Isolation of mycorrhizal fungi from roots of Caribbean pine

[ 201 ]

Trans. Br. mycol. Soc. 68 (2) 201-205 (1977)

Printed in Great Britain

ISOLATION OF MYCORRHIZAL FUNGI FROM ROOTS OF CARIBBEAN PINE By S. A. EKWEBELAM Sa vanna Forestry Research Station, Forestry Research Institute of Nigeria, Samaru, Zaria

Several fungi were isolated directly from mycorrhizal roots of Caribbean pine (Pinus caribaea Mor.), in plantations established on three different sites during a period of 1 year. In all, 105 pure cultures of basidiomycetes representing 10 putative species were recovered, and confirmed as mycorrhizal fungi using the synthetic culture test. The frequency of occurrence and distribution of fungi isolated were independent of the range of sites examined and the age differences of the plantations sampled, but varied considerably according to seasonal conditions. The highest numbers of basidiomycete fungi were recovered in midsummer and the lowest numbers in late winter and early summer. This seasonal effect was probably a function of the growing conditions for the host plants, as reflected by soil moisture status. Despite the widespread occurrence of the eetomycorrhizas of pines, knowledge of the relative frequency of different fungi is limited owing to the difficulties in isolating and identifying them. Such knowledge could be especially important in the introduction of exotic conifers to enable the selection of the most effective and most suitable fungal symbionts. The pure culture technique as devised by Melin (1921) and its adaptations (Norkrans, 1949; Lamb & Richards, 1970) have provided the most suitable means for identification and confirmation of the capacity of fungi to produce mycorrhizas with selected tree species. However, there are relatively few references to the identity and mycorrhizal relationships of Caribbean pine (P . caribaea Mor.). Trappe (1962) lists 19 possible mycorrhiza formers with this species in nature, and Hacskaylo & Vozzo (1971) reported successful establishment of mycorrhizas on P. caribaea with pure cultures of Corticium bicolor, Rhizopogon roseolus and Suillus cothurnatus. The present work describes the direct isolation and identification offungi from mycorrhizas of Caribbean pine, and investigates whether any relationship exists between the frequency and distribution of putative fungal species and seasonal conditions. The criteria used are those embodied in Melin's postulates (Melin, 1936) as elaborated by Harley (1969). MATERIALS AND METHODS

Collection of roots

Mycorrhizal roots of Caribbean pine in established plantations were sampled from four different compartments representing four sampling sites,

designated A,B,C and D and aged 24,21, 19 and 11 years respectively, on 6 Dec. 1973 and subsequently on 8 Mar., 8 Aug. and 14 Nov. 1974, corresponding to four sampling periods (i.e. midsummer, autumn, late winter and early summer respectively) . Three replicate samples were taken from each site. The plantations are in the Beerburrum district of the coastal lowlands of subtropical Queensland on soils derived from coarse-grained Mesozoic sandstones which belong to the Great Soil Group classified as lateritic podzolics (Vallance, 1938). Roots were collected and placed inside selfclosing ,mini-grip' polythene bags to prevent drying out. Samples from the mid-summer sampling were transported to the laboratory, washed, refr igerated overnight and processed the following day. Materials from subsequent samplings were collected and transported in polythene bags in an ice chest, refrigerated and processed 2 days following collection. Isolation of fungi

A modification of the technique of Lamb & Richards (1970) was used in the processing of root materials. Suitable mycorrhizas were excised and transferred to 2 X 1 em specimen tubes (one tube per sampling site) fitted with a cap and fine nylon mesh held by another cap with a hole about 4 em! at the bottom. The individual tubes with contents were then transferred to 200 ml glass jars provided with screw caps and treated for 15 min in each of solutions of '7-X '* detergent and calcium hypo-

* Biological Laboratories N.S.W., Australia.

Limited,

Guildford,

202

Isolation of mycorrhizal fung i

chlorite as described by Lamb & Richards (1970). They were then rinsed in several changes of sterile distilled water and plated out on Hagem agar (Modess, 1941) modified with 0'5 ml Fe-EDTA (2'5 ppm Fe3+). Forty-eight mycorrhizas were plated out from each sample (a total of 576 per sampling period). Plates were incubated at 22 ± 1 °C in the dark. Aseptic sy nthesis of mycorrhizas Pyrex test tubes, 25 A 3 ern, were provided with rubber stoppers fitted with two pieces of 0'5 em diam glass tubing about 3 and 5 em long respectively. They were filled with a mixture of finely ground peatmoss and vermiculite (1:6 v/v) to within a few centimetres of the top and wetted with Melin-Norkrans solution (Norkrans, 1949) modified with 1'0 ml Fe-EDTA (5'0 ppm Fe 3+). Fifty ml of solution was added to 18'5 g ot vermiculite-peatmoss mixture. The narrow tubes attached to the rubber stopper were plugged with cotton wool and sealed with 'alfoil', and the assembly was autoclaved at 103 kN /m 2 for 20 min. After autoclaving the pH varied between 5"4 and 5'6. Seeds of Pinus caribaea var. hondurensis Barr. and Golf. were surface-sterilized in 30 % hydrogen peroxide for 60 min and aseptically transferred to sterile Petri dishes containing sterile nutrient medium (Oxoid CM 1, plus 15 g agar), and incubated at 25 ± 1 °C in the dark. Germinating seeds with radicles approximately 1 em long were removed from the incubator and aseptically transferred to a sterile 8 x 20 cm test tube half-filled with vermiculite wetted with nutrient solution (0'25 g each of KH 2P04 , MgS04.7H20 and NHctartrate, 0'11 g CaCI 2, 0'5 ml Fe-EDTA (2'5 ppm Fe 3+) to 1 I distilled water), When seedlings were approximately 8-10 cm long they were aseptically implanted through the shorter tube ot the prepared culture assembly and bound by cotton wool. One seedling was implanted into each tube. After transplanting, tubes were incubated in a galvanized water bath, insulated by a cold-water jacket contained in another galvanized bath, and maintained at a temperature of 24 ± 1 °C by a thermostatic heater. The bath complex was submerged in a tank maintained at 10 ± 2 °C. The seedlings were thus grown in a controlled environment as described by Lamb & Richards (1970). Seedlings were inoculated 6 weeks following transplanting. One hundred and five basidiomycetes, representing ten putative mycorrhizal species (coded M1-M10), recovered during the four sampling periods, were tested. A hyphal suspension of each isolate was prepared in a glass

macerator, and 10 ml of suspension was asept ically pipetted into each seedling culture, R eisolation of fungi from mycorrhizas After 7 months' growth, the seedlings were harvested. Roots were cleansed of vermiculitepeatmoss particles with running tap water, and examined for the presence of mycorrhizas, Where mycorrhizas were formed, some were excised and fixed in formalin-acetic-alcohol in preparation for embedding, sectioning and microscopic examination. A few were excised and subjected to a quick process of isolation as described by Zak (1969) in an attempt to re-isolate the original fungi. Resulting cultures were tested against original cultures by hyphal fusion tests (Lamb & Richards, 1970). RESULTS

Isolation plates were examined after 4 days and those with contaminating organisms (mainly bacteria and common root-inhabiting moulds) were discarded. Uncontaminated mycorrhizas were transferred to fresh plates and re-incubated, Most isolates emerged from mycorrhizas about 10 days after plating, but some took up to 4 weeks. After 10 weeks, all mycorrhizas which did not produce fungi were discarded and the rest were examined culturally and microscopically. The criterion used for screening the isolates was the presence of clamp connexions on the hyphae and all isolates lacking this feature were rejected, including several dark sterile fungi . Isolates with clamp connexions were arbitrarily classified on the basis of similarities and dissimilarities in morphological and cultural characteristics and designated by numbers (T able 1), The similarities and dissimilarities of isolates were confirmed between sampling periods on the basis of hyphal fusion tests (Lamb & Richards, 1970). Recovery of fungi varied from 1-15 % of the total mycorrhizas plated for each sampling period. On the whole, 105 pure cultures of basidiomycetes representing 10 putative species, confirmed as mycorrhizal fungi by the synthetic culture test, and distributed between sites as shown in Table 1, were obtained on the four sampling occasions. It is possible, however, that more mycorrhizal fungi grew out than were recognized, since several hymenomycetous fungi, such as species of Cortinarius, Sui/Ius, Boletus and Russula produce few or no clamp connexions on agar media (Zak & Bryan, 1963; Lamb & Richards, 1970). The highest numbers of basidiomycetes were recovered during the mid-summer sampling and the lowest numbers during the late winter and early summer samplings respectively. This observation supports the view

s. A. Ekwebelam Table

1.

2°3

Arbitrary species classification and frequency of basidiomycete fungi obtained from caribbean pine (p. caribaea Mor.) mycorrhizas*

Species and number of endophytes" Sampling Sampling period sites Ml Dec

Mar

Aug

Nov

Total

A B C D A B C D A B C D A B C 0

M2

M3

M4

4

3 14

2 2

1

4

M5 1 4

M6 23 2 1 18 1

M7 2

M8

M9

Ml0

Total

3 6

31 22 3 29 1 5 7

2 2

2

1

1 1

1 1

1

2

2

2

4

4

4

22

5

47

4

2

4

2

9

1°5

* All figures represent the total number of basidiomycetes recovered from three sub-sites, out of a total of 144 mycorrhizas plated. that the fungal flora varies not only with locality, but also seasonally (Lamb, 1974). The most interesting aspect of the results, however, was that Rhizopogon roseolus and Suillus granulatus, which produce sporophores regularly in Caribbean pine stands in the area (D. I. Bevege, private communication), were never isolated. This was confirmed by comparisons of the isolates recovered with stock cultures of those species together with R.luteolus and S. luteus, on the basis of cultural characteristics and /or hyphal fusion tests, with negative results. Species groups designated M.4 and M6 were the most dominant, and it is possible that both groups constitute the major mycorrhizal symbionts of Caribbean pine in established plantations in the area studied. DISCUSSION

The results demonstrate that many mycorrhizal fungi could be isolated from roots of Caribbean pine, and sampling site and age differences of plantations had little or no effect on the frequency and distribution of the different fungal species. The effect of site was not unexpected since according to Vallance (1938), there is no large difference in the physical and chemical properties of the sites. The only unexpected result in this instance was the lack of effect due to age. However, the finding that the frequency and distribution of

fungal species in different sites are independent of habitat variations and age differences of plantations is supported by the work of Lamb & Richards (1970 ) . An alternative explanation of the differences found among the four sampling periods is the time lag between collection and processing of mycorrhizas. During the mid-summer sampling, collection and processing were carried out with the minimum of delay whereas in later samplings, there was a difference of 2 days between collection and processing. It is possible, though unlikely, that this time lag may have affected the number of basidiomycetes recovered. The differences in the frequency and distribution of fungal symbionts are believed to be due to seasonal factors. This is supported by reference to the climatic data of the sampling area in 1973 and 1974 (Appendix 1). In general, the climate is humid subtropical, and the mean annual rainfall for the Beerburrum-Beerwah area is between 991 and 1600 mm (Richards, 1961). Most of this rain falls in late summer and autumn, and the driest season of the year is spring; rainfall variability is high especially in the winter. In 1973, unseasonally high rains in July followed by good falls throughout the first half of summer provided soil moisture conditions which would favour good plant growth and mycorrhiza formation. This was borne out by examination of the root system as a whole, which

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Isolation of mycorrhizal fungi

during the mid-summer sampling assumed a rusty REFERENCES brown colour with profuse mycorrhiza developHACSKAYLO, E. ] . & Vozzo, ]. (1971). Inoculation of ment, perhaps leading to the large number of fungi Pinus caribaea with ectomycorrhizal fungi in Puerto being recovered at this time. Rico. Forest Science 17,239--245. In contrast, the late winter and early summer HARLEY, ]. L. (1968). Mycorrhiza. In Recent researches samplings in 1974 had been preceded by a proin plant physiology (ed, W. B. Turrill ), pp. 73-103. longed drought with high moisture deficit, resultVistas in botany, III. ing in dry soil conditions in late winter persisting HARLEY, ]. L. (1969 ). The biology of mycorrhiza. London: Leonard Hill. through the spring to the early summer of the same year. These conditions could be expected to have a LAMB, R.]. (1974). The autecology of ectomycorrhizal fungi of Pinus. Ph.D. Thesis, University of New deleterious effect on water relations and mycorrEngland, Armidale, Australia, 146 pp. hiza formation. The roots collected during these LAMB, R. J. & RICHARDS, B. N. (1970 ). Some mycorrper iods showed predominantly old and black hizal fungi of Pinus radiata and Pinus elliottii var. mycorrhizas. This affected the results since many elliottii in Australia. Transactions of the British dark sterile fungi were isolated with a consequent M y cological Society 54, 371-378. lower recovery of known basidiomycetes. These MARKS, G. C. (1965 ). Pathologicalhistology of root-rot associated with late damping-off in Pinus lamberobservations agree with the view that formation of tia.na. Australian Forestry 29, 238-251. mycorrhizas is dependent on soil-moisture condiMARKS, G. C. & FOSTER, R. C. (1973) . Structions (Wilcox, 1968). ture, morphogenesis and ultrastructure of ectoThe variation in the frequency and distribution mycorrhizae. In Eetomycorrhizae: their ecology and of mycorrhizal fungi is probably also associated physiology (ed. G. C. Marks and T. T. Kozlowski), with seasonal variation in root growth and physiopp. 1-41. New York: Academic Press Inc. logy. For instance, at the end of the growing MELIN, E. (1921). Uber die mykorrhizen pilze von season, roots produce an impervious metacutinized Pinus syloestris L. and Picea abies (L .) Karst. layer over the apex and cease growth. Mycorrhizal Svensk botanisk Tidskrift 15, 192-203. fungi do not penetrate this barrier (Marks, 1965; MELIN, E. (1936 ). Methoden der experimentellen untersuchung mykotropher pflanzen. Handbuch der Wilcox, 1968). Concurrent with the cessation of biologischen Arbeitsmethoden XI 4, 1015-1018. root activity, there would be a fall in the amount of carbohydrates translocated from the leaves (see MODESS, O. (1941 ). Zur kenntnis der mykorrhizabildner von kiefer und Fitche. Symbolae Botanicae Marks & Foster, 1973), and this may diminish Upsalienses 5, 1-147· fungal activity. Wilcox (1968) reported that varia- NORKRANS, B. (1949). Some mycorrhiza-forming tion in the cyclic growth behaviour of tree roots Tri choloma species. Svensk botanisk Tidskrift 43, with the season may lead to a decrease or increase 185-49°· in numbers of mycorrhizal fung i. Harley (1968) RAYNER, M. C. (1927). Mycorrhiza: An account of non-pathogenic infection by fungi in vascular plants observed that although most mycotrophic plants and bryophytes. N ew Phytologist 15, 246 . are infected in their natural habitats, some variations in the intensity of mycorrhizal development RICHARDS, B. N. (1961). Fertilizer requirements of Pinus taeda L. in the coastal lowlands of subtropical are observed. Part of this variation is related to the Queensland. Queensland Forest Bulletin 16, 24. growth cycle of the host and to the season of the TRAPPE, J. M. (1962) . Fungus associates of ectotrophic year, and part is due to habitat variation. Rayner mycorrhizae. Botanical Review 28, 538-606. (1927) concluded that mycorrhizal formation by VALLANCE, L. G. (1938). A soil survey of the Beerconifers is a reciprocal phenomenon conditioned by burrum Glasshouse Mountains-Beerwah pineapple the physiological states of both symbionts, which districts. Queensland Agricultural Journal 49, 554in turn is correlated with external conditions of 579· soil and climate. These ob servations and the evi- WILCOX, H. E. (1968). Morphological studies of the root of red Pinus resinosa. I. Growth characteristics dence from the present investigation therefore sugand patterns of branching. American Journal of gest that the frequency and distribution of fungal Botany 55, 247 · symbionts in any geographical area are related to ZAK, B. (1969). Characterisation and identification of seasonal characteristics. Douglas-fir mycorrhizae. In M ycorrhizae (ed. E. Hacskaylo), pp. 38-53. U.S.D .A. Forestry SerThis work formed part of M .Sc.For. degree at vice Miscellaneous Publications. the University of New England, Armidale, Austra- ZAK, B. & BRYAN, W. C. (1963). Isolation of fungal lia, and was made possible by the financial support symbionts from pine mycorrhizae. Forest Science 9, from the Federal Government of Nigeria. 270-278.

S. A. Ekwebelam

2°5

APPENDIX 1

Rainfall (mm)* for Beerburrum-Beerwah area for 1973 and 1974

.

.

1973

1974

Month

Beerburrum

Beerwah

Beerburrum

Beerwah

January February March April May June July August September October November December

145'8 322'3 72'6 61'2 35'1 21'8 626'1 19'3 65'8 25 1'7 86'4 225'6

208'5 432'1 102'6 68,6 62'2 25'4 690'9 24'4 44'2 167'9 92'7 157'5

1249'6 215'6 398'2 161'6 113'9 88'4 9'6 71'4 44'0 111'8 294'0

1088'2 189'8 442'2 160'0 186,8 60'6 8'2 65'7 45'S 93'7 3 12'0

* Source: Meteorological Records, Queensland Forest

Service, Forest Station, Beerwah.

(Accepted for publication 3 August 1976)