Field trials

Field studies were conducted with the aim of verifying resistance of B. napus accessions under natural conditions, with diversity in terms of soil, climate and disease factors. For this trial, B. napus lines were used that showed a reasonably better resistance than the reference standard varieties under greenhouse conditions. The field experiments were conducted in three locations (Table 2.2) and for three seasons (2010/11, 2011/12 and 2012/13).

2.2.2.1 Plant materials

A total of 80 lines (Table 2.1) selected on the basis of previous greenhouse screening results were evaluated under field conditions. Of these, 61 were double haploid (DH) lines derived from resynthesized VL-resistant B. napus lines. The remaining 19 lines with undisclosed genetic backgrounds were obtained from different breeding companies. Fourteen lines (thirteen DH lines and one accession) were repeatedly tested for three consecutive growing seasons across all locations (Appendix 2.2). During each trial, 40 lines (including the reference varieties) were tested.

Cultivars Falcon, Laser, Oase, and Express were used as a standard check. Detailed descriptions and sources of all plant materials are found in (Appendix 2.2). Similar seed lots of all lines tested in the field were used in the parallel evaluation in the greenhouse.

2.2.2.2 Description of experimental locations

The three locations with field trials were Göttingen (Northern Central Germany), Fehmarn (an island in the Baltic Sea, Northern Germany) and Svalöv (Southern Sweden). These locations were selected on the basis of variation in the level of natural disease infestation, climatic factors and soil conditions (Table 2.2).

2.2.2.3 Experimental design

All experiments were laid down in a randomized complete block design (RCBD) with two (Fehmarn and Svalöv) or four (Göttingen) replications. In all locations, 40 accessions including the reference

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varieties were tested during each growing season. Experimental plot size was 8m x 1.5m with eight rows. Distance between blocks was kept 0.3 meter apart. Plant samples harvested from the central two rows were used for data recordings.

Table 2.2 Field characteristics of the three research locations where field studies were conducted from 2011 to 2013. inside the trial sites. For Fehmarn regional weather data was obtained from http://www.wetterkontor.de.

1Knüffer, 2013; ²Eynck, 2008; ³Johansson et al., 2006.

2.2.2.4 Inoculation and agronomic practices

At Svalöv and Fehmarn, where there is a reasonably high level of inoculum in the soil (Eynck, 2008;

Johansson, 2006), experiments were carried out under natural infestation conditions. In Göttingen, artificial inoculation was performed using chopped VL-infected stubbles collected from previous year experiments. This was done by evenly spreading the inoculum on a prepared seedbed at a rate of 15g microsclerotia infested stubbles/m². The inoculum was applied just before sowing and it was thoroughly incorporated into the soil using a disk plough. Seed rate was adjusted to local recommendations i.e. 50, 60 and 80 seeds/m² in Fehmarn, Göttingen and Svalöv, respectively. Early season insect pests (such as aphids and flea beetles) and seed borne pathogens (Phoma lingam, Alternaria spp. and Pythium spp.), were controlled by seed treatment with ‘CRUISER OSR’ (280 g/l thiamethoxam, 8 g/l fludioxonil and 32.3 g/l metalaxyl-M; Syngenta Crop Protection UK Limited) at a rate of 0.02ml per gram of seed. All other agronomic practices including fertilization and spraying

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against weeds, blackleg (Phoma lingam) and stem rot (Sclerotinia sclerotiorum) were applied following standard recommendations.

2.2.2.5 Disease assessment

To measure VL-resistance of selected B. napus lines under field conditions, molecular and phenotypic disease assessment tools were used. For molecular disease measurements, stem samples were collected at the beginning of the ripening stage (BBCH 80) and from these samples, fungal biomass was quantified by qPCR. For this protocol, sampling was done by taking 10cm long stem sections cut approximately 5cm above the base of the plant (Knüfer, 2013). For each accession, 10 stem samples were collected from each replication. Genomic DNA was extracted from 1 gram of lyophilized and ground stem sample taken from a pool of 10 stem samples. All other subsequent qPCR analysis steps including extraction of genomic DNA, DNA quality check, preparation of VL DNA standards, primers and qPCR conditions were similarly performed as described in section 4.2.7.3.

Figure 2.1 Assessment key (1-4) for evaluation of Verticillium longisporum disease severity on stubble and root of oilseed rape (Brassica napus). 1. Healthy tissue: Surface free of any symptoms and fungal structures; pith white and with cottony appearance; no microsclerotia visible. 2. Slight infestation: Narrow and short longitudinal lesions on the surface; pith still whitish but a few patches of microsclerotia visible. 3. Advanced infestation: Very long lesions and abundant microsclerotia visible under the epidermis; epidermis peeling off;

long patches of black microsclerotia visible in the pith. 4. Severe infestation: Most part of the stem epidermis peeling off and disintegrated; the surface black and covered with microsclerotia; pith becomes dark, disintegrated and filled with microsclerotia. S, stem surface. VS, vertical section.

In order to identify the most suitable stage of plant growth at which VL can be detected (quantified) in stem tissue of field grown plants and to precisely know the plant stage at which field grown susceptible and resistant plants can be best differentiated using this method, VL biomass from the

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four reference varieties was quantified at three different growth stages. This study was conducted in 2012/13. For this study, stem samples from the central four rows of each plot were collected at BBCH 65, 70, and 80 representing 50% flowering, end of flowering, and beginning of ripening stages, respectively. For each variety, 40 stem samples representing four biological replicates (10 plant samples per replication) were collected. In Göttingen, since there were four replications, sampling was done by collecting 10 stem samples per plot. In Fehmarn and Svalöv sites, since there were two replications, sampling was done by dividing each plot into two halves and collecting 10 stem samples from each part.

For phenotypic disease assessment, stubble sample collected after harvest was evaluated for stem and root disease severity. For every season study, a total of 50 (Fehmarn and Svalöv) or 100 (Göttingen) stubble samples were collected from each line (i.e 25 samples per replication). Disease severity evaluation was carried out by visual assessment of VL symptoms as well as microscopic estimation of the amount of microsclerotia both on the stem surface as well as in the pith tissue of stem and root samples using a four scale disease index (Fig. 2.1; Appendix 2.6).