[ 281 ] Trans. Br. mycol. Soc. 63 (2), 281-288 (1974) Printed in Great Britain
THE MYCOFLORA OF AN ALKALINE SOIL OF THE OPEN-SAVANNAH OF THE TRANSVAAL By ALBERT EICKER
Department
ofGeneral Botany,
University of Pretoria
The microfungi of an alkaline soil from an open-savannah in the Pretoria district of the Transvaal were surveyed using the dilution plate technique, the soil plate technique and a soil washing method. Over 14 months 127 species representing 53 genera were isolated. The most prevalent species found were Penicillium spp.; Fusarium spp.; T riclwderma viride; Gliocladium roseum; Aspergillus spp.; Mucor spinosus; Spicaria violaceae and Chaetomium globosum. The results are discussed with particular reference to comparable surveys carried out in the Western Transvaal.
Soil fungi have been extensively studied in many areas of the world and comprehensive reviews of these investigations have been made by Chesters (1949), Saksena (1955), Gilman (1957), Barron (1968) and Domsch & Gams (1970). Up to the present time, relatively few surveys of the microfungal populations of soils in native plant communities of Southern Africa have been undertaken. Papendorf (1967, 1969) investigated the occurrence of soil fungi of an Acacia karroo community, while Eicker (1969, 1970a, b) surveyed the soil mycoflora of forest communities in Zululand. The present investigation will contribute towards increasing this knowledge. MATERIALS AND METHODS
The soil studied The site chosen for this analysis was an area of undisturbed, natural vegetation within the municipal area of Pretoria lying 25° 45' South latitude and 28° 14' East longitude, 1369 m above sea level. The average annual rainfall is 762'5 mm with maximum precipitation during the summer months from November to February. The principal geological formation is Daspoort Quartzite of the Pretoria Series which yields an alkaline loam soil. The mechanical composition of the soil is 19 % coarse sand and gravel, 25 % fine sand, 10 % silt and 46 % clay. The analysis at the time of sampling is given in Table I. The vegetation of this area can be classified as Sourish Mixed OpenSavannah (Acocks, 1953) supporting the following trees: Celtis africana Burm. f.; Acacia caffra (Thunb.) Willd.; Diospyros lycioides Desf.; Maytenus heterophylla Eckl. & Zeyh.) N. Robson and Rhus pyroides Burch. The most important grasses forming the ground layer are Cynodon dactylon (L.) Pers.; Digitaria ternata (A. Rich.) Stapf.; Eragostis curvula (Schrad.) Nees; Schiracbyrium sanguineum (Retz.) Alst.; Setaria perennis Hack. and Themeda 18-2
Transactions British Mycological Society Table 1. Chemical analysis of the soil studied Phosphorus (P20S) Potassium Calcium Magnesium Carbon (Walkey-Black method) Resistance pH
ppm ppm 7400 ppm (37 mg equiv/roo g) 610 ppm 3,83 % (oven dry basis) 580 ohm 7'2 12
200
triandra Forsk. Scattered amongst the grasses are several different shrubs and herbs, of which Rhus ;:::eyheri Sond.; Hypoxis rigidula Bak.; Salvia runcinata L.f.; Vernonia oligocephala (DC.) Sch. Bip. ex Walp and Vigna vexillata (L.) A. Rich are the most common. Collection of soil samples Soil samples were collected monthly from May 1971 to June 1972. At the sampling site, the surface litter was first scraped away to reduce contamination from this habitat. A sample of about 1 kg, comprising at least ten subsamples, was taken over an area of about 10 m 2 using a sterile metal trowel and placed in a sealed plastic bag. Samples were stored at 0 °C until processed, which was always within 24 h of collection. Twenty-nine samples were examined in the course of this investigation. Soil moisture content was determined by drying a sample of 5-10 g in an electric oven at 1 5 °0 for 12 h.
°
Isolation and study offungi A variety of methods have been devised for the isolation and study of soil fungi and all are selective to a greater or lesser degree (Chesters & Thornton, 1956; Warcup, 1960, 1967; Gams & Domsch, 1967; Johnson & Curl, 1972). Warcup (1960) is of the opinion that the use ofa combination of isolation techniques yields the maximum information. In this study, therefore, three cultural methods were employed, the dilution plate method (Waksman & Fred, 1922), the soil plate method (Warcup, 1950) and a soil washing technique of Gams & Domsch (1967). The modified dilution plate technique devised by Menzies (1957), was used. Soil aliquots of 25 g (on a dry-soil basis) were suspended in 250 ml sterile water and shaken mechanically for 30 min. A dilution series was prepared from this primary suspension using a stainless steel dipper of 1 ml capacity. The dilution factor used in all plates was 1: 5000, which gave an average of 10-30 colonies per plate. Czapek-Dox agar with 0'5 % Difco yeast extract, rose bengal, 30 p,g/ml streptomycin and 5 p,g/ml aureomycin was used. The plates were incubated at 25°C and examined periodically. They were kept at least three weeks in order to allow slow-growing species to develop sufficiently. For the preparation of soil plates peptone dextrose agar plus rose bengal, streptomycin (Martin, 1950) and aureomycin was used. The soil washing technique was a modification of the Parkinson &
Soil mycoflora. A. Eicker
283
Williams (196 I) method using a continuous flow of sterile water through the washing boxes, which are shaken mechanically during the process. The apparatus was constructed from stainless steel following the procedure of Gams & Domsch (1967). Washed soil particles and organic fragments remaining on the sieves were then removed by rinsing the sieves in dishes of sterile water. Individual particles were removed with sterile forceps, blotted on sterile filter paper and plated on pre-poured media. Only discrete soil particles, small water-stable aggregates and small organic fragments were selected. The medium used was as for the soil plates. In the transfer, cultivation and identification of the cultures, standard mycological methods were used. The majority of cultures were incubated at 25°C until a suitable stage of development had been reached. Subcultures were grown on potato-dextrose or potato-carrot agars. Where growth on these media was unsatisfactory, 2 % malt extract agar or natural substrates such as sterilized grass debris were used. Penicillia and aspergilli were grown on Czapek-Dox agar. Sterile mycelia were incubated under near-ultraviolet radiation (Leach, 1962) in an attempt to induce sporulation. RESULTS
The isolates (Table 2) were ultimately classified into 127 species or taxonomic entities. On the basis of the frequency of occurrence, the most common fungi of the site could be arranged in the following order of dominance: Penicillium spp.; Fusarium spp.; Trichoderma viride; Gliocladium roseum; Aspergillus spp.; Mucor spinosus; Spicaria violaceae; Dark sterile mycelium UP 457; Chaetomium globosum and Paecilomyces spp. Penicillium multicolor, the most common fungus, was isolated throughout this investigation by all three isolation techniques. Trichoderma viride, Gliocladium roseum and Aspergillus rugulosus were also extremely common on isolation plates. Of the sixteen species of phycomycetous fungi isolated, only one, Saprolegnia sp. UP 812, was an Oomycete. The Zygomycetes were represented by the genera Absidia, Actinomucor, Cunninghamella, Gongronella, Mortierella, Mucor and
Transactions British Mycological Society Table 2, Fungi recovered from a Transvaal open-savannah soil, showing relative frequency ofpositive recordings (+ relative frequency less than I %; + + relative frequency 1-3'9%; + + + relative frequency 4-10%; + + + + relativefrequency higher than 10 %) PHYCOMYCETES
Oomycetes Sabrolegnia UP 812 Zygomycetes Absidia cylindrospora Hagem* A. spinosa Lendner* Absidia UP 613 Actinomucor elegans (Eidam) Benjamin & Hesseltine Cunninghamella bainieri Naumov* C. echinulata (Thaxt.) Thaxt. ex Blakeslee* Gongronella butleri (Lendn.) Peyr. & Dal Vesco*
++ + + + +
Mortierella UP 804 Mucorfragilis Bainier* M. spinosus van Tieghem* M. racemosus Fres. * Mucor UP 444 Mucor UP 683 Zygorhynchus moelleri Yuill. * Sterile phycomycete UP 703
+++
+ + ++++ ++ + + ++ +
+ ASCOMYCETES
Achaetomiella irregulare D. Hawksw. ined.* Chaetomium globosum (Kunze) Fr.* C. globosum var. flavo-viride Novak" C. lusitanicum Gomes C. olivaceum Cooke & Ellis* C. spinosum Chivers. Chaetomidium subjimeti Seth* Cochliobolus geniculatus Nelson C. spicifer Nelson*
+
++++
+ + + ++ + + +
Gymnoascus umbrinus (Boudier) Orr, Kuehn & Pluckett Nectria invecta Pethybr.* Neocosmospora vasinfecta E. F. Smith Neurospora sitophila Shear & Dodge Pleospora infectoria Funckel* Thielaviaterricola (Gilman & Abbott) Emmons
++ + + + ++ +
FUNGI IMPERFECT!
Coniella diplodiella (Speg.) Petrak & Sydow. * Coniothyrium fuckelii Sacco* Phoma UP 502 Phoma UP 819
++ + + +
Sphaeropsidales Stagonospora UP 701 Unidentified Sphaeropsidales UP 790* Unidentified Sphaeropsidales UP 822
+ + +
Melanconiales Pestalotia UP 3 I 3
+
Aspergillus aculeatus Iizuka* A. carneus (Van Tiegh.) Blochwitz* A.flavipes (Bain. & Sart.) Thorn & Church* A.flavus (Link) Fr.* A. fumigatus Fres, * A. nidulans (Eidam) Wint.* A. niger van Tiegh.* A. ochraceus Wilhelm* A. rugulosus Thorn & Raper* A. sydowii (Vuill.) Tiraboschi* A. ustus (Bain.) Thorn & Church* A. versicolor (Vuill.) Tiraboschi* A. wentii Wehmer* Aspergillus UP 410
Moniliales Moniliaceae Aspergillus UP 505* + Aspergillus UP 708 Beauveria bassiana (Bals.) Yuill. ++ Gliocladium roseum Bain. agg.* Gliocladium cr. roseum Bain. agg. * + Gliocladium UP 629 ++ Monilia UP 727 ++ Paecilomyces UP 450 + Paecilomyces UP 497 + Paecilomyces UP 777 + Paecilomyces UP 820 ++++ Penicillium breoicompactum + Dierckx* P. canescens Sopp* + P. chrysogenum Thorn.* ++ P. crustosum Thorn.* + P. cyclopium Westling* +
Pestalotia UP 797
+
+ + + ++++ + + + +++ + ++ + + + ++ + +++
Soil mycoflora. A. Eicker T able
(cont .)
Moniliaceae (cont.) Penicillium U P 793 Scopulariopsis brevicaulis (Bain.) Thoms Scopulariopsis U P 523 Spicaria violaceae Abbott * Tr ichodermakoningii Oud.* T. pseudokoningii R ifai agg. '" Tr ichoderma cf. pseudokoningii Rifai agg.* T . viride (Pers.) Gray'" Tr ichoderma UP 304 Verticillium UP 696
P. decumbens Thorn* Pi frequentans Westling* P. herquei Bain. & Sarto* P. lilacinum T horn* P. multicolor G.M.-P.'" P. rainstrickii Smith* P. thomii Maire* P. waksmanii Zal. * Penicillium UP 458 Penicillium UP 465* Penicillium UP 695 * Penicillium UP 770
+ +++ + + + ++ + ++ + ++ + + + +
Acremoniella UP 373 Alternaria alternata (Fr.) Keissler A . tenuissima (Kunze ex Pers.) Wiltshire Alternaria UP 462 Aureobasidiumpullulans (de Bary) Arn aud* Cladosporium osysporum Berk . & Curt. C. tenuissimum Cooke* Cladosporum UP 428 Cladosporium UP 642'"
+ +
++
+ + + ++ + +
Demaiiaceae Gonytrichum macrocladum (Sacc.) Hughes Helminthosporium UP 66 2 Humicola fuscoatra Traaen. Humicola UP 721 Mammaria UP 835 Periconia UP 496 Rhinocladiella sp. near est to R. elatior Mang enot* Stachybotrys atra Corda T orula U P 327 Tr ichocladium UP 85 2 Tuberculariaceae Fusarium U P 326 Fusarium U P 51 1 Fusarium U P 759* Fusarium U P 761* VoluteUa ciliata (Alb . & Schw. ) Fr.
Epicoccum purpurascens Ehr enb. ex Schlecht. Fusarium equiseti (Corda) Sacc." F. graminearum Schwabe* F. oxysporum Schlecht .* F. sambucinum Fu ckel F. solani (Mart.) Sacc.* Doratomy ces stemonites (Pers. ex Fr.) Morton & Smith
2
++
Stilbaceae Graphium putredinis (Corda) Hughes*
+ ++ + ++++ + ++ + ++++ + +
++ + ++ + + + + ++ + +
}++++ +
+
Mycelia sterilia
Papulospora UP 517 Rhizoctonia solani Kiihn
+ +
Dark sterile mycelia UP 457
++++
* Cultures deposited at CMf.
general isolation techniques do not favour the Basidiomycetes (Gochenaur & Whittington, 1967) and only the more direct methods (Warcup, 1959) ar e able to isolate these fungi in any appreciable quantities. As was expected in an investigation of this nature, the majority of species belonged to the Deuteromycetes (Fungi Imperfecti). Prominent genera were Penicillium with eighteen species, Aspergillus with sixteen species, Fusarium with nine species and Trichoderma with five species. Although Cladosporium and Paecilomyces were each represented by four species, none of these occurred with great frequency on isolation plates. Of th e Penicillium species, P. cyclopium, P. frequentans and P. multicolor showed high percentage frequ ency on all isolation plates with the latter
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being dominant. Of the sixteen species of Aspergillus, only A. rugulosus was at all common. It produces cleistothecia with very prominent, dark brown Hulle cells. Some of the isolation plates were regularly incubated at 45°C in order to try and isolate thermophilic fungi. However, Aspergillus fumigatus was the only thermophile obtained. Apinis (1962) and Eggins & Malik (1949) produced evidence that this ecologically specialized group of fungi does occur in the soil. Eicker (1972) has shown that thermophilic fungi are of widespread occurrence in the subtropical areas of Transvaal. Nine species of Fusarium were isolated, F. oxysporum being by far the most abundant. In the soils surveyed by Gordon (1954), Park (1963) and Warcup (1957) this species was also the most common Fusarium. Fusarium is also considered to be a prominent member of the mycoflora of alkaline soils (Mukerji, 1966; Ranzoni, 1968; Warcup, 1951). Ordin (1957) showed that Fusarium spp., together with Penicillium spp., were abundant in uncultivated land. This author has also found that fewer Trichoderma spp. occur in uncultivated soils. Rao (1970) was not able to isolate Trichoderma viride from alkaline garden soil but found it in nearly neutral, uncultivated soil. It is generally accepted that T. viride is characteristic of acid soils (Rao, 1970). The present investigation shows, however, that T. viride can also be very common in alkaline, natural soils. Pugh & Dickinson (1965) showed that Gliocladium roseum requires an alkaline pH. Bhatt (1970) found G. roseum to be very common in the slightly alkaline white cedar forests in Ontario. In the present investigation, this species was also a very common member of the mycoflora. Dematiaceous species, with the exception of Humicola fuscoatra, were of very low occurrence on isolation plates. In a Rhodesian sandveld soil, Hartill (1966) found 25 species amongst which Hormodendron cladosporiodes, Cladosporium herbarum, Humicola sp. and Cladosporium sp. were very common. All isolations from the Rhodesian soil were made from actively growing mycelia. Sterile dark coloured mycelia were extremely common in the Transvaal open-savannah soil, especially isolate UP 457 which formed a very characteristic dark brown colony. Despite a variety of treatments such as cultivation on many media, inoculation on plant debris, incubation at various temperatures and in the presence of near ultraviolet light, they remained sterile. Bhatt (1970) has expressed the view that these sterile dark mycelial forms, which are so frequently recorded, may represent monokaryotic Basidiomycetes. DISCUSSION
The fungi recorded in this study show little agreement with previous findings in dry subtropical areas of this country. The Papendorf school (du Toit, 1967) found Penicillium paraherquei and P. lilacinum to be the most common fungi, followed by Absidia cylindrospora, Humicola sp., Scopulariopsis humicola, Rhieopus arrhizus, Verticillium chlamydosporum and Aspergillus puniceus. However, the most striking difference between data obtained previously and those from this study is that only nineteen of over 200 species recorded in the soil of the open-savannah of Northern
Soil mycofiora. A. Eicker Transvaal and in the soil of the Acacia karroo community in Western Transvaal were common to both sites, although similar methods and culture media were employed in both investigations. This is a strong indication that soils under very similar climatic conditions and comparable phanerogamic floras show an individuality in their mycoflora. However, as Griffin (1972) has pointed out, species lists for different soils provide a comparison ofrecorded fungi and not necessarily offungi actually present in the soil. I wish to express my sincere gratitude to the Director and Staff of the Commonwealth Mycological Institute, Kew, for aiding in the identification of many of the isolates. I am also indebted to the South African Council for Scientific and Industrial Research and the Research and Publications Committee of the University of Pretoria for financial assistance. REFERENCES
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HARTlLL, W. F. T. (1966). Some fungal inhabitants of a Rhodesian sandveld soil. Kirkia 7, 137- 143. JOHNSON, L. F. & CURL, E. A. (1972). Methods for research on the ecology of soil-borne plant pathogens. Minneapolis: Burgess Publishing Co. LEACH, C. M. (1962). Sporulation of diverse species of fungi under near-ultra violet radiation. Canadian Journal of Botany 40, 151-161. MARTlN,J. P. (1950). Use of acid, rose bengal and streptomycin in the plate method for estimating soil fungi. Soil Science 69, 215-232. MENZIES, J. D. (1957). A dipper technique for serial dilutions of soil for microbial analysis. Soil Science Society of America Proceedings 21, 660. MUKERJI, K. G. (1966). Ecological studies of the microorganic populations ofusar soils. Mycopathologia Mycologia Applicata 29, 339-349. ORDIN, A. P. (1957). The influence of vegetation on the microflora of soils. Bulletin of the Academy of Sciences of the U.S.S.R. 4, 495-5 02. PAPENDORF, M. C. (1967). Two new genera of soil fungi from South Africa. Transactions of the British Mycological Society 50, 69-75. PAPENDORF, M. C. (1969). Leptodiscella africana gen. et comb. nov. Transactions of the British Mycological Society 53, 145- I 47. PARK, D. (1963). The presence of Fusarium oxysporum in soils. Transactions of the British Mycological Society 46, 444-448. PARKINSON, D. & WILLIAMS, S. T. (1961). A method for isolating fungi from soil microhabitats. Plant and Soil 13, 347-355. PUGH, G. J. F. & DICKINSON, C. H. (1965). Studies on fungi in coastal soils. VI. Gliocladium roseum Bainier. Transactions of the British Mycological Society 48, 279-285. RANZONI, F. V. (1968). Fungi isolated in culture from soils of the Sonoran desert. Mycologia 60, 356-371. RAo, P. R. (1970). Studies on soil fungi. III. Seasonal variation and distribution of microfungi in some soils of Andhra Pradesh (India). Mycopathologia Mycologia Applicata 40, 277-2 98. SAKSENA, R. K. (1955). Some aspects of studies on soil fungi. Journal of theIndian Botanical Society 34, 1-10. WAKSMAN, S. A. & FRED, B. (1922). A tentative outline of the plate method for determining the number of micro-organisms in the soil. Soil Science 14, 27-28. WARCUP, j. H. (1950). The soil-plate method for isolation of fungi from soil. Nature, London 166, 117-118. W ARCUP, J. H. (195 I). The ecology of soil fungi. Transactions of the British Mycological Society 34, 376-399' WARCUP,j. H. (1957). Studies on the occurrence and activities offungi in a wheatfield soil. Transactions of the British Mycological Society 40, 237-262. WARCUP,j. H. (1959). Studies on Basidiomycetes in soil. Transactions ofthe British Mycological Society 42, 45-52. WARCUP,j. H. (1960). Methods for the isolation and estimation of activity of fungi in soil. In The ecology ofsoilfungi (ed. D. Parkinson and]. S. Waid) , pp. 3-21. Liverpool: Liverpool University Press. WARCUP, J. H. (1967). Fungi in soil. In Soil biology (ed, A. Burges and F. Raw), pp. 51-1 10. London: Academic Press.
(Accepted for publication 28 January 1974)