Aplicación de la herramienta Origen y Utilización de Fondos

Plant material

2.2.1.1.

Plants of two grapevine rootstock varieties commonly grown in New Zealand vineyards (5C and 101-14) were propagated from the dormant shoots of apparently healthy vines grown in the Lincoln University vineyard. Two node cuttings were grown to rooted plants or callused for use in the infection studies. To obtain rooted plants, the cuttings were placed in trays containing pumice, in a shade house on a heat pad at 25°C for 6 weeks, when the average air temperature was 5.4°C. The rooted plants were carefully removed from the trays and their roots were trimmed to 10 cm before inoculation. To callus the cuttings, they were placed in

greenhouse (14 – 28°C) for 1 – 2 weeks before inoculation, which is the standard nursery protocol (B. Corban, pers. comm. 2006). Plants from callused and rooted cuttings are named “callused” and “rooted” plants, respectively, when referring to propagation methods.

Inoculum

2.2.1.2.

Three isolates from each of the three different species were randomly selected from the collection of identified Cylindrocarpon isolates maintained on Spezieller Nährstoffarmer agar (SNA; Appendix 2) at 4°C in the dark. These isolates were from grapevines from different New Zealand regions (Appendix 1) and had been previously identified as C.destructans (isolates referred to as D1, D2 and D3), C. macrodidymum (isolates M1, M2 and M3) and C. liriodendri (isolates L1, L2 and L3) by molecular methods (Mostert et al., 2006). To obtain conidium suspensions, the isolates were grown on PDA (Oxoid Ltd, Basingstoke, UK) at 20°C for 3 weeks in the dark. The conidia were suspended by adding 5 mL of sterile water with 3 drops /L of Tween 80 (polyoxylethylene (20) sorbitan mono-oleate; BDH Chemicals Ltd, Poole, England) to the plate surface, scraping the culture surface with a sterile glass microscope slide and sieving the solution through a sterile sieve of 150 μm mesh. This procedure was repeated twice per culture plate. The final conidium suspension was adjusted to 106 conidia /mL using a haemocytometer and sterile water. The viability of the conidia was confirmed by spreading 100 μL of each conidium suspension diluted to 102 _{conidia /mL, onto} PDA plates and counting the resulting colonies after 2 days incubation at 20°C in the dark.

Inoculation

2.2.1.3.

Plants were inoculated by soaking plant bases for callused plants, or roots for rooted plants, for 30 min in a 5 cm deep conidium suspension (106 conidia /mL) of one of the isolates prior to planting or in sterile water (control). They were potted into 1.5 L pots with “3 – 4 month potting mix” (Appendix 2). There were seven replicates of each isolate, plant type and rootstock variety. The plants were laid out on mesh tables in a greenhouse in a randomised block design under high pressure sodium lamps (Son-T Agro 400, Philips), and allowed to grow for 4 months (Figure 2.1). The lights were turned on from 4 am to 12 pm and from 4 pm to 8 pm for the duration of the experiment to ensure plants were under adequate light for 16 h per day. Temperatures varied over 14 – 30°C. Plants were watered daily.

Assessment

2.2.1.4.

A few extra plants were infected with the conidium suspension of each isolate. The disease incidences of these vines were assessed at different times (2, 3 and 4 months) to determine the best time for the assessment of the trial. The plants were not watered for 24 h prior to harvesting to facilitate potting mix removal. Plants were lifted out of their pots and their roots were shaken to remove loose potting mix. The roots of each plant were washed under

(70°C) at the Field Service Centre at Lincoln University. The roots were dried to constant weight, which was when the dry weights of some randomly selected root systems were equivalent on two consecutive days (7 days), and the weights recorded.

Figure 2.1. Inoculated plants that developed from rooted and callused cuttings of rootstock varieties 101-14 and 5C after 4 months in a greenhouse. Plants were arranged in a completely randomised design.

The main stems (trunks) were thoroughly washed with running tap water and the lower sections were cut to 20 cm to allow for complete vine immersion in the sterilisation tanks. Using the method developed by Halleen et al. (2003) to reisolate the pathogen from the infected plants, the lower stem sections were surface sterilized in batches, each time working with plants inoculated with the same pathogen isolate. The stems were soaked in 70% ethanol for 30 s, 0.35% sodium hypochlorite for 5 min and 70% ethanol for 30 s. The stems were wrapped in paper towels to absorb the excess ethanol and placed onto a new plastic bag for transfer to a laminar flow cabinet, where they were air dried for 10 min. Before pathogen isolation, the root crown, comprising the lowest 1 – 2 cm of the stem base was discarded. A 1 – 2 mm transverse piece of tissue was sliced from the basal end of the stem and cut into four pieces of approximately 3 mm2, which were placed equidistantly and near the edge of a plate containing PDA with chloramphenicol (250 mg /L). A 1 – 2 mm transverse piece of stem was also sliced at 5 cm above the base, cut into two pieces and one piece was placed in the centre of the same plate. It was then sealed with clingfilm and incubated for 7 days at 20°C in the dark. Cylindrocarpon-like isolates grown from the wood pieces were identified by comparing colony morphology and conidia with the cultures of the Cylindrocarpon isolates used to inoculate the specific plants. The proportion of vines infected

at 1 or 5 cm (disease incidence) and the proportion of infected wood pieces at 1 cm (disease severity) were recorded.

Assessment was based on presence of the pathogen instead of symptoms because tissue discoloration occurs frequently on wounding sites and can lead to confusion when interpreting results. As a result, other researchers (Haleen et al., 2004) have placed most emphasis on their isolation results not the amount of trunk discoloration measures. Gubler et al. (2004) stated that grapevines less than 10 years old inevitably died when infected with Cylindrocarpon spp. Therefore, colonisation and degree of colonisation of grapevine tissues by Cylindrocarpon spp. was interpretated as disease incidence and severity, respectively.

Analysis

2.2.1.5.

Data were analysed with the Statistical Package for Social Sciences (SPSS) version 13.0. Although analysis of variance is commonly used to determine disease incidences, the statistician consulted for the different experiments recommended the use of logistic regressions for the binomial data (presence or absence of the pathogen). Therefore disease incidences at 1 and 5 cm above stem bases were analysed using logistic regressions to test all the different effects. This method also provided for pair-wise comparisons between individual treatments within a tested factor, to determine significance of their differences. For the root dry weight and the disease severity, a general linear model was used with terms appropriate to the design and two-way interactions amongst the factors of interest. Where significant main effects or two-way interactions were identified, the significance of differences between related treatments was further explored using Fisher's protected LSD (least significant difference) tests. A P-value of ≤0.05 indicated statistical significance.