Plant and Soil 254: 110, 2003. 2003 Kluwer Academic Publishers. Printed in the Netherlands. 1
Frankia inoculation, soil biota, and host tissue amendment influenceCasuarina nodulation capacity of a tropical soil
J. F. Zimpfer1, C. M. Kaelke1, C. A. Smyth2, D. Hahn3 & J. O. Dawson1,41University of Illinois, Department of Natural Resources and Environmental Sciences, Urbana, IL 61801, USA.2University of Illinois, Department of Crop Sciences, Urbana, IL 61801, USA. 3Department of Chemical Engin-eering, New Jersey Institute of Technology (NJIT), and Department of Biological Sciences, Rutgers University, 101Warren Street, Smith Hall 135, Newark, NJ 07102-1811, USA. 4Corresponding author
Received 19 July 2002. Accepted in revised form 20 August 2002
Key words: actinorhizae, Casuarina, CjI82 001, Frankia, synergism, symbiosis
The effects of soil biota, Frankia inoculation and tissue amendment on nodulation capacity of a soil was invest-igated in a factorial study using bulked soil from beneath a Casuarina cunninghamiana tree and bioassays withC. cunninghamiana seedlings as capture plants. Nodulation capacities were determined from soils incubated insterile jars at 21 C for 1, 7, and 28 days, after receiving all combinations of the following treatments: steampasteurization, inoculation with Frankia isolate CjI82001, and amendment with different concentrationsof Casuarina cladode extracts. Soil respiration within sealed containers was determined periodically during theincubation period as a measure of overall microbial activity. Soil respiration, and thus overall microbial activity,was positively correlated with increasing concentrations of Casuarina cladode extracts. The nodulation capacityof soils inoculated with Frankia strain Cj82001 decreased over time, while those of unpasteurized soils withoutinoculation either increased or remained unaffected. The mean nodulation capacity of unpasteurized soil inoculatedwith Frankia CjI82001 was two to three times greater than the sum of values for unpasteurized and inoculatedpasteurized soils. Our results suggest a positive synergism between soil biota as a whole and Frankia inoculumwith respect to host infection.
In their natural habitats, actinorhizal plants usuallyform root nodules in symbiosis with the nitrogen-fixing actinomycete Frankia enabling them to growon sites with low nitrogen availability (Chapin et al.,1994; Dawson, 1992; Dommergues, 1997; Shumway,2000). Root nodule formation on actinorhizal plants islargely determined by environmental factors such asthe soil pH (Crannell et al., 1994; Griffiths and Mc-Cormick, 1984; Zitzer and Dawson, 1992); the soilmatric potential (Dawson et al., 1989; Nickel et al.,1999, 2001; Schwintzer 1985); and the availability ofelements such as nitrogen (Kohls and Baker, 1989;Thomas and Berry, 1989) or phosphorus (Sanginga
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et al., 1989; Yang, 1995); and the genotypes of bothpartners of this symbiosis (Hall et al., 1979; Huguet etal., 2001; Prat, 1989).
Frankia strains occupy at least two distinct eco-logical niches, the root nodule and the soil. Whilea considerable amount of information is available onFrankia strains isolated from root nodules and ontheir interaction with their host plants (see Bensonand Silvester, 1993; Huss-Danell, 1996 for reviews),much less research has been conducted on Frankiapopulations in soils. For the frankia/actinorhizal plantsymbiosis to occur the bacteria must maintain in-fective populations within the soil biotic communityand establish themselves competitively in host rhizo-spheres. This process and its underlying mechanismsare poorly understood.
2Studies of Frankia populations in soil have untilrecently been based solely on plant bioassays in whicha quantification of the nodulation capacity on a spe-cific host plant (expressed as nodulation units g1 soil)is used to estimate the infective Frankia population.Such analyses have shown that soil near actinorhizalhosts usually has greater nodulation capacity thansurrounding soils (Jeong and Myrold, 2001; Smolan-der, 1990; Zimpfer et al., 1999). However, infectivefrankiae are found in a wide variety of soils, includ-ing those without actinorhizal plants (Burleigh andDawson, 1994; Lawrence et al., 1967; Maunuksela etal., 1999, 2000; Paschke and Dawson, 1992a; Zimpferet al., 1997). The widespread occurrence of Frankiastrains capable of nodulating Alnus, Myrica, Dryas,and Elaeagnus in many soils lacking an actinorhizalhost suggests that soil biotic communities are not de-leterious to frankiae and that these frankiae have thecapacity to grow saprophytically (Maunuksela et al.,1999, 2000; Nickel et al., 1999, 2001).
Soil properties and compounds in plant tissue havebeen shown to increase the nodule-forming capacityof Frankia in soils (Benson and Silvester, 1993; Bur-leigh and Dawson, 1994; Gauthier, 2000; Nickel,2000; Paschke and Dawson, 1992b; Smolander et al.,1990). For frankiae of the Alnus host infection group,for example, flavonoid-like compounds isolated fromseeds of Alnus have been shown to enhance nodula-tion (Benoit and Berry, 1997). Numerous reports haveindicated that plants can selectively favor growth ofcertain bacteria in soil (Elo et al., 2000; Latour etal., 1996, 1999; Lemanceau et al., 1995; Maunukselaet al., 1999; Wilkenson et al., 1994). Nodule form-ation by frankiae, for example, on axenically grownalder seedlings increased due to co-inoculation withBurkholderia cepacia or other unidentified bacteria(Knowlton et al., 1980).
The purpose of this study was to determine the ef-fects of soil biota, Frankia inoculation and host tissueamendment on the nodulation capacity of a tropicalsoil by frankiae of the Casuarina host infection group.In contrast to frankiae of the Alnus and Elaeagnus hostinfection groups, frankiae of the Casuarina host in-fection group are usually not found outside the nativerange of Casuarina trees. They have also been shownto be localized near host plants when introduced as anexotic (Diem and Dommergues, 1990; Zimpfer et al.,1999). The lack of Casuarina-infective Frankia bey-ond the zone of host influence (Zimpfer et al., 1999)suggests that the Casuarina host may influence growthand infectivity of its symbiont. This assumption is
supported by previous studies in which the nodulationcapacity was higher in a soil inoculated with extractsof Casuarina cladodes and a Frankia isolate than inthe same soil without cladode extracts (Zimpfer et al.,1999). We hypothesized that host tissue amendmentand the soil biotic community would increase infectionof a Casuarina host by Frankia.
We investigated the effect of soil biota, Frankiainoculation and tissue amendment on nodulation ca-pacity of soils in a factorial study using soil samplesfrom beneath a Casuarina cunninghamiana tree andbioassays with C. cunninghamiana seedlings as cap-ture plants. Nodulation capacities were determinedfrom soils incubated in sterile jars at 21 C for 1, 7, and28 days, after receiving all combinations of the follow-ing treatments: steam pasteurization, inoculationwith Frankia isolate CjI82001, and soil amendmentwith different concentrations of Casuarina cladode ex-tracts. Soil respiration within sealed containers wasdetermined periodically during the incubation periodas a measure of overall microbial activity.
Materials and methods
Soil was collected 5 m from a mature C. cunninghami-ana tree located in Robins Bay, St. Mary, Jamaica(76 47 52 W, 18 17 50 N). The soil was a sea-wall stony clay, described as thin brown or reddishsoil on hard coral limestone with poor water reten-tion (Vernon, 1960) and 4% organic matter (Zimpferet al., 1999). At the time of collection, soil sampleswere bulked in sterile plastic bags and air-dried for oneweek by enclosing opened plastic bags within a paperbag to minimize aerial contamination.
After drying, the sterile bags were resealed, re-moved from the paper bags, and stored at room tem-perature for six months. The soil was sieved through a3-mm mesh screen before 25 g were added to sterile473 mL Mason jars (Ball Corp. Muncie, Indiana).In a factorial design, jars filled with soil receivedthe following treatments: steam pasteurization at225 C for 1 h, 0.2 mL packed cell volume ofFrankia strain CjI82001, the addition of 15 mL ofan aqueous Casuarina cladode solution containing thefresh weight equivalent of 0, 5, or 50 g of fresh groundCasuarina cladodes L1.
Frankia strain CjI82001 belonging to the Casu-arina host infection group (Diem et al., 1983), was
3grown in P+N medium with propionic acid as thecarbon source (Burggraaf and Shipton, 1982). Beforeinoculation, Frankia cells were washed three times insterile 1% saline solution and harvested by centrifu-gation at 650 g in a clinical centrifuge for 15 min.Frankia cell clumps were subsequently homogenizedusing a sterile glass tissue grinder.
Aqueous extracts of C. cunninghamiana cladodeswere prepared by homogenizing fresh green cladodesin a blender, followed by filtering through cheese clothand filter paper and sterilized by passing through a0.2 m filter.
Frankia strain CjI82001 and tissue extracts of soilswere inoculated onto soils. Three replicates of eachtreatment combination were incubated for 1, 7, and 28days at 21 C, yielding a total of 108 jars of incubatedsoil samples.
Determination of overall microbial activity
Carbon dioxide concentrations in the incubation jarswere measured according to Zibilske (1994) 1, 3, 7,14, 21, and 28 days after initiation of incubation as ameasurement of overall soil microbial activity. Holesin the lids of the Mason jars were fitted with serumstoppers to allow sealed incubation and sampling ofCO2. The concentration of CO2 in 0.5 cc of gas fromthe sealed jars was determi