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Keywords:

  • azoxystrobin;
  • epoxiconazole;
  • fungicidal effects;
  • saprophytic fungi;
  • winter wheat;
  • yield

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The effects of the fungicides azoxystrobin (a strobilurin) and epoxiconazole (a sterol biosynthesis inhibitor) on phyllosphere fungi, senescence and yield were studied in winter wheat in field trials free of visible disease and under controlled environmental conditions. In two field trials, treatments with each of the two fungicides prolonged green leaf area retention and increased yield compared with untreated control plots. Azoxystrobin maintained green leaf area for longer than epoxiconazole and, in one trial, treatments with azoxystrobin gave a greater yield response than epoxiconazole. Mycelial growth on leaf surfaces, mainly originating from saprophytic fungi, was reduced by each of the fungicides. Papilla formation and hypersensitive reactions, almost exclusively against infection attempts by Mycosphaerella spp. (most probably M. graminicola), occurred with high frequency in the leaves. These defence reactions presumably incurred a significant energy cost, accelerating plant senescence. Fewer defence reactions were recorded in azoxystrobin-treated leaves than in epoxiconazole-treated and untreated leaves. Inoculation in a glasshouse experiment with the saprophytic fungi Alternaria alternata and Cladosporium macrocarpum accelerated wheat senescence. Control of the saprophytes by azoxystrobin or epoxiconazole treatments caused a delay in the accelerated senescence, but without significant increase in above-ground biomass and yield. Neither fungicide influenced senescence, above-ground biomass or yield in noninoculated wheat plants. In growth chamber experiments azoxystrobin inhibited spore germination and mycelial growth of A. alternata and C. macrocarpum. Epoxiconazole had little inhibitory effect on spore germination, but strongly inhibited mycelial growth of both saprophytes. Both fungicides reduced A. alternata-induced papilla formation in wheat leaves, with epoxiconazole being more effective. Inoculation with either of the two saprophytes did not significantly increase wheat leaf respiration, in contrast to inoculation with the nonhost pathogen Erysiphe graminis f.sp. hordei. Treatment with azoxystrobin did not affect this latter increase in respiration whereas it was reduced by epoxiconazole treatment. It is proposed that the greater inhibition of infection attempts from Mycosphaerella spp. by azoxystrobin, compared with epoxiconazole, may account for the greater yield given by azoxystrobin in field plots.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The fungicide azoxystrobin belongs to the strobilurins, a recently introduced group of agrochemical fungicides (Godwin et al., 1992; Ammermann et al., 1992; Masuko et al., 1993; Margot et al., 1998). The development of strobilurin fungicides was inspired by the discovery of the strobilurins, oudemansins and myxothiazols, a group of fungitoxic metabolites produced in nature by certain fungi and myxobacteria (Musilek et al., 1969; Anke et al., 1979; Gerth et al., 1980). These metabolites were all shown to exert their fungicidal action by blocking electron transport in the mitochondrial respiratory chain in fungi. The commercial products share this mode of action, thus the name ‘strobilurins’. More specifically, they bind to the ubihydroquinone reduction site, the Qo-site of complex bc1, thus inhibiting electron transfer between cytochrome b and cytochrome c1 in the respiratory chain (Becker et al., 1981; von Jagow & Link, 1986). This severely reduces the aerobic energy production, thereby inhibiting growth of the fungus (Godwin et al., 1994; Shirane et al., 1994; Leinhos et al., 1997) and thus forming the basis for the use of strobilurins as effective fungicides. With this new mode of action the strobilurins are an important addition to the existing fungicide range, particularly for cereals in which recent broad-spectrum fungicide products have been largely based on sterol biosynthesis inhibitors (SBI).

The first years of use have shown azoxystrobin treatment to provide excellent disease control in cereal crops and high yield increases have been obtained (Cohadon et al., 1994; Jørgensen & Nielsen, 1998; Mercer & Ruddock, 1998). Superior yield increases in winter wheat have been obtained with azoxystrobin treatment in comparison with efficient SBI fungicides, even in crops with very little visible disease (Jørgensen & Nielsen, 1994, 1996; Nielsen & Jørgensen, 1995; Jones & Bryson, 1998) but, as yet, the reasons have not been unequivocally demonstrated. The leaves in azoxystrobin-treated wheat have been described as appearing greener (Konradt et al., 1996; Habermeyer et al., 1998) and as retaining their green leaf area for longer than the leaves of SBI-treated wheat (Jones & Bryson, 1998). Therefore a longer period of photosynthetic active green leaf area has been suggested to be the main factor for yield increases obtained with strobilurin fungicides, because the increased photosynthetic period would increase the quantity of assimilate available for grain filling.

In the laboratory, strobilurins have been shown to inhibit the germination and prepenetration growth of several plant pathogenic fungi (Godwin et al., 1994; Gold & Leinhos, 1994; Leinhos et al., 1997). This is in contrast with SBIs, which generally do not inhibit fungal growth until after initial infection (Buchenauer & Kemper, 1981; Hänssler & Kuck, 1987; Godwin et al., 1994). Cereal plants respond to infection attempts with defence reactions that are energy demanding and potentially hasten plant senescence (Smedegaard-Petersen, 1980; Smedegaard-Petersen & Stølen, 1981; Smedegaard-Petersen & Tolstrup, 1985).

It has been previously suggested that delays in senescence and yield increases obtained by fungicide application to fields with very low levels of visible disease are caused by fungicidal control of saprophytic fungi (Dickinson, 1981; Smedegaard-Petersen & Tolstrup, 1985). It was shown in a glasshouse experiment that saprophytic fungi, without the ability to infect plants, are able to accelerate senescence and reduce grain yield of barley (Tolstrup, 1984). The plants reacted against the saprophytes with defence reactions, and the chlorophyll content in the leaves was reduced, indicating accelerated senescence. Both the energy cost of defence reactions and the earlier senescence probably contributed to the yield reduction (Smedegaard-Petersen & Tolstrup, 1985).

The possibility of yield reduction caused by saprophyte activity has led to the suggestion that broad-spectrum fungicides may increase yield by eliminating saprophytic fungi on the leaves (Dickinson, 1981; Smedegaard-Petersen & Tolstrup, 1985). The inhibition of early fungal development by azoxystrobin treatment (Godwin et al., 1994) may thus reduce the frequency of attempted penetration from filamentous saprophytic fungi in the field and thereby possibly reduce plant defence reactions and postpone the senescence that such fungi induce, increasing yield.

This study, involving glasshouse, growth chamber and field plot experiments, was conducted to examine the effects of azoxystrobin treatment on saprophytic fungi on winter wheat leaves and to study the implications for senescence and yield of the presence of saprophytes and/or fungicide. The yield-improving effect of azoxystrobin treatment is postulated to arise from inhibition of the defence-activating and senescence-promoting activity of saprophytic fungi. This is in contrast with SBI treatments, which inhibit the growth of the saprophytes only after a potential fungus–plant interaction has occurred, thus possibly not influencing defence activation and senescence promotion. The effects of azoxystrobin were compared with those of a representative SBI fungicide, epoxiconazole, a highly efficient triazole fungicide (Ammermann et al., 1990; Mercer & Ruddock, 1996). The interactions between plants, saprophytes and fungicides were examined in glasshouse experiments in order to avoid the influence of pathogens and disease on the results, something that is virtually impossible under field conditions.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Glasshouse experiments

Two glasshouse experiments were carried out at Zeneca Agrochemicals, Jealott's Hill International Research Centre, Berkshire, UK, in the period January–July 1998.

Plant material

Seeds of winter wheat cv. Spark were vernalized and sown in 10-inch-diameter (7·5 L) pots in a mix of JIP3 compost (John Innes compost, UK) and washed grit 15 : 1. After emergence the seedlings were thinned to 15 plants per pot and placed in a glasshouse at minimum temperatures 15/18°C (night/day). 400-W mercury lamps supplemented the sunlight in order to adjust the day-length to 18 h. The pots of each of the two experiments were arranged in three tents (115 cm × 150 cm, ground level; height 130 cm) made from transparent polythene. The pots were watered automatically via the capillary matting on which they were placed and supplemented with top watering. Fertilizer was added twice to supplement the soil nutrients. The fungicides, ‘Milgo E’[active ingredient (a.i.) ethirimol, 280 g L−1] and ‘Fortress’ (a.i. quinoxyfen, 500 g L−1), both found to have minimal effect on the growth of the saprophytes, were applied in the early growth stages to keep the plants free of powdery mildew (Erysiphe graminis f.sp. tritici). Biological control agents were applied regularly to control pests and the insecticides ‘Aphox’ (a.i. pirimicarb 500 g kg−1) and ‘Malathion 60’ (a.i. malathion 600 g L−1) were applied, twice and once, respectively, to eradicate thrips and aphids.

Fungal material and inoculation

All plants in the first glasshouse experiment were inoculated with the saprophytic fungi Alternaria alternata (two different isolates) and Cladosporium macrocarpum (three different isolates) originating from Danish wheat fields not treated with fungicides. The fungi were grown under near-UV light for 7–10 days (12 h day/night cycle) on Petri dishes with V8 juice agar and potato dextrose agar, respectively. Sixty dishes of each species were used to make up the mixed spore suspension for each inoculation. The spore concentration ranged from 1·5 to 3·4 × 105 spores L−1 of each species and the suspension included 0·05% Tween 20. Approximately 50 mL spore suspension was applied to the plants of each pot. A hand-held sprayer was used for inoculation at a pressure of approximately 2 atm. After each inoculation the tents were closed for 48 h, in which period the plants were regularly misted to ensure approximately 100% relative humidity, thus obtaining favourable conditions for spore germination and fungal growth. The plants were inoculated every 7–11 days from growth stage (GS) 31–32 to GS 71 (Tottman, 1987), making a total of six inoculations.

In the parallel glasshouse experiment without fungal inoculation, all plants were sprayed six times with distilled water + 0·05% Tween 20, without fungal inoculum. The tents were closed for 48 h and misted regularly as in the other experiment.

Fungicide treatments

Treatments in both experiments comprised of azoxystrobin, epoxiconazole or untreated, 12 pots of each. In each tent there were 12 pots (four of each treatment) fully randomized within the tent. The experimental sprayings were performed using a tracksprayer (‘Those Engineers’, London, UK) with a Teejet 8002 EVS tip nozzle delivering 200 L ha−1. The fungicides were applied at the manufacturers' recommended field rates: ‘Amistar’ (azoxystrobin 250 g ha−1) and ‘Opus’ (epoxiconazole 125 g ha−1) in a three-spray programme at GS 31–32, GS 37–39 and GS 45–51 (as determined when > 50% of the plants reached the target growth stages).

Fungal growth and defence reactions in the leaves

Two methods were used to examine the growth of the fungi and defence reactions in the leaves: (1) cellulose nitrate impressions of the leaf surface encapsulating those fungi present; and (2) clearing of leaf segments for microscopical examination for cellular defence reactions such as fluorescing cell walls and cells reacting hypersensitively (HR) in the epidermis.

1 At GS 43, one F−1 leaf (the leaf below the flag leaf) central segment per pot was dipped into a cellulose nitrate solution and allowed to dry to a thin impression film (Thordal-Christensen & Smedegaard-Petersen, 1988a). The films from both the adaxial and abaxial leaf surfaces were mounted on slides in lactoglycerol (1 : 2 : 1 lactic acid : glycerol : water) with 0·25% Evans blue. An area of 3·4 mm2 from both sides of each leaf was microscopically examined and the two scores were averaged. The spores were scored as germinated or not and germination was considered evident when a germ tube of a length at least half the width of the spore was visible. At least 25 spores of each species were scored per leaf. Mycelial growth was measured according to Olson (1950).

2 At GS 43, one F−1 leaf segment per pot was sampled to study host reactions to the fungal activity. The leaf segments were cleared in 95% ethanol followed by heating to 60°C for 1 h in lactophenol (1 : 1 : 1 : 1 lactic acid : glycerol : water : phenol) with 0·25% aniline blue. The leaf segments were then mounted in lactoglycerol and studied employing light and fluorescence microscopy, excitation maxima 365, 405 and 430 nm. Fungal-induced plant cell wall appositions could be seen fluorescing, or by the colour of the bound stain.

Green leaf area and yield assessments

In both glasshouse experiments, the green leaf area was assessed every 7–10 days. The green leaf area was assessed as percentage green area of the leaf blade for the uppermost five leaves of six randomly chosen plants from each pot. At the latest five assessment dates in the experiment with inoculated plants, this was increased to 10 randomly chosen plants per pot. All assessments of green leaf area and fungal growth were performed on leaves from the main stems of the plants. At harvest the grain yield and total above-ground biomass of the plants were measured for each pot after drying at 65°C for 2 days.

Growth chamber experiments

Plant material and fungicide treatments

The wheat cultivar Lynx was used in all investigations. The plants were grown in plastic pots (12 × 13·5 cm) in the soil mix ‘Weibulls enhetsjord’ (K jord, Svalöf Weibull AB, Hammenhög, Sweden) in a growth chamber for 21–24 days before experiment commencement. The growth chambers were adjusted to a cycle of 16 : 8 h light/darkness at approximately 20°C, 50–60% relative humidity (RH) and 15°C, 80–90% RH, respectively. The light (∼200 µE m−2 s−1) was supplied by fluorescent tubes (Philips TLD 36 W/83). The second developed leaves were fixed in horizontal position, abaxial side upwards, on bent plastic plates using unbleached cotton strings (Lyngs Jørgensen et al., 1993).

The fungicides were used in their commercial formulations: ‘Amistar’ (azoxystrobin 250 g a.i./L) and ‘Opus’ (epoxiconazole 125 g a.i./L) or distilled water (control), supplemented with 0·05% Tween 20, were sprayed on the leaves until run-off at 1 h before inoculation. Fungicide rates are given in the tables in the Results.

Fungal material and inoculation

A. alternata and C. macrocarpum spores were produced as for the glasshouse experiment. Conidial suspensions or distilled water (control), supplemented with 0·05% Tween 20, were sprayed on the leaves until run-off. After inoculation the plants were incubated in plastic bags with only diffuse light. After 48 h, with regular misting to keep the leaves moist, the bags were removed and normal light intensity re-established. For the nonhost pathogen studies, isolate C–15 of the barley powdery mildew fungus E. graminis f.sp. hordei, maintained on barley line P-02 was used. Inoculation was performed by shaking the inoculum-producing barley plants above a settling tower (Thordal-Christensen & Smedegaard-Petersen, 1988b). After inoculation, the plants were returned to the growth chamber until respiration measurements commenced.

Fungal growth and defence reactions in the leaves

For determination of spore germination, mycelial growth on the leaf surface and papilla formation in the leaves, the sampled leaf pieces were cleared by the method of Carver et al. (1992) and stained for 24 h on filter paper soaked with aniline blue (0·25% in 1 : 1 lactoglycerol : ethanol). The leaf pieces were then mounted in lactoglycerol and studied employing light and fluorescence microscopy, excitation maxima 405 and 430 nm. The hyphae were stained by aniline blue and measured using the method of Olson (1950). At least 50 spores on each of four leaves per treatment were assessed when estimating germination frequencies. Fungal-induced papillae and their surrounding haloes, if present, could be observed by the fluorescence of the aniline blue binding to these structures. The frequency of papilla formation was estimated by counting the number of papillae and relating this to the number of fungal spores. An area of 4·16 mm2 was microscopically examined (corresponding to 40 areas each of 0·104 mm2) on each of four leaf pieces per treatment and assessment time.

Dark respiration measurements

For the dark respiration experiments, two plants were grown in 6 × 6 cm pots and the second developed leaves were fixed, abaxial side up, onto plastic netting using rubber bands. Otherwise, the growth conditions, fungicide application, inoculation and incubation were performed as described previously. Respiration measurements were performed in situ. Before measurement, the leaves were gently stroked with wet cotton to remove the fungal material (Smedegaard-Petersen, 1977) and then kept in darkness for 30 min before measurement. CO2 concentration was measured with an URAS 3G infrared gas analyser (Hartmann & Braun AG, Frankfurt a.M., Germany). Ambient air adjusted to 20°C, 95% RH, was used as both sample and reference air stream. Excess air was supplied to the leaf insert, thereby forming an air-seal at the leaf base (Wolf et al., 1969). Air was pumped through the leaf compartment at a constant rate measured by an electronic mass flowmeter (Aalborg Instruments, New York, USA). The signals from the gas analyser and the flowmeter were digitized and transferred to a PC-based program for storage and calculation. Every measurement was read when the signal had stabilized after acclimatization of the leaf. Dry weight (DW) of the leaves was measured after 24 h at 65°C. Respiration was calculated in µmol CO2 s−1 g DW−1 and the measurements from the two leaves of each pot was averaged. At each time-point, four pots of each treatment were used for measurement.

Field experiments

Trial design and treatments

Two field trials were carried out in the 1997/98 growing season in the UK, at Eriswell in Suffolk and at Maidenhead in Berkshire.

Winter wheat seeds cv. Spark dressed with ‘Sibutol’ (a.i. bitertanol + fuberidazole, 375 + 23 g L−1) were sown at a rate of 215 kg ha−1 (Suffolk) or 185 kg ha−1 (Berkshire). In the Suffolk trial, ‘Bombadier’ (chlorothalonil 1100 g a.i. ha−1) and ‘Fortress’ (quinoxyfen 150 g a.i. ha−1) were applied at GS 26 (Tottman, 1987) to control natural epidemics of septoria tritici blotch (Mycosphaerella graminicola) and powdery mildew (E. graminis f.sp. tritici), respectively. Other agricultural procedures were carried out according to common practice in British farming.

‘Amistar’ (azoxystrobin 250 g ha−1) or ‘Opus’ (epoxiconazole 125 g ha−1) was applied in 200 L ha−1 spray volume with hand-held spray booms in a three-spray programme at GS 31, GS 39 and GS 59. The three plots in each block were treated with azoxystrobin, epoxiconazole or left untreated and were randomized within each block. In Suffolk, the trial consisted of 18 blocks with plots each of 6 × 2·5 m. In Berkshire, the trial was divided into nine blocks with plots 12 × 2·5 m in size.

Fungal development and plant defence reactions

To examine the growth of fungi on the leaf surface and defence reactions in the leaves the same methods were used as for leaves from the glasshouse experiments.

1 F−1 leaf segments were sampled from six blocks in the Suffolk trial. The impression films from the adaxial leaf surface were mounted on slides in lactoglycerol (1 : 2 : 1 lactic acid : glycerol : water) with 0·25% Evans blue. An area of 2·025 mm2 (corresponding to 15 areas each of 0·135 mm2) from each of four leaf segments per plot was microscopically examined. The spores observed in the films were determined to genus level and it was recorded if germination had occurred.

2 F−1 leaf segments were sampled and prepared for studies of host reactions against fungal infection, according to the procedure outlined for glasshouse leaves. The adaxial side of four leaf segments from each of the three treatments in four field blocks (only three blocks in Berkshire) was studied. Forty areas, each of 0·104 mm2, were studied per leaf segment. Cells of the stomatal complex, trichomes or cells lying above vascular bundles were excluded from examination. The defence reactions were recorded as: (i) hypersensitive reactions (HR) appearing as one or a limited number of dead cells (auto-fluorescing and with brown coloured cell contents); and (ii) cell wall changes appearing as papillae and haloes at the sites of attempted fungal penetration. The cell wall appositions could be seen with fluorescence microscopy by the bound aniline blue stain. Furthermore, the association of fungal hyphae with defence reactions was recorded.

Green leaf area, disease and yield assessments

Green leaf area was visually assessed as percentage of the whole leaf area remaining green. Disease coverage was assessed by visual estimation of the percentage leaf area affected by disease symptoms. The assessments were carried out for each of the upper four leaves (flag leaf, F–1, F–2 and F–3) of 10 (Suffolk) or 20 (Berkshire) randomly chosen tillers per plot at 7–10 day intervals from GS 47 (Suffolk) or GS 65 (Berkshire).

The central 2 m of each plot was combine harvested on 14 August 1998 in the Suffolk trial and on 19 August 1998 in the Berkshire trial. The grain yields were adjusted to 85% dry matter.

Statistical analysis

Percentage green leaf area and percentage spore germination were transformed by arcsin√(%/100) and data on mycelial growth and papillae elicitation were log transformed before analysis of variance. Other data were analysed for variance without transformation. All data were analysed in pcsas (release 6·11; SAS Institute, Cary, NC, USA) with P ≤ 0·05 throughout.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Glasshouse experiments

The results of the microscopical examination of the leaf surface impressions from the inoculated plants in the glasshouse are presented in Table 1. Germination of C. macrocarpum spores, but not of those of A. alternata, was reduced by treatment with azoxystrobin compared to the epoxiconazole treatment and the untreated. Treatment with epoxiconazole significantly enhanced the germination of both spore types compared with untreated leaves. The mycelial growth of the fungi was not significantly reduced by either of the two fungicides. During the microscopical examination of the leaf segments only a few defence reactions were observed and no effect of the fungicide treatments in this respect could be discerned (data not shown). There were no significant differences in grain yield or total above-ground biomass between any of the treatments and the untreated in this glasshouse experiment (Table 1).

Table 1.  Effects of azoxystrobin and epoxiconazole treatments on fungal growth, yield and biomass of wheat plants inoculated 6 times with Cladosporium macrocarpum and Alternaria alternata under glasshouse conditions
Fungal growth and host yield and biomass UntreatedAzoxy- strobinEpoxicon- azole
  1. Spore germination and mycelium growth was assessed on F−1 (1st leaf under flag leaf) at growth stage 43. Different letters indicate statistically significant differences between treatment means at the 5% level. DW, dry weight.

Percentage Alternaria germinated75·9a69·6a89·8b
Percentage Cladosporium germinated76·0b54·5a91·7c
Mycelial growth  (mm mycelium per mm2 leaf surface)1·00·61·1
Grain yield (g DW per pot)25·824·726·7
Above-ground biomass (g DW per pot)132141138

The persistence of green leaf area in the inoculated plants was maintained by both fungicide treatments, being significantly superior to the untreated (Fig. 1). The percentage of green leaf area of leaves from treated plants was generally 10–15% higher than that of leaves from untreated plants. This applied to all leaf layers examined, from GS 65–73 to GS 83, and thus corresponded to a delay in saprophyte-induced senescence of approximately 14 days for each of the five upper leaves for more than a month (Fig. 1). The two fungicide treatments performed similarly in this respect and on no assessment date was there a significant difference between the treatments in green leaf area for any of the upper five leaf layers.

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Figure 1. The effect of treatments with the fungicides azoxystrobin and epoxiconazole on green leaf area of the five upper leaves of wheat plants inoculated six times with the saprophytic fungi Alternaria alternata and Cladosporium macrocarpum (glasshouse expt 1). bsl00066, azoxystrobin; ▪, epoxiconazole; ●, untreated. GS, growth stage. F−1, the leaf below the flag leaf, etc. Different letters (a, b) at a particular assessment date indicate statistically significant differences at the 5% level between treatment means.

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The impression film method demonstrated that very few fungi were present on the leaves of the noninoculated plants of the second glasshouse experiment (data not shown). There were no significant differences in green leaf area of the noninoculated plants between any of the treatments and the untreated (Fig. 2). There were also no significant differences in grain yield or total above-ground biomass between any of the treatments and the untreated (Table 2).

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Figure 2. The effect of treatments with the fungicides azoxystrobin and epoxiconazole on green leaf area of the five upper leaves of wheat plants. The plants were not inoculated with saprophytic fungi (glasshouse expt 2). bsl00066, azoxystrobin; ▪, epoxiconazole; ●, untreated. GS, growth stage. F−1, the leaf below the flag leaf, etc. There were no statistically significant differences between means of treatments at any assessment date.

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Table 2.  Effects of azoxystrobin and epoxiconazole treatments on yield and biomass of wheat plants not inoculated with fungi under glasshouse conditions
Host yield and biomass UntreatedAzoxy- strobinEpoxicon- azole
  1. There were no statistically significant differences at the 5% level between the treatment means. DW, dry weight.

Grain yield (g DW per pot)28·231·927·0
Above-ground biomass  (g DW per pot)143151138

Growth chamber experiments

In growth chamber experiments with second developed leaves, treatment with azoxystrobin reduced the germination of C. macrocarpum spores to 26% of the level on the untreated at 1 mg a.i. L−1 (active ingredient per L) and to 11% at 5 mg a.i. L−1 (Fig. 3a). The germ tubes from the germinated spores were short on average and at 1 mg azoxystrobin a.i. L−1 the length of mycelial growth was only 4% of that on the untreated leaves and only 2% at 25 mg a.i. L−1 (Fig. 3b). Epoxiconazole treatment reduced the germination at 25 mg a.i. L−1, but not at the lower concentrations tested (Fig. 3a). Epoxiconazole reduced mycelial growth in a log (concentration) dependent manner, but the reduction was significantly less than treatment with azoxystrobin at the two lower fungicide concentrations tested (Fig. 3b). The hyphae of C. macrocarpum occasionally induced aniline blue stained thickenings of the lateral cell walls between the epidermal cells in untreated leaves. No clearly defined papilla formation occurred in any leaves (data not shown).

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Figure 3. The effect of azoxystrobin or epoxiconazole on (a) germination and (b) mycelial growth of Cladosporium macrocarpum spores on second developed wheat leaves. The leaves were inoculated 1 h after fungicide treatment and the assessments were made 96 h after inoculation. Different letters (a, b, c, d) indicate statistically significant differences at the 5% level between treatment means.

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On the untreated leaves, the final percentage germination of the A. alternata spores was reached by 12 h after inoculation (h.a.i.) (Fig. 4a). The fungus apparently exhausts its reserves for mycelial growth by 24 h, after which time 3·5 mm mycelium per spore had been formed on average (Fig. 4b). Papillae did not appear until 24 h after inoculation but then increased to 83 papillae on average per 100 spores at 96 h.a.i. (Fig. 4c). Azoxystrobin treatment delayed spore germination and the final percentage germination was significantly lower than for untreated or epoxiconazole-treated leaves at the end of the experiment (Fig. 4a). Azoxystrobin also delayed mycelial growth and at 96 h.a.i. the length of mycelium per spore on azoxystrobin-treated leaves was 17% of that recorded on the untreated leaves (Fig. 4b). Azoxystrobin treatment also reduced papilla induction to 16% of untreated by 96 h.a.i. (Fig. 4c). Epoxiconazole treatment did not significantly reduce the percentage germination of A. alternata (Fig. 4a), but mycelial growth was significantly reduced with the length of mycelial growth per spore on epoxiconazole-treated leaves being 9% of that recorded on the untreated leaves (Fig. 4b). Papilla formation was reduced to 3% of untreated on epoxiconazole-treated leaves by 96 h.a.i. (Fig. 4c).

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Figure 4. The effect of treatment with azoxystrobin or epoxiconazole on (a) germination; (b) mycelium growth of Alternaria alternata and (c) papilla elicitation by this saprophytic fungus at 12–96 h after inoculation on second developed wheat leaves. The leaves were inoculated with A. alternata 1 h after treatment. bsl00066, 25 mg L−1 azoxystrobin; ▪, 25 mg L−1 epoxiconazole; ●, untreated. Different letters (a, b, c) on a particular time-point indicate statistically significant differences at the 5% level of treatment means.

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The respiration of wheat leaves did not change after inoculation with C. macrocarpum, compared to noninoculated control leaves (Fig. 5). The respiration of leaves inoculated with A. alternata did not increase significantly either with or without fungicide treatment (Fig. 6). Inoculation of the nonhost pathogen Erysiphe graminis f.sp. hordei significantly increased leaf respiration at 12 h.a.i. and 18 h.a.i. (Fig. 7). Azoxystrobin treatment did not significantly reduce the respiration of inoculated leaves. Also, the respiration of azoxystrobin-treated noninoculated leaves at 12 h.a.i. was significantly higher, compared with untreated noninoculated, although it decreased to the level of untreated noninoculated leaves during the course of the experiment. The respiration of epoxiconazole-treated inoculated leaves was significantly lower than that of untreated inoculated leaves from 18 h.a.i. onwards and the respiration of epoxiconazole-treated noninoculated leaves was significantly lower than that of untreated noninoculated leaves at 24 and 48 h.a.i.. (Fig. 7).

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Figure 5. Effect of inoculation of the saprophytic fungus Cladosporium macrocarpum on respiration of second developed wheat leaves in situ. ○, noninoculated leaves; ●, inoculated leaves. There were no statistically significant differences at the 5% level between treatment means.

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Figure 6. Effect of inoculation of the saprophytic fungus Alternaria alternata on respiration of second developed wheat leaves in situ, with and without fungicides (25 mg L−1) applied 1 h before inoculation. ○, untreated noninoculated leaves; ●, untreated inoculated leaves; bsl00066, azoxystrobin-treated inoculated leaves; ▪, epoxiconazole-treated inoculated leaves. There were no statistically significant differences at the 5% level between treatment means.

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image

Figure 7. Effect of inoculation of the nonhost pathogen Erysiphe graminis f.sp. hordei on respiration of second developed wheat leaves in situ, with and without fungicides (25 mg L−1) applied 1 h before inoculation. ○, untreated noninoculated leaves; ●, untreated inoculated leaves; bsl00084, azoxystrobin-treated noninoculated leaves; bsl00066, azoxystrobin-treated inoculated leaves; bsl00000, epoxiconazole-treated noninoculated leaves; ▪, Epoxiconazole-treated inoculated leaves. Different letters (a, b, c, d) on a particular time-point indicate statistically significant differences at the 5% level of treatment means.

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Field experiments

Fungal development and plant defence reactions

The impression films revealed spores mainly of the genera Sporobolomyces and Cryptococcus occurring more frequently than spores of filamentous fungi (Table 3). There was no significant difference in the abundance of cells of the former species on the leaves from fungicide-treated plots compared to untreated control plots. Cladosporium spp. and Mycosphaerella spp. were the most abundant filamentous fungi (Table 3). The fungicide treatments did not significantly reduce the abundance of these genera of fungal spores on the leaves. Neither azoxystrobin nor epoxiconazole significantly reduced spore germination of Cladosporium or Mycosphaerella. However, in plots treated with either of the fungicides, mycelial growth on the leaves was significantly less, compared with untreated plots (Table 3).

Table 3.  The effects of azoxystrobin and epoxiconazole treatments on fungal growth on the F−1 leaf surface in the Suffolk field trial
Leaf surface fungi UntreatedAzoxy- StrobinEpoxicon- azole
  1. The leaves were sampled at GS 75. Different letters indicate statistically significant differences at the 5% level between means of treatments.

Yeast cells (cells per mm2)144445137
Cladosporium
 (spores per mm2)15·15·36·9
 (% germinated spores)291831
Mycosphaerella
 (spores per mm2)8·25·98·3
 (% germinated spores)746265
Other spores (spores per mm2)8·56·14·9
Mycelial growth
 (mm mycelium per mm2 leaf area)8·8b2·9a2·2a

In leaves from untreated plots defence reactions associated with Cladosporium spp. or other saprophytic fungi were very seldom observed (data not shown). Contrary to this, many host defence responses occurred in the epidermal cell walls beneath germ tubes of Mycosphaerella spp. spores (Tables 4 and 5). The observed spores mostly germinated with a short germ tube from the distal end of one or each of the two cells. Appressoria and penetration attempts were often seen to be formed very close to, or beneath, the spores. Defence reactions in the leaves were seen as strong fluorescence from bound aniline blue in papillae and haloes, indicating callose deposition. The anticlinal epidermal cell walls near such papillae in unstained specimens were often seen to be autofluorescing, indicating accumulation of polyphenolic substances. In some cases following attempted penetration by Mycosphaerella spp., the cell walls delineating a whole cell were fluorescing and the cell contents were granulated and slightly brown, indicative of cell death. Only a small proportion of Mycosphaerella spp. infection attempts were successful and even these were restricted to an infection vesicle or, in some cases, an unbranched internal hypha in a single epidermal cell. No infections were observed to have progressed further than this stage.

Table 4.  The effects of azoxystrobin and epoxiconazole treatments on the occurrence of defence reactions against fungal infection attempts in F−1 leaves in the Suffolk field trial
Defence reactions per mm2 leaf, Suffolk trial UntreatedAzoxy- strobinEpoxicon- azole
  1. The leaves were sampled at GS 75. Different letters indicate statistically significant differences at the 5% level between treatment means. HR, hypersensitive reaction.

Papillae4·9ab2·9a6·6b
 associated with Mycosphaerella spp.2·9a1·6a5·0b
HR-reacting cells1·2ab0·7a1·7b
 associated with Mycosphaerella spp.0·8b0·3a1·2b
Table 5.  The effects of azoxystrobin and epoxiconazole treatments on the occurrence of defence reactions against fungal infection attempts in F−1 leaves in the Berkshire field trial
Defence reactions per mm2 leaf, Berkshire trial UntreatedAzoxy- strobinEpoxicon- azole
  1. The leaves were sampled from at GS 73. There were no significant differences at the 5% level between means of treatments. HR, hypersensitive reaction.

Papillae6·72·15·0
 associated with Mycosphaerella spp.4·41·33·1
HR-reacting cells1·10·81·9
 associated with Mycosphaerella spp.0·70·41·0

In the Suffolk trial (Table 4), the incidence of HR-reactions induced by Mycosphaerella spp. infection attempts was significantly reduced by treatment with azoxystrobin in comparison with the untreated. There were significantly fewer papillae and HR-reactions in azoxystrobin-treated leaves than in epoxiconazole-treated leaves. The frequencies of papillae and HR-reactions positively associated with infection attempts by Mycosphaerella spp. were also lower in azoxystrobin-treated leaves compared with epoxiconazole-treated leaves. The study of defence reactions in leaves from the Berkshire trial (Table 5) showed the same trends in the data as the corresponding trial in Suffolk. However, there were no statistically significant differences between the means of the treatments in the data from the Berkshire trial.

Green leaf area and disease control

In both trials, the two fungicide treatments maintained green leaf area in all leaf layers significantly more than in the untreated (Figs 8 and 9). The untreated plots in both field trials were attacked by brown rust (Puccinia recondita f.sp. tritici) and the effect of this disease on green leaf area and yield made it impossible to quantify the effect of saprophytic activity on these same parameters in the untreated plots. However, in both trials, both fungicide treatments effectively kept disease coverage of the upper three leaves below 1% (data not shown).

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Figure 8. Effect of fungicide treatments on the green leaf area of the four upper leaves in the Suffolk winter wheat field trial. bsl00066, azoxystrobin; ▪, epoxiconazole; ●, untreated. F−1, the leaf below the flag leaf, etc. GS, growth stage. Different letters (a, b, c) indicate statistically significant differences at the 5% level between means of treatments.

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Figure 9. Effect of fungicide treatments on the green leaf area of the four upper leaves in the Berkshire winter wheat field trial. bsl00066, azoxystrobin; ▪, epoxiconazole; ●, untreated. F−1, the leaf below the flag leaf, etc. GS, growth stage. Different letters indicate statistically significant differences at the 5% level between means of treatments.

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In the Suffolk trial (Fig. 8), the green leaf area of F-3 leaves was retained significantly more by treatment with azoxystrobin than by treatment with epoxiconazole, when assessed at both GS 63 and GS 71. This was also observed for the F-2 leaf layer at GS 75. Attacks of sharp eyespot (Rhizoctonia cerealis) and take-all (Gaeumannomyces graminis) made assessments of green leaf area after GS 77 invalid, as the leaves curled up and senesced rapidly, independently of the treatment. Consequently it was not possible to observe treatment effects on green leaf area hereafter and only initial effects in the flag and F–1 leaf layers could be recorded.

In the Berkshire trial (Fig. 9), the green leaf area of F–2 and F–3 leaves was significantly higher in azoxystrobin-treated plots than in epoxiconazole-treated plots, in the measurements made at GS 65 and GS 75, respectively.

Yield

In both field trials the yields were significantly higher in the fungicide-treated plots than in the untreated plots. In the Suffolk trial, the yield of azoxystrobin-treated plots was significantly higher than for the epoxiconazole treatment (Table 6). In the Berkshire trial there was no statistically significant difference between the two treatments although the trends were similar to the Suffolk trial (Table 6).

Table 6.  The effects of azoxystrobin and epoxiconazole treatments on the grain yield in the Suffolk and Berkshire field trials
 Treatment and yield (t/ha)
Location UntreatedAzoxy- strobinEpoxicon- azole LSD0,95
  1. LSD0·95, least significant difference at 5% level of probability.

Suffolk8·689·949·490·40
Berkshire7·418·177·850·42

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The growth chamber experiment with A. alternata showed that azoxystrobin inhibited the development of this fungus and, at the end of the experiment, both the extent of spore germination and mycelial growth were lower on azoxystrobin-treated leaves than on untreated leaves. Also, papilla formation in response to development of A. alternata was reduced by treatment with azoxystrobin. Both germination and mycelial growth of C. macrocarpum in the growth chamber were strongly inhibited by azoxystrobin treatment even at the lowest fungicide rate. These results demonstrate an inhibitory effect of azoxystrobin early in the development of saprophytic fungi, in accordance with studies of the effect of azoxystrobin on fungal pathogens of wheat (Godwin et al., 1994).

Epoxiconazole treatment was inhibitory to germination only for C. macrocarpum and then only at the highest fungicide rate tested in the growth chamber experiment. However, mycelial growth of both saprophytes was inhibited by epoxiconazole treatment. This is in agreement with previous studies demonstrating that epoxiconazole has the same primary mode of action as other triazole fungicides, namely the impairment of membrane production through the inhibition of ergosterol production (Akers et al., 1990). The inhibition of ergosterol production and consequent reduction in fungal growth apparently also deprived A. alternata of the ability to induce papilla formation in the host, as this was strongly inhibited by treatment with epoxiconazole.

The frequency of germination of C. macrocarpum spores in the glasshouse on fungicide-treated leaves was high compared with results from leaves in the growth chamber. In the glasshouse, ungerminated spores from a previous inoculation might have been washed off following the repeated inoculations and consequently the percentage germination may have been overestimated. This would imply that fungicide-treated ungerminated spores are more easily washed off than are untreated ungerminated spores. If this was the case, it might also explain the apparent increased germination of both fungi on epoxiconazole-treated leaves in the glasshouse, compared with untreated leaves, a result that was not reflected in the growth room experiment. Alternatively, it may be explained by differences in technique between the glasshouse and growth chamber experiments, i.e. the results not being fully comparable.

Neither fungicide treatment decreased the extent of mycelial growth in the glasshouse experiment. This is in contrast to the growth chamber experiments and may have been caused by more nutrients being available on the leaves of the glasshouse plants in the form of aphid honeydew, thrip remains, pollen and other debris. Aphids and thrips were present despite the employment of biological control agents and an insecticide treatment. The nutrients for fungal growth thereby provided may have weakened the effect of the fungicides with respect to both inhibition of germination and mycelial growth. Dik et al. (1991a) showed that the presence of aphid honeydew or other nutrients partly neutralized the inhibitory effect of three different SBI fungicides on mycelial growth of Septoria nodorum on wheat leaves. It remains to be shown whether similar antagonism of fungicide action by nutrients applies to strobilurins.

It was previously suggested that extra energy expenditure associated with defence reactions against saprophytic fungi affects plant growth if penetration attempts are frequent (Smedegaard-Petersen & Tolstrup, 1985). However, in the present experiments Cladosporium macrocarpum inoculated on wheat leaves produced little visible host reaction and dark respiration did not increase. Neither did dark respiration increase in leaves after inoculation with A. alternata. Examination of leaves sampled at several growth stages from the inoculated plants in the glasshouse revealed only infrequent papilla formation in response to the inoculated saprophytes (data not shown). The increase in dark respiration of leaves inoculated with barley powdery mildew conidia demonstrated that a significant extra energy expenditure could be induced in the case of plant reactions against a nonhost pathogen unable to infect the plant. However, the results of these experiments also suggested that dark respiration increase in leaves reacting against saprophytes or nonhost pathogens is not significantly decreased by the fungicidal action of azoxystrobin. Interestingly, the experiment with powdery mildew suggested that epoxiconazole may decrease the dark respiration in leaves inoculated with the fungus, compared with untreated inoculated leaves. However, this experiment also indicated that this effect might have been caused by a direct effect of epoxiconazole on plant respiration rather than by inhibition of fungal growth.

It has been suggested previously that delays in senescence and yield increases obtained in cereals by fungicide application to fields with very low levels of visible disease are due to fungicidal control of saprophytic fungi (Dickinson, 1981; Smedegaard-Petersen & Tolstrup, 1985). However, it is difficult to verify that the untreated control plots in field experiments are absolutely disease-free and thus the influence on senescence and yield of disease control and saprophyte control may be impossible to separate. The glasshouse experiment with inoculated saprophytes in this study provided the possibility of examining the influence of saprophyte control without any disease present. This experiment showed that both azoxystrobin and epoxiconazole treatments could delay the senescence of saprophyte-inoculated wheat plants. The corresponding experiment without inoculated saprophytes demonstrated that the fungicide treatments did not influence the senescence of the plants directly. Thus, the results strongly indicate that the positive effect of the fungicide treatments on green leaf area retention was caused by inhibition of senescence-promoting activity of the saprophytic fungi and not by a direct effect on the physiology of the plants. The two fungicides inhibited the growth of the saprophytic fungi in different ways in the growth chamber experiments, but their effects in the glasshouse were similar, namely an impairment of the ability of the saprophytic fungi to induce senescence. Saprophyte-induced accelerated senescence of barley and wheat in glasshouse conditions has been found previously. Jachmann & Fehrmann (1989) observed chlorophyll content decrease in wheat inoculated with C. cladosporioides and C. herbarum in the glasshouse, but no significant effect from A. alternata. Likewise, Tolstrup (1984) observed a decrease in chlorophyll content in leaves of barley caused by multiple inoculations with C. macrocarpum and C. herbarum under glasshouse conditions. The exact mechanism whereby senescence is induced by saprophytes has not yet been elucidated. It is likely that the saprophytic fungi elicit defence responses in the plants, not only by inducing papilla formation, but probably also by inconspicuous activity that is sensed by the plants. Cell wall fragments from the fungi and cell wall fragments from the plant cell walls released by digesting fungal exo-enzymes, enabling a saprophytic lifestyle, may also act as elicitors of plant defence activation (Knogge, 1997). C. macrocarpum was previously found to induce resistance against E. graminis f.sp. hordei in barley without the saprophyte causing more than papilla-like reactions in the leaves (Gregersen & Smedegaard-Petersen, 1989). Activation of the plant defence mechanisms may also be triggering, or coincide with, premature senescence. The mechanisms whereby this happens in highly incompatible fungus–plant interactions such as those in the present study are not well elucidated.

Tolstrup (1984) found that inoculations of the saprophytes C. macrocarpum and C. herbarum on barley grown in glasshouse conditions suppressed the grain yield. In the present glasshouse study, the difference in senescence between fungicide-treated and untreated plants was not associated with a difference in the grain yield or total above-ground biomass of the plants. The reason for this apparent discrepancy was probably that the very dense canopy of leaves in the pots had the potential of compensating for loss of green leaf area (Madeira & Clark, 1995). The photosynthesis of each pot as a unit was not significantly affected in untreated pots, compared with treated pots. Therefore, the green leaf area was not a limiting factor for yield.

In the field experiments, basidiomycetous yeasts were a significant component of the leaf phylloplane fungi, as previously demonstrated in wheat crops (Dickinson & Wallace, 1976; Flannigan & Campbell, 1977; Magan & Lacey, 1986). Earlier studies have proposed that these fungi may have a beneficial effect on yield by acting antagonistically against necrotrophic leaf pathogens (Fokkema, 1973; Fokkema & van der Meulen, 1976; Fokkema et al., 1983; Rabbinge et al., 1984) by competing for nutrients. It was speculated that these ‘yeasts’ would reduce superficial hyphal growth and viability of necrotrophic pathogens, thereby resulting in less disease in the crop (Fokkema, 1973; Fokkema & van der Meulen, 1976; Fokkema et al., 1983). Consequently it has been proposed that broad-spectrum fungicides should be avoided in order to exploit the antagonistic effect of ‘yeasts’ (Dik et al., 1991b). However, in the trials reported herein neither azoxystrobin nor epoxiconazole significantly reduced these fungi.

Spores of Cladosporium spp. were present in large numbers in the field and appeared to contribute most of the mycelial growth on the leaves. However, the growth of these fungi resulted in very few visible defence responses in the leaves, in accordance with the response of plants to C. macrocarpum in controlled environmental conditions. This appears to be different to barley, as saprophytic Cladosporium spp. have been shown to induce papilla formation in barley leaves (Tolstrup, 1984; Gregersen & Smedegaard-Petersen, 1989). In the field the canopy is less dense than in the glasshouse. Therefore, photosynthesis under field conditions may be more influenced by loss of green leaf area than in the glasshouse environment (Bryson et al., 1995). Consequently, a saprophyte-induced accelerated senescence would be more likely to influence yield under field conditions than in the glasshouse. Unfortunately, it was not possible to quantify the effect of saprophyte control on yield in the present field trials, because the plants in the untreated plots were attacked by leaf pathogens, in contrast to the plants in the treated plots.

Neither of the fungicides significantly reduced the number of spores of Cladosporium or Mycospharella on field leaves, or their germination. Treatment with azoxystrobin significantly reduced the growth of superficial fungal hyphae, as also found previously by the authors in a Danish field trial (unpublished results), and the number of defence reactions was reduced compared with untreated plants, albeit significantly reduced only with regard to hypersensitive reactions associated with Mycosphaerella spp. in the Suffolk trial.

The results thus suggest that the fungicidal effect of azoxystrobin in the field was not primarily a reduction of spore germination but rather a reduction in the development of the fungal germ tubes, thus possibly allowing fewer fungal germlings to initiate infection and thereby induce active defence responses. These results are in accordance with the results from the present and previous growth chamber experiments (Godwin et al., 1994) in that the fungicidal effect of azoxystrobin is exerted at an early stage in the fungal development, often before attempted infection. However, the field results differ from those of growth chamber experiments in that we did not observe potent inhibition of spore germination. The seemingly weaker inhibition of spore germination in the field compared with growth chamber experiments, however, may be due to the large difference between the two experimental regimes. The fungi observed were different species and/or sexual types and the conditions for fungal growth and fungicidal action were also different. Moreover, ungerminated spores may be washed off the leaves by rain in the field and thus escape experimental recording.

Treatments with epoxiconazole reduced mycelial growth, as also found previously by the authors in a Danish field trial (unpublished results), in accordance with the fact that the main target of the SBI fungicides is the production of ergosterol, thus inhibiting the formation of functional membranes by the fungus (Siegel, 1981). The number of active defence responses in leaves treated with epoxiconazole was equal to that of the untreated leaves; moreover, the number of papilla reactions associated with Mycosphaerella spp. was even larger in epoxiconazole-treated leaves in comparison with untreated leaves in the Suffolk trial. It has been shown previously that fungal pathogen spores of several species are able to germinate, form germ tubes with appressoria and perform penetration and initial internal growth before the inhibition by SBI fungicides becomes effective (Buchenauer & Kemper, 1981; Hänssler & Kuck, 1987). This has been demonstrated with epoxiconazole against P. recondita and septoria diseases of wheat (Godwin et al., 1994) and Uromyces appendiculatus (Akers et al., 1990). The results from the present field trials are in accordance with earlier work, as the trial data clearly demonstrated that epoxiconazole did not inhibit the infection attempts of fungi on the leaf surface.

The size and morphology of the observed Mycosphaerella spores suggested that they were ascospores of M. graminicola (anamorph S. tritici). The Mycosphaerella spores on the leaves measured 14–18 × 4–5 µm, corresponding to European reports for M. graminicola ascospores (Halama, 1996; Hunter et al., 1999). M. graminicola pseudothecia can be found in the leaves during most of the growth season in England and the ascospores are commonly found in spore traps adjacent to areas with winter wheat (Hunter et al., 1999). Sexual reproduction is considered important in the progress of the septoria tritici blotch disease in England (Hunter et al., 1999), but there is no published information on the infection process by M. graminicola ascospores.

Theories to explain the extra yield benefit of strobilurin treatments emphasize the importance of the prolonged green leaf area duration of strobilurin-treated crops. In the Suffolk field trial, treatments with azoxystrobin preserved green leaf area significantly more than treatment with epoxiconazole in leaf layers F-2 and F-3. This was also the case in the Berkshire field trial, although the superiority of azoxystrobin treatment to epoxiconazole treatment was less. Better preservation of the F-2 and F-3 layers with azoxystrobin treatment than epoxiconazole treatment were also obtained in another UK trial with very low occurrence of visible disease (Jones & Bryson, 1998). It is possible that the later senescence of the lower leaf layers following treatments with azoxystrobin, in comparison with treatments with epoxiconazole, resulted from fewer defence reactions in the azoxystrobin-treated leaves. According to Madeira & Clark (1995) loss of green leaf area is likely to be more important in relation to yield in early growth stages than later on because of the exponential relationship between green leaf area and light interception. The carbohydrate production from lower leaves contributes to the stem carbohydrate reserves and thus forms the basis for the size of the upper leaves, thereby indirectly contributing to grain filling. The differences in green leaf area preservation between the two fungicide treatments in trials reported here may be too small to account alone for the difference in yield between the two treatments. However, the effect on yield from the difference in green leaf area preservation may well have been contributory to the effect on yield resulting from the difference in direct energy expenditure that was suggested by the observed defence reactions in the leaves.

In the present study, the main objective was to examine the effect of azoxystrobin and epoxiconazole on senescence and yield due to their control of saprophytic fungi. However, fungicides may affect senescence and yield by other means, i.e. by direct biochemical/physiological effects on the plants. The strobilurin kresoxim-methyl has been reported to reduce the CO2 compensation point in treated wheat leaves (Retzlaff, 1995), and alterations in the phytohormone balance of wheat shoots have been observed, thereby potentially enhancing the growth and delaying onset of senescence in the plants (Grossmann & Retzlaff, 1997). It is uncertain if these results have relevance for azoxystrobin and the performance of azoxystrobin in the field. Gerhard et al. (1999) observed indications of increased flag leaf size in winter wheat field trials resulting from treatments with azoxystrobin or kresoxim-methyl + epoxiconazole (‘Juwel’) in comparison with treatments with epoxiconazole and, in particular, with untreated plots. Epoxiconazole reduced the growth of small wheat plants (Siefert & Grossmann, 1996) and cleavers (Galium aparine) (Benton & Cobb, 1997), and also decreased ethylene production (Siefert & Grossmann, 1996). The plant hormone ethylene is a regulator of plant senescence (Mattoo & Suttle, 1991). Ethylene production may also have been implicated in the response of plants to pathogen attack (Abeles et al., 1992), and defence responses may thus be associated with accelerated senescence by eliciting ethylene production or coinciding with its increased production. However, the role of ethylene for resistance induction in incompatible pathogen–host interactions is uncertain (Ecker, 1995). Decreased ethylene production in comparison with untreated plots was observed in both Kresoxim-methyl + epoxiconazole-treated and azoxystrobin-treated flag leaves of wheat, thus suggesting a possible senescence-delaying effect (Gerhard et al., 1999). This was interpreted as a result of better fungal control, but with a possible contribution from direct biochemical inhibition of ethylene production in the leaves by strobilurins, as suggested for kresoxim-methyl by Grossmann & Retzlaff (1997). However, these direct physiological effects are not easily distinguishable in the field (Gerhard et al., 1999). The results of the present field experiments clearly demonstrate the difficulties of examining physiological effects of fungicides in field trials without the confounding effects of fungus–plant interactions. The results from the glasshouse experiment with noninoculated plants gave no evidence for direct physiological effects of azoxystrobin or epoxiconazole treatments affecting senescence or yield of healthy wheat plants. However, the existence of such effects cannot be rejected on the basis of this study, as they may only become conspicuous and of importance under more stressful conditions, as pointed out by Gerhard et al. (1999).

The two glasshouse experiments in this study provided the possibility of studying the interactions between plants, saprophytes and fungicides in surroundings where pathogens and disease could be prevented and denied any influence on the results. Saprophytic fungi, without the ability to infect the target wheat plants, were shown to accelerate senescence in the glasshouse. The results lend strong support to the theory that saprophytes can accelerate senescence and possibly decrease yield in field-grown wheat crops. Additionally, the results show that fungicides can alleviate adverse effects of saprophytes in the glasshouse, indicating that this can happen in the field as well. However, there were no indications from these experiments that the difference in yield that has regularly been observed between azoxystrobin-treated and SBI-treated winter wheat can be caused by differential control of saprophytes, as the saprophyte-induced senescence was inhibited equally well by azoxystrobin and epoxiconazole in the glasshouse.

The field trials conducted in this study showed that extensive fungal–plant interactions detectable only by microscopy do occur on fungicide-treated wheat leaves, even though only < 1% leaf area was visibly diseased. The high frequency of defence reactions against attempted fungal infection makes it highly probable that the associated energy expenditure can adversely influence the final yield. The reduction of defence reactions obtained with azoxystrobin treatments, particularly compared with treatment with epoxiconazole, can therefore be part of the explanation for the superior green leaf conservation and yield recorded for azoxystrobin-treated plots compared with epoxiconazole-treated plots. The present results demonstrate an example of fungal control, observable only by microscopy, with a probable influence on yield that otherwise might have been attributed to other factors.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors wish to thank Zeneca Agro, Denmark, for funding this study and Zeneca Ltd, UK, for providing facilities for the glasshouse and field experiments. We thank Dr H. J. Lyngs Jørgensen for carefully revising the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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Footnotes
  1. †E-mail: vs@kvl.dk