Introduction

Sclerotinia stem rot, caused by the fungal pathogen Sclerotinia sclerotiorum, is one of the most serious diseases in rapeseed (Brassica napus) (Dunker and Tiedemann Dunker and von Tiedemann 2004; Hind et al. 2003; Lamey 2003; Pope et al. 1989). In the field, the stem and pod of rapeseed are infected by this pathogen, resulting in yield loss. To breed resistant rapeseed variety is an economical and ecological sustainable way to control this disease, but complete resistance is unavailable in current rapeseed.

Brassica napus (AACC), originated from interspecific hybridization between B. rapa (AA) and B. oleracea (CC). It has a narrow genetic basis partly due to its intensive modern breeding and short history of origination and domestication (Becker et al. 1995, Seyis et al. 2003). In contrast, the parental species B. oleracea, including various cultivated and wild types, shows a much wider genetic diversity (Mei et al. 2011a; Snogerup et al. 1990) and harbors some elite traits such as high resistance against Peronospora parasitica (Mithen et al. 1987; Mithen and Magrath 1992), Aleyrodes proletella (Ramsey and Ellis 1994) and Delia radicum (Ellis et al. 1999). More recently, we have found high levels of resistance against Sclerotinia in B. oleracea cytodeme, such as B. rupestris, B. incana, B. insularis and B. villosa (Mei et al. 2011b).

Resynthesis of a rapeseed line (RS line) is an effective strategy to improve current rapeseed via widening genetic diversity (Girke et al. 2012a), enhancing heterotic potential (Girke et al. 2012b; Udall et al. 2004), and transferring elite traits from parental species (Abawi et al. 1975; Crouch et al. 1994; Diederichsen and Sacristan 1996; Dreyer et al. 2001; Happstadius et al. 2003; Walsh et al. 1999). In order to verify the hypothesis that the Sclerotinia resistance of rapeseed can be improved using resynthesized B. napus, a panel of RS lines derived from various types of B. oleracea was screened and the resistance against S. sclerotiorum was compared with the resistance of the parental B. oleracea as identified in our previous research (Mei et al. 2011b). Our data suggest that the Sclerotinia stem resistance of rapeseed can be improved by using B. oleracea.

Materials and methods

Plant materials

A panel of 55 RS lines, collected from Göttingen University and Southwest University (Supplement Table 1), were sown in two crop seasons, 2010–2011 and 2011–2012, in the experimental field of Southwest University, Chongqing (China). A randomized complete block design with two replications was employed, twenty plants of each plot in two rows, with 30 cm between rows and 25 cm within rows. The donors of C subgenome of the RS lines were seven wild and two cultivated types of B. oleracea, which had been identified as resistant against Sclerotinia in our previous research (Mei et al. 2011b).

Resistance assessment

The S. sclerotiorum isolate utilized in Mei et al. (2011b) was used in this study. The plugs (6 mm in diameter) punched from the growing margin of 3-day-old culture of S. sclerotiorum on PDA (potato dextrose agar, 20 % potato, 2 % dextrose and 1.5 % agar) were used as inoculums in resistance evaluation.

The fourth leaves at nine-to-twelve-leaf stage and stems at flowering stage were detached in the field plots and used to evaluate Sclerotinia resistance according to the method of Mei et al. (2011b, 2012). Briefly, an artificial inoculation was performed in closed inoculation chamber in the laboratory, in which the infection temperature and humidity was maintained at 22 °C and 95 %, respectively. Three individuals of each RS line were tested in each evaluation. The lesion size of inoculated leaves and lesion length of inoculated stems 3 days after inoculation (DAI) were collected.

Statistical analysis

A registered rapeseed cultivar in China, ‘Zhongyou 821’, with partial resistance against S. sclerotiorum was used as resistant control. Relative susceptibility (S) to ‘Zhongyou 821’ was calculated based on the equation S = V/V control , where V is the value of the accession tested for leaf (lesion size) or stem resistance (lesion length), while V control is that of ‘Zhongyou 821’.

Analysis of variance (ANOVA) was conducted using the general linear model procedure with SAS, version 6.07 (SAS Institute 1992). Pearson’s simple correlation coefficients were calculated between variables of interest.

Results

The resistance was screened in stem and leaf among RS lines (Fig. 1). The size of lesion in leaf ranged from 11.53 to 19.60 cm2 in 2010 and from 5.84 to 11.34 cm2 in 2011, while the length of lesion in stem ranged from 3.51 to 7.45 cm in 2010 and from 4.12 to 8.23 cm in 2011. In order to compare the resistance of RS lines across 2 years, a relative susceptibility to ‘Zhongyou 821’ was calculated (supplement Table 1; Fig. 2). The average relative susceptibility of the RS lines was 1.11 and 0.77 in leaf and stem, respectively, ranging from 0.88 to 1.36 in leaf and from 0.42 to 1.02 in stem. It indicates that RS lines have stronger resistance in stem than in leaf relative to ‘Zhongyou 821’.

Fig. 1
figure 1

Symptoms 3 days after inoculation in leaf (A) and stem (B) of partial resistant check ‘Zhongyou 821’, parental line of B. oleracea, resynthesized line of B. napus and parental line of B. rapa from left to right

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Fig. 2
figure 2

The relative susceptibility to ‘Zhongyou 821’ in leaf and stem among resynthesized lines of B. napus

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Table 1 shows the result of ANOVA for the relative susceptibility among RS lines. Significant differences for leaf and stem resistance were detected among RS lines, as well as among types of parental B. oleracea (P < 0.01). Significant differences of resistance for year-by-leaf interaction (P < 0.01), but not for year-by-stem interaction (P = 0.37) were detected, indicating that the stem resistance is more stable than leaf resistance in RS lines. A low and non significant correlation of resistance between leaf and stem (r = −0.18, P = 0.25) suggested that different genetic mechanisms separately control stem and leaf resistance of RS lines.

Table 1 ANOVA of relative susceptibility to ‘Zhongyou 821’ in leaf and stem among resynthesized B. napus
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To investigate the resistance transfer from B. oleracea into B. napus, the mean resistances of all RS lines derived from the same type of B. oleracea were compared with those of corresponding type of B. oleracea as identified in the previous study (Mei et al. 2011b). Although the resistant level of RS lines was lower than that of corresponding parental B. oleracea, a moderate correlation of resistance was found between them in both leaf (r = 0.74, P = 0.02) and stem (r = 0.69, P = 0.04) (Fig. 3).

Fig. 3
figure 3

Relative susceptibility to ‘Zhongyou 821’ in leaf A and stem B for resynthesized lines classified by the type of parental B. oleracea and corresponding types of B. oleracea reported in the previous study (Mei et al. 2011b)

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These results indicate that the resistance of resynthesized rapeseed lines is associated closely with that of the parental B. oleracea type, and that the stem resistance of rapeseed against Sclerotinia can be improved by using B. oleracea.

Discussion

In comparison with ‘Zhongyou 821’, most RS lines exhibited higher resistance level especially in stem, indicating diverse resistance mechanism in RS lines and current rapeseed. More recently, two major quantitative trait loci (QTL) for Sclerotinia resistance from B. incana, a wild of B. oleracea, were mapped on the chromosome C09 (Mei et al. 2013), whereas several independent studies on mapping for Sclerotinia resistance in rapeseed (Yin et al. 2010; Zhao and Meng 2003; Zhao et al. 2006) did not find any major QTL on N19 which corresponds to C09 of B. oleracea. Therefore, the RS lines are important resources to improve Sclerotinia resistance of current rapeseed.

In the previous study (Mei et al. 2011b), the Sclerotinia resistance level of rapeseed was found in the middle of that of its two parental species, B. oleracea with high level of resistance and B. rapa with high level of susceptibility. It was in accordance with the present study, in which the resistance level of RS line was usually lower than that of its parental types of B. oleracea, though the resistance of parental lines of B. rapa was not listed. These findings show that additive genetic effects possibly play important roles in the Sclerotinia resistance of RS lines. If so, it will be an interesting breeding strategy to improve the Sclerotinia resistance of rapeseed by transferring resistance from B. oleracea into B. rapa due to the high colinearity between their genomes (Cheung et al. 2009; Rana et al. 2004), and then resynthesizing rapeseed to pyramid the resistance from B. oleracea and B. rapa.