INDOLE-3-ACETIC ACID PRODUCTION BY MYCORRHIZAL FUNGI DETERMINED BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY

New Phytol. (1983) 94, 401-407 401 INDOLE-3-ACETIC ACID PRODUCTION BY MYCORRHIZAL FUNGI DETERMINED BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY BY M. EK, P. O. LJUNGQUIST AND ELNA STENSTROM* Swedish Forest Products Research Laboratory, Box 5604, S-114 86 Stockholm, Sweden (Accepted 5 March 1983) SUMMARY A method for determining indole-3-acetic acid (IAA) production in mycorrhiza-forming fungi was developed. Culture medium and the fungal mycelium were, extracted with ethyl ether together with deuterated IAA as an internal standard, and the extracts thereafter evaporated to dryness. The dry extracts were then silylated in pyridine with BSTFA and analysed by gas chromatography-mass spectroscopy. Compared to other methods the procedure required a minimum of preparatory work, gave good reproducibility and a standard deviation of acceptable level for studies involving biological material. The method was used to study IAA production in 16 mycorrhiza-forming fungi. Results indicated large differences in the ability of the fungal strains to produce IAA. Pisolithus tinctorius 185, a strain previously shown by other workers to give a strong root infection in field experiments, produced the largest amount of IAA. Several other fungi showing high IAA values also infected plant roots with relative ease in laboratory experiments. INTRODUCTION Auxins are widespread in nature and are produced by plants and many fungi. Their importance as plant hormones is well known and the capacity of fungi to produce auxins has often been reported. Slankis (1948,1951) first demonstrated that auxins were produced by mycorrhizal fungi. Ulrich (1960) reported that only Suillus variegatus and S. granulatus out of 11 fungi produced indole-3-acetic acid (IAA) without tryptophan being present in the culture medium. All other mycorrhizal fungi examined required tryptophan as a precursor. Paper chromatography and biological tests with Avena coleoptiles were used to determine the production of IAA. Tomaszewski and Wojciechowska (1973) also used the Avena coleoptile curvature test to determine the production of auxins in a number of mycorrhizal fungi, and Strzelczyk, Sitek and Kowalski (1977) used paper chromatography combined with the Avena coleoptile test. The significance of auxin production by mycorrhiza-forming fungi is still uncertain (Slankis, 1974). The auxin might affect the infection or the metabolism of the host plant after the infection. Slankis (1958) showed that high concentrations of IAA in the growth medium of pine seedlings produced a similar morphological change in the short roots as did a mycorrhizal infection. The aims of the present study were twofold. Firstly, to develop a specific and relatively simple method to determine the production of IAA. Secondly, adoption To whom correspondence should be addressed. 0028-646X/83/070401 +07 $03.00/0 1983 The New Phytologist

4O2 M. EK et al. of this method to determine the production of IAA by several mycorrhiza-forming fungi. MATERIALS AND METHODS Cultures of mycorrhizal fungi Pure cultures of 19 mycorrhizal fungi were used. Most were isolated in this laboratory, 11 from fruiting bodies which had grown in close proximity to pine {Pinus silvestris) and two from mycorrhizal roots. Six isolates were received as gifts (Table 1). Table 1. Mycorrhizal fungi used in this study Mycorrhiza-forming fungi Suillus luteus Suillus luteus Suillus variegatus Suillus granulatus Suillus bovinus Suillus bovinus Boletus badius Amanita muscaria Amanita muscaria Pisolithus tinctorius Pisolithus tinctorius Tricholoma imbricatum Ecto mycorrhiza (unknown) Ecto mycorrhiza (Thelephora terrestris) Laccaria laccata Hebeloma crustuliniforme Hebeloma crustuliniforme Lactarius rufus Cenoeoccum graniforme Isolated from mycorrhiza. Strains S.I. 78a S.I. 79a S.v. 78c S.g. 79a S.b. lie S.b. 79a B.b. 78d ^.m. 78a A.m. 78c P.t. 185 P.<. 79a T.i. 79a M:l* M:12* L.I. S-238A H.c. (siv) H.c. 81a L.r. 79b e.g. Received from G. Lindeherg, Uppsala, Sweden D. Marx, Athens, USA A. Strandberg, Uppsala, Sweden R. Molina, Corvallis, USA J. Garbay, Champenoux, France G. Chevalier, Clermont Ferrand, France Pure cultures from fruiting bodies were obtained by aseptically cutting small pieces from the inside of the sporocarp with a sterilized knife. The pieces were placed on modified Melin-Norkrans medium (MMN) (Marx, 1969) with 15% agar and on MMN containing 20 mg 1""^ benlate (Du Pont), 50 mg T^ neomycin sulphate and 50 mg 1~^ streptomycin sulphate (MMN-B) and the mycelium was allowed to grow out. When isolating fungi from mycorrhizal roots, the roots were rinsed in water and cleaned under the microscope before being surface sterilized in HgCla (01 %) for 4 s. Thereafter, the roots were washed five times in sterile water, placed on MMN and MMN-B with agar and the mycelium allowed to grow out. One of the isolates from mycorrhizal roots (M:l) was a fungus which sometimes spontaneously infected young pine seedlings in a nursery (Korsnas Marma Nursery, Nassja, Sweden). M:l produces white bifurcated mycorrhiza with a lot of external mycelium and rhizomorphs. The other isolate (M: 12) is very frequently found in Swedish nurseries and is probably Thelephora terrestris.

spectrometry of IAA in mycorrhizal fungi 403 Culture conditions. All fungi were grown in 100 ml MMN medium in 0-05 M KH2PO4 buffer (ph 5-0) in 250 ml Erlenmeyer-flasks. To the medium was added 50 mg 1~^ sterile filtered (Millipore 0-45 /im) tryptophan. Four pieces of mycelium on agar (5 mm) were transferred to each of eight flasks. The fungi were grown at room temperature (23 "C) in darkness for 5 weeks, except for Pisolithus tinctorius 185 and 5. bovinus 79a which were harvested 3, 5 and 7 weeks after they had been transferred to the liquid medium. Determination of IAA Selection of method. The biological determination of auxins using Avena coleoptiles (Larsen, 1955; Nitsch and Nitsch, 1956) and the spectrofiuorometric method (Stoessl and Venis, 1970; Hemberg and Tillberg, 1980) include a rather long preparatory procedure, during which losses of auxins can occur. At the start of the investigation, the spectrofluorometric method described by Hemberg and Tillberg (1980) was chosen but it was found to give poor reproducibility. IAA is an unstable molecule which is easily oxidized in the presence of light and oxygen. This was especially conspicuous in the fungi investigated here. Therefore, it was necessary to develop a method for its determination which comprised as little preparation as possible. A procedure based upon gas chromatography-mass spectrometry (GC-MS) was chosen (McDougall and Hillman, 1978). Such a method requires no purification of the extracts prior to analysis since these contain no substances which might interfere with the GC-MS analysis. Furthermore, the method incorporated deuterated IAA (dj-iaa) as an internal standard reacting in the same way as IAA. This is of particular importance when working with fungi which may both produce and destroy IAA (Strelczyk et al., 1977). The use of dg-iaa as internal standard also made it possible to monitor the amount of IAA degradation which occurred during extraction of fungal mycelium and culture filtrate. Extraction of IAA. When a fungus was harvested pairs of flasks were pooled to give four parallels before filtering through a glass fibre filter. The mycelium was homogenized in 80 % cold methanol in a tissue grinder fitted with a teflon pestle and the internal standard (dj-iaa) (see below) was added. The homogenized mycelium in methanol was then allowed to stand at 5 C for 20 h to facilitate auxin extraction. Thereafter, samples werefiltered through a glass fibre filter and the mycelium dried overnight at 90 C for dry weight determinations. The methanol was evaporated and 50 ml 0 05 M K2HPO4 buffer (ph 8 0) was added to the residual water phase, which contained small particles of undissolved materials, to dissolve the material. The ph was then lowered to 2*7 by addition of 2-5 M H3PO4 and the solution extracted three times with 50 ml ethyl ether. The ether fraction was dried over Na2SO4 and evaporated to dryness. Itnmediately after separating the mycelium from the culture medium, the internal standard was added to the culture filtrate and the ph adjusted to 2-7 with 2 5 M H3PO4. The culture filtrate was then extracted with ethyl ether, the ethereal layer dried over Na2SO4 and evaporated to dryness. Gas chromatography-mass spectrometry. The residues from the ether extractions were dissolved in 0-1 ml pyridine and 0-1 ml Ar,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) to silylate the IAA. After reacting at 50 C for 2 h the IAA was quantified with GC-MS. Each sample was injected three times.

404 M. EK et al. The GC-MS investigations were carried out using a Carlo Erba (Fractovap MOD 2900) gas chromatograph-finnigan mass spectrometer (3200 F) with an online computer system (6000). The column used was an OV 101 glass WCOT (25 mxo27 mm ID) manufactured by LKB, Stockholm, Sweden. The injector temperature was 250 ^^C and the detector temperature was 300 C. The oven temperature was isothermal at 220 C. The carrier gas flow was 0-82mlmin~^ and the split ratio 1:30. Mass spectrometry was operated in the multiple ion detection mode (MID). The mass spectra were all run with 70 ev. The transfer line temperature was 300 C and the ion source temperature 100 C. Molecular ions (319, 321) and the base peaks (202, 204) were chosen, the retention time was 3*45. Deuterated IAA, prepared according to Caruso et al. (1978), was used as internal standard. Response factor was deternnined before each quantification series and was taken from the corresponding calibration curve. Values are expressed in ng IAA mg~^ dry weight of the mycelium. All chemicals were of analytical grade. The ethyl ether was freshly distilled prior to use to eliminate peroxides. RESULTS AND DISCUSSION Analytical method The advantage of using the GC-MS method instead of bioassay methods is that the internal standard reacts in the same way as IAA. The yield of the standard was determined in every sample and was found to vary between 10 and 100%. No clear correlation was found between yield and any other factor such as fungal species, total amount of IAA or date of determination. This means that even during this preparation of the sample unknown factors can cause a breakdown of up to 90 % of the original IAA. With this method, the original amount of IAA in each sample can be calculated since the yield is known in each case. This correction is made in Table 2. Since no simple correction is possible in the coleoptile test or in the spectroruorometric test, the presented method using GC-MS should give more reliable values. The reproducibility was good. For example, the values for P. tinctorius 185 using four parallels harvested after 5 weeks were 16 760 + 5160 ng mg~^ dry weight in the culture medium and 790 + 290 ng mg~^ dry weight of mycelium. When repeated 2 months later with the same growth conditions the corresponding values were 18 970 + 3180 ng mg~^ dry weight in the culture medium and 790 + 100 ng mg~^ dry weight of mycelium (Table 2). The relatively high and standard deviations must be considered as normal for biological material and are not dependent on the method used. However, quantification of IAA by GC-MS is not as sensitive as either the coleoptile or the spectrofiuorometric test. The detection limit on the GC-MS was 10 pg per injection. Finally, this method only gives the IAA content and not that of other auxins that might be produced by the fungi. To determine other auxins, the coleoptile test would be more valid. Auxin content Figures 1 and 2 show how the amount of auxin found in the mycelium and the medium varies for the two fungi, P. tinctorius 185 and S. bovinus 79a, as a function of growth time.the results show that the amount of IAA in the medium is much higher than that in the mycelium (by a factor of 10 to 20). The fact that

spectrometry of IAA in mycorrhizal fungi 405 Table 2. Amounts of IAA in cultures of different mycorrhizal fungi and fungal dry weights after 5 weeks Suillus luteus 78a Suillus luteus 79a Suillus variegatus 78c Suillus granulatus 79a Suillus bovinus 77c Suillus bovinus 79a Boletus badius 78d Amanita muscaria 78a Amanita muscaria 78c Pisolithus tinctorius 185 Pisolithus tinctorius 79a Tricholoma imbricatum 79a M:l M:12 Laccaria laccata S-238A Hebelotna crustuliniforme (siv) Hebeloma crustuliniforme 81a Lactarius rufus 79b Cenococcum graniforme Growth medium (ng IAA mg~* dry weight) 18±5 110 + 40 16 + 2 95 + 120 6530 + 3870 600+120 43 ±17 67 + 24 13 + 13 18970 + 3180 840 + 612 33 + 20 550+150 1010 ±390 79 ±25 380+180 27±2 Mycelium (ng IAA mg~' dry weight) 1 + 2 150+100 79 ±20 4 + 4 790±110 4 + 5 140 + 8 47 ±13 15±8 190+180 80±7 Dry weight of the mycelium (mg 1-') 840±7 840 + 95 1035 ±25 385±1OO 940 ±22 71O±5O 530+62 353 ±72 605 ±66 670+14 255 ±30 265 ±142 1010±44 635±37 400 + 28 586 + 32 383±15 94±13 880 ±76 Each value is the mean of four determinations ±s.d. the fungi are still growing during the whole period of 5 to 7 weeks suggests that the homone in the medium is not a result of lysis. The amount of precursor (tryptophan) available should still be adequate although nitrogen might be limiting and restrict metabolism. To avoid nitrogen depletion cultures of all other fungi were harvested after 5 weeks' growth. Table 2 shows the amounts of IAA in the mycelium and growth medium for the different strains. The values are corrected for losses during preparation. In both cases P. tinctorius 185 produces the highest levels. The other strain of P. tinctorius investigated has a much lower, but still comparatively high, IAA content in the medium but no IAA could be detected in the mycelium, indicating a difference in IAA content by a factor of 1000 between the two strains. The Suillus group is relatively homogeneous with regard to IAA content except for the two strains of 5. bovinus which show much higher contents. The two Hebeloma crustuliniforme strains are very different from each other with one of them showing no detectable IAA. Only in Cenococcum graniforme was the amount in the mycelium higher than that in the medium. Tomaszewski and Wojciechowska (1973) found a direct correlation between dark pigment exuded into the culture medium and the amount of IAA produced. This is true for most of the fungi examined here but there are exceptions. M: 1 gave a dark yellow culture medium but only low levels of IAA. Conversely, M: 12, Laccaria laccata and Lactarius rufus produced relatively large amounts of IAA while the culture medium was uncoloured. There are obviously great differences in the rates of IAA production and degradation among different fungi and even between strains of the same fungus. The high standard deviations in some cases, especially for S. granulatus, also point to either a high genetic variability even within the strain or a strong dependence on cultural conditions, reflecting small differences between parallels. In this study.

4o6 M.EKet al. cn c E D 91 1000-500- 3 Time of growth (weeks) Fig. 1. Amounts of I AA in cultures of Suillus bovinus {S. b. 79a), variation with time in the mycelium ( X ) and in the culture medium (O)- Each point is the mean of four determinations. Bars represent the standard deviation. - 1000 E < < 10 000- -500 T3 E Time of growth (weeks) Fig. 2. Amounts of laa in cultures of P. tinctorius (P. t. 185), variation with time. In the mycelium ( X ) and in the culture medium (O)- Each point is the mean of four determinations. Bars represent the standard deviation. the same cultural conditions have been chosen for all strains, regardless of their growth rate, ph optimum, etc. This was to obtain a rough classification of many strains. It is likely that different strains require different amounts of tryptophan for maximum IAA production. The important question concerns the physiological importance, if any, of the observed large differences in IAA production for the mycorrhiza and for the host plant. Firstly, IAA may influence the infection stage. It is interesting to note that the highly infective P. tinctorius 185 has a very high level of IAA production. L. laccata S-238A, also with high IAA levels, similarly readily infects seedlings of different conifers (Molina, 1980). The same is true for S. bovinus lie, P. tinctorius 79a and M; 12 (probably T. terrestris). The high levels of IAA in these fungi could indicate that IAA has a function in the infection process between plants and fungi. The only obvious exception to this rule is Hebeloma crustuliniforme (siv) that, according to Garbaye (pers. comm. 1981) and our own unpublished experiments, is very infective but does not show a high IAA value, at least not under the conditions used in the present study. The same is true for the other H.

Spectrometry of IAA in mycorrhizal fungi 407 crustuliniforme, strain 81a. However, these fungi might produce other auxins that are not detected by the GC-MS method. Auxin production by the fungi might also affect the growth of the host plant after infection. The possibility of comparing the level of IAA produced by a fungal strain with the growth promoting effect on a mycorrhizal plant infected with the same strain is very limited at present. Most fungi have not been tested in field experiments, mainly because of the difficulties of infecting seedlings in a practical way. But it is interesting to note that very high levels of IAA are produced by P. tinctorius 185 and that a strong positive effect on growth has been demonstrated for this strain by Marx (1977a, b, 1979). ACKNOWLEDGEMENT Financial support from Jacob Wallenbergs Forskningsstiftelse is gratefully acknowledged. The authors wish to thank Dr K. P. Kringstad, Swedish Forest Products Research Laboratory, Professor T. Hemberg and Dr E. Tillberg, University of Stockholm, Sweden for helpful discussions. REFERENCES CARUSO, J. L., SMITH, R. G., SMITH, L. M., CHENG, T.-Y. & DAVES JR., G. D. (1978). Determination of indole-3-acetic acid in Douglas Fir using a deuterated analog and selected ion monitoring. Plant Physiology, 62, 841-845. HEMBERG, T. & TILLBERG, E. (1980). The influence of the extraction procedure on yield of indole-3-acetic acid in plant extracts. Physiologia Plantarium, SO, 176-182. LARSEN, P. (1955). Growth substances in higher plants. In: Modern Methods of Plant Analysis, vol. 3 (Ed. by K. Paech & M. V. Tracey), pp. 565-625. Springer Verlag, Berlin. MARX, D. H. (1969). The influence of ectotrophic mycorrhizal fungi on the resistence of pine roots to pathogenic infections. Phytopathology, 59, 153-163. MARX, D. H. (1977a). Manipulation of selected mycorrhizal fungi to increase forest biomass. Tappi Forest Biology Conference, Wood Chemistry, June 1977, Madison, 139-149. MARX,D. H.,BRYAN, W. C. & CORDELL, C. E.(1977b). Survival and growth of pine seedlings with PWO/I(AJ«ectomyrrhiza after two years on reforestation sites in North Carolina and Florida. Forest Science, 23, 363-373. McDouGALL, J. & HiLLMAN, J. R. (1978). Analysis of indoie-3-acetic acid using GC-MS techniques. Seminar series for the Society for experimental biology, 4, 1-25. MOLINA, R. (1980). Ectomycorrhizal inoculation of containerized western conifer seedlings. US Forest Service Research Note PNW, 357, 1-10. NiTSCH, J. P. & NiTSCH, C. (1956). Studies on the growth of coleoptile and first internode sections. A new, sensitive straight-growth test for auxinl. Plant Physiology, 31, 94-111. SLANKIS, V. (1948). Einfluss von Exudaten von Boletus variegatus auf die diehotomische Verzweigung isolierter Kiefemwurzeln. Physiologia Plantarium, 1, 390-400. SLANKIS, V. (1951). Uberden Einfluss von/?-indolylessigsaure und anderen Wuchstoffen auf das Wachstum von Kiefemwurzeln. In: Symbolae Botanicae Upsalienses, 11, 1-63. SLANKIS, V. (1958). The role of auxin and other exudates in mycorrhizal symbiosis of forest trees. In: The Physiology of Forest Trees (Symposium) (Ed. by K. V. Thiman), pp. 427-443. The Ronald Press Co., New York. SLANKIS, V. (1974). Soil factors influencing formation of mycorrhizae. Annual Review of Phytopathology, 12, 437-457. STOESSL, A. & VENIS, M. A. (1970). Determination of submicrogram levels of indole-3-acetic acid. Analytical Biochemistry, 34, 344-351. STRZELCZYK, E., SITEK, J. M. & KOWALSKI, S. (1977). Synthesis of auxins from tryptophan and tryptophanprecursors by fungi isolated from mycorrhizae of pine {Pinus silvestris). Acta Microbiologica Polonica, 26, 255-264. ToMASZEWSKi, M. & WojciECHOWSKA, B. (1973). The role of growth regulators released by fungi in pine mycorrhizae. Proceedings of the 8th International Conference on Plant Growth Substances, pp. 217-227. Hirokawa Publishing Co., Tokyo. ULRICH, J. M. (1960). Auxin production by mycorrhizal fungi. Physiologia Plantarium, 13,