Specificity and effectivity in nodulation by Frankia on southern hemisphere actinorhiza

ELSEVIER FEMS Microbiology Letters 12.5 (1995) 231-236

Specificity and effectivity in nodulation by Frankia on southern hemisphere actinorhiza

Anita Sellstedt a,b,*

a Plant Molecular Biology, Department of Plant Physiology, Unic,ersily of Lime& 901 X7 UmeB, Sweden ’ CSIRO, Davies Laboratory, Division of Soils, PMB, PO Aitkenl,ale. Queensland 4814, Australia

Received 23 August 1994; revised 25 October 1994; accepted 11 November 1994

Abstract

Nodulation ability was tested for Fran/& strains HFPCcI3 and ELl, and Frankia sources A.t. and G.a. from Allocasuarina torulosa and Gymnostoma australianum, respectively, on A. torulosa Miq., Casuarina cunninghamiana Miq., G. australianum L. Johnson and Elaeagnus triflora Roxb. It was shown that A. torulosa and C. cunninghamiana formed

nodules only with the Frankia sources obtained from their own host plant, while E. triflora formed nodules with three of the four Frankia sources tested. All nodules formed were effectively fixing nitrogen. Specific nitrogenase activity was highest in E. triflorn inoculated with the Frankia strain isolated from nodules of the same species. Identification of Frankia sources in the nodules was performed by use of PCR amplification of DNA with a random primer. PCR amplification of DNA isolated from nodules of G. australianurn and E. triflora inoculated with Frunkia strain ELI revealed, when compared with DNA

amplified from free living Frankia strain ELl, that there was only one Frankia strain causing the observed nodules.

Keywords: Acetylene reduction; Actinorhizal plants; Frankia; Nodulation; PCR amplification

1. Introduction

The actinomycete Frankia forms nodules with a large number of woody species, including temperate, tropical and subtropical species [l]. The most important actinorhizal family in tropical and subtropical areas are the Casuarinaceae. The genus Casuarina is economically important in e.g. wood production and land reclamation. Many species of Casuarinaceae are particularly suitable as they have been shown to grow well on saline and arid as well as on infertile soils.

* Corresponding author (Urnel address).

Several strains have been isolated from nodules of Casuarinaceae from all over the world since 1981 [2]. Studies on cross-infectivity among actinorhizal

species and Frankia have up to very recently been concentrated on temperate species [3-51, with very few southern-hemisphere species and Casuarina-

compatible strains represented [6-81. Baker [3] iden- tified four host infectivity groups amongst Frankia strains: (i) strains which nodulate Alms and Myrica;

(ii) strains which nodulate Casuarina and Myrica; (iii) strains which nodulate Elaeagnaceae and Myrica; and (iv) strains which nodulate only Elaeagnaceae [3]. Other researchers [4] only specified three groups. Studies of cross-infectivity demonstrated that a sin- gle Frankia could infect more than one genus of

0378-1097/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved SSDI 0378.1097(94)00503-6

232 A. Sellstedt / FEMS Microbiology Letters 125 (I 995) 231-236

actinorhizal plants [9-111. Also, it has been sug-

gested that some nodules from Casuarinaceae might contain more than one Frankia strain [12]. This is interesting, as it shows the complexity of symbiotic

systems. In addition, results in inoculation trials where no

nodulation has been obtained are difficult to interpret

[6]. However, growth substrates are believed to play an important role in inoculation trials [6]. Liquid cultures are sometimes found to be difficult in medi-

ating inoculation, while nodulation of roots growing in solid growth substrates is more often successful

[13]. In the present study a solid substrate was used

to enhance the probability for nodulation.

2. Materials and methods

2.1. Plant material

Seeds of A. torulosa, C. cunninghamiana, E.

triflora and G. australianurn (Table 1) were surface-

sterilized (30% H,O,, (v/v), 15 min) and rinsed 3 x 15 min in sterilized deionized water. After ger-

mination of the seeds, the seedlings as well as rooted cuttings from E. triflora and G. australianurn were planted into 600-ml free-draining pots containing a peat/vermiculite (2/l (v/v)) mixture which had

been limed to pH 6.5. One plant was transferred to each pot and was inoculated with a pure Frankia

Table 1 Actinorhizal species and Frunkia sources

Plant species Allocasuarina torulosa

Casuarina cunninghamiana

Eleagnus triflora

Gymnostoma australianurn

Frankia sources HPPCcI3 EL1

strain or a crushed nodule inoculum (Table 1) 21

days after planting. For each species, four control pots containing non-inoculated seedlings were ran-

domly placed amongst the pots with inoculated seedlings. Seedlings were kept in an air-conditioned glass-house (temperature range of 15-20°C during the night to 25-38°C during the day) under natural day light (12 k 1 h). Plants were watered daily with

deionized water and given nutrients every fortnight. Nutrients were supplied in a nutrient solution accord- ing to [14] modified to contain 0.3 pmol Co (as

CoSO, .7H,O) 1-l and 1.0 Fmol MO (as Na,MoO,) 1-l. All plants received a starter application of nitro-

gen (2 mg N per pot) to sustain growth until nodulation occurred.

Seedlings were also kept in a climate chamber with a 12-h day (25-35°C) and 12-h night (15-20°C) cycle, a light source giving 500 pmol E m-’ SK’ and a relative humidity of 80%. The climate in these

climate chambers was programmed to be similar to the natural conditions described above. Nodules from these plants were used for isolation of DNA and

PCR amplifications.

2.2. Nodule inocula

Crushed nodule inocula were prepared from frozen nodules, which were surface-sterilized as described above. Nodules were then ground for 10 s in a mortar with a pestle and subsequently filtered through

G.a.

A.t.

CSIRO, Australia CSIRO, Australia Noah Creek, Daintree, QLD Noah Creek, Daintree, QLD

Pure culture isolated from C. cunninghamiana [13]

Pure culture isolated from Elaeagnus triflora (Reddell, unpublished), nodules collected near Goolagan Creek, Northern Territory, Australia Crushed nodule inoculum from nodules of Gymnostoma

australianurn collected at Noah creek, Daintree, North Queensland, Australia Crushed nodule inoculum from nodules from Allocasuarina

torulosa, collected at Alice Springs, Northern Territory, Australia

A. Sellstedt / FEMS Microbiology Letters I25 (1995) 231 -i36 233

a 11-pm filter. Each seedling, which was kept in the

air-conditioned glass-house described above, was inoculated by syringing 2.5 ml of inocula or 2.5 ml of

a thick suspension of pure culture grown in P-medium [15], next to the base of the stem.

2.3. Assessment of nodulation

Nodulation was assessed after 21, 56, 90 and 217

days from planting by gently removing the upper layer of vermiculite/peat mixture. At day 90 and

217, plants were also analysed for acetylene-depen- dent ethylene production. Ten percent acetylene was added to a gas-tight vessel containing the intact

root-system of the plant. Production of ethylene was studied after 30 min up to 90 min. Gas samples were taken at intervals of 10 min and analysed in a Shimadzu gas chromatograph [ 161.

2.4. DNA extraction

DNA was extracted from nodules of G. aus-

tralianum and E. triji’ora containing Frankia ELl, as well as free-living Frankia strain ELl. Frunkia

preparations were made from nodules as described earlier, with the exception that a 11-pm filter was used in the last filtration step. Free-living Frankia

was grown for 14 days at 28°C in a nitrogen-containing medium [17], and for 5 days in the above-men- tioned medium without nitrogen [17]. Hyphae and

cells of Frankia were harvested by centrifugation (15 340 X g, 10 min), washed once in 1 X TE buffer (10 mM Tris (2-amino-2-(hydroxymethyl)-1,3-pro-

pandiol) pH 8.0 with 1.0 mM EDTA (ethylenedia- minetetraacetic acid). Digestion, lysis and DNA extraction was performed essentially as described be-

fore [18] and the pellet was resuspended in distilled

and sterilized water.

2.5. PCR amplification

Part of the Frank&z genome was randomly amplified using a modification of the polymerase chain reaction (PCR) as described earlier [18]. The primer used was a IO-mer oligonucleotide designated A-05 with the sequence AGGGGTCTI’G (OPERON, Operon Technologies Inc., 1000 Atlantic Ave., Alameda, CA 94501). Frankia DNA (10 ng) was

amplified essentially as described before [18]. 30 ~1 of the PCR product was analysed by electrophoresis on a 1.2% (w/v) agarose (IBI electrophoresis grade) gel.

2.6. Dry weights

After measurements of acetylene reduction plants

were separated into stem, side branches, roots and nodules. The different plant parts were dried (70°C

24 h) and weighed.

3. Results and discussion

In this study all Frankia sources except A.t.

Frankia nodulated E. trifzora (Table 2). Nodules were first observed 21 days from planting (Table 2). Seedlings of A. torulosa, C. cunninghamiana and E.

trif[ora inoculated with Frankia sources from their

own species were well-nodulated. Seedlings of C. cunninghamiana and A. torulosa inoculated with

nodule sources other than those from their own species did not nodulate. In plants of E. triflora

inoculated with EL1 and Gy 75% of the plants were nodulated. Nodulation was also checked up to 217 days after planting, but no further nodulation was observed. I used G. australianum as a representative of the genus Gymnostoma in this study, and it was shown to form nodules only with the Frankia source

isolated from Gymnostoma nodules (Table 2). A specific nodulation pattern in the genus Casuari- naceae has been suggested [6]. They also concluded that the genus Gymnostoma is not useful for cross-

Table 2

Inoculation of actinorhizal species with Frankia sources

Plant species Frankia source

HFPCcI3 A.t. G.a. EL1

A. torulosa _ 6/6 - _

C. cunninghamiana 6/6 _ _ _

E. triflora 4/6 - 4/6 6/6 G. australianurn - _ 2/6 4/6 Non-inoculated _ _ _ _

Six plants from each species were inoculated with the different

Frankia sources. 6/6 indicates that six out of six plants were

nodulated etc.; - indicates no nodules were found. Plants were

observed for nodulation from 21 to 217 days after inoculation.


inoculation trials [6] due to promiscuity. However,

this was not supported by my study. Torrey and Racette [6] explained that the reason for this unsuit- ability of Gymnostoma in cross-inoculation trials is that it represents a partial promiscuous genus within

the Casuarinaceae. Other hosts seems to be more

precise in their specification, e.g. C. cunninghamiana (this study) and A. torulosa (this study; [6].

eating that the occurrence of E. trijlora in the rain

forest is important as it can fix nitrogen and later on deliver it to the ecosystem. It was also shown that the lowest nitrogenase activity was recorded in Gym- nostoma inoculated with Gy. This is also reflected by the slow growth rate of G. australianum in its

natural habitat (data not shown).

Nodule dry weights were highest in C. cunninghamiana inoculated with HFPCcI3 and lowest in E. triflora inoculated with HFPCcI3 and EL1 (Table 3). Reddell and Bowen [12] reported that effective-

ness in nitrogen fixation was reflected by weight of nodules [9], though others [16] and this study have not confirmed this report. Interestingly, it was shown

that Frankia strain EL1 isolated from E. triflora formed nodules on its original host plant (Table 2). This is noteworthy, since it has been shown that

Frankia might lose the ability to nodulate the host

where it was isolated [7]. In addition, an attempt to assess the effectivity of the nodules was made by use of measurements of nitrogenase activities. Our analy- sis showed that the nodules formed were effective (Table 3). Specific nitrogenase activity was highest

in the E. triflora symbioses including the Frankia source isolated from its own host. There was as much as a three-fold difference in specific nitroge-

nase activity between E. triflora inoculated with EL1 and G. australianurn inoculated with Gy (Table 3). The nodules formed in the symbiosis between E. triflora and EL1 must therefore contain a lot of active tissue. This is a very interesting finding, indi-

Some species, e.g. Allocasuarina lehmania were

badly nodulated in liquid culture but well-nodulated on a solid substrate [13]. The discrepancy between nodulation in liquid and solid medium is not yet clear, but might be due to the decrease in survival of Frankia in liquid media. We have therefore used a peat/vermiculite mixture, which has been used suc-

cessfully for Casuarina species. Most species that were nodulated in this study were well-nodulated,

which might reflect the advantage of the substrate

used. Infectivity of Frankia on host plants is, however,

only one characteristic for the systematic classifica-

tion of Frankia [3,6,19]. The use of molecular biology tools could be very useful in characterizing different Frankia strains. Hybridization with nif probes has been used [8] to detect genetic diversity among Frankia isolated from Casuarina nodules.

They found two groups of isolates: some that could not re-infect Casuarina, but were infective on Hip- pophae rhamnoides, and some that could re-infect Casuarina host plants. These two groups were desig-

nated (i) Casuarina-compatible and (ii) Casuarina- isolated Elaeagnus-compatible strains. Additionally, PCR amplification of DNA with arbitrary primers

Table 3 Nitrogenase activities, nodule dry weights and specific nitrogenase activities

N, ase activity Noduie dry weight (g)

(FmolC,H, plant-’ h-‘1

C. cunninghamiana HFPCcI3 8.2 + 0.9 0.153 & 0.011

E. trijbra HFPCcI3 1.4 + 0.1 0.024 + 0.002 EL1 3.9 + 0.2 0.029 f 0.002 G.a. 2.9 + 0.1 0.067 f 0.012

G. australianurn G.a. 0.8 f 0.05 0.055 f 0.010 EL1 1.2 + 0.03 0.033 * 0.003

A. torulosa A.t. 1.9 f 0.04 0.099 + 0.011

Specific N, ase activity

( pmolCZH, (g DW)-1 h-l)

53.5 f 10.0

58.6 f 5.0

132.7 f 21.0

44.0 k 5.6

15.3 f 0.3

36.4 + 4.1

19.1 + 0.23

Values are from 217 days from inoculation (means f SE, n = 6).

A. Sellstedt / FEMS Microbiology Letters 125 (1995) 231-236 235

Kb G E F Kb Acknowledgements

12,o

370

LO

Fig. 1. PCR amplification of Frankia DNA isolated from nodules

of G. australianurn (G), E. triflora (E) and from free-living

Frankia ELI (F).

has been used on eight different Frankia strains isolated from Casuarinaceae [18]. It was clearly shown that the strains were different, and this method is particularly useful in strains where the genome is unknown, such as in newly isolated strains. PCR amplification of total Frankia DNA from isolated Frankia EL1 and Frankia EL1 extracted from nodules was used in order to reveal amount of Frankia strains in the nodules. Here it has clearly been shown that no other strain than Frankia EL1 caused the nodules on G. australianum and E. triflora (Fig. 1).

Thanks are due to Dr. Paul Reddell, for allowing

part of the work to be performed in his laboratory, and for excellent comments on the manuscript; and to the Swedish Natural Research Science Council and Swedish Council for Forestry and Agricultural

Research.

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Specificity and effectivity in nodulation by Frankia on southern hemisphere actinorhiza