Plant and Soil 171: 359-363,1995. © 1995KluwerAcademicPublishers. Printedin theNetherlands.

Short communication

Studies of two Frankia strains isolated from Trevoa trinervis Miers

Alic ia Carrasco , James R. Sa lyards and Al i son M. Ber ry Department of Environmental Horticulture, University of California, Davis, California 95616-8587, USA.

Received 6 May 1994. Accepted in revised form 15 September 1994.

Key words: antibiotic resistance, carbon source, Frankia, Trevoa trinervis

Abstract

The Frankia strains TtI 11 and TtI 12 isolated from T. trinervis Miers were characterized regarding their carbon source utilization, intrinsic antibiotic resistance, infectivity, and effectivity on the original host. Both strains grew on BAP medium supplemented with glucose, maltose, and sucrose, but differed in their ability to use other carbon sources such as propionate, pyruvate, acetate, succinate, citrate, and mannitol.

The isolates were sensitive to five of the twelve antibiotics tested at 1 #g mL-1 concentration: chloramphenicol, tobramycin, eritromycin, streptomycin, and rifampicin. They exhibited a variable degree of resistance at 1 /zg mL- 1 concentraction to penicillin G, 4-fluorouracil, oleandomycin, and lincomycin.

Both isolates were able to infect and nodulate the original host plant, and thus represent the first reported infective and effective microsymbionts for T. trinervis Miers, a rhamnaceous actinorhizal host.

Introduction

Frankia is an actinomycetal genus whose members have the ability to form nitrogen-fixing root nodules in association with genera in eight angiosperm fam- ilies (Baker and Schwintzer, 1990; Newcomb and Wood, 1987). Both the nodules induced by Frankia and the species of plants which bear these nodules are termed actinorhizal (Tjepkema and Torrey, 1979). Seven genera ( Colleta, Trevoa, Retanilla, Talguenea, Kentrothamnus, Ceanothus and Discaria) in the fami- ly Rhamnaceae have been reported to bear actinorhizal nodules (Balboa et al., 1988; Balboa, 1989; Carrasco et al., 1992; Caru, 1993; Longeri and Abarzua, 1989; Medan and Tortosa, 1976; Medan and Tortosa, 1981; Silvester et al., 1985)

Members of the genus Trevoa are restricted to the Pacific coast of South America, with five species in Chile and one in Peru (Navas, 1976). Trevoa trinervis Miers is widespread in the matorral zone of central Chile, being characterized by its fast growth on dis- turbed sites (primary colonizer) and for its nitrogen- fixing capacity, adding nitrogen to the soil (Rundel and Neel, 1978; Silvester et al., 1985).

We have previously reported the isolation of Frankia strains TtI 11 and TtI 12 from T. trinervis Miers nodules (Carrasco et al., 1992). Recently, Caru (1993) reported the isolation of another Frankia strain from the same actinorhinal host. The ultrastructural characterization showed that the microorganisms iso- lated have the morphological features accepted for the genus FranMa, i. e. prokaryotic septate hyphae, stalked spherical symbiotic vesicles, and polymorphic sporan- gia.

In the present communication we report the char- acterization of the strains TtI 11 and TtI 12 regarding their in vitro culture carbon source requirements, the background sensitivity to selected antibiotics, and their infectivity and effectivity on the T. trinervis Miers host plant.

Materials and methods

Bacterial strains Frankia sp TtI 11 and TtI 12, isolated from the root nodules of T. trinervis Miers as described previously (Carrasco et al., 1992) were used in this study. Strain

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BR, isolated from Casuarina equisetifolia and provid- ed by Dr H G Diem was used as standard (unpub- lished).

Inocula Bacteria were grown at 28°C in BAP medium, a defined mineral medium containing 5.0 mM NHaC1 and 5.0 mM Na-propionate as the only nitrogen and carbon sources respectively. Cells were collected by centrifugation (1,700 g, 10 rain, 4°C), washed and resuspended in BAP, homogenized by repetitive pas- sages through a 0.7-mm needle, and used as inoculum at 2/,g protein per mL of culture medium. The basal medium (BAP), modified from Murry et al. (1984) con- tained (g L -1) MgSOa.7H20, 0.05; CaCI2.2H20, 0.01; NH4C1, 0.267; K2HPO4, 0.3; Na2HPO4.2H20, 0.2 and FeNaEDTA, 0.01. Trace elements and vitamins were added as described by Shipton and Burggraaf (1982). The vitamins, trace elements, and phosphate buffer (10 mM) were autoclaved separately; pH of the media was 6.8. All carbon sources (propionate, pyruvate, glucose, maltose, sucrose, mannitol, D-arabinose, acetate, suc- cinate, citrate) were filter-sterilized, excepting soluble starch and added at 3.6 g C L - 1 final concentration.

Growth Cells for growth, background resistance to antibiotics, carbon source requirements, and infectivity experi- ments were grown at 28°C in 100-250-mL cylindrical bottles. Growth was measured as #g protein m L - 1 by the Bradford procedure, using cells that were sonicated (60 s at 100W) with a 250 Branson Sonifier, and boiled for 10min in 0.3 NNaOH.

Nitrogenase activity Nitrogenase activity was measured under air with the acetylene reduction test (Postgate, 1982). Nodules of each root system were incubated in 9.5-mL vials in mixtures of 90% air and 10% acetylene. Acetylene reduction activity was measured after 0.5 and 1 h incu- bation and expressed as #M of C2H4 produced per hour and per mg of dry nodule. Gas chromatography with flame ionization detection was done by injecting 0.2- 0.5-mL samples into a column (1.8m x 3mm) packed with Porapak N (80-100 mesh).

Infection test Seeds of T. trinervis Miers were collected from plants growing in the fields near Santiago, Chile (Caj6n del Maipo, 50 km southeast of Santiago). Seeds were

Table 1. Hydrocarbon effect on growth of Frankia strains TtI 11 and TtI 12 a

Growth (t*g Protein mL - l ) Carbon source TtI 11 TtI 12

None 1.17 4- 0.05 1.33 -4- 0.06 Acetate 4.17 4- 0.09 7.88 4- 0.10 D-arabinose 1.13 4- 0.11 N.D. Citrate 1.42 4- 0.15 3.70 4- 0.21 Glucose 8.06 4- 0.35 6.86 4- 0.29 Maltose 5.08 4- 0.25 5.80 4- 0.19 Mannitol 1.19 4- 0.09 3.56 4- 0.24 Propionate 18.81 4- 0.25 5.32 4- 0.20 Pyruvate 5.86 4- 0.22 12.12 4- 0.20 Soluble starch 7.98 4- 0.29 N.D. Succinate 0.95 4- 0.08 4.86 4- 0.16 Sucrose 5.73 4- 0.21 13.34 4- 0.17

aThe cultures were incubated at 28°C, during six days with occasional agitation. The carbon sources (filter-sterilized) were added at 3.6 g C L -1 to the basal BAP medium. Values are the mean of three independent determinations 4- standard deviation.

germinated and seedlings grown in moist peat moss or vermiculite. About six weeks after sowing the seeds, at the time of differentiation of the second true leaf, seedlings were transferred to pots contain- ing peat-sand-fir bark (1/3 each) (UC mix) and grown in a controlled environment (day temperature 23.8°C, night temperature 18.3°C). The 6-week-old plantlets were supplied with 1/10 strength Hoagland's solution (Hoagland and Arnon, 1938). Each plant was inoculat- ed with 1 mL of a suspension of washed, homogenized hyphae and spores from 4-week-old cultures (1-mL packed cells per 100 mL water). Two to three months later, the root systems were examined for the presence of nodules.

Results and discussion

Carbon source utilization The ability of the Frankia isolates TtI 11 and TtI 12 to utilize individual carbon sources in the basal media is summarized in Table 1. At the concentration tested (3.6 g CL- 1), isolate TtI 11 used acetate, glucose, maltose, propionate, pyruvate, sucrose and soluble starch. Iso- late TtI 12 used acetate, citrate, glucose, maltose, man- nitol, propionate, pyruvate, sucrose and succinate.

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Table 2. Backgroundresistance to selected antibiotics of Frankia strains TtI 11 and TtI 12

Antibiotic Conc(tzg m L - 1 ) Growth a %b Growth a %b

(~g Protein m L - l) Inhibition (#g Protein m L - I) Inhibition

TtI 11 Ttl 12

None 20.43 4- 0.48 0 18.80 4- 0.23 0

Nalidixic acid 5 21.80 4- 0.42 0 18.82 4- 0.25 0

10 21.80 4- 0.42 0 18.65 4- 0.21 0

Cycloheximide 20 21.20 4- 0.26 0 18.30 4- 0.30 0

Chloramphenicol 1 N.D. ° N.D. d 3.46 4- 0.09 81.6

10 0.50 4- 0.035 98.6 1.16 4- 0.08 93.9

Erithromycin 1 1.28 4- 0.02 93.8 2.16 4- 0.37 88.6

5-Fluorouracil 1 18.16 4- 0.02 12.2 3.13 4- 0.04 83.4

Kanamycin c 1 0.87 4- 0.03 92.8 N.D. d N.D. d

10 0.04 4- 0.06 99.8 2.57 4- 0.40 86.4

Lincomycin 1 18.12 4- 0.36 11.5 19.47 4- 0.31 0

10 0.7 + 0.02 92.6 N.D. d N.D. d

Oleandomycin 1 12.28 4- 0.18 40 18.67 4- 0.15 0.7

10 1.49 4- 0.06 92.8 N.Dfl N.D. d

Penicillin G c 1 16.32 4- 0.23 20.1 11.65 4- 0.15 38.3

10 1.43 4- 0.016 93.1 0.93 4- 0.02 95.1

Rifampicin c 1 1.57 4- 0.04 92.4 1.56 4- 0.03 91.8

10 0.05 4- 0.04 97.6 0.03 4- 0.02 99.8

Streptomycin c 1 0.87 4- 0.03 95.2 1.23 4- 0.035 93.5

10 0.87 4- 0.03 95.2 N.D. d N.D. d

Tobramycin 1 0.68 4- 0.01 96.7 1.84 4- 0.018 90.3

10 0.02 + 0.01 99.9 N.D. d N.Dfl

The cultures were grown in BAP medium with Na-propionate (5 mM) and NH4C1 (5 mM) and incubated at 28°C during 6 days with occasional agitation. Antibiotics were added to the medium at the indicated concentration. aGrowth is expressed as total growth measured as total soluble protein by the Bradford method minus inoculum (2 /zgP mL-1) . b% inhibition is expressed as

Growth A 100 - - x 100

Growth B Growth A = growth in the presence of antibiotics. Growth B = growth in the absence of antibiotics. CLysis dN D = not determined.

The isolates were metabolically versatile using a relatively ample range of substrates for growth. They seem to be particularly adept at sugar utilization. In this character, they are similar to the strains EANIpec and EuII studied by Tisa et al. (1983).

Growth was evident at the relatively high substrate concentration used, which may be indicative of either a low affinity of the transport metabolic system or a low efficiency as energy source. Propionate at the concen- tration tested was the best carbon source for strain TtI 11. Strain TtI 12 grew better with pyruvate at the same concentation. We performed a concentration growth experiment with propionate and pyruvate at concen- trations ranging from 0 to 18 g C L - 1 with the two iso-

lares. The results (not shown) confirmed that strain TtI 11 grows better than TtI 12 with propionate in the range of 0.18-0.72 g C L -1 . Strain TtI 12 utilized pyruvate optimally at a higher concentration (> 1.8 g C L-1). Shipton and Burggraaf (1982) showed that growth of Frankia was sensitive to relatively small changes in concentration of propionic acid in the case of strains AvcI 1, CpI 1 and LDagp 1. The optimum concentration was about 0.2 g C L - 1

The hydrocarbon sources used in this study were filter-sterilised so that, apart from the possibility of trace amounts of contaminating sugars, the data relate to sugar utilization and not to degradation products.

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Table 3. Infectity and effectivity of Frankia strains TtI 11 and Tt112 on T. trinervis Miers

Strain % # Nodules/plant a Effectivity b infection

Tt111 95 (n = 20) 6.5 + 0.6 120.1:1:1.9 Tt112 100 (n = 4) 4.5 4- 1.6 54.2 :t: 1.2

aAverage of nodules per infected plant 4- standard error. b Nodule nitrogenase activity expressed as nM C2H4 mg- 1 DW nodule h- l + standard error.

Antibiotic resistance The background resistance of the two Frankia iso- lates to twelve antibiotics is shown in Table 2. Strains TtI 11 and TtI 12 were sensitive to 1 #g mL - l of chloramphenicol, tobramycin, erithromycin, strepto- mycin, and rifampicin. They showed a variable degree of resistance at the 1 #g mL- 1 level concentration to penicillin G, 5-fluorouracil, oleandomycin, and lin- comycin. The background resistance to nalidixic acid was high, thereby making its potential as marker in mutant selection not useful. The standard strain BR was resistant to kanamycin, rifampicin, nalidixic acid, and chloramphenicol at 1 and 5 /~g mL- t (data not shown). As expected for prokaryotic microorganisms, cycloheximide was not inhibitory. Both isolates and the strain BR were resistant to nystatin (10-50/~g mL- 1 ) and chlorpromazine (10-20/~g mL- 1). (Data not shown.)

The background resistance of different Alnus and Elaeagnus Frankia strains to various antibi- otics has been examined (L Simon, in Normand and Lalonde, 1986). A very high background resistance was observed to D-cycloserine, ampicillin, penicillin G, rifampicin, and tetracycline. On the other hand, the background resistance was minimal to gentamycin, kanamycin, streptomycin, vancomycin and thiostrep- ton.

Our results can not be compared with Simon's results because the growth conditions were quite dif- ferent. We tested the background resistance at 6 days of incubation in a defined medium, and their results were obtained after 60 days of incubation in complex QmodB medium. In such a long period of incubation, the antibiotics may have been inactivated or decom- posed. Recently, Caru (1993) reported the background resistance to antibiotics of nine Frankia strains isolated from several genera of the family Rhamnaceae. Those isolates were sensitive to the antibiotics tested at 5 #g mL- 1 concentration.

The knowledge of intrinsic antibiotic resistance is useful in genetic work, specifically in cloning because it allows the researcher to use positive selection. Also, it enables one to look for specific genetic markers in mutagenesis experiments. Recently, we have obtained rifampicin resistant mutants (rif R) after mutagenizing hyphae fragments (strain TtI 11) with nitrosoguani- dine.

Infectivity In Table 3 are listed the results of inoculation of T. trinervis Miers with the Frankia strains TtI 11 and TtI 12. The results indicate that both Frankia strains have the ability to infect and nodulate the original host, the percentage of infection being of 95% for TtI 11, and 100% for the TtI 12 isolate. The nodules induced on T. trinervis Miers root systems were of the same external morphology described in a previous article (Carrasco et al., 1992), i.e. clusters of coralloid root nodules with a dichotomous branching pattern.

Samples of Trevoa nodulated root system showed positive acetylene reduction (Table 3), indicating a clear pattern of active nitrogen fixation. Acetylene reduction rates ranges from 105.8-135 nM C2H4 mg- 1 DW nodule h-1 in the case of strain TtI 11 (based on 1-h subsarnples). Acetylene reduction activity was restricted to nodulated tissue. Non-nodulated root tis- sue of comparable size showed no acetylene reduction activity.

The positive effect of the symbiotic association on T. trinervis Miers was evident by the successful growth of the plants that turned healthy green and showed good shoot elongation (not shown). The total growth of each inoculated plant was at least twice as much as the growth of each control plant.

Since the Frankia isolates TtI 11 and TtI 12 were able to infect the host plant, both can be considered as microsymbionts of T. trinervis Miers. The symbiotic association was also an effective one, since nodules were able to reduce acetylene. To our knowledge, these are the first infective (and effective) isolates from the genus Trevoa.

While isolated strains have been reported to nodu- late effectively in several of the actinorhizal host fam- ilies (Torrey, 1990), only one rhamnaceous Frankia isolate has been shown to nodulate not only its own host but also species of the Elaeagnaceae (Gauthier et al., 1984). Such cross-inoculation studies in the family Rhamnaceae are scanty (Torrey, 1990). The infective strains described by us will be, without doubt, quite

useful in the further definition of cross-inoculation groups. We are currently testing the Trevoa isolates on several other actinorhizal plants within the Rham- naceae, and in other actinorhizal families.

Acknowledgements

Part of this research was done at the Faculty of Sci- ences, University of Chile, Santiago, Chile.

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Section editor: R 0 D Dixon