Introduction

Exotic eucalypts and pines are important plantation species in south China, where the climate is conducive to high growth rates. Due to a large domestic demand for fuelwood, paper pulp, poles, and sawn timber in China, the plantation area of these fast-growing species is continuing to expand. Eucalyptus species were first introduced to south China for large-scale plantations in the 1950s (Qi 2002). Widely planted species in the region include Eucalyptus citriodora, Eucalyptus camaldulensis, Eucalyptus globulus, Eucaplytus grandis, Eucaplytus maidenii, Eucaplytus robusta, Eucaplytus tereticornis and Eucaplytus urophylla. Among them, E. globulus, E. urophylla, and a hybrid known as Eucalyptus leizhou No. 1 are now the main plantation eucalypts in China, and the plantation area is over 1.5 million ha. In addition to eucalypts, some commercial pine species have been introduced to south China, such as Pinus elliottii, Pinus caribaea, and Pinus taeda. It is estimated that there are over 150,000 ha of exotic pine plantations in south China (Jiang and Lu 2000). However, productivity of exotic eucalypts and pines in south China is relatively low compared to plantation productivity outside China and is variable depending on site and silvicultural inputs (Jiang and Lu 2000; Xu et al. 2000). Impediments to productivity include abiotic (e.g., infertile soil and alkaline soil) and biotic (e.g., pathogenic bacteria and fungi) factors (Xu et al. 2000; Hardy et al. 2003).

Inoculating Eucalyptus and Pinus with compatible ectomycorrhizal (ECM) fungi has been shown to be beneficial in many parts of the world (Marx et al. 1985; Thomson et al. 1996; Duñabeitia et al. 2004). It has been suggested that ECM fungi can help improve productivity of eucalypt plantations in China (Chen et al. 2000c; Dell et al. 2002). An early introduction of three exotic pine species in Guangdong Province failed in 1974, and subsequent success in re-establishing plantations with mycorrhizal seedlings suggests that the failure was due to lack of compatible ECM fungi in the soil (Ge and Bi 1989). A number of ECM fungi, collected from under Eucalyptus in Australia, have been introduced in research trials into eucalypt plantations in south China to screen compatible fungi with exotic eucalypt species (Dell and Malajczuk 1997; Chen et al. 2000c; Brundrett et al. 2005). Two genera favored for introduction are Pisolithus and Scleroderma because they readily colonize eucalypt roots in disturbed habitats (Lu et al. 1998). At present, spores of Pisolithus collected from the field in south China are being used to inoculate clonal eucalypts in a commercial nursery. It is also easy to collect spores of Scleroderma from species that form large epigeous basidiocarps and then to produce spore inoculum for nursery inoculation programs. However, Scleroderma spores are not being used commercially in China because of a lack of knowledge regarding its host range and compatibility with the main plantation species. This fungal genus has potential for application in commercial plantation forests in the region, as some beneficial isolates can aggressively compete with other ECM fungi in the field (Garbaye et al. 1988; Hall et al. 1995; Thomson et al. 1996; Cairney and Chambers 1999; Dell et al. 2002; Martin et al. 2003). In plantations with exotic eucalypts and pines, these fungi are desirable as inoculum if they are compatible with the host tree and are effective in promoting survival and growth in the field.

Previously, we determined optimum spore densities and spore storage conditions for mycorrhization of eucalypts with a few collections of Scleroderma (Chen et al. 2006). In this study, we investigate the capacity of Scleroderma to form associations with seedlings of Eucalyptus and Pinus under glasshouse conditions. This work addresses whether there is a need to source Scleroderma fungi from outside China to inoculate these exotic plantation trees and to match inocula with host genera.

Materials and methods

Experimental design

A complete randomized design consisting of 64 host–fungus combinations (four hosts, 15 fungi, and one non-fungus control) was used to compare fungal ability to colonize roots and promote plant growth under glasshouse conditions. There were eight replicate plants for each treatment.

Fungal inoculum

Details of the 15 collections of Scleroderma from eight species used in this trial are given in Table 1. Fungi were identified to putative taxa using morphological features, and voucher specimens are housed at the Research Institute of Tropical Forestry and Murdoch University. Fungal sporocarps were mostly collected from E. globulus plantations in temperate Western Australia, while some that were collected from south China, Indonesia, and Thailand were also used to compare their compatibility. Scleroderma species associated with eucalypts in subtropical or tropical parts of Australia were not included, as there was no difference in the relative success of temperate and tropical fungal isolates in nursery trials in China in an earlier study (Brundrett et al. 2005). The spore mass was dried at 30°C for 48 h and crushed by hand and sieved (400 μm) to produce spore powders. Spores were stored dry at 4°C for 3–18 months before use. The fungal spore mass was blended in distilled deionized (DI) water (1:10, vol.) for 5 s on low speed in a food blender. A drop of Tween 80 was added to assist suspension. The initial spore concentration was measured with a hematocytometer, and the bulk spore suspension was serially diluted to obtain a concentration of 105 spores/ml.

Table 1 Details of fungi used in the host range trial
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Host species and seed germination

Four exotic plantation tree species from two genera were used: E. globulus Labill., E. urophylla ST Blake (Myrtaceae); P. elliottii Engl. and P. radiata D. Don (Pinaceae). Seeds of eucalypts (E. globulus seedlot no. 18398, from Triabunna, Tasmania; E. urophylla seedlot no. 19393, from Pantar Island, Indonesia) were surface-sterilized by shaking in a 1.2% solution of NaOCl for 5 min (E. urophylla) or 10 min (E. globulus). After four rinses in sterile DI water, 20 seeds were transferred to each 90-mM Petri dish with 20 ml of germination medium (0.7% water agar amended with 500 μM CaSO4 and 3 μM H3BO3). Plates were incubated in darkness at 25°C for 8 days and in the light at 25°C for 2 days before planting (Chen et al. 2000a).

Seeds of P. elliottii (seedlot no. 11578, from CSO7E, Woodford) and P. radiata (seedlot no. K109S; from Grimwade, WA) were soaked in 1% H2O2 solution at 4°C for 5 days. Seeds were rinsed in sterile DI water and then surface-sterilized in 30% H2O2 for 25 min. After washing four times with sterile DI water, seeds were placed in 90-mM Petri dishes with germination medium (0.7% water agar amended with 500 μM CaSO4 and 3 μM H3BO3). Plates were incubated in darkness at 25°C for 6 days and in the light at 25°C for 3 days before transplanting.

Seedling growth medium

A nursery potting mix containing expanded coarse perlite, peat moss (natural sphagnum), and river sand (4:2:1 v/v) was used. The mix was moistened and steam-pasteurized twice at 60°C for 2 h. Plastic pots (70×70 mm top diameter, 200 mm height) were surface-sterilized with KClO3 (2–5%) and allocated 500 g each of air-dried soil mix.

Planting, inoculating, and maintenance

Two uniform healthy seedlings were transplanted into each free-draining pot, and seedlings were thinned to one after 2 weeks. Seedlings in each treatment were given 10 ml spore suspension, at a rate of 106 spores/seedling, 1 week after transplanting. The spore slurry was added in a 2-cm deep hole near the plant using a 5-ml pipette. All pots were put on to holding trays and randomly placed on benches in a glasshouse. The glasshouse was evaporatively cooled, and the maximum temperature ranged from 30–42°C and the minimum temperature ranged from 13–16°C during the period of the experiments. The position of each tray was randomly changed biweekly. The potting mix was watered to field capacity (ca. 10% w/w) before planting. Soil moisture levels were then maintained by an automatic overhead watering system (twice a day, 5 min each). A complete liquid fertilizer was applied biweekly to the surface of the containers based on previous experiments for eucalypt mycorrhization (Dell and Malajczuk 1995). The following were supplied at each application: macronutrients (milligrams/plant): N 1.5, P 0.7, K 0.9, Ca 0.5, S 0.5, and Mg 0.2; micronutrients (micrograms/plant): B 1.5, Cu 1.2, Fe 2.9, Mn 2.4, and Zn 0.9.

Data collection

Seedlings were harvested 12 weeks after planting. The shoot of each seedling was removed and its height measured. Mycorrhizal morphological characters of each fungus–host pair were visually assessed. Putative ECM root tips were further examined by transverse section or longitudinal section under light or scanning electronic microscopy and confirmed if both mantle and Hartig net were present. Root and shoot dry weights (60°C, 48 h) were determined. For each fungal treatment, individual root tips were selected to make fresh microscopic preparations. A randomly selected 2 g subsample of roots was preserved in 50% ethanol for mycorrhizal assessment. The samples were cleared in 10% KOH for 30 min at 90°C and stained with Trypan blue. Percentage of ECM colonization was under a dissecting microscope using the gridline intersect method (Brundrett et al. 1996). Colonization data were expressed as relative mycorrhizal abundance in three categories: +, 1–25% root-colonized; ++, 26–50% colonized; and +++, 50–100% colonized.

Statistical treatment of the data

Two separate analyses were performed. Two-way ANOVA was used to test the effect of both tree and fungus on the mean mycorrhizal colonization, shoot height, root dry weight, and shoot dry weight and total dry weight for each host. These variables measured on each seedling were dependent, so the initial significance test used the multivariate Wilk’s Lamba statistic, followed by univariate F test where the initial multivariate analysis was significant. One-way ANOVA was used for possible differences in the above variables. For percentage mycorrhizal colonization, data were transformed by Arc sin × before performing statistic analysis. The significance of mean values was determined using Duncan’s Multiple Range Test (P<0.05) (Gomez and Gomez 1984).

Results

Colonization and specificity

After 12 weeks, Scleroderma ECMs were present on all four hosts; however, the number of effective spore collections and percentage of mycorrhizal seedlings differed between tree genera and between tree species. There were significant differences in mycorrhizal colonization within tree species and between fungus collections (P<0.001). Interactions between tree and fungus were also observed. The majority of spore collections formed characteristic ECMs on pines and eucalypts (Table 2). Fourteen of the 15 fungus collections formed ECMs on pine seedlings and 13 on eucalypt seedlings. Over 93% of the inoculated seedlings of pines and 86% of eucalypts were ECMs. ECMs were extensive on some treatments of pine or eucalypt seedlings. Three spore collections colonized 50–100% of fine roots on P. elliottii seedlings, and two inoculation treatments of P. radiata reached similar levels of colonization. As in eucalypt seedlings, three spore collections resulted in abundant ECMs on E. urophylla, while two collections produced 50–100% ECMs on E. globulus.

Table 2 Relative mycorrhizal abundance on four plantation tree species inoculated with eight Scleroderma species from 15 collections
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Collections of Scleroderma from under eucalypt plantations in Western Australia were capable of colonizing eucalypts and pines tested in this trial. Among them, three collections (SAR-1, SCE-2, and SVE-1) resulted in well-developed ECMs on seedlings across the four hosts. However, there were differences within taxa from Australia. For example, ECMs formed extensively on both eucalypts and pines (50–100% of short roots) with SAR-1 (Scleroderma areolatum), while SAR-2 from the same taxon failed to colonize seedlings of P. elliottii and colonized less than 25% of the short roots of the other three hosts. Two collections of Scleroderma cepa (SCE-1 and SCE-2) formed ECMs on all hosts, while SCE-3 only colonized roots of pines. All four Chinese collections were compatible with P. elliottii; however, only one produced abundant mycorrhizas on the seedlings of other hosts. The two Chinese Scleroderma fungi collected from under secondary pine forests colonized larger component of short roots with pines than with eucalypts. The Scleroderma collected from the nursery bed formed ECMs with both eucalypts and pines, while a collection from under acacias in the field (SPA-1) only colonized roots of P. elliottii. Collection SAL-1 was an effective colonizer (26–50% of short roots colonizeed) for eucalypts only and SCE-1 and SCI-2 for pines. Seedlings of both eucalypts inoculated with SCE-3 and SPA-1 did not form ECMs. SAR-2 and SPA-1 did not form mycorrhizal associations on P. elliottii and P. radiata, respectively. These results suggest that there were some differences between collections in the ability to form ECMs on each host.

Scleroderma ECMs of pines and eucalypts had shiny, white extramatrical mycelia and distinctive white rhizomorphs. Three typical morphological features of Scleroderma ECMs are the dichotomous, multiple dichotomous, or pyramidal branching patterns. Examination of transverse sections of putative ECM roots confirmed that the Hartig net hyphae were present around both epidermal and cortical cells of pine roots but they were present around epidermal cells only of eucalypt roots.

Growth of the hosts

There were significant differences in the height, shoot dry weight, root dry weight, and the total dry weight between hosts, fungus treatment, and their interactions, respectively, when a two-way ANOVA was performed (P<0.001). For each host, effects of fungus treatments were significant (P<0.001) on the above variables, except for root dry weight of E. globulus (one-way ANOVAs). Inoculated seedlings of pines or eucalypts were generally larger than uninoculated controls of each tree (Figs. 1 and 2). Inoculation significantly stimulated the height and total biomass of E. globulus by up to 45 and 60%, and by 70 and 105% for E. urophylla. SAL-1, SCE-2, SCI-2, SFL-2, SPA-1, SSP-7, and SVE-1 extremely promoted the growth of E. urophylla, and SAR-3, SFL-2, SPA-1 and SSP-7 were the better growth promoters for E. globulus (Fig. 1). In terms of the effects on the growth of P. elliottii, seedlings of four fungus treatments (SAL-1, SAL-2, SCI-2, and SFL-1) were significantly taller than the other treatments, and nine of 15 fungus collections increased seedling total dry weight (Fig. 2). All fungus collections, except SCI-1, greatly enhanced the height and total biomass of P. radiata.

Fig. 1
figure 1

Effects of Scleroderma inoculation on shoot height and total dry weight of Eucalyptus globulus (left) and E. urophylla (right). Standard errors are given. For each host, bars with same letters in each parameter are not significantly different by Duncan’s Multiple Comparison (P<0.05). CONT uninoculated control; other codes are given in Table 1

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Fig. 2
figure 2

Effects of Scleroderma inoculation on shoot height and total dry weight of Pinus elliottii (left) and P. radiata (right). Standard errors are given. For each host, bars with same letters in each parameter are not significantly different by Duncan’s Multiple Comparison (P<0.05). CONT unicoculated control; other codes are given in Table 1

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Discussion

Differences between the collections of Scleroderma, in their ability to form ECMs and promote growth, were apparent on seedlings of the four plantation hosts under glasshouse conditions. Fully developed ECM symbioses were present on the roots of 14 (pines) or 13 (eucalypts) of the 15 fungal inoculant treatments, although the proportion of mycorrhizal roots varied between tree species and collections. The results suggest that there is low host specificity of Scleroderma between host genera. However, the collections of Scleroderma from under pines were less aggressive on eucalypts than were some of the Scleroderma collections from under eucalypts in plantations in Australia. Furthermore, there were differences within taxa, even from same collecting region. For example, one collection of Australian S. areolatum (SAR-1) formed extensive ECMs on both eucalypts and pines (over 50% of short roots), while SAR-2 (same storage as SAR-1) failed to colonize seedlings of P. elliottii and colonized less than 25% of short roots of P. radiata and E. urophylla by week 12. By contrast, Scleroderma citrinum and Scleroderma flavidum collected from either south China, Western Australia, or northern Thailand showed no or little host specificity on all hosts in this trial. S. citrinum has been found to be commonly associated with various tree species in nurseries, plantations, and forests (Schramm 1966; Garido 1984; Ingleby et al. 1985; Duñabeitia et al. 2004), and collections of this fungus have been tested to be compatible with many pine species (Takacs 1961; Trappe 1962; Schramm 1966; Azevedo 1982; Parladé et al. 1996; Duñabeitia et al. 2004). The S. areolatum collections used in this trial may be polymorphic, and molecular tools are required to define species boundaries.

Scleroderma fungi, such as Scleroderma bovista, S. citrinum, and S. verrucosum, have been observed to be present in coniferous or eucalypt forests and plantations (Pryor 1956; Takacs 1961; Bakshi 1966; Birch 1973; Guzmán and Varela 1978; Dunstan et al. 1998; Cairney and Chambers 1999; Chen et al. 2000b, 2004). S. bovista, S. cepa, S. citrinum, Scleroderma meridionale, and Scleroderma verrucosum have been previously used to inoculate Eucalyptus and Pinus species (Trappe 1962; Marx 1969; Chu-Chou 1979; Richter and Bruhn 1989; Dell et al. 1994; Parladé et al. 1996; Lu et al. 1998; Rincón et al. 2001; Chen et al. 2000b). While most studies tested the comparability of Scleroderma in one host genus only (Richter and Bruhn 1987; Duponnois et al. 2005), Dell at al. (1994) and Sanon et al. (1997) compared across a few tree genera from Australia or the Northern hemisphere, respectively. Scleroderma has been considered to be incompatible with Casuarina (Dell at al. 1994) or Alnus (Molina 1981). Reddy and Satyanarayana (1998) reported that isolates of S. cepa and S. flavidum failed to form ECMs on E. tereticornis in vitro. Apart from these studies, the literature suggests that Scleroderma generally colonizes a wide range of host trees.

Results on colonization ability of 15 collections of Scleroderma on four plantation trees showed that variable degrees of compatibility of fungal collection–host species influenced successful colonization and development of mycorrhizas. These are consistent with previous findings on eucalypt mycorrhization (Malajczuk et al. 1990; Dell et al. 1994; Parladé et al. 1996). Previous studies conclude that many ECM fungi show specificity at the host genus level rather than at the host species level (Chilvers 1973; Malajczuk et al. 1982). A few broad-host-range fungal symbionts, such as Amanita muscaria and Hebeloma crustuliniforme, are capable of forming ECMs in common on eucalypts and pines (Malajczuk et al. 1982). In this experiment, Scleroderma collected from under eucalypt plantations in Australia formed ECMs on both P. elliottii and P. radiata. By contrast, Scleroderma associated with Pinus massoniana in China was a poor colonizer of eucalypts than pines.

Inoculation with some Scleroderma species generally enhanced the growth of all host plants involved in this trial confirming the potential usefulness of this genus for slow-growing nursery stock. Growth promotion at the nursery stage is not necessarily important for fast-growing species, such as E. urophylla, but is advantageous for most pines. The priority of inoculating containerized-grown seedlings is the extent of mycorrhizal success and the subsequent improvement in survival and growth in the field. Nevertheless, previous work has shown that Scleroderma can enhance the growth of eucalypts (Burgess and Malajczuk 1990; Reddell and Milnes 1992; Dell et al. 1994; Reddy and Satyanarayana 1998), pines (Pinus caribaea, P. contorta, and P. kesiya) (Rangarajan et al. 1990; Rao et al. 1996; Fay et al. 1997), Castanopsis hystrix (Chen et al. 2001), and some dipterocarps (Santoso 1991; Omon 1996). The finding that Scleroderma species promoted plant growth confirms four previous studies where Scleroderma was inoculated onto Afzelia africana (Bâ et al. 1999), E. globulus (Lu et al. 1998), Pinus sylvestris (Colpaert et al. 1992), or Quercus ilex ballota (Seva et al. 1996). The experimental conditions applied in our trial have been previously tested to grow mycorrhizal eucalypts with inoculation of Scleroderma spores (Chen et al. 2006). Results from inoculation of eucalypts at three nurseries in Western Australia (Mediterranean) and south China (subtropical and temperate) with 18 collections of Scleroderma revealed that there were no significant differences in the performance of congeneric fungal collections from different climatic regions (Brundrett et al. 2005). This suggests that fungi of this genus may be particularly suitable for inoculating eucalypts in south China, where climate ranges from lowland subtropical/tropical to highland temperate.

Results on the efficiency in colonizing eucalypts between Australian and Chinese collections of Scleroderma expand previous findings of a field inoculation trial on E. urophylla in south China (Chen et al. 2000c), where 10 of the 11 collections of Scleroderma from Australia were effective in improving tree survival and productivity while there was no response inoculation by a single Chinese collection. While Chinese Scleroderma collected from under secondary pine forests or pine plantations could be used for inoculating seedlings in pine nurseries, there may be a risk to using these fungi for inoculating eucalypts in Chinese nurseries. Hence, it is recommended that Scleroderma be sourced from outside China to inoculate eucalypts until the time basidiomes are sufficiently abundant in eucalypt plantations to enable the local collection of spores. Due to the ease of application and the availability of large quantities of spores from a few sporocarps for nursery inoculation needs, spore inoculum is preferable for application on an operational scale in China.