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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 africanum rooted cuttings using a range of mycorrhizal fungi

Eneke Esoeyang TAMBE BECHEM 1* and Ian James ALEXANDER 2

1 Department of and Animal Sciences, Faculty of Science, University of Buea, P.O. Box 63 Buea, South West Region, . 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 . 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 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 . 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 after every four weeks Watering was done as required. for the rest of the 16 weeks experimental period. Inoculum production and EM inoculation of Gnetum using inoculum on natural Inoculum production and synthesis substrate attempts The substrates were chosen arbitrarily Rooted cuttings were used in synthesis based on availability. In the process of experiments only after they had developed inoculum production, 2 g of barley straw sufficient numbers of lateral roots (visual (about 1 cm in length) moistened with 20 ml appreciation). The origin, host plant and of 1% glucose solution was autoclaved at 121 species of EM fungi used in the inoculation °C for 30 minutes in 100 ml Erlenmeyer trial experiment are shown on table 1. flasks. Prior to autoclaving, the pH of mixture The following carriers and techniques was adjusted to 5.0, flasks capped with cotton were used in the synthesis of ectomycorrhizas wool and aluminium foil. Contents were with Gnetum rooted cuttings; allowed to cool to room temperature before • Inoculum production and synthesis using inoculation under sterile conditions with 8 peat/vermiculite. fungal mycelia discs (5 mm diameter) cut • Inoculum production and synthesis using from the edge of actively growing mycelia natural substrates (barley, rye grain and from each of the different fungal isolates, held baby corn). on MMN agar plates. • Synthesis using mycelium inoculum on 2 g of baby corn was chopped into bits filter paper. of about 0.5-1 cm in diameter and processed • Synthesis using soil inoculum. as described for barley straw above. 2 g of rye • Synthesis using spores, mycorrhiza root grain was also processed in the same manner. tips and mycelium of S. sinnamariense . Incubation was at 30 °C in the dark for 6-8 weeks. Inoculum production and EM inoculation of Gnetum using peat/vermiculite EM synthesis with Gnetum using paper- All the EM fungi: Paxillus , Pisolithus based inoculum in perspex plates and the two Scleroderma isolates were Inoculum production was by the initially held on Modified Melin Norkran method described in Chilvers et al. (1986). ° (MMN) (Marx, 1969) agar plates. Incubation was at 30 C in the dark 580 E. E. TAMBE BECHEM and I. J. ALEXANDER / Int. J. Biol. Chem. Sci. 3(3): 578-586, 2009

Table 1: The species, origin and host of six ectomycorrhizal fungi isolates used in in vitro synthesis trials .

Species Host Origin Ref. code Paxillus involutus Larix sp . United Kingdom P1014 Pisolithus tinctorius Pinus sp . Thailand Pt msn Pisolithus tinctorius Acacia mangium Senegal Pt COI32 Pisolithus tinctorius Pinus sp . Australia Pt John Cairney Scleroderma sinnamariense Gnetum africanum Cameroon Scleroderma Scleroderma sp. Tilia Aberdeen- Scotland Scleroderma fence

A

M

B C

Plate 1: Synthesized Gnetum ectomycorrhizas. ( A) Gnetum africanum + Scleroderma sinnamariense. (B) transverse section of nonmycorrhizal Gnetum root (C) transverse section of ectomycorrhizal Gnetum root. M shows the Mantle and arrow shows the Hartig net. Bar in B is 60 µm while that in C is 20 µm.

for 4 weeks. Inoculation of cuttings was done inoculum. This was to stabilise and allow for in Perspex plates using the method of Finlay further development of lateral roots. and Read (1986). Watering was done as Incubation following inoculation was for 16 required and plants were held in this condition weeks. Eight replicate plates were run. During for 4 weeks before introduction of the fungal observation, upper sheet of plate and 581 E. E. TAMBE BECHEM and I. J. ALEXANDER / Int. J. Biol. Chem. Sci. 3(3): 578-586, 2009 inoculum paper were carefully removed, inoculation, each pot containing an assay plant before examination of roots using stereo and was turned upside down; pot removed leaving compound microscopes where necessary. the plant with the intact root system held in Fungal growth in all carriers was the growth medium. A mycorrhizal system evaluated by visual observation and by the use with viable tips and mycelia collected from of a stereomicroscope. The presence of the ectomycorrhizal plant was placed in close mycelia of the fungus around and within the proximity to ultimate lateral roots (targets for carrier was an indication of growth. In the mycorrhization) of the test plant. Test plants process of assessing growth within the carrier, were then carefully placed into pots and a small portion of the carrier containing fungal watering was done as required. A total of mycelium was aseptically collected, placed in eight plants were inoculated with a Petri dish and observed for the presence of ectomycorrhizal system and mycelium. mycelium within the carrier with the aid of a Incubation was in the greenhouse for 16 stereomicroscope. weeks and watering was done as required. Observation was done as previously EM inoculation of Gnetum using soil explained. inoculum Decomposing material from the soil EM inoculation of Gnetum using spores surface containing abundant ectomycorrhizal Fresh sporocarps of S. sinnamariense roots was collected from beneath birch were collected from the vicinity of the (Betula pendula ), beech ( Fagus sylvatica ) and ectomycorrhizal Gnetum plant. Each Scots ( Pinus sylvestris ) growing in sporocarp was cut into two halves to expose Aberdeenshire, UK. The material was the spores and each half was placed in contact combined and passed through a coarse (1 cm) with lateral roots of Gnetum plant in free- sieve. Soil was processed and used in draining plastic pots (15 × 15 cm F). inoculation, within 72 hours of collection. The Inoculation was also done by use of dried sieved mixture was added to pre-washed sand spores of S. sinnamariense . This was simply in a 1:1 ratio. This was then used to pot rooted by breaking the dried sporocarp containing Gnetum cuttings in 15 × 15 cm F free-draining mature spores and putting both spores and plastic pots. An uninoculated control was run, peridium into the pot close to the roots of test in which a portion of the sieved soil was plants. Four plants were inoculated with fresh sterilised twice at 121 °C for 1 h with a 24 h sporocarps and four with dried spores. This period between the two sterilisations. The brought the total to eight plants in the sterilised soil and washed sand were mixed in experiment. Plants were watered as required a 1:1 ratio. The resulting mixture was used to and incubation was for a 16 weeks period in pot Gnetum cuttings. There were a total of the green house. Observation was done at the eight inoculated and eight control plants. end of the incubation period. Incubation was in the greenhouse for 16 weeks. Watering was done as required. Inoculation of Gnetum with AM fungi During observation, a pot was tipped over and The fungi isolates used here were removed from the soil; roots were then Glomus mosseae (BEG 12) and Gigaspora observed with the aid of a hand lens for any margarita (BEG 34). They were from the signs of colonization. At the end of the European Bank of Glomales. The fungi were experiment, roots were carefully separated held in pot cultures of steamed sand and washed free of soil before observing both (autoclaved twice at 121 °C for 1 hour with 24 visually and microscopically based on method hours between sterilisation) with Sorghum sp. described in Brundett et al. (1996). as host plant. The inoculum obtained therefore consisted of spores held in a fine sand mix. EM inoculation of Gnetum using root The potting medium for the Gnetum plants fragments was peat/sand 1:1. Medium was autoclaved Mycelia and mycorrhizal root systems for 1 h at 121 °C. It was then allowed to cool were harvested from roots and the vicinity of for 24 h, followed by a second autoclaving established Gnetum potted plants, colonized session for the same period of time and by Scleroderma sinnamariense. During temperature. Sterile medium was used to fill 582 E. E. TAMBE BECHEM and I. J. ALEXANDER / Int. J. Biol. Chem. Sci. 3(3): 578-586, 2009 free-draining pots (12.7 × 12.7 cm F). During this period, giving a total rooting percentage inoculation, a planting hole was made in each of 68.3%. Enough rooted cuttings were pot, and one tablespoonful of inoculum mix of therefore available to run the inoculation either Gigaspora or Glomus species was experiment. added into the planting hole prior to the introduction of the plant. Six plants were Inoculum production inoculated with each fungus species resulting At the end of the 8 weeks incubation to a total of twelve plants used in the period, mycelium from each fungus isolate experiment. Incubation was for a 16 weeks had grown into the vermiculite/peat substrate, period in the greenhouse. Plants were watered barley, rye grain, baby corn and nitrocellulose as required. After 16 weeks of growth, root filter paper. However growth of the samples were collected from plants initially Scleroderma isolates was poor relative to the inoculated with Glomus and Gigaspora and Pisolithus and Paxillus isolates. S. observation for AM was as described in sinnamariense showed extremely limited Brundett et al. (1996). growth in barley, rye grain and baby corn, but grew well on nitrocellulose paper placed on EM synthesis between Hopea sp. and S. MMN agar. sinnamariense The purpose of this experiment was to Mycorrhiza synthesis have an idea as to whether S. sinnamariense Inoculation of Gnetum was possible from Gnetum in Cameroon could form with the use of root fragments collected from mycorrhiza with Hopea odorata from the vicinity of already established Malaysia. We used two years old Hopea sp . ectomycorrhizal Gnetum plants. At the end of rooted cuttings. The rooted cuttings were non- the experimental period, all eight plants had mycorrhizal and this was confirmed by formed ectomycorrhizas with the fungus. microscopy of free hand sections as well as Ectomycorrhizas were observed as early as six cleared and stained roots. The experimental weeks after inoculation. Mycorrhiza tips and potting medium was a 1:1 mixture of mycelium had the characteristic bright yellow peat/sand. Potting medium was used to fill coloration (Plate 1) which is usually observed free-draining pots (9 × 9 cm F). Viable on this fungus-plant association in nature. The ectomycorrhizal root systems and mycelia mycorrhiza had a well developed mantle and were collected from a well established Hartig net (Plate 1). Inoculation was also Gnetum plant colonized with S. sinnamariense possible with the use of spores of S. from Cameroon. A combination of sinnamariense . At the end of the experimental ectomycorrhizal root fragments and mycelium period, all eight plants had formed were placed in the vicinity of the roots of the ectomycorrhiza with the fungus. Mycorrhizas Hopea seedlings during potting. Twenty were established as early as 8 weeks. The Hopea seedlings were inoculated in the mycorrhizas had a bright yellow coloration experiment. Incubation was in the greenhouse with a well developed mantle and Hartig net. for 16 weeks. Plants were watered as required. Gnetum did not form mycorrhizas with the At the end all mycorrhizal synthesis Paxillus and Pisolithus isolates used in this experiments, roots were sampled for study. Mycorrhiza synthesis using paper- colonization using method described in based inoculum on Perspex plates was also Brundett et al. (1996). Confirmation of impossible with all the isolates tested. The colonization was by making free hand fungal species present in the soil inoculum sections of suspected EM root tips and were unable to initiate mycorrhiza formation observing for the presence of a mantle and/or with Gnetum . Hartig net. Gnetum did not form arbuscular mycorrhizas with the Glomus and Gigaspora RESULTS species used in this study. The cleared and At the end of the 16 weeks, Gnetum stained roots had no arbuscules, vesicles or cuttings which had not rooted had at least hyphal coils which are characteristic features developed a callus. Out of the 60 cuttings used of this type of mycorrhiza. in the propagation, 41 developed roots within 583 E. E. TAMBE BECHEM and I. J. ALEXANDER / Int. J. Biol. Chem. Sci. 3(3): 578-586, 2009

EM synthesis between Hopea sp. and S. with the fungus depended on the rate of sinnamariense growth of the fungus species. They found that The Hopea plant was unable to form fast-growing fungi like Thelephora terrestris mycorrhizas with S. sinnamariense from and Pisolithus tinctorius totally colonized the Cameroon. All twenty Hopea plants still had substrate in 2-litre containers in 2-3 months non-mycorrhizal roots at the end of the whereas slow-growing fungi such as experiment. Cleared and stained root sections Cenococcum geophilum would take up to 8-10 showed neither a mantle nor Hartig net which months to colonize the same amount of are features characteristic of ectomycorrhizas. substrate. It is also possible that the fungus could not stand the physical handling involved DISCUSSION in processing the inoculum. It was demonstrated during this study In spite of the fact that the Pisolithus that Gnetum spp . are predominantly and Paxillus species in the current study had ectomycorrhizal. Mycorrhiza formation was totally colonized the carrier prior to possible only with Scleroderma inoculation of the plant, there was no EM sinnamariense , a natural fungal partner to the formation. The absence of colonization of plant. Mycorrhization could be achieved prior Gnetum by these species is probably due to to outplanting by a simple method requiring lack of compatibility. Gnetum shows the use of mycorrhiza tips, mycelia and spores seemingly strict host-fungus specificity and of the fungus. The sustainability of this has mostly been associated with S. method is due to the fact that sterile sinnamariense . Nonetheless it was found conditions and other additional technical during a related field survey, that it also does know-how are not necessary. Furthermore, in form EM with another fungus which we this method root tips and mycelia from one suspect to be a Scleroderma sp . But S. mycorrhizal plant could be used to inoculate sinnamariense was the most prevalent and about 30 or more rooted cuttings, resulting in widespread fungal partner of Gnetum spp . in the use of less soil inoculum which is bulky, Cameroon. time consuming and expensive, with possible The limited growth of S. sinnamariense introduction of pathogens. It is important to on rye grain, baby corn and barley straw may note that the use of soil inoculum is not be due to several reasons. One possibility is feasible in large scale production systems. that growth inhibitors may have been released However more of such studies need to be during autoclaving. Similarly a lack of growth carried out before an effective, reproducible by typically fast growing fungi such as and reliable method of inoculum production Pisolithus tinctorius and Thelephora terrestris and inoculation is adopted for Gnetum . on peanut hulls, perlite, corn cobs and pine There are several possible reasons for barks has been observed as reported by Marx the inability of S. sinnamariense grown on and Kenney (1982). nitrocellulose paper and peat/vermiculite to The failure of Gnetum to form colonize Gnetum . The paper may have been arbuscular mycorrhizas with Glomus and rapidly colonized by bacteria hence reducing Gigaspora is surprising. This is because about the inoculum potential of the fungus. The 90% of land plants form AM with these fungus may have lost its viability before fungal species (Smith and Read, 1997). colonization of the roots could commence. Another reason is that Gnetum always The inoculum potential on peat/vermiculite harbours ectomycorrhiza even when growing may have been low due to the poor growth of amongst Chromolaena odorata, which is a the fungus on this carrier. This is possible plant that forms AM. Such an observation, because at the time of inoculation, the fungus points to the fact that Gnetum might be strictly had not thoroughly colonized the carrier. A ectomycorrhizal. Members of the Gnetaceae longer incubation time may be needed for have always been reported to form inoculum production with this fungus since it ectomycorrhizas (Fassi, 1957; St. John, grows slowly. This would allow the fungus to 1980b; Smith and Read, 1997), although thoroughly colonize the substrate. Marx and Onguene (2000) reported the occasional Kenney (1982) reported that the length of observation of AM structures in some Gnetum incubation following inoculation of substrate root fragments collected from South 584 E. E. TAMBE BECHEM and I. J. ALEXANDER / Int. J. Biol. Chem. Sci. 3(3): 578-586, 2009

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