Reproductive significance of feeding on saprobic and arbuscular mycorrhizal fungi by the collembolan, Folsomia candida

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1 Functional Ecology 1999 ORIGINAL ARTICLE OA 000 EN Reproductive significance of feeding on saprobic and arbuscular mycorrhizal fungi by the collembolan, Folsomia candida J. N. KLIRONOMOS, E. M. BEDNARCZUK and J. NEVILLE Department of Botany, University of Guelph, Guelph, Ont., Canada N1G 2W1 Summary 1. Collembolans have often been credited with negatively affecting arbuscular mycorrhizal (AM) symbioses, mainly by grazing and severing the associated external fungal network from host roots. However, most previous experiments were performed using relatively clean systems where other, non-mycorrhizal, fungi were largely excluded. Yet, plant rhizospheres harbour a wide variety of highly palatable non-am fungi, most of which have saprobic lifestyles. 2. In this study we isolated and cultured several rhizosphere fungi, and the collembolan, Folsomia candida, from the Long-Term Mycorrhiza Research Site, University of Guelph, Canada, to test the hypothesis that, given a choice, collembolans would prefer to feed on saprobic fungi and that such a choice is of adaptive significance to the animals. 3. A laboratory food preference experiment revealed that F. candida favours common saprobic fungi over a variety of AM fungi. Coincidentally, fecundity levels across two Folsomia generations were higher when animals fed exclusively on the preferred fungus, Alternaria alternata. When fed less palatable fungi, fecundity was greatly reduced; in fact animals from the F1 generation were unable to produce any eggs when placed on an exclusive diet of one of the following three AM fungi, Acaulospora spinosa, Scutellospora calospora and Gigaspora gigantea. 4. These results indicate that a strict diet of AM fungi by collembolans has reproductive consequences. Therefore, we propose that under natural conditions these animals spend more time feeding on common saprobic fungi rather than their AM counterparts. This suggests that previous clean studies that investigated the interactions between collembolans and AM fungi may have reported exaggerated effects of animal grazing. The influence of collembolans on the functioning of AM symbioses, under more natural conditions, remains not well understood. Key-words: Arbuscular mycorrhiza, collembola, optimal foraging theory, soil fungi Functional Ecology (1999) Ecological Society Introduction Arbuscular mycorrhizal (AM) fungi provide plants with mineral nutrients, improved water uptake, as well as protection from pathogenic fungi, in exchange for photosynthates (Allen 1991; Newsham, Fitter & Watkinson 1995). These symbiotic fungi are common in the soil and are important to the development and maintenance of plant communities (Grime et al. 1987; van der Heijden et al. 1998). Although these fungi are generally considered to be mutualists, they can function anywhere along a mutualism parasitism continuum (Johnson, Graham & Smith 1997). The nature and strength of the interaction can be influenced by many factors, including grazing of the extra-radical mycelium by soil fauna (Fitter & Sanders 1992). Proper functioning of the symbiosis depends on an intact extra-radical mycelium (Friese & Allen 1991) and grazing by mycophagous animals can sever parts or the entire structure from the associated root system. This could push the symbiosis further towards the parasitic side of mutualism parasitism continuum (Johnson et al. 1997). Collembolans (Class Insecta) are largely mycophagous, ubiquitous soil animals, with densities greater than individuals m 2 (Lussenhop 1992; Klironomos & Kendrick 1995a). Through their grazing activities, they can influence litter decomposition rates, nutrient cycling, the distribution of fungal inocula, and plant growth and development (Visser 1985). With regards to grazing on AM fungi, there is ample evidence that they can influence the nature of the symbiosis. Warnock, Fitter & Usher (1982) found 756

2 757 Palatability of mycorrhizal fungi Ecological Society, Functional Ecology, that Folsomia candida decreased the growth of Allium porrum inoculated with an AM fungus by feeding on the extra-radical hyphae. Finlay (1985), McGonigle & Fitter (1988) and Harris & Boerner (1990) reported that decreasing collembolan densities increased both yield and phosphorus inflow to roots, further suggesting that the animals fed extensively on AM fungi. Collembolans were also shown to reduce AM fungal infection when present at the time of soya-bean planting (Kaiser & Lussenhop 1991). One aspect that these studies did not consider, however, is the coexistence of other, non-mycorrhizal, fungi in soil. Saprobic and parasitic fungi are also common in the rhizosphere, and have been shown to be highly palatable to collembolans (Fitter & Sanders 1992). Numerous experiments have demonstrated a food-preference hierarchy for collembolans; they tend to prefer hyphae and spores from members of the Division Dikaryomycota, in particular conidial taxa that are darkly pigmented, such as Cladosporium, Alternaria and Epicoccum (Mills & Sinha 1970; Visser & Whitakker 1977; Shaw 1988). Moore, St John & Coleman (1985) showed that while AM fungal hyphae and spores were used as food sources by soil animals, the hyphae were severed rather than entirely ingested. Furthermore, the AM fungus, Glomus macrocarpum, was found to be unpalatable to many soil animals in comparison to the saprobic, Alternaria alternata and Trichoderma harzianum (Klironomos & Kendrick 1996), and when both, AM and saprobic fungi, were introduced in microcosms plant and mycorrhizal growth and development was typically stimulated or unaffected by soil animals (Klironomos & Kendrick 1995b; Larsen & Jakobsen 1996; Lussenhop 1996). Together, these studies suggest that collembola can graze on AM fungi, but that saprobic fungi are more palatable, and they will avoid AM fungi when a variety of fungal groups are available. However, only one AM fungus has been tested for relative palatability (Klironomos & Kendrick 1996). It is unknown if this selection pattern is consistent across a community of AM fungi and, more important, whether the preference for conidial fungi over AM fungi is an adaptive behaviour by collembolans. In accordance with the optimal foraging model, Collembola should feed on the most energetically rewarding food source, one that will yield the greatest reproductive success (Stephens & Krebs 1986). If there is an abundance of food types to choose from, as is the case with soil fungi, then only the most reproductively profitable foods should be selected. This profitability is still to be determined for AM fungi and compared to that of conidial fungi as it applies to collembolan fitness. The purpose of this study was to test the hypothesis that the collembolan, F. candida, would find conidial fungi more palatable over a range of AM fungi and that this preference has evolved as an adaptation to maximize reproductive success. Materials and methods STUDY SITE AND ORGANISMS All organisms were isolated from the Long-Term Mycorrhiza Research Site (LTMRS), established in 1996 within a 3 ha portion of the Nature Reserve at the University of Guelph Arboretum. The site is located at ' 30 N and ' 00 W and is 335 m above sea level. The soil is a London loam (c. 50% sand, 35% silt, 15% clay) of shallow to moderate depth with a relatively stone-free top-soil with a seasonably high water table. The site was originally farmed but has not been disturbed since at least It is now considered an old-field meadow, dominated by perennial grasses, asters and goldenrods. Microarthropods were extracted from the soil using a Macfadyen funnel (Edwards 1991). Numerous species were found but only the collembolan, F. candida, was successfully cultured in the laboratory. This species was the third most abundant collembolan in the soil, representing 21% of the total extracted population of collembolans at this site. It reaches average yearly densities of c individuals m 2 and with a peak density exceeding individuals m 2 in early autumn. Folsomia candida was chosen for this study because it is predominantly mycophagous and it has been shown to feed on AM fungi. We maintained this species in small jars containing a 25:1 plaster of Paris/charcoal mixture. The substrate was kept moist and the animals were fed baker s yeast on a weekly basis. For AM fungal extraction, soil was collected from various locations within the LTMRS using a 2 cmdiameter corer. The soil was placed in plastic bags and stored at 4 C for a maximum of 2 weeks. AM fungi were isolated from the soil using trap cultures (Brundrett, Melville & Peterson 1994). A band of soil was placed between two layers of Turface within c. 25 cm pots. Allium porrum was used as the bait plant. After 2 months of growth, the pots were cored, AM fungal spores were extracted and identified, and new pot cultures were initiated using single spores. AM fungal spores were extracted using the method which is outlined in Klironomos & Kendrick (1995a) with a modification. Each soil sample was placed in a blender with 250 cm 3 of distilled water and mixed at medium speed for 1 5 min. The soil solution was then sieved through a series of sieves (2000, 500, 250, 45 µm) into a large plastic bin with a high pressure hose. Soil retained on the 45 µm sieve was decanted with 140 cm 3 of distilled water into a 150 cm 3 beaker. This soil solution was left in the in the beaker for at least 4 h to allow all of the soil debris to settle to the bottom of the beaker while the spores remained at the surface of the water. The spore suspension was then decanted through 1 2 µm gridded (3 mm 3 mm) nitrocellulose filter paper. This procedure yielded only a few fungal species, so the original A. porrum shoots were harvested and pots were seeded again,

3 758 J. N. Klironomos et al. and the entire procedure was repeated three times. By the end of this procedure, 23 fungal species (listed in van der Heijden et al. 1998) were isolated and were kept in dual pot culture with A. porrum under greenhouse conditions but only the following six were used in this study: Acaulospora denticulata, Acaulospora spinosa, Gigaspora gigantea, Glomus etunicatum, Glomus intraradices and Scutellospora calospora. For non-am rhizosphere fungi, the serial washing technique (Harley & Waid 1955) was used before planting 2 mm root fragments of Plantago lanceolata, Solidago canadensis and Aster novae-angliae on 2% malt extract agar (MEA), with 50 µg cm 3 Rose Bengal added (Bragulat et al. 1991). The most commonly isolated taxa, in order of frequency, were species of Penicillium, Trichoderma, Acremonium, Gliocladium, Cladosporium, Alternaria, Mucor, Fusarium, a sterile dark morphotype, and Verticillium. The fungi were mantained on MEA under 4 C. The two conidial species, A. alternata and T. harzianum were chosen for this study because they were associated with all three plants and, furthermore, because their palatability to F. candida is known (Klironomos & Kendrick 1996). Alternaria alternata is highly palatable, whereas T. harzianum is relatively unpalatable and typically avoided as a food source. Because these species represent extremes in the palatability continuum for F. candida, they are good reference points for the study of feeding on AM fungi. fungal species listed above. In this experiment, the fungal species were kept separate to test their relative value as a food source. For inoculations, a 2 g root inoculum containing hyphae and spores of the desired fungus was placed directly below the pre-germinated seed. After 8 weeks of growth, all plant roots were well colonized, and extensive extra-radical mycelium was visible. Control treatments received a sterile root inoculum. At this time, 20 1 week-old animals, which had been starved from birth, were added to the pouches. Twenty-eight days later, the animals were removed with a fine brush and the number of eggs produced was recorded. This period was calculated as the time it took for the collembolans to mature from the first day of feeding to the first day of egg laying plus 7 days. After the animals were removed, the eggs were allowed an extra 2 weeks and the number of hatched eggs was recorded. Each fungus treatment was replicated 10 times, for a total of 90 growth pouches. All newly hatched animals (F1 generation) from each of the above growth pouches, were removed with a fine brush and placed in a fresh growth pouch with the same fungal food source and the entire experiment was repeated as described above. The only difference was that the number of animals in each pouch was not necessarily 20 but rather the number of F1 animals that hatched. Ecological Society, Functional Ecology, FOOD-PREFERENCE EXPERIMENT Petri dishes (85 mm 15 mm) were filled with a 25:1 plaster of Paris/charcoal mixture. Each Petri dish contained a 3 3 grid of food stations, placed 2 cm apart. Each food station consisted of three 0 5 cm-long root fragments of P. lanceolata inoculated with one of eight fungal species or an uninoculated control. All fungal species were present in each Petri dish. For inoculations, a 2 g root inoculum containing hyphae and spores of the desired fungus was placed directly below the pre-germinated seed. After 8 weeks of growth, all plant roots were well colonized and extensive extra-radical mycelium was visible. The three root fragments were taken from these roots. Nine Latin-square designs (Moore, Ingham & Coleman 1987) were used, each replicated three times, resulting in 27 treatment replications. Twenty 1 week-old animals which had been starved from birth were added to each dish and, 48 h later, a faecal count was performed within a 0 5 cm radius around each root station. FECUNDITY EXPERIMENT One P. lanceolata seedling was grown from seed in sterile, soil-free, growth pouches (Peterson & Chakravarty 1991). Prior to planting, seeds were surface sterilized with 35% hydrogen peroxide for 10 min. The plants were inoculated with one of eight STATISTICAL ANALYSES The design for the food-preference experiment was a modification of the replicated Latin square described by Moore et al. (1987). Each design comprised different rows and similar columns. Designs were considered fixed effects. The fecundity experiment was established as a completely randomized design. Analysis of variance was used to test design and treatment effects. The Tukey post-hoc test was used on significant treatment F ratios following analysis of variance. Results FOOD-PREFERENCE EXPERIMENT The collembolan, F. candida, was found to be a selective feeder (Fig. 1). The animals deposited significantly (F 8,136 = 53 14; P = ) more faecal pellets around the conidial fungal food sources, than around any of the AM fungi or the root control. The number of faecal pellets deposited around the six AM fungal food sources did not differ significantly from each other or from that of the root control. However, of the two conidial fungi, A. alternata seemed to be preferred. Twice as many faecal pellets were found around stations with A. alternata than T. harzianum. Significant design effects (F 8,17 = 0 77; P = 0 636) or design food interactions (F 64,136 = 0 75; P = 0 90) were not detected.

4 REPRODUCTION EXPERIMENT Generation one Fig. 1. Results of the food preference experiment. Average number of faecal pellets produced by 20 collembolans after 48 h. Different letters represent differences at the significance level P < Error bars represent 1 SE. RC, root control; GG, Gigaspora gigantea; SC, Scutellospora calospora; AS, Acaulospora spinosa; AD, Acaulospora denticulata; GE, Glomus etunicatum; GI, Glomus intraradices; TH, Trichoderma harzianum; AA, Alternaria alternata. The number of eggs laid differed significantly with the fungal food source provided in the growth pouches (F 8,81 = 15 68; P = ; Fig. 2). The animals produced more eggs when fed the conidial fungus, A. alternata. A diet of T. harzianum, however, did not result in an egg production that was significantly different from that achieved on the AM fungi but it was significantly greater than that of the root control diet. Among the AM fungi, G. intraradices and G. etunicatum diets yielded a significantly higher egg production than S. calospora and G. gigantea. Furthermore, the total number of eggs that hatched also differed significantly with food source (F 8,81 = 10 40; P = ; Fig. 2). A diet of A. alternata resulted in the highest number of eggs hatched but a diet of T. harzianum did not yield a significantly greater number of hatchings than any of the remaining diets. More eggs hatched on a diet of G. etunicatum, than that of S. calospora, G. gigantea and the root control. No significant diet effect was found on the proportion of eggs hatched (F 8,55 = 0 90; P = 0 519; Fig. 3). Generation two Again, the total number of eggs laid (F 8,81 = 12 75; P = ; Fig. 2) and the number of eggs hatched (F 8,81 = 12 14; P = ; Fig. 2) differed significantly with food source. The animals raised on a diet of A. alternata produced the highest number of eggs, of which the highest number also hatched. All the other food sources yielded an egg production and hatching not significantly different from zero. Percentage hatching also differed with food source (F 4,29 = 5 28; P = 0 003; Fig. 3). Feeding on a diet of A. alternata resulted in a higher rate of hatching than G. etunicatum but it did not differ from those achieved on T. harzianum, G. intraradices and A. denticulata. Discussion Fig. 2. Number of eggs produced and hatched in the fecundity experiment by (a) 20 parents and (b) the F1 generation over 28 days on specific diets. Different letters represent differences at the significance level P < Error bars represent 1 SE. RC, root control; GG, Gigaspora gigantea; SC, Scutellospora calospora; AS, Acaulospora spinosa; AD, Acaulospora denticulata; GE, Glomus etunicatum; GI, Glomus intraradices; TH, Trichoderma harzianum; AA, Alternaria alternata. These results clearly show that the collembolan, F. candida, is a selective feeder and prefers to graze on conidial fungi over a range of AM fungal species. Folsomia candida, and other collembolans and mites, have been previously shown to display selective feeding, but the vast majority of studies have focused on saprobic members of the Dikaryomycota. The division Zygomycota and, in particular, members of the order Glomales, all of which form AM have been largely ignored. Yet, AM fungi are dominant in most soils, and coexist with soil microarthropods. The one food-preference experiment that included AM fungi (Klironomos & Kendrick 1996) only included one species, G. macrocarpum. It was found to be relatively unpalatable and the present study shows that result to be of broader significance.

5 760 J. N. Klironomos et al. It is not known why AM fungi are unpalatable to F. candida. In a previous study (Klironomos & Kendrick 1996) several collembolan and mite species were attracted to thin hyphal segments (< 5 µm in diameter) and generally avoided coarser hyphae (> 10 µm in diameter). This could be a possible explanation, because a large proportion of the AM hyphal network is coarser that 10 µm, containing thick and multi-layered cell walls. Unlike other fungal groups, AM fungi require a long-lived and intact external mycelium to translocate inorganic nutrients to the host plant efficiently and this hyphal thickness may be an adaptation by the fungi to protect the mycelial network from getting severed. Alternatively, these fungi may contain low concentrations of essential nutrients, such as nitrogen, or they may produce chemical antifeedants. To date, studies have not been performed to evaluate either of these possibilities. Nevertheless, regardless of the mechanism, the present results are not consistent with the hypothesis that soil microarthropods actively graze on the external AM mycelium and are a detriment to mycorrhizal functioning. In light of the present evidence, this needs to be tested in the presence of a more realistic diversity of fungal foods on which the animals can graze. Past studies have possibly exaggerated the negative impacts of collembolan grazing on the AM symbiosis by omitting saprobic and pathogenic groups and thus forcing the insects to feed on AM fungi (Warnock et al. 1982; Finlay 1985; Harris & Boerner 1990). The food preferences shown here may be of adaptive significance to the animals. The preferred foods were also the ones that increased reproductive success. Some exceptions exist: two of the least palatable Fig. 3. Percentage of eggs hatched in the fecundity experiment by parent and F1 generation collembolans on specific diets. Different letters represent differences at the significance level P < Error bars represent 1 SE. RC, root control; GG, Gigaspora gigantea; SC, Scutellospora calospora; AS, Acaulospora spinosa; AD, Acaulospora denticulata; GE, Glomus etunicatum; GI, Glomus intraradices; TH, Trichoderma harzianum; AA, Alternaria alternata. AM fungi, G. intraradices and G. etunicatum, were as profitable as the more favoured conidial fungus, T. harzianum. Still, we would expect collembola to feed primarily on the most beneficial fungi. All optimal foraging models predict that the most profitable prey should be included in the diet (Stephens & Krebs 1986) but evidence for links between diet and fitness are extremely rare. This is because optimal foraging models have typically been tested with mammals and it is difficult to raise such animals on single food sources for multiple generations. 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