ARBUSCULAR MYCORRHIZAS IN PLANTS COLONIZING COAL WASTES IN SCOTLAND

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1 New PhytoL (1974) 73, ARBUSCULAR MYCORRHIZAS IN PLANTS COLONIZING COAL WASTES IN SCOTLAND BY M. J. DAFT AND T. H. NICOLSON Department of Biological Sciences, University of Dundee (Received 18 May 1974) SUMMARY All the grasses and nearly all the dicotyledonous plants growing on three coal tips in Scotland were infected by vesicular-arbuscular mycorrhizal fungi. Infection levels ranged from 20 to 90%. Three main types of fungal endophytes were identified, two being Endogone spp. and one with very narrow hyphae. Mixed infections were present in some plants and there was some evidence that the endophytes occurred in different areas of the tips. The ecological importance of the mycorrhizas in these habitats is discussed. INTRODUCTION Symbiotic associations that participate in the fixation of nitrogen and uptake of phosphorus are involved in pioneer colonization of nutrient-deficient sites (Harley, 1970). In sand dunes, which have a low nutrient status, most of the colonizing grasses are infected with vesicular-arbuscular mycorrhizas caused by Endogone species (Nicolson, i960). It has been suggested that infection with these endophytes could also be of importance in plants colonizing industrial waste (Nicolson, 1967) where plant cover is difficult to establish (Bradshaw, 1970; James, 1966; Schramm, 1966). However, as far as we know, they have not been reported from coal waste material. As coal tips show a number of stages in plant colonization, we undertook a survey of three tips for the presence of vesicular-arbuscular mycorrhizas. MATERIALS AND METHODS Sampling of plants and spoil. Coal tips at the Devon and Glen Ochil collieries in Clackmannanshire (Nat. Grid Ref. NN and NN respectively) and the Balgonie colliery in East Fife (NT990307) were surveyed. Plants growing on the tips in relative abundance were collected in October 1971 for each tip and again at the same time of year in 1972 for the Devon tip. We have found in the past that autumn is a suitable time of year to examine plant material for mycorrhizal infections, as high infection levels normally occur at this time. Between six to ten plants of each species growing in close proximity were carefully dug up with spoil attached to their roots and placed separately in polythene bags for transportation. The number of plants removed was kept to a minimum to preserve the establishment on the tip. After identification of the plants, their roots were carefully washed in water to remove loose detritus and a portion cut off and fixed in formalin/acetic/alcohol. From pooled samples of the roots, mycorrhizal infections were estimated by the methods described previously (Daft and Nicolson, 1129

2 1130 M. J. DAFT AND T. H. NICOLSON 1966, 1969). While such figures may not give the most accurate assessment of infection (Daft and Nicolson, 1972), it was the most convenient method for this investigation. Total phosphate of the spoil samples was determined using the Molybdenum Blue method and extractable phosphate by the calcium chloride method. The ph values of the samples were measured on a Pye model 292 ph meter. Identity of endophytes. Spoil material collected along with the plants was wet-sieved and the fractions remaining on the 250- and i5o-/^m sieves were examined for endophyte spores and mycelium (Gerdemann and Nicolson, 1963). Further identification of the endophytes followed growing three kinds of test plants in the glasshouse: (i) sievings were inoculated on to maize plants grown in sterilized sand; (ii) plants from the tips were grown in sterilized sand; and (iii) maize plants were grown in spoil taken from the plant collection areas. Roots from these test plants were examined microscopically together with sievings from the tests. RESULTS. The Devon colliery tip (Fig. i and Plate i. No. i) consists of an elongated mound running approximately north-south with several slurry lagoons on the east side and a larger lagoon below the north-east face of the tip. It contains 1,250,000 tons of spoil, was last used for tipping in i960, is 53 m high and covers 4.8 ha. The spoil consists of 75% 'runof-mine dirt' and 25% washer discard. Such discard is mainly blaes with some sandstone and fireclay. Total phosphate levels in the spoil (675 ^/g/g spoil) were similar to those found in sand dunes (Nicolson, unpublished), another habitat with pioneering plant species with high mycorrhizal infections, although the amount of extractable phosphate (2.31 /ig/g spoil) was much higher. There was little variation in the ph values of the spoil samples ( ph units). At the Glen Ochil colliery, tipping finished in In 1965, it was levelled and its faces were graded. The spoil contains 55% 'run-of-minc dirt' and 45% washer discard and its composition is similar to that in the Devon tip. Total phosphate levels for this tip were 766 /jg/g and extractable phosphate 1.37 ngjg spoil. The area has an almost complete coverage of grasses and herbaceous ruderals together with some sedges. This tip had the highest ph value (7.4). Very sparse plant colonization had occurred on the tip at Balgonie although tipping ceased in i960. Again the main components were similar in composition but the proportion was 35% 'run-of-mine dirt' and 65% washer discard and these had a ph value of 5.7. Total phosphate was highest on this tip (1087 A'g/g) but extractable lowest (0.35 //g/g spoil). Examination of plant material Four main areas of the Devon tip were sampled (Fig. i). Area 1 on the west side was less steep than the east side but many erosion gullies were present and, in general, the plants grew along these. Area 2 was the ridge on the south side of the tip where some plants were present. The north side of the tip was very steep with little or no established vegetation. Area 3 was on the east side of the tip where there were fewer plants than in Area i. The only plant colonizing the upper unstable slopes of this area was the Rosebay Willowherb [Chamaenerion angustifolium) although, at the base of the slope, there was a variety of plants. In Area 4 on the east side of the tip, vegetation was dense. The gradients on the tip can be seen from the contour lines (Fig. i). From the Glen Ochil and Balgonie collieries, samples oi Dactylis, Deschampsia, Festuca,.

3 Arhuscular mycorrhizas of coal wastes 1131 Agrostis and Holcus were collected. Tbe results for tbe 1971 samples (Table i) sbow tbat all plants examined were mycorrbizal. On tbe Devon tip (Table ia), two types of infection were present, first, tbe usual type of mycorrhiza caused by Endogone spp. wbicb produces tbe characteristic infection of tbe vesicular-arbuscular (VA) type (Nicolson, 1967) and Fig. I. Plan of the Devon tip showing the plant collection areas (1-4). Scale 1:25OO; contours at 4-m intervals. secondly, an unusual type of infection caused by a fungus witb very narrow bypbae (approx. I /(m diameter). Tbe two samples froni Area i (Fig. i) were mostly infected by this endopbyte. CItamaenerion angustifouum, colonizing tbe bighest areas of tbe steep eastern slopes (Area 3, Fig. i) and tbe two grasses from tbe extensively colonized Area 4

4 1132 M. J. DAFT AND T. H. NICOLSON were infected by both types of endophyte. The grasses from the Glen Ochil and Balgonie tips were infected with Endogone alone (Table ib, c). In a more extensive sampling programme undertaken in 1972 and confined to the Devon tip, both grasses and herbaceous dicotyledons were collected, identified and their Table i. Mycorrhizas from three coal tips (sampled November 1971) (plant names after Clapham, Tutin and Warburg (1962) here and in Table 2) (a) Devon tip Area in Fig. i I z 3 Mean* mycorrhizal Host infection (%) Endophyte Agrostis tenuis Hotcus lanatus Chamaenerion angustifolium Festuca ovina agg. Agrostis tenuis Approx (b) Glen Ochil tip Dactylis gtomerata 61 Deschampsia caespitosa 60 Endogone sp. only Festuca ovina 71 Endogone sp. only (c) Balgonie tip Agrostis tenuis 47 Endogone sp. only Holcus lanatus ao Endogone sp. only Deschampsia caespitosa ae Endogone sp. only Mean of six determinations Mainly the narrow endophyte Mainly the narrow endophyte 1 Both narrow endophyte and f Endogone sp. J Endogone sp. only Table 2. Mycorrhizas in grasses and herbaceous dicotyledons from the Devon coal tip (sampled Area in Fig. I I 3 4 I a 3 Host GRASSES Dactylis glomerata Festuca ovina Holcus lanatus Ii. lanatus Agrostis tenuis Holcus lanatus Festuca ovina DICOTYLEDONS Chamaenerion angustifolium Senecio viscosus Cirsium arvense Reseda luteola Senecio viscosus Chamaenerion angustifolium Ruhus fruticosus agg. Chamaenerion angustifolium Senecio viscosus October 1972) Mean* mycorrhizal 7.al infection (%) Fragaria vesca 62.5 Mean of six determinations. Endophyte > Endogone sp. only Narrow endophyte only Mostly narrow endophyte but some Endogone sp. > Endogone only Mostly the narrow endophyte with some Endogone sp. Endogone sp. predominantly hut some narrow endophyte } Narrow endophyte only Mostly narrow endophyte with some Endogone sp. Probably both endophytes Endogone only Endogone sp. predominating but some narrow endophyte Endogone sp. only roots Stained for examination. The grasses were heavily infected from both sites (Table 2). In plants on the more stable western slopes (Area i. Fig. i) where fairly extensive plant colonization had occurred and on Area 4 where plant cover was complete, the high mycorrhizal infection was of the usual Endogone type. Plants from the higher slopes

5 Arbuscular mycorrhizas of coal wastes 1133 (Area 2) where primary colonization was taking place were infected with the endophyte with narrow hyphae. Almost all the dicotyledons sampled had high mycorrhizal infection (Table 2). In most cases both endophytes were present with only two species [Chamaenerion angustifolium at Area 3 in 1972 and Fragaria vesca) showing Endogone alone. In one species. Reseda luteola, no infection was present and the roots of Rumex crispus and Rubus fruticosus agg. were so heavily pigmented that an accurate assessment of infection could not be made. The distribution pattern of the types of endophytes in the dicotyledons was not so clear as in the grasses but the narrow endophyte did tend to occur more on plants growing in the areas of primary colonization on the higher slopes of the tip. Characteristics of the endophytes From direct examination of roots and soil sievings from the field and from material grown in the glasshouse, it was evident that three main types of endophytes were present on the coal tip flora. (a) Chlamydosporic Endogone spp. The majority of infections on the coal tip plants appear to have been caused by small-spored Engogone spp. which form characteristic arbuscules (Plate i. No. 2) and vesicles (Plate i. No. 3). In fractions sieved from the soil, the extensive mycelium was of a typical Endogone nature but no large ectocarpic chlamydospores were found (Nicolson and Gerdemann, 1968). Chlamydospores found fell within the size range for E. fasciculata (J. W. Gerdemann and J. M. Trappe, personal communication) with spore dimensions greater than 50 /(m. The possibility of E. fasciculata being an important endophyte in this habitat was further confirmed by the extensive formation of chlamydospores in the moribund root material from maize plants inoculated with sievings from the coal tip (Plate i. No. 4). Similar spore production by this endophyte species occurs on the roots of sand dune grasses (Nicolson, 1959; Nicolson and Johnson, 1974) and in other plants (J. W. Gerdemann and J. M. Trappe, personal communication). (b) Endogone calospora. In some plants of Dactylis glomerata grown in coal tip soil from Area 3 collected in 1971, there was an extensive development of the azygosporic species, Endogone calospora (Nicolson and Gerdemann, 1968). When the pots were wetsieved, azygospores with typical bulbous attachments were abundant (Plate 2, No. 5). The irregular accessory vesicles, characteristic of this species, were present on mycelium sieved from pots and attached to the roots (Plate 2, No. 6). These were borne singly at the ends of coiled hyphae and not in groups as in other allied species (Nicolson and Gerdemann, 1968; Gerdemann, 1971; Old, Nicolson and Redhead, 1973). Most of the azygospores were formed on the external mycelium around roots but many had obviously been formed inside root tissue (Plate 2, No. 7a) when the bulbous attachments were very closely appressed to the surface of the spore (Plate 2, No. 7b). It could not be determined how these azygospores originated in roots. Previously this group of endophytes has been observed to form only arbuscules on internal hypae (Gerdemann, 1971) but, in the material examined here, many root fragments had vesicular structures in their cortices. These may have given rise to the intracortical azygospores or a chlamydosporic Endogone species may have been also present in the sample. The formation of vesicles within root tissues by E. calospora has been recently reported (Furlan and Fortin, 1973). The differences between chlamydosporic and azygosporic phycomycetous endophytes have been discussed by Old, Nicolson and Redhead (1973). It was apparent in this study that the hyphae of the two groups display different characteristics. The chiamydosporic

6 II34 M. J, DAFT AND T. H. NICOLSON group of endophytes show the irregular hyphal features with conspicuous angular projections frequently recorded since Butler in The hyphae of the other group, however, are much smoother in outline. Other differences between these groups of endophytes will no doubt become apparent as further morphological and ultrastructural studies are made. (c) Endophyte of unknown affinity with narrow hyphae. The internal and external mycelia of chlamydosporic Endogone endophytes are easily recognizable from hyphal characteristics because of such features as irregularity and size. However, fungi of another type with narrow hyphae (i /^m diameter) have been found to form mycorrhizal infections in New Zealand (Greenall, 1963; Baylis, 1969; Crush, 1973a) and in the U,K, (Ali, 1969; Mosse and Hayman, 1971). In addition, one of us (THN) has noted the sporadic occurrence of such an endophyte, particularly in samples of Scottish upland grasses, over the last 20 years. A similar type of endophyte occurred widely in the coal tip flora (Tables i and 2), Its characteristics follow very closely the descriptions given by Crush (1973a) and AH (1969), The external mycelium can form tangled masses on the root surface where appressorial swellings are formed (Plate 3, Nos, 9 and 10). From these, tufts of penetrating hyphae sometimes arose (Plate 3, No. 10). Root penetration occurred either through hairs (Plate 3, No. 8) or through or between epidermal cells (Plate 3, Nos, 9 and 10). In addition, small intercalary or terminal vesicles were formed on the external mycelium (Plate 3, No. 12a and Plate 4, No. 13). Frequently the hyphae followed the interstaces of the epidermal cells with frequent anastomoses, forming H-shaped configurations (Plate 3, No. 12b). This also occurred when the hyphae followed the intercellular spaces in the cortex. Their diameter rarely exceeded i //m with vesicles reaching 5-7 ^m. 'Fhese dimensions are similar to those cited by Crush (1973a) and Ali (1969), although the hyphae reported by Mosse and Hayman (1971) measured 4-6 //m. In the inner cortical layers, more profuse mycelial development occurred, showing as a deeply stained region (Plate 3, No. 11) where arbuscules were formed (Plate 4, No. 13). In the case of the narrow endophyte, these were very similar to those formed by chlamydosporic Endogone sp. (Plate i. No. 2). In some samples, the narrow endophyte was present alone; in others, it occurred with Endogone in different roots or together in the same segments (Tables i and 2). Sometimes (as found by Ali, 1969), the two types of endophytes were very closely intermingled, occupying the same or adjoining cells (Plate 4, Nos. 14 and 115). In such cases when arbuscules were present, it could not be determined which endophyte was forming them (cf. Plate i. No. 2 and Plate 4, No. 13), The identity of this endophyte is unknown. The general appearance is fungal in nature and, although the hyphal diameter is very small, it is unlikely to be an actinomycete. The hyphae are basically coenocytic with occasional septa so it is most likely phycomycetous, A similar endophyte was named Rhizophagus tenuis by Greenall (1963) though there is no indication that it is related to Endogone-Xy^& endophytes in the usually accepted sense (Nicolson, 1967; Gerdemann, 1968; Mosse, 1973). Further indications of the identity must await either its culture or the discovery of some identifiable type of spore. Recently, the terminology regarding symbiotic fungi has been discussed by Lewis (1973)- For mycorrhizas, he convincingly argues for the discontinuance of the terms endo- and ecto- with or without the suffix -trophic. He suggests the specific use of 'vesicular-arbuscular' to describe mycorrhizas induced by Endogone spp, but this may not strictly apply to all the three types of endophytes discussed here, Azygosporic

7 Arbuscular mycorrhizas of coal wastes 1135 endophytes, such as E. calospora may not form vesicles in the root infection phase. Also, the endophyte with narrow hyphae, while apparently forming arbuscules similar to Endogone spp., did not form similar vesicles, although small vesicular structures were present (Plate 5, No. 17). The term 'arbuscular' covers all these three types of endophytes and for that reason it is used in the title of this paper. Root hair production and mycorrhizal infection The possible relationship between vesicular-arbuscular mycorrhizas and root hair development has been discussed by Baylis (1970, 1972a, b). He suggested that the degree to which a plant species becomes dependent on mycorrhiza for the uptake of ions such as phosphate may be inversely related to its root hair development. One striking feature of the plants from the coal tips was their variation in root hair production. Grasses are known for their long root hairs and this was found in the examples from coal spoil. However, in the dicotyledons root hair development varied considerably. In general, there were three main categories: (i) where root hairs were long and abundant, as seen in Senecio viscosus (Plate 4, No. 10), Reseda luteola and Chamaenerion angustifolium; (2) an intermediate group, as in Cirsium arvense and Riimex crispus where root hair growth was limited; and (3) where hairs were either very short and present on a few roots or completely absent as seen in Fragaria vesca, Rulms sp. and Urtica dioica. In the grasses sampled, there was a wide variation in mycorrhizal infection (Tables i and 2). The dicotyledons showed no obvious relationship between root hair development and the degree of infection (Table 2). High infections occurred in both Chamaenerion angustifolium with profuse hairs and in Fragaria vesca where very few are produced. Root hair development may vary depending on microbiological and physical conditions (Rovira and Bowen, i960; Barley and Rovira, 1970). Also Bole (1973) has found that the density of root hair development was not closely related to P uptake and Mosse, Hayman and Arnold (1973) could find no close relationship between mycorrhizal infection and root hair development. Further work is necessary before the relative importance of root hairs and mycorrhizal infection in host nutrition can be assessed fully. DISCUSSION That phycomycete endophytes are abundant in coal tip plants is a further indication of their potential ecological importance. In other similar pioneer habitats, such as the anthracite and bituminous coal spoils in Pennsylvania (Daft and Hacskaylo, 1975), sand dunes (Nicolson, i960) and urban waste areas (Stevenson, 1964), these mycorrhizal infections are also strongly developed. In local dune systems, a single species of endophyte {Endogene fasciculata) is dominant (Nicolson and Johnson, 1974), whereas, in the coal tip fiora, at least three species are present. Here, in addition to E. fasciculata type infections, the azygosporic E. calospora and a further phycomycete endophyte with narrow hyphae have been identified. All three types of endophytes noted in the coal tip flora are potentially beneficial to plant growth. First, chlamydosporic endophytes have been extensively tested and found to stimulate plant growth under a variety of conditions (Mosse, 1973). This is particularly true for E. fasciculata and E. macrocarpa, two species which may grade in to one another as a continuous complex (J. W. Gerdemann and J. M. Trappe, personal communication). One variety of the latter species stimulated the growth of strawberry plants taken from the Devon tip when grown in sterilized coalspoil (Daft and Nicolson, unpublished).

8 II36 M. J. DAFr AND T. H. NiCOLSON Secondly, although azygosporic species have not been so extensively tested, E. calospora markedly enhances the growth oi Allium cepa particularly at higher temperatures (Furlan and Fortin, 1973). Endogene gigantea can stimulate the growth of Liriodendron (Clark, 1969), maize (Daft and Hacskaylo, 1974) and phlox, daisy and viola (Daft and El-giahmi, unpublished). Thirdly, an endophyte with narrow hyphae, called Rhizophagus tenuis by workers in New Zealand, has also been found to stimulate the growth of grasses, particularly under extreme phosphate stress (Crush, 1973b). The distribution of the three endophytes is of interest since the narrow endophyte was generally more prevalent on the pioneer species near the higher regions of the tip (Tables i and 2) and was then gradually replaced by known Endogone species as further plant colonization progressed. Another point of interest is that Reseda luteola showed no trace of mycorrhizal infection even when growing with other plants that were substantially infected. It belongs to a family (Resedaceae) that Gerdemann (1968) lists as being not susceptible to infection by Endogone. This author also lists the Urticaceae as being non or rarely mycorrhizal but in a single plant of Urtica dioica we examined some 29% infection was noted. It would have been desirable to examine further samples but this was not possible as this species is not a usual colonizer on this coal tip. More detailed work is required concerning the dependence of pioneering plants on mycorrhizal infections and, in particular, root-hair development, uptake of nutrients and the relative efficiencies of the endemic endophytes in the uptake of phosphorus. According to Walker (1965) 'phosphorus emerges as perhaps the key element because of its great ecological importance'. Therefore, much of the ecology of plants, particularly in primary habitats may be determined by how this vital element is obtained. As can be seen in the recent review by Mosse (1973), the contention that 'vesicular-arbuscular mycorrhiza may have been evolved as a means for the more efficient extraction of phosphorus from the pedosphere' (Daft and Nicolson, 1969) has been amply substantiated. ACKNOWLEDGMENTS We thank the National Environment Research Council for financial support, Mr E. L. Hindmarch, former Chief Scientist and Mr R. C. Dalrymple, Area Civil Engineer of the National Coal Board for suggesting the sites, for the map used in Fig. i and giving all assistance. REFERENCES ALI, B. (1969). Occurrence and characteristics of the vesicular-arbuscular endophyte of Nardtis stricta. Nova Hedwigia, 17, 409. BARLEY, K. P. & ROVIRA, A. D. (1970). The influence of root hairs on the uptake of phosphate. Soil Sci. Plant Anal., I, 287. BAVLIS, G. T. S. (1969). Host treatment and spore production by Endogone. N.Z.J. Bot., 7, 173. BAYLIS, G. T. S. (1070). Root hairs and phycomycetous mycorrhizas an phosphorus-deficient soil. Plant and Soil, 33, 714. BAYLIS, G. T. S. (1972a). Minimum levels of available phosphorus for non-mycorrhizal plants. Plant and SoiL 36, 233, BAYLIS, G. T. S. (1972b). Fungi, phosphorus and the evolution of root systems. Search, 3, 257. BOLE, J. B. (1973). Influence of root hairs in supplying soil phosphorus to wheat. Can. Jf. Soil Sci., 53, 169. BRADSHAW, A. D. (1970). Plants and industrial waste. Trans. Bot. Soc. Edin., 41, 71. BUTLER, E. J. (1939). 'l"he occurrences and systematic position of the vesicular-arbuscular type of mycorrhizat fungi. Trans. Br. mycol. Soc, zz, 274. CLARK, F. B. (1969). Endotrophic mycorrhizal infection of tree seedlings with Endogone spores. Forest Sci., IS, 134- CRUSH, J. R. (1973a). Significance of endomycorrhizas in tussock grassland in Otago, New Zealand. N.Z.J. Bot., II, 645.

9 Arbuscular mycorrhizas of coal wastes CRUSH, J. R. (1973b). The effect of Rhizophagus tenuis mycorrhizas on ryegrass, cocksfoot and sweet vernal. Neiu Phytol., 72, 965. DAFT, M. J. & HACSKAYLO, E. (197s). Vesicular-arbuscular mycorrhizas in the anthracite and bituminous coal wastes of Pennsylvania (in preparation). DAFT, M. J. & NICOLSON, T. H. (1966). Effect of Endogone mycorrhiza on plant growth. I. New Phytol., 65, 343- DAFT, M. J. & N1C01.S0N, T. H. (1969). Effect of Endogone mycorrhiza on plant growth. II. Influence of soluble phosphate on endophyte and host in maize. New Phytol., 68, 945. DAFT, M. J. & NICOLSON, T. H. (1972). Effect of Endogone mycorrhiza on plant growth. IV. Quantitative relationships between the growth of the host and the development of the endophyte in tomato and maize. New Phytol., 71, 287. FuRLAN, V. & FoRTiN, J. A. (1973). Formation of Endomycorrhizae by Endogone calospora on Alltum cepa under three temperature regimes. Naturaliste Can., loo, 467. GERDEMANN, J. W. (1968). Vesicular-arbuscular mycorrhiza and plant growth. Ann. Rev. Phytopathol., 6, 397' GERDEMANN, J. W. (1971). Fungi that form the vesicular-arbuscular type of endomycorrhiza. In: Mycorrhizae (Ed. by E. Hacskaylo) (Proc. ist N. American Conference on Mycorrhizae), p. 9. U.S.D.A. Misc. Pub. No GEHDEMANN, J. W. & NICOLSON, T. H. (1963). Spore of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Tram. Br. mycol. Soc, 46, 235. GREENALL, J. M. (1963). The mycorrhizal endophytes of Griselinia littoralis (Cornaceae). N.Z. jf. Bot., 1, 389- HARLEY, J. L. (1970). The importance of micro-organisms to colonizing plants. Trans. Bot. Soc. Edin., 41, 65. JAMES, A. L. (1966). Stabilizing mine dumps with vegetation. Endeavour, 15, 154. LEWIS, D. H. (1973). Concepts in fungal nutrition and the origin of biotrophy. Biot. RezK, 48, 261. MOSSE, B. (1973). Advances in the study of vesicular-arbuscular mycorrhiza. Ann. Rev. Phytopathol., 11, 171. MOSSE, B. & HAYMAN, D. S. (1971). Plant growth responses to vesicular arbuscular mycorrhiza. II. In unsterilized field soils. New Phytol., 70, 29. MOSSE, B., HAYMAN, D. S. & ARNOLD, D. (1973). Plant growth responses to vesicular-arbuscular mycorrhiza. V. Phosphate uptake by three plant species from P-deficient soils labelled with '^P. New Phytol., Ti, 809. NICOLSON, T. H. (1959). Mycorrhiza in the Gramineae. I. Vesicular-arbuscular endophytes, with special reference to the external phase. Tram. Br. mycol. Soc, 4a, 421. NICOLSON, T. H. (i960). Mycorrhiza in the Gramineae. II. Development in different habitats, particularly sand dunes. Trans. Br. mycol. Soc, 43, 132. N1C01.S0N, T. H. (1967). Vesicular-arbuscular mycorrhiza a universal plant symbiosis. Sci. Prog., Oxf., NICOLSON, T. H. & GERDEMANTVT, J. W. (1968). Mycorrhizal Endogone species. Mycologia, (to, 313. NICOLSON, T. H. & JOHNSON, C. (1974). Studies on mycorrhiza in sand dunes (in preparation). OLD, K. M., NICOLSON, T. H. & REDHEAD, J. F. (1973). A new species of mycorrhizal Endogone from Nigeria with a distinctive spore wall. New Phytol., 7a, 817. RoviRA, A. D. & BowKN, G. D. (i960). Effect of micro-organisms on the development of roots and root hairs of subterranean clover (T. subterraneum). Nature, Lond., 185, 260. ScHRAMM, J. R. (1966). Plant colonization studies on black wastes from anthracite mining in Pennsylvania. Trans. Am. Phil. Soc, 47, 331. STEVENSON, G. (1964). The growth of seedlings of some pioneer plants and the micro-organisms associated with their roots. Trans. Br. mycol, Soc, 47, 331. WALKER, T. W. (196.';). The significance of phosphorus in pedogenesis. In; Experimental Pedology (Ed. by F. G. Hallsworth & D. V. Crawford) (nth Easter School in Agricultural Sciences, Univ. Notts.), p Butterworths.

10 1138 M. J. DAFT AND T. H. NICOLSON EXPLANATION OF PLATES PLATE I No. I. Coal tip at the Devon colliery. Deep mine tip seen from the east note lower slopes with tree cover. Typical vesicular-arbuscular endophytes. Nos. 2 and 3. Arbuscules (A) and vesicles (V) respectively of an Endogone endophyte in the roots of Holcus lanatus from the Devon tip x 272 and x 176. No. 4. Spores (S) of an Endogone fasciculata type extruding from moribund roots of maize inoculated with a coal spoil fraction, x 160. PLATE Z Endogone calospora developed on the roots of Dactylis glomerata. Taken from the coal tip and grown in sterile sand. No. 5. An azygospore from a sieved fraction showing the bearer hypha (b) with bulbous base. X 250. No. 6. Single warty accessory vesicle (V) borne on a coiled hypha. x 250. No. 7. Outline of an azygospore embedded in cortical tissue (a) and in (b) a spore with adhering root tissue showing attachment appressed to spore wall, x 250. PLATE 3 Endophyte with narrow hyphae in roots of Senecio viscosm. No. 8. Hypha entering the cortex by means of a root hair. X Nos. 9 and 10. Formation of appressorial (A) bodies on the root surface and a group of penetrating hyphae (h). x No. II. An infected root showing a typical inner darkly stained zone (i) where the hyphae and arbuscules are concentrated, x 150. No. 12 (a) and (b). Hyphae running over the root surface and forming vesicular swellings (V) and appressoria (A). In (b) an 'H' configuration can be seen where the hyphae follow the interstaces of the outer cortical cells, x 750 and x 100 respectively. PLATE 4 Endophyte with narrow hyphae together with an Endogone endophyte in roots of Senecio viscosus. No. 13. Hyphae (h) and arbuscules (A) of the endophyte with narrow hyphae. x 900; cf. Plate I, No. 2 for the arbuscule of an Endogone endophyte. No. 14. Comparison between the two types of hyphae (hi) of the narrow and (h2) of an Endogone endophyte separated by one cell, x 900. No. 15. Comparison between the two types of vesicles (Vi) of the narrow and (V2) of an Endogone endophyte produced in the same area of the cortex, x 900.

11 TH1-: NEW PHYTOLOCilST, 73, 6 PLATE I M. J. DAFT \Ni. T. 11. N\CO\.S(.)N~A1WI!SCVI.AR XI Y('()liRHIZ.4S Oh' CO.II. WASTE.S (facing p. 11

12 THE NEW PHYTOLOGIST, 73, 6 PI.ATI' 2 V 7a M. J. DAFT AND T. II. NlCOhSON^ARBUSCULAR MYCORRHIZAS OF COAL WASTES

13 THE NEW PHYTOLOGIST, 73, 6 PLATE 3 M. J. D.M'l'AND r. 11. NlCOhSON ARBUSCULAR MYCORRHIZAS OF COAL WASTES

14 THE NEW PHYTOLOGIST, 73, 6 Pl.ATH 4 15 M. J. DAFT AND T. H. NICOLSON MYCORRAIZAS OF ('OAl\WASTES

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