Plant and Soil 178: 283-286,1996. O 1996 KluwerAcademic Publishers. Printed in the Netherlands.

Alnus rubra nodulation capacity of soil under five species from harvested forest sites in coastal British Columbia

John H. Markham and Chris E Chanway 1 Faculty of Forestry, University of British Columbia, 270 - 2357 Main Mall Vancouver, B.C. V6T 1Z4, Canada * (l Joint appointment with Dept. of Soil Science, Faculty of Agricultural Sciences)

Received 3 May 1995. Accepted in revised form 11 September 1995

Key words: Alnus rubra, Betula papyrifera, Frankia, Rubus spp.,nodulation capacity

Abstract

Nodulation of Alnus rubra seedlings after inoculation with soil from under A. rubra, Betula papyrifera. Rubus lacianutus, R. spectabilis, and R.ursinus on 2 recently harvested sites was compared. Nodulation capacity was low compared to other published reports, ranging from 0 to 18.9 infective units cm -3 of soil and was significantly affected by the site and plant species. Nodulation capacity of soil under alder was significantly higher than under all other species except R. spectabilis, regardless of site. The lowest nodulation capacity was found in soil under B. papyrifera.

Introduction

This paper reports on the abundance of Frankia in soil under some early successional species in 2 recently har- vested conifer stands in British Columbia, Canada. A number of studies have documented low Frankia abun- dance in soils devoid of actinorhizal plants (Paschke and Dawson, 1992; Smolander, 1990: Smolander and Sundman, 1987). Houwers and Akkermans (1981) have also shown that Alnus incana had a low prob- ability of becoming nodulated after 2 years of being planted in such soils. A. rubra is one of a species that invade sites after the harvesting of conifer stands in coastal British Columbia. Mature conifer stands in this region are typically devoid of actinorhizal plants and a preliminary sampling showed no detectable Frankia in the soil (see Methods). We therefore wanted to deter- mine if some early successional species would promote Frankia growth and survival in the soil and therefore facilitate alder nodulation.

The most likely non actinorhizal species to support Frankia populations in the soil are those closely related to actinorhizal species. High inoculation capacity has been found in soil from stands ofBetula nigra (Paschke and Dawson, 1992), B. pendula (Smolander, 1990;

* Fax no: + 1 6048225744

Smolander and Sarsa, 1990; Smolander and Sundman, 1987) and B. pubescens (van Dijk, 1984, in Smolander and Sundman, 1987) which are in the same family as Alnus. Laboratory studies have also shown Frankia is able to grow on the root surface of B. pendula and B. pubescens (R/Snkkt~ et al., 1993). B. papyrifera is the dominant birch in the Pacific Northwest and a com- mon component of disturbed sites. There have been no studies to date on its effect on Frankia abundance in the soil. A number of Rubus species are also common in disturbed forest sites in the Pacific Northwest. Bond (1976) reported that Rubus ellipticus, from Java, had Frankia-like nodules and Becking (1979) later con- firmed that the nodules had an acetylene reduction rate simiilar to other actinorhizal plants growing in the area (but see Stowers, 1985). It is possible then that like Betula species, non actinorhizal Rubus also support significant rhizosphere populations of Frankia. There- fore the objective of this study was to quantify the Frankia populations in soil under B. papyrifera and 3 Rubus species. As direct isolation of Frankia from soil is not generally possible (Lechevalier and Lechevalier, 1990) this was done using a plant bioassay technique with A. rubra as the host.

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Table 1. Sampling site description, pH is the mean pH (and standard deviation) of soil samples used in nodulation capacity tests

Site G Site K

Harvesting 1978 - Trees harvested history 1990 - Vegetation cut, aerial herbicide

application (glyphosate) 1991 - Burned

1985 - Trees harvested 1987 - Burned

Dominant species

Alnus rubra, Betula papyrifera, Fescue spp., Rubus spp., Holcus lantanum. Salix spp.

A. rubra, Betula papyrifera, Gaultheria shalon, Rubus spp., Vacinium parvifolium

pH 5.71 (0.32) 4.55 (0.46)

Materials and methods

Soil was sampled from the rhizosphere ofA. rubra, B.

papyrifera, R. lacianatus, R. spectabilis, and R. ursinus

on 2 recently harvested sites (Table 1) in the University of British Columbia Malcolm Knapp Research Forest (Lat. 49 o 16' N, Long. 122 o 24'). The soil in the area is a hummo-ferric podzol of glaciofluvial origin. Virgin stands of Douglas-fir, Western Hemlock and Western red Cedar were harvested from this area in the 1920's and the sites sampled regenerated naturally with these conifer species. The canopy of these second growth stands is generally closed with understory plants being absent. A preliminary sampling of soil in an unharvest- ed stand next to site K (Table 1) showed no detectable Frankia (minimum detectable nodulation capacity = 1.23 cm -3, See below for methods). Since the infec- tiveness of Frankia strains is strongly influenced by nodule spore type (Torrey, 1987), nodules were sam- pied throughout the region in the fall of 1993 and their spore type determined. No nodules collected within the research forest were found to be spore positive.

Soil samples for nodulation capacity tests were col- lected from under monoculture patches of each species in July and August of 1993 and 1994. Each patch sampled was at least 10 m from the other species sam- pied. Samples were taken by first removing any organic material from the soil surface and driving a 5 cm dia. × 7 cm (125 cm 3) aluminum cup into the mineral soil and extracting a core. Cups were washed before use and a separate cup was used for each sample. Samples were taken from within ca. 20 cm of the base of a stem and always contained a mass of fine roots. Samples

were stored in a refrigerator and used within 24 hours of being collected. Determination of nodulation capac- ity was made on individual soil cores from 4 different patches of each species on each site.

Nodulation capacity was determined by the plant inoculation method (Smolander and Sundman, 1987). A. rubra seeds were surface sterilized with 2.6% NaOC1 for 15 minutes, rinsed 5 times in sterile dis- tilled water and germinated on sterile moistened filter paper. Once germinated, seedlings were transferred to sealed 125 mL polyethylene containers. The lid of each container was fitted with a rubber septum from a 10 mL syringe through which a seedling was inserted using a 18 gauge syringe needle. Containers were filled with half strength nutrient solution (Huss-Dannell, 1978) supplemented with 3 mM KNO3. Seedlings were grown in a growth chamber with a 16 hour photope- riod (light intensity ca. 300 #mol m -2 s - l PAR) and day/night temperatures of 24°/18°C. Nutrient solu- tions were changed every other week and containers were topped up with sterile water every week as need- ed. Plants were grown for 8 weeks before inoculation by which time numerous roots extended to the bot- tom of the containers. One week prior to inoculation, the nutrient solution was changed to a nitrogen free solution which was then used for the duration of the experiment.

The individual soil cores were mixed with 250 mL of water by hand and the pH measured in these sus- pensions. Samples were then made up to 500 mL with N-free nutrient solution and filtered through a 1 mm mesh. From these suspensions 10, 20 and 500 times dilutions were prepared using the N-free nutrient solu-

tion and 4 replicate plants inoculated per dilution by filling the container with 125 mL of the suspension. This corresponded to 3.13, 1.56 and 0.0625 cm 3 of soil applied to a seedling at each dilution and a minimum detectable nodulation capacity of 0.0799 infective units cm -3. Seedlings were exposed to the inoculum for 2 weeks after which the suspension was replaced with N-free nutrient solution every 2 weeks. After 8 weeks the number of nodules which were clearly visible on the plant were counted. Uninoculated controls never developed any nodules.

The nodulation capacity (NC) in each soil sample was calculated using the following equation of Van Dijk et al. (1988):

Nc_- EEx/E p, i j i j

where ~-'~i ~ j X is the nodule count in each jar of the j replicate jars per dilution of inoculum and )~i )-'~j P is the sum of the soil volume used in each replicate of each dilution.

A mixed model ANOVA, with sites as a random factor, was used to analyse the effect of plant species and site on nodulation capacity. In order to homogenize the variances the data was log transformed twice. Post hoc comparisons were made using a Ryan's Q test (Day and Quinn, 1988).

Results

The nodulation capacity varied significantly between plant species and sites (Table 2) with no interaction between these 2 factors. The maximum number of nodules formed on a seedling was 89. The nodula- tion capacity per soil volume was linear over the range of dilutions, with nodules rarely being formed at the 500 times dilution. Soil from site K had on average a lower nodulation capacity by a factor of 0.30. Soil from under A. rubra and R. spectabilis had approxi- mately 5-10 times the nodulation capacity of soil from under the other plant species. Soil under B. papyrifera on site K induced no nodules and on site G had the lowest nodulation capacity. Variability in nodulation capacity within a species was high, as indicated by the coefficient of variation, which was usually greater than 100%.

Mean soil pH was higher on site G than on site K for all species. Individual soil sample pH values ranged from 4.02 (R. spectabilis on site K) to 6.32

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Table 2. Mean nodulation capacity and standard deviation (infective units cm -3) for each species on sites sampled. Letters denote significant differences in nodulation capac- ity between species at alpha = 0.05 using a Ryan's Q test

Species Site G Site K

A. rubra 14.46 (16.09) 18.87 (15.41) a

B. papyrifera 1.05 (1.13) 0 (0) b R. ursinus 1.28 (1.20) 0.44 (0.51) b

R. lacianatus 2.40 (0.91 ) 0.20 (0.24) b

R. spectablis 11.59 (17.42) 2.08 (1.70) ab

(R. spectabilis on site G). A linear regression of mean nodulation capacity versus pH showed pH to be a poor predictor of nodulation capacity (r 2 = 0.175).

Discussion

As expected, soil from under A. rubra had a rela- tively high nodulation capacity compared to the other species tested. However, this is the first report of a high nodulation capacity under a Rubus species (com- pared to the other non actinorhizal species tested) and is further evidence that Frankia can be found in asso- ciation with non actinorhizal hosts. Since only soil under R. spectabilis showed a relatively high nodu- lation capacity, this does not seem to be a general phenomena within the genus. The higher nodulation capacity associated with R. spectabilis could be relat- ed to the fact that R. ellipticus is actinorhizal. This is the first report on the nodulation capacity under B. papyrifera. While all other studies have found high nodulation capacities under other Betula species (with the exception ofaB. pubescens stand in Finland on very acidic soil), sampling was done from under mature Betula stands (Paschke and Dawson 1992; Smolan- der, 1990; Smolander and Sarsa 1990; Smolander and Sundman, 1987;). Further work is needed to deter- mine if mature stands of B. papyrifera also have high nodulation capacities.

The soils in this study showed a nodulation capac- ity at the low end of the range of published reports. For example, Smolander (1990) reviewed the literature and found that the nodulation capacity of soil varied between 0 - 3000 and 10 - 100,000 infective units cm -3 for soil under non actinorhizal and actinorhizal species, respectively. It is unlikely however that the low nodulation capacity values found here are solely

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due to the techniques used. We found soil from an A. sinuata stand in the same region as the sites used in this study had a high (ca. 600 cm -3) nodulation capacity using the same techniques.

In this study, nodulation capacity was determined for individual soil cores. In other studies, soil samples have been pooled from plots within stands (Myrold and Huss-Dannell, 1994; Paschke and Dawson, 1992; Smolander, 1990; Smolander and Sundman, 1987). While such an approach should not have an effect on estimates of mean nodulation capacity it disregards variation within sites. Research into the spatial dis- tribution of Frankia is needed to determine the most suitable sampling scale. As these results show that plant species can have a strong effect on nodulation capacity, the proper scale of soil sampling may be the size of the plants on the study sites.

Acknowledgements

This work was funded by a Natural Sciences and Engi- neering Council of Canada Research Grant to C P C. We would like to thank Laura Lazo for her suggestions on the manuscript.

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Section editor: F R Minchin