Arboriculture & Urban Forestry 39(4): July 2013 polymer on resistance of horsechestnut (Aesculus hippocastanum L.) against the foliar pathogen Guignardia aesculi and ii) dura- tion of any resistance conferred by monitoring Guignardia leaf blotch severity over two growing seasons under field conditions. MATERIALS AND METHODS Experiments were conducted in 2007 and repeated in 2008. The experiment used bare-rooted stock of horsechestnut obtained from a commercial supplier. To ensure uniformity of stock for experimental purposes, trees were graded and used only if con- firming to the physical characteristics specified: height 100.0±7.0 cm, stem diameter 3.5±0.30 cm, root area 400.0±50.5 cm2 . Trees were then sealed in plastic bags, placed inside larger paper bags, and stored at 6°C±0.5°C (a standard storage temperature for trees in the UK) in a refrigerated cold store in darkness. Follow- ing six weeks at 6°C±0.5°C dark storage, trees were removed from cold store (January 28, 2007; February 1, 2008) prior to fungicide and polymer treatments on the same day as removal. All fungicides used for experimental purposes (Table 1) were diluted with water to achieve a concentration of 0.15 g and 0.30 g active ingredient (a.i.) per liter of water. Manufacturers generally recommend 0.15 g a.i. per liter of water for plant protection pur- poses when fungicides are applied as a foliar spray. The water- retaining polymer Aquastore F was hydrated with each fungicide solution at 5 g polymer per liter of solution. Following hydra- tion of each polymer, the root systems of ten trees were dipped for 30 seconds and gently agitated throughout the polymer to ensure maximal contact with the root system. The influence of the water-retaining polymer alone (no fungicide) on Guignardia leaf blotch severity was also investigated and bare-rooted stock dipped for 30 seconds in water only (no fungicide treatment or polymer) acted as controls. In addition, a comparative evaluation of the fungicide penconazole, commercially used for Guignar- dia leaf blotch control, was conducted by spraying trees at the manufacturers recommended rate of 1.5 ml l-1 of water. Penco- nazole sprays were applied at four growth stages namely: bud break (March 19, 2007; March 23 2008), flower cluster formation Table 1. The influence of water-retaining polymer (WRP) and fungicide combinations applied at the time of plant- ing for the control of Guignardia leaf blotch on leaves of horsechestnut (Aesculus hippocastanum L.) as measured by observed pathogen severity. Treatment Water (control) WRP WRP + Penconazole (0.15 g) WRP + Penconazole (0.30 g) WRP + Thiabendazole (0.15 g) WRP + Thiabendazole (0.30 g) WRP + Propiconazole (0.15 g) WRP + Propiconazole (0.30 g) WRP + Myclobutanil (0.15 g) WRP + Myclobutanil (0.30 g) WRP + Epoxiconazole (0.15 g) WRP + Epoxiconazole (0.30 g) WRP + Potassium Phosphite (0.15 g) WRP + Potassium Phosphite (0.30 g) Penconazole (spray) Leaf blotch severity 2007 4.6a 4.5a 2.5b 2.2b 1.8b 2.0b 1.8b 1.7b 1.6b 2.1b 2.5b 1.6b 2.4b 2.5b 0.0c 4.4a 4.4a 4.2a 4.7a 4.7a 4.3a 4.5a 5.0a 4.9a 4.2a 4.0a 4.4a 4.6a 4.1a Note: All values mean of ten trees. Also, lowercase letters indicate significant differences between means for each evaluation date (P = 0.05). ©2013 International Society of Arboriculture 2008 4.6a 183 (April 21, 2007; April 27, 2008), 90% petal fall (May 18, 2007; May 22, 2008), and full leaf expansion (June 21, 2007; June 25 2008). Prior to the first penconazole spray application, trees were inspected and no visible symptoms of Guignardia leaf blotch were apparent. Following dipping, trees were immediately plant- ed out into field trial plots at the University of Reading, Shin- field Experimental Station, Reading (51°43N, -1°08W) at 1.5 m spacing. A randomized complete design was used. There were fifteen treatments; 6 fungicide × 2 concentrations, 1 water-retain- ing polymer, 1 control, and 1 penconazole foliar sprayed com- parative analysis with ten trees per treatment to provide a total of 150 trees used in each experimental year. The soil was a sandy loam, containing 5%–7% organic matter with a pH of 6.4. Weeds were controlled chemically using glyphosate (Roundup® ) prior to planting and by hand during the trial. No irrigation was required and no fertilizer was applied to trees during each experiment. Tree Vitality Five leaves per tree were randomly selected throughout the crown and used for chlorophyll fluorescence and chlorophyll content measurements. Leaves were then tagged to ensure only the same leaf was measured throughout. Each five fluo- rescence and chlorophyll content values per tree were pooled to provide one value per tree for statistical analysis purposes. Chlorophyll Fluorescence Chlorophyll fluorescence was used as a measure of damage to the leaf photosynthetic system and to identify potential phyto- toxicity effects. Leaves were adapted to darkness for 10 minutes by attaching light exclusion clips to the leaf surface and chloro- phyll fluorescence was measured using a Handy PEA portable fluorescence spectrometer (Hansatech Instruments Ltd, King’s Lynn, UK). Measurements were recorded up to 1 second with a data acquisition rate of 10ms for the first 2 milliseconds and of 1 millisecond thereafter. The fluorescence responses were induced by a red (peak at 650 nm) light of 1500 mmol m-2 Photosynthetically s-1 Active Radiation intensity provided by an array of six light emitting diodes. The ratio of variable (Fv = Fm - Fo) to maximal (Fm) fluorescence—i.e., Fv/Fm where Fo = minimal fluorescence, of dark-adapted leaves was used to quantify the detrimental effects of Guignardia leaf blotch infec- tion on leaf tissue. Fv/Fm is considered a quantitative measure of the maximal or potential photochemical efficiency or optimal quantum yield of photosystem II (Willits and Peet 2001). Like- wise Fv/Fm values are the most popular index used as a measure of plant vitality (Maxwell and Johnson 2001; Percival 2004). Leaf Chlorophyll Concentration Data on degradation of the leaf chlorophyll molecule as a result of Guignardia leaf blotch infection were recorded using a Minolta chlorophyll meter SPAD-502. Chlorophyll was measured at the midpoint of the leaf next to the main leaf vein. Calibration was obtained by measurement of absor- bance at 663 and 645 nm in a spectrophotometer (PU8800 Pye Unicam, Portsmouth, UK) after extraction with 80% v/v aqueous acetone (regression equation = 6.00 + 0.058x; r2 adj = 0.88, P ≤ 0.001) (Lichtenthaler and Wellburn 1983).
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