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342-IJBCS-Article-Dr E E Tambe Bechem Available online at http://www.ajol.info Int. J. Biol. Chem. Sci. 3(3): 578-586, 2009 ISSN 1991-8631 Original Paper http://indexmedicus.afro.who.int Inoculum production and inoculation of Gnetum africanum rooted cuttings using a range of mycorrhizal fungi Eneke Esoeyang TAMBE BECHEM 1* and Ian James ALEXANDER 2 1 Department of Plant and Animal Sciences, Faculty of Science, University of Buea, P.O. Box 63 Buea, South West Region, Cameroon. 2 University of Aberdeen, School of Biological Sciences, Plant and Soil Sciences, Cruickshank Building, Aberdeen AB24 3UU. * Corresponding author, E-mail: [email protected] ABSTRACT We studied the ability of peat/vermiculite, barley, rye grain, baby corn and nitrocellulose filter paper to serve as carriers for inoculum production using an isolate of Scleroderma sinnamariense , Scleroderma sp., Paxillus involutus and three isolates of Pisolithus tinctorius . Soil collected from the vicinity of some ectomycorrhiza tree species, mycorrhiza root tips, mycelium and spores of S. sinnamariense were also tested as inoculum. Inoculation of Gnetum rooted cuttings was by the use of both dried and fresh sporocarps of the fungus as well as by use of inoculum prepared on the carriers mentioned above. All fungal isolates showed some growth on peat/vermiculite, barley, rye grain, baby corn and nitrocellulose filter paper. The Paxillus and Pisolithus isolates grew better on these carriers as compared to the Scleroderma isolates. The use of viable root tips, mycelium and spores of S. sinnamariense led to the formation of ectomycorrhiza on the previously uncolonized plants. This method can be successfully used to produce ectomycorrhizal Gnetum rooted cuttings, which may be used for outplanting thereby increasing their chances of survival. © 2009 International Formulae Group. All rights reserved. Keywords : Scleroderma sinnamariense, Pisolithus, Paxillus, ectomycorrhiza, inoculum production. INTRODUCTION America. This plant which is in high demand Gnetum africanum and G. yields a profit margin of about $2,630 per year buccholzianum are gymnosperm lianas found per household (Fondoun and Manga, 2000). in the forests of Cameroon as well as in other The overexploitation of the plant and the countries of Central and West Africa. The unsustainable harvesting methods puts this leaves are used as a vegetable and are a good plant at risk of extinction. With the current source of proteins and mineral salts (Fokou and steady decline in area of forests in West and Domngang, 1989; Mialoundama, 1993). and Central Africa, supplies of Gnetum spp. These plants have also been shown to have are becoming scarce (Shiembo et al., 1996). important ethnopharmacological uses There is a need therefore, to look for ways to (Robyns, 1948; Watt and Breyer-Brandwijk, improve cultivation of the plant. 1962; Bouquet, 1969; Lowe, 1984; Schultes Gnetum spp. are reported to form and Raffauf, 1990; Paru et al., 1995). ectomycorrhiza (EM) (Mialoundama and The collection and sale of Gnetum Mbou, 1992; Ingleby, 1999; Onguene and leaves is important in the cross border trade Kuyper, 2001). Mycorrhiza is a symbiotic between Cameroon and its neighbours as well association formed by some soil inhabiting as with Europe and the United States of fungi and the roots of higher plants. In this © 2009 International Formulae Group. All rights reserved. E. E. TAMBE BECHEM and I. J. ALEXANDER / Int. J. Biol. Chem. Sci. 3(3): 578-586, 2009 relationship, the fungus receives published literature exists on the production of photosynthetically derived carbon compounds inoculum using Gnetum ectomycorrhiza fungi from the plant. The plant in turn benefits by associates and synthesis of EM with Gnetum . increased uptake of mineral nutrients, possibly Presently in some research institutions in improved disease and toxin resistance as well Cameroon, for example the Limbe Botanic as water relations (Smith and Read, 1997). Garden and at International Centre for Two major types of mycorrhizal associations Agroforestry Research (ICRAF), Gnetum exist depending on the position of the fungus rooted cuttings used in domestication trial in relation to the root surface: ecto- experiments are inoculated by planting in mycorrhizas with intercellular colonization rhizosphere and bulk soil collected from the and endomycorrhizas with both inter- and natural habitat of the plant for some time in intracellular colonization (Smith and Read, the nursery, prior to outplanting. With this 1997). method of inoculation only a limited number Establishment of Gnetum plantations in of mycorrhizal plants can be produced Cameroon, which are expected to reduce because of the difficulties involved in pressure on the wild stock, is desirable. It is obtaining inoculum. It is therefore imperative envisaged that successful domestication will that other methods of inoculation be studied. depend partly on mycorrhization of the rooted Pisolithus tinctorius and Paxillus cuttings before outplanting. However for this involutus , two broad host range EM fungi to occur, information on the potential and were included in these inoculation actual fungal partners is needed. A method of experiments. inoculation will also be vital. The main objective was to synthesize Devising methods to synthesise mycorrhizas by inoculating rooted Gnetum mycorrhizas is also essential to provide cuttings with pure mycelial cultures or spores experimental material to investigate the of EM. physiology of the symbiosis, and the role of The specific objectives were to evaluate; EM colonization in the growth and nutrition • The ability of Gnetum to form of Gnetum . Attempting inoculum production mycorrhizas with broad host range EM on different substrates and the use of different fungi. techniques would give an indication of the • The potential of different substrates to capacity of S. sinnamariense to withstand the support growth of the EM fungal physical, chemical and biological associates of Gnetum (inoculum manipulations involved. production). Mycorrhizas can be synthesised by • Different methods of inoculation. bringing seedlings of host plants and EM • The possibility of forming arbuscular fungi inoculum close to each other in a mycorrhiza (AM) on Gnetum using favourable growth environment. Synthesis has Gigaspora margarita and Glomus been achieved through several different mosseae, which are broad host range and methods, using both sterile and non sterile widespread fungi. techniques (Fortin et al., 1983; Wong and • EM synthesis attempts between Hopea Fortin, 1989; Burgess et al., 1994). The odorata and Scleroderma sinnamariense. advantages and shortcomings of the different techniques have been discussed by Peterson MATERIALS AND METHODS and Chakravarty (1991). These experiments were run in the In all the inoculation trials described in screen house at the Department of Plant and this paper, non-sterile techniques were used. It Soil Sciences, University of Aberdeen, was difficult to get sterile plants because of Scotland, United Kingdom. the method of propagation employed in their production. Our major interest was to look at Production of Gnetum rooted cuttings compatibility and mycorrhiza formation under The propagation method adopted here circumstances as close to that in nature as was based on Shiembo et al . (1996) but with possible. Cuttings were used in this slight modifications. The Gnetum stock plants experiment because of the difficulties in were kept in a phytotron with a relative germinating Gnetum seeds. To date, no humidity of 85%, temperature 26 °C and a 579 E. E. TAMBE BECHEM and I. J. ALEXANDER / Int. J. Biol. Chem. Sci. 3(3): 578-586, 2009 photoperiod of 8 h darkness and 16 h light. The methodology used in inoculum Plants were watered as required. The plants production was based on that of Marx and were held under these conditions for about Bryan (1975). Each isolate was run in three two months prior to the propagation replicates. The pH of the carrier prior to and experiment. Propagation was carried out in a after autoclaving was 5.4. Incubation was at heated bench top non-mist propagator. The 30 °C for all Pisolithus isolates and S. propagator consisted of a tray about 60 cm sinnamariense whilst the Paxillus involutus long, 32 cm wide and a depth of 8 cm. The and Scleroderma collected from Aberdeen tray had a lid made of transparent polythene. were incubated at room temperature (10-15 The rooting medium was peat °C) for 6-8 weeks. A peat/sand mixture in a (SHAMROCK ) mixed with coarse sand 1:1 ratio was used as potting medium during (washed concrete sand) in a 1:1 ratio. The inoculation. Two methods of inoculation were coarse sand had initially been washed several carried out. One in which the inoculum was times with tap water followed by decantation incorporated in an 8:1 ratio through out the between successive washings. The rooting potting medium and another in which the medium was used to fill the tray to the brim mycorrhizal inoculum was banded in the and watered to field capacity. The temperature potting medium. Each fungus isolate was run in the propagator was set at 25 °C. A total of in three replicates for each inoculation 60 half-leaf cuttings were used in the procedure. Plants were kept for 16 weeks on a propagation. Watering was done as required bench top in the greenhouse with temperature by use of a fine sprayer. Plants were observed of 25-30 °C, relative humidity of about 85% for any signs of rooting fortnightly, thereafter and a photoperiod of 8 h dark and 16 h light. observations continued
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