- Email: [email protected]
Aphids on Norway spruce and their effects on forest-floor solution chemistry
Forest Ecology and Management 118 (1999) 1±10
Aphids on Norway spruce and their effects on forest-¯oor solution chemistry Beate Michalzika,*, Thomas MuÈllerb, Bernhard Stadlera b
a Bayreuth Institute for Terrestrial Ecosystem Research, University of Bayreuth, 95440 Bayreuth, Germany Centre for Agricultural Landscape and Land Use Research, MuÈncheberg, Institute of Microbial Ecology and Soil Biology, 14641 Paulinenaue, Germany
Received 8 April 1998; accepted 19 August 1998
Abstract Many species of aphids on spruce excrete large quantities of honeydew. As providers of carbohydrates, it is likely that they affect several ecosystem functions by fueling biological processes which are energy-limited. In a rainfall simulation experiment, we manipulated the level of aphid infestation on spruce and studied the effects of honeydew on forest-¯oor solution chemistry collected underneath infested and uninfested trees. A non-destructive sampling method was used to allow repeated measurements using the same forest ¯oor during the experimental period. Although the input of dissolved organic carbon (DOC) underneath infested trees to the forest ¯oor was considerably higher compared to uninfested trees, carbon concentrations of forest-¯oor leachates did not differ between treatments. Concentrations of dissolved organic nitrogen (DON), NO3±N and Ntotal of forest-¯oor leachates, underneath infested and uninfested trees followed a marked seasonal trend with low concentrations recorded in June and after-frost treatment of the forest ¯oor, while the highest concentrations were recorded in July/August. Statistically signi®cant differences in soil solution properties underneath infested and uninfested trees were recorded in July when honeydew-affected forest-¯oor leachates had lower concentrations of NH4±N, Ntotal, a lower conductivity and a higher pH. Despite a large input of honeydew, no pronounced seasonal trend was found in the carbon leachate concentrations of the forest ¯oor (e.g. DOC, hexose-C). Discriminant function analysis showed that forest-¯oor leachates can be classi®ed according to the experience of frost and nitrogen concentrations, which were affected by the honeydew of aphids. At the end of the experiment, the number of micro-organisms present in the forest ¯oor was not signi®cantly different between treatments but was higher in the Oh compared to the Olf horizon. Microbial communities did not appear to be severely in¯uenced by deep temperatures. It seems likely that, in coniferous forests, aphids can considerably reduce local ¯uxes of nitrogen from the forest ¯oor. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Aphid honeydew; Cinara; Forest-¯oor leachates; Norway spruce
1. Introduction *Corresponding author. Fax: +1-49(0)921/55-5799; e-mail: [email protected]
Aphids on trees often excrete copious amounts of honeydew, which mainly consists of sugars such as
0378-1127/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0378-1127(98)00481-2
2
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
fructose, glucose and melezitose (Maurizio, 1985). On a per-hectare basis, it has been reported that an annual average of 400±700 kg fresh mass of honeydew can be expected in a coniferous forest (ZwoÈlfer, 1952; Zoebelein, 1954). Estimates for lime aphids on Tilia spp. even amount to 800 g mÿ2 aÿ1 (Llewellyn, unpubl. PhD thesis, Glasgow University 1970) or range between 2 and 50 kg honeydew dry mass for a single mature lime tree (Heimbach, 1986). Aphid population densities usually ¯uctuate spatially and temporally. Thus, although locally abundant, the availability of honeydew as a source of energy is unpredictable for consumers of carbohydrates. This unpredictability also makes it dif®cult to investigate the effects of honeydew for ecosystem processes, such as nutrient cycling or solute export in leachates from the forest ¯oor of a natural forest stand. In the phyllosphere of Norway spruce, we found that honeydew signi®cantly promotes the growth of micro-organisms and affects throughfall carbon and nitrogen chemistry (Stadler et al., 1998). Therefore, the population dynamics of aphids is a key to an understanding of the temporal and spatial variability in aphid±micro-organism interactions and throughfall ¯uxes. The effect of honeydew on soil biology and chemistry has already received some attention. Bacteria and fungi readily respond to the availability of carbohydrates, either by increasing in numbers or respiration, depending on soil type (Dighton, 1978a, b; Petelle, 1980). Besides its effects on the soil biota, it is conceivable that the honeydew of aphids also affects nutrient cycling or soil-leachate chemistry, which is, however, much more dif®cult to demonstrate. From an investigation of mineral soil in an Alnus rubra Bong. plantation (Grier and Vogt, 1990) it appears that honeydew does in¯uence ammoni®cation and nitri®cation, although the applied method of repeated insecticide application (Malathion) for removing aphids from the control treatment could have adverse effects on the soil fauna. Furthermore, a repeated sampling of soil introduces an uncontrolled variability to an experiment because small-scale differences in soil structure and chemistry are the rule rather than the exception, thus obscuring effects (Manderscheid and Matzner, 1995; GoÈttlein and Stanjek, 1996). Another limitation in studies on the effects of honeydew on soil micro-organisms is that they typically cover only a short period of time. This might be
due to the fact that aphids and micro-organisms respond quickly to resources which are only temporarily available; thus, most experimenters ignore seasonal aspects, such as winter frost. In the ®eld, however, concentrations of nutrients in forest-¯oor leachates are sometimes found to increase in early spring after snow melt (Yavitt and Fahey, 1985) which seems to indicate a fatal effect of frost on the biota and/or inhibition of decomposition processes. We account for this effect by studying the population densities of micro-organisms and forest-¯oor leachate chemistry before, and after, arti®cial frost application, both when honeydew was present and absent during summer. In particular, the objectives of this paper were: 1. to produce in a controlled rainfall and aphid infestation experiment carbon-rich throughfall; 2. to investigate the effects of different levels of Cinput on forest-floor leachate chemistry; and 3. to test the hypothesis that frost disrupts the microbial populations in the soil and induces changes in leachate chemistry. We discuss the results with respect to the effects imposed by above- and below-ground biotic interactions on plant nitrogen availability and ecosystem processes. 2. Materials and methods 2.1. Experimental design In order to exclude unknown variability from the study, we standardised our experiment with respect to plant quality, nutritional supply, rain chemistry, amount of rain applied and microclimatic differences between trees. In a rainfall simulation experiment, six 10-year-old spruce trees (Picea abies (L.) Karst.) were covered with a transparent roof (20 m2) at an experimental ®eld close to the university. All trees were grown in containers 50 cm in diameter, 30 cm deep, ®lled with compost soil. Above each tree an irrigation system was installed to spray the rain solution onto the trees with the help of a ®ne axial ¯ow, full cone nozzle (Lechler, Metzingen), which released the water homogeneously at a 608 angle. Because we considered the volumes of rain sprayed onto the trees and irrigation time schedule as important parameters, which might
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
affect throughfall concentrations and biotic interactions, the quantities of rain applied to the trees followed the daily rain volumes recorded from May to August at the Waldstein site the previous year. The Waldstein site is located in the Fichtelgebirge in the northeastern part of Bavaria at an altitude of 800 m asl, where biogeochemical processes and ¯uxes are routinely measured. Thus, we used the information on real precipitation patterns for our manipulation experiment. For technical reasons, it was not possible to immediately use the records of the same year in our experiments. The irrigation solution matched throughfall solution recorded for the Waldstein site (e.g. in April/May 1996) (mg/l): 10.03 NH4±N, 10.18 NO3±N, 11.33 K2SO4, 3.55 Na2SO4, 4.93 MgSO47H20, 7.33 CaCl2, and 286.2 mg/l (1 N) H2SO4 with pH 3.61 and conductivity of 254 mS/cm. To ensure the population growth of aphids and honeydew production, three of these trees were enclosed in an insect-proof net while the others were prevented from being colonised by aphids. At the beginning of May, each tree in the cage was infested with 100 individuals of Cinara pilicornis (Hartig), which started to feed on one- and two-year-old twigs of spruce and 100 individuals of C. costata (Zett.) which prefer to feed on older twigs, closer to the trunk. Both species are seasonally abundant at the Waldstein site. The aphid infestation paralleled the population increase on spruce trees at the Waldstein site, where the population dynamics of these aphids was monitored for several years (Stadler et al., 1998). After the aphid populations peaked at the end of June/beginning of July and natural enemies started to feed on the aphids at the Waldstein site, we lifted the insect net and gave natural predators access to the aphid populations. Winged aphids were able to disperse. Thus, we simulated a ®eld situation with a heavy infestation of spruce during June/beginning of July and a subsequent decline in aphid numbers from mid-July onwards. The colonisation of the control trees was prevented by rearrangement of the net to separate control from infested trees. Underneath each tree we placed two soil lysimeters (diameter 17 cm), one located at the periphery, the other next to the trunk to account for a possible spatial variability in aphid distribution within a tree and quantities of honeydew, which are likely to arrive at the soil lysimeters via throughfall. The forest ¯oor
3
(Ol±Oh horizon) was carefully cut out from the Waldstein site and transferred undisturbed into the lysimeters one week prior to the start of the experiment. Forest-¯oor solutions were collected by providing a permanent suction to the lysimeter (250±300 mbar). Next to each soil lysimeter we placed a throughfall sampler (diameter 15 cm) to monitor the amount of DOC and hexose-C in throughfall that entered the soil lysimeters. Each week we collected the solutions and estimated the number of aphids above the lysimeters. During the experiment the position of the lysimeters and rain samplers was not changed. In early September, the ®eld experiment was ®nished after the aphids had completely disappeared from the infested trees. We then transferred the lysimeters to a growth chamber, where they were stored for two weeks at 28C. After that time, we cooled the lysimeters down to ÿ208C for another two weeks, simulating deep frost. For defrosting the soil was transferred back to the 28C growth chamber. To simulate snow melt we carefully ¯ushed the soil body with one liter of water. The chemical composition of the water was (mg/l): 4.76 Ca2, 11.30 K, 1.04 Mg2, 9.59 Clÿ and 32.00 SO2ÿ 4 . Snow-melt solution consisted of the same chemicals as the irrigation water, however, without the nitrogen compounds. It was further adjusted with 1 N H2SO4 to the average conductivity (190 mS/cm) and pH (3.65) recorded at the Waldstein site from rain samplers during January± April. While ¯ushing, the leachates were again collected from the forest ¯oor by applying a permanent negative pressure. As in the ®eld upper soil horizons are frequently freezing and melting during early spring, we repeated the freezing/leaching procedure to follow delayed changes in ¯oor solution chemistry (if any). 2.2. Chemical analyses Forest-¯oor solutions were immediately ®ltered with a cellulose-acetate membrane (0.45 mm, Sartorius). Dissolved organic carbon was determined as CO2 after persulfate-UV-oxidation (Foss Haereus liqui TOC) and Hexose-C in throughfall and forest ¯oor solution was measured colorimetrically by the anthrone method (Jermyn, 1975) at a wavelength of 630 nm. Ammonia nitrogen (NH4±N) and NO3±N were measured by ion chromatography. Dissolved
4
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
organic nitrogen (DON) was calculated by subtracting NH4±N and NO3±N from total dissolved nitrogen (Ntotal). Total nitrogen was measured after thermooxidation at 7008C as NOx (Abimed: TN-05).
above the samplers, sampling date, collecting site, leachate volume, conductivity, pH, NO3±N, NH4±N, DON, DOC and total N. The statistical analyses followed that of Sokal and Rohlf (1995).
2.3. Determination of the population density of micro-organisms
3. Results
To determine the effect of frost on the soil biota, soil samples were collected from each lysimeter prior to, and after, frost application by cutting out a soil cylinder (diameter: 2.5 cm) across the soil pro®le. It was not possible to take soil samples during the complete course of the experiment without destroying the forest-¯oor matrix in the lysimeters. To test whether micro-organisms in upper soil horizons were more affected by the availability of carbohydrates compared to those of lower horizons, we separated the forest ¯oor into Olf and Oh horizons, which were analysed for the number of colony forming units (CFU) of bacteria, yeast and ®lamentous fungi. From each sample, 5 g were shaken in Erlenmeyer's ¯asks for 30 min together with 45 ml of sterile sodium-pyrophosphate solution (8 g NaCl and 1 g Na2P2O710H2O in one-liter distilled water) and 5 g of small pebbles (ca. 3 mm in diameter). Immediately after shaking, the samples were logarithmically diluted in quarter-strength Ringer's solution and analysed by spread plating. Total aerobic, heterotrophic bacteria populations were enumerated on Standard II nutrient agar (Merck), supplemented with 0.4 g/l cycloheximide (Merck) to inhibit the growth of fungi. Sabouraud±1% dextrose±1% maltose agar (Merck) was used to enumerate total yeasts and ®lamentous fungi which were easily distinguished on the basis of their colony forms. Chloramphenicol (Berlin-Chemie) was added at 0.4 g/l to suppress bacterial growth in this medium. All media plates were incubated at 258C for six days.
Aphid density increased during June and was the highest at the beginning/mid-July with an average of >1000 individuals above the soil lysimeters (Fig. 1(a)). The increase was much more pronounced in the centre of the trees compared to the periphery which is due to the structure of trees, as more twigs covered the lysimeters closer to the stem. After the insect cages were removed, aphid numbers declined on account of alatae dispersal and predators, which started to feed on the aphids. DOC and hexose-C concentrations in throughfall of infested trees mirrored the population dynamics of aphids with
2.4. Statistical analysis Using multivariate discriminant analysis as a tool to summarise the results of the experiment, we compared forest-¯oor leachates, separated by summer temperatures or exposure to frost collected underneath infested and uninfested spruce. The following variables were included in the analysis: Number of aphids
Fig. 1. Seasonal dynamics in (a) the number of aphids above the soil lysimeter located at the centre (&, solid line) and periphery (}, dotted line) underneath Norway spruce and (b) in DOC (!, solid line) and hexose-C (5, dashed line) concentrations in throughfall of infested Norway spruce or from uninfested trees (*, DOC; *, hexose-C) (data from centre and periphery combined, meansS.E.) The arrow indicates the time when the insect nets were removed.
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
highest concentrations in July, prior to the removal of the cage (Fig. 1(b)). Carbohydrate input underneath uninfested trees was close to zero. Large standard errors, both in aphid numbers above the lysimeters and carbohydrates concentrations in throughfall, were largely due to the high degree of mobility of aphids. In forest-¯oor leachates, sugar concentrations did not follow the above-ground seasonal dynamics and no statistically signi®cant difference was found in hexose-C concentrations, either between infested and uninfested treatments (Mann and Whitney U-test: U469.5, W1064.5, P0.132, n69), or between the centre and periphery (U563.5, W1221.5, p0.7053, n69). The average hexose-C concentration in the soil leachates was 2.9 mg/l. The seasonal dynamics in forest-¯oor solution chemistry, separated for soil in¯uenced by honeydew and unaffected controls, is shown for several chemical parameters in Fig. 2. The general trend is that, at the beginning of the experiment and after frost application, both treatments did not differ in the concentration of nutrients or other soil properties, while during July forest-¯oor leachates of the different treatments contained different concentrations of NH4±N (U83.0, W412.0, p0.012, n36) and Ntotal (U88.5, W406.5, P0.02, n36). Large standard errors prevented the demonstration of statistically signi®cant differences in concentrations between other compounds. However, conductivity and pH also were signi®cantly different between treatments during July (conductivity: U63.0, W432.0, p0.002, n36; pH: U81.0, W252.0, p0.010, n36). Just as with hexose-C (not shown here), no signi®cant difference between treatments was found in DOC concentrations. Frost had no signi®cant effect on leachate concentration for any parameter with respect to differences between the two treatments. However, the leachate chemistry was often markedly different before, and after, snow melt simulation. The largest drop in concentration was recorded for NO3±N (66.4%), while NH4±N concentration was only reduced by 22.9% after frost. DON concentrations of honeydew-affected and unaffected forest-¯oor leachates declined by 62.8% already at the end of July. Although constant concentrations of inorganic N were applied during irrigation, the average solute concentrations in the leachates decreased in the order NO3±N>NH4±
5
N>DON. DOC did not show a pronounced decline in leaching concentrations after frost. Leachates were classi®ed by multivariate discriminant analysis into four distinct groups (Fig. 3). With discriminant function one (DF1), variables like NO3± N, sampling date, pH and conductivity contributed to group separation which mirrored the frost effect (eigenvalue6.52, canonical correlation0.93, 2222.89, p<0.0001). The number of aphids above the samplers and different nitrogen compounds (NH4± N, Ntotal, DON) had the greatest in¯uence on discriminant function two (DF2) (eigenvalue0.63, canonical correlation0.62, c247.41, p0.0002) (Table 1). The other variables were of lower importance for group separation and correlated with DF3 (not shown here). Comparing the numbers of micro-organisms present in the honeydew-affected forest ¯oor with those of the unaffected, before and after exposure to frost, did not reveal any statistically signi®cant differences for any horizon (Fig. 4(a and b)). Thus, it seems likely that the availability of carbohydrates during summer does not affect the population density of micro-organisms for a long period of time. However, signi®cantly higher numbers of bacteria yeast and ®lamentous fungi were found more often in the Oh-horizon compared to the Olf-horizon, especially before frost application (Fig. 4(a)). Thus, our expectation that micro-organisms of the upper horizon will be more positively affected by honeydew compared to those of lower soil horizons does not seem to be true. No consistent pattern emerged for particular types of micro-organisms with respect to the number of CFU's developing before, and after, frost. In some compartments bacteria yeast and ®lamentous fungi developed better after frost (mostly in Olf), while in others they did worse (mostly Oh), regardless of whether honeydew was present or absent. 4. Discussion The in¯uence of herbivores on ecosystem processes is usually dif®cult to demonstrate although there are some good examples for large grazing mammals which may affect ecosystem structure and functioning (McNaughton et al., 1982; Huntly, 1991; Pastor and Naiman, 1992; Pastor et al., 1993; see Jones et al.,
6
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
Fig. 2. Seasonal dynamics in different chemical properties of forest-floor leachates collected underneath infested (*, dashed line) and uninfested (&, solid line) Norway spruce from June to August (meansS.E.). Arrows indicate the time when the soil samples were frozen.
1997 for a review on ecosystem engineers). Similarly, the plant±soil system may be affected by plant allocation processes to different organs in response to
grazing (Jaramillo and Detling, 1988; Polley and Detling, 1988; Jones and Last, 1991). Holland and Detling (1990) suggested carbon allocation to roots,
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
Fig. 3. Classification of forest-floor leachates subjected to different treatments. For each group the 95% confidence ellipses are shown. Group centroids mark forest-floor leachates collected underneath infested (*, dashed line) and uninfested (&, solid line) trees during the growing season and after frost application: (5), control; and (~), infested. The analysis included the variables given in Table 1. Table 1 Pooled within-group correlation between discriminant variables and canonical discriminant function (DF) 1 and 2 for the classification of forest-floor leachates according to the presence/ absence of honeydew as source of energy and different temperature exposure a Variable NO3±N (mg/l) Sampling date pH Conductivity (mS/cm) Number of aphids NH4±N (mg/l) Ntotal (mg/l) DON (mg/l) Collecting site Leachate volume DOC (mg/l) % of variance
DF1
DF2 b
ÿ0.483 0.450 b 0.306 b ÿ0.248 b ÿ0.071 0.076 ÿ0.197 ÿ0.038 ÿ0.003 0.305 0.036 90.5
ÿ0.037 ÿ0.196 0.235 ÿ0.238 0.620 b ÿ0.463 b ÿ0.255 b ÿ0.117 b 0.005 b ÿ0.003 0.003 8.7
a
Percentages of correct classification of leachates receiving different treatments: control, summer (88.6%); infested, summer (77.8%); control, frost (91.7%); infested, frost (91.7%). b Denotes largest absolute correlation between each variable and a particular discriminant function.
which is a possible response of grasses to herbivory, as a key link between above-ground herbivory and below-ground microbial metabolism. Soil microorganisms also may be affected by carbon-based plant secondary chemicals during litter decomposition, which ultimately could affect plant nitrogen availability (Horner et al., 1988). However, there are further
7
mechanisms conceivable that link above-ground herbivory with below-ground biogeochemical processes without plants as direct mediators. Aphids, in particular, appear to be a promising group to study the effects of insect herbivores on soil microbes and ecosystem processes, because they not only can in¯ict direct damage on trees (Johnson, 1965; Dixon, 1971a, b) but often excrete large quantities of honeydew, especially on conifers, which are consumed by micro-organisms (Stadler and MuÈller, 1996). Micro-organisms play a key role for nitrogen cycling in many soil systems where they can serve as sources and sinks for nutrients (Coleman et al., 1983) with nitrogen availability and the type and amount of carbohydrates present as critical parameters determining whether nitrogen is immobilised or mineralised (Aber and Melillo, 1982; Pastor et al., 1987). If soil microbial communities are carbon limited (Dommergues et al., 1978; Coleman et al., 1983), the excreta of aphids should positively affect their growth and/or metabolism. As a result, nitrogen immobilisation/mineralisation as well as leachate chemistry of forest ¯oors should be affected. Our results showed that the mineralization±immobilization balance is signi®cantly affected by the availability of honeydew. During the infestation period in June and July, average above-ground concentrations of hexose-C were >100 times higher than the concentrations that left the forest ¯oor with the leachates, indicating an almost complete utilization of this type of energy for microbial growth or respiration. Soil solution properties differed signi®cantly during July when most honeydew reached the forest ¯oor with throughfall. The concentrations of NH4±N and total N were signi®cantly lower during that time and although large standard errors prevent the demonstration of statistically signi®cant differences among other N compounds between the two treatments their values were, on average, lower in the honeydew-affected forest ¯oor leachates during July (Fig. 2). Therefore, honeydew availability seems to be an important control variable over soil microbial activities and nitrogen mobilization/immobilization. As a consequence, it seems likely that plants, which compete with soil micro-organisms for nutrients, will be affected in terms of a reduced nitrogen availability. The application of frost to the soil body affected both treatments in the same way, irrespective of a
8
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
Fig. 4. Number of colony forming units (CFU)/(g soil) fresh mass of bacteria, yeast and filamentous fungi in different forest-floor horizons (Olf, 0±5 cm; Oh, 5±10 cm) cultivated from honeydew-affected and unaffected forest floor. Samples were taken after (a) the aphid population disappeared in September, and (b) after frost treatment. Columns placed side-by-side compare different forest-floor horizons within an organism group. Pairs of columns marked with an asterisk differ significantly at p<0.05 (Mann and Whitney U-test).
preceding in¯uence through carbohydrates. Frost decreased leachate concentrations of all nitrogenous compounds between 22.9 and 66.4% compared to leachates collected at the end of August. However, the relatively higher leachate concentrations of NH4± N after frost as against NO3±N may indicate weaker retention mechanisms for this nitrogen compound, especially if biological processes are interrupted, because often NH4±N is the preferred N source for the biota (Brock and Madigan, 1991). Contrary to the ®eld observations of Yavitt and Fahey (1985), we did not ®nd increased solute concentrations from the
forest ¯oor under standardised conditions. It is likely that the effect of snow melt on leachates is dependent on the precipitation patterns during summer and winter as well as forest type (Currie et al., 1996) and the importance of such seasonal ¯ushing may vary annually as well as with the region. An indication of the importance of particular parameters for the chemistry of forest-¯oor solutions is given by the discriminant function analysis which emphasises the effects of low temperatures on nitrogen mobilisation and carbohydrate input to the forest ¯oor. Leachates collected after frost differed from those collected
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
during summer mostly in NO3±N-concentrations, while leachates collected during summer were affected by the honeydew of aphids (indexed by aphid number) and subsequent processes which affected NH4±N availability. After the aphid populations declined and the input of honeydew to the soil lysimeters ceased, we found no statistically signi®cant differences in the number of bacteria, yeast and ®lamentous fungi between treatments (Fig. 4). It seems likely that micro-organisms either were highly sensitive to changes in carbohydrate availability responding with immediate ¯uctuations in their population densities or by adjusting their metabolic rates without increase in numbers. Differences in the number of micro-organisms present in different forest-¯oor horizons argue for habitat preferences which may not only be determined by the availability of carbohydrates, but also by soil moisture, oxygen content, etc. We gained no direct evidence for the role of micro-organisms on forest-¯oor leachate chemistry once the input of honeydew had ceased. But the fact that large quantities of carbohydrates, reaching the forest ¯oor during the infestation period, did not affect output leachate concentrations indicates that microbial activities may ultimately be responsible for differences in leachate chemistry between treatments. The physiological mechanisms of plants to allocate carbon to their roots in response to herbivory (Holland and Detling, 1990) is but one mechanism that in¯uences micro-organisms and soil chemical properties. Effects via the faeces of herbivores or excreta of aphids are equally conceivable. Our results support the hypothesis that areas with a high carbon input to the forest ¯oor will show an increased nitrogen immobilization and, most likely, decreased plant nitrogen availability (Grier and Vogt, 1990). There is growing evidence that insect herbivores have a measurable effect on ecosystem processes beyond outbreak situations and they may in¯uence nutrient cycling in a number of ways. The diversity of effects includes selective foraging which alters plant community composition and structure, frass, excreta, changes in leaf-litter chemistry, canopy leaching, plant resource allocation, herbivore±micro-organism interactions, to name but a few (Tukey, 1970; Swank et al., 1981; Schowalter et al., 1986; Seastedt and Crossley, 1984; Choudhury, 1988; Pastor et al., 1993). The
9
distribution and abundance of herbivores are, therefore, likely to be important features, which determine the impact on ecosystem functions, such as nutrient cycling or biological productivity. These effects are, however, often dif®cult to demonstrate, especially for herbivores in non-outbreak situations. Whether the effects we found underneath individual trees will contribute to measurable effects on a larger scale, such as a forest stand or forest watershed, is not clear at the moment. Long-term effects of different degrees of chronic herbivory on ecosystem function therefore deserve closer inspection. Acknowledgements We wish to thank E. Matzner, C. Matthies for critical comments on an earlier draft of the manuscript. A. Glaûer, K. Moser, B. Popp and C. StoÈcker helped with chemical analyses. Financial support was given by the German Ministry for Research and Technology (FoÈrdernummer: BMBF No. PT BEO 51-0339476B). References Aber, J.D., Melillo, J.M., 1982. Nitrogen immobilization in decaying hardwood leaf litter as a function of initial nitrogen and lignin content. Can. J. Bot. 60, 2263±2269. Brock, T.D., Madigan, M.T., 1991. Biology of microorganisms, 6th edn., Prentice Hall, Englewood Cliffs, NJ. Choudhury, D., 1988. Herbivore induced changes in leaf-litter resource quality: a neglected aspect of herbivory in ecosystem nutrient dynamics. Oikos 51, 380±393. Coleman, D.C., Reid, C.P.P., Cole, C.V., 1983. Biological strategies of nutrient cycling in soil systems. Adv. Ecol. Res. 13, 1±55. Currie, W.S., Aber, J.D., McDowell, W.H., Boone, R.D., Magill, A.H., 1996. Vertical transport of dissolved organic C and N under long-term N amendments in pine and hardwood forests. Biogeochem. 80, 1±35. Dighton, J., 1978a. In vitro experiments simulationg the possible fates of aphid honeydew sugars in soil. Soil Biol. Biochem. 10, 53±57. Dighton, J., 1978b. Effects of synthetic lime aphid honeydew on populations of soil organisms. Soil Biol. Biochem. 10, 369± 376. Dixon, A.F.G., 1971a. The role of aphids in wood formation. I. The effect of the Sycamore aphid, Drepanosiphum plantanoides (Schr.) (Aphididae), on the growth of Sycamore, Acer pseudoplantanus (L.). J. Appl. Ecol. 8, 165±179. Dixon, A.F.G., 1971b. The role of aphids in wood formation. II. The effect of the lime aphid, Eucallipterus tiliae L. (Aphidi-
10
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
dae), on the growth of lime, Tilia vulgaris Hayne. J. Appl. Ecol. 8, 393±399. Dommergues, Y.R., Belser, L.W., Schmidt, E.L., 1978. Limiting factors for microbial growth and activity in soil. In: Alexander, M. (Ed.), Advances in Microbial Ecology, Plenum Press, New York, London. pp. 49±104. GoÈttlein, A., Stanjek, H., 1996. Micro-scale variability of solid phase properties and soil solution chemistry in a forest podzol and its relation to soil horizons. Eur. J. Soil Sci. 47, 627±636. Grier, C.C., Vogt, D.J., 1990. Effects of aphid honeydew on soil nitrogen availability and net primary production in an Alnus rubra plantation in western Washington. Oikos 57, 114±118. Heimbach, U., 1986. Freilanduntersuchungen zur Honigtauabgabe zweier Zierlausarten (Aphidina). J. Appl. Entomol. 101, 396± 413. Holland, E.A., Detling, J.K., 1990. Plant response to herbivory and belowground nitrogen cycling. Ecology 71, 1040±1049. Horner, J.D., Gosz, J.R., Cates, R.G., 1988. The role of carbon based plant secondary metabolites in decomposition in terrestrial ecosystems. Am. Nat. 132, 869±883. Huntly, N., 1991. Herbivores and the dynamics of communities and ecosystems. Ann. Rev. Ecol. Sys. 22, 477±503. Jaramillo, V., Detling, J.K., 1988. Grazing history, defoliation, and competition: effects on shortgrass production and nitrogen accumulation. Ecology 69, 1599±1608. Jermyn, M.A., 1975. Increasing the sensitivity of the anthrone method for carbohydrate. Analyt. Biochem. 68, 332±335. Johnson, N.E., 1965. Reduced growth associated with infestations of Douglas-fir seedlings by Cinara species (Homoptera: Aphidae). Can. Ent. 97, 113±119. Jones, C.G., Last, F.T., 1991. Ectomycorrhizae and trees: implications for aboveground herbivory. In: Barbosa, P., Krischik, V.A., Jones, C.G. (Eds.), Microbial Mediation of PlantHerbivore Interactions, pp. 65-103. Wiley & Sons, New York. Jones, C.G., Lawton, J.H., Shachak, M., 1997. Positive and negative effects of organisms as physical ecosystem engineers. Ecology 78, 1946±1957. Manderscheid, B., Matzner, E., 1995. Spatial and temporal variation of soil solution chemistry and ion fluxes through the soil in a mature Norway spruce (Picea abies (L.) Karst.) stand. Biogeochem. 30, 99±114. Maurizio, A., 1985. Honigtau-Honigtauhonig. In: Kloft, W.J., Kunkel, H. (Eds.), Waldtracht und Waldhonig in der Imkerei. Ehrenwirth, MuÈnchen. pp. 267±295. McNaughton, S.J., Coughenour, M.B., Wallace, L.L., 1982. Interactive processes in grassland ecosystems. In: Estes, J.R.,
Tyrl, R.J., Brunken, J.R. (Eds.), Grasses and grasslands, systematics and ecology. University of Oklahoma Press, Oklahoma. pp. 167±193. Pastor, J., Stillwell, M.A., Tilman, D., 1987. Nitrogen mineralization and nitrification in four Minnesota old fields. Oecologia 71, 481±485. Pastor, J., Naiman, R.J., 1992. Selective foraging and ecosystem processes in boreal forests. Am. Nat. 139, 690±705. Pastor, J., Dewey, B., Naiman, R.J., McInnes, F.P., Cohen, Y., 1993. Moose browsing and soil fertility in the boreal forests of Isle Royale national park. Ecology 74, 467±480. Petelle, M., 1980. Aphids and melezitose: a test of Owen's 1978 hypothesis. Oikos 35, 127±128. Polley, H.W., Detling, J.K., 1988. Defoliation, nitrogen, and competition: effects of plant growth and nitrogen nutrition. Ecology 70, 721±727. Schowalter, T.D., Hargrove, W.W., Crossley Jr., D.A., 1986. Herbivory in forested systems. Ann. Rev. Entomol. 31, 177± 196. Seastedt, T.R., Crossley Jr., D.A., 1984. The influence of arthropods on ecosystems. Bioscience 34, 157±161. Sokal, R.R., Rohlf, F.J., 1995. Biometry, 3rd edn. Freeman and Company, New York. Stadler, B., MuÈller, T., 1996. Aphid honeydew and its effect on the phyllosphere microflora of Picea abies (L.) Karst. Oecologia 108, 771±776. Stadler, B., Michalzik, B., MuÈller, T., 1998. Linking aphid ecology and ecosystem processes: the effect of spatial and temporal variability in aphid abundance on nutrient fluxes in a coniferous forest. Ecology (in press). Swank, W.T., Waide, J.B., Crossley Jr., D.A., Todd, R.L., 1981. Insect defoliation enhances nitrate export from forest systems. Oecologia 51, 297±299. Tukey, H.B., 1970. The leaching of substances from plants. Ann. Rev. Plant Physiol. 21, 305±329. Yavitt, J.B., Fahey, T.J., 1985. Organic chemistry of the soil solution during snowmelt leaching in Pinus contorta forest ecosystems, Wyoming. In: Caldwell, D.E., Brierley, J.A., Brierley, C.L. (Eds.), Planetary Ecology, pp. 485±495. Van Nostrand Reinhold Company, New York. Zoebelein, G., 1954. Versuche zur Feststellung des Honigtauertrages von FichtenbestaÈnden mit Hilfe von Waldameisen. Z. Ang. Ent. 36, 358±362. ZwoÈlfer, W., 1952. Die Waldbienenweide und ihre Nutzung als forstentomologisches Problem. Verh. deut. Ges. angew. Entomol. Frankfurt (1952) 164±168.
Aphids on Norway spruce and their effects on forest-¯oor solution chemistry Beate Michalzika,*, Thomas MuÈllerb, Bernhard Stadlera b
a Bayreuth Institute for Terrestrial Ecosystem Research, University of Bayreuth, 95440 Bayreuth, Germany Centre for Agricultural Landscape and Land Use Research, MuÈncheberg, Institute of Microbial Ecology and Soil Biology, 14641 Paulinenaue, Germany
Received 8 April 1998; accepted 19 August 1998
Abstract Many species of aphids on spruce excrete large quantities of honeydew. As providers of carbohydrates, it is likely that they affect several ecosystem functions by fueling biological processes which are energy-limited. In a rainfall simulation experiment, we manipulated the level of aphid infestation on spruce and studied the effects of honeydew on forest-¯oor solution chemistry collected underneath infested and uninfested trees. A non-destructive sampling method was used to allow repeated measurements using the same forest ¯oor during the experimental period. Although the input of dissolved organic carbon (DOC) underneath infested trees to the forest ¯oor was considerably higher compared to uninfested trees, carbon concentrations of forest-¯oor leachates did not differ between treatments. Concentrations of dissolved organic nitrogen (DON), NO3±N and Ntotal of forest-¯oor leachates, underneath infested and uninfested trees followed a marked seasonal trend with low concentrations recorded in June and after-frost treatment of the forest ¯oor, while the highest concentrations were recorded in July/August. Statistically signi®cant differences in soil solution properties underneath infested and uninfested trees were recorded in July when honeydew-affected forest-¯oor leachates had lower concentrations of NH4±N, Ntotal, a lower conductivity and a higher pH. Despite a large input of honeydew, no pronounced seasonal trend was found in the carbon leachate concentrations of the forest ¯oor (e.g. DOC, hexose-C). Discriminant function analysis showed that forest-¯oor leachates can be classi®ed according to the experience of frost and nitrogen concentrations, which were affected by the honeydew of aphids. At the end of the experiment, the number of micro-organisms present in the forest ¯oor was not signi®cantly different between treatments but was higher in the Oh compared to the Olf horizon. Microbial communities did not appear to be severely in¯uenced by deep temperatures. It seems likely that, in coniferous forests, aphids can considerably reduce local ¯uxes of nitrogen from the forest ¯oor. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Aphid honeydew; Cinara; Forest-¯oor leachates; Norway spruce
1. Introduction *Corresponding author. Fax: +1-49(0)921/55-5799; e-mail: [email protected]
Aphids on trees often excrete copious amounts of honeydew, which mainly consists of sugars such as
0378-1127/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0378-1127(98)00481-2
2
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
fructose, glucose and melezitose (Maurizio, 1985). On a per-hectare basis, it has been reported that an annual average of 400±700 kg fresh mass of honeydew can be expected in a coniferous forest (ZwoÈlfer, 1952; Zoebelein, 1954). Estimates for lime aphids on Tilia spp. even amount to 800 g mÿ2 aÿ1 (Llewellyn, unpubl. PhD thesis, Glasgow University 1970) or range between 2 and 50 kg honeydew dry mass for a single mature lime tree (Heimbach, 1986). Aphid population densities usually ¯uctuate spatially and temporally. Thus, although locally abundant, the availability of honeydew as a source of energy is unpredictable for consumers of carbohydrates. This unpredictability also makes it dif®cult to investigate the effects of honeydew for ecosystem processes, such as nutrient cycling or solute export in leachates from the forest ¯oor of a natural forest stand. In the phyllosphere of Norway spruce, we found that honeydew signi®cantly promotes the growth of micro-organisms and affects throughfall carbon and nitrogen chemistry (Stadler et al., 1998). Therefore, the population dynamics of aphids is a key to an understanding of the temporal and spatial variability in aphid±micro-organism interactions and throughfall ¯uxes. The effect of honeydew on soil biology and chemistry has already received some attention. Bacteria and fungi readily respond to the availability of carbohydrates, either by increasing in numbers or respiration, depending on soil type (Dighton, 1978a, b; Petelle, 1980). Besides its effects on the soil biota, it is conceivable that the honeydew of aphids also affects nutrient cycling or soil-leachate chemistry, which is, however, much more dif®cult to demonstrate. From an investigation of mineral soil in an Alnus rubra Bong. plantation (Grier and Vogt, 1990) it appears that honeydew does in¯uence ammoni®cation and nitri®cation, although the applied method of repeated insecticide application (Malathion) for removing aphids from the control treatment could have adverse effects on the soil fauna. Furthermore, a repeated sampling of soil introduces an uncontrolled variability to an experiment because small-scale differences in soil structure and chemistry are the rule rather than the exception, thus obscuring effects (Manderscheid and Matzner, 1995; GoÈttlein and Stanjek, 1996). Another limitation in studies on the effects of honeydew on soil micro-organisms is that they typically cover only a short period of time. This might be
due to the fact that aphids and micro-organisms respond quickly to resources which are only temporarily available; thus, most experimenters ignore seasonal aspects, such as winter frost. In the ®eld, however, concentrations of nutrients in forest-¯oor leachates are sometimes found to increase in early spring after snow melt (Yavitt and Fahey, 1985) which seems to indicate a fatal effect of frost on the biota and/or inhibition of decomposition processes. We account for this effect by studying the population densities of micro-organisms and forest-¯oor leachate chemistry before, and after, arti®cial frost application, both when honeydew was present and absent during summer. In particular, the objectives of this paper were: 1. to produce in a controlled rainfall and aphid infestation experiment carbon-rich throughfall; 2. to investigate the effects of different levels of Cinput on forest-floor leachate chemistry; and 3. to test the hypothesis that frost disrupts the microbial populations in the soil and induces changes in leachate chemistry. We discuss the results with respect to the effects imposed by above- and below-ground biotic interactions on plant nitrogen availability and ecosystem processes. 2. Materials and methods 2.1. Experimental design In order to exclude unknown variability from the study, we standardised our experiment with respect to plant quality, nutritional supply, rain chemistry, amount of rain applied and microclimatic differences between trees. In a rainfall simulation experiment, six 10-year-old spruce trees (Picea abies (L.) Karst.) were covered with a transparent roof (20 m2) at an experimental ®eld close to the university. All trees were grown in containers 50 cm in diameter, 30 cm deep, ®lled with compost soil. Above each tree an irrigation system was installed to spray the rain solution onto the trees with the help of a ®ne axial ¯ow, full cone nozzle (Lechler, Metzingen), which released the water homogeneously at a 608 angle. Because we considered the volumes of rain sprayed onto the trees and irrigation time schedule as important parameters, which might
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
affect throughfall concentrations and biotic interactions, the quantities of rain applied to the trees followed the daily rain volumes recorded from May to August at the Waldstein site the previous year. The Waldstein site is located in the Fichtelgebirge in the northeastern part of Bavaria at an altitude of 800 m asl, where biogeochemical processes and ¯uxes are routinely measured. Thus, we used the information on real precipitation patterns for our manipulation experiment. For technical reasons, it was not possible to immediately use the records of the same year in our experiments. The irrigation solution matched throughfall solution recorded for the Waldstein site (e.g. in April/May 1996) (mg/l): 10.03 NH4±N, 10.18 NO3±N, 11.33 K2SO4, 3.55 Na2SO4, 4.93 MgSO47H20, 7.33 CaCl2, and 286.2 mg/l (1 N) H2SO4 with pH 3.61 and conductivity of 254 mS/cm. To ensure the population growth of aphids and honeydew production, three of these trees were enclosed in an insect-proof net while the others were prevented from being colonised by aphids. At the beginning of May, each tree in the cage was infested with 100 individuals of Cinara pilicornis (Hartig), which started to feed on one- and two-year-old twigs of spruce and 100 individuals of C. costata (Zett.) which prefer to feed on older twigs, closer to the trunk. Both species are seasonally abundant at the Waldstein site. The aphid infestation paralleled the population increase on spruce trees at the Waldstein site, where the population dynamics of these aphids was monitored for several years (Stadler et al., 1998). After the aphid populations peaked at the end of June/beginning of July and natural enemies started to feed on the aphids at the Waldstein site, we lifted the insect net and gave natural predators access to the aphid populations. Winged aphids were able to disperse. Thus, we simulated a ®eld situation with a heavy infestation of spruce during June/beginning of July and a subsequent decline in aphid numbers from mid-July onwards. The colonisation of the control trees was prevented by rearrangement of the net to separate control from infested trees. Underneath each tree we placed two soil lysimeters (diameter 17 cm), one located at the periphery, the other next to the trunk to account for a possible spatial variability in aphid distribution within a tree and quantities of honeydew, which are likely to arrive at the soil lysimeters via throughfall. The forest ¯oor
3
(Ol±Oh horizon) was carefully cut out from the Waldstein site and transferred undisturbed into the lysimeters one week prior to the start of the experiment. Forest-¯oor solutions were collected by providing a permanent suction to the lysimeter (250±300 mbar). Next to each soil lysimeter we placed a throughfall sampler (diameter 15 cm) to monitor the amount of DOC and hexose-C in throughfall that entered the soil lysimeters. Each week we collected the solutions and estimated the number of aphids above the lysimeters. During the experiment the position of the lysimeters and rain samplers was not changed. In early September, the ®eld experiment was ®nished after the aphids had completely disappeared from the infested trees. We then transferred the lysimeters to a growth chamber, where they were stored for two weeks at 28C. After that time, we cooled the lysimeters down to ÿ208C for another two weeks, simulating deep frost. For defrosting the soil was transferred back to the 28C growth chamber. To simulate snow melt we carefully ¯ushed the soil body with one liter of water. The chemical composition of the water was (mg/l): 4.76 Ca2, 11.30 K, 1.04 Mg2, 9.59 Clÿ and 32.00 SO2ÿ 4 . Snow-melt solution consisted of the same chemicals as the irrigation water, however, without the nitrogen compounds. It was further adjusted with 1 N H2SO4 to the average conductivity (190 mS/cm) and pH (3.65) recorded at the Waldstein site from rain samplers during January± April. While ¯ushing, the leachates were again collected from the forest ¯oor by applying a permanent negative pressure. As in the ®eld upper soil horizons are frequently freezing and melting during early spring, we repeated the freezing/leaching procedure to follow delayed changes in ¯oor solution chemistry (if any). 2.2. Chemical analyses Forest-¯oor solutions were immediately ®ltered with a cellulose-acetate membrane (0.45 mm, Sartorius). Dissolved organic carbon was determined as CO2 after persulfate-UV-oxidation (Foss Haereus liqui TOC) and Hexose-C in throughfall and forest ¯oor solution was measured colorimetrically by the anthrone method (Jermyn, 1975) at a wavelength of 630 nm. Ammonia nitrogen (NH4±N) and NO3±N were measured by ion chromatography. Dissolved
4
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
organic nitrogen (DON) was calculated by subtracting NH4±N and NO3±N from total dissolved nitrogen (Ntotal). Total nitrogen was measured after thermooxidation at 7008C as NOx (Abimed: TN-05).
above the samplers, sampling date, collecting site, leachate volume, conductivity, pH, NO3±N, NH4±N, DON, DOC and total N. The statistical analyses followed that of Sokal and Rohlf (1995).
2.3. Determination of the population density of micro-organisms
3. Results
To determine the effect of frost on the soil biota, soil samples were collected from each lysimeter prior to, and after, frost application by cutting out a soil cylinder (diameter: 2.5 cm) across the soil pro®le. It was not possible to take soil samples during the complete course of the experiment without destroying the forest-¯oor matrix in the lysimeters. To test whether micro-organisms in upper soil horizons were more affected by the availability of carbohydrates compared to those of lower horizons, we separated the forest ¯oor into Olf and Oh horizons, which were analysed for the number of colony forming units (CFU) of bacteria, yeast and ®lamentous fungi. From each sample, 5 g were shaken in Erlenmeyer's ¯asks for 30 min together with 45 ml of sterile sodium-pyrophosphate solution (8 g NaCl and 1 g Na2P2O710H2O in one-liter distilled water) and 5 g of small pebbles (ca. 3 mm in diameter). Immediately after shaking, the samples were logarithmically diluted in quarter-strength Ringer's solution and analysed by spread plating. Total aerobic, heterotrophic bacteria populations were enumerated on Standard II nutrient agar (Merck), supplemented with 0.4 g/l cycloheximide (Merck) to inhibit the growth of fungi. Sabouraud±1% dextrose±1% maltose agar (Merck) was used to enumerate total yeasts and ®lamentous fungi which were easily distinguished on the basis of their colony forms. Chloramphenicol (Berlin-Chemie) was added at 0.4 g/l to suppress bacterial growth in this medium. All media plates were incubated at 258C for six days.
Aphid density increased during June and was the highest at the beginning/mid-July with an average of >1000 individuals above the soil lysimeters (Fig. 1(a)). The increase was much more pronounced in the centre of the trees compared to the periphery which is due to the structure of trees, as more twigs covered the lysimeters closer to the stem. After the insect cages were removed, aphid numbers declined on account of alatae dispersal and predators, which started to feed on the aphids. DOC and hexose-C concentrations in throughfall of infested trees mirrored the population dynamics of aphids with
2.4. Statistical analysis Using multivariate discriminant analysis as a tool to summarise the results of the experiment, we compared forest-¯oor leachates, separated by summer temperatures or exposure to frost collected underneath infested and uninfested spruce. The following variables were included in the analysis: Number of aphids
Fig. 1. Seasonal dynamics in (a) the number of aphids above the soil lysimeter located at the centre (&, solid line) and periphery (}, dotted line) underneath Norway spruce and (b) in DOC (!, solid line) and hexose-C (5, dashed line) concentrations in throughfall of infested Norway spruce or from uninfested trees (*, DOC; *, hexose-C) (data from centre and periphery combined, meansS.E.) The arrow indicates the time when the insect nets were removed.
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
highest concentrations in July, prior to the removal of the cage (Fig. 1(b)). Carbohydrate input underneath uninfested trees was close to zero. Large standard errors, both in aphid numbers above the lysimeters and carbohydrates concentrations in throughfall, were largely due to the high degree of mobility of aphids. In forest-¯oor leachates, sugar concentrations did not follow the above-ground seasonal dynamics and no statistically signi®cant difference was found in hexose-C concentrations, either between infested and uninfested treatments (Mann and Whitney U-test: U469.5, W1064.5, P0.132, n69), or between the centre and periphery (U563.5, W1221.5, p0.7053, n69). The average hexose-C concentration in the soil leachates was 2.9 mg/l. The seasonal dynamics in forest-¯oor solution chemistry, separated for soil in¯uenced by honeydew and unaffected controls, is shown for several chemical parameters in Fig. 2. The general trend is that, at the beginning of the experiment and after frost application, both treatments did not differ in the concentration of nutrients or other soil properties, while during July forest-¯oor leachates of the different treatments contained different concentrations of NH4±N (U83.0, W412.0, p0.012, n36) and Ntotal (U88.5, W406.5, P0.02, n36). Large standard errors prevented the demonstration of statistically signi®cant differences in concentrations between other compounds. However, conductivity and pH also were signi®cantly different between treatments during July (conductivity: U63.0, W432.0, p0.002, n36; pH: U81.0, W252.0, p0.010, n36). Just as with hexose-C (not shown here), no signi®cant difference between treatments was found in DOC concentrations. Frost had no signi®cant effect on leachate concentration for any parameter with respect to differences between the two treatments. However, the leachate chemistry was often markedly different before, and after, snow melt simulation. The largest drop in concentration was recorded for NO3±N (66.4%), while NH4±N concentration was only reduced by 22.9% after frost. DON concentrations of honeydew-affected and unaffected forest-¯oor leachates declined by 62.8% already at the end of July. Although constant concentrations of inorganic N were applied during irrigation, the average solute concentrations in the leachates decreased in the order NO3±N>NH4±
5
N>DON. DOC did not show a pronounced decline in leaching concentrations after frost. Leachates were classi®ed by multivariate discriminant analysis into four distinct groups (Fig. 3). With discriminant function one (DF1), variables like NO3± N, sampling date, pH and conductivity contributed to group separation which mirrored the frost effect (eigenvalue6.52, canonical correlation0.93, 2222.89, p<0.0001). The number of aphids above the samplers and different nitrogen compounds (NH4± N, Ntotal, DON) had the greatest in¯uence on discriminant function two (DF2) (eigenvalue0.63, canonical correlation0.62, c247.41, p0.0002) (Table 1). The other variables were of lower importance for group separation and correlated with DF3 (not shown here). Comparing the numbers of micro-organisms present in the honeydew-affected forest ¯oor with those of the unaffected, before and after exposure to frost, did not reveal any statistically signi®cant differences for any horizon (Fig. 4(a and b)). Thus, it seems likely that the availability of carbohydrates during summer does not affect the population density of micro-organisms for a long period of time. However, signi®cantly higher numbers of bacteria yeast and ®lamentous fungi were found more often in the Oh-horizon compared to the Olf-horizon, especially before frost application (Fig. 4(a)). Thus, our expectation that micro-organisms of the upper horizon will be more positively affected by honeydew compared to those of lower soil horizons does not seem to be true. No consistent pattern emerged for particular types of micro-organisms with respect to the number of CFU's developing before, and after, frost. In some compartments bacteria yeast and ®lamentous fungi developed better after frost (mostly in Olf), while in others they did worse (mostly Oh), regardless of whether honeydew was present or absent. 4. Discussion The in¯uence of herbivores on ecosystem processes is usually dif®cult to demonstrate although there are some good examples for large grazing mammals which may affect ecosystem structure and functioning (McNaughton et al., 1982; Huntly, 1991; Pastor and Naiman, 1992; Pastor et al., 1993; see Jones et al.,
6
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
Fig. 2. Seasonal dynamics in different chemical properties of forest-floor leachates collected underneath infested (*, dashed line) and uninfested (&, solid line) Norway spruce from June to August (meansS.E.). Arrows indicate the time when the soil samples were frozen.
1997 for a review on ecosystem engineers). Similarly, the plant±soil system may be affected by plant allocation processes to different organs in response to
grazing (Jaramillo and Detling, 1988; Polley and Detling, 1988; Jones and Last, 1991). Holland and Detling (1990) suggested carbon allocation to roots,
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
Fig. 3. Classification of forest-floor leachates subjected to different treatments. For each group the 95% confidence ellipses are shown. Group centroids mark forest-floor leachates collected underneath infested (*, dashed line) and uninfested (&, solid line) trees during the growing season and after frost application: (5), control; and (~), infested. The analysis included the variables given in Table 1. Table 1 Pooled within-group correlation between discriminant variables and canonical discriminant function (DF) 1 and 2 for the classification of forest-floor leachates according to the presence/ absence of honeydew as source of energy and different temperature exposure a Variable NO3±N (mg/l) Sampling date pH Conductivity (mS/cm) Number of aphids NH4±N (mg/l) Ntotal (mg/l) DON (mg/l) Collecting site Leachate volume DOC (mg/l) % of variance
DF1
DF2 b
ÿ0.483 0.450 b 0.306 b ÿ0.248 b ÿ0.071 0.076 ÿ0.197 ÿ0.038 ÿ0.003 0.305 0.036 90.5
ÿ0.037 ÿ0.196 0.235 ÿ0.238 0.620 b ÿ0.463 b ÿ0.255 b ÿ0.117 b 0.005 b ÿ0.003 0.003 8.7
a
Percentages of correct classification of leachates receiving different treatments: control, summer (88.6%); infested, summer (77.8%); control, frost (91.7%); infested, frost (91.7%). b Denotes largest absolute correlation between each variable and a particular discriminant function.
which is a possible response of grasses to herbivory, as a key link between above-ground herbivory and below-ground microbial metabolism. Soil microorganisms also may be affected by carbon-based plant secondary chemicals during litter decomposition, which ultimately could affect plant nitrogen availability (Horner et al., 1988). However, there are further
7
mechanisms conceivable that link above-ground herbivory with below-ground biogeochemical processes without plants as direct mediators. Aphids, in particular, appear to be a promising group to study the effects of insect herbivores on soil microbes and ecosystem processes, because they not only can in¯ict direct damage on trees (Johnson, 1965; Dixon, 1971a, b) but often excrete large quantities of honeydew, especially on conifers, which are consumed by micro-organisms (Stadler and MuÈller, 1996). Micro-organisms play a key role for nitrogen cycling in many soil systems where they can serve as sources and sinks for nutrients (Coleman et al., 1983) with nitrogen availability and the type and amount of carbohydrates present as critical parameters determining whether nitrogen is immobilised or mineralised (Aber and Melillo, 1982; Pastor et al., 1987). If soil microbial communities are carbon limited (Dommergues et al., 1978; Coleman et al., 1983), the excreta of aphids should positively affect their growth and/or metabolism. As a result, nitrogen immobilisation/mineralisation as well as leachate chemistry of forest ¯oors should be affected. Our results showed that the mineralization±immobilization balance is signi®cantly affected by the availability of honeydew. During the infestation period in June and July, average above-ground concentrations of hexose-C were >100 times higher than the concentrations that left the forest ¯oor with the leachates, indicating an almost complete utilization of this type of energy for microbial growth or respiration. Soil solution properties differed signi®cantly during July when most honeydew reached the forest ¯oor with throughfall. The concentrations of NH4±N and total N were signi®cantly lower during that time and although large standard errors prevent the demonstration of statistically signi®cant differences among other N compounds between the two treatments their values were, on average, lower in the honeydew-affected forest ¯oor leachates during July (Fig. 2). Therefore, honeydew availability seems to be an important control variable over soil microbial activities and nitrogen mobilization/immobilization. As a consequence, it seems likely that plants, which compete with soil micro-organisms for nutrients, will be affected in terms of a reduced nitrogen availability. The application of frost to the soil body affected both treatments in the same way, irrespective of a
8
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
Fig. 4. Number of colony forming units (CFU)/(g soil) fresh mass of bacteria, yeast and filamentous fungi in different forest-floor horizons (Olf, 0±5 cm; Oh, 5±10 cm) cultivated from honeydew-affected and unaffected forest floor. Samples were taken after (a) the aphid population disappeared in September, and (b) after frost treatment. Columns placed side-by-side compare different forest-floor horizons within an organism group. Pairs of columns marked with an asterisk differ significantly at p<0.05 (Mann and Whitney U-test).
preceding in¯uence through carbohydrates. Frost decreased leachate concentrations of all nitrogenous compounds between 22.9 and 66.4% compared to leachates collected at the end of August. However, the relatively higher leachate concentrations of NH4± N after frost as against NO3±N may indicate weaker retention mechanisms for this nitrogen compound, especially if biological processes are interrupted, because often NH4±N is the preferred N source for the biota (Brock and Madigan, 1991). Contrary to the ®eld observations of Yavitt and Fahey (1985), we did not ®nd increased solute concentrations from the
forest ¯oor under standardised conditions. It is likely that the effect of snow melt on leachates is dependent on the precipitation patterns during summer and winter as well as forest type (Currie et al., 1996) and the importance of such seasonal ¯ushing may vary annually as well as with the region. An indication of the importance of particular parameters for the chemistry of forest-¯oor solutions is given by the discriminant function analysis which emphasises the effects of low temperatures on nitrogen mobilisation and carbohydrate input to the forest ¯oor. Leachates collected after frost differed from those collected
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
during summer mostly in NO3±N-concentrations, while leachates collected during summer were affected by the honeydew of aphids (indexed by aphid number) and subsequent processes which affected NH4±N availability. After the aphid populations declined and the input of honeydew to the soil lysimeters ceased, we found no statistically signi®cant differences in the number of bacteria, yeast and ®lamentous fungi between treatments (Fig. 4). It seems likely that micro-organisms either were highly sensitive to changes in carbohydrate availability responding with immediate ¯uctuations in their population densities or by adjusting their metabolic rates without increase in numbers. Differences in the number of micro-organisms present in different forest-¯oor horizons argue for habitat preferences which may not only be determined by the availability of carbohydrates, but also by soil moisture, oxygen content, etc. We gained no direct evidence for the role of micro-organisms on forest-¯oor leachate chemistry once the input of honeydew had ceased. But the fact that large quantities of carbohydrates, reaching the forest ¯oor during the infestation period, did not affect output leachate concentrations indicates that microbial activities may ultimately be responsible for differences in leachate chemistry between treatments. The physiological mechanisms of plants to allocate carbon to their roots in response to herbivory (Holland and Detling, 1990) is but one mechanism that in¯uences micro-organisms and soil chemical properties. Effects via the faeces of herbivores or excreta of aphids are equally conceivable. Our results support the hypothesis that areas with a high carbon input to the forest ¯oor will show an increased nitrogen immobilization and, most likely, decreased plant nitrogen availability (Grier and Vogt, 1990). There is growing evidence that insect herbivores have a measurable effect on ecosystem processes beyond outbreak situations and they may in¯uence nutrient cycling in a number of ways. The diversity of effects includes selective foraging which alters plant community composition and structure, frass, excreta, changes in leaf-litter chemistry, canopy leaching, plant resource allocation, herbivore±micro-organism interactions, to name but a few (Tukey, 1970; Swank et al., 1981; Schowalter et al., 1986; Seastedt and Crossley, 1984; Choudhury, 1988; Pastor et al., 1993). The
9
distribution and abundance of herbivores are, therefore, likely to be important features, which determine the impact on ecosystem functions, such as nutrient cycling or biological productivity. These effects are, however, often dif®cult to demonstrate, especially for herbivores in non-outbreak situations. Whether the effects we found underneath individual trees will contribute to measurable effects on a larger scale, such as a forest stand or forest watershed, is not clear at the moment. Long-term effects of different degrees of chronic herbivory on ecosystem function therefore deserve closer inspection. Acknowledgements We wish to thank E. Matzner, C. Matthies for critical comments on an earlier draft of the manuscript. A. Glaûer, K. Moser, B. Popp and C. StoÈcker helped with chemical analyses. Financial support was given by the German Ministry for Research and Technology (FoÈrdernummer: BMBF No. PT BEO 51-0339476B). References Aber, J.D., Melillo, J.M., 1982. Nitrogen immobilization in decaying hardwood leaf litter as a function of initial nitrogen and lignin content. Can. J. Bot. 60, 2263±2269. Brock, T.D., Madigan, M.T., 1991. Biology of microorganisms, 6th edn., Prentice Hall, Englewood Cliffs, NJ. Choudhury, D., 1988. Herbivore induced changes in leaf-litter resource quality: a neglected aspect of herbivory in ecosystem nutrient dynamics. Oikos 51, 380±393. Coleman, D.C., Reid, C.P.P., Cole, C.V., 1983. Biological strategies of nutrient cycling in soil systems. Adv. Ecol. Res. 13, 1±55. Currie, W.S., Aber, J.D., McDowell, W.H., Boone, R.D., Magill, A.H., 1996. Vertical transport of dissolved organic C and N under long-term N amendments in pine and hardwood forests. Biogeochem. 80, 1±35. Dighton, J., 1978a. In vitro experiments simulationg the possible fates of aphid honeydew sugars in soil. Soil Biol. Biochem. 10, 53±57. Dighton, J., 1978b. Effects of synthetic lime aphid honeydew on populations of soil organisms. Soil Biol. Biochem. 10, 369± 376. Dixon, A.F.G., 1971a. The role of aphids in wood formation. I. The effect of the Sycamore aphid, Drepanosiphum plantanoides (Schr.) (Aphididae), on the growth of Sycamore, Acer pseudoplantanus (L.). J. Appl. Ecol. 8, 165±179. Dixon, A.F.G., 1971b. The role of aphids in wood formation. II. The effect of the lime aphid, Eucallipterus tiliae L. (Aphidi-
10
B. Michalzik et al. / Forest Ecology and Management 118 (1999) 1±10
dae), on the growth of lime, Tilia vulgaris Hayne. J. Appl. Ecol. 8, 393±399. Dommergues, Y.R., Belser, L.W., Schmidt, E.L., 1978. Limiting factors for microbial growth and activity in soil. In: Alexander, M. (Ed.), Advances in Microbial Ecology, Plenum Press, New York, London. pp. 49±104. GoÈttlein, A., Stanjek, H., 1996. Micro-scale variability of solid phase properties and soil solution chemistry in a forest podzol and its relation to soil horizons. Eur. J. Soil Sci. 47, 627±636. Grier, C.C., Vogt, D.J., 1990. Effects of aphid honeydew on soil nitrogen availability and net primary production in an Alnus rubra plantation in western Washington. Oikos 57, 114±118. Heimbach, U., 1986. Freilanduntersuchungen zur Honigtauabgabe zweier Zierlausarten (Aphidina). J. Appl. Entomol. 101, 396± 413. Holland, E.A., Detling, J.K., 1990. Plant response to herbivory and belowground nitrogen cycling. Ecology 71, 1040±1049. Horner, J.D., Gosz, J.R., Cates, R.G., 1988. The role of carbon based plant secondary metabolites in decomposition in terrestrial ecosystems. Am. Nat. 132, 869±883. Huntly, N., 1991. Herbivores and the dynamics of communities and ecosystems. Ann. Rev. Ecol. Sys. 22, 477±503. Jaramillo, V., Detling, J.K., 1988. Grazing history, defoliation, and competition: effects on shortgrass production and nitrogen accumulation. Ecology 69, 1599±1608. Jermyn, M.A., 1975. Increasing the sensitivity of the anthrone method for carbohydrate. Analyt. Biochem. 68, 332±335. Johnson, N.E., 1965. Reduced growth associated with infestations of Douglas-fir seedlings by Cinara species (Homoptera: Aphidae). Can. Ent. 97, 113±119. Jones, C.G., Last, F.T., 1991. Ectomycorrhizae and trees: implications for aboveground herbivory. In: Barbosa, P., Krischik, V.A., Jones, C.G. (Eds.), Microbial Mediation of PlantHerbivore Interactions, pp. 65-103. Wiley & Sons, New York. Jones, C.G., Lawton, J.H., Shachak, M., 1997. Positive and negative effects of organisms as physical ecosystem engineers. Ecology 78, 1946±1957. Manderscheid, B., Matzner, E., 1995. Spatial and temporal variation of soil solution chemistry and ion fluxes through the soil in a mature Norway spruce (Picea abies (L.) Karst.) stand. Biogeochem. 30, 99±114. Maurizio, A., 1985. Honigtau-Honigtauhonig. In: Kloft, W.J., Kunkel, H. (Eds.), Waldtracht und Waldhonig in der Imkerei. Ehrenwirth, MuÈnchen. pp. 267±295. McNaughton, S.J., Coughenour, M.B., Wallace, L.L., 1982. Interactive processes in grassland ecosystems. In: Estes, J.R.,
Tyrl, R.J., Brunken, J.R. (Eds.), Grasses and grasslands, systematics and ecology. University of Oklahoma Press, Oklahoma. pp. 167±193. Pastor, J., Stillwell, M.A., Tilman, D., 1987. Nitrogen mineralization and nitrification in four Minnesota old fields. Oecologia 71, 481±485. Pastor, J., Naiman, R.J., 1992. Selective foraging and ecosystem processes in boreal forests. Am. Nat. 139, 690±705. Pastor, J., Dewey, B., Naiman, R.J., McInnes, F.P., Cohen, Y., 1993. Moose browsing and soil fertility in the boreal forests of Isle Royale national park. Ecology 74, 467±480. Petelle, M., 1980. Aphids and melezitose: a test of Owen's 1978 hypothesis. Oikos 35, 127±128. Polley, H.W., Detling, J.K., 1988. Defoliation, nitrogen, and competition: effects of plant growth and nitrogen nutrition. Ecology 70, 721±727. Schowalter, T.D., Hargrove, W.W., Crossley Jr., D.A., 1986. Herbivory in forested systems. Ann. Rev. Entomol. 31, 177± 196. Seastedt, T.R., Crossley Jr., D.A., 1984. The influence of arthropods on ecosystems. Bioscience 34, 157±161. Sokal, R.R., Rohlf, F.J., 1995. Biometry, 3rd edn. Freeman and Company, New York. Stadler, B., MuÈller, T., 1996. Aphid honeydew and its effect on the phyllosphere microflora of Picea abies (L.) Karst. Oecologia 108, 771±776. Stadler, B., Michalzik, B., MuÈller, T., 1998. Linking aphid ecology and ecosystem processes: the effect of spatial and temporal variability in aphid abundance on nutrient fluxes in a coniferous forest. Ecology (in press). Swank, W.T., Waide, J.B., Crossley Jr., D.A., Todd, R.L., 1981. Insect defoliation enhances nitrate export from forest systems. Oecologia 51, 297±299. Tukey, H.B., 1970. The leaching of substances from plants. Ann. Rev. Plant Physiol. 21, 305±329. Yavitt, J.B., Fahey, T.J., 1985. Organic chemistry of the soil solution during snowmelt leaching in Pinus contorta forest ecosystems, Wyoming. In: Caldwell, D.E., Brierley, J.A., Brierley, C.L. (Eds.), Planetary Ecology, pp. 485±495. Van Nostrand Reinhold Company, New York. Zoebelein, G., 1954. Versuche zur Feststellung des Honigtauertrages von FichtenbestaÈnden mit Hilfe von Waldameisen. Z. Ang. Ent. 36, 358±362. ZwoÈlfer, W., 1952. Die Waldbienenweide und ihre Nutzung als forstentomologisches Problem. Verh. deut. Ges. angew. Entomol. Frankfurt (1952) 164±168.