Association of fusarium wilt susceptibility with clubroot resistance derived from winter Brassica napus L. ‘Mendel’

Abstract

The development of canola cultivars with resistance to soil-borne diseases, such as clubroot and fusarium wilt, is important for successful production of this crop. A clubroot resistance gene on the A3 chromosome of the European winter Brassica napus L. cultivar ‘Mendel’ has been introgressed into Canadian spring canola for the development of clubroot resistant cultivars. However, the spring canola lines carrying this resistance were found to be susceptible to fusarium wilt disease, which is an impediment for use of this resistance source in breeding. Clubroot and fusarium wilt resistance were analysed using a doubled haploid (DH) population derived from crossing spring canola lines carrying ‘Mendel’-based clubroot resistance with canola lines resistant to fusarium wilt. This analysis showed that a Mendelian gene was involved in the control of resistance to each disease. However, testing for independent assortment of these two traits showed a significant deviation from the expected 1:1:1:1 segregation, indicating that a genetic linkage exists between the loci governing resistance to these two diseases. The occurrence of about 10% of the lines with recombinant phenotypes suggested that these two loci are located about 10 cM apart; this demonstrates the possibility of developing a canola line carrying resistance to both diseases.

Résumé: Le développement de cultivars de canola résistants aux maladies terricoles, telles que la hernie et la fusariose, est essentiel aux bons rendements de cette culture. Un gène de résistance à la hernie, situé sur le chromosome A3 du cultivar d’hiver européen de Brassica napus L. ‘Mendel’, a été introgressé dans le canola de printemps canadien dans le but de développer des cultivars résistants à la hernie. Toutefois, les lignées de canola de printemps porteuses de la résistance se sont avérées réceptives à l’égard de la fusariose, ce qui constitue un obstacle à l’utilisation de cette source de résistance dans le cadre du processus de sélection. La résistance à la hernie et à la fusariose a été analysée à l’aide d’une population dihaploïde dérivée du croisement des lignées de canola de printemps porteuses de la résistance à la hernie issue du cultivar ‘Mendel’ avec les lignées de canola résistantes à la fusariose. Cette analyse a permis de démontrer qu’un gène mendélien était impliqué dans la gestion de la résistance à chaque maladie. Par ailleurs, un test visant l’assortiment indépendant de ces deux traits a affiché une déviation significative du taux de ségrégation attendu de 1:1:1:1, indiquant qu’une liaison génétique existe entre les locus régissant la résistance à ces deux maladies. L’occurrence d’environ 10% des lignées comportant les phénotypes recombinants a suggéré que ces deux locus sont situés à environ 10 cM l’un de l’autre, ce qui prouve la possibilité de développer une lignée de canola porteuse de la résistance aux deux maladies.

Keywords:

• Clubroot resistance
• fusarium wilt resistance
• linkage
• recombination

Mots clés:

• liaison
• recombinaison
• résistance à la fusariose
• résistance à la hernie

Introduction

Among the different soil-borne diseases of oilseed Brassica napus, clubroot disease caused by Plasmodiophora brassicae Wor. (Howard et al., 2010) and fusarium wilt disease caused by Fusarium oxysporum f. sp. conglutinans (Wollenweb.) (Snyder & Hansen, 1940) are two of the most important. Infection of canola roots by P. brassicae results in the formation of galls which restrict the supply of water and nutrients to the above-ground parts of the plant and this can result in yield loss of about 30% (Tewari et al., 2005); however, almost complete loss of the crop has been reported in a few severely infested fields (for review, see Strelkov & Hwang, 2014). Fusarium wilt in canola causes chlorosis, wilting and stunted growth, and may result in severe yield loss and plant death. Lange et al. (2007) reported that yield loss due to this disease in spring B. napus canola can be about 15–75%, depending on the cultivar and the extent of infection.

Clubroot resistance in Brassica oilseed and vegetable crops is often controlled by a major dominant gene located on the A genome (for review, see Piao et al., 2009). The major dominant gene of the A3 chromosome in the European winter B. napus cultivar ‘Mendel’ has been extensively used in Canada for the development of clubroot resistant cultivars (Rahman et al., 2011; Fredua-Agyeman & Rahman 2016). Genetic analysis of fusarium wilt resistance in B. napus suggests that a major dominant gene is involved in the control of this trait (Klassen & Fernando, 2007; Lange et al., 2010). Genetic analysis and molecular mapping of resistance in B. oleracea also showed that resistance to fusarium wilt is controlled by a major dominant gene (Pu et al., 2012; Lv et al., 2014). Pu et al. (2012) mapped the major dominant gene to chromosome C7 (≈ O7); they also mapped a minor QTL to chromosome C4 (≈ O4). On the other hand, Lv et al. (2013, 2014) mapped the major dominant gene to C6. Fusarium wilt resistance has also been reported in B. rapa (A genome) (Fjellstrom & Williams, 1997). According to Shimizu et al. (2014), resistance to this disease in B. rapa is also controlled by a single dominant gene; however, knowledge of the genomic location of this resistance in the A genome is scarce in the literature. Lange et al. (2010) provided evidence that the gene controlling this trait in B. napus is located in the A genome chromosome A3.

The objective of this study was to understand the genetic basis of resistance to clubroot and fusarium wilt in spring B. napus canola utilizing populations segregating for resistance to these two diseases.

Materials and methods

Plant materials

A total of 80 doubled haploid (DH) lines, derived from crossing two advanced generation spring B. napus canola breeding lines carrying clubroot resistance and fusarium wilt susceptibility with a single spring B. napus canola breeding line susceptible to clubroot but resistant to fusarium wilt, were used in this study. The details regarding the production of the DH lines are described in Fredua-Agyeman & Rahman (2016). In addition to these DH lines, 51 advanced generation breeding lines derived from crossing clubroot resistant fusarium wilt susceptible lines with clubroot susceptible fusarium wilt resistant lines were also included. Clubroot resistance in all parental lines was derived from the European winter B. napus cultivar ‘Mendel’ (Rahman et al., 2011).

Clubroot resistance evaluations

All 131 lines were evaluated for clubroot and fusarium wilt resistance in a greenhouse or growth chamber, or in field plots. For the clubroot disease reaction testing in the greenhouse (20–22ºC day/15ºC night and 16 h photoperiod), the P. brassicae single spore isolate SACAN-ss1 (classified as pathotype 3 following Williams (1966) and obtained from Dr Stephen Strelkov, University of Alberta) was used. The details of plant growth medium, pot size, watering, fertilization, inoculation and disease scoring are reported elsewhere (Fredua-Agyeman & Rahman, 2016). For field testing, the experimental materials were grown in a P. brassicae infested field located at Leduc, Alberta either in 2013 or in 2014; the predominant pathotype in this field was pathotype 3. Seeding was done in 3-m-long one-row plots with the susceptible check cultivar ‘Hi-Q’ in every 10th plot. Scoring for clubroot resistance was done at the flowering stage; for this, 25 plants from each plot were uprooted and scored on a 0–3 scale as described in Rahman et al. (2011) and the lines were categorized as resistant or susceptible.

Fusarium wilt resistance evaluations

Testing for fusarium wilt resistance in growth chamber assays included only DH lines, while field testing was done on both DH and advanced generation breeding lines. Growth chamber testing was carried out following the Screening for Fusarium Wilt Disease in Canola – Indoor Screening (Method 1) protocol listed in Appendix B of the Procedures of the Western Canada Canola/Rapeseed Recommending Committee (WCC/RRC) incorporated for the Evaluation and Recommendation for Registration of Canola/Rapeseed Candidate Cultivars in Western Canada (Franke et al., 2010). Ten seedlings of each DH line were grown in fine-grained industrial quartz sand (Granusil® 4020, Unimim Canada Ltd, Quebec) contained in a metal steamer tray which was placed in a 24°C water bath inside a growth chamber (PGR15, Conviron, Winnipeg, MB) under controlled conditions (16 h photoperiod with temperatures of 22°C day/20°C night). At 10–12 days after seeding, seedlings were gently removed from the sand, roots rinsed with sterile deionized water, and inoculated by submerging roots in a 5 × 10⁶ conidia mL⁻¹ spore suspension of F. oxysporum f.sp. conglutinans (a mixture of equal concentrations of F. oxysporum stock cultures F05 and F08, accessions 65 and 70, respectively, kindly provided by Dr Ralph Lange of InnoTech Alberta, Vegreville, AB) for one hour prior to gently re-planting in the sand. The cultivars ‘Lolinda’ and ‘SP Banner’ were used as susceptible and resistant checks, respectively. The inoculated seedlings were then returned to the growth chamber and watered as required until scoring. Disease severity scoring was done as described by Franke et al. (2010) approximately 2 weeks after inoculation on a 0–9 scale, where 0 = no stunting and no disease symptoms on the seedlings; 1 = slight stunting and slight chlorosis of leaves; 3 = moderate stunting and most tissue chlorotic; 5 = moderate stunting, severe chlorosis and some necrosis; 7 = severe stunting and most tissue necrotic; 9 = dead plants. The average disease severity score for each line was calculated, and lines with a disease score of less than 3.0 were considered as resistant.

Field testing for fusarium wilt resistance was carried out in a naturally infested fusarium wilt disease nursery located near Star City, Saskatchewan. Trials were planted in a Randomized Complete Block Design (RCBD) with a minimum of three replicates and with a plot size of 2 × 3 m rows for each entry, and included susceptible check cultivar 45A55 and resistant check cultivar ‘SP Banner’. Trials were seeded in mid to late spring either in 2012 or in 2013, and plants were visually assessed for fusarium wilt resistance at the four-leaf stage, and then again after silique set, using the following classifications: S (susceptible) = conspicuous wilt symptoms equal to or worse than those on the S check in all replicates; I = inconsistent disease reaction among replicates; R (resistant) = no wilt symptoms observed in all replicates. Entries classified as ‘I’ in the field test were re-tested in a growth chamber assay as described above to confirm disease reaction.

Results

A total of 57 DH lines were evaluated in a greenhouse or growth chamber for resistance to clubroot and fusarium wilt resistance. Of these, 32 were resistant (disease score 0 or 1) and 25 were susceptible (disease score 2 or 3) to clubroot disease. In the case of fusarium wilt, 22 lines were resistant (average disease score < 3.0) and 35 were susceptible (disease score > 3.0). Thus, segregation for resistance to these two diseases, independently, followed a 1:1 segregation (P > 0.05), as could be expected for a trait controlled by a Mendelian gene (Table 1). However, in testing for independent assortment of these two traits, segregation deviated significantly from the expected 1:1:1:1 ratio (χ² = 36.26, P < 0.001), where about 90% of the lines exhibited the parental phenotypes, i.e. clubroot resistant + fusarium susceptible or clubroot susceptible + fusarium resistant. Recombination between these two loci, however, occurred showing the possibility of recovering about 5% lines with resistance to both diseases.

Table 1. Segregation for clubroot and fusarium wilt resistance in a doubled haploid (DH) population derived from a cross between a clubroot susceptible (CS) fusarium wilt resistant (FWR) Brassica napus line and a clubroot resistant (CR) fusarium wilt susceptible (FWS) B. napus line, and tested in greenhouse and growth chamber experiments.

CR		CS		CR: CS 1:1		FWR: FWS 1:1		Independent assortment 1:1:1:1
FWR	FWS	FWR	FWS	χ^2	P	χ^2	P	χ^2	P
2	30	20	5	0.86	0.35	2.97	0.09	36.26	< 0.001
(3.5%)	(52.6%)	(35.1%)	(8.8%)

In addition to the above-mentioned DH lines, a total of 74 DH and advanced generation breeding lines were also tested for resistance to these two diseases in the greenhouse and in field plots. In this case also, about 90% of the lines were found to be of the parental type and about 10% were recombinants (Table 2). Thus, the results from growth chamber, greenhouse and field experiments demonstrate that resistance to P. brassicae pathotype 3 in the winter B. napus cultivar ‘Mendel’ is linked to fusarium wilt susceptibility and these two loci are located about 10 cM apart.

Table 2. Clubroot and fusarium wilt resistance in a doubled haploid (DH) and different advanced generation populations, derived from cross between clubroot susceptible (CS) fusarium wilt resistant (FWR) Brassica napus and clubroot resistant (CR) fusarium wilt susceptible (FWS) B. napus lines, tested in greenhouse (GH) and field plots.

Population	Clubroot/ fusarium test	CR		CS
		FWR	FWS	FWR	FWS
DH	GH/Field	0	13	8	2
Adv. lines	GH/Field	3	14	3	0
Adv. lines	Field/Field	1	5	24	1
Total		4	32	35	3
		5.4%	43.2%	47.3%	4.1%

Discussion

The canola cultivars currently grown commercially in Canada and Europe are mainly F₁ hybrids. For the development of a clubroot and fusarium wilt resistant F₁ hybrid, theoretically, a parent line carrying the dominant clubroot resistance gene from ‘Mendel’ in the homozygous state but susceptible to fusarium wilt can be combined with a parent line carrying a dominant gene for fusarium wilt resistance in the homozygous state but susceptible to clubroot. This will result in the F₁ hybrid heterozygous for both disease resistance genes which should exhibit resistance to both diseases. However, use of these types of parental lines in commercial hybrid seed production will leave the production field at risk of crop loss due to the occurrence of fusarium wilt disease. Data presented from this study demonstrate that the clubroot resistance from ‘Mendel’ can be combined with fusarium wilt resistance, and use of this type of recombinant inbred line in hybrid breeding will eliminate the risk of crop loss due to fusarium wilt disease.

The clubroot resistance gene from the cultivar ‘Mendel’ is located on the A3 chromosome of B. napus (Fredua-Agyeman & Rahman, 2016). This chromosome has been reported to carry multiple clubroot resistance loci, such as CRa, CRb, CRk, Crr3, CRd and Rcr1/Rcr2. However, recent studies have shown that CRa and CRb are the same locus (Kato et al., 2013; Hatakeyama et al., 2017) and Rcr1 (≈ Rpb1)/Rcr2 is located in the same genomic region as CRa (Huang et al., 2017). On the other hand, the CRk and Crr3 loci are located at different positions, in proximity to each other, about 35 cM apart from CRa/CRb (Sakamoto et al., 2008), while CRd is located about one cM from Crr3 (Pang et al., 2018). The clubroot resistance from ‘Mendel’ is located in the CRa/CRb region (Fredua-Agyeman & Rahman, 2016); the linkage association of this resistance with fusarium wilt susceptibility indicates that the genomic region of A3 carrying the CRa/CRb also carries a locus associated with resistance to fusarium wilt; however, these two loci are about 10 cM apart, which allows potential recombination to occur between the genes.

Previous studies on molecular mapping of fusarium wilt resistance in the Brassica A genome is scarce. According to Lange et al. (2010), the chromosome A3 of B. napus carries a locus controlling resistance to this disease. Mapping of this trait in the C genome (Pu et al., 2012; Lv et al., 2013, 2014) showed that the genomic region of C7 (≈ O7) carrying the major dominant gene for fusarium wilt resistance (Pu et al. 2012) is co-linear with the genomic region of A3 (≈ R3) of B. rapa where the clubroot-resistance gene CRb is located (Nagaoka et al., 2010), and this provides further evidence that chromosome A3 carries a fusarium wilt resistance gene. However, linkage association of clubroot resistance from ‘Mendel’ with fusarium susceptibility, as reported in the present study, provides evidence that the genomic region of A3 carrying clubroot resistance also carries a fusarium wilt resistance gene.

Acknowledgements

The authors thank Drs Rudolph Fredua-Agyemann and Abdus Shakir and Mr Jose Salvador Lopez for assistance in disease resistance tests in greenhouse and field, and other staff of the Canola Program of the University of Alberta and Crop Production Services for assistance in other routine operations.

Additional information

Funding

This work was supported by the Alberta Canola Producers Commission [2007F066R] and [2010C014R] and Alberta Crop Industry Development Fund [2007F066R] and [2010C014R].