Methods

Experiment design

Winter oilseed rape cultivars are ideally sown in late August at Rothamsted. Seed should be ordered well in advance. Sufficient replication of plots should be made to account for experimental error and to allow statistical separation of assessments or sampling made on individual blocks of plots on different days or with different assessors. If yields are required, plots should be at least 3m x 10m and at least 3 plots per cultivar x treatment. A statistician should be consulted during experiment design. The field experiment should be agreed by the oilseeds, legumes and forage crop commodity group at Rothamsted. Cultivar choice should consider resistance to the target disease(s) and other naturally occurring diseases.

Inoculation techniques

(A) Stubble - The type of stubble (cultivar, fungicide exposure and whether upper or lower stems) and the conditions it has been subjected to will affect the pathogen population released from it and the timing of release. For phoma canker experiments, stem-base material, collected after harvest and kept outdoors in natural conditions should be applied soon after drilling if the intention is to supplement naturally occurring ascospore inoculum. For light leaf spot, upper stem and pod debris is more appropriate. The inoculum should be placed around plots i.e. in a line around the experiment, c. 3 m from the plots, if airborne spore, i.e. ascospore-based inoculum is intended. Debris applied directly to plots may result in unusually early and severe infections caused by rain-splashed conidia or direct contact – which is OK only if early infection is required.

(B) Where artificial inoculation is required e.g. spray application of a spore suspension, UV light is often used to stimulate sporulation in Petri-dish cultures. Purpose-built ventilated cabinets are used (currently in Bawden basement or 10 C room Bawden032), containing 2 UV tubes (Philips TLD 36W/08) or sometimes one UV and one cool-white tube (OSRAM L36W/23) as these produce a little near-UV. These are usually used with a timer to provide a cycle of 12 hours UV/ 12 hours dark. Check that the tubes are working – but use UV-blocking glasses to view them! Spore suspensions are made typically by flooding the Petri-dish with 10ml water (to which a surfactant may be required e.g. 0.01% Tween), agitating the surface to release spores and pouring the spore suspension into a container. The final concentration should be measured with a haemocytometer slide and diluted to the desired concentration with water. Plants will need to be covered with plastic bags or cloche(s) to maintain high humidity required for infection where artificial inoculation has been made.

Crop density and growth measurements

Crop density should be measured in November and after harvest to provide data on establishment, winter kill and to relate yield to crop density. This is calculated by counting the number of plants in 4 x 0.5 m rows and noting the distance between rows (usually 12.5 cm at Rothamsted).

Each month, the growth stage (GS) of the crop should be evaluated using the scale derived by Sylvester-Bradley & Makepeace(1985). Leaf position, if measured should be based on the number of leaf scars present in addition to the presence of existing leaves.

Disease assessments

A minimum of 10 plants per plot should be assessed (more is ideal at the start of epidemics when incidence is low). Plants should be sampled at random from the sampling area of each plot, and individual data should be collected for each plant, recording incidence (% plants affected) and severity of all diseases present.

It should be made clear whether severity measurements are % areas affected per plant or % of number of tissues, e.g. pods or leaves, affected or if a scale is used, it should be defined. Examples: phoma leaf spotting severity may be for each plant the % area of leaf spotting or the number of leaves affected. Phoma stem canker (crown cankers and phoma stem lesions, as defined by West et al., 2001) may be scored on a 0-5 scale (where 0 = uninfected, 1 = < 25% stem circumference girdled, 2 = 25-50% girdled, 3 = > 50% girdled, stem firm, 4 = > 50% girdled, stem weak, 5 = plant dead or lodged), or the ADAS 0-4 scale (which combines scores 1 and 2 in the above scale), or the HGCA RL 0-6 score (0 = uninfected, 1 = < 25% stem circumference girdled, 2 = 26-50% girdled, 3 = > 51-75% girdled, 4 = > 76-100% girdled, 5= 100% girdled + stem weak, 6 = plant dead or lodged). If results will be compared with data from other sites/organisations it is best to standardise the assessment method.

Where disease indexes are calculated it should be clear how these are calculated and ideally the proportion of uninfected plants should score zero towards the final disease index.