In order to test the influences of multiple AMF communities on pollinator visitation and plant reproductive traits across several strawberry cultivars, a four (AMF community) by three (strawberry cultivar) factorial randomised glasshouse experiment was conducted over two years. In September 2013, strawberry plants for the main experimental stage (misted tips that had been rooted into coir) were purchased from Nessen BV, Netherlands (a supplier which was able to provide plants free of previous AMF colonisation). Three cultivars were used - Elsanta and Sonata were selected to represent the two main varieties used in commercial production in the UK, and Darselect in order to provide a more distantly related cultivar grown
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in the EU, along with having different growth habits, such as flower phenology, and typical yields.
Before planting, roots from three plants from each cultivar were stained using Trypan Blue (Koske and Gemma, 1989) and assessed using the grid-line intersect method to confirm that they were free of AMF from the propagation process. Plants were then potted in 0.5 L sterile pots with strawberry mix coir (Bulrush Horticulture Ltd, Londonderry, UK), containing 100 ml of the appropriate treatment inocula (substrate and root fragments mix) produced in the inocula production phase (described above) (Table 2.1). Sixty plants of each strawberry cultivar were used across each of the four AMF communities, creating a replication level of 15 plants per treatment and 180 plants in total. Plants were grown under cool house conditions, with no supplemental lighting or heating, greenhouse fans permanently on, and a temperature range of 2-5°C for 6 months over winter to establish full AMF colonisation before the flowering period in the following year. In April 2014, at the end of this overwintering period, plants were repotted into sterile 3 L pots with strawberry mix coir containing 1/3 of the recommended application rate of fertiliser for strawberries to create a low P environment (2.7 g/L – Osmocote 14-16M, Geldermalsen, Netherlands). Plants were arranged into three blocks, each containing an equal number of plants from each treatment, and the location of each plant within the block was completely randomised. There were 20 rows of plants within each block, each containing three plants (Figure 2.1). Each pot was separated by 20 cm gap between the adjacent pot. Plants were grown for a total of two years over the course of the study, and during winter months (October to March) each year, environmental conditions in the glasshouse were altered to reflect outdoor conditions as described above. During the summer months, plants had 18 hours of light per day, with supplemental lighting when light levels fell below 150 W/m2, temperatures maintained at 21°C during the day, 16°C during the night, and an overhead screen closed when light values exceed 450 W/m2. Additional fertiliser was not applied at any stage, as Osmocote is a slow release fertiliser. Fungicides were not used at any stage, and any evidence of powdery mildew was treated with a mixture containing rapeseed oil (15.6 ml/L), anionic and non-ionic surfactants (7.8 ml/L) (Ecover Zero Washing Up Liquid, Ecover UK Ltd., Richmond, UK), and bicarbinate of soda (15.6 g/L) in sterile distilled water. Chrysoperla rufilabris (Green lacewing) larvae were used to control aphids, and Phytoseiulus persimilis were used to control Tetranychus urticae (Red spider mite) if either pest was sighted in the glasshouse.
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At the initiation of flowering, a commercial hive of Bombus terrestris Audax (Standard Hive, BioBest, Netherlands), containing approximately 80 workers was placed within the greenhouse in order to provide pollinators for the experiment. Visitation rates and foraging behaviour were determined by releasing 15 bees from the hive and walking a 1-hour transect through the greenhouse. Transects were conducted daily from Monday to Friday during the flowering period, and each transect was started at a randomly allocated location in the greenhouse. An observer would slowly walk the transect along each block of plants, observing the three rows (9 plants) directly adjacent (Figure 2.1). When a bee was observed to land on a flower, a timer was started to measure the duration of the visit, and the foraging behaviour was observed. Foraging behaviour was classed as 1) foraging for pollen, 2) foraging for nectar, 3) foraging for both pollen and nectar, or 4) non-foraging visits (when a bee would land on a flower but collecting neither pollen or nectar). The total number of visits per flower were calculated by combining these measures. Two measures of the duration were used for analysis – the total duration of each visit type observed during all transects a plant received, and the average duration of visits (calculated by dividing the total duration by the number of visits).
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Figure 2.1: Illustration of the layout of plants, block, transect route, and observation window in the greenhouse. Black rectangles show each block, containing 60 plants each (green circles). Transects were started at a random location, and the observer (blue circle) monitored 9 plants from the three rows adjacent as passed (light blue square), walking in the direction indicated in by the orange arrows. The direction walked (in the direction illustrated, or the reverse) was determined randomly using a random number generator.
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To determine male and female reproductive capacity, individual flowers were enclosed in Enviromesh (Ultrafine, Agralan Ltd, UK), until petals had opened. Nectar quantity was then measured by probing the nectaries with a 0.1 μl capillary tube, and measuring the quantity of nectar extracted with digital callipers. Nectar was harvested at two points in the day, during the morning (10-11 am), and the afternoon (2-3 pm), as nectar is secreted throughout the day, and the timing of the secretion may depend on AMF influences or strawberry cultivar. Anthers were then harvested from the same flowers as a proxy metric to determine male reproductive capacity, before being counted, freeze-dried, and weighed on a micro-balance.
A subsample of plant roots were stained with Trypan Blue (Koske and Gemma, 1989) at the end of the experiment, and assessed using the grid-line intersect method (McGonigle et al., 1990). Results can be found in Section 3.3.3.