Plant and Soil 164: 283-289, 1994. (~ 1994 Kluwer Academic Publishers. Printed in the Netherlands.

Occurrence of Myrica-nodulating Frankia in Hawaiian volcanic soils

Stephen H. Bur l e igh and Jef f rey O. D a w s o n University of Illinois, Department of Forestry, W503 Turner Hall, 1102 S. Goodwin Ave., Urbana, IL 61801, USA

Received 22 March 1994. Accepted in revised form 9 July 1994

Key words: Actinomycete, actinorhizae, Frankia, Myrica, nodulation, volcanic soils

Abstract

The ability of Hawaiian volcanic soils to nodulate actinorhizal Myrica cerifera, Casuarina equisetifolia, and Alnus glutinosa was determined using a host-plant bioassay. Myrica-nodulating Frankia occurred in five volcanic deposits with depositional ages ranging from 20 to 162 years before present. The oldest deposit had a mean estimated nodulation capacity from 450 to 1200 times greater than those of the younger deposits. Only the oldest deposit had high moisture content, high organic matter content, and increased vegetative cover, including an abundance of actinorhizal M. faya. Casuarina- and Alnus-nodulating Frankia were not detected in any of these volcanic deposits.

Introduction

Myrica faya is a nitrogen-fixing, actinorhizal tree indigenous to the Azores, Canary Islands, and Madeira. M. faya was introduced to Hawaii around the turn of the century and is now considered a noxious weed because of its aggressive growth, its tendency to form monospecific stands, and its dominance over native plants (Smith, 1985). Ecosystem level properties such as productivity and nutrient cycling have been influ- enced by this species (Vitousek and Walker, 1989). While much of the success of this species is due to its rapid growth, early and prolific reproduction, wind pollination, and avian dispersal, the nitrogen-fixing symbiosis with the actinomycete Frankia is ultimate- ly responsible for the devastating effect M. faya has had in Hawaii (Vitousek, 1990; Vitousek and Walker, 1989). Through this symbiosis, M. faya can colonize nitrogen-deficient deposits to the exclusion of native species.

Little is known about the occurrence of Frankia in volcanic soils. Several species of actinorhizal plants nodulate on newly formed soils, such as Myricajavan- ica on volcanic soils in Java (Becking, 1970), M. fava on volcanic soils in Hawaii, USA (Doty and Mueller- Dombios, 1966; Turner and Vitousek, 1987), Dryas drummondii on newly formed glacial soils in Alaska, USA (Lawrence et al., 1967), Hippophae rhamnoides

on coastal sand-dunes in The Netherlands (Oremus, 1980), and M. cerifera on newly formed coastal soils in Virginia, USA (Young et al., 1992). These obser- vations indicate that the dispersal of Frankia to newly formed soils and subsequent host nodulation occurs commonly.

Frankia may be moved in the environment by biotic agents such as birds (Paschke and Dawson, 1993) and earthworms (Redell and Spain, 1991) and by abiotic agents such as water (Arveby and Huss-Danell, 1988). Wind may also disperse Frankia, since this organism can remain viable when air-dried (Burleigh and Torrey, 1990; Oremus, 1980; Sougoufara et al., 1989). Howev- er, Frankia does not produce an above-ground sporo- phyte to facilitate aerial dispersal of spores and Paschke (1993) did not find significant levels of Frankia in hail, rainwater, or air samples.

Once deposited on soils, Frankia may be able to survive depending on environmental conditions. The abundance of infectious Frankia in soil has been asso- ciated with the presence of actinorhizal plants (Arveby and Huss-Danell, 1988; Dawson and Klemp, 1987; Oremus, 1980; Smolander, 1990) and their removal can result in a decrease in infectious Frankia (Wol- lum et al., 1968). The abundance of infectious Frankia near roots of actinorhizal plants is probably due to rhi- zosphere interactions (Diem et al., 1982; Vergnaud et al., 1985) and root-nodule decomposition (Ore-

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mus, 1980), which may release infectious Frankia. Frankia can also survive in the soil in the absence of actinorhizal plants (Dawson and Klemp, 1987; Houwers and Akkermans, 1981; Paschke and Dawson, 1992b; Young et al., 1992) and can apparently flourish under certain non-host plants such as birch (Paschke and Dawson, 1992a; Smolander, 1990; Smolander et al., 1990), indicating that the ability of vegetation to enhance the Frankia infectious capacity of soil is not limited to actinorhizal plants.

Frankia might possibly grow in soil saprophytical- ly. Evidence for saprophytic growth includes the ability of pure-cultured strains to grow in vitro, the ability of Frankia to produce siderophores (Aronson and Boy- er, 1992), and the correlation of Frankia nodulation capacity with soil properties such as moisture content (Dawson et al., 1989; Righetti et al., 1986), organic matter content (Becking, 1970; Houwers and Akker- mans, 1981; Young, 1992), micronutrients (Righetti et al., 1986), and biotic activity (Smolander, 1990).

Frankia may survive in soils by remaining in a dormant form. Dormancy would enable Frankia to inhabit soil environments that are too harsh for vegeta- tive growth. Burleigh and Dawson (1994) and Burleigh and Torrey (1990) found that hyphal and spore prepa- rations can remain infectious when air-dried and Ore- mus (1980) found that actinorhizal nodule powders can remain infectious for several years.

Quantification of Frankia populations in soil remains problematic. The infectious capacity of soil is not necessarily related to the quantity of Frankia in the soil, since the infectious capacity can vary seasonally, even though the actual population of Frankia as mea- sured by genomic units can remain the same (Myrold and Huss-Danell, 1994). Hence, only large differences in infectious capacity are likely to be indicative of dif- ferences in Frankia population.

We chose Hawaiian volcanic soils to study the dis- tribution of infectious Frankia because these soils have been deposited at periodic intervals and are colonized by nodulating M. faya. We determined the infectious capacity of five Hawaiian volcanic deposits using a host-plant bioassay to describe chronological patterns of occurrence of infectious Frankia.

Materials and methods

The study was conducted in the Hawaii Volcanoes National Park on the island of Hawaii, Hawaii, USA. Five sites with deposition dates of 1974, 1959, 1921,

1894, and 1832 were chosen within or neighboring the Kilauea Caldera. All sites were located within 2.5 km of one another. The 1974, 1921, and 1894 deposits were lava flows within the caldera. The 1959 deposit was the Pu'u Pua'i ash field located approximately 0.8 km from the caldera rim and the 1832 deposit was Byron Ledge located on the eastern rim of the caldera. No vegetation occurred on the 1974 and 1921 deposits, sparse vegetation, consisting primarily of grasses and small shrubs was present on the 1959 and the 1894 deposits, while dense vegetation had developed on the 1832 deposit. The actinorhizal plant M. faya was observed only on the 1832 deposit (Table 1).

Surficial soils (l-5 cm in depth, including that part of the "O" horizon of the 1832 deposit, which was root-bound and inseparable from its substrate), were aseptically collected from five random locations with- in each deposit. The samples were placed in sterile plastic bags and exposed to the air, unsealed, in a room free of Frankia at 25°C for 24 hours to elimi- nate condensation and thus minimize fungal and bac- terial growth during transport and storage. The bags were subsequently sealed and stored at 20°C for two months prior to the baiting-study. Because air-dried samples remain infectious for months (Tortosa and Cusato, 1991), the use of dried preparations is stan- dard practice for assaying relative infectious capacities of soil. Surficial soils commonly remain infectious in spite of fluctuating moisture content. Furthermore the physiological status of Frankia, rather than popula- tion size, seems to be the primary determinant of soil infectious capacity (Myrold and Huss-Danell, 1994). Hence, short-term drying and storage may make infec- tious capacities of soil samples more comparable than those obtained from fresh soils varying in soil moisture content.

Myrica cerifera, Casuarina equisetifolia, and Alnus glutinosa seedlings were used in the host- plant bioassay. These species were selected because Myrica, Casuarina, and Alnus span the known host- specificity spectrum for Frankia isolates (Baker, 1987) and species from these genera grow in Hawaii. M. cer- ifera was chosen instead of M. faya, because M. cer- ifera has proven to be virtually a universal acceptor of Frankia species (Baker, 1987), while the variety of Frankia strains capable of nodulating M. faya is unknown.

Seeds of each species were surface-sterilized for 20 minutes using 30% hydrogen peroxide, and then sown in a twice-pasteurized sand:fine gravel:vermiculte (1:1:1) mix in 4-cm-wide × 14-cm-deep sterile plastic

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Table 1. Characteristics of five volcanic deposits from the Hawaii Volcanoes National Park

Moisture Organic Year of Location within content con ten t Vegetative deposition the park (%)z (%)z cover

1974 Kilauea Caldera 0.5 B 0.3 B none

(19 ° 24' 8" N 155 ° 17' 37" W)

1959 Pu'u Pua'i ash field 0.3 B 0.2 B sparse

(19 ° 24' 55" N 155 ° 15' 28" W)

1921 Kilauea Caldera 1.3 B 1.6 B none

(19 ° 24' 16" N 155 ° 17' 25" W)

1894 Kilauea Caldera 0.6 B 0.5 B sparse

(19 ° 24' 37" N 155 ° 16' 55" W)

1832 Byrons Ledge 18.8 A 27.8 A denseY

(19 ° 24' 50" N 155 ° 15' 54" W)

ZMean of five randomly selected, surficial soil samples, Means in a column with the same capital letter are not significantly different (a=0.05). YThe actinorhizal species Myricafaya was observed only at this site.

tubes. The germinated seedlings were fertilized once every three days with a complete, 1/8-strength nutrient solution (Huss-Danell, 1978) and grown for 8 weeks in a greenhouse room with a filtered air supply follow- ing the methods of Paschke and Dawson (1992b). The N-level was then reduced to 1 mM ammonium nitrate and the seedlings were grown for two additional weeks prior to inoculation, because higher levels of substrate nitrogen can inhibit infection (Kohls and Baker, 1989).

Fifty grams of each soil sample were rehydrated in 50 mL of sterile BAP medium (Murry et al., 1984), sonicated three times at 20 watts for 10 seconds using a Fisher Model 300 sonic disrupter and then shaken for one hour at 100 rpm. Particulate debris were allowed to settle, then four 10-fold dilutions were made from the supernatant. Five tubes of each species were individ- ually inoculated with two mL of each serial dilution. Ninety-eight uninoculated seedlings of each species were included to detect contamination. While there was a variable number of seedlings per tube (Myri- ca, 54-3 (#+SD); Alnus, 65:3; Casuarina, 2+1), all tubes had a dense and uniform root mass at the time of inoculation.

Ten weeks after inoculation, each tube of seedlings was scored for nodule number and a subset of tubes from each species was assayed for seedling number, nodule, shoot, and root dry weight, shoot and root length and acetylene-reduction activity following the methods of Paschke and Dawson (1992b). The water content of the remaining samples was determined by

dehydration at 70°C for 3 days and the organic con- tent determined by soil combustion using a muffle fur- nace at 500°C for 12 hours. Estimates of the infectious capacity of the soils were calculated using most prob- able number statistics (Alexander, 1982) and analy- sis of variance was employed to determine treatment variation using the Statistical Analysis System (SAS Institute Inc., 1985). Duncan's multiple-range test was employed to determine differences among treatment means.

Results

M. cerifera became nodulated when inoculated with soil suspensions from each of the five deposits (Table 3). The nodulation capacity of the 1832 deposit was significantly greater than that of the 1974, 1959, 1921, and 1894 deposits (a=0.05). There was also a signif- icant difference in the nodulation capacity between samples taken within deposits (a=0.05). The oldest deposit had an average most probable number esti- mate of 36 per cm 3 of soil, which was approximately 700-times greater than the overall mean of the other four deposits. Neither A. glutinosa nor C. equisetifolia nodulated when inoculated with any of the soils. The uninoculated M. cerifera and C. equisetifolia control plants did not become nodulated, and only one out of 98 tubes of the uninoculated A. glutinosa control plants

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Table 2. The nodule, shoot, and root dry weights, and shoot and root lengths of nodulated and unnodulated Myrica cerifera seedlings

Seedling Unnodulated Nodulated

Characteristic z Myrica cerifera Myrica cerifera

Seedlings per tube 5.8 A 6.2 A

Nodule number per tube 0.0 B 22.5 A

Nodule dry weight (g) 0.0 B 0.06 A

Shoot dry weight (g) 0.40 B 1.31 A

Root dry weight (g) 0.45 A 0.49 A

Root/shoot dw ratio 1.13 A 0.37 B

Shoot length (cm) 6.5 B 11.2 A

Root length (cm) 10.3 A 10.9 A

Root/shoot length ratio 1.6 A 1.0 B

ZAverage value of six randomly selected nodulated or unnodulated con- tainers of M. cerifera previously inoculated with Hawaiian volcanic soils. Means in a row with the same capital letter are not significantly different (a=0.05).

developed nodules, indicating that the background lev- el of Frankia contamination was negligible.

Both the moisture content and the organic matter content of the 1832 deposit was significantly greater than that of the 1974, 1959, 1921, and 1894 deposits (Table 1). There was also a significant difference between samples taken within deposits for both mois- ture and organic matter content (a=0.05). Ten weeks after inoculation the nodulated Myrica were dark green and reduced acetylene to ethylene at a rate of 1.9-t-0.8 (#±SD) umol tube - l hr -1, whereas the unnodulat- ed Myrica, Alnus, and Casuarina were yellow with a mean acetylene reduction rate of 0.5+0.2 umol tube- hr - l . Nodulated M. cerifera had lower root:shoot ratios for both length and dry weight relative to unn- odulated M. cerifera (Table 2).

Discussion

Myrica-nodulating Frankia occurs in volcanic soils deposited as recently as 20 years ago. Soils with depo- sitional dates ranging from 1974 to 1894 had relatively- low, uniform nodulation capacities. These open soils could be characterized as having high surficial temper- atures, frequently low soil moisture, low organic matter content, and sparse vegetative cover. These conditions do not favor the growth of most soil microbes. Frankia dispersal to these deposits apparently has occurred within the last 20 years, however there was no differ- ence in the infectious capacity of neighboring deposits

up to 80 years older. It may be possible that these soils experience periodic deposition of Frankia, but their survival after deposition is of short duration. Burleigh and Dawson (1994) and Oremus (1980) found that the viability of desiccated Frankia preparations can decrease with age.

The oldest, most-infectious deposit had a relatively high soil moisture and organic matter content and was the only site where M. faya was observed. Aplet (1990) found that Hawaiian volcanic deposits dominated by M. faya had increased populations of earthworms, indi- cating increased soil fertility and biotic activity. We speculate that the abundance of infectious Frankia in this soil is due to a favorable rhizosphere environment, M. faya root-nodule decomposition, and saprophytic growth as a result of suitable soil conditions.

There was considerable variation in the nodulation capacity between soil samples taken within the oldest deposit (Table 3). Paschke and Dawson (1992a, b) and Oremus (1980) also observed considerable differences in the populations of infectious Frankia in otherwise uniform soils. Such variation in nodulation capacity may result from scattered favorable soil microhabitats, localized deposition by dispersal agents, or localized release of Frankia from degrading root-nodules of M.

faya. There are no isolates of Myrica-nodulating Frankia

from Hawaii, hence little is known about their mor- phology and their effects on actinorhizal plants. Mor- phologically atypical, ineffective Frankia are com- monly isolated from actinorhizal nodules gathered

Table 3. Most probable number estimate of Myrica-nodulating Frankia in Hawaiian volcanic soils of different depositional ages

Year of Sample MPN Mean MPN

deposition number per ccm soil z per ccm soil y

1974 1 0.2 0.08 B

2 0

3 0.22

4 0

5 0

1959 1 0.13 0.03 B

2 0

3 0

4 0

5 0

1921 1 0 0.04 B

2 0.19

3 0

4 0

5 0

1894 1 0 0.04B

2 0

3 0

4 0

5 0.22

1832 1 123.71 36.39 A

2 7.22

3 l 1.34

4 6.7

5 32.99

ZMost probable number estimate derived following Alexander (1982). YValues with the same capital letter are not significantly different (a=0.05).

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from widespread locations (Valdes et al., 1993). How- ever, the Myrica-nodulating Frankia in this study were typical in their influence on Myrica growth as indicat- ed by the reduced root:shoot length and dry weight ratios of nodulated plants (Table 2) and by the acety- lene reduction values. Low levels of ethylene produced by the pots of unnodulated plants are attributable to non-symbiotic nitrogen fixation by soil microorgan- isms and ethylene evolution by plants. Background values are provided to adjust acetylene reduction val- ues for nodulated plants.

All M. faya in the Hawaii Volcanoes National Park are reported to be nodulated (Vitousek and Walker,

1989), suggesting that the relatively small amount of infectious Frankia present in the undeveloped soils is adequate for Myrica nodulation. Low levels of soil Frankia may be sufficient to support M. faya coloniza- tion, since the nitrogen-fixing ability of actinorhizal plants is a function of nodule mass rather than nod- ule number (Dawson and Gordon, 1979). Alternative- ly, codispersal of both Frankia and Myrica seed may explain the consistent nodulation ofM. faya on unde- veloped volcanic deposits with relatively low Frankia infectious capacities. Walker (1990) found that the exotic bird Zosteropsjaponica acts as an agent of dis- persal for Myrica in Hawaii and Paschke and Dawson

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(1993) found significant levels of Frankia in the nests of several bird species, suggesting that birds may be an agent of dispersal for both of these organisms. The absence of Casuarina- and Alnus-infective Frankia on these volcanic deposits illustrates the distinctive nature of infectious Frankia populations at this location.

Acknowledgements

This project was supported in part by the Jonathan Baldwin Turner Graduate Fellowship administered by the College of Agriculture, University of Illinois at Urbana-Champaign. We would like to thank the National Park Service for permitting soil sampling at the Hawaii Volcanoes National Park.

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Section editor: R 0 D Dixon