Indian Journal of Experimental Biology Vol. 47, April 2009, pp. 289-297

In vitro synthesis of Frankia and mycorrhiza with Casuarina equisetifolia and ultrastructure of root system

Sanniyasi Elumalai† & Nanjian Raaman*

Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, India

Received 12 November 2007; revised 1 December 2008

Casuarina equisetifolia is one of the ecologically and economically important tropical coastal trees nodulated by nitrogen-fixing actinomycete Frankia and forming symbiotic associations with both ecto- and endomycorrhizal fungi. The present study aims at the ultrastructural study of interactions between C. equisetifolia, Frankia, and mycorrhiza. C. equisetifolia seeds were sterilised and germinated under in vitro condition. The seedlings were transferred to conical flasks containing vermiculite and saw dust with Hoagland’s solution. After 30 days, the inoculum of AM fungus ⎯ Glomus fasciculatum (A), ectomycorrhizal fungus⎯Pisolithus tinctorius (E) and actinorhizal Frankia (F) were inoculated individually and in various combinations, (A+E), (A+F), E+F) and (A+E+F). After 90 days, the experimental plant roots and nodules were harvested for assessment of growth characters of mycorrhizal and actinorhizal association by light and scanning electron microscope methods. C. equisetifolia roots were infected with arbuscles and vesicles of G. fasciculatum; P. tinctorius formed fungal sheath but no Hartig net. Large number of cortical cells were seen infected with Frankia, hyphae of Frankia were frequently seen penetrating from cell to cell directly through cell walls and Frankia occupied majority of the cell volume.

Keywords: Actinorhiza, Casuarina equisetifolia, Frankia, Glomus fasciculatum, Mycorrhiza, Pisolithus tinctorius, Ultrastructure

Casuarina equisetifolia is an exotic tree from Australia, with unquantified ecological impacts. These plantations may obliterate the natural sand dune ecosystems along the Indian Peninsular coast, which are very much important for Tsunami protection. C. equisetifolia provides a range of economical and ecological goods and services1-3. Casuarina species are unique trees, possessing mycorrhizal and actinorhizal symbionts in their roots system4. It is widely used for stabilization and restoration of soils and sand dune stabilization, tsunami protectant, as a bio-shield (or) vegetative shelter belt tree, and as a source of timber and fuel3. The mycorrhiza of actinorhizal plants is essential to obtain higher yields especially when the plants grow in the phosphorus deficient soils and coastal saline sandy soils. Atmospheric dinitrogen fixation rates of these plants are of the same order as those observed in legumes5-7. Thus rates of over 100 kgN/ha/yr, which is

approximately the amount of nitrogen added as fertilizer in intensive forest exploitations have been reported for C. equisetifolia. Conversion of coastal sand dunes to plantations has intensified dramatically after the tsunami of December 2004, driven largely by the belief that bio-shields mitigated tsunami inundation.

The roots of C. equisetifolia have been found to support ectomycorrhizae or arbuscular mycorrhizae (AM) in addition to nitrogen fixing nodules simultaneously. The presence of AM hyphae in nodular tissues of C. equisetifolia, and an ectomycorrhizal sheath around young nodules and roots improve the nitrogen fixation and phosphorus uptake. Globally, they have potential for integrating into schemes for addressing issues of shelter belts in the coastal area, reforestation, and tsunami protection8,9 .

In some actinorhizal nodules, there is no diffusion barrier between the soil atmosphere and the infected zone2,3. In nodules, intercellular air spaces extend into the zone of infected tissue and are connected to the soil atmosphere through lenticels or nodule roots. As a result, the PO2 in the infected zone may be too close to that of the atmosphere2. Casuarina may be unique among actinorhizal plants in its small number of

_________________ *Correspondent author Telephone: 91-44-22202755 E-mail: raaman55@gmail.com †Present address: Post Graduate & Research Department of Plant Biology and Biotechnology, Presidency College, Chennai 600 005, India E-mail: ananandal@yahoo.com

INDIAN J EXP BIOL, APRIL 2009

290

intercellular air spaces in the zone of initiation in Gymnostoma (Casuarinaceae) induced by Frankia10. Evidence that initial penetration occurs through root hair deformation, first presented in Alnus glutinosa11,12 showed root hair infection in the nodulation of Comptonia peregrina. This was also confirmed for two species of Myrica and Casuarina11. The nodule is bounded by a cork periderm, which contains lenticels through which gases can exchange with atmosphere.

Ectotrophic mycorrhizal association of Pislithus tinctorius with roots of Casuarina distyla has been reported13. A survey made in C. glauca and C. equisetifolia roots in both wet and dry sites exhibited the presence of both ectotrophic and endotrophic mycorrhizae14. The role of Casuarina as a pioneering plant and Tsunami protecting tree may be assisted by the presence of mycorrhiza on the root system8,9. The results of dual inoculation of Frankia (crushed nodules) and Pisolithus tinctorius or Rhizopogon luteolus (spores) in C. equisetifolia revealed that nodulation and biomass production could be increased than the individual spore inoculation4. Growth and root structure of Eucalyptus, Allocasuarina and Casuarina seedlings inoculated with ectomycorrhizal fungi, Pisolithus tinctorius and Scleroderma cepa were reported5. Therefore, the present investigation aims at the effect of an endomycorrhizal fungus Glomus fasciculatum, an ectomycorrhizal fungus Pisolithus tinctorius and Frankia on the growth performance of C. equisetifolia seedlings under controlled conditions and ultrastructure of root system. Materials and Methods

In vitro synthesis of mycorrhiza and Frankia with C. equisetifolia—Healthy seeds of C. equisetifolia obtained from Forest Department, Government of Tamil Nadu were surface sterilized with 30% H2O2 for 15 min and thoroughly washed thrice with distilled water. The seeds were kept in moist petri plates. Seeds were germinated in a chamber 16:8 hr L: D photoperiod at 24° ± 1ºC in a controlled environment. After 20 days, the seedlings were transferred to 500 ml conical flasks containing sterilized vermiculite: sawdust (6:1) and half strength Hoagland’s solution. After 30 days, the inoculum of Glomus fasciculatum (A), Pisolithus tinctorius (E), and Frankia (F) were inoculated individually and in various combinations: (A+E), (A+F), (E+F), and

(A+E+F). Distilled water (1 ml) containing 100 spores of G. fasciculatum , 8 mm diam of P. tinctorius fungal mycelium and 1 ml pack cell volume of Frankia cells was used as inocula (all the cultures used in this study were taken from Dr. N. Raaman’s Lab). AM (arbuscular mycorrhizal) spores were isolated by wet sieving and decanting method followed by Schenck and Perez15. Ectomycorrhizal fungus was identified based on the morphological characters of fruit bodies16,17. The flasks were incubated at 25° ± 1°C with 16 hr of light. Suitable controls were maintained. Five replicates were kept for each treatment. After 3 months, the plants were harvested for further ultrastructural studies.

The data were analysed in PC/AT computer using statistical packages for social sciences (SPSS Version:11) software. 23 – Factorial data were subjected to analysis of variance. The influences of treatments of G. fasciculatum, P. tinctorius, Frankia, A+F, A+E, E+F, and A+E+F on C. equisetifolia were analysed. The significant effects were calculated by least square different (LSD) test.

Pure culture synthesis of P. tinctorius with C. equisetifolia—One set of P. tinctorius inoculated plants was tested for pure culture synthesis of ectomycorrhizal fungi with the C. equisetifolia. After 3 months (90 days), the ectomycorrhizal roots formed by P. tinctorius on C. equisetifolia were removed for further investigation. They were washed thoroughly in running tap water, then with distilled water and cut into 1 cm pieces. The root pieces were surface sterilized with 6% sodium hypochlorite and 95% ethanol and rinsed in sterile distilled water. The root pieces were plated on MMN (Modified Melin Norkrans) medium in sterilized petri plates18. The plates were incubated at 25° ± 1ºC for 5 days. The emerging hyphae from the root pieces were subcultured, identified and compared with original P. tinctorius culture.

Endomycorrhizal synthesis and recovery—Spores of AM fungus Glomus fasciculatum were extracted from C. equisetifolia plants, which were inoculated with G. fasciculatum; spores were isolated by wet sieving and decanting method19.

Recovery of Frankia—After 3 months, the root nodules were removed and washed thoroughly with running tap water. Frankia was isolated from nodules as described by Wheeler et al3.

Preparation of sections for light microscopy—Frankia and mycorrhizal fungi associated roots and

ELUMALAI & RAAMAN: IN VITRO SYNTHESIS OF FRANKIA

291

nodules of C. equisetifolia were washed to remove soil and other debris adhering to them. They were fixed in formalin acetic acid (FAA) for 48 hr, samples were dehydrated in tertiary butyl alcohol (TBA) and embedded in paraffin wax (melting point 58°-60ºC). Sections (8-10 µm thick) were cut with a rotary microtome. Dried sections were passed through xylol-alcohol series for dewaxing, stained with aniline blue and saffranin, and examined under a light microscope (Olympus).

Scanning electron microscopy studies—Healthy nodules and roots were collected and rinsed in cold water to remove soil particles. The cleaned nodules and roots were cut into pieces and observed under scanning electron microscope20.

Fixation—The pieces were fixed in glutaraldehyde (1-6%) in 0.05-0.1M phosphate buffer (pH 6.8-7.4) as the initial fixative21,22 at 4°C for 1-4 hr. Osmium tetroxide (1-2%) in the buffer was used as post fixative for 4 hr.

Dehydration—Water was removed from the sample to minimize extraction of cytoplasmic components from the sample. Acetone was used for dehydration. To

ensure total dehydration of the sample, three changes of 100% acetone were done for not less than 20 min each.

Critical point drying—During critical point drying process, the acetone in the specimen was removed. Thoroughly dehydrated specimens were placed in the chamber of the critical point dryer (Polaron) and immersed in liquid CO2. The specimen chamber was heated to convert the liquid CO2

to gas; the gas pressure was released slowly over 20-30 min period. Then, the specimen was removed from the chamber and mounted on the stubs using double side adhesive tape.

Sputter coating—The samples were coated with a thin film of gold to provide uniform electrical conductivity from all parts of the sample in sputter coating unit (Polaron). The coated stubs were taken out and observed under SEM (Jeol) at Indian Institute of Technology (IIT), Chennai. Results

Effect of mycorrhizal and actinorhizal inoculation on C. equisetifolia—The mycorrhizal and actinorhizal inoculation on C. equisetifolia in laboratory condition showed good growth in 90 days old plants (Table 1).

Table 1—Effect of mycorrhizal and actinorhizal inoculation on C. equisetifolia under laboratory condition at 90 d. [Value are mean ± SD]

Length (cm) Dry weight (mg) Treatment

Shoot Root Shoot Root

AM infection

(%)

Ecto roots (no)

Nodules(no)

Prot roots (no)

N content (μg/g dry wt)

P content(μg/g

dry wt)

Control 9.30 ±0.90 b

9.27 ±0.12 a

3.93 ±0.15 a

2.67 ±0.23 a

0 0 0 0 116.00 4.00 a

31.00 ±1.00 a

G. fasciculatum (A) 11.37 ±0.21 c

10.20b

±0.10 4.47

±0.12 b3.07

±0.12 b50.00

±2.00 b0a 0 5.00

±1.00 b126.00 ±2.00 b

36.00 ±2.00 b

P. tinctorius (E) 11.67 ±0.12 c

10.63 ±0.21 c

5.00 ±0.26 c

3.27 ±0.1bc

0.00 0.00

14.23 ±1.53 b

0 6.67 ±1.15 b

131.00 ±1.00 cd

42.00 ±2.00 c

Frankia (F) 12.00 ±0.17 cd

11.17 ±0.15 d

5.27 ±0.12 cd

3.70 ±0.10 c

0.00 0.00

0.00 0.00

2.33 ±0.58 b

6.33 22.89 b

130.33 ±2.08 c

46.67 ±1.15 d

A + E 12.43 ±0.12 de

11.98 ±0.25 e

5.47 ±0.12 de

3.67 ±0.25 c

64.33 ±2.31 c

15.33 ±2.31 bc

0.00 0.00

12.33 ±2.00 c

134.67 ±1.15 d

53.00 ±1.00 e

A + F 12.97 ±0.26 ef

11.93 ±0.12 e

5.60 ±0.20 e

4.00 ±0.10 d

65.33 ±2.52 c

0.00 0.00 a

4.00 ±1.00 c

16.00 ±2.00 d

147.00 ±3.61 e

58.00 ±2.00 f

E + F 13.30 ±0.26 f

12.80 ±0.26 f

5.33 ±0.12 de

4.23 ±0.06 d

0.00 0.00

16.67 ±3.06 bc

4.33 ±0.58 c

16.00 ±1.53 d

154.33 ±1.53 f

64.00 ±1.00 g

A + E + F ±3.67 =0.31 a

13.10 ±0.17 g

6.30 ±0.10 f

4.60 ±0.20 d

76.00 ±2.00 d

20.67 ±9.58 c

5.30 ±0.58 d

21.67 ±2.08 e

163.67 ±1.53 g

65.00 ±1.00 g

F value P value

207.59 0.000*

156.72 0.000*

63.19 0.000*

44.55 0.000*

1486.300.000*

18.37 0.000*

62.16 0.000*

48.86 0.000*

13.51 0.000*

222.410.000*

Note: 1) Significant at 1% Level 2) Different alphabet between treatment denotes significant at 5% level Since P value is less than 0.01, there is significant difference between treatments with regard to biomass parameters based on least

square difference (LSD) test. The treatment of A+E+F is significant with other treatment.

INDIAN J EXP BIOL, APRIL 2009

292

The shoot length (13.67 cm), root length (13.10 cm), shoot dry weight (6.30 mg), and root dry weight (4.60 mg) were higher in plants with triple inoculation (A+E+F). High percentage of AM infection (76%) was recorded in triple inoculation plants. Ectomycorrhizal roots were more in triple inoculated plants (20.67) compared to P. tinctorius (E) inoculated plants (5.30) than in Frankia (F) inoculated plants (2.33). More number of proteoid roots was observed in triple inoculated plants (21.6) and least in G. fasciculatum (A) inoculated plants (5.00). High levels of nitrogen (163 µg/g dry wt) and phosphorous (65 µg/g dry wt) were observed in G. fasciculatum inoculated plants. Both, ecto and endomycorrhizal infection increased the growth significantly when the seedlings were grown together with Frankia.

G. fasciculatum infection in C. equisetifolia—In the present study, spores of G. fasciculatum were isolated from the rhizosphere and vermiculite-sand mixture (Fig. 1a). C. equisetifolia roots were found to infected with arbuscles and vesicles of G. fasciculatum (Figs. 1b, c and d) when stained with tryphan blue. Almost all the roots were infected with G. fasciculatum.

Pure culture synthesis of P. tinctorius with C. equisetifolia—Under controlled conditions, P. tinctorius isolated from Kovalam (near Chennai) formed well developed ectmycorrhizae, with a well developed fungal sheath (Figs. 1e and f) but no Hartig net. The surface of the fungal sheath was yellow, felty, with numerous emanating hyphae. The fungal sheath was prosenchymatous with radiating hyphae (Figs 2a and b). P. tinctorius isolated from the root pieces agreed with the originally inoculated culture of P. tinctorius.

Recovery of Frankia—Frankia associated nodules were collected and Frankia cultures were reisolated following the procedure described by Racette and Torry10. It (Frankia) was similar in characters to Frankia inoculated initially. During the isolation of Frankia from nodules, after 2-3 weeks of incubation, white globoid colonies appeared with a maximum diameter of 0.3 mm. After 4 weeks, the colonies turned orange. In the colonies, septate hyphae and unsmooth vesicles were observed under light and scanning electron microscopes. The hyphae had terminal or intercalary multilocular sporangia. Biochemical tests such as nitrate and nitrite reduction tests gave positive results for Frankia.

Ultrastructure of Frankia nodule—Cortical cells near the infected hair showed both cell division and hypertrophy resulting in the initial swelling of the root. One to several nodule lobes developed at each infected site (Fig. 2c). Hyphal growth proceeded from the infected hair into cells of the prenodule, with penetration of the hyphae from cell directly through the cell wall.

In nodules collected on 90th day of in vitro synthesis, large number of cortical cells were infected with Frankia. Near the tip of the nodule lobes, Frankia was seen in the early stages of new cell infection with only a small portion of the cell occupied by the hyphae. Whereas, along the length of the nodule lobes, Frankia occupied majority of the cell volume (Fig. 3). Secondary wall thickening was commonly seen in these cells which was distinguished by differential, blue-red-green staining with toluidine blue, saffranin and fast green. Closer to the base of the nodule, the Frankia hyphae were quit senescent.

Symbiotic vesicles of Frankia were seen within the cells of C. equisetifolia. The vesicles were mostly located at the periphery of infected cells and were more or less spherical in shape. Unlike the mature vesicles, there were no pronounced void spaces seen around these vesicles.

Frankia and endomycorrizal associated C. equisetifolia roots and nodules stained with tryphan blue showed dark blue colour indicating the Frankia and arbuscular mycorrhizal infection. The size and shape of cortical cells of C. equisetifolia root infected with Frankia and endomycorrhiza were generally larger than the uninfected ordinary cortical cells. Hyphae and vesicles were seen in the cytoplasm of Frankia (Fig. 2d) and the endomycorrhiza associated cells were packed with spores and sporangia, arbuscles and vesicles. The Frankia spores and sporangia were highly irregular in shape (Fig. 2e) and more in number than arbuscles and vesicles. In the longitudinal sections, Frankia infected C. equisetifolia root nodules clearly showed the dichotomously divided two lobes. Sections of C. equisetifolia root nodules and endomycorrhizal roots showed extensive infection in cortical cells by filamentous hyphae, vesicles, arbuscules, spores and sporangia.

Discussion The importance of mycorrhizal association to the

members of the Casuarinaceae has been reported8,9. This family includes two genera, Allocasuarina and

ELUMALAI & RAAMAN: IN VITRO SYNTHESIS OF FRANKIA

293

Casuarina and much of the recent debate has focused on the relative importance of the arbuscular mycorrhiza and / or ectomycorrhizal association in members of each genus. Allocasuarina forms ectomycorrhizas more commonly than Casuarina species5,7

. In China, Casuarina species are

interplanted with Eucalyptus in costal regions to maximize the growth17.

In Australian isolates of Pisolithus and Scleroderma, it was observed that ectomycorrhizae either failed to form or formed infrequently with Casuarina compared to Eucalyptus and Allocasuarina7. Inoculated plants

Fig. 1—(a) Chlamydospore of Glomus fasiculatum × 300; (b) Vesicles in C. equisetifolia roots × 120; (c) Arbuscles in Casuarina equisetifolia root × 120; (d) Arbuscules and vesicles in Casuarina equisetifolia × 120; (e) Cross section of C. equisetifolia root showing ectomycorrhizal fungal sheath × 120; and (f) Cross section of C. equisetifolia root with ectomycorrhizal association (enlarged view) × 300.

INDIAN J EXP BIOL, APRIL 2009

294

of three isolates of Pisolithus in test tubes containing perlite – peat – Melin - Norkrans agar formed ectomy- corrhizas on Casuarina but these lacked Hartig net. The sheathed roots that formed with Casuarina were typical of incompatible associations. Those hyphal envelopes may simply be the result of the growth on

root excludes without requiring fungus-root symbiosis. However, the hyphal envelopes that formed on Casuarina with Scleroderma and Pisolithus were compact. The Casuarina sheaths are mantles, a tissue that is specific to ectomycorrhizal devlopment18,19. This suggests that the mantle between the roots of

Fig. 2—(a) Scanning electron micrograph of hyphae of ectomycorrhiza on the surface of C. equisetifolia roots × 1000; (b) Enlarged view of the hyphae of ectomycorrhiza in C. equisetifolia × 2000; (c) Longitudinal section of the Frankia nodule lobes showing the infection of Frankia. × 80; (d) Scanning electron micrograph of endomycorrhiza hyphae in root nodule cells × 1000; and (e) Spores of Frankia under scanning electron microscope (SEM) × 2000.

ELUMALAI & RAAMAN: IN VITRO SYNTHESIS OF FRANKIA

295

Casuarina and the hyphae of Pisolithus and Scleroderma are incompatible for ectomycorrhizal development. Mycorrhizal fungal progression in roots provokes changes in cell shape and cytoplasmic

organization. Root cells undergoing ectomycorrhiza formation elongate, whereas arbuscles differentiation in AM root cells involves complete reorganization of the cytoplasm.

Fig. 3—(a) Enlarged view of the Frankia hyphae in C. equisetifolia root nodule cell × 1500; (b) Scanning electron micrograph of infected cells with Frankia at sporangium stage, in C. equisetifolia roots × 1500; (c) Scanning electron micrograph of infected cells with Frankia showing numerous spores in the sporangium in C. equisetifolia roots × 3500; (d) Scanning electron micrograph of Frankia infected root nodule cells of Casuarina equisetifolia root showing infection in entire wall × 3500; (e) An enlarged view of a single cell with infection of Frankia in C. equisetifolia roots × 5000; and (f) An enlarged view of spores and sporangium of Frankia in C. equisetifolia × 8000.

INDIAN J EXP BIOL, APRIL 2009

296

The importance of dual symbiosis in primary colonizers like Casuarina which contained root nodules infected with actinomycetes and roots with AM infection19 and the phosphate solubilization20 has been stressed. The occurrence of ectomycorrhiza in C. equisetifolia has been observed earlier23. In soil under C. cunninghamiana, ectomycorrhizal fungal spores have been observed but C. cunninghamiana did not support AM infection22,23. In some species of Casuarina, ectomycorrhizal association has not been found. Rose8 noted the presence of arbuscular mycorrhizal structures in roots of C. cunninghamiana obtained from sand woodlands of Florida and Japan and in roots of C. equisetifolia from coastal area. The spores of AM fungi, Gigaspora gigantea and Gigaspora nigra were found in the soils of C. cunninghamiana. AM fungal associations with roots and actinorhizal plants have been shown in several cases to yield enhanced phosphorus uptake, increased nodulation and nitrogen fixation. After six months of growth, mycorrhizal inoculated plants contained twice as much nitrogen as seedlings that had been inoculated with only Frankia21.

C. equisetifolia roots were infected with arbuscles and vesicles of G. fasciculatum. Almost all the roots were infected by G. fasciculatum and spores of G. fasciculatum were isolated from the rhizosphere soil of C. equisetifolia. Root nodules with actinomycetes and roots with AM association were observed in coastal vegetation of C. equisetifolia19,24. The results showed that P. tinctorius formed ectomycorrhizal association with C. equisetifolia with a well developed fungal sheath, but no Hartig net formation, (failed to form Hartig net)6,7.

Actinomycetes are filamentous microorganisms with its terminal ends in the form of swollen vesicles3. Casuarina may be unique among actinorhizal plants in its small number of intercellular air spaces in the zone of infected cells and the lack of continuity between these spaces4. Frankia, living within the cells of the root nodules of A. glutinosa, produced sporangia both intercalary and terminally, forming large number of thick walled spores within sporangia. Sporangia and spores are most clearly visualized at the ultrastructure25. Only bacteria-like cells are occurring in host cells of C. equisetifolia but no vesicles26. In the present study, the procedures of Zhang et al22 were followed for the isolation and culture of a strain of Frankia that effectively nodulated species of Casuarina. Frankia strains isolated from the other host plants27 always exhibited hyphae and sporangia but

vesicles appeared on some of the media like Quispel Modified Medium (QMOD) and the nitrogen free medium27. Many Frankia strains have been isolated from the nodules of trees of Casuarinaceae.

After 90 days, in nodules that were apparently healthy and growing, large number of cortical cells was seen infected with Frankia. Near the tip of the nodule lobes, Frankia was seen in the earlier stages of new cell infection with only a small portion of the cell occupied by hyphae. Frankia hyphae were frequently seen penetrating from cell to cell directly through cell walls. Further, along the length of the nodule lobes, Frankia occupied majority of the cell volume.

Nodulated C. equisetifolia was infected by both ecto and endomycorrhizal fungi within the same root system. This suggests that tetrapartite symbioses are a general phenomenon in Casuarina sp. However, no attempt has been made to study the effect of these tetrapartite symbioses on the growth performance and ultrastructure of C. equisetifolia. The present study reports the effect of an endomycorrhizal fungus G. fasciculatum, an ectomycorrhizal fungus P. tinctorius and Frankia on the growth performance for C. equisetifolia seedlings under controlled conditions. Both ecto and endomycorrhizal infection increased biomass significantly when the seedlings were grown together with Frankia. The similar phenomenon was observed earlier also27,28.

The benefits of tripartite symbioses of C. equisetifolia with symbiotic microorganisms are also known20. The multiple symbioses of alders is a likely strategy that allows host tree to grow in extreme environmental conditions. Both ecto and endomycorrhizal fungi with plants are known for their ability to absorb more nutrients and water, producing growth regulators, resist root pathogens, and survive in adverse soil conditions such as low or high pH, high soil temperature15,27,29. The production of growth regulators by Frankia is also known30,31.

The formation of root nodules by Frankia and ecto and endomycorrhizal association is considered as essential for the survival and establishment of C. equisetifolia (tsunami protecting plants) in nitrogen poor sites such as coastal sandy and gravely soils, raw mineral soils, and wet soils.

Acknowledgement The financial assistance to one of the authors (SE)

by CSIR, New Delhi, is gratefully acknowledged. Thanks are due to Dr. (Mrs.) Shanthi, Technical

ELUMALAI & RAAMAN: IN VITRO SYNTHESIS OF FRANKIA

297

Officer, Material Science Department, IIT, Madras for scanning the specimens.

References 1 Rodriguez Barruco G, Effecto del N-amino sobre la

fijaconde N–atmosferrico en nodules de Casuarinn, Annal. Edafol. Agrob, 31 (1972) 905.

2 Tjepkema J D, Oxygen regulation in leguminous and actinorhizal nodules in symbiotic nitrogen fixation in the management of temperate forests edited by J C Gordon, C T Wheeler & D A Perry (Oregon State University, Corvallis ) 1979, 175.

3 Wheeler C T, Henson I E & Mclaughlin M E, Hormones in plants bearing actinomycete nodules, Bot Gaz (Chicago), Suppl 140 (1979) S 52.

4 Rangarajan M, Kandasamy D, Oblisami G & Narayanan R, Variation in responses to inoculation with Frankia and two ectomycorrhizal fungi on growth and nodulation in Casuarina equisetifolia, in Mycorrhiza for green Asia, First Asian conference on mycorrhizae, Jan 29-31, Madras India, edited by A Mahadevan, N Raman and K Natarajan (University of Madras) 1988, 291.

5 Bougher N L, Dell B & Malajczuk B, Growth and root structure of Eucalyptus Allocasuarina and Casuarina seedlings inoculated with ectomycorrhizal fungi, in Proceedings of the 8th North American Conference on Mycorrhizae edited by M F Allen and S E Williams 5-8 September 1990 (Jackson Wyoming University of Wyoming, Jackson ,Wyoming) 1990, 32.

6 Theodorou C & Reddell P, In vitro synthesis of ectomycorrhizas on Casuarinaceae with a range of mycorrhizal fungi, New Phytol, 118 (1991) 279.

7 Dell B, Malajczuk N, Bougher N L & Thomson G, Development and function of Pisolithus and Scleroderma ectomycorrhizas formed in vitro with Allocasuarina, Casuarina and Eucalyptus, Mycorrhiza, 5 (1994) 129.

8 Rose S L, VA Mycorrhizal association of some desert plants of Baja California, Can Bot J, 59 (1980) 1056.

9 Gardner L C, Mycorrhizal status of actinorhizal plants MIRCEN - J Appl Microbiol Biotech, 5 (1986) 147.

10 Racette S & Torrey J G, The isolation, culture and infectivity of a Frankia strain from Gymnostoma papuanum (Casuarinaceae), Plant Soil, 118 (1989) 165.

11 Callaham D, Newcomb W, Torrey J G & Peterson R L, Root hair infection in actinomycete–induced root nodule initiation in Casuarina myrica and Camptonia, Bot Gaz. (Chicago), 140 (1979) (Suppl) SI-S9.

12 Callaham D & Torrey J G, Prenodule formation and primary nodule development in roots of Camptonia (Myricaceae), Can J Bot, 55 (1977) 2306.

13 Bamber R K, Mulette K & MacKowski C, Mycorrhizal studies, in Research Report 1977-1978 (Forest Commission of New South Wales Sydney, Australia) 1980, 79.

14 Torrey J G, Root development and root nodulation in Casuarina, in Casuarina ecology, management and utilization, edited by S J Midgley, J W Turnbull and R D

Johnson (CSIRO, Melbourne) 1983, 180. 15 Schenck N C & Perez Y, Manual for the identification of VA

mycorrhizal fungi, (Synergestic publications, Gainsville, USA) 1990, 286

16 Grand L F, Distribution, plant associates and variation in the basidiocarps of Pisolithus tinctorius in the USA, Mycologia, 68 (1976) 672.

17 Marx D H, Tree host range and world distribution of the ectomycorrhizal fungus Pisolithus tinctorius, Can J Microbiol, 23 (1977) 217.

18 Harley J L, The importance of microorganisms to colonizing plants, Trans Bot Soc. (Edinburgh) 41 (1970) 65.

19 Gullan P K, Vegetation of Crassbourne, Ph.D thesis, Monash University, Victoria, Australia. 1975.

20 Harley J L & Smith S E, Mycorrhizal symbiosis (Academic Press, London) 1983, 483.

21 Diem H G, Gauthier D & Dommergues Y, An effective strain of Frankia from Casuarina sp, Can Bot J, 61 (1983) 2815.

22 Zhang Z, Lopex M F & Torrey J G, A comparison of cultural characteristics and infectivity of Frankia isolates from root nodules of Casuarina species, Plant Soil, 78 (1984) 78.

23 Diem H G, & Dommergues Y R, Current and potential uses and management of Casuarinacease in the tropics and subtropics, in Biology of Frankia and Actinorhizal plants, edited by C R Schwintzer & J D Tjepkema (Academic Press, New York) 1990, 317.

24 Raman N & Elumalai S, Studies on mycorrhizal and actinorhizal association in Casuarina equisetifolia in Coramandal coastal region, J Trop For, 7 (1991) 138.

25 Becking J H, Frankiaceae FAM Nov. (Actinomycetales) with one new combination and six new species of the genus Frankia Brunchorst 1886 174, Int Syst J Bacteriol 20 (1970) 201.

26 Chatrapaul L, Chakravarthy P & Subramaniam P, Endomycorrhizal effects on nodulation, nitrogen–fixation, and growth of Alnus glutinosa as affected by glyphosate, Proc Inter Workshop on appl of Pl. Biotech. in For and Horti New Delhi, India (1990).

27 Baker D, Torrey J G & Kidd G H, Isolation by sucrose–density fractionation and cultivation in vitro of actinomycetes from nitrogen fixing root nodules, Nature 281 (1979) 76.

28 Diem H G & Gauthier D, Isolement et culture in vivo d’ une souche infective et effective de Frankia isolee de nodules de Casuarina sp Hebd C R, Séances Acad Sci Paris Ser C 295 (1982) 759.

29 Raman N & Elumalai S, Influence of phosphorus on cluster root formation by Casuarina equisetifolia in water culture, Indian J Exp Biol 30 (1992) 928.

30 Raman N & Elumalai S, Presence of cytokinin in the root nodules of Casuarina equisetifolia, Indian J Exp Biol, 34 (1996) 577.

31 Raman N & Elumalai S, Gibberellic acid in mycorrhizal and actinorhizal inoculated Casuarina equisetifolia, Indian J Exp Biol, 36 (1998) 703.