Mature Plant Experiments: 1 and 2
Experiments were conducted with a standard growing medium: a 50:50 (v/v) mix of sieved compost (John Innes No. 3, Erin, UK) and horticultural grade sharp sand (Westland, UK). This mixture was selected as this substrate is free of large particulates and free-draining, but still retains adequate water for carrot growth, whilst the high compost content is nutrient rich to support long-term plant growth. Solid substrate
inoculum of P. violae isolate P10 (4-8 months old) was prepared as outlined above (Section 3.2.1) in 1 L flasks. The contents of four flasks were decanted into a large sterile bag to give approximately 2 kg of starting material and thoroughly mixed by hand for 10 minutes to ensure the mix was homogenous. Oospore concentration was initially estimated by vortexing 1 g of this inoculum in 10 ml SDW for 1 minute and oospores counted in a 1 ml counting chamber (Sedgewick-Rafter). Two replicate counts for each of four 1 g samples were recorded. This starting inoculum was diluted appropriately in horticultural sand and mixed in a cement mixer to obtain a bulk batch of diluted inoculum at a concentration of approximately 5000 oospores g-1. This was checked by a further
oospore count, after which a final dilution was made to obtain another bulk batch of inoculum with a concentration of approximately 1000 oospores g-1. Appropriate amounts
of the 1000 and 5000 oospore g-1 inoculum were then mixed with the compost/sand
growing medium in a cement mixer to obtain final P. violae oospore concentrations of 5, 10, 25, 50 and 75 oospores g-1 (5.4, 10.8, 27.0, 54 & 81.0 oospores/cm3). All growing
medium/inoculum mixtures were prepared such that the compost/sand ratio was always 50:50 (v/v). Plastic pots (5.5 L capacity, 20 cm diameter, 27.5 cm high) were filled with the inoculated growing medium (5.65 kg/pot) at each of the five oospore concentrations. Control pots containing a mixture of 50:50 (v/v) compost/sand only were also included. In total over 600 litres of mixture was prepared for each experiment.
Pots were placed in deep saucers within a glasshouse compartment (max. 18˚C, min. 16˚C; supplementary lighting from 05:00-20:00 h when day length was reduced) and watered to a high moisture content before sowing with untreated carrot seed (10 seeds/pot, cv. Nairobi, Elsoms Seeds Ltd, UK). Initially, the growing medium was kept damp on the surface by gentle overhead watering while carrot seedlings emerged, with additional weekly watering in the saucers. After 6-7 weeks, where there were more than five seedlings remaining, seedlings were thinned to five per pot. From 7-8 weeks onwards, watering was increased from both above and below via the saucers to keep the growing medium damp. Carrot plants also received watering with a nutrient solution via the saucers from nine weeks post sowing (w.p.s), alternating weekly with 2N:1P:4K (Vitax Vitafeed) and 0N:1P:3K (Solufeed) feeds. Aphiline, Amblyline, Encarline, Exhibitline & Hypoline biocontrol agents (Bioline AgroSciences, UK) were routinely used for controlling aphids, thrips, whitefly and sciarid flies. In total, 16 replicate pots were prepared for each oospore concentration in a randomised block design consisting of four blocks each containing four replicate pots of each treatment. Two experiments were set
up (Figure 3.3): Experiment 1 on 28/04/2016 (21 weeks duration) and Experiment 2 on 16/06/2016 (23 weeks duration).
Mature Plant Experiment: 3
This experiment was designed to test the effect of both P. violae oospore concentration and a metalaxyl seed treatment on cavity spot lesion development. Experimental set up was as described above for Experiments 1 and 2, but with oospore concentrations of 1, 10, 50 and 100 oospores g-1 and using both untreated and metalaxyl-treated carrot seed
(cv. Nairobi, Elsoms Seeds Ltd, UK). Eight replicate pots for each P. violae oospore concentration for each seed type were included, arranged in 12 blocks over two benches in a randomised block design (Figure 3.4).
Figure 3.3 Pot-grown carrots inoculated with P. violae in Experiment 1 (right) and 2 (left) at 13 and 6 w.p.s respectively; July 2016.
3.2.6 Assessment of carrot plant growth, cavity spot symptoms and infection by P. violae
Mature Plant Experiments: 1, 2 and 3
In each experiment, carrot seedling emergence (germination) and seedling mortality (damping-off), were recorded weekly for the first 6-7 weeks. The fresh and dry weight of the seedlings that were removed as part of thinning were recorded to assess the effect of
P. violae on seedling growth at an early stage. Both mean shoot fresh and dry weight per plant, and total shoot fresh and dry weight per pot, were recorded. Carrot plants were harvested at 21, 23 and 26 w.p.s for Experiments 1, 2 and 3 respectively. The top-growth (foliage) from the five carrots in each pot was removed and the total fresh and dry weight per pot, as well as the mean fresh and dry weight per plant, was recorded. Carrots were gently loosened and removed from the growing medium to reduce damage to the long tap roots, washed, and both mean weight per root and total root weight per pot were recorded. Each carrot was assessed and scored for cavity spot incidence (presence of one or more cavity spot lesions on each carrot) and cavity spot severity (the number of lesions per carrot). Severity was measured across all roots (including those unaffected) as well as across infected roots only.
To confirm infection by P. violae, one carrot was selected from two of the four pots from each oospore concentration treatment in each block (total eight roots per treatment), the long thin tap root excised and stored at 5˚C in SDW until used. Roots were surface sterilised in 70% ethanol for 1 minute and washed twice in SDW after which they were cut into three sections of roughly equal length (top: nearest the base of the carrot, middle and bottom; total 24 root pieces per treatment). A 1-2 cm length of each section was plated onto CMA amended with rifampicin (30 mg L-1; pH 6), incubated in the dark at
17˚C and examined for P. violae growth after 5-10 days. Furthermore, small pieces of carrot tissue excised from selected cavity spot lesions in each treatment were plated directly onto CMA amended with rifampicin and also monitored for P. violae growth.
Statistical analysis was carried out with the support of Andrew Mead (Rothamsted Research) in Genstatâ (18.1 edition, VSN International Ltd). All data recorded were subjected to statistical analysis using ANOVA with angular transformation of percentage germination, percentage seedling mortality (damping-off, calculated as a percentage of those which germinated) and cavity spot incidence. A log10 transformation of cavity spot
severity was performed for severity across all roots. No statistical analysis was possible for severity across infected roots only due to a large number of missing values (i.e. unaffected roots). In each of Experiment 1 and 2, one pot was removed for mean shoot weight per seedling and total shoot weight per pot at seedling stage due to very poor germination affecting the growth of the remaining seedling(s). Further outliers and anomalies were removed from the analyses as necessary. Linear and quadratic polynomial contrasts were included within the oospore concentration effect to assess for smooth trends in the response across concentration (i.e. regression lines were fit to assess if there was a dose-response relationship). Assessment of P. violae infection from the subset of root pieces excised from the carrot tap roots was carried out using a Chi-squared (c2) analysis. Where applicable, all data presented in figures are the back-transformed
values, and data presented in tables are the transformed values used for statistical analysis.