Ectomycorrhization of Acacia mangium, Willd. and Acacia holosericea, A. Cunn. ex G. Don in Senegal....

Journal of Arid Environments (2002) 50: 325}332doi:10.1006/jare.2001.0800, available online at http://www.idealibrary.com on

Ectomycorrhization of Acacia mangium, Willd.and Acacia holosericea, A. Cunn. ex G. Don inSenegal. Impact on plant growth, populations ofindigenous symbiotic microorganisms and plant

parasitic nematodes

H. Founoune*-, R. Duponnois*? & A. M. Ba( A

* IRD, Laboratoire de Bio-PeHdologie, BP 1386, Dakar, SeHneHgal-UniversiteH Moulay IsmanK l, Laboratoire de Biotechnologie et d’ameH lioration

des plantes, BP 4010, Meknes, MarocAISRA, Direction des Recherches sur les Productions Forestieres,

BP 2312, Dakar, SeHneHgal

(Received 28 April 2000, accepted 6 February 2001)

The ectomycorrhization of two Australian Acacia species (A. mangium and A.holosericea) with two basidiomycetes (Pisolithus and Scleroderma) was studied insterilized and unsterilized soils. The three fungal isolates, two exotic strains(Pisolithus COI 007 and COI 024) and one indigenous (Sclerodermal) en-hanced the development of the Acacia species. In the unsterilized soil, thenumber of nodules per plant of indigenous rhizobia was increased when theplants had associated mycorrhizae. The plant parasitic nematode communitywas reduced by the fungal strains, especially for H. pararobustus withA. holosericea. The ectomycorrhizal associations of the Australian Acacias,mostly unknown in West Africa, appears to be very promising for use of thesetrees in afforestation programs.

� 2002 Elsevier Science Ltd.

Keywords: Acacia; reforestation; plant parasitic nematodes; Mycorrhizae;Rhizobia; Senegal

Introduction

The soil microorganisms which are known to play vital roles in ecological processes areparticularly present in the root region (rhizosphere) of plants (Garbaye, 1991). Theserhizospheric bacteria, actinomycetes or fungi carry out a range of physiological activitiessuch as breakdown of organic matter, mobilization of mineral nutrients, nitrogenfixation, secretion of growth substances and antagonism against plant pathogens, whichare of great relevance to plant growth (Linderman, 1988). It is usually assumed that themost beneficial contribution of soil microorganisms to plant development is the supplyof nutrients, particulary nitrogen and phosphorus. These are the two major elementswhich limit plant development in tropical areas.

?Corresponding author. Fax: (221) 832 16 75. E-mail: robin.duponnois@ird.sn

0140-1963/02/020325#08 $35.00/0 � 2002 Elsevier Science Ltd.

326 H. FOUNOUNE ET AL.

During the last decades, drought and over-exploitation of the natural resources haveinvolved a dramatic deforestation in sahelian regions of West Africa. Different solutionshave been proposed to rehabilitate these areas, and in particular, the cultural practice ofagroforestry. However the choice of the tree species in these cultural systems must considertheir tolerance to the harsh environmental conditions encountered in these regions suchas the mineral deficiencies and plant pathogens (i.e. plant-parasitic nematodes).

N�-fixing leguminous trees have a promising future in forestry and agroforestry in dry

and humid tropical lowlands. Among this group, Acacia species are associated withendomycorrhizal and ectomycorrhizal fungi as well as rhizobia in tropical ecosystems(Reddell & Warren, 1987; Ba( & PicheH , 1991). It is well known that the inoculation ofefficient bacterial or fungal symbionts can dramatically increase the growth of thesetrees (Brgess et al., 1994; Galiana et al., 1990, 1994) and consequently optimize theirbeneficial effects in agroforestry systems. However, these plant-parasitic nematodescan inhibit both tree and adjacent crops development. Particularly, Meloidogyne spp., themajor pests of vegetables and important problem affecting the production ofsubtropical and tropical crops ( Johnson & Fassuliotis, 1984; Prot, 1986; Duponnoiset al., 1995).

The principal aim of agroforestry is to create positive interactions between woodyperennials, herbaceous plants and their biotic and abiotic environments in order toincrease the overall productivity of the system. It is important to understand theseinteractions in order to predict the behaviour of agroforestry systems following man-agement practices such as tillage, pruning, fertilization or microbial inoculation (rhi-zobia, mycorrhizal fungi).

The aims of this study were to determine the impact of ectomycorrhizal associationsby Pisolithus sp. and Scleroderma dictyosporum, on two Acacia species, Acacia mangiumand A. holosericea. Seedling growth, ectomycorrhizal rate and ectomycorrhizal depend-ency were measured on plants growing in both sterilized and unsterilized soils. Inaddition, ectomycorrhizal associations of the two Acacia species growing in unsterilizedsoils was monitored for the establishment of rhizobial symbiosis from indigenousrhizobia, internal colonization by indigenous mycorrhizal fungi in the roots, and for thenumber of indigenous plant-parasitic nematodes, Hoplolaimus pararobustus, Scutel-lonema cavenessi and Tylenchorynchus germanii.

Materials and methods

Plants

Seeds of A. mangium (provenance Kolda) and A. holosericea (Provenance Bel Air) weresurface-sterilized in 95% sulfuric acid for 60 min. The acid solution was then decantedand the seeds rinsed and imbibed for 12 h in four changes of sterile distilled water. Seedswere then transferred aseptically into Petri dishes filled with 1% (w : v) water agarmedium. These plates were incubated for 2 days at 253C. The germinating seeds wereused when rootlets were 1–2 cm long.

Preparation of the fungal inoculum

The two strains of ectomycorrhizal fungus Pisolithus sp. (COI 007, COI 024) and theScleroderma dictyosporum isolate (Sd 109) were maintained on Melin Norkrans modifiedby Marx (MNM) agar medium (Marx, 1969). The strains COI 007 and COI 024 wereisolated in southern Senegal from sporocarps sampled under monospecific forest planta-tion of A. mangium and Eucalyptus camaldulensis, respectively. Scleroderma dictyosporum,Sd 109, was isolated in Southern Burkina Faso from fresh sporocarps under a Afzelia

ECTOMYCORRHIZATION OF A. MANGIUM AND A. HOLOSERICEA 327

africana plantation (Sanon et al., 1997). The solid inoculum was prepared in 1)6-l glassjars containing 1)3-l vermiculite-peat mixture (4 : 1, v : v) moistened with liquid MNMmedium (Duponnois & Garbaye, 1991). This substrate was inoculated with fungalplugs taken from the margin of the fungal colonies and incubated for 6 weeks at 283C inthe dark.

Plant culture

The sandy soil used in this experiment was sampled from a 17-year-old plantation ofA. holosericea at Sangalkam (50 km at the east of Dakar). It was highly colonised withrhizobia but without ectomycorrhizal fungi (Duponnois, unpublished data). It was passedthrough a 2-mm sieve and autoclaved for 1 h at 1403C to eliminate the native microfloraand fauna. After autoclaving, its physicochemical characteristics were as follows: pH H2O6)5; clay 3)6%; fine silt 0)3%; coarse silt 1)0%; fine sand 55)5%; coarse sand 39)4%; totalcarbon 0)54%; total nitrogen 0)06% and soluble phosphorus 8)8 mg kg!1. This soil wasmixed with 10% (v : v) fungal inoculum or 10% vermiculite-peat mixture (4 : 1, v : v) forthe control treatments (without fungus). Then pre-germinated seeds of A. mangium andA. holosericea were planted one per plastic bag (1 dm3) filled with autoclaved soil. Theseedlings were placed in a glasshouse (323C day, 283C night, daylength approximatively12 h) and watered twice weekly without fertiliser. There were 14 replicates per treatmentarranged in a completely randomized design.

After 3 months, seven plants from each treatment were harvested. The root systemswere removed from the soil and gently washed. For each plant, the roots were cut into2–3-cm-long pieces and mixed in a bucket of water. Lateral roots were randomlysampled and observed under a stereomicroscope (magnification�160) until at least 100root segments had been counted as ectomycorrhizal or not. The ectomycorrhizal rateswere determined as (number of ectomycorrhizal root segments/total number of countedroot segments)�100. Dry weight of shoots and roots were measured after oven-dryingat 703C for 1 week. Ectomycorrhizal dependency (MD) was calculated according toPlenchette et al. (1983) as follows: (shoot biomass of ectomycorrhizal plants—shootbiomass of the non ectomycorrhizal plants)/(shoot biomass of ectomycorrhizalplants)�100.

The remaining seven plants were transferred into 10-l pots filled with the sameunsterilized soil and placed in the glasshouse. The experimental design was a completetwo-way factorial with two Acacia species and three fungal treatments (control withoutfungal inoculation, COI 024, Sd 109 for A. holosericea and control without fungalinoculation, COI 007, Sd 109 for A. mangium).

The height of the plants was measured after 6 months culturing. Then they wereuprooted and the root systems gently washed. The soil from each pot was mixed,a 250-g sub-sample was taken and the nematodes were extracted using Seinhorst’s(1962) elutriation technique. The leaves and the stems of each plant were divided andtheir oven-dried weights (2 weeks at 653C) were measured.

Root nodules induced by indigenous rhizobia were detected and counted. Two gramsof fresh root were randomly sampled along the root system of each plant to determinethe intensity of mycorrhizal symbiosis and kept in alcohol (703). The root samples wereobserved under a stereomicroscope (magnification: �160) to determine the ectomycor-rhizal rates as before. The same samples were used to quantify the internal colonizationof indigenous arbuscular mucorrhizal fungi in the roots. The roots were clarified andstained according to the method of Phillips & Hayman (1970). The rate of colonizationwas estimated in terms of percentage of mycorrhizal root pieces. The roots were cut intoapproximately 1-cm pieces which were preserved in 50% glycerine on a slide formicroscopic observation (Brundrett et al., 1985). About one hundred 1 cm-root pieceswere observed per plant. The endomycorrhizal rates were expressed as (number of

328 H. FOUNOUNE ET AL.

mycorrhizal fragments/number of non-mycorrhizal fragments)�100. Then the entireroot systems were washed, cut into short pieces and placed in a mist chamber for 2 weeksto recover nematodes (Seinhorst, 1950). The nematodes were counted and identifiedunder a stereomicroscope (magnification: �120). The root systems were over dried (1week, 603C) and weighed. Ectomycorrhizal dependency (MD) was calculated as before.

Statistical analysis

Data were treated with two-way analysis of variance. Means were compared usingPLSD Fisher test (p(0)05). The percentages of mycorrhization were transformed byarcsin (sqrt) before statistical analysis and for nematode populations, data were trans-formed by log(x#1) prior to analysis.

Results

For A. holosericea, shoot biomass was significantly increased with both fungal strains inthe sterilized soil (Table 1). However no significant effect was recorded for rootbiomass (Table 1). In the case of A. mangium, only S. dictyosporum Sd 109 significantlystimulated shoot and root development of seedlings (Table 1). Ectomycorrhizal rateswere significantly lower with Sd 109 than with the two Pisolithus strains (COI 007 andCOI 024) for both Acacia species (Table 1). However mycorrhizal dependency wassignificantly higher for A. holosericea than for A. mangium. No nodules induced bycontaminating rhizobia were detected in any treatment.

When the plants were transferred to the unsterilized soil, the data recorded for thegrowth of A. holosericea (shoot and height) were generally higher than for A. mangium(Table 2). On the contrary, the root growth of A. mangium was more important than forA. holosericea. The beneficial effects of the ectomycorrhizal symbiosis on thedevelopment of A. holosericea occured with both fungal treatments except for leafbiomass and for height in Sd 109 fungal treatment (Table 2). However no significanteffect of the fungal symbiosis has been measured for height, total shoot biomass andstem biomass of A. mangium plants (Table 2). Compared with the control, treatmentswith COI 007 and Sd 109 respectively, significantly increased root and leaf biomass ofA. mangium plants. The ectomycorrhizal rates were not significantly differentamong the fungal treatments (Table 2). For ectomycorrhizal dependency, the highestdependencies were obtained with A. holosericea. No endomycorrhizal infections havebeen recorded inside the root systems of the plants in any treatment.

The number of rhizobia nodules per plant was significantly higher with COI 024 forA. holosericea and with COI 007 for A. mangium than in the not inoculated treatment(Table 3). The total dry weight of the nodules was also higher in the fungal treatments,excepted with Sd 109.

Three main nematode genus were identified in the soil: Hoplolaimus pararobustus,Scutellonema cavenessi and Tylenchorynchus germanii (Table 4). The ectomycorrhizalassociation with Sd 109 decreased the number of nematodes per pot for H. pararobustuswith A. holosericea. On the contrary, the total number of S. cavenessi parasitizingA. mangium was significantly higher when the fungal strain COI 007 was inoculated(Table 4). For the other data, no significant differences were recorded.

Discussion

Leguminous trees can grow on infertile soils in part because of their symbiotic microor-ganisms such as mycorrhiza and rhizobia (dela Cruz & Garcia, 1992). Research suggests

ECTOMYCORRHIZATION OF A. MANGIUM AND A. HOLOSERICEA 329

that the main mycorrhizal association with these Australian Acacia species is endomycor-rhizal symbiosis (Reddell & Warren, 1987). For example, Acaulospora spp. and Glomusspp. were the dominant strains of arbuscular mycorrhizal fungi associated withA. mangium in acid soils (Yantasah, 1989; Poonsawad & Yantasath, 1991). Regardingthe endomycorrhizal symbiosis, it has been shown that A. mangium was mycorrhizal-dependent (Tambalo-Zarate & dela Cruz, 1991). A study performed in a sterilizedP-deficient soil in Senegal concluded that the mycorrhizal dependency of A. holosericeafor a strain of Glomus sp. was at least 70% (Senghor, 1998). However, in our experi-ment, we have not observed endomycorrhizal infections. The soil collected in Sangal-kam is known to be slightly colonized by endomycorrhizal fungi which could explain thelack of endomycorrhizal structures inside the roots recorded in these experimentalconditions (Duponnois, pers. comm.). However the knowledge about the dependencyof Australian Acacia species with ectomycorrhizal fungi is very scarce. Lee (1990)reported the presence of fruiting bodies of Thelephora ramariodes in A. mangium planta-tions in Malaysia and the Philippines. However evidence that this fungus forms trueectomycorrhizal associations (fungal sheath and Hartig net) is lacking. In Senegal,Ducousso (1990) reported the presence of Pisolithus spp. sporocarps in A. holosericeaplantations. Pisolithus genus is distributed world-wide in association with a wide range ofhost genera (Molina et al., 1992). It is generally assumed that this fungal genus wasintroduced in Africa through exotic tree species such as Eucalyptus, Melaleuca,Allocasuarina and Acacia. However the influence of these ectomycorrhizal associationson the growth of the host plant has rarely been assessed with Acacia spp. Duponnois& Ba( (1999) demonstrated that a strain of Pisolithus increased the growth of A. mangiumis several senegalese soils. They concluded that A. mangium was ectomycorrhizal-dependent. All these soils were characterised by a low content of Phosphorus. In ourexperiment, it was demonstrated that A. holosericea is also ectomycorrhizal dependent.However all these results were obtained from experiments performed with sterilized soilswithout indigenous microflora. Our results show that the positive effect of theectomycorrhizal symbiosis on the growth of A. holosericea can also be obtained in anunsterilized soil. This suggests that some of the ectomycorrhizal strains used in theseexperiments are very competitive with the indigenous microflora. It is well known thatthe soil microorganisms interact with the fungal symbiosis. For example, Kabre (1982)showed that microflora isolated from a South Senegal soil involved a large inhibition ofthe ectomycorrhizal association of Pinus caribaea with Pisolithus tinctorius. In contrast,a strain of soil microorganisms called ‘Mycorrhization Helper Bacteria’ increased thefungal establishment (Duponnois et al., 1993; Frey-Klett et al., 1997). The com-petitiveness of fungal symbionts has been recorded with A. mangium but to a lowerextent. As the development of the fungus is dependent on the host plant species, it is notsurprising to record these differences. One of the conclusions is that the ectomycor-rhizal dependency of A. mangium is less important than that of A. holosericea under theseexperimental conditions.

Another important finding from these experiments is the beneficial effect onplant growth of an ectomycorrhizal strain, S. dictyosporum, isolated in West Africa. Thecompatibility between this fungal strain and A. holosericea has been already described inaxenic conditions (Ducousso, 1990; Ba( , 1990). However, the ectomycorrhizal symbio-sis with S. dictyosporum (Sd 109) in non axenic conditions (sterilized or unsterilized soil)has never been assessed with these two Acacia species. The fungal strains is pre-adaptedto the environmental conditions encountered in West Africa. Its ecological character-istics and its beneficial effects on the plant could be very useful in controlledmycorrhizal associations in the Sahel.

The ectomycorrhizal symbiosis increased the number of rhizobial nodules and/ortheir dry weight. Several published reports have demonstrated the positive effect ofthe dual inoculation (rhizobial and mycorrhizal) on Acacia which enhances seedlinggrowth and nodulation compared to plants inoculated with rhizobia only (Cornet

330 H. FOUNOUNE ET AL.

& Diem, 1982; Senghor, 1998). In this study, the ectomycorrhizal symbiosis withScleroderma and Pisolithus increased the development of nodules. It is well known thatthe ectomycorrhizal fungi can stimulate rhizogenesis (Linderman & Paulitz, 1990). Thiseffect could increase the number of nodulation sites and consequently the numberof nodules per plant. However this present data only concerns one step of the rhizobialsymbiosis and studies of interactions between these two microorganisms must beundertaken.

In this experiment, three main nematode genera, parasitising A. holosericea andA. mangium, were identified: S. cavenessi, T. germanii and H. pararobustus. The Hop-lolaimus species have been particularly recorded in sugarcane fields (Spaull & Cadet,1990). Scleroderma cavenessi is considered to be the most harmful species to peanuts(Germani, 1981). The antagonistic effect of endomycorrhizal symbiosis on plant-parasitic nematodes (especially Meloidogyne spp.) is well documented (Thomson Casonet al., 1983; Duponnois & Cadet, 1994). On the contrary, no reports indicated theeffect of ectomycorrhizal associations on the multiplication of nematodes. In thepresent study, mycorrhizal association of A. holosericea with Sd 109 decrease H. para-robustus population. No effect has been recorded with A. mangium associated withthe same fungal strain. Moreover, Sd109 has caused an increase of the S. cavenessipopulation on A. mangium. The antagonistic effect of this fungal symbiosis isspecific to the host plant.

In conclusion, it appears that ectomycorrhizal symbiosis can enhance the growth ofA. holosericea and A. mangium. It can increase the nodulation of the root systems andcould act against plant parasitic nematodes. Moreover these plant species are compatiblewith an indigenous fungus S. dictyosporum. The controlled ectomycorrhizal symbiosiscould be very useful in afforestation programs in order to facilitate the growth ofthese Acacia speices.

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