The effect of inoculation with either cultured Frankia or crushed nodules on nodulation and growth of Alnus rubra and Alnus glutinosa seedlings in forest nurseries

Forest Ecology and Management, 43 ( 1991 ) 153-166 Elsevier Science Publishers B.V., Amsterdam

153

The effect of inoculation with either cultured Frankia or crushed nodules on nodulation and growth ofAlnus rubra and Alnus glutinosa seedlings in forest nurseries C.T. Wheele#, M.K. Hollingsworth b, J.E. Hooke#, J.D. McNeill b, W.L. Mason b, A.J. Moffat c and L.J. Sheppard d aDepartment of Botany, The University of Glasgow, Glasgow G12 8QQ, UK bForestry Commission, Northern Research Station, Roslin EH25 9SY, UK CForestry Commission, Alice Holt Lodge, Farnham GUIO 4LH, UK dlnstitute of Terrestrial Ecology, Bush Estate, Penicuik EH26 OQB, UK (Accepted 3 September 1990 )

ABSTRACT Wheeler, C.T., Hollingsworth, M.K., Hooker, J.E., McNeill, J.D., Mason, W.L., Moffat, A.J. and Sheppard, L.J., 1991. The effect of inoculation with either cultured Frankia or crushed nodules on nodulation and growth ofAlnus rubra and Alnus glutinosa seedlings in forest nurseries. For. EcoL Manage., 43:153-166. The growth of seedlings of Alnus rubra and Alnus glutinosa in chemically sterilised seed beds in forest nurseries in Scotland and southern England was enhanced considerably after inoculation with Frankia in comparison with uninoculated seedlings receiving PK and, in most experiments, NPK fertiliser. Alnus rubra seedlings inoculated with an elite Frankia strain, UGL Ar 1.2.5q, isolated from nodules from a Scottish plantation of A. rubra and characterised previously in climate chamber experiments, showed greater growth than seedlings inoculated either with crushed A. rubra nodules from the same plantation or with Ar 14, a North American Frankia strain. After inoculationwith Ar 1.2.5q, seedlings showed faster growth, were more robust and had a higher N con[ent than plants from seed beds receiving standard nursery fertilisers ( basal NPK or basal NPK + NK top-dressing). Inoculation ofA. glutinosa seed beds with Frankia UGL Ag 1.1.8 also stimulated greater growth of seedlings than fertiliser treatments. With this species, pre-germination application of inoculum promoted seedling growth more than application post-germination. For both species, foliar nutrient analysis showed that concentrations of phosphorus and calcium were influenced strongly by seedling inoculation and were inversely correlated with growth and foliar nitrogen concentrations. The results emphasise the importance of inoculation for the production of robust, rapidly growing seedlings in nurseries and illustrate the advantages of inoculation with selected strains of Frankia.

INTRODUCTION

Inoculation o f nursery beds either with soil on which alders have grown or

with nodules collected from trees is a practice which usually ensures good 0378-1127/91/$03.50 © 1991 Elsevier Science Publishers B.V. All rights reserved.

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C.T. WHEELER ET AL.

nodulation with the nitrogen-fixing actinomycete Frankia. This input of fixed nitrogen can improve seedling growth, especially on nitrogen-deficient soils (Wheeler and Miller, 1990). The opportunity to enhance symbiotic fixation of nitrogen in actinorhizal plants by endophyte selection has arisen relatively recently, with the development of techniques for the isolation of Frankia and for its routine culture in defined media (Callaham et al., 1978 ). Tests carried out under controlled conditions have shown wide variability in the effectiveness of Franlda strains for nodulation and nitrogen fixation in plants such as alders (Normand and Lalonde, 1982; Stowers and Smith, 1985 ). Hooker and Wheeler (1987) showed that Frankia strains, isolated from nodules of the North American red alder (Alnus rubra Bong. ) growing in Scotland, varied both in the mass of nodules induced on the host plant and in the specific nitrogen-fixing activity of the nodules (nitrogen fixed per unit mass of nodules). The most effective strains gave rise to a good nodule mass of high specific activity in nitrogen fixation. Some isolates were more effective in symbiotic nitrogen fixation than strains from the normal geographic range ofA. rubra. In addition, evidence was obtained for the multiple occupancy of nodules by Frankia strains that differed widely in symbiotic effectiveness. Inoculation of plants with crushed nodule preparations produced nodules that were less effective in nitrogen fixation than those formed after inoculation with some of the cultured Frankia strains, isolated from the same source of nodules; strain infectivity does not necessarily parallel effectivity in nitrogen fixation. Cultured Frankia has been used on a large scale for the inoculation of greenhouse-grown, containerised alders in North America (Berry and Torrey, 1985; Perinet et al., 1985 ). A positive response during subsequent growth in the field of Casuarina seedlings that were inoculated while containerised with alginate-immobilised Frankia has been noted (Sougoufara et al., 1989) but the effect of inoculation with superior strains of Frankia on alder seedling growth in open ground nursery beds has not been evaluated. Two sets of experiments to investigate this are described. The first, at the Forestry Commission Northern Research Station nursery, near Edinburgh, compared, under standard nursery conditions, the effect on red alder seedling growth and nitrogen accretion of the inoculation of sterilised seed beds either with selected strains of cultured Frankia, isolated from A. rubra nodules, or with crushed nodule preparations. Nodules from the same location near Glasgow from which one of the Frankia strains originated were crushed to prepare an inoculum directly. The other strain was from North America. Further comparisons of inoculated plants with seedlings which had received fertiliser nitrogen were also included, to assess the relative benefits for seedling growth in the nursery of inoculation and fertiliser treatment. A similar experiment was carried out at the Forestry Commission Headley nursery in East Hampshire, which examined the effect on the growth, nodu-

FRANKIA INOCULATION OF ALNUS SEEDLINGS

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lation, nitrogen content and the content of other nutrients of A. rubra and Alnus glutinosa, inoculated with cultured Frankia under standard nursery conditions. The timing of inoculation was also studied, a comparison being made between inoculation before germination soon after seed sowing and when seedlings had reached the two-leaf growth stage. MATERIALS AND METHODS

The origin and culture of Frankia The North American strain DDB 01310210 (synonym Ar I4 ), isolated from nodules ofA. rubra, was kindly supplied by Dr. Dwight Baker, Yale University, USA. Isolation ofFrankia U G L 013103 (synonym Ar 1.2.5q ) from nodules of A. rubra from a plantation at Lennox Forest, near Glasgow, and of Frankia U G L 010708 (synonym Ag 1.1.8 ) from nodules ofA. glutinosa from a coppice at Conic Hill, Balmaha, Loch Lomond, has been described previously (Hooker and Wheeler, 1987). Strains were grown for 3 months at 25-28 °C in static liquid culture in propionate m e d i u m (Malcolm et al., 1985) containing casamino acids (1.0 g l - t ) , as the N source and supplemented with Tween 80 (0.5 g 1-1 ).

Inoculum preparation Crushed nodule inocula were prepared from nodules harvested from A.

rubra growing at Lennox Forest. Nodulated roots were harvested on the day of use or were kept at 4°C after harvest the previous day. Before use, the nodules were washed thoroughly with tap-water. The young nodule lobes were separated and homogenised in water in an 'Omnimix' homogeniser at threequarter speed for 20 s. The homogenate was filtered through muslin, suspended in water and applied to the seed bed with a clean, methanol-sterilised watering can at the rate of 4.5 g m -2 fresh weight equivalent of nodules in 2-3 1 of water. Cultured Frankia inoculum was prepared by filtering and washing the mycelium with water, before homogenisation as described above. The homogenate was applied to the seed beds with a washed watering can, as above, at a rate of 40 ml gravity-sedimented mycelium volume m -2, dispersed in 2-3 1 of water.

Source of seed and seed bed preparation Experiments at Bush Experiments 1-3 were carried out at the Forestry Commission nurseries, Bush Estate, Roslin. The soil at Bush is a sandy/clay loam, pH 4.8--5.0, or-

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ganic matter content 6%. Seed o f A l n u s rubra Bong. was collected from a stand at Lennox Forest that originated from a Vancouver, British Columbia, seed provenance and was stored at 4 °C until required. Seed beds were sterilised chemically in the a u t u m n with dazomet (tetrahydro-3,5-dimethyl- 1,3,5-thiadiazine-2-thione ) applied as 'Basamid' at 380 kg h a - ~. Beds were prepared and hand sown in April/early May (see below ) and were gritted immediately after sowing. Two alternative basal fertiliser regimes were incorporated before sowing. These were: ( 1 ) N P K - a slow-release N P K Mg fertiliser (5.5% N; 20% P205; 10.5% K20; 8.5% Mg: 'Enmag', Scottish Agricultural Industries (SAI) Fertilisers Ltd. ) incorporated at 1300 kg product h a - ~; (2) PK - a combination of potassic superphosphate (20% P205, 20% K20: SAI Fertiliser Ltd. ) and granular superphosphate (21% P205: SAI Fertilisers Ltd. ) incorporated at 630 kg product ha-~ and 600 kg product ha-~ respectively. These regimes followed standard prescriptions for the nursery based on routine soil analysis (Aldhous, 1972 ). The detailed arrangement of the experimental plots, sowing and inoculation dates were as follows. E x p e r i m e n t 1 - 1985. Unreplicated plots were 3 m × 1 m and were separated

by a 1 m unplanted buffer, with a physical barrier of 30 cm depth, placed in the middle of each buffer to reduce seepage. N P K fertiliser was applied as a basal dressing before sowing one of the non-inoculated control plots. A second non-inoculated plot and three other plots, which were inoculated with crushed nodules, F r a n k i a Ar I4, or Ar 1.2.5q respectively, received a basal dressing of PK fertiliser before sowing. Seed was sown on 22 April ( 800 gerruinable seed m -2) and plots were inoculated with F r a n k i a immediately after sowing. Cold, dry weather delayed germination and the beds were re-inoculated 6 weeks later. None of these plots was top-dressed with fertiliser. Plants were harvested for analysis on 26 October. E x p e r i m e n t 2 - 1986. Five unreplicated plots ( 12 m × 1 m) were located in

different parts of the nursery to avoid cross-contamination. N P K fertiliser was applied to one of the non-inoculated plots before sowing. A second noninoculated plot received in addition a top dressing of NK fertiliser (25% N; 16% K20; 'Kaynitro', ICI Fertilisers Ltd. ) at 150 kg product h a - 1 l, 2 and 3 months after inoculation. Three further plots were inoculated with the Frankia sources noted above. These plots received a basal dressing of PK fertiliser before sowing. Seed was sown on 2 May ( 800 germinable seed m - 2 ) and plants were inoculated on 6 June, when the seedlings had produced one or two true leaves, and plants were harvested for analysis on 25 September. E x p e r i m e n t 3 - 1987. Five treatments were replicated four times in 2 m × 1

FRANKIA INOCULATION OF ALNUS SEEDLINGS

15 7

m plots in a randomised block design with a 2.0 m buffer between plots. Inoculated plots were further isolated using lawn edging of 30 cm depth. The experiment compared inoculation with Ar 1.2.5q, which consistently was the most effective treatment in the previous two experiments, with a range of nursery fertiliser regimes. Treatments were: N P K basal dressing; N P K basal followed by top dressing with N K (product and rates as in Experiment 2) in late June, late July and late August; PK basal dressing; PK basal dressing with a top dressing of NK, applied as in the N P K treatment; PK basal dressing and inoculation with Ar 1.2.5q. Seed was sown on 22 April (500 germinable seed m -2) and seedlings were inoculated on 26 June at the first/second true leaf stage. Plants were harvested for analysis on 26 October. Experiments at Headley The soil at the Forestry Commission nurseries at Headley, Surrey, is a loamy sand o f p H 5.2-5.5 and organic matter content 2.0-2.2%. Experiment 4 - 1988. This experiment was sited on soil sterilised with methyl bromide (300 kg product ha -l ) on 28 March 1988. It examined the effect and time of inoculation factorially with three fertiliser regimes: (a) 0-20-20 PK fertiliser (Sheppy 207 ) at 875 kg h a - ~before sowing; (b) as (a) but with an additional 200 k g h a -x ICI Nitrochalk (25% N); (c) as (b) but with 100 kg ha-~ ICI 'Kaynitro' as a top dressing, applied monthly from June to August. The A. glutinosa seed was collected from the Marches, UK, in 1986; the A. rubra seed was from Washington, USA (Ident 87 ( 7 9 7 ) ) . Seed was sown at approximately 600 germinable seed m - 2 o n 9 May 1988 onto treatment plots of 1 m E, separated by unplanted buffers of I m 2 and further isolated by wooden boards sunk 40 cm into the soil between plots. Each treatment plot was replicated in three blocks. Inoculation of plots before seed germination was on 23 May, and of plots at the two-leaf stage on 28 June. Harvest analysis

At Bush, assessment of numbers of seedlings per plot and plant height was carried out using five grids l 0 cm X 100 cm wide systematically located on a central 1 m E plot. All plants within the grid were counted and every tenth tree was measured for height. In 1987, five seedlings per plot were harvested at random from the central grid for leaf and stem nutrient analysis. Analysis of nodule numbe~, plant dry weight and nitrogen content was carried out on plants harvested at random from the centre of the beds in the numbers indicated in the tables. Plants were carefully dug from the plots before significant leaf fall and the roots were worked free of soil, taking care to retain detached fragments. Nodules were counted and detached and the plants and nodules were dried at 80°C to constant weight. Assay of total nitrogen content of the

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ground material was by a semi-micro Kjeldahl procedure (Hooker and Wheeler, 1987). At Headley, approximately 50 seedlings were excavated from each assessment plot in September 1988, using random number tables to select the site of excavation within the plot. Tree roots were carefully removed and remaining soil sieved to retrieve broken root fragments. The roots were washed free of soil, stem heights were measured and nodule sites counted. Shoots were detached from roots and each was dried at 80°C to constant weight. Concentrations of N, P, K, Mg and Ca were determined for both leaf and root samples using standard laboratory procedures. RESULTS

In all experiments, plants which were not inoculated showed low levels of nodulation at harvest. These nodules normally had a peripheral distribution on the root system, whereas inoculated seedlings showed heavy nodulation at the root crown. This difference in nodulation patterns suggests that contamination of uninoculated nursery seedlings probably occurred late in the growing season, as a result of the lateral spread of indigenous Frankia into the seed beds from adjacent areas. In 1985 (Table 1a), the average dry weight of inoculated plants was almost three times greater than that of the uninoculated plants that had received either PK or N P K basal fertiliser. Although the weights of inoculated seedlings were rather variable at harvest, the higher nitrogen content and high plant dry weight of plants inoculated with Ar 1.2.5q suggested that this was the most effective inoculum. Further, the dry weight and nitrogen contents of Ar 1.2.Sqinoculated plants were about 20% greater than for plants inoculated with crushed A. rubra nodules, collected from the same area as the nodules from which this Frankia strain was isolated. In 1986, the height and nitrogen content at harvest of plants inoculated with Ar 1.2.5q were about three and 1.5 times, respectively, the values for uninoculated plants receiving basal N P K (Table lb). The height of plants inoculated with Ar 1.2.5q was also greater than for plants that had received both basal N P K fertiliser and N top dressing. Although the dry weight of Ar 1.2.5q-inoculated plants was not significantly greater than that of fertilisertreated plants, in general the latter were of rather weaker growth than the former plants. In this experiment, both the height and dry weight of plants inoculated with crushed nodules (LCR) or with Ar I4 were inferior to those of fertilised plants, even though the total nitrogen content of the inoculated plants was higher. In both experiments, the percentage of useable seedlings in the autumn after sowing was high, with 96% ofA. rubra inoculated with Ar 1.2.5q being useable (height over 15 cm) in 1986 compared with 80% receiving N P K fertil-

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TABLE 1 The effect of inoculation with different Frankia strains or of fertiliser treatments on the growth of Alnus rubra (a) 1985 harvest; n=20 (standard error)

PK NPK LRC ArI4 Arl.2.5q

Total plant dry wt. (g)

Nodule dry wt. (g)

No. of N (mg g- ~) infection sites

N (mg plant-~ )

1.0 (0.08) 1.0 (0.07) 2.7 (0.19) 2.8 (0.34) 3.2(0.21)

0.016 0.007 0.18 0.11 0.19

2.0 (0.4) 1.0 (0.2) 69.0 (5.7) 21.0 (2.6) 11.0(1.3)

12.9 10.5 54.3 60.7 75.3

(0.003) (0.002) (0.015) (0.015) (0.025)

12.9 10.5 20.1 21.7 23.5

(b) 1986 harvest; n = 15 or 3 for nitrogen analyses (standard error)

NPK NPK+TD LCR Arl4 Arl.2.5q

Total plant dry wt. (g)

Nodule dry wt. (g)

No. of N (mg g- ~) infection sites

N (mg plant- t )

Plant height (cm)

1.9 (0.14) 1.9 (0.14) 1.3 (0.08) 1.4 (0.14) 2.0(0.15)

0.007 0.005 0.16 0.09 0.10

2.2 (0.66) 2.4 (0.82) 47.7 (5.51) 9.8 (1.53) 13.9(0.09)

17.1 26.8 25.0 33.7 51.8

19.2 (0.47) 25.8 (0.72) 14.7 (1.44) 20.4 (0.97) 30.2(0.79)

(0.003) (0.002) (0.02) (0.01) (0.011)

9.0 (0.16) 14.2 (0.53) 19.2 (0.96) 24.1 (1.0) 25.9(0.28)

PK and NPK treatments involved basal fertiliser application to seed beds before sowing. Treatments' + TD' received additional top dressings with NK fertiliser. Plants were inoculated as shown with LCR (Lennox A. rubra crushed nodules) or cultured Frankia ArI4 or Ar 1.2.5q. Nitrogen analyses in 1985 were for five samples from 20 bulked and ground plants. In 1986, 15 plants were bulked into three groups of five for separate analysis. TABLE 2 The effect of inoculation with Frankia Ar 1.2.5q or of fertiliser treatments on the growth ofAlnus rubra

PK NPK PK+TD NPK+TD Ar 1.2.5q

Total plant drywt. (g)

Nodule dry wt. (g)

No. of N (mg g- ~) infection sites

2.97 (0.15) 3.08(0.2) 3.4 (0.18) 3.37 (0.21) 4.1 (0.20)

0.14 (0.001) 5.8 (0.49) 0.12(0.10) 6.6(1.17) 0.06 (0.008) 3.1 (0.43) 0.03 (0.007) 1.1 (0.21) 0.16 (0.02) 41.4 (4.0)

17.3 (0.33) 17.4(0.32) 15.7 (0.28) 13.5 (0.18) 20.9 (0.16)

N (mgplant -~)

Plant RCD height (cm) (mm)

51.4 53.4 53.3 45.5 86.5

24.4 (0.61) 29.4(1.14) 35.3 (0.87) 41.8 (1.70) 40.3 (0.69)

6.60 6.72 6.40 6.40 7.26

Measurements of dry weight, infection and height were for 60 plants in each treatment. These plants were bulked into 15 groups of four for separate analysis for N content. Root collar diameter (RCD) was measured on a further five plants from each plot. Results were significantly different at P<0.1 and at P<0.05 for RCD.

i s e r + N K top dressings. These experiments showed that growth of A. rubra seedlings inoculated with Ar 1.2.5q is likely to be superior not only to that of plants inoculated with many other Frankia strains or with crushed nodules, but also to that of seedlings receiving fertiliser treatment. In 1987, where the growth of fertiliser-treated seedlings was compared specifically with that of plants inoculated with Ar 1.2.5q in a fully replicated

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experiment (Table 2 ), chance infection of non-inoculated plants was greater in plots receiving PK or N P K basal dressing than was experienced previously. The nodules that formed in such treatments were large, fewer in number than on the inoculated plants and had a peripheral distribution on the roots. It is probable that they formed later in the growth of the seedlings but their formation may have reduced the differences in plant growth between treatTABLE 3 Effect of inoculation with Frankia Ar 1.2.5q (Alnus rubra) or with Frankia Ag 1.1.8 (Alnus glutinosa) on plant growth Fertiliser treatment

No inoculation

Inoculation before germination

Inoculation at two-leaf stage

PK NPK NPK+TD PK NPK NPK+TD PK NPK NPK+TD PK NPK NPK+TD PK NPK NPK+TD

2.14 2.27 2.30 0.0 0.1 0.0 0.01 0.01 0.01 0.03 0.04 0.03 0.32 0.47 0.42

16.66 19.36 13.11 2.1 2.0 0.8 0.28 0.35 0.33 0.09 0.11 0.12 3.31 3.15 2.90

7.06 6.15 5.31 1.5 0.7 0.4 0.10 0.07 0.11 0.05 0.05 0.08 2.30 1.48 1.35

PK NPK NPK+TD PK NPK NPK+TD PK NPK NPK+TD PK NPK NPK+TD PK NPK NPK+TD

1.87 1.82 2.00 0.0 0.0 0.0 0.02 0.01 0.01 0.10 0.06 0.06 0.18 0.19 0.21

13.70 17.56 16.30 3.1 4.4 3.3 0.18 0.29 0.33 0.08 0.11 0.18 2.27 2.63 1.90

5.31 6.39 5.44 1.9 2. I !.8 0.06 0.17 0.08 0.05 0.12 0.09 1.28 1.39 1.04

A. rubra Mean height (cm) (17.4) Mean number of nodule sites (0.09) Mean shoot weight (g) (0.003) Mean root weight (g) (0.001) Shoot/root ratio (0.32) A. glutinosa

Mean height (cm) (25.8) Mean number of nodule sites (0.53) Mean shoot weight (g) (0.002) Mean root weight (g) (0.001) Shoot/root ratio (0.08)

Seed beds were inoculated either with Frankia Ar 1.2.5q (Alnus rubra) or with Frankia Ag 1.1.8 (Alnus glutinosa), dispersed in water in a watering can. Measurements were made on approximately 50 plants in each treatment. Figures in parenthesis are residual mean squares from the analysis of variance.

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TABLE 4

The effect of time of inoculation on foliar concentrations of nutrients (% oven dry weight) of Alnus rubra and A. glutinosa from Experiment 4

A. rubra Foliar N (0.157)

Foliar P (0.008) Foliar K (0.071 ) Foliar Mg (0.001) Foliar Ca (0.010)

No inoculation

Inoculation before germination

Inoculation at two-leaf stage

PK NPK NPK+TD PK NPK NPK+TD PK NPK NPK+TD PK NPK NPK+TD NP NPK NPK+TD

1.20 1.10 1.11 0.49 0.68 0.69 0.89 1.19 1.32 0.26 0.25 0.24 1.10 1.10 1.02

2.73 3.16 3.53 0.21 0.25 0.21 1.09 1.22 1.50 0.24 0.25 0.24 0.66 0.71 0.65

2.50 3.15 3.26 0.25 0.27 0.30 1.15 1.06 1.54 0.22 0.18 0.21 0.65 0.50 0.55

PK NPK NPK+TD PK NPK NPK+TD PK NPK N P K + TD PK NPK NPK+TD PK NPK NPK+TD

2.13 2.07 2.11 0.70 0.56 0.68 1.62 1.41 1.80 0.08 0.09 0.10 0.61 0.63 0.73

2.70 2.86 3.54 0.21 0.22 0.23 1.17 0.96 1.26 0.14 0.15 0.16 0.49 0.59 0.71

3.28 2.63 3.59 0.34 0.28 0.31 1.47 1.31 1.41 0.14 0.14 0.16 0.51 0.56 0.53

A. glutinosa Foliar N (0.074) Foliar P (0.002) Foliar K (0.077) Foliar Mg (0.003) Foliar Ca (0.023)

Figures in parenthesis are residual squares from the analysis of variance.

ments. It is notable that chance infection of non-inoculated plants receiving top dressing of'Kaynitro' was reduced by more than 50% compared with PK and NPK basal treatments alone. The results showed clearly that inoculation with Ar 1.2.5q enhanced seedling dry weight and nitrogen content, which were at least 20% and 80% greater, respectively, than the plants from the fertilised plots. The height and root collar diameter of inoculated plants were also greater than for plants receiving fertiliser treatments, with the exception of seedlings that had received basal N P K + N K top dressing. Analysis of total N content

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TABLE 5 Correlation matrixes showing relationships of foliar nutrient concentrations and shoot height for AInus rubra and A. glutinosa from Experiment 4

A. rubra Shoot height %N %P %K %Mg %Ca A. glutinosa Shoot height %N %P %K %Mg %Ca

Shoot height

%N

%P

%K

%Mg

%Ca

1.00

0.57 1.00

-0.92 -0.23 1.00

-0.80 -0.32 0.92 1.00

0.68 0.85 -0.75 -0.58 1.00

-0.42 -0.43 0.53 0.32 -0.30

1.00 1.00

0.75 1.00

-0.85 -0.73 1.00

0.25 0.43 -0.05 1.00

0.38 -0.57 0.33 -0.22 1.00

-0.57 -0.73 0.56 -0.27 0.89

1.00

showed a large increase in N content, both per gram of dry matter and per plant, of up to 55% and 90% respectively, over uninoculated, fertiliser-treated plants. Separate analyses (not shown) of the mineral content of foliage and of stems confirmed these higher levels of N in nodulated plants, but also showed a small reduction in leaf P and a larger reduction of from 30 to 90/tg p g-i stem dry weight of Frankia-inoculated plants (total P content 0.19 mg g-~ stem dry weight). No evidence of mycorrhizal infection was found on inspection of the root systems of l 0 sample plants from each treatment (C. Walker, Forestry Commission Research Division, personal communication, 1987). The development of the roots system of plants relying on nitrogen fixation for growth has often been reported to be less than that of mineral-Nfed plants and, although not measured specifically in the Bush experiments, was evident in the high shoot/root ratio of nodulated A. rubra, receiving PK fertiliser alone, in the Headley experiments (Table 3 ). The results of Experiment 4 at Headley were broadly comparable with those from the Bush nursery experiments. Nodulation of plants in the uninoculated seed beds was lower than experienced at Bush and may reflect either the lower rainfall at Headley, with less movement of Frankia in soil water from adjacent areas, or the effectiveness of methyl bromide as a soil sterilant. Mean height, mean number of infection sites, mean shoot and root weights and shoot/root ratios were all significantly greater ( P < 0.001 ) in inoculated plots than controls for both A. glutinosa and A. rubra (Table 3). The increase in growth of inoculated compared with uninoculated plants was much greater than in the Bush experiments. Tree seedling performance was also substantially better for those plots inoculated around the time of seed sowing than

FRANKIA INOCULATION OF ALNUS SEEDLINGS

16 3

for those inoculated when seedlings had reached the two leaf stage - differences which again would be most apparent on soil of relatively low fertility. There was some evidence that the applied nitrogen in the N P K plots inhibited nodulation in A. rubra but not in A. glutinosa (Table 3 ). Analysis of variance of nutrient concentrations (Tables 4 and 5 ) show pronounced and significant ( P < 0.001 ) effects of inoculation on nitrogen in both foliage and roots for both species; in A. glutinosa, foliar concentrations were below o p t i m u m levels found by other workers (Burg, 1985 ), even from plots which had received nitrogen in Nitrochalk and Kaynitro. Almost all other significant effects resulted from larger nutrient concentrations in foliage and roots of uninoculated seedlings compared with inoculated ones. DISCUSSION Even though the soils of the nurseries used for these experiments have been well worked for many years and are of moderate fertility, inoculation of A. rubra with at least one of the Frankia strains tested enhanced seedling growth in all of the experiments, and the nitrogen content of all plants was increased by inoculation. Early nodulation of seedlings stimulated growth, and in experiments at Bush produced robust seedlings suitable for planting out at l + 0 years, rather than 1 + 1 years as currently recommended in the U K (Evans, 1984). The poor nodulation of the uninoculated plants observed in these experiments reflects the levels of nodulation that would be found normally on alder seedlings in this nursery at the end of the growing season. A second year in the nursery will result in increased nitrogen input from nodules that develop from chance infection of the seed beds during the first year. The occurrence of such infection obviously depends on the presence of compatible, indigenous strains of Frankia in the nursery soil, and the speed at which infection occurs will depend on the efficiency of sterilisation of the seed beds and factors such as soil type and rainfall or irrigation, which may transfer infective propagules to the seed bed from adjacent areas. Because of the precautions taken to separate beds, it is thought unlikely that in the present experiments the inoculum was a source of contamination, particularly in Experiment 2 where the beds were widely separated in the nursery. The high effectiveness of Frankia strain Ar 1.2.5q for nodulation and nitrogen fixation in A. rubra, demonstrated originally in a screen of A. rubra isolates on A. rubra grown in mineral nitrogen-free media under controlled conditions (Hooker and Wheeler, 1987 ), was also apparent in the nursery. The growth differences between plants inoculated with the various Frankia sources were less in the nursery at Bush than in the controlled tests, presumably as a result, in particular, of the effect of soil nitrogen on plant growth and the development of symbiosis, and to more general effects of a less favourable environment on plant growth. It is notable that, as in previous tests under con-

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trolled conditions, the effectiveness of the symbiosis ofA. rubra with Frankia Ar 1.2.5q, isolated from A. rubra nodules from Lennox Forest, was greater than that given by inoculation with crushed nodules, collected from the same area. It is unlikely that compounds released by crushing nodules affected strain effectiveness as addition of filter sterilised, crushed nodule extracts to Ar 1.2.5q inoculum had no effect on the development of symbiosis. It was found in earlier work that Lennox A. rubra nodules contain several Frankia strains of lower effectiveness than Ar 1.2.5q (Hooker and Wheeler, 1987 ). Some of the strains present in crushed nodule inoculum in addition to Ar 1.2.5q are likely to be highly infective but of low effectiveness in nitrogen fixation. The much poorer growth of uninoculated seedlings at Headley compared with those at Bush may be due to the greater speed at which nutrients and applied fertilisers, especially nitrogen, leach through the sandy soil at Headley - the soil at Bush is a relatively heavy clay loam which is more retentive of soil nutrients. The reasons for the variability in the growth of seedlings receiving similar treatments in different experiments at Bush are unknown. However, some of the variation may stem from differences in the mineral content or other properties of soil at different locations in the nursery or from differences in seed germination times. This may be the case in particular for the 1986 experiment, where experimental plots were widely separated in the nursery. The 1987 experiment showed clearly that inoculation produced robust seedlings, with greatly increased root collar diameter compared with the fertiliser-treated seedlings and, in comparison with most treatments, an increase in seedling height at the end of the first season's growth. The Headley experiment showed that inoculation had a large effect on shoot/root ratios and relative nutrient concentrations in plant material. The increase in shoot/root ratios in inoculated seedlings reflects the increase in nitrogen available for plant assimilation; correlations of 0.57 and 0.75 were obtained for the relationship between shoot/root ratio and root nitrogen concentrations across the range of treatments for A. rubra and A. glutinosa respectively. Small shoot/root ratios in uninoculated plots probably reflect the very low levels of soil available nitrogen: total soil nitrogen averaged 0.053% and C : N ratio averaged 24 from bulked soil samples taken from each replicated block at the beginning of the experiment. As mentioned above, the effect of the addition of Nitrochalk (NH4NO3) and Kaynitro (KNO3) was probably short-lived because of rapid leaching of these compounds, especially the former, from the sandy nursery beds. The foliar concentration of phosphorus and calcium (and potassium in A. rubra) were strongly influenced by inoculation treatment and are inversely correlated with seedling growth and nitrogen foliar concentration (Table 5 ). Inoculation, or lack of nodulation, also influenced the balance between nutrients; N: P ratios were very small in uninoculated treatments from both species (range 1.6-2.4 and 3.0-3.7 for A. glutinosa and A. rubra respectively),

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but were much closer to the norm of 10 (Evans, 1984) in inoculated plots (range 10.0-16.8 and 9.4-15.4 ). These results suggest that unnodulated seedlings take up nutrients other than nitrogen in excess amounts, but only when nitrogen is supplied symbiotically can they make use of them in growth. Plants produced in the Bush experiments have been planted out subsequently in a series of experiments on both second rotation forest sites and nutrient-deficient mineral reclamation sites. Although few differences were found at the first site type, inoculated plants have consistently shown superior growth on reclamation sites (McNeill et al., 1989). This result is of considerable practical importance as Alnus species are frequently planted on reclamation sites in Britain. Such results and the improvements in seedling growth that were obtained after inoculation with the elite Frankia strain UGL Ar 1.2.5q indicates a potential for commercial development of an inoculum suitable for distribution to nurseries. Such an inoculum would be of particular value when there are low levels of indigenous Frankia in the soil or when the strains present are of poor effectiveness. Rigorous programmes of seed bed sterilisation are often carried out before sowing alder because of the sensitivity of the seeds to seed bed herbicides. However, further work is required to establish whether a single strain such as Ar 1.2.5q is of universal effectiveness for nodulation ofA. rubra and perhaps other species of alder on all soil types (Sheppard et al., 1988). ACKNOWLEDGEMENTS

We wish to thank P.D. Howard for his help in establishing and maintaining the nursery experiment at Headley, and A. Sutcliffe and J.S. Wright for assistance in inoculum preparation and in the harvest and analysis of plants at Bush and Headley, respectively. The participation of J.E.H. was aided by a research grant to C.T.W. from the Natural Environment Research Council. REFERENCES Aldhous, J.R., 1972. Forest Nursery Practice. For. Comm. Bull. 43. HMSO, London, 184 pp. Berry, A.M. and Torrey, J.G., 1985. Seed germination, seedling inoculation and establishment ofAlnus spp. in containers in greenhouse trials. Plant Soil, 87:161-173. Burg, J. van den, 1985. Foliar analysis for determination of tree nutrient status - a compilation of literature data. Rijksinstituut voor Onderzoek in de bos- en landschapsbouw "de dorschkamp". Wageningen Rapp. 414, 615 pp. Callaham, D., Torrey, J.G. and del Tredici, P., 1978. Isolation and cultivation 'in vitro' of the actinomycete causing root nodulation in Cornptonia. Science, 199: 899-902. Evans, J., 1984. Silviculture of Broadleaved Woodlands. For. Comm. Bull. 62. HMSO, London, 232 pp. Hooker, J.E. and Wheeler, C.T., 1987. The effectivity of Frankia for nodulation and nitrogen fixation in Alnus rubra and A. glutinosa. Physiol. Plant., 70:333-341. McNeill, J.D., Hollingsworth, M.K., Mason, W.L., Moffat, A.J., Sheppard, L.J. and Wheeler,

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C.T., 1989. Inoculation ofAlnus rubra seedlings to improve seedling growth and forest performance. For. Comm. Res. Inf. Note 144. Forestry Commission, Edinburgh, 3 pp. Malcolm, D.C., Hooker, J.E. and Wheeler, C.T., 1985. Frankia symbiosis as a source of nitrogen in forestry: a case study of symbiotic nitrogen fixation in a mixed Alnus-Picea plantation in Scotland. Proc. R. Soc. Edinburgh, 85B: 263-282. Normand, P. and Lalonde, M., 1982. Evaluation of Frankia strains isolated from provenances of two Alnus species. Can. J. Microbiol., 28:1133-1142. Perinet, P., Brouillette, J.G., Fortin, J.A. and Lalonde, M., 1985. Large scale inoculations of actinorhizal plants with Frankia. Plant Soil, 87: 1 7 5 - 1 8 3 . Sheppard, L.J., Hooker, J.E., Wheeler, C.T. and Smith, R.I., 1988. Glasshouse evaluation of the growth ofAlnus rubra and Alnus glutinosa on peat and acid brown earth soils when inoculated with four sources of Frankia. Plant Soil, 110:187-198. Sougoufara, B., Diem, H.G. and Dommergues, Y.R., 1989. Response of field-grown Casuarina equisetifolia to inoculation with Frankia strain ORS 021001 entrapped in alginate beads. Plant Soil, 118: 133-137. Stowers, M.D. and Smith, J.E., 1986. Inoculation and production of container-grown red alder seedlings. Plant Soil, 87:153-160. Wheeler, C.T. and Miller, I.M., 1990. Current utilisation of actinorhizal plants in Europe. In: C. Schwintzer and J.D. Tjepkema (Editors), The Biology of Actinorhizal Plants. Academic Press, New York, pp. 365-389.