Species-specific responses of a root- and shoot-feeding insect to arbuscular mycorrhizal colonization of its host plant

Authors

  • Alan C. Gange

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    1. School of Biological Sciences, Royal Holloway University of London, Egham Hill, Egham, Surrey TW20 0EX, UK
      Author for correspondence: Alan C. Gange Fax: +01784 470756 Email:a.gange@rhul.co.uk
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Author for correspondence: Alan C. Gange Fax: +01784 470756 Email:a.gange@rhul.co.uk

Summary

  •  The responses of root-feeding black vine weevil (Otiorhynchus sulcatus) larvae and leaf-feeding adults to colonization of strawberry (Fragaria x ananassa) plants by one or two species of arbuscular mycorrhizal fungi are reported here.
  • Glomus mosseae and Glomus fasciculatum were isolated from a commercial field and used to colonize strawberry plants, singly and in combination. Vine weevil larvae were reared on roots of colonized and uncolonized plants. When the larvae were mature, leaves from all plants, with and without larvae, were fed to adult weevils.
  •  Colonization by either fungus reduced larval survival and biomass. However, colonization by both fungi had no effect on the larvae. These effects were manifested in changes in plant performance; weevil feeding decreased plant foliar and root biomass as well as runner production, but only when mycorrhizas were absent or inoculated together. Mycorrhizas also mitigated the effects of larval feeding on adult weevils.
  •  The response of root-feeding insects to arbuscular mycorrhizal colonization depends on which fungi are present in the root system. Furthermore, arbuscular mycorrhizal fungi might play a critical role in mitigating interactions between phytophagous insects.

Introduction

The black vine weevil (Otiorhynchus sulcatus) is an unusual insect in many respects. It is extremely polyphagous in both the larval and adult stages, having been recorded as feeding on plants in 46 families (Masaki et al., 1984). Furthermore, larvae and adults can feed on the same host plant, and while these stages are not necessarily temporally separated they are spatially so, as the larvae are subterranean and feed on roots, while the wingless, all-female adults eat foliage. O. sulcatus is the only root-feeding insect whose interactions with arbuscular mycorrhizal (AM) fungi have been studied (Gange et al., 1994; Gange, 1996).

There is increasing evidence that AM fungal colonization of plant roots can affect the growth and reproduction of insects feeding on the host plant (Gange & Bower, 1997). If one considers foliar-feeding insects, then (with caution) a general pattern seems to be emerging that chewing insects are negatively affected by the presence of the mycorrhiza (e.g. Gange & West, 1994; Gange & Nice, 1997), while sucking insects appear to be positively affected (Borowicz, 1997; Gange et al., 1999a). In the only two examples of studies with a root-feeding insect (Gange et al., 1994; Gange, 1996), chewing larvae of O. sulcatus were also negatively affected by the mycorrhiza. The negative effects were quite dramatic, with a 40% reduction in the survival and size of larvae being found.

Such negative effects may clearly have important consequences for the insect in question, but they may also be of importance to other insects, feeding on the same host plant, but spatially separated from the root-feeder. It has been shown that root- and foliar-feeding insects interact in a ‘+/−’ fashion, termed contramensalism (Masters et al., 1993; Masters & Brown, 1997). Root feeding and the subsequent imposition of water stress (Gange & Brown, 1989) appear to affect the growth of foliar-feeding insects positively, while foliage removal results in reduced root growth and a decreased performance in root-feeding insects (Masters & Brown, 1997). However, this work has involved two-species situations, and the simple experiment of investigating the effects of a root-feeding larva on the performance of its foliar-feeding adult has never been attempted (Masters & Brown, 1997). In natural situations, contramensalistic relations may not be stable and could lead to the local extinction of the root-feeding insect, if foliar-feeding insect populations were high. However, the presence of a mycorrhiza, by having negative effects on both the root- and foliar-feeder, could reduce the strength of the insect–insect interaction and thus help to maintain an equilibrium situation. This is a complex problem, and one aim of this paper is to make a preliminary study by investigating whether any AM–root-feeding-insect interactions can be seen in the host acceptance of a foliar-feeding insect. The black vine weevil is ideal for this type of study, with its spatially separated larval and adult stages.

A feature of the laboratory-based insect–mycorrhizal studies to date is that, with one exception (Gange, 1996), they have involved one species of insect and one species of fungus (Gange & Bower, 1997). Field studies may be a little more realistic as they have involved the reduction of naturally occurring colonization levels by the use of fungicide (Gange & West, 1994; Gange & Nice, 1997). However, in neither of these two studies were the mycorrhizal species colonizing the roots identified. Evidence suggests that plants may be colonized by a number of different mycorrhizal species at the same time, or at different times during a season (Clapp et al., 1995; Merryweather & Fitter, 1998), and that it is unrealistic to imagine that plant root systems are colonized by one fungus at a time. In the only study to have investigated the responses of an insect to two fungi, Gange (1996) found that O. sulcatus larvae were negatively affected by Glomus mosseae and Glomus intraradices N.C. Schenck & G.S. Sm. when either colonized the roots of strawberry (Fragaria×ananassa Duch.), but inoculation with both fungi produced no effect. This was a surprising result, and it is the main aim of this paper to document whether this also happens with fungal species known to occur in a commercially planted field of strawberry.

Strawberry is a strongly mycorrhizal plant, owing to its high demand for phosphate (Dunne & Fitter, 1989), and there are several studies documenting enhanced growth and reproduction in response to mycorrhizal colonization (e.g. Vestberg, 1992; Williams et al., 1992). Furthermore, AM fungi have been shown to increase the resistance of this plant to infection of the roots by the pathogenic fungus Phytophthora fragariae Hickman (Norman et al., 1996). The black vine weevil is a major pest of strawberry in the United Kingdom (Moorhouse et al., 1992), and growers realize the urgent need for a control system that can be effective and reduce the reliance on chemicals (Anonymous, 1995). Entomopathogenic nematodes are an excellent example of one such system (e.g. Wilson et al., 1999), and AM fungi have the potential to be another, if they reduce growth and survival of larvae. However, before mycorrhizal control of this pest becomes a reality, we must determine which AM fungi are present in strawberry fields and what effects they have on both larvae and adults of the weevil.

Materials and Methods

Spore numbers in the field

A commercial strawberry field, near Mereworth, Maidstone, Kent, UK, measuring 105 m × 70 m was selected for the study. The field had been sterilized with methyl bromide and subsequently planted 3 yr earlier with cv. ‘Elsanta’, and had not received any fungicide application during this time. The bicarbonate-extractable phosphate (P) content (Allen, 1989) was 66.8 ± 5.1 µg g−1 (mean of 20 random samples). The field was sampled systematically, with transects taken across the shorter axis of the field. There were seven transects down the long axis, each 15 m apart. In each transect, a soil sample was taken from every other row of plants. Each sample was approximately 1 m apart, giving a total of 245 (7 × 35 rows) samples. Each soil sample was taken with a corer measuring 5 cm × 10 cm deep, and was positioned next to the crown of the plant, to ensure that it contained some root material. Spores of AM fungi were extracted from the soil by wet sieving and sucrose centrifugation (Brundrett et al., 1996). Identification of spores was attempted using Schenck & Perez (1990), Morton (1988, 1993) and Yao et al. (1996).

Laboratory experiment

Eighty 13-cm-diameter flower pots were each filled with 500 g of horticultural compost (J. Arthur Bowers, Sinclair Horticulture, Lincoln, UK). This medium was chosen because it provided a bicarbonate-extractable P level (62.4 ± 4.6 µg g−1) similar to that of the field soil. Every pot received 100 ml of a 45-µm filtrate of field soil to correct for the background microbial community (Koide & Li, 1989). Mycorrhizal inoculum was provided by spreading 4 g of inert clay granules containing a mixture of root fragments, spores and hyphae in a layer 4 cm below the surface. The two commonest AM species (as measured by spore extraction) in the field were Glomus mosseae (Nicol. & Gerd.) and Glomus fasciculatum (Thaxt.) Gerd. & Trappe, and commercially available single-species cultures of these two species were used (Vaminoc, MicroBio Ltd, Hemel Hempstead, UK). The infectivity of each inoculum was: G. mosseae, 396 ± 86 propagules g−1; G. fasciculatum, 486 ± 76 propagules g−1 (measured by the most probable number method). Four fungal treatments were established: controls (which received 4 g of irradiated inoculum), either fungus inoculated singly, and dual inoculation. In the last case, pots received 2 g of each species inoculum. There were 20 pots of each fungal treatment and these were placed in a randomized block arrangement in a constant-environment room, set at 20°C and 16 : 8 light : dark (L : D).

Immediately after inoculation, a single strawberry runner (cv. ‘Elsanta’) was pegged out into each pot. The plants producing the runners had been created the previous year in the same way, thus resulting in clonal material. Runners were allowed to root and become established for 10 wk before they were severed from the parent plants. At this stage, half of the pots in each fungal treatment were inoculated with 10 mature (brown) eggs of Otiorhynchus sulcatus (Fab.) (Coleoptera: Curculionidae). There were therefore 10 replicates of each of the eight (four fungal × two weevil) treatments, in a fully factorial design.

Twenty ‘spare’ plants, infested with weevil eggs but no mycorrhiza, were set up at the same time and these were used to check when the larvae were fully grown. After 10 wk, it was apparent that this was the case and at this point the final stage of the experiment commenced. Each of the 80 experimental plants was confined within a Sunbag (Sigma Chemical Co., Poole, UK) and a single adult weevil introduced on to the foliage of each plant. Adults were taken from culture and starved for 48 h prior to this experiment. Adults were left on the plants for 2 d, after which time the number of leaves upon which they had fed was recorded. Leaf area eaten was also measured by image analysis.

Plants were then harvested, the number of leaves and runners produced recorded, and the foliar parts dried to constant weight at 80°C. Roots and weevil larvae were separated from the soil medium by immersion in water. Larval number per pot and the live biomass of each were recorded. Mean larval weight per pot was calculated for analysis. A 2-g sample of roots from each plant was taken, washed free of soil and mounted in water. Slide preparations were examined at ×200 using a Zeiss Axiophot (Welwyn Garden City, UK) epifluorescence microscope to reveal arbuscules (Gange et al., 1999b). Colonization levels were assessed using the cross-hair eyepiece intersection method of McGonigle et al. (1990). Approx. 200 intersections per slide were recorded, to give a measure of percentage root length colonized (%RLC). Total root dry biomass was estimated by weighing the main part of the root system and correcting for the 2-g sample loss in each case. The P content of dry foliage of each plant was measured by the molybdenum blue method, following acid digestion (Allen, 1989).

Statistical analysis

All data sets were tested for normality and homogeneity of variances. Percentage data (%RLC, percentage weevil survival and percentage of leaves attacked by the adult weevils) were subjected to the angular transformation prior to analysis (Zar, 1996). Count data (number of leaves and runners per plant) were subjected to square root transformation. Plant performance parameters and attack by adult O. sulcatus were analysed by three-way factorial ANOVA, employing G. mosseae, G. fasciculatum and O. sulcatus larvae as main effects, with the UNISTAT® statistical package (London, UK). Weevil growth and survival were analysed by two-factor ANOVA with each fungal species as the main effect.

Results

Spore numbers in the field

The spore populations that could be identified were dominated by G. mosseae and G. fasciculatum, with 91% of samples containing either or both of these two species. Morisita’s Index of Dispersion (Elliott, 1993) was calculated as 3.37 for G. mosseae and 5.42 for G. fasciculatum, indicating highly clumped distributions. Overall, G. mosseae accounted for 63% of all spores identified and G. fasciculatum accounted for 15%, while 15% of spores could not be identified to species with certainty. As 91% of all samples contained G. mosseae and/or G. fasciculatum, these two species of fungi were chosen for the laboratory trial.

Laboratory experiment

No colonization was detected in any of the 20 plants that received irradiated inoculum. There was no effect (Fig. 1) of weevil larvae on colonization (F1,54 = 0.67, P > 0.05). Addition of both species did not alter colonization levels from those achieved by either of the single inoculations.

Figure 1.

Colonization of strawberry roots, measured as percentage root length colonized (%RLC) (arbuscules only). Values are means ± one standard error. Open bars, no weevil herbivory; shaded bars, weevils present.

Colonization by G. mosseae elevated leaf P content (F1,72 = 11.99, P < 0.001), as did colonization by G. fasciculatum (F1,72 = 57.45, P < 0.001) (Fig. 2). However, the P content of leaves on plants colonized by both species was not higher than that of either single inoculation, leading to a significant interaction term (F1,72 = 37.96, P < 0.001). Weevil larvae had no effect on plant P levels (Fig. 2).

Figure 2.

P concentration of leaf material per plant in each fungal and weevil treatment. Values are means ± one standard error. Open bars, no weevil herbivory; shaded bars, weevils present.

The presence of either species of fungus had a significant negative effect on the survival and final size of weevil larvae (Table 1, Fig. 3a). However, of more interest was the strong interaction between the fungi. Addition of both species appeared to counteract the negative effect of single inoculation, so that survival in this treatment was not different from that in the control (Fig. 3a). There was also an interaction between the fungi in their effect on larval biomass (Table 3, Fig. 3b). Each species when inoculated alone reduced plant biomass, but in the dual-inoculation treatment, biomass was the same as that in the control treatment (Fig. 3b).

Table 1.  Summary of results from ANOVA, testing for the effects of mycorrhizal colonization by Glomus mosseae and/or Glomus fasciculatum on the survival and growth of larvae of Otiorhynchus sulcatus
 SurvivalLarval weight
 FPFP
  1. Degrees of freedom for all F-values: 1, 36.

G. mosseae (M) 4.78< 0.0511.29< 0.01
G. fasciculatum (F) 6.01< 0.05 4.77< 0.05
M × F76.48< 0.00161.65< 0.001
Figure 3.

Effect of arbuscular mycorrhizal colonization on weevil larval performance. Values are means ± one standard error. (a) Mean larval survival. (b) Mean larval biomass.

Table 3.  Summary of results from ANOVA, testing for the effects of feeding by Otiorhynchus sulcatus larvae and mycorrhizal colonization by Glomus mosseae and Glomus fasciculatum on percentage of leaves attacked and leaf area eaten by adult O. sulcatus
 Percent leaves attackedLeaf area eaten
 FPFP
  1. Degrees of freedom for all F-values: 1, 72. n.s, not significant at P = 0.05.

O. sulcatus (W)2.75n.s.15.73< 0.001
G. mosseae (M)0.05n.s. 0.03n.s.
G. fasciculatum (F)0.03n.s.22.29< 0.001
W × M0.32n.s. 0.11n.s.
W × F0.01n.s. 1.38n.s.
M × F0.22n.s.67.09< 0.001
W × M × F0.01n.s. 6.86< 0.05

Larval feeding reduced foliar biomass (Fig. 4a), runner production (Fig. 4b) and dry root biomass (Fig. 4c) (Table 2). Inoculation with G. mosseae increased foliar and root biomass, but had no effect on runner production (Table 2). Inoculation with G. fasciculatum increased all three plant parameters (Table 2). Interactions were found between the two fungal species in their effects on both foliar and root biomass. In both cases this was because single inoculations of either species, irrespective of weevil presence, significantly increased biomass, but when both fungi were present no extra increase was seen. This was entirely due to the three-way interactions between the two fungi and the weevil (Table 2). Thus single inoculations of fungi resulted in lower weevil survival and consequent higher plant biomass (Fig. 4a,c). However, if both fungi were present there was no effect on weevil survival, and consequently biomass of plants in this treatment was similar to that in the treatments where the weevil was present but the fungi were absent. In summary, colonization by either fungus alone reduced weevil performance and its effect on the host plant, but the negative effects on the insect disappeared when both fungi were present.

Figure 4.

Effect of arbuscular mycorrhizal colonization and feeding by vine weevil larvae on strawberry growth. Values are means ± one standard error. (a) Mean dry foliar biomass. (b) Mean number of runners produced per plant. (c) Mean dry root biomass. Open bars, no weevil herbivory; shaded bars, weevils present.

Table 2.  Summary of results from ANOVA, testing for the effects of feeding by Otiorhynchus sulcatus larvae and mycorrhizal colonization by Glomus mosseae and Glomus fasciculatum on plant dry foliar biomass, runner production and root dry biomass
 Foliar biomassRunnersRoot biomass
 FPFPFP
  1. Degrees of freedom for all F-values: 1, 72. n.s., not significant at P = 0.05.

O. sulcatus (W)45.68< 0.00120.01< 0.00129.26< 0.001
G. mosseae (M) 4.09< 0.05 0.67n.s. 3.99< 0.05
G. fasciculatum (F) 9.64< 0.01 7.99< 0.01 5.15< 0.05
W × M 0.02n.s. 4.55< 0.05 0.24n.s.
W × F 1.22n.s. 0.37n.s. 1.01n.s.
M × F88.82< 0.001 1.49n.s.22.19< 0.001
W × M × F15.14< 0.001 2.38n.s. 6.22< 0.05

Neither fungus nor the presence of O. sulcatus larvae had an effect on the percentage of leaves attacked by adult weevils (Table 3). However, the amount of leaf material consumed by adult O. sulcatus was significantly increased if larvae of this species were also feeding on the plant (Table 3, Fig. 5). Colonization by G. fasciculatum significantly reduced the amount of leaf material consumed. There was a strong interaction between the two fungal species; irrespective of weevil presence, colonization by either fungus alone decreased the amount eaten, while colonization by both species had no effect. Therefore the effects were similar to those seen on the root-feeding larvae; the presence of both fungi cancelled out the negative effects on the insect elicited by either fungus alone. Finally, there was also an interaction between G. mosseae, G. fasciculatum and O. sulcatus larvae. This was because when either fungus was present alone, larval feeding had no effect on that of the adult weevil. However, when both fungi were present together, the presence of larvae increased the amount of leaf eaten by the adult weevils.

Figure 5.

Effect of arbuscular mycorrhizal colonization and feeding by vine weevil larvae on leaf area consumption by adult vine weevils, over a 2-d period. Values are means ± one standard error. Open bars, no weevil herbivory; shaded bars, weevils present.

Discussion

Both AM fungi reduced the performance of root-feeding larvae. In the control treatment, about 50% of the eggs produced full-grown larvae, but in either fungal treatment this was reduced to about 12%. Furthermore, larval biomass was effectively halved by the presence of either fungal species. The effect of G. mosseae on O. sulcatus larvae is similar to that previously reported (Gange et al., 1994; Gange, 1996), but this is the first record of a similar effect being caused by G. fasciculatum.

The mechanism by which AM fungi can negatively affect rhizophagous insect herbivores is unknown. The three possible explanations are a physical, nutritional and/or a chemical effect. It is believed that root-feeding insects may be limited more by the quantity of root available for consumption than by its quality. This is because roots are nutritionally poor and are generally less well defended by secondary metabolites than foliar tissues (McKey, 1979; Brown & Gange, 1990). However, mycorrhizal fungi usually stimulate root production, as found in this study and by Norman et al. (1996) who reported that G. fasciculatum increased root biomass of strawberry. If the quantity of root was limiting to the insect, one might expect a positive effect of the fungi on the insect, which clearly did not occur. While it is accepted that root quantity may not have been limiting in this study as the larvae were confined in pots, it is also likely that any increase in root biomass in a field situation would have little effect on O. sulcatus. This is because the larvae are mobile and polyphagous (Masaki et al., 1984) so they are unlikely ever to be short of food.

A nutritional explanation based on food quality is unlikely, because of the lack of response of rhizophagous insects to changes in root quality (Brown & Gange, 1990). Furthermore, mycorrhizal-induced changes in plant P content were not responsible for the effects seen in this experiment. Because the study was conducted in a soil of high P availability, designed to mimic the field situation, treatments consisting of low and high P were not included in the design. The AM fungi successfully colonized the plants and elevated leaf P, even though soil P was high. This result was not unexpected, as Werner et al. (1990) obtained high AM colonization of strawberry in even higher soil P conditions, attributable to the exceptionally high demand for P by this species (Dunne & Fitter, 1989). Critically in the current study, the P content of plants inoculated with both fungi was not different from that of either single inoculation, yet the performance of the larvae differed between dual and single fungal treatments. The increased performance of weevil larvae on plants without AM fungi and on those with both species cannot therefore be attributed to a difference in P content, relative to the singly inoculated plants.

Colonization by mycorrhizal fungi may result in chemical changes in roots, and it is the alteration of root biochemistry that provides the most likely explanation for the detrimental effects on the larvae. A wide variety of chemicals may be present in mycorrhizal roots that could have activity against insect herbivores. Some examples are phenolics (Morandi, 1996), terpenoids (Maier et al., 1995; Peipp et al., 1997) and isoflavonoids (Vierheilig et al., 1998). All of these compounds have been shown to exhibit activity against phytophagous insects (Mullin et al., 1991; Dakora, 1995). Mycorrhizal fungi are well known as bioprotectants of roots against soil-borne plant pathogens (Azcón-Aguilar & Barea, 1996), and the defences in roots elicited by mycorrhizas against plant pathogen invasion could equally have activity against insects.

In the experiment reported by Gange (1996), an interaction was found between G. mosseae and G. intraradices in that dual inoculation of these species appeared to counteract the negative effect seen with either fungus alone. It is interesting that the same effect has been found in this study. An extensive body of literature exists on competition between mycorrhizal fungi for colonization of the root and the consequences for the host plant (Wilson & Tommerup, 1992). The outcome of any interaction depends on the particular combination of fungal species used, but, in general, multiple inocula often fail to result in colonization above that of a single inoculation or to provide synergistic effects on host plant growth (Hepper et al., 1988; Pearson et al., 1994). In the current study, interactions were recorded between the two fungal species, as either alone significantly increased foliar and root biomass, but the addition of both fungi resulted in no further increase. Colonization levels in the dual-inoculation treatment were equal to, or lower than, those obtained in either single inoculation. Furthermore, it appears that if any chemical changes are elicited by either fungus when alone, these disappear when both fungi are present. It is therefore possible that insects such as O. sulcatus could be extremely useful bioassays of chemical changes in host plants in future studies of the interactions between mycorrhizal species. The measurement of colonization by each fungal species in the dual treatment was beyond the scope of this study, but, as molecular methods become widely available, exciting opportunities will open up for studying the consequences of different mycorrhizal combinations on insect herbivores. We know that, naturally, roots can be colonized by a number of mycorrhizal fungi (Clapp et al., 1995), and these methods will enable laboratory experiments to become more realistic mimics of field situations.

In virtually all field situations, root-feeding insects display highly aggregated spatial distributions, often conforming to the negative binomial (Brown & Gange, 1990). Previous explanations have included the oviposition behaviour of the adult, plant preferences and the heterogeneous nature of the soil environment (Brown & Gange, 1990). The data reported here indicate that another reason for these aggregated distributions may be the occurrence of mycorrhizal fungi. Mycorrhizal populations are also aggregated in field soils (Brundrett, 1991), and the spore densities found here were no exception. Therefore, if weevil eggs are deposited near plants that are colonized by either of these two fungi, subsequent larval survival will be much less than if the eggs are deposited near plants with neither or both fungi. Aggregated mycorrhizal distributions, coupled with this differential survival, could then lead to an aggregated distribution of larvae, commonly seen in field situations (e.g. Hanula, 1993). If the spore populations recorded are an indication of colonization of the roots, then it can be suggested that, in 76% of the plants sampled (which had spores of only one of the species used in the experiment), larval survival and growth would be less than on the 21% of plants that had both species or no spores at all.

The nature and quality of the host plant have a major effect on adult oviposition in O. sulcatus (Nielson & Dunlap, 1981). The data reported here suggested that plants that were colonized by either of the two fungi resulted in less leaf material being consumed, implying a degree of mycorrhizal-induced resistance, confirming that generalist chewing insects appear to be negatively affected by the presence of mycorrhiza (Gange & Bower, 1997). It was interesting that the amount of leaf eaten by adults was positively affected by the presence of the larvae. Similar interactions between a root- and a shoot-feeding insect have been reported before (Masters & Brown, 1997), but these involved two species, rather than adults and larvae of the same species. The positive effects of a rhizophagous insect on a foliar-feeding one have generally been attributed to drought stress imposed by root removal and the subsequent improvement of foliar food quality through soluble nitrogen and carbohydrate mobilization (Masters & Brown, 1997). One amino acid that has been cited as being instrumental in this interaction is proline, which acts as a phagostimulant for some insects (Gange & Brown, 1989). However, there is an extensive body of literature on how mycorrhizal fungi, such as G. fasciculatum, can alleviate drought stress in plants (e.g. Aguilera-Gomez et al., 1998), and it has also been shown that colonization of roots by G. mosseae and G. fasciculatum can lead to decreases in proline levels (Ruiz-Lozano & Azcón, 1997). Therefore the mechanism by which root-feeding insects affect foliar-feeding insects may be reversed by the presence of mycorrhiza. Furthermore, as the mycorrhiza has been shown to have a negative effect on the root feeder, this will help to lessen the impact on the foliar-feeding species. Patchy distribution or co-occurrence of mycorrhizal fungi may thus serve to introduce variation into experiments designed to investigate the insect–insect interactions (e.g. Masters & Brown, 1992). To an extent, these interactions between insect and fungi were seen in the experiment with adult O. sulcatus feeding. This was designed as a preliminary study and could not be continued for longer, as adult feeding may have then affected performance of the larvae (Masters & Brown, 1997) and colonization by the mycorrhiza (Gange & Bower, 1997). However, a three-way interaction between the two fungi and the insect was observed, because adult feeding was affected by larval presence only when both fungi were present and not when either was inoculated singly. Competition between the fungi therefore not only resulted in a lessening of the detrimental effect on the larvae, but also reduced the effect that these larvae had on adult feeding. Clearly, a more detailed experiment is required to determine the exact nature of the interactions in a four-species system such as this.

The simple experiment reported here has resulted in some complex outcomes, and suggests that understanding the interactions between insect herbivores and mycorrhizal fungi in field situations will be far from easy. Factors to be considered must include the species identity of the fungi involved and their relative effects on insects. Nevertheless, these data do suggest that the contramensalistic relation between spatially separated herbivores outlined by Masters et al. (1993) may be stabilized by the presence of mycorrhizal fungi, leading to the coexistence of the species in natural communities.

Acknowledgements

I am grateful to Hugh Lowe Farms Ltd for permission to sample in their field. Mycorrhizal inoculum was generously provided by MicroBio Ltd, Hemel Hempstead, UK.

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