Plant and Soil 210: 273–283, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
273
The effect of regeneration burns on the growth, nutrient acquisition and mycorrhizae of Eucalyptus regnans F. Muell. (mountain ash) seedlings T. M. Launonen1 , D. H. Ashton2 and P. J. Keane1 1 Department
of Botany, La Trobe University, Bundoora, 3083 Victoria, Australia and 2 School of Botany, The University of Melbourne. Parkville, Victoria 3052, Australia Received 27 August 1998. Accepted in revised form 10 May 1999
Key words: ectomycorrhizae, Eucalyptus regnans, forest burns, nitrogen, phosphorus
Abstract This study was conducted to compare the effects on the growth of Eucalyptus regnans seedlings of unheated soil and soil heated to different extents (as indicated by soil colour–bright red or black) in burnt logging coupes, and to separate the effects of heating of the soil on direct nutrient availability and on morphotypes and effectiveness of ectomycorrhizae. Burnt soils were collected from three logging coupes burnt 2, 14 and 25 months previously and unbumt soil from adjacent regrowth forests. Compared to unburnt soil, the early seedling growth was stimulated in black burnt soil from all coupes (burnt 2, 14 and 25 months previously). Seedling growth was generally poor in red burnt soil, especially in soil collected 2 months after burning. However, the concentration of extractable P was extremely high in red burnt soil, especially in soil collected 2 months after burning. In black burnt soil, extractable P was increased in soil 2 months after burning, but not in the soils collected 14 or 25 months after burning. However, both total P content and concentration in seedlings were increased in all collections of black burnt soil. Frequency of ectomycorrhizae was high in seedlings grown in all black burnt soils, but the mycorrhizal mantles were poorly developed in seedlings in black burnt soil collected 2 months after burning. Seedlings were also ectomycorrhizal in red burnt soil, except in soil collected 2 months after burning. Fine root inocula from seedlings grown in black burnt soils collected 14 and 25 months after burning significantly stimulated both seedling growth and P uptake compared with the uninoculated control, whereas the fine root inocula from the seedlings grown in all the other soils did not. These results suggest that, in black burnt soil, both direct nutritional changes and changes in the ectomycorrhizae may contribute to seedling growth promotion after regeneration burns. The generally poor seedling growth in red burnt soils is likely to have been due to N deficiency as the seedlings in these soils were yellow-green and the tissue concentrations of N were significantly lower than in other treatments. Abbreviations: GR – growth ratio Introduction Reliance on forest fire and the consequent enhancement of seedling growth in the ash-bed (the ‘ash-bed effect’) is fundamental to the ecology of E. regnans forests (Ashton and Attiwill, 1994). Without fire, these forests fail to regenerate adequately (Ashton and Willis, 1982). To ensure rapid seedling growth after clear felling, silvicultural practices commonly involve burning slash and litter on the surface of soil (Tomkins et
al., 1991). The quantity and quality of fuel is known to strongly influence the duration and the temperature of the burn and therefore chemical properties of the soil following burning (Humphreys and Craig, 1981; Tomkins et al., 1991). Soils become orange red when heated at 600◦C (Chambers and Attiwill, 1994). In red baked soils, which have generally high ash content (if formed during forest burns) and therefore high pH (Attiwill and Leeper, 1987), soil oxidation is predominant (King et al., 1993) and haematite is formed
274 (Chambers and Attiwill, 1994). Temperatures close to and above 600◦C are reached when heavy slash or logs are burnt on soil (Humphreys and Craig, 1981). Black burnt soils form under moderate intensity fires and have charcoal or ash present (King, 1993). The uneven distribution of fuel within the coupe results in a mosaic of differentially heated seedbeds with various amount of ash accumulation. The red burnt soil may be 1–2 cm deep above black burnt soil or 10–20 cm deep at sites of sustained high temperatures during the burning (due to high fuel loads). Black burnt soils are several cm deep above normal red-brown loam. Unburnt patches are few and recognised by intact litter. Following forest burns, changes occur in the pH, the concentrations of exchangeable cations and organic carbon, and the water content of the soil (Chambers and Attiwill, 1994; Tomkins et al., 1991). As noted by Ashton and Kelliher (1996a), in addition to the effects of ash and heating, the ash-bed effect may involve the effect of soil drying. Marked increases in soil pH after burning are due to the presence of ash, in which CaO and MgO are dominant oxides (Attiwill and Leeper, 1987). In Australia, the mobility and the availability of P is known to be restricted in forest soil (Brundrett et al., 1996). It has been shown that, in mature forest soil, which is commonly acidic, the first limiting nutrient is P, and P and N are the only limiting nutrients for E. regnans seedling growth (Ashton and Kelliher, 1996b). During forest fires, large amounts of nutrients, especially P (Chambers and Attiwill, 1994) and inorganic N (Weston and Attiwill, 1990), become available for seedlings. In addition to direct nutritional effects, ectomycorrhizae may indirectly influence nutrient availability for plants in soil (McLaughlin, 1996). It is known that ectomycorrhizae increase the surface area of roots for nutrient absorption (Bowen, 1973) and may also produce phosphatases, phytases and proteases, which enable the mycorrhizal roots to access organic forms of both P and N in soil (Abuzinadah and Read, 1986; Bartlett and Lewis, 1973; Dighton, 1983). Heating of soil may result in selection and promotion of mycorrhizae beneficial for plant growth (Warcup, 1981, 1983) as recovery times for different fungi may vary. Warcup (1991) found that after regeneration burns the roots of eucalypt seedlings were colonised predominantly by ascomycetes. In this study, two pot experiments were conducted to determine whether the stimulation of seedling growth in heated soils collected from burnt logging
coupes can be solely explained by the increase in concentration of readily available P in soil, whether the quality of mycorrhizal infection of the seedlings is different in heated soils than in unheated soil, and whether ectomycorrhizae in heated soils differ from those in unheated soils in their ability to contribute to the nutrient uptake and the growth promotion of seedlings. Ectomycorrhizal infection was assessed by counting the number of root tips ectomycorrhizal with each morphotype and by determining the concentration in the roots of seedlings of a fungal sterol, ergosterol, which is an indicator of viable fungal biomass (Nylund and Wallander, 1990). This method has been commonly used to quantify viable fungal biomass in ectomycorrhizal mantle and Hartig net (Antibus and Sinsabaugh, 1993; Ekblad et al., 1998; Salmanowicz and Nylund, 1988).
Materials and methods The source and preparation of soils, seedlings and root inoculum Soils were collected from three burnt logging coupes and adjacent regrowth forests (58-year-old stands, burnt in 1939) near Noojee, Victoria, Australia. The coupes had been burnt 2, 14 or 25 months prior to soil collection and they were located at altitudes of 850 m (37◦8 S, 145◦90 E), 500 m (37◦8 S, 146◦ 00 E) and 300 m (37◦9 S, 145◦90 E) above sea level, respectively. In each coupe, soil from the top 0–5 cm (depending on the depth of burnt soil) was collected and mixed from four or five randomly selected sites where heating had caused it to become either red (red burnt) or black (black burnt). Care was taken to avoid mixing underlying unheated soil with soil in which a colour change had been induced by heating. No attempt was made to separate ash from soil. For unburnt controls, the top 15 cm of soil was collected, sieved through 1 cm mesh and mixed from four or five sites in regrowth E. regnans forest adjacent to each burnt coupe. Soils were stored in plastic bags at 4◦ C for about 2 weeks before experiments were established. Pot experiments Experiment 1: Bioassay of soils The first pot experiment was carried out in a glasshouse under ambient conditions (April–August 1997; minimum temperature 2–14◦C, maximum temperature 24–42◦C, photosynthetically active radiation
275 2000–6000 µE/m2 /s). Eucalyptus regnans seedlings (3 per pot) were grown in black plastic pots (10 cm in diameter), each filled with burnt and unburnt soils from different coupes (10 replicates per treatment). The soils used were red burnt soil (R2, R14 and R25) and black burnt soil (B2, B14 and B25) from coupes burnt 2. 14 and 25 months previously, and unburnt soil (U2, U14 and U25) from regrowth forests adjacent to each coupe. Unfertilised sand/vermiculite 5:1 (vol:vol) mix (SV) was used to obtain a nutrient-poor control. The experiment was set out on the glasshouse bench in a completely randomised design. At harvest (after 15 weeks), the leaves, stems and roots of seedlings were separated and a subsample of roots from four replicate plants was frozen in liquid N2 and then freeze-dried for ergosterol analysis. Another root subsample was taken from each of the 10 replicate seedlings for mycorrhizal assessment with a light microscope. The weights of the remaining root tissues and all the leaves and stems were determined after drying at 80◦ C for 4–7 days. The dried material was analysed for P and N. Experiment 2: Inoculation experiment In the second experiment (August–December, 1997; at 22◦ C in a temperature-controlled glasshouse, photosynthetically active radiation 2600–6100 µE/m2 /s), all pots were lined with clean plastic bags and filled with soil/sand mix (two parts of unburnt forest soil with three parts of coarse sand (vol/vol), autoclaved twice with 3 days between autoclaving treatments to ensure that all fungi with heat-resistant spores were killed). To promote drainage, two holes were cut in the plastic bags. Inoculum consisting of 0.25 g (fresh weight) of surface-sterilised (2 min in a mixture of 1.5% NaHClO2 and 15% ethanol, rinsed in 4 changes of sterile distilled water) fine roots of 3-month-old E. regnans seedlings grown in pots of differentiallyheated field soils was placed on the surface of the mix and covered with approximately 5 mm depth of the soil/sand mix. Surface sterilised (5 min in 30% H2 O2 , rinsed in 4 changes of sterile distilled water) E. regnans seed was sown in the pots and the seedlings were later thinned out to two per pot, with eight replicate pots. The pots were watered with de-ionised water when necessary. Seedlings were harvested 5 months after sowing. At harvest, a random subsample of each root system was taken for mycorrhizal assessment with a light microscope and the remainder of the root material and the shoots were dried at 80◦C for 6
days to obtain dry weights, and the dried material was analysed for P. Mycorrhizal assessment The frequency of different ectomycorrhizal morphotypes in each root subsample was determined by examination of 100 fine root tips (or all root tips if the sample had less than 100 tips) under a dissecting and compound light microscope. Root tips were assigned to different morphotypes on the basis of the branching pattern and colour of the mycorrhizae, the diameter of hyphae and their cell wall thickness, the presence of clamp connections, and the size of the cells in the mantle. Mycorrhizal status was confirmed by the presence of a Hartig net. The number of fine root tips with each ectomycorrhizal type and the number of nonmycorrhizal root tips were recorded for each replicate root subsample. Determination of P in plant tissue Oven-dried leaf, stem and root material from two or three replicates was pooled and ground. Either whole samples or subsamples (usually 20–50 mg) were then digested in 1 ml of HNO3 /HClO4 (4:1, v/v) in glass tubes in a heated aluminium block. The temperature was increased over a period of 11.75 h to a maximum of 230◦C which was maintained for 30 min before cooling. After adjusting the volume of the digests to 5 ml with Milli-Q water, the concentration of P was determined by inductively coupled plasmaoptical emission spectrometry (ICP-OES, GBC Scientific Equipment, model Integra XM) at 177.495 nm, calibrated with a matrix-matched standard. The results were adjusted for the dry weight of the plant part and expressed either as µg g−1 of plant tissue or as total P content per plant part. Determination of total N Oven-dried shoots from two replicates were pooled and ground. Total nitrogen was determined by digesting the whole samples or subsamples of pooled material (usually 20–50 mg) with 5 ml H2 SO4 (98%) in the presence of a Kjeldahl catalyst tablet (Ajax Chemicals, Auburn, NSW) in calibrated Kjeldahl tubes. The tubes were heated for 2 h at 390◦C in a programmable heating block. After cooling, the total nitrogen content of each sample was measured by steam distillation using a Kjeltec 1035 nitrogen analyser (Tecator). The res-
276 ults were adjusted for the dry weight of the shoot and expressed as µg N mg−1 plant tissue. Determination of ergosterol in the roots Freeze-dried root samples (3–20 mg dwt) from four replicates in each treatment were ground in liquid N2 , extracted and ergosterol determined following the method described by Martin et al. (1990), with some modifications. The homogenates were centrifuged at 4◦ C for 5 min at 13 000 rpm using a bench centrifuge and the supernatants were filtered with 0.45 µm acetate filters. Ergosterol in the prepared samples was determined by high-performance liquid chromatography (HPLC) and UV detection. Aliquots (l0–100 µl) were passed through a reverse-phase, C18 colunm (4.6×250 mm, Hypersil, 5 µm) in a column heater at 37◦C. Methanol (100%) was delivered at 1 ml min−1 by a GBC pump (LC 1150). Samples were injected every 30 min by an autosampler (GBC, LC 1650). Ergosterol was detected by absorbance at 280 nm (GBC UV-VIS detector, LC 1210). The ergosterol in the samples was quantified using an ergosterol standard (Sigma Chemical Co., St. Louis, Missouri). Soil P, pH and water content Soil pH was measured using a glass electrode in 1/5 (vol/vol) suspension of soil and distilled water. The water content of soil was obtained by determining the percentage of water in an oven-dried (105◦C for 3 days) soil sample. Soil P was determined following the method of Mitchell (1964). Non-sieved soil samples (15 g fresh weight; ca. 10 g of oven dry weight) were digested by shaking for 16 h in 400 ml dilute acetic acid (2.5%) and the digests were then filtered (Whatman No. 1) into glass tubes (20 ml) for analysis using ICP as described above. The results were expressed on an oven dry weight basis.
transformed before analysis. If there was a significant interaction, each set of data (using soil from only one coupe and from adjacent forest) was analysed both with and without the additional control (SV) using one-way ANOVA. The means were separated by Fisher’s protected least significant difference (lsd).
Results Seedling growth and nutrient acquisition After 15 weeks, seedling growth was significantly (p≤0.0001) greater in all black burnt soils (B2, B14 and B25) and in R14 than in unburnt soils (Table 1). In all red burnt soils, in particular in R2, the leaves of the seedlings were pale yellow-green, whereas in all black burnt soils the leaves were fully green. In unburnt soils, the leaves were dark green with purple tips and margins. The growth in burnt soil as a proportion of that in unburnt soil collected at the same time was greatest in B14 (GR 9.8) and slightly less in B2 (GR 8.8). The GR was 4.1 in B25 and 4.5 in R14. The absolute increase in seedling dry weight was greatest in B2. The growth of the seedlings in R2 was less than in unburnt soil. The growth of the seedlings in all unburnt soils was poor and similar to that in SV. In black burnt soil, the root:shoot ratio (dry weight) was less than in unburnt soil (Table 1). The seedlings in all black burnt and red burnt soils (except for R2) had a higher concentration of P in leaf, stem and root tissues than those grown in unburnt soils (Table 1). The total seedling P content was 34, 21 and 6 times higher in B2, B14 and B25, respectively, than in unburnt soil from adjacent forest. The concentration of N in leaf tissue was significantly (p≤0.0001) lower in all red burnt soils than in black burnt and unburnt soils (Table 1). Extractable P, pH and water content of soils
Statistical analyses The growth ratio (GR: the ratio of mean dry weight of seedlings grown in burnt soil or in soil inoculated with roots from burnt soil to mean dry weight of seedlings in unburnt soil or in soil inoculated with roots from unburnt soil) was determined (Ashton and Kelliher, 1996a). Raw data were examined for homogeneity of variances and, when necessary, transformed before statistical analysis using two-way ANOVA (Zar, 1984). Data expressed as percentages were arcsin
The concentrations of extractable P in all red burnt soils (R2, R14 and R25) and in B2 were significantly higher (p≤0.0001) than in unburnt soil collected from adjacent forest at the same time (Table 1). Concentrations of P were similar in B14 and B25 and in unburnt soils (controls). The R2 soil had the highest pH (8.5) and the highest water content (55%). The water contents of all other soils were 25.0%–41.5%. The pH was 7.0–7.5 for R14 and R25, 6.4–7.4 for all black burnt soils, 5.l–5.9 for unburnt soils, and 6.3 for SV.
277 Table 1. Extractable P in differentially heated soils (R, red burnt; B, black burnt; U, unburnt), and the dry weight and concentration of P, N and ergosterol in tissue of E. regnans seedlings grown in red burnt, black burnt and unburnt soil collected from logging coupes (unburnt soil from adjacent regrowth forest). The soils were collected from logging coupes which had been burnt 2, 14 and 25 months previously. Control seedlings (SV) were grown in unfertilised sand/vermiculite mix. Seedlings were grown in pots in a glasshouse for 15 weeks before harvesting. Means are followed by s.e.m. in parentheses and n is indicated in a separate column. All concentrations are on a dry weight basis
R2
B2
U2
R14
Seedling dry weight (mg)
15.2 (0.8)
343.6 (29.4)
39.3 (7.6)
88.4 (4.3)
Dry weight root/shoot ratio
0.6 (0.03)
0.2 (0.02)
0.4 (0.04)
0.5 (0.09)
Soil treatment B14 U14
R25
B25
U25
20.0 (2.0)
29.2 (1.4)
115.0 (9.0)
0.2 (0.01)
0.5 (0.02)
0.6 (0.06)
195.8 (16.4)
SV
n
27.8 (1.4)
10.0 (1.2)
10
0.3 (0.02)
0.4 (0.03)
0.5 (0.03)
10
Extractable soil P (µg g−1 soil)
230.1 (2.4)
54.2 (2.3)
5.6 (1.6)
94.7 (6.3)
7.9 (4.1)
6.3 (3.2)
92.2 (5.8)
11.0 (4.9)
2.6 (1.6)
NA
3
Seedling P content (µg)
10.8 (0.9)
640.8 (37.3)
18.9 (1.5)
116.6 (2.7)
201.8 (6.7)
9.3 (1.0)
37.8 (2.7)
134.8 (6.0)
22.8 (1.9)
11.9 (1.1)
3
Concentration of P in leaf (mg g−1 )
0.46 (0.04)
1.71 (0.11)
0.60 (0.04)
1.42 (0.04)
1.03 (0.04)
0.36 (0.02)
1.48 (0.12)
1.12 (0.05)
0.51 (0.01)
0.48 (0.03)
5
in stem (mg g−1 )
0.50 (0.03)
1.59 (0.03)
0.51 (0.03)
1.48 (0.08)
0.93 (0.05)
0.41 (0.03)
1.18 (0.12)
0.98 (0.02)
0.47 (0.04)
0.58 (0.06)
3
in root (mg g−1 )
0.96 (0.20)
2.37 (0.24)
1.08 (0.16)
1.03 (0.06)
1.24 (0.05)
0.63 (0.06)
1.30 (0.06)
1.34 (0.05)
0.83 (0.06)
0.78 (0.10)
5
Concentration of N in leaf (mg g−1 )
12.80 (0.66)
17.80 (1.39)
28.80 (1.98)
8.40 (0.24)
19.20 (1.56)
15.20 (0.92)
12.20 (1.24)
23.60 (1.03)
19.00 (0.45)
7.60 (0.24)
5
Concentration of ergosterol in root (µg g−1 )
5.22 (0.73)
3.87 (0.55)
4.57 (0.75)
5.18 (0.43)
9.76 (0.88)
4.55 (0.65)
5.50 (0.75)
10.48 (1.56)
6.85 (1.16)
3.80 (0.18)
4
Root ergosterol concentration and mycorrhizal infection of seedlings The seedlings grown in B14 had a significantly (p≤0.0004) higher ergosterol concentration in the total roots than the seedlings in unburnt soil (Table 1). The root ergosterol concentration of the seedlings grown in B25 did not differ from that in unburnt soil from adjacent regrowth forest, but was significantly (p≤0.0085) higher than that of the seedlings in SV. At 15 weeks, the seedlings grown in all black burnt soils had a higher incidence of mycorrhizal root tips than the seedlings grown in red burnt and unburnt soils (Table 2). In R2 the seedlings were non-mycorrhizal. The dominant mycorrhizae present on the seedlings in black and red burnt soils differed morphologically from those in unburnt soil. The seedlings in B14
had no mycorrhizal morphotypes in common with the seedlings growing in control soil. However, when grown in B25, the seedlings developed some dark mycorrhizae (not dominant) similar to those on the seedlings in the controls. The dominant mycorrhiza of the seedlings in B2 was different from mycorrhizae in all other treatments. The mycorrhizal roots of these seedlings (in B2) were light in colour with a relatively large diameter (200–350 µm), and hyphae varied in diameter, lacked clamp connections and formed large cells in the mantle or on the root surface (Table 3). Root epidermal cells were not always elongated and the Hartig net was not always well developed. Sometimes root hairs were present and often roots were not fully covered by mantle. Seedlings grown in R14, B14 and B25 were dominated by a relatively thin (l00– 150 µm in diameter), light-coloured mycorrhiza with
278 Table 2. Incidence of ectomycorrhizal root tips in E. regnans seedlings grown in differentially heated soils (R, red burnt; B, black burnt; U, unburnt) collected from burnt logging coupes and adjacent forest 2, 14 and 25 months after regeneration burns. Unfertilised control seedlings were grown in sand/vermiculite mix (SV). Seedlings were grown in pots in the glasshouse for 15 weeks. Different morphotypes of ectomycorrhiza (for descriptions refer to Table 3) are indicated by T1 to T9. Means are followed by s.e.m. in parentheses (n=10)
Soil treatment
R2
B2
Mycorrhizal type T1
0
T2
0
T3
0
T4
0
21.7 (4.2) 0
T5
0
0
T6
0
0
T7
0
0
T8
0
0
29.4 (4.4) 17.0 (7.3) 20.6 (6.7) 0
T9
0
0
Other
0
Total
0
No. of types
0
59.0 (5.8) 0
U2
Frequency of root tips ectomycorrhizal (%) in: R14 B14 U14 R25
0
0
0
0
0
26.4 (7.2) 0
28.1 (3.6) 0
0
36.4 (34) 0
50.4 (4.2) 0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.2a
0
80.7 (3.4) 2
67.2 (6.4) 4
62.9 (6.6) 2
2.0 (0.7) 80.5 (1.8) 3
0 0
0
8.6 (3.3) 0 18.2 (3.9) 1.3 (0.8) 41.0 (6.9) 3
0 4.9 (2.5) 0
B25
U25
SV
0
0
0
0
0
0
0
5.0 (0.6) 0
0
0
0
46.9 (5.0) 0
0
0
0
0
0
0
14.1 (2.1) 18.8 (3.2) 24.5 (3.6) 0
19.1 (6.6) 0
35.1 (5.3) 0
0
0
27.4 (5.8) 0.8a
0
63.3 (5.1) 4
1.1a
62.3 (3.7) 3
6.9 (1.4) 77.9 (3.7) 6
0
1.1a
1
a Only one replicate infected
a well-developed Hartig net and thin, regular hyphae with no obvious clamp connections. Seedlings grown in unburnt soils or in R25 or B25 had a higher incidence of dark and pyramidal or clublike mycorrhizae than seedlings in other soils. Clamp connections were present in association with dark mycorrhizae on seedlings grown in two of the unburnt soils. The number of mycorrhizal morphotypes on seedlings was less in R2 and B2 than in U2, but increased in heated soils with increasing time after burning. Growth, nutrient acquisition and mycorrhizal infection of seedlings inoculated with mycorrhizal fine roots of seedlings grown in dfferentially heated soil Compared with mycorrhizal inocula from unburnt control soil, those from B14 and B25, but not from B2, stimulated seedling growth significantly (p≤0.03) (Table 4). The root inocula from B14 promoted seed-
ling growth more (GR 7.0) than the inocula from B25 (GR 5.0). All inocula from black burnt soils resulted in a higher incidence of mycorrhizal root tips than inocula from unburnt and red burnt soils (Table 4). When inoculated with mycorrhizal roots from B14 and B25, the seedlings grown in sterile sand/soil mix formed mycorrhizae with well-developed Hartig nets and these were dominant in the seedlings inoculated with mycorrhizal roots from B14. In all other treatments, including inoculation with mycorrhizal roots from B2, the seedlings were either poorly mycorrhizal or the dominant mycorrhizae had poorly developed Hartig nets. Inoculation with mycorrhizal roots from B14 and B25 enhanced P uptake by seedlings significantly compared with the uninoculated controls; the seedlings inoculated with roots from B25 had a significantly higher concentration of P in the leaves (p≤0.004) and roots (p≤0.008) than seedlings inocu-
279 Table 3. Selected characteristics of some mycorrhizal types (T1–T9) occurring in seedlings grown in differentially heated soils collected from burnt logging coupes Characteristic
Diameter of mycorrhiza (µm) 100–130 130–170 170–250 250–350 Colour Light Medium Dark Presence of mantle Tips covered Always Usually Rarely Hartig net Partially developed Fully developed Epidermal cells Elongated Not elongated Hyphae Irregular Regular Diameter of hyphae (µm) <1 1–2 2–5 5–9 Diameter of cells in mantle (µm) <4 4–6 6–10 External hyphae Clamp connections Growth habit Pyramidal Club-like Long, scarcely branched
Mycorrhizal type T5 T6
T1
T2
T3
T4
T7
T8
T9
− − + +
− + + −
+ − − −
+ − − −
− + + −
+ − − −
− + + +
− + + +
+ − − −
+ − − +
+ + − +
+ − − +
+ + − +
+ + − +
− + − +
− + + +
− + + +
+ + +
− + +
− + −
− + +
− + −
− + −
− + −
+ − −
+ − −
+ − −
+ +
− +
+ +
− +
− +
− +
− +
− +
− +
+ +
+ −
+ +
+ −
+ −
+ −
+ −
+ −
+ −
+ −
− +
− +
− +
+ −
− +
+ +
− +
− +
− + + +
+ + − −
− − + −
− + − −
− + + −
− + − −
− + + +
+ − − −
+ + − −
+ + + − −
+ − − − −
+ + − + −
+ − − − −
− + + + −
+ − − + −
+ + + − ±a
+ + − − −
+ − − ±a −
− − +
− − +
− − +
− − +
− − +
− − +
+ + −
− − +
− − +
a Not always present
lated with roots from unburnt soil (Table 4). Overall, the leaf and root P concentrations of inoculated seedlings were either greater than or equal to those of uninoculated control seedlings.
Discussion Phosphorus is the most limiting nutrient for growth of E. regnans seedlings in unburnt forest soil (Ashton and Kelliher, 1996b). After a forest burn, large quantities of nutrients become readily available for plants (Chambers and Attiwill, 1994; Polglase et al., 1986;
280 Table 4. Seedling dry weight, frequency of ectomycorrhizal root tips and phosphorus content and concentration in the leaves and roots of E. regnans seedlings grown in sterile unburnt soil/sand mix inoculated with 0.25 g (fresh weight) of fine roots from seedlings grown in differentially heated soils. All treatments were the same, except for the root inoculum, which was from R, red burnt; B, black burnt; or U, unburnt soil collected from burnt logging coupes and from adjacent unburnt forest 2, 14 and 25 months after burning. Control seedlings (C) were left uninoculated. Seedlings were grown in pots in the glasshouse and harvested 5 months after sowing seeds. Means followed by s.e.m. (in parentheses), n is indicated in a separate column
Seedling dry weight (mg) Seedling P content (µg) P concentration in leaf (mg g−1 ) P concentration in root (mg g−1 ) Frequency of ectomycorrhizal root tips (%)
R2
B2
U2
R14
34.4 (4.2) 15.4 1.9 0.46 (0.06) 0.38 (0.03) 4.7 (3.1)
153.8 (40.3) 86.8 22.8 0.55 (0.04) 0.61 (0.05) 59.9 (11.9)
97.2 (42.5) 60.7 26.4 0.60 (0.05) 0.70 (0.04) 32.0 (10.6)
110.3 (48.2) 65.1 28.1 0.59 (0.06) 0.59 (0.08) 23.8 (12.3)
Source of root inoculum B14 U14 R25 502.1 (68.8) 303.1 41.5 0.58 (0.07) 0.69 (0.03) 74.0 (6.2)
72.2 (25.6) 39.1 13.6 0.51 (0.06) 0.66 (0.07) 15.8 (9.8)
87.7 (39.4) 46.5 20.8 0.52 (0.03) 0.55 (0.07) 12.0 (10.7)
B25
U25
253.5 (67.1) 185.4 49.2 0.69 (0.08) 0.88 (0.05) 72.3 (6.5)
50.1 (13.5) 22.8 6.1 0.44 (0.01) 0.50 (0.07) 14.8 (10.2)
C 47.8 (5.6) 28.0 1.4 0.44 (0.02) 0.47 (0.04) 6.4a
n 8 4 4 4 8
a Only one replicate infected
Weston and Attiwill, 1990). In the present study, the remarkably high concentration of chemically extractable P in red burnt soil, even 25 months after burning, highlights the role of red burnt soil as a potential source of P for seedlings that are able to access this seedbed in burnt coupes. In the field, we have observed that the E. regnans seedlings on black burnt soil adjacent to deep deposits of red burnt soil outgrew (both in height and stem diameter) their neighbours during the second and third year after burning. The concentration of extractable P in black burnt soil, which was initially high, had returned to the same level as in unburnt soil after 14 months, indicating that it may have been used up by plants and microorganisms. Chambers and Attiwill (1994) suggested that the uptake of nutrients by rapidly regenerating seedlings, together with its immobilisation by the microorganisms recolonising the soil, may help conserve the nutrients mobilised by the burn. Although the results of our study are limited in that only one coupe for each time period after burning was sampled, they agree with the trends reported elsewhere. The improvement in soil nutrient status has been shown to last for a variable period following forest burns, on some occasions for over 4 years (Adams and Attiwill, 1991; Adams et al., 1989; Ellis et al., 1982; Polglase et al., 1986; Tomkins et al., 1991; Weston and Attiwill, 1990). Chambers and Attiwill (1994) investigated changes in the availability of nutrients for a 5-month period
following different sterilisation treatments of mature E. regnans forest soil and concluded that the increase in the availability of N and P is the main reason for the seedling growth stimulation after burning in E. regnans forest. The results of our study are consistent with this conclusion in that the enhanced P acquisition and growth stimulation of the seedlings in black burnt soil collected 2 months after burning seems to be primarily due to an increase in the availability of soil nutrients. However, 14 and 25 months after burning, the concentration of chemically extractable P in black burnt soil was relatively low (the same as in unburnt control soils), but the P absorption and the seedling growth were markedly promoted in these soils compared with unburnt controls. Although limited by the fact that only one extraction method was used to determine available P in soil, our results suggest that the level of chemically extractable P may not necessarily correlate well with biological availability in the heated soils. Indeed, some eucalypts have been shown to be able to extract nutrients from insoluble sources (Ashton, 1976; Mullette et al., 1974). Ashton and Kelliher (1996b) suggested that the ability of a plant to extract P (as indicated by the P content of the plant) is a better measure of availability of P for plants than chemical extractability of P from soil. In red burnt soils, which had high concentrations of extractable P, the inhibition of seedling growth was apparently due to the low N availability – the
281 concentration and content of N in the foliage were significantly lower in seedlings grown in red burnt soil than in black burnt and unburnt soils and the uniform yellow green colour of the foliage also suggested N deficiency. As N is known to be volatilised at temperatures above 100◦C (Attiwill and Leeper, 1987), some N was probably lost when the soil was heated during the regeneration burn. Weston and Attiwill (1990) reported marked increases in the concentration of both − soil NH+ 4 and NO3 after forest burns, but the concentrations of both forms of inorganic N decreased to the concentration found in unburnt forest soil within 16 months after burning. In this study, the dominant ectomycorrhizae. as indicated by morphology, formed on seedlings in unburnt soils differed from those in heated soils, and this difference was sustained for up to 25 months. Although the seedlings in all black burnt soils were highly ectomycorrhizal, the poorly-developed mantles and Hartig net of the dominant morphotype in soil burnt 2 months previously may have been a result of prolific root growth in this soil, in which extractable P was also increased. Thus, the mycorrhizal contribution may have been less important for seedling growth in this soil. The significant growth promotion and the enhanced P acquisition of seedlings in black soils burnt 14 and 25 months previously could be largely due to increased mycorrhizal uptake rather than direct uptake of P, since chemically extractable P in these soils was as low as in unburnt soils. The apparent inability of the ectomycorrhizal seedlings in unburnt soils to take up P in sufficient amounts to overcome P deficiency (as indicated also by purple colouration of the foliage and low tissue concentration of P) and growth inhibition may be partly due to the quality of the mycorrhizae formed on seedlings in this soil. The non-mycorrhizal status of the seedlings in R2 may indicate a delay in recolonisation of soil sterilised during the burn. Warcup (1991) found that seedlings growing in deep deposits of ash in burnt coupes were non-mycorrhizal. However, as one morphologically homogenous ectomycorrhiza may contain one or more species of fungus (Beenken et al., 1998), further work is required to confirm that the different morphotypes consist of different species of fungi. In the present study, an ergosterol assay was used to determine differences in the concentration of living fungi in the roots from seedlings grown in different soils and an inoculation study was used to confirm differences between fine root inocula from different soils in their effect on seedling growth.
The results of the ergosterol assay provide evidence that, although the seedlings in black burnt soils grew extremely well and the mycorrhizal mantles were generally thin, the relative amount of living fungal tissue in the roots of the seedlings grown in black soils burnt 14 and 25 months previously was greater than that in the sand/vermiculite control. In these two burnt treatments, the incidence of ectomycorrhizal root tips was relatively high (ca. 80% of root tips infected), the mantles were complete (although thin) and Hartig nets penetrated into the epidermal layer. We have observed generally thin mantles in mycorrhizae of E. regnans, and the Hartig net penetrates only into the epidermal cell layer. Although the incidence of ectomycorrhizal root tips was high in black soil burnt 2 months previously, the mantles were patchy and the Hartig net poorly developed, and this may have contributed to the low ergosterol concentration (no different from the control). The concentrations of ergosterol were considerably lower than reported previously (usually ca. 1 mg g−1 ) for mycorrhizal root tips (Antibus and Sinsabaugh, 1993; Bermingham et al., 1995; Martin et al., 1990; Ekblad et al., 1998), although Ekblad et al. (1998) measured concentrations as low as 0.06 mg g−1 in synthesized Pinus mycorrhiza. The low concentrations in our study are likely to be a result of dilution of fungal tissue by root tissue. Whole roots, instead of just mycorrhizal fine root tips, were used in the present assay, and the presence of older root material could have contributed to the relatively low ergosterol concentrations measured. The results of the inoculation experiment provide further evidence that the rapid seedling growth in black soils burnt 14 and 25 months previously is primarily due to the beneficial mycorrhizae formed on these seedlings. Inoculation of seedlings (in sterile soil/sand mix) with the mycorrhizal roots from seedlings grown in black soils burnt 14 and 25 months previously stimulated seedling growth and significantly enhanced P acquisition of the seedlings, compared with uninoculated controls. Overall, the results of the present study show that the enhancement of seedling growth in seedbeds following slash burning may be a result of both an improvement in the availability of inorganic soil nutrients and changes in the mycorrhizal communities that are important in P uptake by the seedlings. Seedbed conditions following fire are likely to vary greatly due to variation in the duration and temperature of the burn at particular sites. The red and black burnt soils form a mosaic and the extensive lateral roots of
282 E. regnans seedlings can eventually exploit pockets of increased soil P and N. In the second growing season, E. regnans lateral roots extend for 20–40 cm (Ashton 1975). This explains the observed initial poor growth in deep deposits of red burnt soil in the first 1–2 years after burning, and the much improved growth at these sites during the second year. Initially seedlings with their roots entirely within the red burnt soil are limited by low N availability, but once their roots access black burnt soil adjacent to or below the red burnt soil this deficiency is overcome. Although seedling growth is initially vigorous in black burnt soil, the greatest growth occurs on the margin of red and black burnt soils where older seedlings have access to both N and P. In the case reported here, the seedlings in black burnt soil from a recently burnt coupe seemed to benefit primarily from the flush of available nutrients whereas the growth promotion of the seedlings in black burnt soil collected 14 and 25 months after burning was mainly due to development of a beneficial mycorrhizal community on the seedlings. The results of this case study suggest that the stimulation of the growth of E. regnans seedlings in ash-beds following fire is likely to involve both direct nutritional and indirect mycorrhizal effects, with the nutritional effects being more prominent directly after fire and the mycorrhizal effects becoming important in the following years.
Acknowledgements We wish to thank Mr L B Edwards, Department of Agriculture, La Trobe University for assistance with P analysis; Mr P J McMahon, Department of Botany, La Trobe University for assistance with ergosterol analysis; Mr M J Bartley, Department of Botany, La Trobe University for assistance with P and analysis; Dr N C Uren, Department of Agriculture, La Trobe University for assistance with P analysis and valuable comments on the manuscript; Mr P Dignan, Centre for Forest Tree Technology, Department of Natural Resources and Environrnent for providing E. regnans seed and Dr M J Keough, Zoology Department, Melbourne University for advice on statistical analysis. The improvements on the manuscript suggested by the two anonymous referees were most helpful. The senior author was supported by an Australian Postgraduate Award.
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