NewForests 5: 35--42, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Inoculation of fumigated nursery soil increases nodulation and yield of bare-root red alder (Alnus rubra Bong.)

A. B. HILGER 1, Y. TANAKA 2 and D. D. MYROLD l* Department of Crop and Soil Science, Oregon State University, Strand Agriculture Hall

202, Corvallis, OR 97331-2213, USA; 2 Weyerhaeuser Co., Western Forestry Research Center, 505 North Pearl Street, Centralia, WA 98531, USA (*requests for offprints)

Received 1 February 1989; accepted 11 March 1991

Key words: actinorhizae, fertilization, Frankia, nitrogen, nodulation

Application. Inoculation of a fumigated nursery bed with alder soil (1.9 kg m -z) resulted in earlier and greater nodulation of bare-root red alder seedlings, and increased yield of packable seedlings by 43%.

Abstract. To determine if inoculation increases nodulation and yield of bare-root red alder (Alnus rubra Bong.), fumigated nursery plots were treated with inoculum and ammonium sulfate (28 kg N ha -a) in a factorial experiment. Inoculum was alder soil with 100 infective units of Frankia g-L Seedlings were evaluated for nodulation at age 10 wk and when lifted, at age 9 mo. Inoculation produced earlier and more extensive nodulation and increased seedling root collar diameter, height, and dry weight. Fertilization decreased seedling height, but did not decrease nodulation. No interaction of fertilization with inoculation was found. Inoculated unfertilized plots had the highest yield of packable seedlings (257 m-2), and uninoculated fertilized seedlings had the lowest yield (126 m-2).

Introduetion

Red alder (Alnus rubra Bong.) is a potentially valuable species in inten- sively managed forests (Tarrant et al. 1983). Until recently, efforts to control this species, as a competitor of softwoods, have exceeded efforts to promote its culture. Quality red alder planting stock is needed but no guidelines exist for raising bare-root red alder.

The purpose of this study was to determine if inoculation increased nodulation and growth of red alder seedlings in fumigated nursery beds. Alder seedlings are normally supplied with nitrogen fixed symbiotically, by Frankia in root nodules; alder is always nodulated in natural habitats (Bond 1976). Frankia spores are produced within sporangia in root nodules (Torrey 1987), with no mechanism for spore release into air; thus, long-range dispersal probably requires movement of soil. This process can

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be very slow; Arveby and Huss-Danell (1988) found no evidence of Frankia dispersal into peat-bog plots from nearby alder stands over several years. Because fumigation of nursery beds might reduce the Frankia population below that required to nodulate seedlings, inoculation may be necessary.

Nursery beds were amended with inoculum and ammonium sulfate, in factorial combinations. Inoculation increased seedling nodulation and growth. Fertilization reduced growth but did not inhibit nodulation. We concluded that inoculation was necessary for adequate nodulation and growth of seedlings in fumigated beds.

Methods and materials

Experimental design

A randomized complete block design was used in a factorial experiment, with four treatments: inoculated, fertilized; not inoculated, fertilized; inoculated, not fertilized; and not inoculated, not fertilized. There were 4 replicates. The bed was divided into four blocks and the treatments randomly assigned within each block to plots 0.61-m long and 1.2-m wide, with a 15-cm buffer zone between each plot.

Plot preparation

Bed space at a nursery near Mima, Washington, was supplied by Weyerhaeuser Co. Beds were fumigated with methyl bromide-chlorpicrin at 389 kg ha -1, injected at 15 to 20-cm depth in September 1986, then covered with a tarpaulin for 1 week after fumigation.

Inoculum soil was collected from the surface horizon in a young red alder stand near Corvallis, Oregon, and stored at 4 °C (field-moist) for about 1 mo until used. Uninoculated bed soil and inoculum soil were bioassayed for Frankia infective units, as described below. Inoculum soil (1.9 kg m -2) was spread over each plot, and raked in to 5-cm depth, just before seeding.

Red alder seeds, collected locally, were germinated to determine the number of pure live seed (PLS) per gram. Plots were broadcast-seeded with about 990 PLS m - 2 o n 29 April 1987. To protect seed from desiccation, Reemay polyester sheet (Ken-Bar Inc., 24 Gould St., Reading, Mass. 01867) was applied after sowing, and removed 19 June 1987. This cover is permeable to light, water, and air. Plots were irrigated when

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necessary. Ammonium sulfate (13.5 g m -z, 28 kg N ha-l), dissolved in water (1 1 plot-~), was applied 10 wk after seeding.

Frankia bioassay

Soil samples were bioassayed for Frankia infective units (IU) by a plant infection method, using most probable number (MPN) technique. Seeds were surface-sterilized (30% hydrogen peroxide, 20 min), sown on auto- claved acid-washed quartz sand, stratified at 4 °C for 1 wk, and germi- nated at 27 °C, with light. Germinants, with a radicle at least 2.5 cm long, were transferred to 50-ml centrifuge tubes containing 45 ml of 1/4- strength, N-free Hoagland's solution (Hoagland and Arnon 1950). The stem was held in a hole in the tube cap by a piece of foam stopper. Seedlings were grown for 2 wk (27 °C day/22 °C night, 16-h daylength) before inoculation with soil dilutions.

Ten grams of sieved (2-mm mesh), field-moist soil, was suspended in 95 ml sterile distilled water, and ten glass beds added. After this dilution was shaken 100 times by hand, successive 10-fold dilutions were made in sterile distilled water, down to 10-Sg m1-1. Ten seedlings were inoculated, with 1 ml each, for each dilution, by running the inoculum over the root into the bioassay tube. Ten control seedlings received no inoculum. Nutrient solution was replaced every 2 wk, and at 6 wk, seedlings were examined for nodules. The numbers of nodulated seedlings at each of three successive dilutions were used to calculate the MPN (Koch 1981) of Frankia IU g-lsoil (dry weight). Uninoculated nursery soil (0--10 cm) contained fewer Frankia than bioassay can detect (1 IU g-~). The inoculum contained 100 IU g-l; thus, inoculated soil contained about 2 IU g-l, in the upper 0--5 cm.

Seedling evaluations

Early nodulation, at seedling age 10 wk, was estimated by randomly selecting 5 seedlings per plot, and examining roots for nodules. On 19 January 1988, all seedlings were lifted by hand, counted, and measured for height between the apical bud and the soil surface. A randomly selected 20% subsample of seedlings from each plot were also evaluated for root collar diameter (RCD, measured 2 cm above the soil surface), number of nodules, and dry weight. Roots were washed before counting the total number of nodules and the number of large (diameter > 1 cm) nodules. Roots and shoots were dried (70 °C) and weighed. Seedlings were arbitrarily considered packable if at least 20 cm tall.

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Statistical methods

Three-way analyses of variance (ANOVA) of block x fertilizer x inoculum were done, with fertilizer x inoculum interaction included as a source of variation. Two-way ANOVAs (treatment x block) were also done to allow comparisons of treatment means. Logarithm (ln) transformation of RCD, height, dry weight, and number of nodules per plant were necessary to normalize these data; thus, plot means of log values were used in ANOVAs for these parameters. Means separation was done with Fisher- protected LSD.

Results

No significant (P < 0.05) fertilization x inoculation interactions were found; therefore, the effects of these two factors were evaluated independ- ently. Inoculation increased seedling nodulation, growth and yield of packable seedlings (Table 1). Inoculation increased yield by an average of 43% _ 18% (P -- 0.05). Fertilization decreased yield, but had no effect

Table 1. Inoculation and fertilization effects on mean a number, size and nodulation of bare-root red alder seedlings, n = 8.

Inoculation Fertilization

Parameter (_) (+) pb (_) (+) pb

Number at lifting Total (m -z) 443 428 0.5338 461 412 0.0898 Packable c (m -z) 159 227 0.0005 225 161 0.0007

Size at lifting Root collar (mm) 3.2 4.1 0.0001 3.8 3.5 0.1720 Height (cm) 21.2 33.5 < 0.0001 29.5 25.2 0.0195 Shoot (g) 1.1 2.1 0.0001 1.8 1.4 0.3771 Root Dry (g) 1.0 1.4 0.0018 1.3 1.1 0.4712

Nodulation At 10 weeks (%) 10 80 < 0.0001 43 48 0.6665 At lifting (%) 77 97 < 0.0001 89 85 0.1984 At lifting (plant - l) 1.8 4.9 < 0.0001 3.3 3.4 0.8157

" ANOVAs for mean root collar diameter, height, dry weight, and nodules plant -~ used mean In(value) to determine main effects. Arithmetic means shown here. b Significance of factor main effect, in ANOVA. c Seedlings at least 20 cm tall.

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on other parameters. Inoculated unfertilized seedlings were tallest; thus, this treatment produced the most packable seedlings (Table 2).

Inoculation greatly improved early nodulation; eight times more nod- ulated seedlings were found in the inoculated than in uninoculated treat- ments, at 10 wk after seeding (Table 1). At this stage of growth, inoculated plants were already greener than the uninoculated plants. At lifting, nodulation of inoculated seedlings was still greater than of uninoculated seedlings, but 77% of the uninoculated seedlings were nodulated. Inocu- lated seedlings had more nodules per plant than did uninoculated seed- lings (Table 1). Inoculated seedlings often had nodules close to the root collar, in contrast to more distal nodules on uninoculated seedlings. We speculate that uninoculated roots only became nodulated when they penetrated soil that contained infective Frankia, below the fumigated surface. Because plots were small and close, cross-contamination might also account for the nodulation of unlnoculated seedlings.

Table 2. Mean a number s and nodula t ion of red alder seedlings on plots t reated with (+) or wi thout ( - ) inoculat ion with alder soil (I) or ferti l ization with a m m o n i u m sulfate (F).

N u m b e r of seedlings lifted (m -2) Nodula t ion (%)

T rea tmen t Total Packable b At 10 wk At lifting

+ I - F 448 a 258 a 75 a 97 a + I + F 408 a 197 b 85 a 97 a - I - -F 472 a 192 b 10 b 80 b - I + F 416 a 126 c 10 b 74 b

a Values sharing the same lower-case letter are not different (P = 0.05). b Seedlings at least 20 cm tall, at lifting.

Fertilization had no significant effect on the percentage of nodulation or the number of nodules per plant (Table 1). If fertilization reduced nodule specific activity, the growth response to nodulation might be reduced. To test this, linear regressions of the number of large nodules (N) on seedling height (H) were calculated for fertilized and unfertilized plots. For fertilized plots, In H = 12.2 In N + 18.3 (r 2 = 0.52), and for unfertilized plots, In H -- 12.3 In N + 21.6 (r 2 = 0.48); both regressions were significant at P < 0.0001. Neither slopes nor intercepts differed (P < 0.05) between fertilized and unfertilized plots; thus, no difference in nodule activity was indicated.

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Discussion

The surface soil of these fumigated beds apparently lacked infective Frankia, because no Frankia was detected by bioassay, nodulation of uninoculated seedlings was delayed, and nodules were distal on uninocu- lated roots. Inoculation was necessary and effective for promoting early nodulation of red alder seedlings in fumigated nursery beds. In the Netherlands, inoculation of nursery soil with Frankia-containing soil has been advised, especially after soil sterilization (Hahn and vanElk 1978; as cited by Houwers and Akkermans 1981). Based on studies in the United Kingdom, McNeill et al. (1989) suggest that inoculation of red alder with Frankia strains is a possible standard nursery technique.

Early formation of effective nodules gave inoculated seedlings a growth advantage that persisted until lifting. Benefits of inoculation may extend beyond promoting seedling growth in the nursery. Burgess et al. (1986) studied the effects of Frankia inoculation on a variety of alder species including red alder; containerized seedlings were inoculated, transferred to nursery beds for 1 year and then outplanted. The effects on first-year growth in nursery beds varied among species, but inoculated trees were larger than uninoculated trees by the second year after outplanting.

Because we used soil inoculum, other soil components (e.g., mycorrhi- zal fungi or beneficial associative bacteria) may have produced the growth response. In a subsequent study at Mima nursery in 1988, however, inoculation with pure-culture Frankia in peat carrier similarly enhanced alder nodulation and growth; inoculation doubled mean seedling heights (Martin et al. 1990). Koo (1990) reported that nodulation is more impor- tant for mycorrhiza formation, than mycorrhizae are for nodulation, in container-grown red alder. He also reported that growth of containerized alder increases more by nodulation than by mycorrhiza formation; and even at high rates of N fertilization, non-nodulated seedlings do not grow as well as nodulated seedlings. Thus, enhanced nodulation by Frankia rather than effects of mycorrhizae or other soil microorganisms was the more likely cause of the enhanced growth response to soil inoculation.

Fertilization with ammonium sulfate did not substitute for beneficial effects of successful inoculation; yield of packable seedlings was de- creased. Inorganic nitrogen can inhibit nodulation of container-grown red alder seedlings (Stowers and Smith 1985). Timing of nitrogen application, as well as rate, can determine whether inhibition will occur (Granhall et al. 1983). We found no evidence that fertilization inhibited nodulation or nodule activity. Other effects of ammonium sulfate may account for inhibition of seedling growth (e.g., soil acidification from uptake of ammonium ions).

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Inoculation with alder soil was an effective, economical method to test if alder production would benefit from bed inoculation. For routine inoculation on a large scale, however, soil is impractical and might introduce disease organisms. We have tested several other methods to inoculate nursery beds with Frankia; results are reported in Martin et al. (1990). Preliminary testing of inocula is advisable because effectiveness can vary with Frankia strain (Carpenter et al. 1984) and host-plant genetics. Teissier du Cros (1984) reports that inoculation can suppress growth of red alder, but enhance growth of A. cordata. Control of both Frankia strain and alder stock increases the chance of improving red alder planting stock quality and growth.

Conclusions

-- Early nodulation of red alder bareroot seedlings required inoculation. -- Inoculation increased yield of packable seedlings by 42%. -- Ammonium sulfate (28 kg N ha -1) decreased yield, but did not affect

nodulation.

Acknowledgments

This is technical paper 8707 of the Oregon Agricultural Experiment Station. Financial support for this research was provided by the Agricul- tural Research Foundation of Oregon State University and by a Presi- dential Young Investigator award from the National Science Foundation, BSR-8657269, to D.D.M. Weyerhaeuser Co. generously provided space in their Mima nursery and technical support of the field operations. The field and laboratory assistance of Pam Brotherton, John Dodd, Timo Hokkanen, Kendall Martin, Michael Mungoven, and Ted Nason are greatly appreciated. We thank Randy Molina, Robin Rose, Carolyn Carpenter, Will Littke, Tom Terry, and several anonymous reviewers for their comments on earlier drafts of this paper.

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