Alarmas

Apple bait

5.2.1.1.

The apple bait method used by Mwanza and Kellas (1987), which succeeded in isolating C. destructans from soil, was tested. Potting mix (1 L) was placed into a double layer of autoclavable bags (305 x 660 mm) and autoclaved at 121°C for 15 min at 15 psi. A conidium suspension from C. destructans isolate D2 (Appendix 1) was made as described in Section 2.2.1.2 with a final conidium concentration of 106 conidia /mL. The suspension was diluted to 105 and 104 conidia /mL using sterile distilled water and 1 mL of each of the three conidia suspensions was mixed with 60 g of autoclaved potting mix. Apple (Malus domestica) varieties Granny Smith and Braeburn were used as bait with three apples for each treatment. The apples were surface disinfected under a laminar flow hood by spraying 70% ethanol onto their skins until each surface was completely covered. A 3 cm diameter slot cut to a 1 cm depth was made on the surface of each apple and 3 g of infested potting mix was then placed into the slot which was covered with the apple piece. The apples were placed in a 23 × 16 × 8 cm box without a lid, covered with a plastic bag and incubated at 20°C in the dark. After 6 days, 8 pieces (2 × 2 × 2 mm) were cut from the lesion areas in each apple and they were plated onto PDA (Oxoid Ltd, Basingstoke, UK) amended with 250 mg /L of chloramphenicol (Sigma Sigma-Aldrich, St. Louis, USA) within a laminar flow hood. The plates were wrapped with cling film and incubated at 20°C in the dark for 1 – 2 weeks. The number of apples infected with C. destructans and other fungal species (out of the three replicate apples) for each concentration and apple variety was recorded.

The experiment was repeated as described previously using the same conidium concentrations mixed with 60 g of non autoclaved garden soil instead of potting mix. Three replicates were used for each time and apples were assessed 5 and 7 days after infestation. Apples incubated for 5 days were assessed as previously described using ten pieces of apple for each replicate, however apples incubated for 7 days were cut in two and ten pieces of apples were taken from lesions in the apple flesh and ten pieces were taken from underneath the apple skin of the lesions. The number of apples infected with C. destructans and other fungi for each concentration, apple variety and sample location was recorded. Control plants were not used in these experiments as only the suitability of apple bait to recover C. destructans was tested.

Seedling bait

5.2.1.2.

A number of vegetable species were selected for their use as seedling bait based on the reported incidence of Cylindrocarpon spp. in these hosts and the likely ease of growth by wine growers. Control seedlings were not used for the following experiments as seedlings were being tested for their ability to be infected with C. destructans.

A conidium suspension from C. destructans isolate D2 was made up as described in Section 2.2.1.2 at a concentration of 106 conidia /mL and 100 mL of the suspension was mixed in a 35 × 27 × 7 tray with 6 L of potting mix which was previously autoclaved as described in Section 5.2.1.1. A preliminary experiment used seeds of spinach (Spinacea oleracea) hybrid n°7, red clover (Trifolium pratense) and pea (Pisum sativum) variety Easy Peasy. The seeds were sowed in 3 rows, 3 cm apart in trays with the infested potting mix. The seedling trays were kept for 18 to 26 days at room temperature and were watered frequently, as needed. Symptomatic seedlings were harvested by gently uprooting them, the roots were washed with tap water and the seedlings were placed on paper towels. They were surface sterilised by soaking in 70% of ethanol for 30 s, 0.35% of sodium hypochlorite for 1 min and 70% of ethanol for 30 s under a laminar flow hood. The seedlings were then placed onto a glass Petri dish. Four 2 mm transversal pieces of root and stem were cut and placed onto PDA with chloramphenicol. The plates were sealed with cling film, incubated at 20°C in the dark for 7 days and the proportion of seedlings infected with C. destructans and other fungal species was determined.

The experiment was repeated using tomato (Solanum lycopersicum) variety Moneymaker, pea and spinach seeds as above. The spinach and tomato seedlings were harvested and assessed as previously after 21 and 43 days, respectively, while pea seedlings were assessed after 18 and 21 days, by taking root pieces as above and four cotyledon pieces.

The experiment was repeated using tomato, spinach and pea seeds (same varieties as above) sowed in a 50/50 v/v mixture of non autoclaved garden soil and autoclaved potting mix, infested with C. destructans and assessed as above.

A second vegetable bait experiment was conducted using a 50 /50 v /v mixture of garden soil and potting mix with seedlings of tomato variety Moneymaker and bean (Phaseolus vulgaris) variety Dwarf French Tenderness. The tomato and bean seeds were germinated on filter paper soaked with sterile water in Petri dishes. The seedlings were then grown in vermiculite until roots reached 5 – 10 cm long. A conidium suspension using a mixture of three isolates for each species (C. liriodendri isolates L1, L2 and L3 , C. destructans isolates D1, D2 and D3, and C. macrodidymum isolates M1, M2 and M3; Appendix 1) were obtained as in Section 2.2.1.2, with a final concentration of 105 conidia /mL. The roots of each seedling were wounded by cutting off root tips before transplanting one seedling per 500 mL pot containing a 50/50 v/v mix of autoclaved garden soil and autoclaved potting mix. Beside each plant stem base, a hole was made in the soil and 1 mL of the spore suspension was poured. The treatments (control, C. liriodendri, C. destructans and C. macrodidymum) were replicated five times and laid out in a randomised block design. The seedlings were left to grow for a month at room temperature and watered regularly. They were surface sterilised as previously and a 1-2 mm piece was taken from the upper and lower stem parts, the base, upper, intermediate and lower root parts of each plant. The pieces were plated onto PDA with chloramphenicol and incubated as previously. The seedlings were assessed for the presence of Cylindrocarpon spp. The infection incidences were analysed using a general linear model with a binomial error distribution with Genstat version 12.

The experiment was repeated with 12 week old tomato plants purchased from a nursery. The plants were wounded and planted in 500 mL pots with a 50/50 mix v/v of non sterile garden soil and potting mix. The soil beside each plant was infested with 1 mL of 107 conidia /mL as previously. Five replicates were used for the different species and the control in a randomised block design.The plants were harvested 12 days after inoculation and assessed as above for the presence of C. destructans.

Parsnip assay

5.2.1.3.

Conidium suspensions of C. liriodendri isolates L1, L2 and L3, C. macrodidymum isolates M1, M2 and M3 and C. destructans isolates D1, D2 and D3 were made as in Section 2.2.1.2 at concentrations of 102 and 104 conidia /mL. Parsnip roots (Pastinaca sativa) were cut transversally into 5 mm deep pieces, which were each placed onto a 10 cm diameter filter paper, for which 10 were arranged on paper towels in a 31 × 42 cm tray. The filter paper and

surface with 100 μL of conidium suspension. Treatments were replicated 5 times per conidium concentration and laid out in randomised blocks. The trays were covered with a plastic bag containing wet paper towels to maintain high humidity and the parsnips incubated at 20°C in the dark. After 8 days, the parsnip pieces were assessed visually for infection. Samples that visually appeared to be infected by fungi were plated onto PDA with chloramphenicol (250 mg /L) and incubated for 7 days at 20°C in the dark. The plates were assessed for the presence of Cylindrocarpon spp.