changes in lipid composition during ascocarp ...

Mycologia, 80(6), 1988, pp. 900-903. © 1988, by The New York Botanical Garden, Bronx, NY 10458

CHANGES IN LIPID COMPOSITION DURING ASCOCARP DEVELOPMENT OF THE TRUFFLE, TUBER MELANOSPORUM 1 M. SANCHOLLE, 2 ,4

J.D.

WEETE, 3 •5 M. KULIFAJ, 2 AND

C.

MONTANT 2

Laboratoire de Clyptogamie, Universite Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse Cedex, France 3 Department ofBotany and Microbiology, Alabama Agricultural Experiment Station, College of Sciences and Mathematics, Auburn University, Auburn, Alabama 36849-5407 2

.r

Species of the genus Tuber are mycorrhizal ascomycetous fungi with subterranean fruiting bodies. Most research on this genus has focused on systematics and attempts to induce ascocarp formation (Montant eta!., 1983; Parguey-Leduc et a!., 1984), and more recently on the morphology of mature ascocarps (Parguey-Leduc et a!., 1987b) and the ultrastructure of ascospore development (Parguey-Leduc et a!., 1987a). In recent years the demand for truffles has increased, but yields have decreased dramatically for unknown reasons. In spite of the commercial importance of truffles, e.g., Tuber melanosporum Vitt. (black truffle of the Perigord), in France and elsewhere, relatively little is known about the biochemistry and physiology of these fungi, particularly as they relate to ascocarp development. Kulifaj (1984) reported changes in various ascocarp constituents as a function of development, and we have identified the sterol components of ascocarps of several Tuber species (Weete eta/., 1985). This communication is an extension of our research on T. melanosporum in which we report changes in the total lipid, fatty acid, sterol and lipid phosphorus contents of ascocarps as a function of development with the aim of identifying chemical characteristics that might signify ascocarp maturity. The fruiting bodies used in the present study were collected, as for previous studies (Montant eta/., 1983), in southeastern France, near Valreas (Vaucluse). Ascocarps were collected from truffle-bearing oak trees as described previously (Montant eta!., 1983; Kulifaj, 1984). The char1

AAES publication No. 6-881530P.

This work was conducted while the first author was on sabbatical leave at Auburn University. 5 Corresponding author. 4

acteristics (weight, time of harvest, morphological and cytological) used to distinguish the six stages of ascocarp development for this study are summarized in TABLE I. The tissues were dried by lyophilization and stored at -10 C prior to analysis. Lipid was extracted from the fungal material according to the Bligh and Dyer (1959) procedure, as reported by Kates (1972) for microorganisms. For each ascocarp stage of development, portions of several ascocarps were pooled prior to extraction. Total lipid content was measured gravimetrically. A portion of the total lipid was subjected to alkaline hydrolysis, and the total sterols and fatty acids were isolated as described previously (Weete et a/., 1983). Fatty acids were analyzed as their methyl ester derivatives by capillary gas-liquid chromatography (GLC) (Weete eta!., 1983), and sterols were analyzed as their acetate derivatives by GLC using a 25-m, fused-silica, DB-5 glass capillary column (J. and M. Scientific) at 280 C isothermal and with a carrier gas (He) flow rate of about I m!lmin (Weete et al., 1985). Fatty acids and sterols were identified by comparison of retention times with authentic standards (Applied Science Laboratories, State College, Pennsylvania), and quantitated by the internal standard method using methyl docosanoate and 5-dihydrocholesteryl acetate as standards, respectively. Lipid phosphorus was measured colorimetrically by the modified Bartlett micro procedure (Bartlett, 1959). The total lipid content of Stage I ascocarps was relatively high at about 25% of the dry weight and decreased progressively to a relatively low lipid content of3.6% in those at Stage V (TABLE II). The most mature ascocarp (Stage VI) contained about 9% lipid. In spite of the substantial decrease in total lipid content over the course of

901

BRIEF ARTICLES

TABLE

I

CHARACfERIZATION OF SIX STAGES DURING DEVELOPMENT OF THE FRUIT-BODY OF TUBER MELANOSPORUM

Stage

Weight

Cytological characters

Aroma

B/Ed

Saprophytic cells developing axially in the fruit-body No asci yet Sporophytic cells in centrum and periphery First asci developing Many asci

0

1.8

0

1.5

0 0

1.5 2.0

Ascus development completed but some more asci can still develop under the peridial scurf The whole fruit body is like a "spore bag"

+

0.5

++

0.6

Morphological characters

I•

0.3 g

Peridial scurf orange-red Flesh white

II•

0.3-0.75 g

Peridium orange-red

Ill"

IVl'

0.8-2 g 2.5 g

Vc

Variable

Same aspect as Stage II Peridium reddish-brown (light to dark) Flesh white Substantial weight increase Peridium black with reddishbrown shades Flesh cream to grey

VIc

Variable

Peridium and flesh black (melanization completed)

• Stages I to III can be collected using a special method of raking described by Montant et a!. (1983). b Stage IV can be detected by a light swelling and cracking of the ground above the fruit-body. c Stage V and VI detectable by dogs, pigs and flies. d B = brassicasterol; E = ergosterol.

ascocarp development (80% reduction from Stages I to IV), the phospholipid content remained relatively constant on a dry weight basis from Stages I to IV, but progressively decreased to 5% at Stage VI (TABLE II). Fatty acids detected in the ascocarps were essentially those that might be expected for Ascomycetes (Weete, 1980). The major fatty acid throughout development was linoleic acid (5077% of the total), with the highest levels occurring during Stages II to IV (TABLE III). Myristic acid was one of the major fatty acids of the Stage

I ascocarp at 18%; it was not detected in Stages II to IV but was a minor component of the mature fruiting bodies (Stages V and VI). Otherwise, palmitic acid was the predominant saturated fatty acid detected at each of the six developmental stages. Linolenic acid (19%) was also a major component of the total fatty acid fraction of the Stage I ascocarps, but was either absent or a minor component in ascocarps in the other stages of development. It was shown previously (Weete eta!., 1985) that ergosterol (24,6-methyl-cholesta-5, 7 ,22E-

TABLE

II

CHANGES IN THE TOTAL LIPID AND STEROL CONTENTS OF ASCOCARPS OF TUBER MELANOSPORUM AS A FUNCfiON OF DEVELOPMENT

Stage of development I (very young) II III IV

v

VI (mature)

Total lipid

Total phospholipid•

Total sterol

(f.!g/mg DW)

(f.!g/mg DW)

(f.!g/mg DW)

251.9 212.0 149.8 50.1 36.0 89.8

± ± ± ± ± ±

lO.P 1.0 3.0 1.0 0.7 1.0

13.8 14.9 16.5 14.3 10.2 4.9

± ± ± ± ± ±

0.5 0.6 0.4 0.4 0.3 0.6

0.77 1.17 0.98 1.01 0.56 0.32

± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.0 0.0

Ergosterol

Brassicasterol

(%)

(%)

65.3 60.7 60.8 66.9 33.2 37.5

± ± ± ± ± ±

5.2 5.1 4.7 11.0 2.6 10.2

34.7 39.3 39.2 33.1 66.8 62.5

• Phospholipid was calculated by multiplying the lipid phosphorus content by 25. Standard deviation values are based on the average of two replicates.

b

± ± ± ± ± ±

5.2 5.1 4.7 11.0 2.6 10.2

Sterol/phospholipid molar ratio 0.11 0.16 0.12 0.14 0.11 0.13

·,•'

MYCOLOGIA

902

. M

;;

...-

r:--:.ni.C)~~.....: ............................ .-.~

.0

......

i