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 determined by gas chromatography(Hewlett-Packard, 5890 GC fitted with a TCD, Avon,PA) on a Porapack N 80/100 mesh column (AlltechAssociates, Deerfield, IL). After each sampling, jarswere opened, equilibrated with air, and resealed.
Determination of nodulation capacity
Seeds of Casuarina cunninghamiana were surfacesterilized in an aqueous mixture of 0.5% v/v NaOClfor 3 min followed by 5 rinses with deionized H2Oprior to planting into 414 cm Cone-tainers (Stueweand Sons, Corvallis, OR) filled with a 1:1:1 mixtureof fine vermiculite, mixed sand, and fine gravel. Theseedlings were grown in the greenhouse at a tem-perature of about 23 C and a photoperiod extendedto 16 h using 1000-watt high-pressure sodium lightsplaced 1.5 m above the plants when ambient lightintensity fell below 400 mole m2 s1 PPFD. Theplants were watered weekly with 1/8-strength nutrientsolution containing 0.179 m NH4NO3 (Huss-Danell,1978). Two weeks prior to inoculation, the nitrogensupply was removed from the nutrient solution to fa-cilitate nodulation. After 12 weeks, at which time the
roots of the seedlings fully occupied the growing me-dium within each tube, the plants were inoculated withserial dilutions of incubated soil.
For the serial dilutions, 300 mL of deionized H2Owas added to the content of each jar and the result-ing slurry was mechanically stirred for 5 min, afterwhich the contents were filtered through cheese-cloth.From these solutions six ten-fold dilutions were pre-pared and applied to the capture plants. Ten ml of theinoculum were applied to each plant, with three plantsinoculated at each dilution level.
Twelve weeks after inoculation the plants wereharvested and the root nodules counted. Standard re-gression with the intercept forced through 0 was usedon the linear portion of the data to determine the num-ber of nodules formed per gram of substrate applied.One nodule was assumed to represent one infect-ive unit of Frankia. From these data the number ofnodules formed per gram of incubated soil in each in-cubated jar was calculated according to the methods ofPaschke and Dawson (1992b).
Analysis of variance (ANOVA) using SAS ProcMixed (Littell et al., 1996) was performed to detectsignificant treatment and interaction effects for eachincubation period. Proc Mixed with the repeated op-tion and elimination of control treatments with meansof 0.0 were necessary to comply with the statisticalrequirement that treatment variance not be hetero-genous. Differences between treatment combinationmeans were determined using the least squares meansmethod (p < 0.05).
Determination of overall microbial activity
Carbon dioxide evolution was positively correlatedwith the concentration of cladode tissue present in theextracts, with highest values measured 1 day after ini-tiation of the experiment (Figure 1). For unpasteurizedsoils incubated for one day, soils amended with C.cunninghamiana cladodes in aqueous solution at con-centrations of 5 or 50 g L1 evolved approximately16 and 33 mmol of CO2 [kg soil]1 day1, respect-ively. Soils not amended with cladode tissue extractshad a much lower rate of CO2 evolution (11 mmol[kg soil]1 day1). Pasteurized soils had the lowestvalues of CO2 evolution with less than 1 mmol kg1of soil day1 (Figure 1). Inoculation of Frankia strainCjI82001 did not have a significant effect on carbon di-
4Figure 1. Periodic carbon dioxide evolution rates of soil amended with C. cunninghamiana cladodes in aqueous solution (0,5, or 50 g L1)(n=3). Additional treatments included soil pasteurization and subsequent inoculation of Frankia strain CjI82001, or unpasteurized soil with orwithout inoculation of Frankia strain CjI8200l.
oxide production. Values of carbon dioxide productiondecreased in all treatments over time (Figure 1). After7 days of incubation, the carbon dioxide evolutionrate from unpasteurized soils without cladode tissueextracts was only 80% of the initial CO2 evolutionrate, while that from unpasteurized soils with aqueouscladode extracts at concentrations of 5 and 50 g L1was reduced to 67% and 60% of initial values, respect-ively. After incubation for 28 days, CO2 evolutionfrom unpasteurized soils without cladode tissue ex-tract amendment was 50% of the initial value and thatfrom soils incubated with extracts from solutions of5 and 50 g of cladode tissue L1 was 50% and 35%,respectively.
Determination of nodulation capacity
At the time of harvest the C. cunninghamiana seed-lings used as bioassay capture plants displayed darkgreen cladodes when they were nodulated, indicat-ing the presence of nitrogen-fixing Frankia strains.Cladodes of unnodulated plants, however, werechlorotic indicating nitrogen deficiency. None of theplants used for the analysis of pasteurized soils be-came nodulated, except for those for which soils wereinoculated with Frankia strain CjI82001. Therefore,
the nodulation capacity of pasteurized soils withoutinoculated Frankia strain CjI82001 could not be in-cluded in the statistical analyses which cannot haveheterogenous variation about treatment means.
For soils incubated for one day, the presence of anintact soil biotic community (p
5Table 1. Summary ANOVA table of Casuarina-infecti...