THE INSTITUTES FOR APPLIED RESEARCH BEN-GURION UNIVERSITY OF THE

BGUN-ARI-68-95] Limited

CULTIVATION OF PFEILII- THE KALAHARI DESERT

Final report for the period March 1991 - February 1995

V. Kagan-Zur,l N. Roth-Bejerano 2 and F.W. Taylor 3

1The Institutes for Applied Research 2 Ben-Gurion University of the Negev 3Veld Product Research, Gaborone, Botswana

Submitted to the Office of the Science Advisor U.S. Agency for Interantional Development (File No. 86382101)

Beer-Sheva, October 1995 FINAL REPORT (MARCH 1991 - FEBRUARY 1995)

Submitted to the Office of the Science Advisor U.S. Agency for Interantional Development

CULTIVATION OF TERFEZIA PFEILII-THE KALAHARI DESERT TRUFFLE

V. Kagan-Zur and N. Roth-Bejerano Ben-Gurion University of the Negev Beer-Sheva, Israel

F.W. Taylor Veld Product Research, Gaborone, Botswana

Project Number: C9 - 114 Grant Number: 86382101

A.I.D. grant project officer:

Project duration: March 1991 - February 1995 Tab'e of contents

Executive summary 2

Research Objertives 3 Methods and Results 5 Impact, Relevance and Technology Transfer 13 Project Activities 13

Project Productivity 13

Future work 13

Literature cited 14 Executive summary

This report summarizes the research on Terfezia pfeilii-the Kalahari desert truffle, funded by the AID-CDR. The Terfezia pfeilii is one of the most highly sought-after

food sources in the Kalahari, particularly among marginal groups such as the Basarwa (Bushmen) for whom these fungi represent an important source of

nutrients-as well as food security, since they may be dried for later consumption.

Due to the environmental degradation caused by livestock and drought conditions,

have become much less abundant in the vicinity of their villages than they

used to be. The purpose of the research was to study the ecology and biology of the truffle in an effort to develop methods for its agricultural cultivation.

During the 4 years research period we organized three expeditions into the

Kalahari to study the Terfezia pfeilii Henn. Members of both the Is'aeli and the

Botswana teams participated in the first two, only members of the Botswana team

took part in the third. Ascocarps (fruiting bodies) of the truffle were collected during the first two trips, no truffles were found on the third. Truffles were discovered attached to a 'stalk' consisting of sand particles and a mesh of mycelial tissue.

Rhizomorphs were found in fhe vicinity of this 'stalk'. The ascocarps generally occurred on the southern (shady) side of shrubs and trees, only a few of which had roots that appeared to be slightly mycorrhized. For the most part, plants found to be mycorrhized in such locations were both herbaceous monocotyledons and dicotyledonous shrubs and trees.

Soils at T. pfeilii sites had a lower than neutral pH and a low calcium content. Results of experiments conducted in Israel with the aim of effecting mycorrhization of potential symbiont plants such as Acacia species, Grewiaflava or Cynodon dactylon by Terfezia pfeilii were never as clear cut as experiments using

Terfezia leonis (an Israeli desert truffle), T. boudieri and Tuber melanosporum, which developed abundant specific mycorrhiza. Though two types of mycelium

2 were isolated from T. pfeilii fruiting bodies, which resembled the Terfezia leonis, their mycorrhization ability was limited. Isolation of mycelium from truffle 'stalks' in Botswana seemed at first to be successful, but later turned out to be a contamination. Grewia flava, Acocia hebeclada and Acacia melifera seedlings inoculated with mycelium and spores failed to develop mycorrhiza. Cynodon dactylon seedlings inoculated with spores and putative mycelium resulted in mycorrhiza formation, mainly endo-mycorrhiza. Results of small-scale experiments aimed at mimicking bush conditions in Botswana, were ambiguous, as plants in control plots were also mycorrhized, and

similar local mycorrhizal fungi - probably not T. pfeilii - may have mycorrhized the plants in inoculated plots. In spite of the ambiguous results drawn from the experimental part, the ecology and biology of T. pfeilii in its natural habitat was learned and may be used in future domestication projects. Mr. Douglas Thamage, who was trained in Israel within the framework of this project, undertook the research work in Botswana using the skills obtained in Israel.

Research Objectives In rainy years desert truffles (Terfezia and Tirmania) appear throughout the Kalahari desert (1), which covers two thirds of Botswana. They cor.stitute an important element in the diet of the desert dwellers (2,3). The surplus is sold in the town markets, providing gatherers with a crucial source of cash income. The desert truffles are consumed as a side or a main dish. The nutritional value of truffles was found to be high (2,3,4). Terfezia may serve as a source of proteins (20-27% of dry matter), fibers (7-13%) and ascorbic acid, which reaches 2-5% of dry matter (2,3). High potassium levels, fair levels of iron, copper, zinc and manganese as well as essential amino acids were also found (4).

3 Terfezia and Tirmania generally form symbiosis with members of the Cistaceae (5,6,7). In South Africa no members of this family have been found (8), hence other plants enter symbiosis with Terfezia and Tirmania. One of the Kalahari truffles is Terfezia pfeilii identified by Marasas and Trappe (1). As other desert truffles, Terfezia pfeilii is also appreciated by the lo:al inhabitants. The yields of truffles depend strongly upon the rains, which are not regular in the desert. Moreover, recently uncontrolled grazing has disturbed the sandy soil of the Kalahari desert and made it difficult to find truffle sites around Kalahari villages. The objective of this project was to domesticate the locally prized Botswana truffle Terfezia pfeilii and thus to develop an indigenous cash crop that requires very little water and can be grown on Kalahari soils using relatively simple agricultural techniques. We figured that agricultural projects designed for developing countries should follow certain guidelines: a. they should avoid depending on expensive materials and machines which are generally unavailable; b. they should use, as a starting point, materials known to the inhabitants; c. they should avoid damage to the environment. Thus, in the short run indigenous crop may produce better results than new unfamiliar introductions. The Kalahari desert truffle is such a crop, the cultivation of which will require very little fertilizer if at all at the first stage, nor will it require the use of insecticides, a very important factor in developing countries. In addition, this type of agriculture does not call for the cleaning of vast areas of lands for cultivation with its attendant de-stabilizing effect on the fragile ecology. As only indigenous plants will be grown (those having a symbiotic relationship with the truffles), truffle growing will constitute no threat to the environment; on the contrary it could rehabilitate run-down areas while providing a crop much appreciated by the potential consumers. Cultivation of truffles seemed to have a major impact on the improvement of the quality of life on remote area dwellers. 1. The supply of a known, much appreciated

4 important food source-the truffle-could be virtually guaranteed in any suitable area of the Kalahari. 2. Markets could be developed in the less remote towns and villages at more advantageous prices, thus providing a cash income to rural area dwellers. With more research concerning storage, preservation and conservation, this crop may be exported. This kind of treatment could be undertaken by a pilot food processing factory that has already been established on the edge of the Kalahari specifically to process indigenous (wild) fruit, etc. The development of cultivation of Terfezia pfeilii besides being innovative and consonant with research conducted elsewhere in the world today, weuld be also in agreement with the policy of the Botswana government to give priority to development in rural areas, where more than 80% of the population live but less than 5% have gainful employment. As far as we can ascertain different species of the desert truffie Terfezia or Tirmania are not at present agriculturally cultivated. Attempts in this direction have been, or are being, made in some desert countries like (5,9,10). We are working in Israel on the cultivation of the Israeli desert truffle Terfezia leonis and have had some initial success (11,12,13). More advanced is the research and cultivation state of the European truffle - Tuber, which is cultivated in Italy and in

Sou h France, (recently reviewed by Giovannetti et al. (4),.

Methods and Results

The research concentrated on three directions: a. Field expeditions b. Laboratory experiments c. Field experiment

5 a. There were three expeditions to Botswana. Their aim was to learn the ecology and biology of T. pfeilii in the wild. These included a thorough study of the , its attachment to the plant, identification of the symbiont plants, chemical analysis of the soils in which T. pfeilii was found. Another important aim of these trips was collecting of fungal fruiting bodies and seeds of putative plant symbionts for future laboratory experiments. T. pfeilii truffles were not restricted to soils of alkaline pH, as other Terfezia species (15). They were found mainly on the southern, shady side of shrubs and trees, such as Acacia (liebeclada and melifera) and Grewia flava. Perennial Gramineae plants were also present at all the truffle sites. Of all the plant samples taken from the vicinity of the ascocarps and examined under the microscope, only Cynodon dactylon (L.) Pers. and Stipagrostis uniplumis (L.) de Winter seemed to be well mycorrhized. C. dactylon is used in South Africa as a lawn plant. These results are summarized in the attached paper (16).

b. The aim of the laboratory experiments was to mycorrhize potential symbionts with T. pfeilii. We hoped to learn from these experiments the nature of the symbiont plant(s) and the optimal conditions for mycorrhization, which could be used later fcr field experiments.

Mycelium isolation: Truffle mycelium, which resembled the mycelium of Terfezia leonis was isolated from fruiting bodies. Two kinds of mycelia were obtained, a white (w) and a yellow (y) one. The mycelia were regularly grown on Fontana medium (17). Terfezia pfeilii spore germination was induced either in sterilized sand or sterilized vermiculite, saturated with either liquid Fontana medium or liquid MMN (18) or on roots of putative symbiont plants.

6 Inoculation experiments: Inoculation experiments in vitro with isolated mycelium were carried out in solidified (0.8% agar) half-strength Hoagland solution 2 (19). A ca cm piece of Fontana agar containing growing peripheral mycelium was added to a test tube containing 5 ml of the above medium. One seed was then placed in each tube. Inoculation experiments with spores were performed in sterilized soil. Seeds of some potential symbionts were placed in moist vermiculite to germinate. Seeds of Acacia were treated before germination with sulfuric acid, seeds of Grewiaflava were mechanically de-coated to induce germination (Tables 1,2). Germinated seedlings were transferred into a soil mixture containing sterilized sand:vermiculite:Finnish peat in various proportions. Fruiting bodies, containing ripe spores were superficially sterilized, ground and added to the soil mixture. Seedlings were irrigated with half-strength Hoagland solution (19) as seemed necessary. Soil was kept moist at all times.

Mycorrhiza measurement: For ectomycorrhiza the roots were stained by boiling in trypan blue in lactic acid (12). For endomycorriza the root bleaching method (20) was used before staining.

Results: T. pfeilii spores failed to germinate on agar media, but germinated in semi-sterile conditions on sand and vermiculite (table 3) and on Grewia flava roots (fig. 1). Spore germination was dependent upon the culture medium and the substrate (table 3). Throughout the project spore germination decreased, apparently due to their aging. Pre-treatment with acetone, or presence of C. dactylon roots did not improve germination of aged spores. No infection of either Acacia or Grewiaflava seedlings was obtained. However, when Cynodon dactylon was sown together with Acacia species or with Grewia flava some mycorrhization could be found in semi-sterile cultures (table 4). Some

7 mycorrhization of Cynodon dactylon was obtained without the dicotyledon seedlings in soil mixture, which contained 80% sand and 20% peat (table 5), indicating that C. dactylon is a putative symbiont plant. Inoculation was stronger at the higher light intensity than at the lower one, indicating the importance of nutritional state for mycorrhiza formation. Mycorrhization by mycelia isolated fron T. pfeilii was higher than mycorrhization by spores (table 5). The ambiguous results concerning inoculation by spores may stem from the small number of spores used, as found in the case of mycorrhization with Tuber (21), or from a loss of germineability due to spore aging. Bleaching the roots before staining revealed a higher mycorrhization percentages than without bleaching (table 6), indicating that T. pfeilii is an endo-mycorrhitic fungus under the test conditions. Spores of T. pfeilij improved the development of C. dactylon (fig. 2), suggesting that mycorrhiza, if formed, is beneficial to the host plant. The above experiments indicate that Cynodon dactylon is the putative symbiont, which can be mycorrhized by T. pfeilii. It also appears that inoculation by spores is possible, though the exact optimal conditions for mycorrhization remains still unk nown.

c. The field experiments were performed in Botswana. Their aim was to grow mycorrhized plants in situ. Grewiaflava, Acacia hebeclada, Acacia melifera, Eragrostis tef and Cynodon dactylon seeds were sown in beds and inoculated with spores. The roots were examined for mycorrhitic association. The inoculated roots of G. flava were nicely mycorrized, but so were the non-inoculated roots, apparently due to contamination of the soil used. No mycorrhiza was observed in roots of other plants, and also no spores were found in the vicinity of the roots, pointing to the possibility that too small amounts of spores were used. These experiments show the difficulty in successful massive inoculation of suspected symbionts to allow cultivation of T. pfeilii.

8 Table 1. Acacia hebeclada germination treatments.

Treatment % swollen seeds % germination

None 0 0 4 h in H2SO4 100 61 24 h in H2SO4 100 0

Table 2. Grewia flava germination.

Treatment % Germination

None 0

4 h in H2SO4 0

24 h in H2SO4 0

De-coating 10

De-coating + 48 h rinsing in running tap water 30

9 Table 3. Terfezia pfeilii spore germination.

Nutritient Ca 2 + /oTest-tubes containing solution mg/1 germinated spores

Sand Vermiculite

Fontana 16.67 0 0 Fontana 50.00 0 6.25

MMN 16.67 37.5 50 MMN 50.00 56.25 50 MMN 100.00 75 n.d.

n.d. - not determined.

Table 4. Effect of co-cultivation of Cynodon dactylon and three dicotyledonous plants inoculated with T. pfeilii spores on mycorrhization of plant roots.

Dicotyledonous Fungal Control Spores plant relation to root Infected % infected % pots* pots* Acacia Hyphae l\2 50 5\13 38 hebec. Mycorrhiza 0\2 - 8\13 62**

Acacia Hyphae 0\2 - 0\5 - melif. Mycorrhiza 1\2 50 4\5 80**

Grewia Hyphae 0\3 - 2\9 22 flava Mycorrhiza 2\3 67 4\9 44** *Proportion of infected pots of totai pots. ** 1-2 pots showed endomycorrhiza.

10 Table 5. Effect of soil composition and light intensity on association between Cynodon dactylon and Terfeziapfeilii. Results are presented as percent of total number of pots per treatment.

60% shade 30% shade Soil type Fungal type Hyphae Mycor. Hyphae Mycor. No. 1* Control 0 0 0 0 Spores 20 0 11 0 Mycelium (y) 27 0 40 0 Mycelium (w) 18 0 60 0

No. 2** Control (0) 0 0 n.d. Spores I1 0 Mycelium (y) 44 22 67 33 Mycelium (w) 11 11 67 22 *Sand:peat:vermiculite, 1:1:1. **Sand:peat, 4:1. n.d.=not done.

Table 6. Micorrhization of Cynodon dactylon seedlings by Terfezia pfeilii.sporesas revealed by bleaching the roots before staining. (Soil composition: 75% sand, 20% peat and 5% vermiculite). Results are presented as percent of total number of pots per treatment.

Non-bleached roots Bleached roots

Treatment Soil pH ± SE Hyphae Mycor. Hyphae Mycor.

Control 7.69± 0.057 n.d. n.d. 0 0

Spores 7.37 ± 0.032 47 12 0 53 n.d. = not done.

11 Fig. 1. Terfezia pfeilii spore germinating on Grewia flava root. Magnification x400.

.

.,P,2. Effect of Terfezia pfeilii spores added to soil mixture on development of

Cynodon dactylon seedlings.

Cynodon dactylon

Cynodon dactylon Cynodon dactylon + T. pteiIll spores control + T.pfeilI spores + P. Fluorescens

12 Impact, Relevance and Technology Transfer

During the research period both groups took part in expeditions into the desert in

pursuit of truffles, their ecology and biology. This knowledge can be used in future truffle research in general and domestication of T. pfeilii in particular. Mr. Douglas

Thamage was trained in Israel and undertook research in Botswana. Purchase of

some minor equipment and materials using funds made available to this project, along with the utilization of University facilities, represented a strengthening of research capabilities of Veld Products Research.

Project Activities

The Israeli partner participated in two expeditions to Kalahari desert.

The Botswana principal investigator visited Israel twice. Information was flowing

constantly between the partners. One paper, which acknowledges AID-CDR grant, was published (included).

Project Productivity

Regrettable, the project did not accomplished the proposed goal. namely cultivation of Terfezia pfeilii. The atypical characteristics of Terfezia pfeilii, different from those of other Terfezia species were an obstacle to massive mycorrhization and cultivation of mycorrhized plants.

Future Work

We are planning to continue the research after seeking funds from other sources. Further investigation of establishment of mycorrhization and cultivation of mycorrhized plants is needed. Transfer of plants yielding truffles from the wild to a cultivation field may help understand the ways to domesticate the crop.

13 Literature cited 1. Marasas, W.F.O. and Trappe, J.A. 1973. Notes on southern African Tuberales. Bothalia 11, 139-141. 2. Ackerman, L.G., Van Wyk, P.J. and Plessis, L.M du. 1975. Some aspects of the composition of the Kahuhari truffle or N'abba. S.A. Food Review pp. 145-146. 3. Sawaya, W.N., AI-Shalhat, A., Al-Sogar, A. and Al-Mohammed, M. 1985. Chemical composition and nutritive vlaue of truffles of Saudi-Arabia. J. Food Sci. 50, 450-453. 4. Al Delaimy, K.S. 1977. Protein and amino acid composition of truffle. Can. Inst. Food Sci. Technol. J. 10, 221-222. 5. Dexheimer, J., Gerard J., Leduc, J-P and Chevalier, G. 1985. Etude ultrastructurale comparee des associations symbiotiques mycorhiziennes Helianthemuin salicifoliuni-Terfezia claveryi, salicifolium- Terfezia leptoderma. Can. J. Bot. 63, 582-591. 6. Binyamini, N. 1980. Addenda to the hypogeous mycoflora of Israel. Nova Hedwigia Band XXXII Braunschweig pp. 9-20. 7. Chevalier, G., Mousain, D. and Couteaudier, Y. 1975. Associations ectomycorhizrennes entre de Tuberaceae et des Cistaceae. Ann. Phytopathol. 7, 355-356. 8. Riley, H.P. 1963. Families of flowering plants in Southern Africa. University of Kentlickey Press. 9. Awameh, S.M. and Alsheikh, A. 1980. Ascospore germination of black kam6 (Terfezia boudieri). Mycologia LXXII, 50-54. 10. Awameh, M.S. 1981. The response of Helianthemrum salicifolium and H. ledifoliutn to infection by the desert truffle Terfezia boudieri. Mushroom Science XI, 843-853. 11. Kagan-Zur, V., Roth-Bejerano, N. and De Malach, Y. 1986. Truffle cultivation on marginal lands in Israel. Report No. BGUN-ARI-73-86 of the Institutes for Applied Reserch, Ben-Gurion University of the Negev. (In Hebrew). 12. Roth-Bejerano, N., Livne, D. and Kagan-Zur, V. 1990. Helianthemum-Terfezia relations in different growth media. New Phytol. 114, 235-238. 13. Kagan-Zur, V., Raveh, E., Lischinsky, S. and Roth-Bejerano, N. 1994. Heliantheinun-Terfezia association is enhanced by lack of iron in the growth mediums. New Phytol. 127, 567-570. 14. Giovannetti, G., Roth-Bejerano, N., Zanini, E. and Kagan-Zur, V. 1994. Truffles and their cultivation. Hort. Rev. 17:71-107.

14 15. Poitou, N., Villenave, P. and Delmas, J. 1983. Croissance du mycelium de Tuber melanosporum Vitt et de certains competiteurs en fonction du pH du milieu. C.R. Sc. de l'Acad d'Agric. France 69 (16), 1363-1369. 16. Taylor, F.W., Thamage, D.M., Baker, N., Roth-Bejerano, N. and Kagan-Zur, V. 1995. Notes on the Kalahari Desert Truffle, Terfezia pfellii (Terfeziaceae). Mycol. Res. 99, 874-878. 17. Bonfante, P.F. and Fontana, A. 1972-1973. Sulla nutrizione del micelia di Tuber melanosporuiw Vitt. in coltura. At. Acad. Sci. Torino 731-741. 18. Marx, D.H. 1969. The influence of ectotropic mycorrhizal fungi on the resistance of pine roots to pathogenic infection. I: Antagonism of myccorhizal fungi to root pathogenic fungi and soil bacteria. Phytopathol. 59, 153-163. 19. Hoagland, D.R. and Arnon, D.I. 1950. The water culture method for plants without soil. Circular 374, Univ. of California, Agriculture Experimental Station, Berkeley. 20. Koske, R.E. and Gemma, J.N. 1989. A modified procedure for staining roots to detect VA mycorrhizas. Mycol. Res. 92:486-488. 21. Roth-Bejerano, N. and Kagan-Zur, V. 1994. Introduction of Tuber nelanosporum the black Perigord truffle as a potential crop for calcareus lands in Israel. Report No. BGUN-ARI-8-94 of the Institutes for Applied Research, Ben-Gurion University of the Negev. (In Hebrew).

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