Mycorrhizal associations in some Nigerian forest trees

[ 377 ] Trans. Br. mycol, Soc. 51 (3 and 4),377-387 (1968) Printed in Great Britain

MYCORRHIZAL ASSOCIATIONS IN SOME NIGERIAN FOREST TREES By J. F. REDHEAD

Department of Forestry, University

ofIbadan, Nigeria

(With 4 Text-figures) The incidence of mycorrhizal associations in fifty-one tree species indigenous to the Lowland Rain Forest (Keay, 1959) of Nigeria and in fifteen exotic tree species was investigated. All theexotic tree species and forty four ofthe indigenous species were found to have endotrophic mycorrhizal associations, and three indigenous species had ectotrophic associations. The significance of this is discussed. The types of association and their occurrence are described. The amount of external mycelium on the root was positively correlated with the incidence of the internal associate but neither was correlated with the presence or absence of root hairs and both were negatively correlated with rootlet depth in the soil.

The association of fungi with the roots of higher plants has been extensively studied and there are many records of the distribution in temperate regions. Trappe (1962) has published a list of species forming ectotrophic associations, and Butler (1939) has reviewed the morphology, anatomy, and distribution ofthe vesicular-arbuscular type ofendotrophic mycorrhiza. There have been few records from the lowland tropics, notably the extensive study by Janse (1896) in Java and more recently by Johnston (1949) in Trinidad, and Peyronel & Fassi (1957,1960) and Fassi & Fontana (1962) in the Congo. Janse found that sixty-nine of seventy-five species studied, including all the woody dicotyledons, had characteristic endotrophic mycorrhizas. Johnston examined ninety-three species, including thirteen species of forest trees, and observed that eighty, including all the forest trees, had endotrophic mycorrhizal associations. The reports by Peyronel, Fassi and Fontana were the first records of ectotrophic mycorrhiza in the tropical Lowland Rain Forest. MATERIALS AND METHODS

Collection of root material Specimens of root material were collected from Lowland Rain Forest and from plantations and nurseries in Western Nigeria. In order to retain mycelium on the root surface, specimens were washed by shaking gently in water and immediately placed in Karpechenko's fixative in sealed tubes. The composition of the fixative is: Solution A Chromic acid, 5 g Glacial acetic acid, 50 ml Distilled water, 425 ml

Solution B Formaldehyde (36%), 134 ml Ethyl alcohol (95%), 45 ml Distilled water, 246 ml

Equal parts of the fixative were mixed immediately before use.

Transactions British Mycological Society Examination of root material The fixed root material was placed in water and examined microscopically for external hyphae, root hairs and nodules. Species showing ectotrophic mycorrhizal association were sectioned by means of a freezing microtome. Both transverse and longitudinal sections were cut and stained with either cotton blue in lactophenol, or trypan blue according to the method of Minderman (1956). Longitudinal sections of the roots of other species were prepared using a hand razor. Sufficiently thick sections were cut to avoid the loss of stout intracellular hyphae and large hyphal inclusions, which tended to wash out of the cells when very thin sections were cut. Some roots were soaked in 15 % potassium hydroxide for 2-3 days, as described by Dowding (1959) before teasing out gently with a needle on a microscope slide. This method separated the cortical cells and enabled the pathway of the larger hyphae to be followed more easily than by sectioning. This material was stained with cotton blue in lactophenol and mounted in lactophenol. RESULTS

Over 100 specimens were collected from sixty-six species and these included fifty-eight genera and twenty-five families. Fifty-one species were indigenous trees of the Lowland Rain Forest and the remainder were exotic trees. Ectotrophic mycorrhizal associations Ectotrophic mycorrhizal associations were found in three species, Afzelia africana Sm., A. bella Harms var. bella and Brachystegia eurycoma Harms; all members of the Caesalpiniaceae. The specimen of A. africana was an 18 in tall seedling growing beneath the parent tree. All the fine rootlets were covered by a sheath 30 It thick made up of very fine hyphae. There was extensive formation of branched rhizomorphs up to 60 It diam. In A. bella all the young roots were swollen and greyish due to the presence of very large mantles offine compact septate hyphae. In the youngest lateral rootlets hyphae penetrated between the cells of the piliferous layer. The thickest mantle was observed on the tap root and this entirely covered the root cap (cf. Clowes, 1954). In order to verify the identity and to confirm a general occurrence of an ectotrophic association in this species further investigations were made. Seeds were collected from a mature tree of A. bella and sown in six isolated batches in a nursery in the Sapoba Forest Reserve. Soil from below the parent tree was added to three of the batches. Growth in the six plots was identical and the plants were lifted at 4 months old when about 18 in high. Microscopic examination showed the presence of a thick fungal mantle on the rootlets from all six plots. The mantles were very similar to those observed in the original specimen of A. bella, collected 20 miles away at the Obaretin Forest Reserve. Two collections of root material were made from isolated specimens of Brachystegia eurycoma. The first was a 9 in tall seedling growing on a fairly recently deposited bank of pure mineral sand, the second was a similar

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seedling in dark sandy soil very rich in leaflitter and rotting plant remains. Both were under the shade of parent trees. All the fine rootlets in both specimens were covered by a fungal mantle, but these were quite different. In the specimen growing on mineral sand the mantle was 35 It in a.verage thickness and composed of fine, almost colourless hyphae. Rhizomorphs up to 70 It thick were numerous and so were extensive masses of loose hyphae. Clamp connexions were not observed. The mantle ofthe specimen growing in soil rich in leaflitter was 40 It thick but composed of very stout deep brown hyphae 3-4 It diam and showing clamp connexions. Rhizomorphs up to 70 It diam occurred but the individual hyphae were more conspicuous, ramifying in the soil in loose masses and binding the soil particles together. Endotrophic mycorrhizal associations No fewer than eighty-eight specimens of root material, including all those from exotic trees, showed fungal hyphae within the root. Their morphology corresponded closely to endotrophic mycorrhizas, described by others in a wide range of plants (Hawker, Harrison, Nichols & Ham, 1957; J anse, 1896; Johnston, 1949; Lihnell, 1939; Mosse, 1963; Nicolson, 1959)·

100 1'

A

100 1'

Fig. I. Mycorrhiza of Khaya ioorensis. (A) L.S. outer cortex of root showing external and internal phases ofmycorrhizal fungus; p, piliferous layer; e, external hypha; i, intracellular hypha; T, 'resin' filled cells of cortex. (B) Root surface showing thick-walled external hyphae with finer branches.

The amount of external mycelium on the roots varied greatly and so did the number of root hairs, even on the same individual. In contrast to the observations of other workers (cf. Nicolson, 1959) hyphae could only be seen to enter the roots through the piliferous layer with certainty relatively rarely. Where they did the intracellular hyphae were typically extremely coiled (Fig. I). In an attempt to ascertain whether there was any correlation between

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the amount of external mycelium and (i) the distribution of root hairs, (ii) the depth in the soil of the rootlet sampled, and (iii) the amount of internal fungal associate, data were analysed for 105 seedlings of Khaya ivorensis A. Chevalier. These were grown in wooden tubs from a single batch of seed from the same parent tree. Rootlets from each seedling were inspected microscopically, the presence or absence of root hairs was recorded and the incidence of internal and external mycelium assessed as follows: five classes ofinfection by internal mycelium (0,5,25,60, go) were scored to correspond with the percentage ofcortical cells infected, excluding

A

Fig. 2. (A) External spores on mycelium ofrhizosphere of Khaya ivorensis; (B, C) Cortical cells of Nauclea diderrichii with intracellular vesicles.

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those full of resin which never appeared to be infected in Khaya, and four classes of infection by external mycelium (0, 10, 30, 50). Using the X2 test no significant correlation between presence or absence of root hairs, and, either the incidence of internal fungal associate, or the external mycelium, could be demonstrated. The coefficients of correlation (r) obtained were: between depth of rootlet examined and incidence of external mycelium - 0'2446, significant at the 0'02 P level (0'230) ; between depth of rootlet and incidence ofinternal fungal associate - 0'2 712, significant at the 0'01 P level (0'254) ; between incidence of external mycelium and incidence of the internal fungal associate +0'579, significant at the o'oolPleve1 (0'321).

100 Jl

Fig . 3. L.S. cortex of Khaya ioorensis showing vesicles (v), intracellula r hyphae (i) , and ' resin ' filled cells (r).

The morphology of the external hyphae (Fig. 1) was very similar to that described by Butler (1939), Peyronel (1924) and more recently by Nicolson (1959), who emphasized the dimorphic character of the mycelium. The thick-walled, stout hyphae averaged 7 It diam. and had granular contents usually staining deep blue with cotton blue in lactophenol, in contrast to the fine hyphal branches only 2 It diam, which were hyaline and did not stain, Only two spore-like bodies have been observed on the external mycelium and these were both on the same rootlet of Khaya ivorensis. They were terminal on fine hyphal branches and measured 36 x 24 It and 40 x 29 It respectively (Fig. 2A). It was not clear whether there were basal septa to

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the spores or not, but both had dense granular contents and one showed fine lines which appeared to be internal striations. They were similar in size and appearance to intracellular vesicles found in K. ivorensis and Nauclea diderrichii Merrill (Figs. 2B, C, 3). The incidence ofinternal fungal associates varied from individual hyphae to extremely congested associations where every cortical cell contained fungal materiaL The internal hyphae were usually stout (about 6 fl diam.) and possessed granular contents. They were normally aseptate but septa did occur occasionally as with the external hyphae. They usually stained blue with cotton blue in lactophenol but not invariably. Occasionally finer hyphae occurred but these were exceptional. Longitudinal sections of roots gave the impression that the hyphae were usually intracellular but the ease with which the larger hyphae teased out from the cortical cells after treatment with 15 % KOH suggested that the stout straight hyphae were often intracellular. Inside the cells the hyphae coiled intensively and often filled the cell space (Figs. I, 3). When hyphae passed through a cell wall they were usually unconstricted at this point. Often only fragments of hyphae could be discerned amongst a mass of granular material which appeared to be a product of hyphaI disintegration (Fig. 4A). In other cases hyphae branched frequently to form arbuscules (Fig. 4B, C) but these rarely appeared as finely branched structures and normally had the appearance of a cauliflower with the branching details obscured in a mass of granular material. Arbuscules were present in forty specimens of root material representing twenty-nine genera and nineteen families; most frequently in the roots of young plants. The lateral roots of eleven trees over 14 ft in height were examined and only those from one species, an exotic, Jacaranda ovalifolia R. Br., showed arbuscule formation. In only one case, a specimen of Tectona grandis L. with severely checked growth, were arbuscules found in the outer cortex. Three other specimens of root material from different species had some arbuscules in the mid-cortex but in all other cases arbuscules were segregated in a restricted zone in the inner cortex. Hyphae were not observed to enter the endodermis. Structures corresponding to the ,sporangioles' of J anse (1896) were found in only one species, Dalbergia sissoo Roxb., an exotic showing unhealthy growth on a poor site. Vesicles (Figs. 2 B, C, 3) were only found in three species Guarea cedrata Pellegr. ex A. ChevaL, Khaya ivorensis and Nauclea diderrichii. They were always intracellular and terminal with dense granular contents and usually a thick wall. In N. diderrichii many of the vesicles showed the fine lines or striations which have already been referred to in describing the external spore-like structures on K . iuorensis. Twelve specimens of root material did not show any fungal associate and of these, four were from young seedlings, three were from recent transplants just forming new roots, four were from healthy naturally regenerated saplings and one was from a z-year-old transplant showing poor growth. All the larger plants examined showed some degree ofincidence ofinternal fungal association, but this seemed to bear no relation to the health or vigour of the tree or to the intensity ofcompetition from other plant species. There was much variation between the development of the association in

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various tertiary roots of the same plant and between similar roots on different plants of the same species. Four species of the family Mimosaceae and one species of the family Papilionaceae had root nodules: all but one had some degree of internal fungal association as well. }.....

A

100 !'

50 II

Fig. 4. (A, B) L.S. cortex of Khaya ioorensis. (A) Intracellular hyphae in various stages of disintegration; (B) mainly intercellular hyphae; (C) L.S. cortex of Nauclea diderrichii with arbuscules and stages of hyphal 'digestion'.

DISCUSSION

Ectotrophic mycorrhizal associations The three species found to develop ectotrophic mycorrhizal associations were members of the Caesalpiniaceae, though four other species in this family possessed endotrophic mycorrhizal associations. It is interesting that the only other records of ectotrophic mycorrhizas from the Lowland Rain Forest were in this family and also from Africa. Obaretin Forest Reserve, where the first specimen of root material of Afzelia bella was collected, was 20 miles from Sapoba where seed was collected and sown to produce plants bearing an apparently identical ectotrophic mycorrhizal association. Material from A. africana in Ibadan 150 miles away appeared superficially very similar, though the hyphae from this host stained deeply with cotton blue whereas the hyphae from 25

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A. bella did not. On the other hand, the two associations on different specimens of Brachystegia eurycoma from different sites were quite dissimilar, one having very fine hyphae the other stout hyphae. These associations appear to be formed by more than one species of fungus. The hyphae from A. bella were septate and the stout hyphae from B. eurycoma also bore prominent clamp connexions. It is most probable that these ectotrophic mycorrhizal associations are formed by basidiomycetes. If this proves to be true then they must be capable of withstanding higher temperatures than those optimum for the basidiomycete species normally forming ectotrophic mycorrhizal associations in temperate regions, many of which have to be kept at low temperatures for continued growth in culture. Harley (1959) quoted an optimum temperature of zo °C for most species, but Hackskaylo, Palmer & Vozzo (1965) found that for some species the optimum temperature was as high as 29° which is the mean maximum temperature near ground level in the Lowland Rain Forest of Nigeria (Evans, 1939). Endotrophic mycorrhizal associations Occurrence It is apparent that almost all woody species of the Lowland Rain Forest form endotrophic mycorrhizas. The formation of this type of mycorrhiza did not appear to be associated with the vigour of the tree, its place in the canopy with regard to light, and the nutrient status of the soil although these factors may affect the association. Most of the specimens lacking the association were very young and had grown on newly cleared and burnt nurseries. It is possible that changes in the creation of a new nursery, probably the fierce burning in clearing forest debris make the soil a less favourable environment for endotrophic fungi which could infect the young seedlings. Variation in anatomy In several specimens of root material two types of internal hyphae were observed. Sometimes the hyphae differed in their degree of stoutness and sometimes in their capacity to take up the cotton blue stain. This may possibly be due to more than one species of fungus forming associations within the same root. In almost all cases there was a definite pattern of distribution of fungal mycelium within the root tissues, especially when arbuscules were present. Arbuscules were most frequently found in young plants and were usually restricted to rows of cells in the inner cortex (cf. Johnston, 1949). It has not been possible to identify hyphae in cortical cells nearer than the row of cells containing arbuscules. This may suggest that the endodermis or inner cortical cells produce some factor controlling hyphal spread. The correlation offungi with the presence of root hairs If the external mycelium of the fungal partner of the endotrophic association absorbs nutrients, trees forming the association might be

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expected to have fewer root hairs or no root hairs when compared with trees which have not formed the association. This expected correlation was not found in Khaya ivornesis nor apparent in the general survey.

The significance of mycorrhizal associations There are many reports that under certain conditions the formation of an ectotrophic mycorrhizal association is beneficial or even essential to tree growth (Gendina, 1960; Klotz, 1956; Raets, 1962; Van Suchtelen, 1962; Wright, 1957; Tserling, 1960). Melin & Nilsson (1950, 1953) have demonstrated that the hyphae growing out from an ectotrophic mantle can have a function similar to that of root hairs in that mineral nutrients can be passed from the external environment via fungal hyphae to the higher plant, whilst Harley & Brierley (1954), Harley, Brierley & McCready (1954) and Harley & McCready (1950, 1952a, b), using radioactive isotopes and detached beech roots, found that the fungal mantles of mycorrhizal roots absorbed phosphate five times and potassium twice as rapidly as non-mycorrhizal roots. Experimental work on endotrophic mycorrhizal associations, other than in orchids, has been restricted by the difficulty ofisolation of the endophyte in order to synthesize the association under experimental conditions. Recent work has been discussed by Mosse (1963) and several workers in temperate regions have demonstrated that a wide range of plants show improved growth under certain conditions when they have developed an endotrophic mycorrhizal association (Baylis, 1959,1961; Baylis, McNabb & Morrison, 1963; Daft & Nicolson, 1966; Gerdemann, 1964, 1965; Meloh, 1961; Mosse, 1957). Baylis, McNabb and Morrison found that, after two years, seedlings of Podocarpus totara G. Berm, ex D. Don. infected by a phycomycetous mycelium weighed seven times those grown in a fungusfree condition. The external mycelium appears to provide a highly efficient absorption mechanism for nutrients, particularly for phosphorus. Mycorrhizal plants are able to grow on soils which would normally prove too deficient in phosphorus for non-mycorrhizal plants. This may have great significance in the ecology of the Lowland Rain Forest where heavy rainfall causes rapid leaching, and where phosphorus is often in short supply. The present study in Nigeria confirms the almost universal occurrence of mycorrhizal associations in species examined from lowland tropical forests. Further investigations are needed to ascertain the nature of the fungi forming these associations and the effect of these associations on the growth of trees under rain forest conditions. REFERENCES

BAYLIS, G. T. S. (1959). Effect of vesicular-arbuscular mycorrhizas on growth of Griselinia littoralis (Comaceae). New Phytol. 58, 274-280. BAYLIS, G. T. S. (1961). The significance of mycorrhizas and root nodules in New Zealand vegetation. Proc. roy. Soc. N·Z· ag, 45-50. BAYLIS, G. T. S., McNABB, R. F. R. & MORRISON, T. M. (1963). The mycorrhizal nodules of Podocarps. Trans. Br. mvcol. Soc. 46, 378-384. BUTLER, E. J. (1939). The occurrence and systematic position of the vesicular-arbuscular type of mycorrhizal fungi. Trans. Br. mycol. Soc. 22, 274-301.

Transactions British Mycological Society CLOWES, F. A. L. (1954). The root cap of ectotrophic myccorhizas. New Phytol. 53, 5 25-529. DAFT, M.J. & NICOLSON, T. H. (1966). Effect of Endogone mycorrhiza on plant growth. New Phytol. 65, 343-350. DOWDING, E. S. (1959). Ecology of Endogone. Trans. Br. mycol. Soc. 42, 449-457. EVANS, G. C. (1939). Ecological studies on the rain forest of Southern Nigeria. II. The atmospheric environmental conditions. J. Ecol. 27, 436-482. FASSI, B. & FONTANA, A. (1962). Micorize ectotrofiche di Brachystegia laurentii e di alcune altre Cesalpiniaceae minori del Congo. Allionia 8, 12 I- 131. GENDINA, S. B. (1960). [Mycorrhiza accelerates growth of Pine.] (Russian.) Les. Khoz, I2, 39-41. GERDEMANN, J. W. (1964). The effect ofmycorrhiza on the growth ofMaize. Mycologia 56, 34 2-349. GERDEMANN, J. W. (1965). Vesicular-arbuscular mycorrhizae formed on Maize and Tulip Tree by Endogonefasiculata. Mycologia 57, 562-575. HACSKAYLO, E., PALMER,J. G. & Vozzo,J. A. (1965). Effect of temperature on growth and respiration of ectotrophic mycorrhizal fungi. Mycologia 57, 748-756. HARLEY. J. L. (1959). The biology if mycorrhiza. London. HARLEY,J. L. & BRIERLEY,J. K. (1954). The uptake of phosphate by excised mycorrhizal roots of the Beech. VI. Active transport of phosphorus from the fungal sheath into the host tissue. New Phytol. 53, 240-252. HARLEY,J. L., BRIERLEY,J. K. & MCCREADY, C. C. (1954). The uptake of phosphate by excised mycorrhizal roots of the Beech. V. The examination of possible sources ofmisinterpretation of the quantities ofphosphorus passing into the host. New Phytol. 53,92-g8. HARLEY,J. L. & MCCREADY, C. C. (1950). The uptake of phosphate by excised mycorrhizal roots of the Beech. New Phytol. 49, 388-397. HARLEY,J. L. & MCCREADY, C. C. (I952a). The uptake of phosphate by excised mycorrhizal roots of the Beech. II. Distribution of phosphorus between host and fungus. New Phytol. 49,388-397. HARLEY, J. L. & MCCREADY, C. C. (I952b). The uptake of phosphorus by excised mycorrhizal roots of the Beech. III. The effect of the fungal sheath on the availability of phosphate to the core. New Phytol. 5I, 342-348. HAWKER, L. E., HARRISON, R. W., NICHOLS, V. O. & HAM, A. M. (1957). Studies on vesicular-arbuscular endophytes, I. Trans. Br. mycol. Soc. 40, 375-390. JANSE, J. M. (1896). Les endophytes radicaux des quelques plantes Javanaises. Annls Jard. bot. Buitenz, I4, 53-2 I2. JOHNSTON, A. (1949). Vesicular-arbuscular mycorrhiza in sea island cotton and other tropical plants. Trop, Agric., Trin. 26, I 18-12 I. KEAY, R. W.J. (1959). An outline if Nigerian vegetation, 3rd ed. London: Crown Agents. KLOTZ, K. (1956). Praktische Auswertung des Mykorrhiza-Problems in der Forstwirtschaft, AUg. Forstz, n, 362-363. LIHNELL, D. (1939). Untersuchungen uber die Mycorrhizen und die Wurzelpilze von Juniperus communis. Symb. bot. upsal, 3, 1-141. MELIN, E. & NILSSON, M. (1950). Transfer of radioactive phosphorus to Pine seedlings by means of mycorrhizal hyphae. Physiologia Pl. 3, 88-92. MELIN, E. & NILSSON, M. (1953). Transfer of labelled nitrogen from glutamic acid to Pine seedlings through the mycelium of Boletus variegatus. Nature, Lond. I7I, 134. MELOH, K. A. (1961). Untersuchungen zur Biologie und Bedeutung der endotrophen Mycorrhiza bei Zea Mays L., und Avena sativa L. Dissertation Universitat Koln. pp. 1-77· MINDERMAN, G. (1956). New techniques for counting and isolating free living nematodes from small soil samples and from oak forest litter. Nematologia I, 216-226. MOSSE, B. (1957). Growth and chemical composition of mycorrhizal and non-mycorrhizal Apples. Nature, Lond. I79, 922-g24. MOSSE, B. (1963). Vesicular-arbuscular mycorrhiza: an extreme form offungal adaptation. Symbiotic Associations, pp. 146-170. London: Cambridge University Press. NICOLSON, T. (1959). Mycorrhiza in the Graminae. I. Vesicular-arbuscular endophytes with special reference to the external phase. Trans. Br. mycol. Soc. 42, 421-438.

Mycorrhizal associations. J. F. Redhead PEVRONEL, B. (1924). Specie di' Endogene' produtti de micorizie endotrofiche. Boll. Staz, Patol. veg. Roma 5, 73-75. PEVRONEL, B. & FASSI, B. (1957). Micorrize ectotrofiche in una Cesalpiniacea del Congo belga Atti Acad. Torino 9I, 8 pp. PEVRONEL, B. & FASSI, B. (1960). Nuovi casi de simbiosi ectomicorrizica in Leguminose della famiglia delle Cesalpiniacea. Atti Acad. Torino, 94, 3 pp. RAETS, G. H. (1962). Apuntes prelirninares sobre el desarrollo del Pinus caribae en el vivero en relacion con la presencia 0 ausenciade la micorriza, Bol. Inst. For. Lat. Am. 9,1-15. TSERLING, G. I. (1960). [Mycorrhiza formation in Larch under the conditions of Transvolga chernozem soils and modes of its stimulation.] (Russian.) Microbiology, Moscow 29, 401-407· TRAPPE, J. M. (1962). Fungus associates of ectotrophic mycorrhizae. Bot. Rev. 28, 538606. V AN SUCHTELEN, N. L. (1962). [Mycorrhiza on Pinus spp. in the tropics.] (Dutch.) Meded. Land. Hogesch. Gent. 27, 1104-1106. WRIGHT, E. (1957). Importance ofmycorrhizae to Ponderosa Pine seedlings. For. Sci. 3, 275-280.

(Accepted for publication 25 April 1967)