Arbuscular mycorrhizas of Vangueria infausta Burch. subsp. infausta (Rubiaceae) from South Africa Academic research paper on "Biological sciences"

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S. Afr. J. Bot. 1999, 65(5 & 6): 434-436

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Arbuscular mycorrhizas of Vangueria infausta Burch. subsp. infausta (Rubiaceae) from South Africa

A. Gaur, J.V. van Greuning* R.C. Sinclair and A. Eicker

Department of Botany, University of Pretoria, Pretoria, 0002 Republic of South Africa

Received 19 March 1999: revised I (¡August 1999

Rhizosphere soil samples and root pieces of Vangueria infausta were collected from three different sites. Arbuscular mycorrhiza! (AM) fungus spores of four species were isolated from the soil. Roots were examined for the presence and density of AM fungal structures AM fungal colonization ranged from 66-79% with intracellular hyphal coils forming the major component of AM fungal infection. No vesicles were observed.

Keywords Arbuscular mycorrhiza, Vangueria infausta, Glomus, Gigaspora-

*To whom correspondence should be addressed (E-mail: jvgreun@scientia.up.ac.za)

Arbuscular mycorrhiza! (AM) fungi occur in mutualistic symbiosis in the roots of many plant species and their rote in plant growth and nutrition, water stress, and resistance to disease are well established (Lynch 1990). In this association, the AM fungi, after colonizing the host root cortex, develops a hyphal network Forming various structures in the cortical cells. The intricate architecture shown by mycelium highlights the potential for enhanced nutrient exchange that is beneficial to both AM fungi and host plants (Harley & Smith ¡983).

AM fungi are widespread in horticultural crops. There are reports of improved growth and disease resistance of fruit crops (Lovato et til. 1994) due to mycorrhizal associations. Wild medlar ( Vangueria infausta) is one of the indigenous fruit trees with commercial potential occurring in abundance in woodlands, scrub, or in sandy valleys from the Eastern Cape, Free State to KwaZulu-Natal and Swaziland, and westward to North West Cape. Its natural range extends to Zimbabwe, Mozambique and Malawi (Palmer & Pitman 1972). The wild medlar fruits are eaten raw, stored as dried fruits or used as flavouring agents in porridge.

Vangueria infmtsta is a small deciduous tree, a member of the family Rubiaceae. The fruit is a globular drupe up to 3.8 cm in diameter and light brown when ripe. The seed kernel is also eaten. In addition to its food value, decoctions of different parts of the plants are also used by Africans as a remedy for menstrual troubles, cough, pneumonia and roundworm (Watt & Breyer-Brandwijk 1962).

The occurrence of AM fungi in relation to indigenous fruit trees has not yet been examined in South Africa. In the present investigation, a survey was conducted to determine the association between Vangueria infausta and AM fungi from several sites in the Pretoria area

Plant roots and soil samples of V, infausta were collected in three regions in and around Pretoria (Table t). At all sites the soil type was sandy loam and the average rainfall is from 100-210 mm per annum.

At each site five samples were taken at random, 7 cm in diameter and 30 cm deep, 0.5 m from the base of the tree. The samples were thoroughly mixed and the roots removed, washed under running tap water and fixed in formaldehyde acetic acid alcohol (formaldehyde 5 ml, acetic acid 5 ml, 50%ethanol 90 ml constituting FAA). Roots of V. infausta were readily distinguished from other roots by their reddish colour. A subsample from each sampling was separated for analyses of spore population and the rest was used to produce trap pot cultures as described by Morton et at. (1993).

Before staining, the roots were washed under running tap water and cut into pieces I cm long. The root pieces were cleared by boiling in 10% KOH for 30 minutes. After a gentle wash in tap water, they were bleached in 35% H203 for 40 minutes and stained in 0.1% Clilorazol black E (Brundrett et ai 1984). The roots were then destained in 50% glycerol, mounted on slides in glycerin jelly and gently squashed with a coverslip. The AM fungal colonization was determined using the method of Biermann and Linderman (1981) and the values expressed as percentage of total root length colonized by AM fungi. The stained roots were examined and photographs taken using a Nikon Optiphot microscope equipped with a Nikon HFX-II photographic system.

Pieces of root tip, 5 mm long, were dehydrated, embedded in wax, sectioned and stained with Saffranin and fast green (Sass 1966). The thin (8 jim) sections were examined for the presence of AM fungal structures.

The spores of AM fungi were isolated from 100 g soil samples (every sample replicated 8 times) from each collection site by wet sieving and decanting (Gerdman & Nicolson 1963) using 212, 106 and 53 p.m sieves. The spores retained on different sieves were collected in a beaker and recovered by sucrose cen-trifugation (Daniels & Skipper 1982), Total counts from the

Table 1 Arbuscular mycorrhizal colonization and associated spore population in V. infausta at three localities in South Africa

Site Locality (Grid reference) % colonization * Spore count Associated vegetation

Site A ((.¡rysslarid) Lrasmuskloof, 2528 CC 66.0 5b Themeda triandra, Melinu spp.. Setaria spp. Protea caffra

Site B (Savanna) 30 km North of Pretoria 2528 HC 71.6 7a Acacia cqffra, Burkea africana, pappen capensis, Panicum maximum

Site C (Savanna) Roodeplaat Dam 2528 C'B 78.8 7a tiyparrhenia hirta

I SD - - 1.47 -

LSI). Least Significant Difference. Values are means of eight replicates. 'Spore counts expressed in spiires per tO g soil sample

Figure 1 Sectioned and squashed roots of V. infausta. A. Transverse section of the root. B. squashed root, and C. cortical cells with arbus-cules. showing epidermis (ep), exodermis, (ex), cortex (co). Scale bar = 30 pm.

sievings were made on filter paper under dissecting microscope illumination at 20 x (Gaur & Adholeya 1994). For identification, spores were isolated from the trap cultures, picked up using a micropipette and mounted in polyvinyl lactic acid glycerol (PVLG, polyvinyl alcohol 1.66 g, water 10.0 ml, lactic acid 10.0 ml and glycerol 1.0 ml) for observation through the compound microscope. Spore morphology was examined using intact and squashed spores both in water and Melzer's reagent (chloral hydrate 100 g, distilled water 100 ml, iodine 1.5 g, potassium iodide 5.0 g). The AM fungal species were identified by spore colour, size, wall structure and other morphological characteristics (Schenck & Perez 1990). Spore count was subjected to one-vvay analysis of variance (ANOVA) and Duncan's Multiple Range Test (DMRT) using Costat Statistical software (Cohort Berkeley, Calif). A significance level of 95% was applied.

All the f infausta trees sampled were found to be highly endomycorrhizal. The root epidermal cells of V. infausta have particularly thick lignified walls and an exodermis with well-suberized walls (Figure 1A). The AM fungai structures in the cortical tissues were only visible after squashing or sectioning the root material (Figure IB). Despite this difficulty, extensive hyphal infections were visible. Hyphal coils were abundantly present in the root cortex and were the main component of the AM fungal infection. No vesicles were observed in any root segment. The major characteristic of AM fungal root colonization in

V. infausta was the extensive development of intracellular coils and varied from 66-79% of the total root length (Figure IC) (Table 1). These intracellular coils continue to grow from one cell to the next and form arbuscules as a series of separate branches. This is similar to the Paris series pattern described for Clintonia borealis (Widden 1996), Erythronium americaman and Trillium species (Brundret & Kendrick 1990).

Chlamydospores were isolated from the soil in very low numbers (5-7 spores 10 gm 1 of soil), (Table 1). Spores of the genera Glomus and Gigaspora were recovered from rhizosphere soil of V. infausta, Most of the spores belonged to the genus Glomus (75%). Spores of AM fungal species Glomus etunicatum Becker & Gerdemann, G. intraradices Schenck & Smith, G. occultum Walker and Gigaspora albida Schenck & Smith were recovered (Figure 2). The low spore numbers in the soil samples contrasts with the high percentage of root colonization and may result from the infrequent sporulation in these habitats. Factors such as soil moisture and temperature may influence the rate of sporulation (Reid & Bowen 1979). The present study is the first report of the association of AM fungi with V. infausta. The extent of association of AM fungi in V. infausta shows considerable promise for selection of suitable endomycorrhizal fungi for improving the establishment and productivity of this native tree, especially in nutrient deficient soils. The current study revealed that I' infausta was highly mycorrhizal in natural conditions. Future

S. Afr. J. Bot. 1999, 65(5 & 6)

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Figure 2 Spores of various AM fungal species collected in the rhizosphere of V. infausta. A. Glomus etunicatum Becker & Gerdemann. B. G, intraradiccs Schenck & Smith. C G. occulfum Walker, D. Gigaspora albida Schcnck & Smith.

efforts will he aimed at documenting site variability in mycor-rhizal status and inoculation of this tree species in the nursery with various isolates of indigenous AM fungi.

Acknowledgements

We thank the National Research Foundation and the research fund of University of Pretoria for financial support of this project.

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