On the physiology of a fungus symbiotic with orchids

GAETAN HARVAIS AND AIN RAITSAKAS Department of Biology, Lakehead Universify, Thunder Bay, Ontario P7B 5E1

Received April 11, 1974

HARVAIS, G., and A. RAITSAKAS. 1975. On the physiology of a fungus symbiotic with orchids. Can. J. Bot. 53: 144-155.

A fungus (RslO) symbiotic with orchids was grown in axenic liquid-still culture on mineral-sugar media. Dextrose was limiting at 1% and 2%, with mycelium dry weights directly proportional to the amount of sugar in the medium, but not at 3%. Sucrose gave similar results and was converted into reducing sugars, in the medium, inexcessof utilization. With 3% sugar growth was not much better than with 2%, and daily dry weight values were more variable after the active growth phase. With 1% and 2% dextrose in the media, and 10,20, and 30 mllflask, dry weight was not affected by the different aeration conditions. Inocula failed to grow on media with pH 3.0, but growth seemed normal with pH 4.5. Ninhydrin-positive substances were not released in the culture media but were found in ethanol extracts of the mycelium. They were more abundant during the active growth phase and on media with 1% vs. 2% dextrose; with less sugar, high levels were maintained for longer periods. They consisted mainly of glutamic acid, glutamine, and aspartic acid, with some fluctuations in concentration related to growth phase. Metabolism seemed different on media with ammonium + nitrate vs. nitrate alone as nitrogen source. The hyphal wall contained chitin.

Protocorms of Dacfylorhiza purpurella (an orchid with which RslO is symbiotic) were grown on eight nutrient media with one amino acid in each, until only the latter were depleted. The best results were with glutamic acid, ornithine, and arginine, followed by aspartic acid and glycine. Alanine, serine, and lysine were toxic. N-acetyl-D-glucosamine derived from chitin was drip-fed to other cultures. It was ineffective at low and toxic at high concentrations.

Implications of the results relating to mycorrhizal associations are briefly discussed.

HARVAIS, G., et A. RAITSAKAS. 1975. On the physiology of a fungus symbiotic with orchids. Can. J. Bot. 53: 144-155.

Un champignon (RslO) symbiotique des orchidBes a Ctk cultivk axkniquement en milieu liquide non agitk contenant du sucre et des sels minkraux. Le dextrose limitait lacroissance et le poids sec du mydlium Btait proportionnel la concentration, a 1% et 2%, mais non 3%. Le sucrose a donne des rksultats semblables et Btait converti en sucres rkducteurs dans le milieu, et ceci en excks par rapport a I'utilisation Avec 3% de sucre, la croissance n'ktait pas beaucoup amBliorBe par rapport a 2%, et les valeurs quotidiennes du poids sec Btaient plus variables aprks la +r ide active de croissance. Avec 1 et 2% de dextrose dans les milieux et avec 10,20, et 30 ml par fiole, le poids sec n'Btait pas affect6 par les diffkrentes conditions d'dration. Les inoculums n'ont pas poussk sur les milieux i pH 3.0 mais la croissance semblait normale i pH 4.5. Des substances positives a la nihydrine n'ont pas kt6 iibkrkes dans les milieux de culture, mais elles ont Btk repbres dans les extraits Bthanoliques du mydlium. Elles Btaient plus abondantes pendant la phase de croissance active et sur les milieux ayant 1% (versus 2%) de dextrose; avec des quantitBs moindres de sucre,. leurs concentrations ont BtB maintenues BlevBes pendant des +rides plus longues. Ces substances comprenaient surtout I'acide glutamique, la glutamine et I'acide aspartique avec quelques Auctuations de concentration relikes 9. la phase de croissance. Le mBtabolisme semblait diffkrent sur les milieux contenant de I'ammonium et du nitrate, comparativement i ceux qui ne contenaient que du nitrate comme source d'azote. La paroi cellulaire contenait de la ch~tine.

Desprotocormes de Dacfylorhiza purpurella (une orchidBe symbiotique avec la souche RslO) ont Bte cultivds sur huit milieux nutritifs contenant chacun un acide amink diffkrent et ceci, jusqu'aBpuisement dece dernier. Les meilleurs rBsultats ont 6tB obtenus avec I'acideglutamique, I'ornithine et I'arginine, suivis par I'acide aspartique et laglycine. L'alanine, la sbrine et la lysine Btaient toxiques. De la N-adtyl-D-glucosamine dbrivbe de chitine a 6tB ajoutee goutte i goutte dans d'autres cultures. Cette substance Btait inefficace a faible concentration et toxique aux concentrations BlevBes.

Les auteurs discutent les implications de ces rbsultats en rapport avec les associations mycorhiziennes. [Traduit par le journal]

Introduction by digestion of endophytic fungi, but the neces- Orchid seeds have insufficient food reserves sity of the fungi endophytically and that of

to attain the photosynthetic stage and require digestion of the fungus for growth stimulation symbiotic fungi to enable them to do so in have been questioned (Knudson 1922; Harvais nature and sometimes even in culture. The 1965; Harley 1969; Hadley and Williamson orchid is believed to derive its necessary nutrients 1971). On the other hand, many symbiotic

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HARVAIS AND RAITSAKAS: A F1

endophytic fungi are believed to be soil sapro- phytes capable of existing independently of the orchids (e.g., Downie 1943). The work of Smith (1966) and (1967) also supports this view and strongly suggests that the orchid may derive greater benefit from active, translocating endo- phytes than from digestion of their mycelia (see also Hadley and Williamson 1971). Although it is realized that the host-fungus interaction is complex, whether in symbiotic or pathogenic associations (see Wood 1967; Harley 1969), it seemed feasible that a good symbiotic fungus could produce in axenic culture, metabolites that may be beneficial to its compatible orchid host. Harvais (1972) found that in axenic cul- tures of Dactylorhiza purpurella, growth on a medium consisting of minerals, dextrose, casa- mino acids, and yeast extract was as good as that in dual culture with one of the best symbionts, RslO, but inferior in the absence of yeast ex- tract, and worse on mineral-dextrose alone. It was then suggested that some, if not all, of the amino acids and vitamins present in those organic supplements may be provided to the orchid by its symbionts.

The main aims of this investigation were to establish the conditions under which RslO could be grown reproducibly in liquid culture, the effect of changing the conditions on its pro- duction of amino acids, and the effect of the dominant amino acids identified on the growth of D. purpurella in axenic culture. (Vitamin production will be treated in a separate com- munication.) Since RslO, originally identified as a strain of Rhizoctonia solani Kiihn, is dikaryotic and is considered to be a basidio- mycete, one might expect its hyphal wall to contain chitin (Aronson 1965 ; Burnett 1968 ; Jones et al. 1972), which upon digestion might produce substances of greater biological signil ficance than would e.g. glucans. Therefore, a secondary aspect of this work was to verify the presence of chitin and test the effects of its digestion product on D. purpurella in axenic culture.

Axenic Fungal Cultures Materials and Methods

Since some of the conditions and procedures were varied in later experiments, only general ones most frequently used are described here. Specific treatments will be mentioned when deal- ing with the respective experiments.

JNGUS SYMBIOTIC WITH ORCHIDS 145

All cultures were incubated at 25OC in the dark in chambers with forced air circulation. For each experiment, RslO was inoculated from stock slopes onto plates of potato-dextrose-agar (PDA, see Harvais 1973). After 5-6 days, active growing fronts were subcultured on PDA plates again. Five to 6 days later, inocula for the experiments were taken from the active fronts on those plates, as 5-mm-diam discs.

The mineral medium used for most experi- ments, medium I, was the same as that used in dual cultures. It consisted, in milligrams per liter, of (BDH AnalaR): Ca(N03),-4H,O, 400; NH4N03, 400; KH2P04, 200; MgS04.7H20, 200; KN03, 200; KCl, 100; MnS04.4H20, 0.5; H3B03, 0.5; ZnS04.7H,0, 0.5; CuS04.5H,0, 0.5; Na,Mo04.2H,0, 0.02; Co(N03),.6H20, 0.025; ammonium ferric citrate, 25 (-5 Fe). It was supplemented with sugar, dispensed in 125-ml Erlenmeyer flasks (generally 10 mllflask), autoclaved at 15 lb/in.2 for 15 min, inoculated, and kept in still culture.

For dry weight determinations, perforated nickel crucibles were lined with absorbent cotton 'wool,' oven-dried at 100°C for 22 h, and cooled in a desiccator over silica gel for 1 h before weigh- ing. Mycelial mats were then vacuum-filtered through the crucibles and each was washed with 10 ml distilled water and processed as above. Filtrates from replicates were mixed together and stored at -20°C until the end of the individual experiments. They were then thawed and tested for sugars and ninhydrin-positive substances after their volumes were measured and adjusted. Sugar was assayed quantitatively after the phenol-sul- phuric acid colorimetric method described and discussed by Hodge and Hofreiter (1962). For the ninhydrin tests, color standards were first prepared using dilutions of DL-glutamic acid ranging from 25 to 150 mglliter. These solutions were mixed with 1-ml aliquots of a 5% solution of ninhydrin in acetone (w/v), heated in a water bath at 85°C for 10 min, and then diluted with 10 ml distilled water. Their absorbance was recorded at 570 mp. For the initial crude assays of ninhydrin- positive substances in the samples, colors were matched with the glutamic acid standards before concentration and chromatography were at- tempted.

Experiments, Results, and Discussion In preliminary experiments lasting 24-30 days,

10 ml of medium I + 1% dextrose and one, two,

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CAN. J . BOT. VOL. 53, 1975

FIG. 1. Dry weights of mycelium (continuous lines) and levels of reducing sugars in culture filtrates (broken lines) of a strain of Rhizoctonia solani (RslO) grown in liquid culture on mineral medium I + three concen- trations of dextrose. 0, 1% dextrose; 0, 2%; A 3%.

three, and four inoculum discs were used per flask, and treatments were triplicated. The cul- tures with three and four inoculum discs gave consistent results, and three replicates seemed adequate. In all treatments there was an initial sharp increase in dry weight followed by a very gradual drop. The most active growth phase seemed to occur generally during the first 6 days. The sugar levels of the filtrates dropped sharply to zero during that period, in inverse relation to the dry weights, indicating that sugar was limiting. The filtrates were ninhydrin-negative.

The aim of the next experiment was to in- crease the yield of mycelium and hopefully amino acids too. In this and subsequent experi- ments the inocula were standardized at four discs per flask and replicates a t three per treat- ment.

Experiment 1 Medium used: medium I + 1%, 2%, and 3%

dextrose. Data recorded daily: mycelium dry weights, sugar levels, and ninhydrin reaction of filtrates. Duration: 28 days.

Figure 1 shows dry weights and sugar levels in filtrates with 1%, 2%, and 3% dextrose. The close match of the dry weight curves during the active growth periods suggests that the culture conditions were adequately uniform and the experiment was highly reproducible. With 2% dextrose the dry weight was double that with 1% dextrose. In both media sugar became limiting by days 5-6. With 3% dextrose, growth was not much better than with 2% and was not limited by sugar, and the dry weight values after the active growth phase were rather erratic. With 1% and 2% dextrose the standard error of the means (SE,, P = 0.05) was remarkably sma!! after the active growth phase. For example, the values with 2% dextrose were, in centigrams, as follows.

Days 3 4 5 6 7 8 9 Means 4.5 8.8 12.2 12.5 11.6 11.1 10.8 SEm k 0 . 7 k 1 . 8 - + 1.1 kO.1 k 0 . 2 k 0 . 3 k 0 . 2

This medium seemed adequate for a relatively 1 except that sucrose was used instead of dex- high yield of mycelium with sugar only just trose. Except for +he levels of reducing sugars, limiting. All the filtrates gave negative ninhydrin the results were remarkably similar in both reactions. experiments, showing once again their high

reproducibility. Figure 2 shows the dry weights Experiment 2 and levels of reducing sugars on medium 1 + This experiment was identical with experiment 2% sucrose. By day 3 the level of reducing sugars

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HARVAIS AND RAITSAKAS: A FUNGUS SYMBIOTIC WITH ORCHIDS

FIG. 2. Dry weights of mycelium (continuous lines) and levels of reducing sugars in culture filtrates (broken lines) of a strain of Rhizoctonia solani (RslO) grown in liquid culture on mineral medium I + 27, sucrose.

in the filtrates had risen sharply in excess of utilization. This rapid and perhaps total con- version of sucrose in the medium by RslO seems common in many fungi (Burnett 1968). Never- theless, substituting sucrose for dextrose im- proved neither growth nor liberation of amino acids in the culture media. In other ex~eriments not detailed here, where nitrate was the sole source of nitrogen, the results were similarly negative. It must be concluded that this approach is unlikely to be fruitful.

In subsequent experiments attention was directed to amino acids in free pools in the mycelium. The ethanol extraction recommended by Sherma and Zweig (1971) was adopted. Repli- cates were doubled. Each day, for each treat- ment, in addition to the data recorded in experi- ment 2, three mycelial mats were dropped into 75 ml of boiling aqueous ethanol (70% EtOH). This large volume ensured an insignificant tem- perature drop after immersion of the mats. After 5 min the mats were removed, ground in a mortar, and returned to the boiling solution for 5 more min to facilitate extraction. The solutions were then filtered, boiled down to 50 ml, and tested with ninhydrin. When the colors were faint. volumes were reduced further and the extracts tested again. The extracts were then stored at -20°C for later use. Note that the original ethanol extracts were colloidal, and it was feared that if this was because of proteins, the ninhydrin reactions would be misleading. The thawed extracts were found to contain

precipitates. When they were membrane-filtered (pore size 0.2 microns (p)), they were quite clear and their ninhydrin reactions were essentially unchanged. This proved to be a simple and con- venient way of eliminating presumably polysac- charides and was adopted routinely for the extracts. In this paper, the values given for the ninhydrin reactions refer to the membrane- filtered extracts after storage at - 20°C.

In an experiment designed to investigate how the source of nitrogen used may affect amino acid production, the ammonium nitrate in medium I was replaced by nitric acid to give the same amount of total nitrogen, exclusively as nitrate. RslO failed to grow on this medium (plus sugar), which after autoclaving had a pH of 3. Apparently normal growth was later ob- tained with mineral medium I1 (pH 4.5), in which, to give the same amount of total nitrogen as in medium I, the ammonium nitrate in medium I was replaced by 705 mglliter KNO, + 425 mglliter NaNO,. This was to ensure that the additional Kf and Naf would be at equal con- centrations and hopefully would not interfere with the uptake and metabolism of the original K f .

The next experiment was concerned with in- creasing the yield of mycelium further. From the earlier results, it seemed unnecessary to record the data daily for long periods. Instead, they were recorded daily during and shortly after the expected active growth period, and intermittently afterwards.

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CAN. J. BOT. VOL. 53, 1975

TABLE 1 pH of culture filtrates, and ninhydrin reactions (nrn)* of ethanol extracts of mycelia, in liquid

cultures of a strain of Rhizoctonia solani (RslO)

Medium I + Medium I + 2% dextrose 1% dextrose Medium I1 + 2% dextrose

A, B, C, D, E, 10 mllflask 20 mllflask 20 mllflask 10 ml/flask 20 mllflask

Day PH nrn pH nrn pH nrn pH nrn pH nrn

0 4.5 4.5 4.5 4.5 4.5 3 4.5 3.0 37.2

*The ninhydrin reactions were compared with glutamic acid standards and expressed as milligrams per gram dry my- celium.

Experiment 3 Treatments were as follows. A and B : medium

I + 2% dextrose, 10 and 20 mllflask. C: medium I + 1% dextrose, 20 mllflask. D and E: medium I1 + 2% dextrose, 10 and 20 mllflask. Data recorded: cf. those in experiment 1, plus the ninhydrin reaction of ethanol extracts of fresh mycelium.

The dry weight values appear in Fig. 3, A to E, and some other data are given in Table 1. Figure 3 shows that particularly in treatments A, B, and C, the growth curves were as expected. In all treatments the drop to zero of sugar levels in the media coincided with the maximum dry weights. All filtrates were ninhydrin-negative. The results for the comparable treatments are as follows.

A us. B-In A, maximum dry weight was half that in B and was obtained a day earlier. In those terms data from A day 4 (Table 1) are com- parable with those for B, day 5, etc. The pH of the filtrates was significantly higher in B. The strong ninhydrin reaction of the ethanol extract in B, day 5, suggests differences in nitrogen metabolism with the two volumes of media, but values for A, day 3, might have been equally high. In both treatments values were higher during the active growth phase than later.

D us. E-The comments made about the dry weight curves for A vs. B apply equally here except that in D and E, events happened a day earlier. Also, for comparable days, pH was

FIG. 3. Dry weights of mycelium of a strain of Rhizoc- tonia solani (RslO) grown in liquid culture. A and B, me- dium I + 2% dextrose, 10 and 20 ml/flask. C, medium I +1% dextrose, 20 mllflask. D and E, medium I1 + 2% dextrose, 10 and 20 mllflask.

initially higher in E, but the ninhydrin reaction of the extracts did not differ appreciably, indi- cating that on medium 11, RslO was less affected by aeration than on medium I.

A us. D-The dry weight curves were very similar, but values for D were slightly higher and were obtained a day earlier. Comparing data for

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HARVAIS AND RAITSAKAS: A F1. JNGUS SYMBIOTIC WITH ORCHIDS

A, day 5, with those for D, day 4, etc. shows no significant differences except for a higher pH in D.

B us. E-Essentially the same comments as above apply here except that B had the higher pH.

B us. C-For the dry weight curves essentially the same comments apply here as for B vs. A. In B, pH values were high and fairly constant, but varied more sharply in C, with a drop on days 3 and 4 and a sudden rise on day 5 when sugar became limiting. More striking are the very high and relatively persistent levels of ninhydrin- positive substances in the mycelial extracts. It should be noted that the dry weight curves for A and C matched closely. Obviously, under the experimental conditions, the total amount of sugar in each medium determined growth (dry weight), but its concentration was much more important with respect to free pools of amino acids.

In this experiment metabolism seemed dif- I

ferent on both media when the volumes per flask differed. This could relate to aeration and

1 the reduced and oxidized forms of nitrogen used. In the next experiment the ammonium and ~ nitrate levels of the filtrates were also assayed using, at this exploratory stage, simple, even if less accurate, methods. These were, for 'ammo- nia'-ammonium, direct nesslerization (section 132B) without preliminary distillation (section 132A), and for nitrate, ultraviolet spectro- photometry (section 133B), described and dis- cussed in Standard Methods for the Examination of Water and Wastewater, 13th edition (1971).

Experiment 4 Treatments were as follows. A and B: medium

I + 1 % dextrose, 10 and 30 mllflask. C and D : medium I1 + 1% dextrose, 10 and 30 mllflask. Data recorded: as in experiment 3, plus 'ammo- nia' and nitrate levels in filtrates.

Figure 4, A to D, shows the dry weight curves, and some other data are given in Table 2. The growth curves were as expected, except that for D. In all treatments the drop of sugar levels to zero coincided with maximum dry weights. All filtrates were ninhydrin-negative. Therefore, some substances that could interfere with the ammonium test can be ruled out. Direct nessler- ization of medium I gave ammonium values somewhat greater than the theoretical ones. The data given in Table 2 for ammonium have been

0 1 0 2 4 6 8 1 0 1 2 1 4

Days

FIG. 4. Dry weights of mycelium of a strain of Rhizoc- ronia solani (RslO) grown in liquid culture. A and B, medium I + 1% dextrose, 10 and 30 ml/flask. C and D, medium I1 + 1% dextrose, 10 and 30 ml/flask.

compensated for this discrepancy. Also, for the nitrate assays, spectrophotometric comparisons of filtrates from successive days gave equivocal compensation factors for organic contents. Therefore, the nitrate values given in Table 2 were derived using the formula given for the method adopted. The actual nitrate (and ammo- nium) values may be lower than those tabulated. The results for comparable treatments are as follows.

A us. B-Maximum dry weight in B was about three times that in A and was obtained 2 days later. Data for A, day 4, are comparable with those for B, day 6, etc. The values for the ninhy- drin reaction of the extracts were initially higher in A but dropped more sharply with time. In the filtrates, pH values were higher in B, and the drastic drop in nitrate (and ammonium) levels was also greater. These suggest a greater net anion uptake and possibly utilization under restricted aeration. Nitrate could be the domi- nant anion, but since ammonium uptake also seems greater in B, other anions would be in- volved too.

C us. D-The comments on A vs. B apply equally here except that in C and D the original

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TABLE 2

pH, ammonium, and nitrate levels (milligrams per liter) in culture filtrates, and ninhydrin reactions (nrn)* of ethanol extracts of mycelia, in liquid cultures of a strain of Rhizoctonia solani (RslO)

Medium I + IS , dextrose Medium I1 + 1% dextrose

Day pH NH,+ NO,- nrn pH NH,+ NO3- nrn pH N H 4 + NOo- nrn pH N H I + NO3- nrn -

0 4.5 85 644 4.5 85 644 4.5 0 953 4.5 0 953 4 5.0 27 49 26.8 6.0 24 54 25.1 5 5.5 6 4 17.5 4.0 6.8 15 27 19.2 7.0 6 6.6 8 23 20.9 7.1 19 16 20.9 7 6.3 6 0 7.6 6.6 4 4 24.2 6.8 8 21 10.5 7.1 4 19 24.6 9 6.4 2 3 19.0 7 .1 3 17 19.0

11 6.4 1 1 17.3 7.1 4 17 17.8

*The ninhydrin reactions were compared with glutamic acid standards and expressed as milligrams per gram dry mycelium.

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HARVAIS AND RAITSAKAS: A FUNGUS SYMBIOTIC WITH ORCHIDS 151

medium contained no ammonium. (This is dis- cussed below.)

A us. C-The dry weight curves matched fairly closely. The ninhydrin reactions were essentially the same in both treatments for com- parable days, indicating that nitrogen metab- olism was little affected bv the two forms of inorganic nitrogen used. Filtrate pH and levels of nitrate (and ammonium?) were higher in C. It may be that from day 5 nitrate was limiting in A. (Note that in C, by day 5, over 900 mg/liter appears to have been utilized.)

B us. D-The dry weight curve for B, but not D. was normal. The comments for A vs. C on ninhydrin reaction and pH apply equally here. Except for day 6, nitrate levels remained higher in D, but this could be because dry weight was less. The level of 'ammonium' (?) was higher in D on day 6 but essentially the same in both sub- sequently. In D, day 6, the low nitrate value may be related to the high one for 'ammonium.'

It will be noted that medium I1 (used in treat- ments C and D) originally contained no ammo- nium. The 'ammonium' levels detected subse- quently could be due to contaminants not eliminated by direct nesslerization or could indicate the ability of RslO to reduce nitrate to ammonium in the medium, in excess of utiliza- tion, and more so during the active growth phase. This ability is known in other fungi (Nicholas 1965). To verify this, preliminary dis- tillation (section 132A, Nicholas 1965) was done before nesslerization, using filtrates of A and C, day 4, D, day 6, and medium I as control. The ammonia values obtained were, in milligrams per liter, 3.2, 0.5, 1.1, and 85, respectively. The control gave exactly the theoretical value; the method is very reliable. This indicates that in A, and probably B, all the ammonium was utilized before the active growth phase was completed and this could have been limiting. It also sug- gests that reduction of nitrate takes place in the mycelium and not the medium.

In this experiment there seemed to be some differences in growth and metabolism on the two mineral media under different aeration. On both media higher levels of ninhydrin-positive sub- stances were maintained for long periods under restricted aeration. On the other hand, since levels of ammonium and nitrate in medium I appear suboptimal, some reservation is necessary when comparing the media. In future work, more frequent assays during the active growth period

may prove more fruitful. Higher levels and more critical analyses of ammonium and nitrate in the media would clarify some points more con- clusivelv.

The fact that greatest amounts of ninhydrin- positive substances were obtained with low sugar levels under restricted aeration leads to some speculation. In nature, carbohydrate levels may be expected to be low. If endophytic conditions are regarded as being relatively anaerobic, this is when the fungus may be most beneficial to the orchid through its nitrogen metabolism.

Chromatography In experiment 3 the greatest yield of ninhy-

drin-positive substances was obtained in treat- ment C and was maintained for a long period (see Table 1). The extracts for each day were concentrated as required and used in one- dimensional ascending chromatography on Whatman No. 1 paper. Initially the solvent used was 1% diethylamine in 11 : 5 : 5 2,6-lutidine- ethanol-water (v/v) (Block et al. 1958). Spraying with solutions of ninhydrin followed by copper sulphate showed some substances with close R, values, being possibly: aspartic-glutamic acids, lysine-ornithine, ct-alanine, arginine, glycine, and serine. Subsequently, better separation and identification were obtained using the following two solvent systems in parallel runs: 12:3: 5 (v/v) n-butanol - glacial acetic acid - water and 5 : 1 (v/v) liquid phenol - water + HCN (Sherma and Zweig 1971). Since the same substances seemed to occur consistently in most extracts, these two solvents proved adequate. At this stage the assays were mainly qualitative, but the relative intensity of the spots (as an indication of concentration) was also noted. The results for experiment 3 are shown in Table 3, where 1,2,3, etc., indicate the more intense spots in decreasing order. It is significant that the same amino acids and amine were present over a 22-day period, differing only in their relative abundance, with presumably no appreciable autolysis occurring. Glutamine was dominant during the active growth phase and again on days 18 and 22, and glutamic acid was so in the interim. Although this could be a result of interconversion of glutamine and glutamic acid, it seems to follow a pattern related to growth phase (and (or) time). In experiment 5, the results for treatments A and C, and B and D (Table 3) were identical for comparable days. In A and C, glutamine was

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TABLE 3 Relative abundance of the major ninhydrin-positive components of ethanol extracts of mycelia in liquid cultures of a strain of Rhizoctonia solani (RslO)

Experiment and Aspartic Gluta- treatment Day acid mic acid Glutamine Serine Alanine

4C 3 3 2 1 4 4 3 2 1 4

NOTE: The numbers 1 to 5 indicate the more intense spots o n paper chromatograms, in decreasing order.

dominant on day 4, and glutamic acid on days 5 and 7. The fluctuations seem related to the growth phases and independent of the media and aeration conditions. In this experiment, quantita- tive assays were done by densitometry (Shellard 1968). The values for, e.g., treatment D, day 6, were, in milligrams per gram of dry mycelium, glutamic acid, 5.3; aspartic acid, 4.7; glutamine, 3.4; serine, 1.2; and alanine, 0.8. These account, respectively, for 26, 22, 16, and 4% (total 74%) of the ninhydrin-positive substances in the extract. Other extracts gave comparable total values. Allowing for experimental error, these five substances may be regarded as the major, and perhaps the only, ninhydrin-positive con- stituents of the free pools.

In experiment 3, free amino acids were particu- larly scanty when the medium contained 2% dex- trose. In treatment E, day 5, after ethanol extraction, the residual mycelium was hydrolyzed to release the bound amino acids following the method adopted by Durzan (1973). Chroma- tography of the ethanol extracts showed, in that order of abundance, aspartic acid, glutamic acid, serine, alanine, and very small amounts of leucine and valine, while the hydrolyzate showed

large quantities of glutamic acid, leucine, and valine, each about 5-10 mg/g of dry mycelium. The apparent absence of glutamine and the presence of leucine and valine seem closely linked with high sugar levels. These aspects deserve further investigation.

Chitin Detection The main aim here was to establish the pres-

ence of chitin in the hyphal wall of RslO. The fungus was grown in liquid culture and har- vested after the active growth phase. Initially, drastic chemical treatments were avoided (see Aronson and Machlis 1959; Aronson 1965). In attempts to remove the protoplasm, digestion with trypsin and pepsin was unsatisfactory and was abandoned. Subsequently, the chemical methods of Fuller and Barshad (1960) proved very suitable and were adopted, since our interest was mainly in qualitative assays. The mycelial mats were prepared for these tests by blending at high speed in a Waring blendor, followed by mortar and pestle grinding. The autoclaved samples gave a strong chitosan reaction (see Jensen 1962). Removal of the chitosans with 2% acetic acid left a very small residue, which could

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HARVAIS AND RAITSAKAS: A FUNGUS SYMBIOTIC WITH ORCHIDS 153

be cellulose. After autoclaving, only 3% (dry their relative merits were more apparent. weight) of the original protoplasm-free wall Nicotinic acid, at over three times its concentra- material was recovered, but at least that 3% tion in medium I11 (Harvais 1972), did not seem seemed predominantly chitin. limiting here. The cultures were assessed fre-

For the X-ray diffraction powder analyses quently, and the experiment was finally recorded commercial chitin (Sigma Chemical Co., Nos. after 28 weeks, when mortality became too high. C 3132 and C 9752) was used per se and after The best results were obtained with glutamic the purification treatment of Fuller and Barshad acid, arginine, and ornithine and were equally (1960). The samples were exposed for 90 min to good on all three. The protocorms differentiated CuK,,,, radiation, 50 kV, 18 mA. In all and grew well, producing active, healthy roots samples the d-spacing lines were, in decreasing and shoots and normal plantlets (Fig. 7). Never- order of intensity, at 4.56, 9.53, 3.38, 4.22, and theless, activity was significantly reduced towards 6.98 A, (Fig. 5a). These matched more closely the end of the experiment, suggesting a con- the values given for chitin by Fuller and Barshad tinued requirement for the amino acids, even by (1960) than by Jones et al. (1972). The method photosynthetic plantlets. Dactylorhizapurpurella used here to purify the chitin seems satisfactory. may be unable to synthesize these substances. Cleaned hyphal walls of RslO were treated The next-best results were obtained equally similarly. The results (Fig. 5b) closely matched with aspartic acid and glycine. Generally, the those obtained previously, confirming satis- same comments as above apply here, except that factorily the presence of chitin. the loss of activity occurred earlier and the

Axenic Orchid Culture

I From the early stages of the chromatographic work, seeds of Dactylorhiza purpurella were not available. There was only a limited supply of 10-month-old protocorms all at the same stage of development, pregrown on medium V (see Harvais 1972). Although a balanced mixture of amino acids (and vitamins) may be important in the orchid's nutrition, this could not be investi-

I

gated here. The approach was to determine what I minimum nutrients would suffice. In the early

chromatograms there was conflicting evidence about the presence, identity, and abundance of some amino acids in the free pools of the sym- biotic fungus. Hence the following were chosen : aspartic and glutamic acids, alanine, arginine, glycine, lysine, ornithine, and serine. Nicotinic acid, the dominant vitamin in yeast extract (see Harvais 1972), was also included. Agar slopes were made of eight nutrient media (one with each amino acid) consisting of medium I + 1 mglliter nicotinic acid + 1 glliter amino acid + 1% dextrose + 1% agar (except with aspartic and glutamic acids which required 2% agar). The amino acids were in the DL form except L-arginine and glycine. The pregrown proto- corms (Fig. 6) were transferred to the eight media and incubated at 25°C in a diurnal 12-h

I light regime (see Harvais 1972 for details). As I growth progressed the cultures were drip-fed I with medium I + dextrose only until the amino

acids were depleted from the media. In this way

plantlets were smaller. The results on alanine and on serine were by

far the worst. Besides little to no growth taking place, the protocorms were moribund after 2 months and dead after 4. These amino acids were useless nutritionally and were toxic.

During the first 2 months lysine had produced the healthiest protocorms of all, but within the next 2 months they all died rapidly. Lysine may have been at a considerably supraoptimal level.

It is interesting to note that glutamic acid, arginine, and ornithine gave the best results. Nicholson (1970) and Turner (1971) indicate that they are linked in closely related pathways. This suggests that once D. purpurella is provided with one of the amino acids in those pathways, it is capable of effecting the necessary transami- nations. It may not be surprizing to find other compounds in those pathways, e.g., citrulline, proline, to be equally utilizable. Also, glutamic acid is recognized as playing a key role in the assimilation of inorganic nitrogen in plants (Devlin 1969) and in transamination processes leading to the formation of as many as 17 amino acids (Wilson et al. 1954). Dactylorhiza pur- purella seems unable to synthesize e.g. glutamic acid or adequate quantities of it, but the results suggest that as long as this amino acid is avail- able the orchid may be able to effect the other transaminations. In nature one might expect the orchid to depend on symbiotic fungi for such supplies. It is significant that glutamic and aspartic acids were the dominant amino acids in

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154 CAN. J. BOT. VOL. 53, 1975

FIG. 5. X-ray diffraction patterns of purified chitin powders: a, from crustacean; b, from hyphal walls of a strain of Rhizoctonia solani (RslO). FIGS. 6 and 7. Axenic protocorms and plantlets of Dactylorhiza purpure!!a on a nutrient medium containing glutamic acid. Fig. 6, a t the time of transfer. Fig. 7, 28 weeks later. FIG. 8 and 9. Axenic plantlets of Dactylorhizapurpurella on a nutrient medium after 37 weeks of drip feedings. Fig. 8, with distilled water (control). Fig. 9, with N-acetyl-D-glucosamine.

the free pools of the symbiotic fungus RslO. free pools) are difficult to reconcile. They could Glutamine may also be found to be equally be due to supraoptimal levels and (or) to the beneficial to the orchid. The markedly adverse isomeric forms used. By analogy, in a recent effects of alanine and serine (also present in the experiment with Cypripedium reginae 50 mg/liter

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HARVAIS AND RAITSAKAS: A FUNGUS SYMBIOTIC WITH ORCHIDS 155

of L-glutamic acid was very beneficial while 500 BURNETT, J. H. 1968. Fundamentals ofmycology. Edward Arnold, London. was These aspects need further DEVLIN, R. M. 1969. Plant physiology. Van Nostrand

investigation. Reinhold, New York. In another test, l-year-old plantlets of D. DOWNIE, D. G. 1943. Source of the symbiont of Goodyera

repens. Trans. Roc . Bot. Soc. Edinb. 33: 383-390. purpurella, pregrown on a suitably rich medium DURZAN, D. J. 1973. The incorporation of tritiated water not detailed here, were drip-fed with N-acetyl- into amino acids in the presence of urea by white spruce

seedlings inlightanddarkness. Can. J. Bot. 51: 351-358. D-glucosamine (Sigma CO" No' A FULLER, M. S., and I. BARSHAD. 1%0. Chitin and cellulose 8625), a breakdown product of chitin, to give in the cell walls of Rhizidiomyces sp. Am. J. Bot. 47: 1-1 1 mglliter. Even at 11 mglliter this amine had 105-109.

HADLEY, G., and B. WILLIAMSON. 1971. Analysis of the no physiological effects comparable with growth post-infection growth stimulus in orchid mycorrhiza. regulators, vitamins, etc. To investigate its New Phytol. 70: 445455. nutritional value, two more drip feedings were HARLEY, J. L. 1%9. The biology of mycorrhiza. ~ d i t e d by

N. Polunin. Leonard Hill, London. done, each at 1 g/liter. After the first of these, HARVAIS, G. 1965. Some aspects of symbiosis in Orchis some harmful effects were manifest and were p~rrpurella. Ph.D. Thesis, University of Aberdeen, Scot-

accentuated after the second. The leaves were very chlorotic; the protocorm body and then the roots and older leaves turned brown and died. Figures 8 and 9 show, respectively, 20-month-old controls and treated plantlets. Also, in 2-month- old cultures of C. reginae on a suitable rich medium not detailed here, 1 mglliter of the amine caused high protocorm mortality in less than 2 months. Although the two orchids dif-

L 2

fered in their sensitivity to the amine, they were both adversely affected by it. These results support Harley's view (1969) that in mycor- rhizae the benefits derived from digestion of endophytic mycelia may have been overrated.

Acknowledgments We are particularly grateful to Dr. D. Orr for

his unfailing readiness to help with the chemical problems. We are also indebted to Mr. S. Spivak and Mr. G. Hashiguchi for their help with the plates and figures, Ms. Susan Pelky and Mrs. Betty Salo for the typing, and the National Research Council of Canada for partial finan- cial support. ARONSON, J. M. 1%5. The cell wall. In The fungi, an

advanced treatise. Vol. 1. Edited by G. C. Ainsworth and A. S. Sussman. Academic Press, New York. p. 49.

ARONSON, J. M., and L. MACHLIS. 1959. The chemlcal composition of the hyphal walls of the fungus Allomyces. Am. J. Bot. 46: 292-300.

BLOCK, R. J., E. L. DURRUM, and G. ZWEIG. 1958. A manual of paper chromatography and paper elec- trophoresis. 2nd ed. Academic Press, New York.

land. 1972. The development and growth requirements of

Dactylorhiza purpurella in asymbiotic cultures. Can. J. Bot. 50: 1223-1229.

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HODGE, J. E. , and B. T. HOFREITER. 1%2. Determina- tion of reducing sugars and carbohydrates. In Methods in carbohydrate chemistry. Vol. 1. Edited by R. L. Whistler and M. L. Wolfrom. Academic Press, New York. p. 380.

JENSEN, W. A. 1%2. Botanical histochemistry. W. H. Freeman, San Francisco and London.

JONES, D., V. C. FARMER, J. S. D. BACON, and M. J. WILSON. 1972. Comparison of ultrastructure and chemi- cal components of cell walls of certain plant pathogenic fungi. Trans. Br. Mycol. Soc. 59: 11-23.

KNUDSON, L. 1922. Nonsymbiotic germination of orchid seeds. Bot. Gaz. 73: 1-25.

NICHOLAS, D. J. D. 1%5. Utilization of inorganic nitrogen compounds and amino acids by fungi. In The fungi, an advanced treatise. Vol. 1. Edited by G. C. Ainsworth and A. S. Sussman. Academic Press, New York. p. 349.

NICHOLSON, D. E. 1970. Metabolic pathways 1970. Koch-Light, Colnbrook.

SHELLARD, E. J. 1%8. Quantitative thin-layer chromatog- raphy using densitometry. In Quantitative paper and thin-layer chromatography. Edited by E. J. Shellard. Academic Press, London. p. 51.

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SMITH, S . E. 1%6. Physiology and ecology of Orchis mycorrhizal fungi with reference to seedling nutrition. New Phytol. 65: 488499.

1%7. Carbohydrate translocation in orchid mycor- rhizas. New Phytol. 66: 371-378.

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