Abstract
The influence of six commercially available fungicides incorporated into a water-retaining polymer and applied to the root system of horsechestnut (Aesculus hippocastanum L.) as a dip at the time of planting was conducted. Potential increases in resistance against the foliar pathogen Guignardia leaf blotch (Guignardia aesculi) was then monitored over two growing seasons. Trials were conducted in 2007 and duplicated in 2008. A comparative evaluation of the fungicide penconazole commercially used for Guignardia leaf blotch control was studied by spraying trees at the manufacturer’s recommended rate of four times during the first growing season but none in the second. None of the treated or control trees died as a result of Guignardia leaf blotch attack during the course of the study and none of the fungicide and water-retaining polymer combinations evaluated was phytotoxic to the test trees. Efficacy as Guignardia leaf blotch protectant compounds over the first growing season was demonstrated when fungicides were incorporated into a water-retaining polymer. Reductions in Guignardia leaf blotch severity were mirrored by increases in leaf chlorophyll fluorescence as a measure of leaf photosynthetic activity and leaf chlorophyll content SPAD values. There were little differences in the magnitude of control efficacy between the fungicides evaluated. Limited efficacy of any of the fungicide and water-retaining polymer combinations as Guignardia leaf blotch protectant compounds was, however, demonstrated the following year after application indicating a fungicide and water-retaining polymer root dip provided one growing season protection only. Application of a water-retaining polymer alone had no effect on reducing Guignardia leaf blotch severity. Based on visual Guignardia leaf blotch severity ratings, greatest protection in both the 2007 and 2008 trial was provided by the synthetic fungicide penconazole applied as a foliar spray four times during the growing season. No efficacy of penconazole foliar sprays as leaf blotch protectant compounds was demonstrated the following year, indicating annual sprays against Guignardia leaf blotch are required for control.
- Aesculus hippocastanum
- Disease Management
- Chlorophyll Fluorescence
- Leaf Chlorophyll Content
- Plant Health Care
- Tree Planting
Guignardia leaf blotch of horsechestnut (Aesculus spp.) occurs in Europe, North America, and Asia (Pastirčáková et al. 2009). Leaf blotch caused by the fungus Guignardia aesculi (Peck) Stewart on Aesculus is diagnosed by identifying the conidial state of the pathogen, Phyllosticta sphaeropsoidea Ellis et Everh (Pastirčáková 2004). The symptoms of leaf blotch disease are brown or reddish-brown lesions that often cover a large portion, or even the entire leaf. Leaves curl and brown, and the tree often appears to be suffering from severe leaf scorch (Percival et al. 2006; Percival 2008). Premature leaf drop normally follows infection. Susceptible species include California, Ohio, red, and yellow buckeyes; several less-known buckeye species; and common, Japanese and red horsechestnuts (Sinclair et al. 1987; Pastirčáková et al. 2009). In terms of Guignardia leaf blotch control, frequent fungicide sprays have a known efficacy in reducing severity (Plenk 1996; Pastirčáková 2003).
Creation of amenity woodlands and urban treescapes in the UK are predominantly established using bare-rooted stock of deciduous tree species. Poor growth and survival following out-planting is common during the first few years of establishment (Watson and Himelick 1997). An important cause of poor growth and death in transplanted trees can be attributed to root loss following lifting from the nursery bed, since as little as 5% of a tree’s root system may be moved with a tree (Davies et al. 2002). Following leafing out, the capacity of the roots to supply the leaves with water is severely restricted. This leads to water stress that with time may be characterized by reduced shoot growth, branch dieback, and possibly death; a concept widely described as transplant stress (Fraser and Percival 2003; Percival and Barnes 2004).
Stress is generally recognized as a prerequisite for disease attack with higher rates of attack and/or increased severity of pathogen infection associated with trees of lower vitality. Consequently, trees suffering from transplant stress following out-planting will be especially susceptible to attack. Water-retaining polymers are polymers that absorb large quantities of water and once incorporated into a growing medium are capable of releasing at least 95% to plants. As the polymers dehydrate, discrete non-toxic granules are formed. Consequently, water-retaining polymers are recommended as a means of reducing root desiccation and post-planting mortalities (Johnston and Pipper. 1997; Grazia et al. 2004). The use of a water-retaining polymer in combination with a fungicide at the time of planting has not been investigated. Such a combination may provide a means of increasing the pathogen resistance of trees over a growing season. Aims of this investigation were to determine the influence of i) a range of commercially available fungicides incorporated into a water-retaining polymer on resistance of horsechestnut (Aesculus hippocastanum L.) against the foliar pathogen Guignardia aesculi and ii) duration 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 confirming 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. Following 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 purposes 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 hydration 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 Guignardia leaf blotch control, was conducted by spraying trees at the manufacturers recommended rate of 1.5 ml l−1 of water. Penconazole sprays were applied at four growth stages namely: bud break (March 19, 2007; March 23 2008), flower cluster formation (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 planted out into field trial plots at the University of Reading, Shinfield Experimental Station, Reading (51°43N,fl-1°08W) at 1.5 m spacing. A randomized complete design was used. There were fifteen treatments; 6 fungicide × 2 concentrations, 1 water-retaining polymer, 1 control, and 1 penconazole foliar sprayed comparative 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.
The influence of water-retaining polymer (WRP) and fungicide combinations applied at the time of planting for the control of Guignardia leaf blotch on leaves of horsechestnut (Aesculus hippocastanum L.) as measured by observed pathogen severity.
Tree Vitality
Five leaves per tree were randomly selected throughout the crown and used for chlorophyll fiuorescence and chlorophyll content measurements. Leaves were then tagged to ensure only the same leaf was measured throughout. Each five fiuorescence and chlorophyll content values per tree were pooled to provide one value per tree for statistical analysis purposes.
Chlorophyll Fluorescence
Chlorophyll fiuorescence was used as a measure of damage to the leaf photosynthetic system and to identify potential phytotoxicity effects. Leaves were adapted to darkness for 10 minutes by attaching light exclusion clips to the leaf surface and chlorophyll fiuorescence was measured using a Handy PEA portable fiuorescence spectrometer (Hansatech Instruments Ltd, King’s Lynn, UK). Measurements were recorded up to 1 second with a data acquisition rate of 10μs for the first 2 milliseconds and of 1 millisecond thereafter. The fiuorescence responses were induced by a red (peak at 650 nm) light of 1500 μmol m−2 s−1 Photosynthetically Active Radiation intensity provided by an array of six light emitting diodes. The ratio of variable (Fv = Fm − Fo) to maximal (Fm) fiuorescence—i.e., Fv/Fm where Fo = minimal fiuorescence, of dark-adapted leaves was used to quantify the detrimental effects of Guignardia leaf blotch infection 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). Likewise 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 absorbance 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).
Guignardia Leaf Blotch Severity
Guignardia leaf blotch severity was assessed visually in September 2007, 2008, and 2009. Each tree was rated on a 0 to 5 rating scale, using a visual indexing technique and ratings on the scale: 0 = no leaf blotch observed; 1 = less than 5% of leaves affected and no aesthetic impact; 2 = 5%–20% of leaves affected with some yellowing but little or no defoliation; 3 = 21%–50% of leaves affected, significant defoliation (30%–50%) and/or leaf yellowing; 4 = 51%–80% of leaves affected, severe foliar discoloration and defoliation (51%–90%); 5 = 81%–100% of foliage affected with 91%–100% defoliation.
The individual ratings for each tree in each treatment were used as a Guignardia leaf blotch severity index for statistical analysis.
Mean Guignardia leaf blotch severity values for treatments were transformed using the arcsine−1 transformation. All data were analyzed using ANOVA after checks for homoscedasticity were met using an Anderson-Darling test and the differences between means were separated by the Least Significance Difference (LSD) at the 95% confidence level (P = 0.05) using the Genstat for Windows 14th Edition program. Back transformed Guignardia leaf blotch severity values are presented here to ease interpretation of data (Blaedow et al. 2006).
RESULTS
Damaging outbreaks of Guignardia leaf blotch were recorded on control trees in both the 2007 and 2008 trials as indi cated by leaf blotch severity ratings of 4.6 on leaves of horsechestnut at the cessation of each growing season, respectively (Table 1; Table 2). None of the treated or control trees died as a result of leaf blotch attack during the course of the study, and none of the fungicide and water-retaining polymer combinations evaluated was phytotoxic to the test trees (data not shown). Efficacy as leaf blotch protectant compounds was demonstrated when fungicides were incorporated into a water-retaining polymer over the first growing season—i.e., observed pathogen severity was, in all cases, significantly lower (P < 0.05) compared to water dipped controls. In these cases, leaf blotch severity was reduced by 46%–65% (2007 growing season) and by 41%–67% (2008 growing season), respectively. Significant reductions in leaf blotch severity were mirrored by significant increases in leaf chlorophyll fiuorescence as a measure of leaf photosynthetic activity (20%–29%, 2007 growing season; 32%–43%, 2008 growing season) and leaf chlorophyll content SPAD values (40%–91%, 2007 growing season; 25%–58%, 2008 growing season); (Table 3; Table 4; Table 5; Table 6). There were little differences in the magnitude of efficacy between fungicides and concentration applied where leaf blotch severity, leaf chlorophyll fiuorescence, and leaf chlorophyll content were statistically similar between treatments in both the 2007 and 2008 studies (Table 3; Table 4; Table 5; Table 6). Limited efficacy of any of the fungicide and water-retaining polymer combinations as leaf blotch protectant compounds was demonstrated the following year after application. In most cases, observed leaf blotch severity, leaf chlorophyll fiuorescence Fv/Fm values, and leaf chlorophyll content were statistically comparable to water-treated controls. This indicates a fungicide and water-retaining polymer combination applied as a root dip at the time of planting provides one growing season’s protection. Application of a water-retaining polymer root dip alone (i.e., no fungicide) had no effect on reducing leaf blotch severity at the cessation of both the 2007 and 2008 study. In all cases, observed leaf blotch severity, leaf chlorophyll fiuorescence Fv/Fm values, and leaf chlorophyll content were statistically comparable to water-treated controls. Based on visual observation of leaf blotch severity, the greatest protection in both the 2007 and 2008 trials was provided by the synthetic fungicide penconazole applied as a foliar spray four times during the growing season. In both the 2007 and 2008 studies, leaf blotch severity was reduced by 100% (Table 1; Table 2). In addition, the highest chlorophyll fiuorescence Fv/Fm and SPAD values as measures of leaf photosynthetic activity and chlorophyll content respectively were recorded in penconazole foliar spray treated trees compared to water-treated controls and fungicide and water-retaining polymer combinations (Table 3; Table 4; Table 5; Table 6). No efficacy of penconazole foliar sprays as leaf blotch protectant compounds was, however, demonstrated the following year after application. In all cases, observed leaf blotch severity, leaf chlorophyll fiuorescence Fv/Fm values, and leaf chlorophyll content were statistically comparable to water-treated controls.
The influence of water-retaining polymer (WRP) and fungicide combinations applied at the time of planting for the control of Guignardia leaf blotch on leaves of horsechestnut (Aesculus hippocastanum L.) as measured by observed pathogen severity.
The influence of water-retaining polymer (WRP) and fungicide combinations applied at the time of planting for the control of Guignardia leaf blotch on leaves of horsechestnut (Aesculus hippocastanum L.) as measured by leaf chlorophyll fluorescence Fv/Fm values.
The influence of water-retaining polymer (WRP) and fungicide combinations applied at the time of planting for the control of Guignardia leaf blotch on leaves of horsechestnut (Aesculus hippocastanum L.) as measured by leaf chlorophyll fluorescence Fv/Fm values.
The influence of water-retaining polymer (WRP) and fungicide combinations applied at the time of planting for the control of Guignardia leaf blotch on leaves of horsechestnut (Aesculus hippocastanum L.) as measured by leaf chlorophyll content (SPAD) values.
The influence of water-retaining polymer (WRP) and fungicide combinations applied at the time of planting for the control of Guignardia leaf blotch on leaves of horsechestnut (Aesculus hippocastanum L.) as measured by leaf chlorophyll content (SPAD) values.
DISCUSSION
Results of this study show that use of a fungicide and water-retaining polymer combination applied to bare-rooted stock of horsechestnut (Aesculus hippocastanum L.) at the time of planting can result in significant reductions in outbreaks of Guignardia aesculi, the causal organism of Guignardia leaf blotch, over a single growing season. However, no significant degree of control was manifest the following year after planting, indicating that foliar sprays of an appropriate fungicide would be required to keep Guignardia leaf blotch severity to acceptable levels.
Little differences in the magnitude of efficacy between fungicides and concentration applied were recorded. This indicates that where reductions in leaf blotch severity are required for the first year after planting, several commercially available fungicides exist for this purpose. The range of fungicides evaluated for test purposes in this study are extensively used to control key fungal pathogens of woody plants, agricultural and horticultural crops, and orchard and forest trees. Results presented here, however, are the first to show efficacy of these fungicides against Guignardia leaf blotch. Although this study showed no significant influence of the type of fungicide used, previous evaluation of fungicides against Guignardia leaf blotch under in vitro and in situ conditions demonstrated marked sensitivity to individual products and concentration (Plenk 1996; Zimmermannová-Pastirčáková 2003). Under in vitro conditions fungicide efficacy was in the order mancozeb > fenarimol > benomyl > dodine > iprodine. Under in situ conditions (i.e., foliar sprays of each fungicide), efficacy was in the order fenarimol > benomyl > mancozeb > dodine > iprodione. Previous research has demonstrated that the efficacy of injected pesticides is related to uptake and translocation from injection site to target, which in turn is dependent on pesticide solubility, health of transport tissues within the vascular system, and tree species (Tattar et al. 1998; Young, 2002; Doccola et al. 2003). Although the mode of application differed in this study (root polymer dip versus injection), results demonstrate that irrespective of the fungicide used, sufficient quantities are translocated to foliar tissue to confer Guignardia leaf blotch protectant properties throughout the canopy over an entire growing season.
The impact of plant pathogen infection on leaf photosynthetic structure and processes can be monitored through changes in chlorophyll fiuorescence kinetics to provide an insight into plant responses to pathogen invasion (Berger et al. 2007a; Berger et al. 2007b; Chaerle et al. 2007); in some circumstances, changes in fiuorescence can be detected before symptoms of pathogen infection become visibly apparent (Bonfig et al. 2006; Berger et al. 2007a). Chlorophyll fiuorescence Fv/Fm ratios are regarded as a highly sensitive measure of damage to photosystem II and therefore, indirectly the leaf photosynthetic apparatus. Consequently, Fv/Fm ratios have been used to quantify damage and/or impairment of the leaf photosynthetic apparatus following fungal invasion and colonization of the leaf surface (Percival and Fraser 2002). Guignardia leaf blotch is regarded as a foliar blight with enzymatic degradation damage of the leaf, chlorophyll molecule, and photosynthetic system as a consequence of infection (Pastirčáková 2003). Fv/Fm ratios ≥0.75 are associated with healthy plants. Only trees sprayed with penconazole four times throughout the growing season had values higher than 0.75 at the cessation of each experiment. Affected leaves of controls with heavy infection and fungicide/water-retaining polymer treated trees displaying milder infection symptoms were characterized by decreased Fv/Fm, below the 0.75 threshold. The brown or reddish-brown lesions that cover a large portion or even the entire leaf indicate Guignardia leaf blotch exerts its influence over the host via vascular connections (Pastirčáková 2003). It is known that Guignardia leaf blotch produces hydrolytic enzymes that degrade the epidermis, cell wall and necrosis symptoms develop as intercellular mycelium spreads rapidly within the leaf (Schlösser 1983; Pastirčáková 2004). The consequence of such interaction distresses the photosynthetic mechanism directly or indirectly and impairs its ability to quench excitation energy. In normal situations, light capture is accompanied by photochemical and non-photochemical quenching mechanisms that balance photon utilization for electron transport purposes and repair of oxidative damage or heat dissipation (Anderson et al. 1997; Cruz et al. 2004). When absorbed light energy exceeds the leaf’s capacity to use trapped energy through photosynthesis or dissipate it by heat, damage to PSII occurs. Results of this study indicate that symptom development in Guignardia leaf blotch infected foliage is associated with increased excitation pressure at PSII centers, followed by oxidative damage and irreversible destruction of centers (Baker et al. 2007; Horton and Ruban 2005; Muller-Moule et al. 2004). Such action leads to loss of chlorophyll and chlorosis (visible Guignardia blotch infection severity) as observed in this study.
Penconazole, when applied four times during the growing season, proved 100% effective for Guignardia leaf blotch control. The effectiveness of penconazole against several other fungal pathogens under laboratory and field conditions has been confirmed by other authors (Kenyon et al. 1997; Mmbaga and Sauve 2004; Percival and Boyle 2005; Schnabel and Parisi 1997). Results of this study support these conclusions with repeat penconazole sprays proving to be the optimal treatment in terms of reduced Guignardia leaf blotch severity, improved photosynthetic efficiency (Fv/Fm), and higher leaf chlorophyll content (SPAD).
Results regarding the use of a water-retaining polymer alone on reducing Guignardia leaf blotch development conclude no significant benefit as observed Guignardia leaf blotch severity, leaf chlorophyll fiuorescence Fv/Fm values, and leaf chlorophyll content SPAD values were in both the 2007 and 2008 trials comparable to water treated controls. Consequently, use of water-retaining polymer alone as Guignardia leaf blotch protectant compound appears limited based on results of this study and are not recommended.
In conclusion, results provide evidence that use of commercially available fungicides products in combination with a water-retaining polymer applied at the time of planting as a root dip to bare rooted stock can be used to reduce Guignardia leaf blotch severity over a single growing season. However, foliar sprays of an appropriate fungicide would be required the year after planting to keep Guignardia leaf blotch severity to acceptable levels. Further research is ongoing, evaluating fungicide and water-retaining polymer root dips against several other key fungal pathogens of trees.
Acknowledgments
The author is grateful for funding in part from the TREE Fund (Hyland Johns Grant).
- © 2013, International Society of Arboriculture. All rights reserved.
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