New Phytol. (1982) 92, 533-537 MYCORRHIZAL SYNTHESIS BETWEEN CISTACEAE ANJy TUBERACEAE BY G. GIOVANNETTJ AND A. FONTANA Centra di Studio sulla Micologia del Terreno del C.N.R. 10125 Turin, Italy (Accepted 12 August 1982) SUMMARY Cistus incanus ssp. incanus was tested with six Tuber species to prove its ectomycorrhizal development; the sanne was done with Tuber melanosporutn and five Cistus speeies. Cistus incanus L. ssp. incanus formed eetomycorrhizas with Tuber albidum Pico, T. brumale Vitt., T. aestivum Vitt. and T. rufum Pico. The five Cistus species {Cistus albidus L., C Iciurifolius L., C salvifolius L., C crispus L. and C. monspeliensis L.) developed ectomycorrhizas with Tuber melanosporutn Vitt. These Cistaceae may be used as shrubby plants and as colonizers in truffle cultivations; the variety of their environmental and ecological requirements is such that it would be possible to increase the types of soil and number of places where truffles can be grown. INTRODUCTION Symbiotic association between Cistus incanus L. ssp. incanus and Tuber nielanosporum Vitt. was observed in nature and mycorrhizal synthesis has already been obtained in artificial conditions by Fontana and Giovannetti (1978-^79). After 1 year, infected C. incanus ssp. incanus seedlings were planted out in an experimental plot; 2 years later they had grown in height to 40 cnn and were surrounded by circular characteristic ' burnt' areas which normally precede truffle fructifications (Delmas, 1978). In fact one peculiar feature of T. melanosporutn biology is that, when its mycelium develops in the soil to form mycorrhizas and sporocarps, almost all the herbaceous vegetation around the mycorrhizal plant dies. These encouraging results led us to believe Cistaceae might enhance truffle growth. We decided to test the capacity of C. incanus ssp. incanus to form ectomycorrhizas with other species of edible Tuber and with T. rufum. In addition to C. incanus we used five other Cistus species with a range of different ecological requirements, to test their ectomycorrhizal capacity with T. melanosporum. MATERIALS AND METHODS The method closely resembles that used to obtain ectomycorrihizas with C. incanus ssp. incanus and T. melanosporum. We carried out two tests in semi-aseptic conditions. During the first, C. incanus ssp. incanus was tested for synthesis with six Tuber species (T. aestivum Vitt., T. brumale Vitt., T. macrosporum Vitt., T. mesentericum Vitt., T. albidum Pico and T. rufum Pico); during the second test five Cistus species (C. albidus L., C. laurifolius L., C. salvifolius L., C. crispus L. and C. monspeliensis L.) were tested in relation to T. melanosporum. Cistus seeds were delicately abraded with emery paper and then soaked for 10 min in a 1 % HgCl^ solution. After sterilization they were rinsed three times in sterile HjO. The fungal inoculum consisted of a carpophore fragment from the inner part which had been pounded in a mortar. This was mixed with the seeds and put into perforated plastic bags containing 50 % marly soil and 50 % limy sand 0028-646X/82/120537 + 05 S03.00/0 1982 The New Phytologist
534 G. GlOVANNETTI AND A. FoNTANA mixture (ph 7) previously sterilized in a steam flow sterilizer. Nine plastic hags containing fungus and seeds and three containing seeds only (as controls) were prepared for each species of Tuber of the first set (Test 1) and for each species of Cistus of the second set (Test 2). All plastic bags were kept in a greenhouse, under a drip irrigation system. Temperatures ranged hetween 15 and 25 to 30 C according to the season. After seed germination, ten seedlings (chosen from amongst the most vigorous) were left in each plastic bag. These synthesis tests began in June 1980 and were checked after 6 and 12 months for the mycorrhizal formation. RESULTS Test 1 At 6 months the faster growth of the inoculated seedlings indicated mycorrhizal development [Fig. l(a)]. At 12 months root and stem growth of seedlings inoculated with T. albidum, T. brumale, T. aestivum and T. rufum was considerably more advanced than that of the controls [Fig. 1 (c)]. However, inside a single plastic bag not all the seedlings were mycorrhizal. Seedlings inoculated with T. macrosporum and T. mesentericum had only grown as fast as the controls. Stereoscopic and cross-section microscopy of C. incanus roots inoculated with the first four Tuber species mentioned above revealed simple and branched mycorrhizas with occasional clusters. Colours ranged from light to dark amber. The surface was either smooth or bore hyphae or setae. There was a pseudoparenchimatous fungal sheath, and a Hartig net between the two or three layers of host cells. These host cells had not apparently undergone any morphological change [Fig. l(b)]. The above general characteristics are found in mycorrhizas formed by fungi of the genus Tuber but the specific mycorrhizal characteristics varied according to the Tuber species and were consistent with previous observations of these same mycorrhizal fungi on other plants. T. albidum ectomycorrhizas were characterized hy the presence of elongated hyphae with septa and no branches (Fontana and Palenzona, 1969), T. brumale ectomycorrhizas by thick setae with no branches (Fontana and Fasolo-Bonfante, 1971; Chevalier, 1973), T. aestivum ectomycorrhizas by wavy hyphae of constant diameter (Palenzona, 1969; Chevalier et al., 1979) and T. rufum ectomycorrhizas by the presence of a smooth sheath or one with a few thin and winding hyphae (Palenzona, Chevalier and Fontana, 1972). Test 2 Six months after the inoculation with T. melanosporum, the C. albidus, C. laurifolius and C. salvifolius seedlings possessed the characteristic black truffle ectomycorrhizas recognizable at macro and microscopic levels. These were similar to those described in C. incanus ssp. incanus (Fontana and Giovannetti, 1978-79). Furthermore, the seedlings had grown faster than the uninoculated ones (Fig. 2). C. crispus and C. monspeliensis also developed T. melanosporum ectomycorrhizas but only after 12 months from inoculation. CONCLUSIONS Mycorrhizal synthesis between hypogeous fungi and Cistaceae was obtained for thefirst time by Chevalier, Mousain and Couteaudier (1975) using T. melanosporum, T. aestivum and T. brumale with Fumana procumbens and C. salvifolius. The present
535 Cb) ^.= i, J....i... ' - ' 1 ; (c) i^ 1.,-;!>''_Li _. ii_:i! j^! ' ' "! ' ' ' < ' ^' "' ' ' '- *dling inoculated with T'. atbidum and uninfected controil at 6 months; (b) ectomycorrhizal section x400. (c) C. incanus seedlings inoculated with T. aestivum and controls at 12 months, (Test 1),
S36 G. GlOVANNETTI AND A. FONTANA (a) (b) Fig. 2. C. atbidus (a) and C. salvifolius (b) seedlings inoculated with T. melanosporum and uninfected controls at 6 months. (Test 2). results demonstrate that mycorrhizal synthesis hetween T. aestivum, T. albidum, T. brumale, T. rufum and C. ineanus ssp. incanus may occur in semi-aseptic environments. This is also true for C. salvifolius, C. albidus, C. laurifolius, C. crispus, C. monspeliensis and T. melanosporum. However, it is important to determine the possible existence of the mycorrhizal association between Cistus and Tuber in nature.
Mycorrhizas between Cistus and Tuber 537 Furthermore, it is observed that the morphology of the ectomycorrhizas of the various species of Tuber seen on shrubby plants such as Cistus sp.pl. is the same as that found in arborescent plants, at least as regards the fungal sheath and its ornaments. The negative results we have obtained so far with T. mesentericum and T. macrosporum can be traced to the difficulty of mycorrhizal synthesis with Cistaceae; undoubtedly, further tests are required. Our results suggest that C incanus ssp. incanus could be used as a shrubby plant for T. melanosporum cultivation as well as for T. aestivum, T. brumale and T. albidum, all edible truffles. Cistus incanus and the other Bve tested Cistus species could be used to grow T. melanosporum. C. incanus, along with C. salvifouus and C. monspeliensis, is among the most common plants of the Mediterranean Hora. C. incanus is ver>' easily found in the Eastern areas, C. salvifouus and C. monspeliensis are distributed all over the Mediterranean area. All three species grow in limy and siliceous soils. C. crispus, C. albidus and C. laurifolius are rare in Italy; the first two can be found in the Western Mediterranean areas, while the third is unevenly scattered in all the Mediterranean Basin. C. albidus is a calcifuge; C. crispus grows both in limy and siliceous soil; C. laurifolius in siliceous soil only (Rizzotto, 1979). The use oicistus spp. to form mycorrhizas, and subsequently truffle carpophores, has many possible applications such as: (1) Cistus spp. may colonize poor, marginal soils, waste land and land not suitable for woody plants; (2) they may be used as shrubby plants in artificial truffle sites; (3) they can be employed on their own or together with the trees usually used for truffle cultivation; (4) they, as shrubby plants need smaller areas than woody plants and therefore,, in artificial truffle grounds, it is possible to increase the number of plants per hectare. This ought to increase the yield of truffle carpophores per unit surface. REFERENCES CHEVALIER, G. (1973). Synthese axenique des mycorhizes de Tuber brumale Vitt. a partir de cultures pures du champignon. Annales de Phytopathologie, 5 (2), 163-182. CHEVALIER, G., DESMAS, C, FKOCHOT, H. & RIOUSSET, L. (1979). L'espece Tuber aestivum Vitt.: I. Definition. Mushroom Science, 10 (part I), 957-975. CHEVALJER, G., MOUSAIN, D. & COUTEAUDIER, Y. (1975). Associations ectomycorhiziennes entre Tuberacees et Cistacees. Annales de Phylopathologie 7 (4), 355-356. DELMAS, J. (1978). Tuber spp. In: The biology and cultivation of edible mushrooms (Ed. by S. T. Chang & W. A. Hayes), pp. 645-681. Academic Press, New York, San Francisco and London. FoNTANA, A. & FASO'LO-BONFAN-TIE, P. (1971). Sintesi naicorrizica di Tuber brumale Vitt. con Pinus nigra Arnold. Allionia, 17, 15-18. FoNTANA, A. & GlovANNE-m, G. (1978-79). Simbiosi micorrizica fra Cistus incanus L. ssp. incanus e Tuber melanosporum Vitt. Allionia, Zi, 5-11., FoKTANA, A. & PALENZOKA, M. (1969). Sintesi micorrizica di Tuber albidum in coltura pura, con Pinus strobus e pioppo euroamericano. Allionia, IS, 99-104. PALENZONA, M. (1969). Sintesi micorrizica tra Tuber aestivum Vitt., T. brumale Vitt., T. melanosporum Vitt. e semenzali di Corylus avellana L. Allionia, 15, 121-131. PALENZONA, M., CHEVALIER, G. & FONTANA, A. (1972). Sintesi micorrizica tra i miceli in coltura pura di Tuber brumale, T. melanosporum, T. rujum e semenzali di conifere e latifoglie. Allionia, 18, 41-52. RIZZOTTO, M. (1979). Ricerche tassonomiche e corologiche sulie Cistaceae. 1. I! genere Cistus L. in Italia. Webbia, 33, 343-378.