizontally expand the area under mustard cultivation in the non-traditional areas (eastern and southern parts of the country) under a rice-fallow system with a long-term objective of diversifying the existing cropping system.It, therefore, necessitates continuous availability of donor parents with a high level of resistance to white rust.This objective might be accomplished by screening a large set of diverse genotypes/germplasm accessions for different isolates/races of the pathogen under controlled conditions followed by field experiments.Pyramiding the resistance genes would help in developing durable resistance for most of the A. candida races infecting the rapeseed mustard cultivars grown in different states of India.More resistance genes in the host would delay the appearance to new races of the pathogen as the pathogen needs more virulent genes to overcome the resistance level of the host due to its low fitness and reproductivity [51,52].It is well accepted and proven throughout the world that genetically resistant genotypes are a more durable, reliable, cost effective, and environment-friendly approach to manage biotic stresses, including diseases and pests that adversely affect crop production and quality [18,53].

Conclusions
The presence of more than one resistance gene in the Donskaja genotype, as identified in this study, will be useful in breeding for durable resistance by discovering new genes for resistance and further pyramiding them.However, there is a dire need to study the virulence spectra and diversity in A. candida with respect to these host resistant genes under controlled condition.The present study also revealed that the resistance to Ac-Dli isolate of A. candida is monogenically inherited in both the resistant sources, i.e., BEC-144 and BIO YSR, and the resistance genes present in them are allelic.The backcross breeding program would be rewarding for easy transfer of single genes providing white rust resistance to the well-adapted, high-yielding genotypes lacking disease resistance.Therefore, the information generated in the present study could be used in mustard breeding programs for the development of cost-effective and durable resistance cultivars.

Figure 1 .
Figure 1.Life cycle of white rust (Albugo candida) on Indian mustard.(A) Asexual phase and (B) Sexual phase.

3 5 FewFigure 3 .
Figure 3. Photographs depicting the disease rating scales (0-9 scale) used for screening parental genotypes and segregating populations against white rust at the cotyledonary (A) and true leaf stage (B).Values in parentheses denote the percent area of leaf covered by white blisters of disease.

Figure 3 .
Figure 3. Photographs depicting the disease rating scales (0-9 scale) used for screening parental genotypes and segregating populations against white rust at the cotyledonary (A) and true leaf stage (B).Values in parentheses denote the percent area of leaf covered by white blisters of disease.

Table 1 .
Screening of Indian mustard genotypes against Delhi isolate (Ac-Dli) of Albugo candida under artificial epiphytotic and natural conditions.

Table 2 .
[29]ng scale (0-9) for measuring disease severity of white rust disease at cotyledonary and true leaf stage in Indian mustard (modified from Fox and Williams)[29].

Table 2 .
[29]ng scale (0-9) for measuring disease severity of white rust disease at cotyledonary and true leaf stage in Indian mustard (modified from Fox and Williams)[29].

Table 3 .
Segregation pattern of resistance to Albugo candida isolate Ac-Dli in the crosses involving susceptible and resistant parents under natural and artificial epiphytotic conditions at IARI, New Delhi, India, during 2019-2022.