STUDIES IN THE PHYSIOLOGY OF LICHENS

Transcription

1 New Phytol. (1967) 66, STUDIES IN THE PHYSIOLOGY OF LICHENS VII. THE PHYSIOLOGY OF THE NOSTOC SYMBIONT OF PELTIGERA POLYDACTYLA COMPARED WITH CULTURED AND FREE-LIVING FORMS BY E. A. DREW* AND D. C. SMITH Department of Agriculture, University of Oxford (Received 3 February 1967) SUMMARY A method for directly isolating cells of the algal symbiont (Nostoc) in quantity from the thallus of Peltigera polydactyla is described. Such directly isolated algae excrete much of the photosynthetically fixed carbon into the medium as glucose, but they lose this property after only 48 hours in culture, at which time the cells begin to release only small amounts of a compound which may be a polysaccharide. These results are discussed in the context of the transfer of fixed carbon from alga to fungus in the lichen thallus during photosynthesis. After the lichen algae had been in pure culture for some time, they showed a carbohydrate metabolism intermediate in a number of respects between the directly isolated Nostoc and a strain of free-living N. muscorum. All three kinds of algae were able to utilize externally supplied glucose. The Nostoc from lichens could not utilize mannitol. INTRODUCTION Smith and Drew (1965) concluded that during photosynthesis in the thallus of the lichen Peltigera polydactyla, substantial amounts of fixed carbon could move rapidly from the algal symbiont {Nostoc) to the fungus. It was estimated that over a 4-hour period, approximately 40% of the carbon fixed in photosynthesis moved into the fungal medulla; mannitol was the major compound which accumulated fixed carbon in the thallus during this time. These observations indicate that the Nostoc symbiont in the lichen has the following characteristics: (a) one or more products of photosynthesis can be rapidly released from its cells in substantial amounts, and (b) the major product released is either mannitol or a substance that can be rapidly converted to it. Comparable features are not found either in free-living Nostoc spp. or in symbiotic forms which have been in pure culture for some time. Thus, Norris, Norris and Calvin (1955) and Linko et al. (1957) found that the major soluble products of photosynthesis with '"^COj in free-living A'^. muscorum and some other Cyanophyceae were amino acids, particularly citrulline; soluble carbohydrates were formed only in small amounts. Henriksson (1961) found that a species of Nostoc isolated from the lichen Collema tenax excreted a variety of compounds into the medium, including polypeptides, polysaccharides and vitamins; however, the polysaccharides were not able to support the growth of the fungus. These differences in physiology between the Nostoc symbiont in the thallus of Peltigera polydactyla and the other forms of Nostoc could be a consequence either of * Present address: Botany Department, The University, Leeds

2 380 E. A. DREW AND D. C. SMITH existence in the symbiotic state or of some genetic difference, or of both. It would clearly be advantageous to have some way of studying the algal symbiont in the condition in which it occurs within the thallus. A technique has therefore been developed for isolating algal cells in quantity from homogenates of the lichen, and their physiology was compared with that of the same Nostoc symbiont after a period in pure culture as well as with a strain of free-living N. muscorum. Such comparisons allow the effect of existence in the symbiotic state upon the algal component of the lichen to be assessed. In conformity with the terminology now generally used in the study of lichens, the lichen alga will be termed the 'phycobiont', and the fungus the 'mycobiont' (Scott, 1957)- MATERIALS AND METHODS Isolation of algae in quantity from thallus homogenates Preparation of thallus homogenate The collection and cleaning of material of Feltigera polydaetyla has previously been described by Harley and Smith (1956). Cleaned healthy thallus lobes were cut into pieces and gently ground and squeezed in a mortar; no abrasive was added. Small quantities of the medium No. 32 of Zehnder and Gorham (i960) (modified by the omission of nitrogen sources) were added and when the liquid was dark green the whole mass in the mortar was filtered through a plastic strainer. The grinding process was repeated twice more for each batch of material, and the resultant filtrates were bulked. Dijferential centrifugation of thallus homogenates The bulked filtrates contained four main types of debris: (a) fragments of thallus, (b) whole and broken algal cells, (c) fragments of fungal hyphae, and (d) subcellular particles. These were fractionated by low speed centrifugation in an MSE Minor Centrifuge according to the following schedule: 375 Sf*^'' 3 """"'f^- This precipitated fractions (a), (b) and (c) above, but prolonged centrifugation at this speed failed to separate further debris from the brown supernatant. The precipitate consisted of a dark green zone of algal cells clearly delineated between a thick bottom layer of massive debris and a thin top layer of fine white debris. The algal region together with the fine debris above it was scraped off with a spatula and resuspended in nutrient medium. 125 g for periods 0/30-90 seconds. The suspension of algal cells and fine fungal debris obtained above was centrifuged for 30 seconds at 125 g. The supernatant from this was then centrifuged at the same speed for 40 seconds, and the procedure repeated, centrifuging each supernatant for 10 seconds longer than in the preceding run; this was continued until a 90-second run. Microscopic examination showed that the shorter runs precipitated mainly fungal debris, whilst later on progressively cleaner precipitates of algal cells were obtained. By repeating these centrifuge regimes on the cleanest algal precipitates reasonably pure algal suspensions were obtained. The timing of centrifuge runs was taken from switching on to switching off the centrifuge; seconds usually elapsed before 125 g was reached. Manual braking was applied after switching off.

3 Physiology o/nostoc/rom Peltigera 381 Yield of algae Using this procedure about 30 mg dry weight of algal cells was obtained from 5 g dry weight of thallus. Estitnates based on chlorophyll content showed that algae comprised approximately 9% of the dry weight of the intact thallus, i.e. 450 mg per 5 g of thallus. Losses of algae during isolation were therefore substantial. It is probable that they occurred partly as a result of retention in thallus fragtnents after grinding, partly in the discarded 'dirty' fractions of centrifuge regitiies, and partly by cell breakage. Conditions and purity of algal preparations The algal suspensions obtained by this procedure contained mainly single ovoid cells of Nostoc averaging 6.0 X7.5 /;. Some short chains of cells were also found but never exceeding four cells in length. Cell size and shape compared well witb the algal cells observed in thin sections of intact thalli, indicating little mechanical or osmotic damage, although a greater degree of aggregation into filaments was found in the thallus; these were presumably dispersed mechanically during the isolation procedure. Three types of contatiiinant were found in the algal preparations. Algae. A very small amount of green algae epiphytic upon the thallus and not removed hy surface washing was unavoidably included in these preparations. However, they were only of potential itnportance when the algal preparations were used to initiate pure cultures, but here it was found that their growth could be repressed by omitting combined nitrogen from the tnedium, and by frequent subculturing. Bacteria. No attempt was made to maintain bacteria-free conditions during isolation and the presence of bacteria in the preparations became apparent if they were incubated with glucose. However, the population was maintained at a low level by normal culture methods in which sources of carbohydrate and combined nitrogen were absent. Fungi. Unlike the other contaminants, the fungal fragments present in the preparations soon died out in subsequent culture. They were only of importance in certain experiments carried out with the algae immediately after isolation, when the utilization of externally supplied carbohydrates of high specific activity was studied. In some of experiments, the results are somewhat distorted by the presence of these fragments of fungi and such cases are individually noted. These slight limitations on the purity of the algal suspensions and cultures derived hy the isolation procedure above must necessarily be borne in mind in assessing the results obtained. Howe\'er, it must be remembered that the great excess of Nostoc present in all the experiments ensured only small discrepancies due to contamination, and this is especially true of experiments on photosynthesis. Pttie cultures of Nostoc symhiont and N. muscorum Cultures of the Nostoc symbiont were started with inocula of cells from the isolated algal suspensions prepared as above. In this way large inocula could be used to reduce the lag period usually encountered with cultures inoculated with a very few cells. The medium used was No. 32 of Zehnder and Gorham (i960) but with combined nitrogen omitted in order to suppress growth of any green algae. The cultures were maintained at 20" C under 425 ft-candles illumination from daylight fluorescent tubes and were continuously shaken. Subcultures were prepared every 8 weeks because soon after this period green algae began to grow in the cultures, presumably since they were

4 382 E. A. DREW AND D. C. SMITH beginning to obtain substances excreted into the medium by the Nostoc cells. Sterile culture techniques were used. A culture of free-living A'^. muscorum was obtained from the Cambridge Collection of Algae and Protozoa (No. 1453/12) and maintained under identical conditions to those described above for the Nostoc isolated from Feltigera. The morphology of the free-living Nostoc muscorum in culture was markedly different from that of the symbiotic Nostoc. The former consisted of the uniseriate filaments typical ofthe genus and termed 'nostocacean'. Heterocysts were present and a mucilaginous sheath to the filament was detectable. By contrast, in cultures of the phycobiont, the typical structures present after 6 weeks were apparently multiseriate ovoid units which careful examination revealed to consist of closely spiralled filaments in a mucilaginous envelope; several of these units were frequently strung together with the filament continuous between them. Also present were some contorted uniseriate filaments. This morphology did not substantially change during 6 months culture involving repeated subculturing. Thus, the cultured phycobiont differed from the free-living strain of A'^. muscorum in the following respects: (a) lack of heterocyst formation, (b) lack of typical 'nostocacean' filaments, (c) production of mainly the apparently aseriate structures, and (d) greater development of the mucilaginous sheaths. Experimental procedures Three types of algal material were used in these experiments: (a) the directly isolated phycobiont cells, used immediately after separation from the thallus of the lichen; (b) cultured phycobiont cells, used after 6-8 weeks pure culture; and (c) A^. muscorum, used after 6-8 weeks culture under the same conditions as (b). In the case of both kinds of cultured algae, the gelatinous colonies were broken up by agitation with a needle so as to yield dense suspensions of aggregates of cells for further use. Aliquots of suspensions were placed in 3 x i in. glass specimen tubes which were shaken continuously at 20^ C under 425 ft-candles illumination from daylight fluorescent tubes. Dark fixation control samples were incubated under the same conditions but the vessels were wrapped in aluminium foil throughout. For photosynthesis experiments incubation media consisted of i ml of a simple, unbuffered solution of NaH'^^COj in distilled water adjusted to ph 7.0. Each vessel contained either 2.5 or 5.0 /<Ci ^*C. No carrier sodium bicarbonate was added, but at no time did availability of carbon appear to become limiting. Analytical procedures The algae were killed with boiling 80% ethanol and then extracted with three changes of hot 80% ethanol. The ethanol-insoluble material was hydrolysed for 3 hours at 100 C with 1.5 N sulphuric acid. Radioactivity in the extracts was estimated by the modified technique described by Smith and Drew (1965) in which aliquots were dried down directly onto aluminium planchettes. The distribution of radioactivity amongst the individual compounds in the ethanol soluble and acid hydrolysate fractions was investigated by paper chromatography, using a variety of solvent systems. For one-dimensional chromatograms the following solvents were used: ethyl acetate-acetic acid-water (14:3:3) (Smith, 1960); ethyl methyl ketone-

5 Physiology o/nostoc/rom Peltigera 383 acetic acid-water saturated with boric acid (9:1:1) (Rees and Reynolds, 1958); t- butanol-picric acid-water (2.2 g picric acid in 100 ml 80% <-butanol) (Hanes and Isherwood, 1949). Two-dimensional separations were made with butanol-propionic acid-water (3 :1.5 :2) and phenol-water (72:28) (Calvin and Bassham, 1962). The distribution of radioactivity on the chromatograms was detected either with a strip scanning device for the one-dimensional papers (Loughman and Martin, 1957) or hy autoradiography with X-ray film for the two-dimensional separations. The positions of authentic marker carbohydrates on the chromatograms were determined by the silver nitrate method of Trevelyan, Proctor and Harrison (1950), prior treatment with hydrofluoric acid (Britton, 1959) being given when papers had been developed with the solvent containing boric acid. RESULTS Photosynthesis The directly isolated phycobiont (i.e. the algal cells directly isolated from thallus homogenates) fixed carbon at a more or less constant rate during a 3-hour experiment in 30 Time { minutes) Fig. I. Distribution of fixed '*C between the medium, and the ethanol-soluble and ethanolinsoluble fractions of the three types of algae, (a) Directly isolated phycobiont, (b) cultured phycobiont, and (c) Nostoc muscorum. Incubation at 20" C, 425 ft-candles illumination, in I ml distilled water containing 5 /vci carrier-free NaH'^COj per sample., Medium; X, ethanol-soluble; O, ethanol-insoluble. which it was incubated in a solution of NaH'^^COj in distilled water in the light (Fig. ia. Table i). More than 50% of the fixed ^'^C was released to the medium during this time, while the rest was about equally divided between intracellular soluble and insoluble material. By contrast, Fig. i(b) and (c) show that both the cultured phycobiont and Nostoc muscorum released very little fixed ^^C into the medium. In the latter alga, incorporation of '"^C into the soluble fraction reached a constant value after 30 minutes, but incorporation into insoluble material was still rising after 3 hours a pattern similar to that found

6 384 E. A. DREW AND D. C. SMITH Table i. Release of photosynthetically fixed ^^C into the medium % of total fixed '*C released minutes minutes minutes hours Directly isolated phycobiont Cultured phycohiont Nostoc muscorum Incubation at 20" C, 425 ft-candles illumination in i ml distilled water containing 5 //Ci carrier-free NaH'^COa. in other free-living algae (e.g. Calvin and Massini, 1952). The cultured phycobiont was intermediate in character and continued to incorporate carbon into both soluble and insoluble material beyond the first 30 minutes of the experiment. The constituents of the various fractions were further analysed by paper chromatography with the following results. Ethanol-soluble fraction. Fig. 2 illustrates the distribution of '"^C amongst the various classes of compounds in the soluble extracts of the three kinds of algae. It will be seen Sugar phosphates Carbohydrates Amino and organic acids Lipids Fig. 2. Chromatogram scans of the ethanol-soluble extracts of the three kinds of algae after 3 hours incubation under the experimental conditions in Fig. i. (a) Directly isolated phycobiont, (b) cultured phycobiont, and (c) Nostoc muscorum. Solvent system: f-butanol (80 ml)-\vater (20 ml)-picric acid (2.2 g). Solvent front at lipid peak.

7 Physiology of Nostoc from Peltigera 385 that the directly isolated phycobiont incorporated proportionally more "^C into carbohydrates than the other algae, and less into acids and lipids. The carbohydrate areas of the chromatograms shown in Fig. 2 were eluted and then investigated by further paper chromatography in other solvents. It was found that the principal carbohydrate in the extract of the directly isolated phycobiont was glucose, although in the other two types of algae it was present only in scarcely detectable amounts. The main components of the soluble carbohydrate fraction of A^. muscorum were fructose and an unknown compound running in the disaccharide region of chromatograms developed with ethyl acetate-acetic acid-water (but with a mobility corresponding neither to sucrose nor to trehalose). This unknown compound was also found in the other two algae, and was in fact the only major soluble carbohydrate detectable in the cultured phycobiont. Fructose occurred in small amounts in the directly isolated and cultured phycobionts. Traces of mannitol were also detected in the directly isolated phycobiont, but this can be attributed to the activity of contaminant fungal fragments which presumably had access to the ['*C]glucose excreted into the medium by the algal cells. The proportionately smaller amount of '"^C in nucleotides and phosphorylated sugars in A^. mttscorum compared with the other algae may reflect the more efficient synthesis of polysaccharides. ^*C released into the medium. The fixed '"^C excreted into the medium by the directly isolated phycobiont was found to consist exclusively of glucose. The fact that none of the other radioactive compounds accumulated within the cells was detected in the medium indicates that the loss of glucose from the cells involved some selective process and was not just a 'leakiness' resulting from an increase in the passive permeability of the cells. Since glucose was also one of the main compounds incorporating '*C within the cells of the directly isolated phycobiont, it is evident that free glucose must be the major product of photosynthesis in this type of alga. Ethanol-insohtble fraction. All three algae were similar in that the major neutral product of hydrolysis of the insoluble fraction which contained '*C was glucose. Other sugars (mannose, fructose and pentoses) incorporated only small amounts of ''^C and were also quantitatively less important than glucose. Since insoluble compounds were the major products of photosynthesis in the cultured phycobiont and in TV. muscorum, it follows that in fact glucose may be the major product of photosynthesis in all three types of algae, but the directly isolated phycobiont differs from the others in incorporating relatively little of the glucose into polysaccharides. Dark fixation of ^*C. Dark fixation never exceeded 10% of the total light fixation of ^'^C in experiments with any of the three algae, and was rarely as high as this. About 70% of the ^*C fixed in the dark was retained in the soluble fraction, mostly as aspartic and glutamic acids. Changes in the directly isolated phycobiont after isolation Marked difi^erences were described above between the directly isolated and cultured phycobionts. Since the latter originates directly from the former, it was of interest to determine how rapid are the changes in the directly isolated phycobiont when it is brought into pure culture. Immediately after preparation, a stock of directly isolated phycobiont cells was kept under constant illumination in distilled water at room temperature. Every 24 hours, a sample of cells was removed and incubated for 3 hours in the light in a solution of

8 386 E. A. DREVV^ AND D. C. SMITH NaH'^COj and the subsequent distribution of fixed ^^C in the cells and in the medium was determined. Data in Table 2 shows that the release of fixed ^"^C into the medium fell off suddenly between 24 and 48 hours after isolation. At the same time, ^"^C no longer appeared as glucose in the medium but as a compound which remained at the origin of chromatograms run in ethyl acetate-acetic acid-water. This could have been some form of polysaccharide; however, it resisted mild acid hydrolysis, although traces of [^*C]- glucose were released during this treatment. It seems unlikely that the difference between the two kinds of isolate arise as a result Table 2. Release of photosynthetically fixed ^^C into the medium by the directly isolated phycobiont determined at intervals over a period of 3 days Time from % total fixed '*C in medium after isolation (hours) 3 hours photosynthesis o IS Algal suspensions maintained on distilled water at 20" C under continuous illumination at 425 ft-candles. Every 24 hours, sample of cells removed and resuspended in an aqueous solution of NaH'^COj (2.5 //Ci/ml, carrier-free) for 3 hours in the light. of selection of one or two genotypes from the mass of algal cells used to initiate the growth of the cultured phycobiont. The changes shown in Table 2 are very rapid, and during the first 48 hours of the experiment, no growth or other changes visible to the eye were apparent in the cultures. Utilization of exogenously supplied carbohydrates It will be shown in the next paper that in the thallus of Peltigera polydactyla, the fungus converts the glucose released by the Nostoc into mannitol. Lewis and Harley Table 3. Uptake of glucose and mannitol by the three types of algae in the dark % ' 'C lost from medium Glucose Mannitol Directly isolated phycobiont Cultured phycobiont Nostoc muscorum 100* 23 Media were carrier-free aqueous solutions of 5 f^c [''''C]- sugars in distilled water; glucose approximately 30 //g per algal sample, and mannitol approximately 50 fig per algal sample. Uptake values corrected to 5 mg dry weight algae in all samples. Incubation 3 hours at 20 C in the dark. * Medium exhausted during experiment. (1965) have postulated that the conversion of carbohydrates derived from the host into polyols by symbiotic or parasitic fungi has the advantage not only of maintaining a concentration gradient into the mycelium, but also of rendering the carbohydrate unavailable for re-use by the host (since many hosts are unable to utilize polyols such as mannitol). In order to study the utilization of carbohydrates by the three kinds of algae, samples were incubated in the dark for 3 hours in solutions of either [^"'CJmannitol or [^*C]- glucose. Table 3 shows that in all cases uptake of mannitol was substantially less than

9 Physiology of Nostoc from Peltigera 387 uptake of glucose. Unfortunately it was not possible in this experiment to adjust the sample sizes of the different Nostoc preparations to be equal, so that direct quantitative comparisons of uptake by the different types of algae could not be made. Analysis of the soluble extracts of cells showed that in both the directly isolated and the cultured phycobiont all the ^^C accumulated from the mannitol medium was present only as mannitol and so had evidently not been metabolized. In the case of A^. muscorum, a small amount of mannitol metabolism had evidently taken place, though it is not knowri to what extent this might have been due to bacteria. On the other hand, glucose was obviously actively metabolized by all three types of algae, since ''^C appeared in CO^ and also in a range of intermediary metabolites within the cells. Thus it appears that the ability of both the directly isolated and the cultured phycobiont to utilize mannitol is negligible. DISCUSSION The investigations described in this paper have demonstrated that it is possible to carry out physiological experiments on algae directly isolated from the lichen thallus and before they are subjected to prolonged pure culture. The need for such studies is illustrated by the marked differences found between Nostoc directly isolated from the thallus and that which had been in pure culture for several weeks. In the case of the directly isolated phycobiont, free glucose was one of the major products of photosynthesis, much of it heing released from the cells into the medium. However, after a period in culture, hardly any free glucose was found in the cells, and none was released into the medium; instead, more carbon was incorporated into insoluble compounds, especially glucose polysaccharides. It is of interest to note that Muscatine (1965) was able to isolate Zoochlorellae from Chlorohydra viridissima by a similar technique to that described here for the isolation of Nostoc. He found that such 'directly isolated Zoochlorella' released to the medium as much as 85% of the total carbon fixed in photosynthesis, principally as maltose. By contrast, free-living Chlorella released very little fixed carbon, and none of it as maltose. The fact that the directly isolated Nostoc phycobiont released substantial amounts of fixed ^*C to the medium has an obvious significance for the efficiency of the symbiotic association, and bears out earlier conclusions of Smith and Drew (1965) about the speed and amount of carbon movement from alga to fungus in the thallus. However, the nature of the processes involved in the loss of glucose from the Nostoc cells is not clear. Since a variety of soluble compounds incorporated ^"'C within the cells during photosynthesis but only glucose appeared in the medium, it is implied that loss of glucose is not a consequence of any simple 'leakiness' of the cells. Free-living Nostoc spp., and also the cultured phycobiont in these experiments, typically form a thick mucilaginous sheath, but this is lacking in the alga in the thallus. Thus, release of glucose might well result from some modification of the mechanism of mucilage production or of some other aspect of cell wall metabolism. It is significant in this respect that within 48 hours of isolation from the thallus the main product excreted by the Nostoc cells ceased to be glucose and it was replaced by less amounts of another compound, possibly a polysaccharide. The experiments described here underline the need for care in assuming that the physiological properties of a symbiont in pure culture are the same as they are in the symbiotic association. Mannitol is the main product accumulating in the thallus of Feltigera polydactyla

10 388 E. A. DREW AND D. C. SMITH during photosynthesis (Smith, 1961). This paper has shown that the principal photosynthetic product of the algal component is free glucose, and that this is released from the cells. It thus seems likely that glucose is the form in which fixed carbon moves from alga to fungus in the thallus, and that the fungus converts the glucose to mannitol. The movement of glucose between the symbionts in the thallus will be considered in the next paper. ACKNOWLEDGMENT One of us (E.A.D.) acknowledges receipt of a D.S.I.R. Research Studentship which enabled this work to be carried out. REFERENCES BRITTON, H. G. (1959). Detection of carbohydrates with silver in the presence of borate. Biochem. J., 73, 191^ CALVIN, ]\I. & BASSHAM, I. A. (1962). Tlie Photosynthesis of Carbon Compounds. Benjamin, New York. CALVIN, M. & MASSINI, P. (1952). The path of carbon in photosynthesis. Experientia, 8, 445. HANFS, C. S. & IsHERWOOD, F. A. (1949). Separation of phosphoric esters on filter paper chromatograms. Nature, Lond., 164, HARLEY, J. L. & SMITH, D. C. (1956). Sugar absorption and surface carbohydrase activity of Peltigera polydactyla (Neck.) Hoffm. Ann. Bot., N.s., 20, 513. HENRIKSSON, E. (1961). Studies in the physiology of the lichen Collema. IV. The occurrence of polysaccharides and some vitamins outside the cells of the phycobiont, Nostoc sp. Physiologia PL, 14, 813. LEWIS, D. H. & HARLEY, J. L, (1965). Carbohydrate physiology of mycorrhizal roots of beech. III. Movement of sugars between host and fungus. Neiv PhytoL, 64, 256. LiNKO, P., HOLM-HANSEN, O., BASSHAM, I. A. & CALVIN, M. (1957). Formation of radioactive citrulline during photosynthetic C'^'^OT fixation by blue-green algae. _7. exp. Bot., 8, 147. LOU(;HMAN, B. C. & MARTIN, R. P. (1957). Methods and equipment for the study of the incorporation of phosphorus by intact barley plants in experiments of short duration. J. exp. Bot., 8, 272. MuscATiNE, L. (1965). Symbiosis of Hydra and algae. III. Extracellular products of the algae. Comp. Biochem. Physiol., 16, 77. NoRRis, L., NoRRis, R. E. & CALVIN, M. (1955)..A sur\ey of the rates and products of short-term photosynthesis in plants of nine phyla. J. exp. Bot., 6, 64. REES, W. R. & REYNOLDS, T. (1958). A solvent for the paper chromatographic separation of glucose and sorbitol. Nature, Lond., 181, 767. SCOTT, G. D. (1957). Lichen terminology. Nature, Lond., 179, 4S6. SMITH, D. C. (1961). The physiology oi Peltigera polydactyla (Neck.) Hoffm. Liclienologist, i, 209. SMITH, D. C. & DREW, E. A. (1965). Studies in the physiology of lichens. V. Translocation from the algal layer to the medulla in Peltigera polydactyla. Nezc PhxtoL, 64, 19^. SMITH, I. (i960). Cliromatographic and Electrophoretic Techniques. Xo\. 1. Chromatography. Heinemann, London. TREVELYAN, W. E., PROCTOR, D. R. P. & HARRISON, J. S. (1950). Detection of sugars on paper chromatograms. Nature, Lond., 166, 444. ZEHNDER, A. & GORHAM, P. R. (i960). Factors influencing the growth of Microcvstis aeruginosa Kutz. Can. J. MicrohioL, 6, 645.

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