Annals of Botany 77: 389–397, 1996

Nitrogen and Phosphorus Nutrition in Mycorrhizal Epacridaceae of South-west Australia

TINA L. BELL and JOHN S. PATE Department of Botany, UniŠersity of Western Australia, Nedlands WA 6907, Australia

Received: 2 February 1995 Accepted: 31 July 1995 Downloaded from https://academic.oup.com/aob/article/77/4/389/2587438 by guest on 27 September 2021

Xylem transport of nitrogen and phosphorus was examined in mature mycorrhizal plants of 41 species in 15 genera of Epacridaceae in native habitat in south-west Australia. Glutamine was the principal nitrogenous solute of xylem of all but four species. In the latter species, arginine or asparagine predominated. Nitrate and ammonium comprised minor fractions of xylem (tracheal) sap N, except in two species in which nitrate contributed over half of the N. Ratios of total-N:phosphate-P in xylem sap varied widely (mean 67p18, range 0n2–495) between species and habitats. Plants of Croninia kingiana (syn. Leucopogon kingeanus) from the one habitat showed higher levels of N and P in xylem early than late in the mycorrhizal season, but there was no consistent evidence of higher N and P levels from upper than deeper parts of their root systems. Study of juvenile populations of four species of epacrids indicated that substantial fractions of the yearly increment of N, P and dry matter was accumulated during the three summer months when infected mycorrhizal hair roots were absent. Glasshouse culture of mycorrhizal plants of Epacridaceae in habitat soil "$ "& enriched with decomposed and leached double ( C, N)-labelled dry matter of wheat showed substantial labelling "& "$ "& "& of shoots with N but not with C. Plants fed similarly treated N-labelled root residues of maize acquired N but "$ failed to generate c C values different from those of control plants. Possible avenues of mycorrhizal and non- mycorrhizal nutrition of Epacridaceae are discussed. # 1996 Annals of Botany Company

Key words: Epacridaceae, nitrogen, phosphorus, amino acids, mycorrhizal nutrition, xylem transport, amino acids, south-west Australia.

both nitrogenous ions were transported to and assimilated INTRODUCTION by seedling shoots, although it was impossible to determine In view of the close taxonomic relationships of Epacridaceae the extents to which fungus and host roots had been and Ericaceae (Watson, 1964; Powell, 1983), their frequent involved in uptake, transfer and partial assimilation of the association with acidic nutrient-poor habitats and their ammonium and nitrate. common development of specialized hair roots harbouring This paper extends the use of root xylem sap analyses to ericoid-type mycorrhizas (Pearson and Read, 1975; Harley provide information on the export of nitrogenous solutes and Smith, 1983; Reed, 1987; Hutton, Dixon and and phosphate from roots of mycorrhizal plants of 41 Sivasithamparam, 1994), one might reasonably conclude species of 15 genera of Epacridaceae sampled across a wide that benefits through mycorrhizal nutrition might be closely range of native habitats in south-west Australia. A detailed similar for members of the two families. Mycorrhiza of a study of sap composition within a single species is described number of Northern Hemisphere Ericaceae have been for an autumn-winter-spring cycle of hair root production suggested to function principally by providing their hosts and infection, and the information obtained related to with nitrogen derived from direct uptake of amino acids or measurements of increments in total N and P of young following enzymatic breakdown of otherwise intractable plants of four species during and after a seasonal cycle of forms of soil organic nitrogen such as peptides or mycorrhizal symbiosis at the same study site. A glasshouse proteinaceous materials (e.g. Bajwa, Abuarghub and Read, study is described in which possible acquisition of carbon to 1985; Bajwa and Read, 1986; Read, Leake and Langdale, host epacrid through mycorrhizal breakdown of organic 1989; Leake and Read, 1990). In a number of studies (e.g. matter is examined in juvenile plants supplied with organic Stribley and Read, 1976, 1980; Read, 1978) measurable matter derived from decomposed and leached plant material "$ "& benefit from presence of mycorrhiza in terms of growth and labelled with C and N. increased N uptake has been observed. Equivalent in- formation on Epacridaceae is simply not available although the recent report of Bell, Pate and Dixon (1994) on response MATERIALS AND METHODS of mycorrhizal seedlings of four south-west Australian Collection and analysis of xylem sap epacrids to added nutrient supplements under glasshouse conditions has indicated primary limitation by nitrogen Xylem sap was collected from roots of a range of south-west rather than phosphorus. Analyses of root bleeding (xylem) Australian epacrids growing in native habitats ranging from "& "& sap of NH%NO$-orNH% NO$-treated plants showed that kwongan sandplain, dunal heath, swamp, Banksia wood- 0305-7364\96\040389j09 $18.00\0 # 1996 Annals of Botany Company 390 Bell and Pate—Nitrogen and Phosphorus Nutrition in Epacridaceae land, and jarrah (Eucalyptus marginata Donn ex Smith) and length per plant and percentage of hair root infected at karri (E. diŠersicolor F. Muell.) forests. For each species at times of sampling for each species. least three adult plants were utilized for sap collection. In certain cases plants of the same species were sampled from two or more widely separated habitats. Sampling was Glasshouse study of utilization by mycorrhizal plants of "$ "& conducted in autumn, winter and spring of 1993 or 1994 and C- and N-labelled plant organic matter all plants from which sap was collected showed well infected Two sources of labelled plant residues were used in the hair roots. Sap collection involved displacement of xylem "$ study, one comprising finely ground material of double ( C, (tracheal) fluid by mild vacuum from freshly excavated "& N)-labelled wheat, Triticum aestiŠum L., the other root 10–20 cm segments of root using the apparatus and "& techniques described by Pate et al. (1994). In three cases material of sand-cultured N-labelled maize, Zea mays L. roots were of insufficient diameter for sap collection so (C% species), decomposing in situ after removal of shoot material. Prior to feeding, samples of each labelled material

lower regions of the stem were sampled. Downloaded from https://academic.oup.com/aob/article/77/4/389/2587438 by guest on 27 September 2021 Detailed study of xylem composition through a single were mixed into habitat soil and allowed to decompose for season was undertaken on an adult population of Croninia a 6 month period under conditions of frequent leaching with kingiana (F. Muell.) J. Powell [syn. Leucopogon kingeanus deionized water to remove mineralized N. (F. Muell.) C. A. Gardner] growing in deep infertile sand in Juvenile plants of Leucopogon striatus R. Br., L. open Banksia woodland on private property near Gingin conostephioides, Astroloma xerophyllum and Croninia (31m08h S, 115m35h E). At each of four sampling periods kingiana and the non-epacrid species Banksia attenuata R. during the mycorrhizal season (May, Jul., Sep. and Nov. Br. (Proteaceae) and Acacia pulchella R. Br. (Mimosaceae) 1993) five to seven plants were progressively excavated to utilized for the feeding study were harvested in Jul.–Aug. the full extent of their root systems (1n5–2n0 m depth) and 1992 or 1993 from natural habitats at approximately 3–10 lateral roots on upper, mid and lowest parts of each root months of age, using the intact root core (15 cm height, system harvested for sap collection. The water table at the 7n2 cm diameter) procedure described by Bell et al. (1994) to study site was at 1n8 m depth in winter retreating to 2n4mby preserve as carefully as possible their complements of hair the end of summer. Specific amino compounds, ammonium, roots or other specialized root structures. After 2 months of nitrate and phosphate were identified and measured in sap acclimitization in the glasshouse, plants and their intact root samples using the HPLC-based assays detailed by Bell et al. cores were transferred to larger pots (22 cm height, 10n7cm (1994). diameter) and each core surrounded by habitat soil into which a labelled residue had been incorporated. Plants were grown for a further 10 month period during which they were watered regularly with deionized water but received no Seasonal studies of increases in total plant N and P nutrient supplements. Shoots of all plants were harvested Populations of juvenile plants of four species of together with those of control plants of all species grown Epacridaceae [Astroloma xerophyllum (DC.) Sond., under the same conditions but without labelled residues. Andersonia gracilis DC., Leucopogon conostephioides DC. Harvested plants were examined for presence of infected and Croninia kingiana] recruiting after a hot wild fire (May hair roots (Epacridaceae), cluster roots (Proteaceae) and 1991) at the Gingin site (see above) were successively nodules (Mimosaceae) in the original soil and in those parts sampled over 1 year at a sequence of harvest times to enable of the root systems which had penetrated into outer regions increments in plant dry matter N and P to be assessed of the pot where the labelled residues were located. Dried, finely ground shoot material of all species and treatments between the following times; (a) the commencement of the "$ "& was analysed for C and N by high resolution mass annual cycle of hair root formation (end of Mar. 1992), (b) "$ spectrometry [see Pate et al., 1990 ( C) and Unkovich, Pate the senescence of hair roots at the end of the subsequent "& season of mycorrhizal activity (end of Dec. 1992), and (c) and Sanford, 1993 ( N)]. the end of the ensuing non-mycorrhizal season (end of Mar. 1993). During the non-mycorrhizal season [interval between (b) and (c)] roots were almost devoid of hair roots and the RESULTS few hair roots which were present showed negligible Xylem sap composition of selected epacrids from south- infection. west Australia Each harvest of a species consisted of 50 plants. Following dry weight determination, N and P levels in all samples of Table 1 shows the order of abundance of major amino dry matter were analysed as outlined in Bell et al. (1994). compounds, proportions of total N as nitrate or ammonium Monthly measurements of soil moisture and levels of nitrate and ratios of total-N:phosphate-P in the root xylem sap and ammonium in the 0–20, 20–50 and 50–150 cm layers of samples of the taxa examined from native habitat. All but the soil profile at the site were carried out during the year of four species transported glutamine as their major organic study. Quantitative assessments of seasonal changes in solute and this amide usually comprised 75% or more of the length and infection status of hair roots of the study species sap organic N. Arginine, asparagine, aspartic acid, glutamic and adult plants of Croninia kingiana used for xylem sap acid and, less commonly, serine, alanine, glycine and gamma collection (see above) were as described in Bell et al. (1994). amino butyric acid comprised the bulk of the remaining Data obtained included information on total hair root amino N. Species in which compounds other than glutamine Bell and Pate—Nitrogen and Phosphorus Nutrition in Epacridaceae 391 T 1. Amide and amino acid composition of xylem sap collected from a range taxa of south-west Australian Epacridaceae in natiŠe habitat and related information on xylem composition with respect to nitrate, ammonium and phosphate

Nitrate-N Ammonium-N Total-N: Soil Order of abundance of major as % of as % of phosphate- Species type* xylem amino acids (N basis) total-N total-N P

Acrotiche cordata D (Site1) GLN"""ASP,GLU,ARG† 0n10 18 A.cordata D (Site 2) GLN"""ARG,GLU,ASP 0n20 2 Andersonia axilliflora G GLN""ASP,SER,GLU 1n03n211 Andersonia aff. caerulea A GLN"""GLU,ASP"SER,ALA,GABA 0n32n025 Andersonia echinocephala G GLN""GLU,ASP,SER,GABA 0n20 8 Andersonia grandiflora G GLN"GLU,ASP"SER,GLY,ALA,ARG 0n17n8 422 Andersonia simplex G GLN""GLU,SER,ASP"GLY,ALA,ARG 0n10 17

Astroloma macrocalyx§ S GLN""ARG,GLU,VAL,ASP 1n4ND‡ND Downloaded from https://academic.oup.com/aob/article/77/4/389/2587438 by guest on 27 September 2021 Astroloma pallidum L GLN,ARG""GLU,ASP"SER,ALA 0n20n53 Astroloma serratifolium A (Site 1) GLN""ASP,GLU,ARG 0n21n381 A.serratifolium S (Site 2) GLN""GLU,ASP,ARG 1n98n914 Astroloma tectum G GLN,GLU"ASP""ARG"SER,GLY,ALA 0n46n931 Astroloma xerophyllum S GLN""ASP,GLU,GABA 0n63n9 260 Brachyloma sp. nov. 1 Al GLN""ARG"ASP,GLU 0n82n039 Brachyloma sp. nov. 2 A GLN""ARG"ASP,GLU 0n60 73 Brachyloma sp. nov. 3 A GLN"ASP,GLU"ARG,GABA,ALA,SER ND 1n8ND Coleanthera myrtoides A GLN""ASP,GLU"ARG 54n10 0n2 Conostephium drummondii A ASN"GLN,ASP,GLU 0n91n511 Conostephium pendulum S (Site 1) ARG""ASN,GLU,GLN,ASP 9n21n025 C.pendulum§ S (Site 2) ARG"ASN,GLU,ASP 8n5NDND Conostephium roei A GLN"ARG,GLU"ASP 0n10 17 Conostephium sp. nov. 1 A GLN"""GLU,ASP,ARG 0n41n314 Cosmelia rubra L GLN""GLU,ASP"SER,ALA,GABA,ARG 0n16n616 Croninia kingiana S GLN""GLU,ASP"SER,ALA 0n73n54 Leucopogon conostephioides G GLN""ASP,GLU"GABA,ARG,SER ND 0 ND Leucopogon cordifolius S GLN""GLU,ASP"GABA,ARG,SER 1n24n8 116 Leucopogon aff. cordifolius S GLN""ARG,GLU,ASP 0n61n9 495 Leucopogon crassiflorus S GLN""ASP,GLU"GABA,SER 1n42n37 Leucopogon aff. crassifolius A GLN""GLU,ASP"SER,GABA,ARG 0n31n323 Leucopogon dielsianus A GLN"ARG,GLU,ASP 0n28n479 Leucopogon insularis S (Site 1) GLN"GLU,ASP"SER,ALA,GABA 0n83n414 L.insularis D (Site 2) GLN"ARG""GLU,ASP 0n10n75 Leucopogon parŠiflorus G (Site 1) ASN"GLN,ASP,GLU 0n44n519 L.parŠiflorus D (Site 2) GLN""GLU,ARG,ASP 0n10 5 Leucopogon planifolius S GLN"ASP,GLU"SER,ALA,GABA 0n15n7 489 Leucopogon strongylophyllus S GLN"ASP,GLU"ARG,SER 0n14n513 Leucopogon Šerticillatus L GLN""ASP,GLU,ARG 56n42n00n3 Lysinema ciliatum A (Site 1) GLN"ARG"ASP,GLU 0n21n639 L.ciliatum A (Site 2) GLN"ARG"ASP,GLU 0n10 48 L.ciliatum S (Site 3) GLN""ARG,ASP,GLU 0n63n525 L.ciliatum G (Site 4) GLN,ARG"GLU,ASP"GABA,ALA 0n21n828 L.ciliatum G (Site 5) GLN,ARG"ASP,GLU"GABA,SER 0n14n0 298 Monotoca tamariscina L GLN"""ASP,GLU"GABA,SER 0n20 40 Needhamiella pumilio G GLN,SER"GLY,ASP,GLU 1n58n721 Oligarrhena micrantha G GLN""GLU,ASP,ARG,SER 1n57n7 116 Sphenotoma capitatum L GLN"GLU,ASP"SER,GLY,ALA ND 15n0ND Sphenotoma drummondii G GLN""ASP,SER,GLU,ARG 0n10 12 Sphenotoma gracile Sw (Site 1) GLN""ALA,GLU,SER 3n211n353 S.gracile P (Site 2) GLN""GLU,SER,ASP 0n32n91 Styphelia intertexia A GLN""ASP,GLU,ARG"GABA,SER 0n51n821 Styphelia tenuiflora L GLN"GLU,ASP"ARG"SER,ALA 0n18n419 Mean (ps.e.) 3n2(1n6) 3n2(0n5) 67 (18)

* Key to soil types: A, arid, salt-laden sandplain; Al, alluvial; D, dunal sand; G, granitic; L, lateritic; P, pasture; S, kwongan sandplain; Sw, swamp. † Key to abbreviations for nitrogenous solutes: ASN, asparagine; ASP, aspartic acid; ARG, arginine; ALA, alanine; GABA, gamma amino butyric acid; GLN, glutamine; GLU, glutamic acid; GLY, glycine; SER, serine; VAL, valine. ‡ ND: not determined. § Data for these samples as indicated by Stewart, Pate and Unkovich (1993). predominated were Conostephium pendulum Benth. (two 57% of total sap N), Leucopogon parŠiflorus (Andr.) Lindl. populations with arginine comprising 35% or more of total (one of two populations with asparagine as 39% of total sap sap N), C. drummondii (Stschegl.) C. A. Gardner (asparagine N) and Lysinema ciliatum R. Br. (two of five populations 392 Bell and Pate—Nitrogen and Phosphorus Nutrition in Epacridaceae within the range of from 13–25 during the sampling May period. Samples of sap collected from upper, mid and lower N:P = 18 laterals and ‘sinker-like’ tap roots did not differ appreciably N:P = 25 Jul. in total-N level and total-N:phosphate-P ratio with depth (e.g. see typical data for two single plants, Fig. 1B and C). Sep. N:P = 13 All parts of root systems were well endowed with heavily infected hair roots at all times of sampling. Nov. N:P = 21 A 0 5 1015 20253035 Increments of plant total N and P during and following an Upper lateral annual cycle of mycorrhizal actiŠity (15–25 cm) N:P = 19 N:P = 30 Seasonal changes at the Gingin study site in soil moisture

Lower lateral and levels of ammonium and nitrate with soil depth were as Downloaded from https://academic.oup.com/aob/article/77/4/389/2587438 by guest on 27 September 2021 (30–40 cm) shown in Fig. 2A and B, respectively. Surface soil layers consistently showed lower moisture levels than at depths of Lower roots N:P = 35 50 cm or more. Top layers of the profile were almost (50–105 cm) B completely dried out during the hot summer months of Jan. 0 5 1015 20253035 to Apr., whereas lower regions of the profile (below 50 cm) remained well charged with water throughout summer. Upper lateral N:P = 17 (10–15 cm) Roots of all species penetrated to at least 70 cm depth so would have had continuous access to these moisture- Mid lateral N:P = 24 (20–35 cm) enriched layers. Nitrate was consistently at low levels (1 µg −" NO$-N g d. wt of soil ) throughout the soil profile Lower lateral N:P = 23 (35–55 cm) compared to ammonium which built up to high levels in the Sinker top 20 cm in the soil profile during late winter and early N:P = 20 −" (>60 cm) C spring (Jul. maximum of 12 µgNH%-N g d. wt of soil , Fig. 0 5 1015 20253035 2B). µg N ml–1 xylem sap Hair root infection of all four species in Mar. was 0 to 10%, depending on the species (Fig. 2C), increased to F. 1. Nitrogenous components of xylem (tracheal) sap of adult plants of Croninia kingiana from a single population from Gingin, W.A. values of approximately 30% by May, then increased more during the 1993 season. A, Mean concentrations of N-containing slowly to maximum values in the range 40–50% in spring solutes in xylem sap for four different collection periods (five to seven and early summer (Sep. to Nov.). Comparable values for plants sampled on each occasion) during the mycorrhizal season of total hair root length per plant (Fig. 2D) showed a slow 1993. B and C, Single plants sampled from lateral or sinker roots situated at different depths down the root system in Jul. (B) and Nov. increase through the winter, the maintenance of the peak (C) of 1993 to illustrate minimal variation in xylem sap composition length over the period Aug. to Dec. followed by a rapid and concentration of solutes with depth of root system. Ratios of total- senescence and loss of hair roots. Based on the above N:phosphate-P (N:P) in xylem are shown against each sap sample. , information, late Mar. 1992 to the end of Dec. 1992 was Asparagine; *, glutamic acid; , glutamine; , other amino acids; designated the ‘mycorrhizal season’ of the study period, 6, ammoniumjnitrate. Jan. 1993 to the end of Mar. 1993 the ensuing ‘non- mycorrhizal season’. with arginine slightly exceeding glutamine). Nitrate-N and Successive sampling of young plants across the study ammonium-N generally comprised only minor proportions period gave values for increments in total plant dry matter, (average of 3n2%) of total sap N (see Table 1), exceptions N and P for the mycorrhizal and non-mycorrhizal seasons being 11–15% sap N as ammonium in two species of as shown in Table 2. In most cases substantial proportions Sphenotoma and nitrate exceeding 50% of total sap N in (ranging from 17 to 85%) of the yearly increment in the Coleanthera myrtoides Stschegl. and Leucopogon Šerticillatus above quantities by a species occurred during the three hot R. Br. Ratios of total-N:phosphate-P in sap varied widely summer months of the non-mycorrhizal season, suggesting around a mean value of 67p18% (see Table 1). Of 46 that growth and uptake of N and P by juvenile plants at the samples analysed, ten showed ratios less than 10 while in site must have been considerably independent of the presence another seven samples unusually high ratio values (100–500) of infected hair roots and associated mycorrhizal activity. were recorded. Disregarding the latter high values the mean total-N:phosphate-P ratio becomes 23 3. p Acquisition of C and N by mycorrhizal plants from Where more than one sap sample was available for a labelled organic residues of decomposed plant material species, amino acid balance was in most cases similar for each set of samples whereas nitrate and ammonium levels Shoots of the fed plants of all species became heavily "& and total-N:phosphate-P ratios varied widely. enriched with N from either the labelled wheat or maize The seasonal study of xylem sap composition in the residues (Table 3). Comparing the atom % excess values "& mature population of Croninia kingiana at Gingin bore of the epacrid shoots with the original N enrichment evidence of declining levels of sap N with season (Fig. 1A). of the supplied labelled material, most species appeared to Total-N:phosphate-P (N:P) levels of xylem sap varied have derived approximately one quarter of their N from the Bell and Pate—Nitrogen and Phosphorus Nutrition in Epacridaceae 393

12 60 A C 10 >50 cm 50

8 40

d. wt soil) 6 0–20 cm 30 –1

4 20 % Soil moisture 20–50 cm

(g water g 2 10 % Hair root infection per plant 0 0 M AMJ JA SOND J F M AMJ JA SOND J F Downloaded from https://academic.oup.com/aob/article/77/4/389/2587438 by guest on 27 September 2021 14 300 B D 12

10 0–20 cm 200 8

d. wt soil) 6 –1

g 20–50 cm 100

µ 4 ( >50 cm Ammonium and nitrate

2 Nitrate (all depths) hair root length (cm) Total

0 0 M AMJ JA SOND J F M AMJ JA SOND J F F. 2. Annual variation (1992–1993 mycorrhizal season) in percentage soil moisture (A), soil nitrate plus ammonia (B), hair root infection status (#, Andersonia gracilis; =, Leucopogon conostephioides; *, Astroloma xerophyllum; , Croninia kingiana) (C) and total hair root lengths (#, Leucopogon conostrephioides; =, Astroloma xerophyllum; *, Andersonia gracilis; , Croninia kingiana) (D) per plant of Astroloma xerophyllum, Andersonia gracilis, Croninia kingiana and Leucopogon conostephioides from Gingin W.A.

T2. Increments in dry matter, total nitrogen and phosphorus of juŠenile plants of four species of south-west Epacridaceae from natiŠe habitat at Gingin. Values are giŠen for the mycorrhizal and subsequent non-mycorrhizal season of the second year of growth of the plants

Increments per plant (mg)*

Dry matter Nitrogen Phosphorus

Non- Non- Non- Mycorrhizal mycorrhizal Mycorrhizal mycorrhizal Mycorrhizal mycorrhizal Species season† season season season season season

Andersonia gracilis 0n12 0n12 0n55 0n80 0n01 0n05 Astroloma xerophyllum 0n18 0n12 0n95 0n90 0n10 0n02 Croninia kingiana 0n24 0n11 1n80 0n65 0n02 0n04 Leucopogon conostephioides 0n31 0n09 1n95 0n85 0n09 0n03

* Based on harvests of 50 plants per species at each time of sampling. † Mycorrhizal season late Mar. 1992–late Dec. 1992, non-mycorrhizal season early Jan. 1993–late Mar. 1993. See Fig. 1 for seasonal patterns of change in soil moisture, soil inorganic N and hair root infection status of juvenile plants at the study site.

"& "$ labelled source. N labelling of epacrids (Table 3) was not from that of controls (Table 3). Similarly, c C values of noticeably greater than that of the non-epacrid species plants fed residues of the C% species maize showed typical C$ "$ Banksia attenuata, indicating more or less equal dependence c C values (k27n0tok30n1), as opposed to less negative "$ on labelled N amongst the species. However, shoot dry values closer to that of the maize c C value (k14n1). matter of the effectively nodulated legume Acacia pulchella "& was less enriched with N, as might have been expected "$ DISCUSSION from its potential capacity to fix nitrogen. C enrichment of "$ dry matter of all species having access to C-labelled This study demonstrates how root xylem sap analyses of residues of wheat (Table 3) was not appreciably different epacrids can be used to provide definitive information on 394 Bell and Pate—Nitrogen and Phosphorus Nutrition in Epacridaceae "& "$ T 3. N-enrichments and c C Šalues of glasshouse grown mycorrhizal epacrid juŠenile plants and other juŠenile plants of non-epacrid species following 10 months of growth in habitat soil enriched with preŠiously decomposed and well leached "$ "& "& organic matter deriŠed from double ( C, N)-labelled whole plant dry matter of wheat or root residues of N-labelled maize. Controls relate to similarly grown material not proŠided with labelled residues

"& "$ Species Atom % excess N c C(=)

Seedlings cultured in presence of wheat residues (initial labelling of dry matter 1 59 atom % "& "$ n excess N, 1n32 atom % excess C) Epacrid Astroloma xerophyllum 0n561 k28n5 Croninia kingiana 0n415 k28.4 Leucopogon striatus 0n652 k30n1 L. conostephioides 0n528 k28n6 Non-Epacrid Banksia attenuata 0n526 k27n0 Acacia pulchella 0n104 k29n5 Downloaded from https://academic.oup.com/aob/article/77/4/389/2587438 by guest on 27 September 2021 "& Seedlings cultured in presence of N-labelled root residues of maize [initial labelling of dry "& "$ matter 1n71 atom % excess N, c C14n1(=)] Epacrid Astroloma xerophyllum 0n542 k28n9 Croninia kingiana ND* ND Leucopogon striatus 0n719 k29n8 L. conostephioides 0n518 k30n0 Non-Epacrid Banksia attenuata 0n547 k28n6 Acacia pulchella ND ND Controls Epacrid Astroloma xerophyllum 0 k28n6 Croninia kingiana 0 k27n6 Leucopogon striatus 0 k29n5 L. conostephioides 0 k28n9 Non-Epacrid Banksia attenuata 0 k27n9 Acacia pulchella 0 k27n9

* ND: not determined.

which forms of N and P are exported to shoots from system which may be present in the root. As shown by Bell mycorrhizal roots and the extent to which the balance of the et al. (1994) for pot-cultured epacrids receiving high amounts solutes involved differs between species, site and season. As of ammonium nitrate, shoots are capable of utilizing the far as organic forms of N are concerned, glutamine emerges nitrogen of unreduced nitrate spilling over from the root as the major exported compound in virtually all species, but system for growth. since certain taxa show other compounds such as arginine It is difficult to find any definite patterning of root xylem or asparagine as their principal component, and in all taxa sap composition that reflects habitat type and this is not presence or absence and order of abundance of minor suprising considering the heterogeneity of soil as a growing components differs appreciably between species, one would medium. Little or no ammonium in root xylem sap tends to conclude that xylem sap composition is to an extent taxon be associated with species growing on dunes and highest specific. total-N:phosphate-P ratios tend to be associated with As might be expected of the wide range of habitats species growing in sandplain or granitic habitats but these examined and the likelihood of wide variation in the conditions are by no means the rule. Detailed analyses of respective soil types in terms of available N and P, xylem soil nutrients of each habitat type would be required to levels of the inorganic components, nitrate, ammonium and begin to find stronger correlations between root xylem sap phosphate and their proportionality relative to total N of composition and habitat type. the sap, vary up to several orders of magnitude. Thus, there Study of the single species Croninia kingiana indicates are xylem samples with less than 0n5% of sap N as that seasonal variations in sap compositional qualities are ammonium plus nitrate, others with over 50% as nitrate or not particularly great at a single site and that deeper parts 15% as ammonium, and total-N:phosphate-P sap ratios of the root export sap of closely similar composition to that encompass a suprisingly broad range from 0n2 to 495. This of roots confined to upper layers of the profile. Soil at the suggests highly flexible performance on the part of mycor- site in question was consistently low in nitrate but much rhizal roots in absorbing and exporting N and P. Such higher in terms of ammonium, suggesting that the latter ion ability is likely to confer special advantage under conditions might be the principal form of N on which host and possibly in which inorganic N and phosphate levels vary widely with mycorrhizal component were feeding. However, at no time soil type and vacillate with idiosyncrasies of season. did ammonium and nitrate comprise more than a few Situations in which nitrate comprises a significant pro- percent of sap-N in this species, suggesting that the portion of sap N imply that where soil nitrate is high, much mycorrhizal roots were consistently able to assimilate of the absorbed nitrate circumvents any nitrate reducing virtually all the inorganic N absorbed, while also possibly Bell and Pate—Nitrogen and Phosphorus Nutrition in Epacridaceae 395

Glutamine and other amino compounds Soil organic N NH+ 4 – NO3 Amino acids Protein peptides

Extracellular Infected cells peptidases proteinases ? Downloaded from https://academic.oup.com/aob/article/77/4/389/2587438 by guest on 27 September 2021

+ NH4 ? Extramatrical ? X hyphae

? ? Uninfected cells

Soil ammonium X

NR

Soil nitrate

Ammonium- Host root assimilating xylem X system Amino compounds of host cytoplasm Ammonium

Nitrate NR reductase Nitrate of host

F. 3. Possible pathways of uptake, metabolism and transfer of nitrogen in the mycorrhizal root system of an epacrid. Ammonium, residual organic matter, low levels of nitrate and free amino acids are depicted as potential sources of N and mechanisms of assimilation are indicated for extramatrical hyphae and for the fungal and host components of hair roots. The scheme is consistent with information on xylem sap composition and data of labelling studies presented in this paper and by Bell et al. (1994). Elements of the scheme for which information is not yet available are labelled with question marks. deriving a proportion of the amino-N component of their terms of compositional characteristics with depth of xylem sap from the organic N component of the soil via the collection from single plants, despite noticeably higher extramatrical component of the fungal associate. Somewhat levels of ammonium and total soil-N in the organic matter- suprisingly, sap composition was remarkably uniform in rich 0–20 cm upper layer than further down the soil profile. 396 Bell and Pate—Nitrogen and Phosphorus Nutrition in Epacridaceae The study following seasonal changes of the contents of N interchanges of fungus and host in hair roots, before these and P in young epacrids showed substantial increments in possibilities can be explored further. Detailed comparison these quantities during the three hot summer months when of mycorrhizal and non-mycorrhizal epacrid plants would hair roots were virtually absent from the roots. It is not be particularly instructive in this connection. known whether continued growth and nutrient uptake during summer is a regular feature of south-west Australian ACKNOWLEDGEMENTS sandplain epacrids, or merely idiosyncratic of the Gingin study site, where the water table was relatively shallow and We thank Dr Kingsley Dixon for advice on selection of field soil moisture remained high at depths to which roots of the sites and assistance in collection of xylem sap, Ed Raisins juvenile plant had already penetrated. In any event nutrient for xylem sap analyses, Murray Unkovich and Lidia "$ "& uptake continued and kept pace with dry matter increases at Bednarek for C and N analyses and Mr and Mrs the Gingin site after the bulk of the seasonal complement of Kinsella for permitting access to field sites on their property.

hair roots had been sloughed off the roots. A somewhat Dr J. Palta, CSIRO, Floreat Park, kindly provided the Downloaded from https://academic.oup.com/aob/article/77/4/389/2587438 by guest on 27 September 2021 "$ "& similar situation has been noted by Read (1978) for Erica double ( C, N)-labelled residues of wheat. This study was spp. in which N mobilized from digestion of intracellular supported by a grant from the Australian Research Council hyphal material was suggested as a possible source during for study of nitrogen nutrition of native Australian flora. periods of N stress. Equivocal results were obtained in the studies feeding "& LITERATURE CITED decomposed, well leached organic residues of N-labelled matter to the rooting media of mycorrhizal epacrid and Bajwa R, Abuarghub S, Read DJ. 1985. The biology of mycorrhiza in non-epacrid plants. All non-legume species derived sub- the Ericaceae. X. The utilization of proteins and the production of proteolytic enzymes by the mycorrhizal endophyte and by stantial and closely similar proportions of their N re- mycorrhizal plants. New Phytologist 101: 469–486. quirement from the labelled sources and since ability to Bajwa R, Read DJ. 1986. Utilization of mineral and amino N sources break down complex residues would not have been expected by the ericoid mycorrhizal endophyte Hymenoscyophus ericae and of the non-mycorrhizal cluster (proteoid) roots of Banksia by mycorrhizal and non-mycorrhizal seedlings of Vaccinium. attenuata, little additional benefit to the epacrids appears to Transactions of the British Mycological Society 87: 269–277. Bell TL, Pate JS, Dixon KW. 1994. Response of mycorrhizal seedlings have accrued through direct access of mycorrhiza to organic of SW Australian sandplain Epacridaceae to added nitrogen and forms of N. Nitrogen labelling of all species would then be phosphorus. Journal of Experimental Botany 45: 779–790. envisaged to have derived principally from mineralization Harley JL, Smith SE. 1983. Mycorrhizal symbiosis. New York: "& of N-labelled residues by general soil microflora. Academic Press. "$ Hutton BJ, Dixon KW, Sivasithamparam K. 1994. The virtual absence of C enrichment of all species when Ericoid endophytes "$ "& of Western Australian heaths (Epacridaceae). New Phytologist given access to double ( C, N)-labelled organic residues of "$ 127: 557–566. wheat or C-labelled root residues of the C% species maize Leake JR, Read DJ. 1990. Chitin as a nitrogen source for mycorrhizal can be interpreted in either of two ways. On the one hand, fungi. Mycological Research 94: 993–994. the result would be consistent with all N coming from Pate JS, Davidson NJ, Kuo J, Milburn JA. 1990. Water relations of the root hemiparasite Olax phyllanthi (Labill.) R. Br. (Olacaceae) and mineralization and being absorbed directly by root or its multiple hosts. Oecologia 84: 186–193. mycorrhizal partner as ammonium or nitrate. On the other "& Pate JS, Woodall G, Jeskhe WD, Stewart GR. 1994. Root xylem hand, at least a fraction of the N accumulated by the transport of amino acids in the root hemiparasitic shrub Olax epacrids might have come from breakdown of complex phyllanthi (Labill.) R. Br. (Olacaceae) and its multiple hosts. Plant, organic N residues by the mycorrhizal partner, but in such Cell and EnŠironment 17: 1263–1273. Pearson V, Read DJ. 1975. The physiology of the mycorrhizal a manner that the resulting organic solutes of N were endophyte of Calluna Šulgaris. Transactions of the British My- catabolized strictly within the fungal component of the cological Society 64: 1–7. infected cells of the hair root. Only the resulting ammonia- Powell JM. 1983. Epacridaceae. In: Morley BD, Toelken HR, eds. N would then be available to the host cytoplasm (see Flowering plants in Australia. Australia: Rigby Publishers, 111–114. discussions of similar mechanisms of N transfer suggested Read DJ. 1978. The biology of mycorrhizas in heathland ecosystems for mycorrhizal Ericaceae, e.g. Stribley and Read, 1974, with special reference to the nitrogen nutrition of the Ericaceae. 1980; Read et al., 1989). These uptake possibilities and In: Loutit MW, Miles JAR, eds. Microbial ecology. New York: associated assimilation pathways into fungal and epacrid Springer-Verlag, 324–328. components are as illustrated in Fig. 3. In this scheme, the Read DJ, Leake JR, Langdale AR. 1989. The nitrogen nutrition of mycorrhizal fungi and their host plants. In: Boddy L, Marchant large organic-N component of root xylem sap of the R, Read DJ, eds. Nitrogen, phosphorus and sulphur utilization by epacrids is pictured as being exclusively synthesized by the fungi. Cambridge: Cambridge University Press, 181–204. host root, whether from inorganic-N (a) derived directly Reed ML. 1987. Ericoid mycorrhizas of Epacridaceae in Australia. In: from the soil as ammonium and to a lesser extent as nitrate, Sylvia DM, Hung LL, Graham JH, eds. Mycorrhiza in the next decade. Gainesville: Institute of Food and Agricultural Sciences, (b) obtained indirectly via mycorrhizal hyphae following 335. fungal uptake of these ions or (c) as suggested above, Stewart GR, Pate JS, Unkovich M. 1993. Characteristics of inorganic following enzymatic breakdown of complex organic residues nitrogen assimilation of plants in fire-prone Mediterranean-type by extramatrical hyphae and subsequent release of am- vegetation. Plant, Cell and EnŠironment 16: 351–363. monium to the host in infected cells of the hair root. Clearly, Stribley DP, Read DJ. 1974. The biology of mycorrhiza in the Ericaceae. IV. The effects of mycorrhizal infection on the uptake "& much more definitive information needs to be obtained on of N from labelled soil by Vaccinium macrocarpon Ait. New the catabolic activities of extramatrical hyphae and solute Phytologist 73: 1149–1155. Bell and Pate—Nitrogen and Phosphorus Nutrition in Epacridaceae 397

Stribley DP, Read DJ. 1976. The biology of mycorrhiza in the and the capacity to utilize simple and complex organic nitrogen Ericaceae. VI. The effects of mycorrhizal infection and con- sources. New Phytologist 86: 365–371. centration of ammonium nitrogen on growth of cranberry Unkovich M, Pate JS, Sanford P. 1993. Preparation of plant samples (Vaccinium macrocarpon Ait.) in sand culture. New Phytologist 77: for high precision nitrogen isotope ratio analysis. Communications 63–72. in Soil Science and Plant Analysis 24: 2093–2106. Stribley DP, Read DJ. 1980. The biology of mycorrhiza in the Watson L. 1964. The taxonomic significance of certain anatomical Ericaceae. VII. The relationship between mycorrhizal infection observations on Ericaceae. New Phytologist 63: 274–280. Downloaded from https://academic.oup.com/aob/article/77/4/389/2587438 by guest on 27 September 2021