Changes in carbohydrate composition of cucumber leaves during the development of powdery mildew infection

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*To whom correspondence should be addressed.

Abstract

Within 3 days after inoculation, greater rates of decrease of hexoses, and of increases in inositol and α-glucans, were detected in leaves of cucumber leaves infected with Sphaerotheca fuliginea (now reclassified as Podosphaera xanthii) than in healthy leaves, together with synthesis of fungal mannitol, arabitol and trehalose. With the onset of sporulation (5 days after inoculation, dai), glucose, polyols, trehalose and glycogen content, as well as fresh and dry weight of leaf tissue, increased more rapidly than in healthy plants, while sucrose, raffinose and starch declined. Starch, glycogen and other α-glucans, extracted from ethanol-insoluble tissue fractions of different water-solubility from healthy and infected leaves, were quantified after amyloglucosidase treatment. Infection-induced alterations in partitioning of photosynthate during a 14 h photoperiod at 7 dai, including a close correspondence in patterns of synthesis of glucose, polyols and glycogen, further clarified the process of diversion of host assimilate to the pathogen. The presence of glucose, but only traces of sucrose in spores, supported evidence from other powdery mildew infections for photosynthate being transferred to S. fuliginea as glucose.

Introduction

Although infection of cucumber plants by the powdery mildew fungus Sphaerotheca fuliginea (reclassified as Podosphaera xanthii; Braun et al., 2002) rarely kills its host, the resulting loss of nutrients, increased respiration and decreased photosynthesis seriously reduce growth and yields compared with uninfected plants (Agrios, 1997). As with other biotrophic pathogens, disease development is likely to induce substantial changes in the carbohydrate content of host plants, and metabolic alterations that may favour fungal development (Hwang & Heitefuss, 1986; Ayres et al., 1996). Some environmental factors have been found to affect the carbohydrate composition of healthy cucumber plants (Matsumota & Teroaka, 1980; Schäffer et al., 1991; Janoudi et al., 1993; Todaka et al., 2000). However, the only investigation of carbohydrate metabolism during powdery mildew infection of this host appears to be that of Kabsch (1982), who recorded decreases in glucose and fructose content of cucumber cotyledons and primary leaves, accompanied by increases in glucose-6-phosphate, fructose-6-phosphate, and the activities of hexokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, at early stages of sporulation by S. fuliginea.

No further information seems to be available concerning changes in carbohydrate composition and partitioning of photosynthate during disease development in crop plants of the Cucurbitaceae. In particular, polyols and trehalose, the major soluble carbohydrates of higher fungi (Lewis & Smith, 1967; Cooke & Whipps, 1993), and characteristic constituents of powdery mildew-infected plants of the intensively studied cereals (Edwards & Allen, 1966), have received less attention in herbaceous dicotyledons, although they were investigated in powdery mildew-infected leaves of oak (Hewitt & Ayres, 1976) and grapevine (Brem et al., 1986).

This study was designed to address the lack of information on the carbohydrate metabolism of dicotyledon host plants during powdery mildew infection, already noted by Kabsch (1982). Despite intensive studies on Erysiphe pisi infection of pea leaves (Manners & Gay, 1982; Aked & Hall, 1993a; Aked & Hall, 1993b), various aspects of the interaction of powdery mildew pathogens with noncereal hosts are still unresolved, including changes in ethanol-insoluble carbohydrates.

Following previous investigation of lipid changes during powdery mildew infection of cucumber (Abood & Lösel, 1989; Abood et al., 1990; Lösel et al., 1994), the present study aimed to increase understanding of the role of carbohydrates in this biotrophic system by examining the partitioning of photosynthate into sugars, polyols and α-glucans at successive stages of disease development, following inoculation of cucumber leaves with S. fuliginea, and also during a single photoperiod.

Materials and methods

Growth and inoculation of host plants

Seeds of Cucumis sativus cv. Beit alpha M2/E6811-42191 were grown in a mixture of Levington's compost and sand (1 : 1) in growth room conditions (20 ± 2°C, 16 h irradiance, 80 µmol m−2 s−1). Uniformly developed seedlings were transplanted into plastic pots (12 cm diameter) containing compost, and allowed to continue growth under similar conditions. When the first leaf was fully expanded, plants were placed in the base of a settling tower (diameter 60 cm; height 80 cm) and inoculated by exposing them to an even distribution of S. fuliginea conidia from heavily infected leaves of cucumber, which had been shaken 6 h previously to remove older spores (Eyal et al., 1968; Abood et al., 1992). Primary germ tubes emerged within 3–6 h, first haustoria within 12 h after inoculation, and a second germ tube developed during the following 6 h. Within 24 h after inoculation, septate hyphae were initiated from the primary and secondary germ tubes, giving rise to surface mycelium from which conidiophores began to develop by 5 days after inoculation (dai), with conidia densely covering the leaf surface by 7 dai.

Determination of ethanol-soluble carbohydrates in healthy and infected tissues

Unless stated otherwise, samples of 12 leaf discs (12 mm diameter) were cut from the youngest fully expanded leaf of replicate healthy and infected plants at midday. Immediately after harvesting, fresh weights of each sample were quickly recorded and the tissue transferred to boiling 80% ethanol for extraction of ethanol-soluble carbohydrates. The extracted tissue was retained for later analysis of insoluble carbohydrates. Sugars and polyols in the ethanol extracts were quantitatively analysed by gas–liquid chromatography (GLC), using the methods of Lewis & Smith (1967) and Holligan & Drew (1971), on 1·5 m glass columns of internal diameter 4 mm, packed with Chromasorb (100–120 mesh coated with SE 52; Supelco, Poole, UK), with nitrogen (45 mL min−1) as carrier gas.

Sampling of cucumber leaf tissue for carbohydrate analysis

To study changes in soluble and insoluble carbohydrates during the development of powdery mildew infection, discs were cut from the youngest expanded leaf of healthy and infected plants, on the day of inoculation (day 0) and at 3, 5, 7 and 9 dai.

Diurnal changes in soluble sugars were investigated by harvesting batches of discs from the youngest expanded leaf of replicate plants, which had been kept in the dark overnight and then exposed to light for periods of 2, 4, 6, 10 and 14 h, under growth room conditions. These samples were immediately weighed and extracted, as above.

Sugar composition of fungal tissue

In order to determine whether mannitol, arabitol and trehalose were derived from the fungus or from the host, discs were cut at 7 dai from the youngest expanded leaf of four replicate healthy and infected plants. Similar samples were taken, at the same time, from corresponding infected leaves from which the surface mycelium was carefully peeled, using a sharp razor, after fresh weights had been quickly recorded. Samples of each tissue and of spores, shaken from several leaves of infected plants grown in similar conditions, were extracted as above.

Ethanol-insoluble carbohydrates of host leaves

The difficulties of determining fungal glycogen in the presence of soluble starch in host tissue from infected leaves were overcome by adopting the following procedures, used by Holligan et al. (1974) in a similar investigation of rust-infected leaves of Tussilago farfara. (i) On the assumption that starch synthesized in host tissue, in light, declines during darkness, while glycogen synthesis continues within the fungal tissue, healthy and infected cucumber plants were partially destarched by keeping them in the dark overnight (6–7 dai), after which glucan synthesis was compared at intervals during the light period in replicate sets of plants. (ii) Discs were cut from healthy and infected leaves when sporulation was active and after extraction of chlorophyll with 80% ethanol, and treated with iodine solution to detect any differential staining of starch or glycogen. (iii) Release of glucose from α-glucans, glycogen and starch after degradation by amyloglucosidase (AMG; Sigma-Aldrich, Poole, UK) was investigated in ethanol-insoluble residues of healthy and infected cucumber leaves which had been harvested at 7 and 9 dai, both with mycelium and after removal of surface mycelium, as well as from S. fuliginea spores. All samples were extracted twice in cold water and after further incubation in hot water at 90°C for 1 h. After centrifugation at 8000 g for 15 min, the supernatant from each extract was decanted, then reduced to 2 mL (water-soluble α-glucan fraction). Distilled water (2 mL) was added to the water-insoluble residue from each replicate sample, followed by 2 mL AMG (2 mg mL−1 in citrate buffer at pH 4·5, giving a final AMG concentration of 1 mg mL−1). The samples were then incubated at 45°C for 2 h. Glucose yields from hydrolysis of soluble α-glucans and starch were determined by the glucose oxidase method (Lloyd & Whelan, 1969), and expressed as a percentage of the total yield of glucose from all washings plus that from the residue.

Statistical analysis

In all experiments, unless stated otherwise, data from the youngest expanded leaves from four replicate healthy and infected plants were analysed for each time and treatment. Means, standard error (SE) and least significant differences (LSD), calculated using genstat, are shown where appropriate.

Results

Changes in soluble and storage carbohydrates of cucumber leaves during the development of powdery mildew infection

The total amounts of ethanol-soluble carbohydrate in discs excised from healthy and infected leaves, at intervals of 2 days from the time of inoculation until 9 dai, varied with time and with development of the pathogen. In healthy leaves, both total soluble sugars and extracted dry weight decreased at similar rates from 3 dai, while fresh weight changed relatively little (Fig. 1). In contrast, the total ethanol-soluble carbohydrate content, fresh weight and extracted dry weight of infected leaves rose throughout this period and, from the onset of sporulation at 5 dai until the final harvest, increasingly exceeded the corresponding values for healthy leaves.

Figure 1.

Effects of powdery mildew infection on (a) total sugars and polyols; (b) leaf fresh weight; (c) extracted dry weight of youngest expanded leaves of healthy (▪) and S. fuliginea-infected (▴) cucumber plants, sampled at midday. Means of four replicate plants ±SE.

Individual soluble carbohydrates exhibited different patterns of change which were markedly altered by infection. During the first 3 days sucrose, the major sugar of healthy leaves, increased at a similar rate in both healthy and infected leaves to a peak at 3 dai, then decreased until 5 dai, more rapidly in infected than in healthy tissue. The decline in sucrose then continued at a slower, constant rate in infected leaves until the final harvest, whereas sucrose remained unchanged from 5 to 7 dai in healthy leaves before falling to a lower value, similar to that of infected tissue, at 9 dai (Fig. 2a). In the first 3 days of the experiment there was a continuous decrease in monosaccharides, which was more rapid in leaves from inoculated than from control plants, with fructose at much lower concentrations than glucose in all samples. However, from 5 dai, when the amounts of both monosaccharides were significantly lower than in healthy tissue (Fig. 2b,c), glucose increased steadily in infected leaves to a value, at 9 dai, similar to the initial level of young healthy tissue, whereas fructose increased only until 7 dai and thereafter did not exceed control values. In contrast, the glucose content of healthy leaves remained stable from 3 to 5 dai, with a further decline between 5 and 7 dai, while the always lower fructose content changed little from 3 to 9 dai.

Figure 2.

Changes in (a) sucrose; (b) glucose; (c) fructose; (d) inositol; (e) raffinose; (f) fungal metabolites (mannitol, arabitol, trehalose); (g) α-glucan in youngest expanded leaves of healthy (▪) and powdery mildew-infected (▴) cucumber plants, sampled at midday during 9 days’ development of infection. Means of four replicate plants ± SE.

Among the ethanol-soluble carbohydrates normally found in healthy leaves, only inositol increased continuously above control values throughout the development of infection (Fig. 2d), whereas the inositol content of healthy tissue, after rising more slowly until 5 dai, subsequently remained constant until the final harvest. Raffinose showed a very similar pattern to that for sucrose in both healthy and infected tissue, but at lower concentrations, declining from an early peak at 3 dai to values which remained relatively stable but were lower in infected than in healthy tissue only from 5 to 7 dai (Fig. 2e).

Mannitol, arabitol and trehalose, which were detectable in healthy tissue at only trace levels (Table 1), accumulated increasingly, throughout the development of powdery mildew infection. Mannitol was present in the largest amounts and, like inositol, increased from 3 dai, while trehalose and arabitol, at much lower concentrations, showed a clear rise only after 5 dai (Fig. 2f). These fungal carbohydrates increased substantially as infection progressed, contributing more than 50 and 70% of the total ethanol-soluble carbohydrate of infected leaves by 7 and 9 dai, respectively (Table 1). In contrast sucrose, which accounted for 65% of this fraction in infected tissue at 3 dai, fell to approximately 15 and 4%, respectively, by 7 and 9 dai. Although decreasing with time, the proportion of sucrose in the soluble fraction of healthy leaves remained sevenfold higher than in infected tissue at the end of the study period. Fructose percentages decreased markedly following infection and, like raffinose, persisted at low values.

Table 1.  Effect of powdery mildew infection on composition of sugars and polyols (percentage of ethanol-soluble carbohydrate) in cucumber leaves during disease development
CarbohydrateDays after inoculation
03579
  • a

    Mean of four replicates.

Healthy
Sucrose36·6a67·549·453·231·0
Glucose29·013·919·211·921·0
Fructose13·2 2·9 4·7 3·7 5·7
Inositol13·3 9·020·021·431·5
Raffinose 5·9 6·7 6·6 8·0 5·3
Mannitol 0·0 0·0 0·0 0·7 2·4
Arabitol 0·0 0·0 0·0 0·6 3·0
Trehalose 0·0 0·0 0·0 0·5 0·0
Infected
Sucrose36·665·135·414·7 4·3
Glucose29·0 8·8 6·511·0 9·3
Fructose13·2 2·1 1·4 2·0 1·5
Inositol13·310·626·217·613·0
Raffinose 5·9 6·4 5·0 3·2 1·4
Mannitol 0·0 2·715·828·032·5
Arabitol 0·0 1·8 5·410·715·8
Trehalose 0·0 2·4 4·212·822·1

These changes in individual sugars and polyols have to be considered in relation to the accompanying alterations in ethanol-insoluble carbohydrates from the same leaf samples, particularly α-glucans (Fig. 2g). In both healthy and infected tissue, the initial rise in α-glucans during the first 3 days of the experiment resembled the pattern of changes in sucrose and raffinose (Fig. 2a,e), apart from the much greater amounts of α-glucans than of total ethanol-soluble carbohydrates present. By 3 dai, synthesis of α-glucans was more rapid in infected than in healthy tissue, where similar accumulation began only at 7 dai, but from 5 dai α-glucans increased in infected tissue at a rate similar to that for glucose content (Fig. 2b).

Diurnal changes in soluble sugars, polyols and α-glucans

To improve understanding of the infection-induced alterations in partitioning of photosynthate, the carbohydrate composition of leaves of healthy and powdery mildew-infected cucumber plants was compared at intervals during a single 14 h light period at 7 dai, when development and sporulation of the pathogen were well established. As in the preceding 9-day study, the ethanol-soluble carbohydrate content was greater in infected than in healthy leaves, increasing during the first 6 h of photosynthesis to an amount double that of healthy leaves, then remaining stable for 4 h before falling to a value similar to that recorded 2 h after the start of photosynthesis (Fig. 3a). In healthy tissue, after a slower rise during the first 6 h, the ethanol-soluble carbohydrate content remained constant until the end of the light period.

Figure 3.

Diurnal alterations, during a 14 h light period, in amounts of (a) total sugars and polyols; (b) sucrose; (c) glucose; (d) fructose; (e) inositol; (f) raffinose; (g) fungal metabolites (mannitol, arabitol, trehalose); (h) α-glucan in leaves of healthy (▪) and powdery mildew-infected (▴) cucumber plants at 7 dai. Means of four replicate plants ± SE.

The sucrose content of infected tissue, although always significantly less than in healthy tissue (Fig. 3b), increased continuously during the photoperiod to a final concentration close to that found initially in healthy tissue. In healthy leaves, however, after a rapid rise in the first 6 h, sucrose remained stable throughout the light period. In contrast to sucrose, glucose concentrations were significantly higher in infected than in healthy leaves at each sampling time, increasing slowly for 10 h then declining to a level not significantly different from that maintained in healthy leaves (Fig. 3c). As before, fructose occurred in much smaller amounts than glucose and, apart from a lower content in infected than in healthy tissue after 2 h of photosynthesis, no clear changes in fructose were associated with infection (Fig. 3d). Throughout the light period inositol maintained a uniform concentration in healthy leaf tissue, significantly lower than in infected leaves, where a slow but constant rise in inositol during the first 10 h of sampling was followed by a slight decrease during the final 4 h (Fig. 3e). Raffinose, like sucrose, accumulated at similar rates in healthy and infected leaves throughout the light period, but at much lower concentrations than the disaccharide. Although raffinose appeared to be reduced by infection, this difference became significant only after 10 h (Fig. 3f).

Diurnal changes in mannitol and arabitol (at about one-third of the concentration of mannitol) in infected tissues showed similar patterns (Fig. 3g) to those of inositol and total ethanol-soluble carbohydrate in infected tissue, reaching maximal concentrations after 6 h of light, then declining to levels lower than at the beginning of the photosynthetic period. Trehalose, which occurred in greater amounts than arabitol, increased only slightly in the first 10 h of illumination, then decreased similarly to less than the initial value.

At the time of this diurnal study, 7 dai, the α-glucan content of infected leaves was already more than double that of healthy leaves (compare midday values here with those in Fig. 2). The high α-glucan content of infected tissue did not change significantly during the light period. In healthy leaves, however, this fraction started from a relatively low level at the end of the dark period, rising only slowly in the first 6 h of photosynthesis, then increased rapidly and stabilised after 10 h of photosynthesis at a value similar to that of infected leaves.

Distribution of carbohydrates between host and fungal tissues

Comparison of the soluble and storage carbohydrate content of healthy leaves (Fig. 4a) with that from infected leaves, with and without surface mycelium (Fig. 4b,c), and from spores (Fig. 4d) provided information about the distribution of ethanol-soluble carbohydrates between host and fungal tissues which was important for understanding their metabolic interaction. Sucrose, which accounted for approximately 53% of the ethanol-soluble fraction in healthy tissue at a time corresponding to 7 dai (Fig. 4a), had fallen by this time to 15% in infected tissue with or without surface mycelium (Fig. 4b,c), while the proportion of raffinose had decreased to about half that found in healthy leaves. Percentages of mannitol, inositol and trehalose in infected leaves were not altered markedly by removal of mycelium, while that of arabitol was reduced. In spore extracts, mannitol, arabitol and trehalose comprised about 86% (approximately 45% mannitol, 30% trehalose and 10% arabitol) of total ethanol-soluble carbohydrates, which included glucose, at a similar percentage to arabitol, very low proportions of fructose and inositol, negligible amounts of sucrose and no detectable raffinose.

Figure 4.

Percentages of sugars and polyols in healthy and infected youngest expanded leaves of cucumber at 7 dai: (a) healthy tissue; (b) infected leaf plus surface mycelium; (c) infected leaf without surface mycelium; (d) fungal spores. Means of four replicates ± SE. Al, arabitol; Fr, fructose; Gl, glucose; Ml mannitol; In, inositol; Su, sucrose; Tr, trehalose; Rf, raffinose.

To assess the relative contributions of the shorter-chain, more water-soluble constituents (including glycogen) and of water-insoluble starch to the total carbohydrate content of healthy and S. fuliginea-infected cucumber leaves, the α-glucan content of cold- and hot-water extracts and water-insoluble material from 80% ethanol-extracted residues from healthy and infected tissues or fungal spores were compared at 7 and 9 dai (Table 2). The proportions of glucose released by AMG from α-glucans in these fractions of infected tissue varied little between these two sampling dates (Table 2). At 7 dai, only 14% of total glucose was released from healthy tissue during all extraction steps, whereas that derived from the residue, assumed to be mainly starch, amounted to 86%. The glucose yields from starch-containing water-insoluble residues from all samples of infected leaves were much lower than from healthy tissue, and declined further between the two sampling dates. In contrast, the glucose yield from shorter-chain, water-soluble fractions of infected leaves, presumably mainly fungal glycogen, amounted to 51% from leaf tissue plus mycelium, 59% from infected tissue without mycelium, and 41·5% from fungal spores (Table 2). Two days later, the corresponding percentages from cold- and hot-water extraction of healthy tissue had each decreased only slightly, while those from soluble fractions of infected tissues and spores had increased further (Table 2).

Table 2.  Percentages of glucose released after amyloglucosidase treatment of fractions of differing water solubility from ethanol-extracted residues of healthy and powdery mildew-infected cucumber leaves and spores of Sphaerotheca fuliginea
ExtractsHealthy leavesInfected leavesFungal spores
+mycelium–mycelium
  • a

    Mean of four replicates.

7 days after inoculation
Cold water 6·4a32·835·027·2
Cold water 0·7 4·6 3·2 1·3
Hot water 4·510·518·0 6·8
Hot water 2·4 3·4 2·8 6·2
Residue86·048·741·058·4
9 days after inoculation
Cold water 6·037·440·430·0
Cold water 0·5 4·2 5·7 2·0
Hot water 3·517·410·7 9·4
Hot water 1·8 3·5 3·6 2·5
Residue88·037·439·055·7

Discussion

Despite its restriction to the aerial surfaces of the host, with haustoria penetrating only epidermal cells, development of the powdery mildew pathogen S. fuliginea significantly altered the carbohydrate composition of cucumber leaves. As quantitatively similar effects were observed in leaves from which the surface mycelium had been carefully removed, it is likely that the metabolic changes induced were largely confined to epidermal cells and the haustoria within them.

The earliest responses to the pathogen appeared within 3 dai as a more rapid decline of monosaccharides, particularly glucose, and a significantly greater increase in total glucan than in corresponding healthy leaves. The fall in monosaccharides, like similar decreases in sucrose and raffinose from 3 dai (when these oligosaccharides had peaked in both healthy and infected leaves), continued for longer and reached significantly lower values in infected than in healthy tissue, while inositol and fungal polyols began to accumulate. These observations, and a slight decrease in α-glucan content, between 3 and 5 dai, suggested an increasing demand on host photosynthate for fungal development before the growth of surface mycelium and conidiophores became visible. Synthesis of inositol, fungal polyols, trehalose, glucose and α-glucans continued subsequently, throughout the further growth and sporulation of S. fuliginea. Similar alterations in hexoses and sucrose were noted in pea leaves during 7 days of infection by Erysiphe pisi (Aked & Hall, 1993b). The substantial synthesis of polyols and trehalose, all virtually absent from healthy leaves, during the development of S. fuliginea in cucumber, is consistent with previous observations on powdery mildew infection of leaves of barley (Edwards & Allen, 1966); oak (Hewitt & Ayres, 1976); pea (Manners & Gay, 1982); and grapevine (Brem et al., 1986), although the relative proportions of individual fractions sometimes differed. As in other biotrophic infections, polyols provide the fungal partner with a substantial mobile sink of reduced carbon, which is not normally accessible to host metabolism (Lewis & Smith, 1967; Lewis, 1970; Holligan et al., 1973; Holligan et al., 1974).

The early acceleration of α-glucan synthesis immediately after inoculation, and its further dramatic rise between 5 and 7 dai, with marked alterations in the proportions of fractions of differing solubility, indicated rapid incorporation of photosynthate into fungal glucans, particularly glycogen, which then increased more slowly to a final value close to that attained by total glucans in healthy leaves.

Although, in both healthy and infected cucumber leaves, the amounts of α-glucan greatly exceeded those of all sugars and polyols, difficulties in biochemically distinguishing fungal and host polysaccharides in biotrophic systems may have discouraged investigation of these fractions in powdery mildew infections. The above analysis of α-glucans from ethanol-insoluble tissue residues of differing solubility in water, which allowed separate quantification of starch and glycogen, indicated that glycogen formed the major part of the significantly increased α-glucan content of infected tissue. This was supported by glycogen-like staining reactions with iodine of storage deposits in hyphae and spores of S. fuliginea, and previous electron microscope cytochemical evidence for the presence of substantial deposits of glycogen in E. pisi mycelium and spores (Martin & Gay, 1983).

Diurnal differences in partitioning of photosynthate into individual soluble and insoluble carbohydrate fractions of healthy and infected leaves, in the middle phase of disease development, provided a more detailed picture of metabolic changes induced by S. fuliginea. The similar patterns of increases in total sugars, glucose, inositol, polyols and α-glucans in infected tissues in the first 6 h of photosynthesis, and of decreases in the final 4 h, with each of these fractions maintaining higher concentrations than in healthy leaves, are consistent with some previous reports of other powdery mildew infections (Brem et al., 1986; Scholes et al., 1994; Wright et al., 1995). Although at lower concentrations than in healthy leaves, the patterns of sucrose and raffinose synthesis appeared little altered by infection. On the other hand, comparison of the relatively stable concentrations maintained by sucrose in healthy leaves, following a rapid rise during the first 6 h of illumination, and by glucose and inositol, throughout the light period, highlights the different factors and thresholds determining the utilization of photosynthate following infection.

The rapid rise in mannitol, during the first 6 h of photosynthesis in infected leaves, to a concentration very close to that of sucrose in corresponding healthy leaves, and an approximately parallel time course for the smaller amounts of arabitol, strongly support mannitol being the primary fungal sink for host photosynthate. This is consistent with the demonstration by Brem et al. (1986) of a more rapid flux of 14C-labelled photosynthate from vine leaves to mannitol than to arabitol in the powdery mildew pathogen Uncinula necator (reclassified as Erysiphe necator; Braun et al., 2002). The rapid decline in mannitol, arabitol, inositol, glucose and trehalose during the final 4 h of the light period, which did not appear to be accounted for by the relatively slow changes in the high and fairly stable content of α-glucans at this time, is likely to correspond to incorporation of these metabolites into other sinks for the growth requirements of the pathogen. The much lower starch content of infected leaves, and greater proportion of water-soluble glucan (mainly glycogen) than in healthy tissue, agreed with observations at the same stage of disease development in the previous 9-day experiment.

In healthy leaves the stabilization of sucrose content, following its initial rise in the first 6 h of photosynthesis, coincided with a dramatic increase in the rate of α-glucan synthesis, suggesting that sucrose had reached a threshold level, triggering the diversion of photosynthate into starch accumulation. The levelling out of starch concentrations in the final 4 h is likely to be accompanied by transfer of assimilate to other biosynthetic pathways and translocation to growing points of the healthy host. In contrast, parallel decreases of sugars, inositol and other polyols in infected tissue during the final 4 h of the light period may indicate adjustment of partitioning and translocation, in the dual system, to the pathogen's requirements for biosynthetic intermediates to support growth and sporulation (Kosuge, 1978; Ayres et al., 1996).

Increases in sugar content have been linked to invertase activity during powdery mildew infections of barley and wheat (Scholes, 1992; Heisteruber et al., 1994; Wright et al., 1995), grapevine (Brem et al., 1986) and pea leaves (Aked & Hall, 1993b). Furthermore, downregulation of the Calvin cycle, resulting from such accumulation of sugar, decreases the availability of assimilate for export in powdery mildew infected barley (Scholes et al., 1994) and wheat (Heisteruber et al., 1994; Wright et al., 1995; Keutgen & Roeb, 1996). The alterations in sugar content recorded here during S. fuliginea infection of cucumber leaves would be consistent with similar metabolic interactions, although these do not yet appear to have been investigated in cucurbit hosts.

A virtual absence of sucrose from S. fuliginea spores, in which glucose, like arabitol, accounts for 10% of total ethanol soluble carbohydrate, as well as close parallels in patterns of accumulation of glucose, polyols and glycogen in infected cucumber leaves, both diurnally and throughout 9 days’ development of S. fuliginea, are consistent with glucose being the sugar transferred from the host tissue to the pathogen, as previously concluded from E. pisi infections of pea (Aked & Hall, 1993a; Clark & Hall, 1998). Sutton et al. (1999) demonstrated rapid hydrolysis of sucrose in powdery mildew-infected wheat leaves, without sucrose or fructose being detected in mycelial extracts. Their elegant investigation of uptake of various 14C-labelled sugars (including asymmetrically labelled sucrose) by infected leaf pieces and isolated mycelial suspensions provided strong evidence that sucrose is cleaved prior to uptake by the pathogen, and that glucose is the major carbon and energy source transferred to the fungal mycelium.

The above interpretation of diurnal and longer-term variations in sugars, polyols and glucans during the biotrophic interaction between cucumber leaves and S. fuliginea may contribute to better understanding of the efficiency of powdery mildew systems, particularly on herbaceous dicotyledons, which differ from cereal hosts in characteristics of carbohydrate composition and thresholds of sugar and starch synthesis. In both categories of host, translocation is diverted to infection sites and assimilate, probably as glucose, is rapidly transferred to the pathogen. However, biochemical differences between them may influence the pathways exploited in mobilizing the carbon and energy flux from host to pathogen in infected cells.

Although other fractions into which photosynthate may be partitioned are not included in the present study, cucumber leaves in early stages of infection by S. fuliginea have previously been shown to incorporate twice as high a proportion of 14C-labelled photosynthate into both storage and membrane lipids as healthy leaves (Abood & Lösel, 1989; Abood et al., 1990). Thus in this system, as with other biotrophic fungal pathogens, conversion of photosynthate to fungal polyols, trehalose, glycogen (Lewis, 1970; Long et al., 1975), lipid and other fungal constituents facilitates a high rate of transport across the host–pathogen interface to support the growth and sporulation of the mycelium.

Acknowledgements

The completion of this paper was made possible by a Developing World Study Visit Award from the Royal Society to J.K.A., which is gratefully acknowledged. We also wish to thank Professor A.J. Willis and Dr J.D. Scholes for their critical reading of the manuscript and valuable comments.

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