Diversity of Ectomycorrhizal Fungi Associated with Eucalyptus in Africa andMadagascar

1 Laboratoire des Symbioses Tropicales et Méditerranéennes, Département BIOS, CIRAD, UMR 113, TA10J, 34398 Montpellier Cedex 5, France 2 Laboratoire des Symbioses Tropicales et Méditerranéennes, Département Environnement et Ressources, IRD, UMR 113, TA10J, 34398 Montpellier Cedex 5, France 3 Département des Sciences et Gestion de l’Environnement, Fondation Universitaire Luxembourgeoise, 6700 Arlon, Belgium


Introduction
Since colonial times, the African woodlands have been subjected to irreversible management policies that have resulted in the planting of large areas with exotic trees, for instance Pinus and Eucalyptus.In Mediterranean and tropical countries eucalypts are planted because they are generally fast-growing trees that make for a profitable business.Nowadays, eucalypt plantations cover approximately 1,8 Mha in Africa and are the most important planted genus on that continent [1].In order to extend plantations and increase profits, it appeared necessary to find and select new eucalypt genotype adapted to different ecological situations.To that end, foresters started to test different Eucalyptus species of different origins, notably in Africa and Madagascar (e.g., [2,3]).As a result, the number of introduced eucalypt species in African plantation trials now exceeds 150.Some of them are used in commercial plantations as pure species or as hybrids [4].The mass introduction of these exotic species in Africa definitely has consequences for local plant biodiversity and also for soil microbial communities such as ectomycorrhizal (ecm) fungi.Indeed, the ability of eucalypt species to form ectomycorrhizae, which are ecologically important symbioses that mainly associate homobasidiomycetous fungi, was observed for the first time in South Africa by Van der Bijl [5].Since then, the ecm status of eucalypt species has been clarified [6] notably in Australia where a large diversity of fungal partner has been described [7][8][9][10][11][12].Until now, ecm fungi found in Africa and Madagascar have resulted putatively from chance introductions, from compatibility with native ecm fungi, or from compatibility with ecm fungi from other exotic plantations.Indeed, the use of soil as an inoculant or any other source of inoculation that might bring ecm fungi from the tree's native area has not been reported when establishing eucalypt plantations.Some controlled field inoculation trials using pure strains were set up, but only on very limited areas (e.g., [14]) and, in this case, the invasion of neighboring plantations by the introduced strains was not reported.Controlled field inoculation trials provide ways of entry for new ecm fungi that cannot be neglected, as is the case for China [15,16].However, the fungal species or even the strain reference might be known, enabling tracking in the plantation or in surrounding plantations.In Africa, we are in a situation where we have no proof of any propagation of a fungal species after the controlled inoculation of a eucalypt plantation.However, we are in a particular case where human activity, eucalypt plantations, has direct and mostly uncontrolled consequences for another compulsorily related biological community: ecm fungi.
Published papers, unpublished information, and authors personal data formed the basis of our work to synthesize the knowledge available on putative ecm fungi of eucalypt plantations in Africa and Madagascar.An annotated list of putative ecm fungi of eucalypts is given for more than 20 African countries and Madagascar.

Materials and Methods
A bibliographical search covering 30 years was carried out enabling us to uncover all the published papers and also a large share of the "grey literature" directly linked to our topic: ecm fungi associated with eucalypts in Africa and Madagascar.Written reports and all the oral reports of ecm partnerships have always to be considered with great caution and skepticism.Indeed, proving the ability of a tree species to form ectomycorrhizae with one particular fungal species is always a difficult task; the existence of other local or introduced ecm tree species in the vicinity of eucalypt plantations as well as the mistaken identification of the fungal partner can lead to misinterpretation.Definitely doubtful data are not considered in the rest of the paper and unreliable data are presented with a special mention of what needs to be considered with caution.
In addition, field surveys were organized in eucalypt plantations, notably in different African countries and in Madagascar; fungal fruiting bodies were systematically collected from the plantations.Litter and a few centimeters of topsoil were removed to search for hypogeous fungi on at least 4 m 2 in each plantation.Fungal specimens were deposited at the Museum National d'Histoire Naturelle de Paris (PC), France.In order to prove the ecm partnership, careful mycelium and root tracking were carried out in the field enabling us to assess physical links between fruiting bodies, ecm root tips, and the bearing trees.
Ectomycorrhizae histological studies were also carried out on materials collected in the field to reveal the fungal sheath and the Hartig net (Table 2).

Results
In Africa, 35 ecm fungal species are mentioned as being associated with 15 identified eucalypt species and a range of unidentified species (at least five), along with hybrids (Table 1).Pisolithus was present and often highly dominant or even alone in almost all the plantations, as is the case in the West African dry tropics with Pisolithus albus in E. camaldulensis plantations (Figure 1(a)).The importance of the genus Pisolithus and to a lesser degree, the genus Scleroderma, was noteworthy (Figures 1(b) and 1(c)).The presence of the ubiquitous ecm fungus Thelephora sp. in the wet tropics was also noteworthy (Figure 1(d)).During the surveys ( [13] in Table 1), ectomycorrhizae with a typical fungal sheath and a Hartig net (Figures 1(e) and 1(f)) were found in the vicinity of fruiting bodies.
In Madagascar, we collected 38 putative ecm fungal species from nine E. robusta plantations on the outskirts of Antananarivo (Table 3 At these nine survey sites, Pisolithus was found only once and was far from dominant.In addition to this surprising diversity associated with E. robusta, two different Pisolithus species were found at two other sites in the southeast of Madagascar (Figure 2(f)).

Discussion
Ectomycorrhizal deficiency never seems to have been a problem in the early establishment of eucalypts.Ryvarden et al. [17] noted that selected trees form satisfactory partnerships with some introduced ecm fungi, notably Pisolithus arhizus which is now extremely abundant under eucalypts in Africa, and also with some elements of the indigenous ecm mycoflora.It is interesting to note that renantherous species of Eucalyptus known for their high dependence on ecm symbiosis [6] have been excluded by foresters due to their poor growth performance even in a Mediterranean climate (North Africa and South Africa), where the range of putatively adapted species is wide.During the process of eucalypt introduction and selection, foresters have neglected ecm symbiosis.This fact has probably led to the selection of tree species dependent little on ectomycorrhizae for early survival and growth.South America is also a place where eucalypts were abundantly planted and are found to be ectomycorrhizal with a range of ecm fungi ( [13, 18-20, 42, 43] and Table 4).Some similarities with the African ecm fungi have to be mentioned, notably, the importance of genera Scleroderma and Pisolithus.On another hand, importance and diversity of genera Descomyces, Hydnangium, Hymenogaster, Hysterangium, and Setchelliogaster seems scarcer in Africa rather than in South America.

Fungal Diversity in Africa.
According to Buyck [44], the native ecm fungi are unable to form ectomycorrhizae with exotic trees and consequently, eucalypts ecm fungi are limited to a few ecm fungal species that are now cosmopolitan.The observation made by Härkönen et al. [23] [17], and 20: Authors' personal observations.* Pisolithus arhizus is a temperate European species described from acidic soils and associated with trees like Quercus, betulus, or pinus.Its presence in Africa associated with introduced Australian species is unlikely.Our assumption is that the specific epithet "arhizus" is erroneous.* * The systematic of Scleroderma are complex and the erroneous use of specific epithets in the identification of this species has to be kept in mind.* * * In the absence of any indication on the subspecies, the possibilities of synonymy with other species are multiple.# Rhizopogon is a genus mostly associated with pines and some temperate deciduous trees.A poor mycorrhizal colonization of eucalypt roots by these Rhizopogon can be considered; indeed Rhizopogon spp.differed markedly in their ability to form ectomycorrhizas according to their taxonomical section and host plant [41].
§ The presence of these temperate taxa in tropical countries has to be carefully considered; confusion with local tropical taxa might be possible.interesting area to improve our knowledge on the role played by ecm diversity in the sustainability of eucalypt plantations.
On the other hand, the presence of ecm fungi associated with eucalypts has not yet been reported under native ecm trees.Presumably there is incompatibility between eucalypt ecm fungi and those of the indigenous African ecm trees (e.g., Afzelia spp., Brachystegia spp., Berlinia spp., and Isoberlinia spp.among the Caesalpiniaceae; Monotes spp., Marquesia spp.among the Dipterocarpaceae; Uapaca spp.among the Euphorbiaceae).

The Case of Madagascar.
In Madagascar 80% of the vascular plants were considered to be endemic [46].It also has one of the highest concentrations of endemic plant families: nine families represented by a total of 19 genera ca.90 species [47,48].Of these, the ectotrophic Sarcolaenaceae [49] represent 56 species.Plantations of the Central Plateau are widely dominated by one single species: E. robusta.Among the 38 putatively ecm fungal species found associated with E. robusta, some red cap Russula sp. and Cantharellus eucalyptorum are marketed locally as edible, and the latter is exported to the surrounding countries.The number of ecm fungi found associated with E. robusta in a one-year survey was larger than the number of ecm fungi found in the whole of continental Africa since more than 80 years of observations.Amanita, Boletus (s.l.), Cantharellus, and Russula are the dominating genera found under E. robusta; on the other hand, members of the Sclerodermataceae were rarely observed.The presence of Madagascan endemic plant taxa able to form ecm in eucalypt plantations or in the nearby surroundings as been carefully explored and none of the already known ecm families such as Sarcolaenaceae, Rhopalocarpaceae, Caesalpiniaceae, or Euphorbiaceae has been found.Other grass and shrubs growing under the canopy of eucalypts plantations were arbuscular mycorrhizal or for a few of them, nonmycorrhizal (unpublished).To conclude, this striking difference has yet to be elucidated.

Hypotheses on the Possible Origins of ECM Fungi in Eucalypts Plantations.
Until now, the rational use of ecm fungi by eucalypt plantations has been poorly developed in Africa.Inoculations with selected ecm strains are still restricted to some experimental plantations [14].The origin of the ecm fungi found associated with eucalypts in Africa and in Madagascar has yet to be elucidated.At least, three hypotheses can be put forward: (1) chance introductions from Australia of (fully) compatible ecm fungi (e.g., Pisolithus spp.), (2) compatibility with some "broad host" ecm fungi growing in native African forests, and (3) compatibility with some ecm fungi growing in other exotic plantations (e.g., pine plantations).In any event, further studies are necessary to unravel the origins of the fungi in eucalypt plantations.Curiously, in Madagascar, the level of fungal diversity observed under E. robusta is comparable to the diversity level of native ectotrophic forests [49].The possibility that ecm fungi associated with E. robusta in Madagascar are of Australian origin has to be explored.Comparisons of our material collected under eucalypts (Table 2) with native Australian specimens and with samples of ecm fungi from native Madagascan ectotrophic forests are necessary to unravel the origins of the fungi observed in eucalypts plantations in Madagascar.

Table 1 :
[21,23,32,33]Buyck's observation: the indigenous fungi cannot grow in symbiosis with the introduced trees.These assertions seem rather excessive; indeed records of typical European species such as Amanita muscaria, A. phalloides, Paxillus involutus, or Rhizopogon luteolus[21,23,32,33]or typical African List of ectomycorrhizal fungi found associated with eucalypts in Africa with an indication of the country where the observation was made and the reference.

Table 2 :
Color, diameter, fungal mantle thickness, and Hartig net depth of ectomycorrhizae in situ collected in Senegal on 3 species of Eucalyptus.
[5] some native fungi, or for fungi introduced with other exotic species such as pines, to form ectomycorrhizae with eucalypts.The presented data clearly show that the level of fungal diversity in Africa remains very low under eucalypts: only 34 ecm fungal species were recorded since 1918[5]!The easily disseminated (by wind and rain splashing) and largely distributed genus Pisolithus is present and dominates in most plantations; other records always seem exceptional.A regular increase in fungal diversity, due to new uncontrolled introductions, to the extension of plantations to new climatic and soil conditions, and to the ageing of some plantations creating favorable conditions for the development of late stage ecm fungi is unavoidable.With the increasing importance of eucalypt plantations in Africa, we have here an

Table 3 :
List of ectomycorrhizal fungi found associated with Eucalyptus robusta and Eucalyptus sp. in Madagascar.Herbarium samples have been deposited at the Museum National d'Histoire Naturelle de Paris (PC), France.# Indicates when the observation was carried out by tracking the mycelium from fungus fruiting bodies up to fine ectomycorrhizal roots and the trunk.

Table 4 :
List of some ecm fungi and associated eucalypt hosts spontaneously found in South America: Brazil (BR), Uruguay (UR), and Argentina (AR).