1 Introduction

Actinorhizal plants usually form root nodules in association with the nitrogen-fixing actionmycete Frankia, which helps them to survive even in nutrient-poor soils by nitrogen (N) fixation. Frankia is a filamentous soil bacteria that interacts with the roots of appropriate host plants to form nitrogen-fixing nodules, also called actinorhizae (Benson and Silvester 1993). Actinorhizal plants include Casuarinaceae, which is a major family of trees that has been disseminated throughout the tropics owing to their ability to grow in adverse conditions (Echhab et al. 2007). Casuarina equisetifolia Forst. belongs to Casuarinaceae and due to its high economic value, farmers are interested in planting this tree as an agroforestry crop in Tamil Nadu and Pondicherry (India). It is useful as wind break, an ornamental plant, soil improvement, live fencing and building material (Nicodemus 2009). Frankia is associated with C. equisetifolia for N fixation and it has been estimated that Frankia fixes atmospheric nitrogen up to 362 kg N/ha/year, which is an essential nutrient for all plant metabolic activities and growth (Shantharam and Mattoo 1997).

To inoculate Frankia generally farmers used to collect the root nodules from mature trees of C equisetifolia and then crush and add them at the time of planting in new sites along with seedlings/cuttings of C. equisetifolia. This practice is often unsuccessful if the crushed root nodules contain dead or inactive Frankia. Further, for pulp and paper production high-yielding genetically superior trees of C. equisetifolia are selected and multiplied by rooted stem cuttings by the farmers of Tamil Nadu and Pondicherry . But the rooted stem cuttings are being propagated in an inert material (vermiculite), and so there is no chance for Frankia association. Although inoculation of Frankia is essential in rooted stem cuttings of C. equisetifolia there is no report found on nodulation of rooted stem cuttings in C. equisetifolia. Further, farmers of Pondicherry and Tamil Nadu (India) apply 150 kg of DAP/year/ha after planting of C. equisetifolia seedlings or rooted stem cuttings in their farmlands. Hence, there is an urgent need to find an alternate solution for use of these chemical fertilizers for the rooted stem cuttings of C. equisetifolia. The effect of inoculation of cultured Frankia strain in rooted stem cuttings of C. equisetifolia on growth, biomass and nodulation was studied, and this could reduce the use of chemical fertilizers. Further, it is intended to decide the effect of Frankia on the efficiency of N uptake of C. equsetifolia rooted stem cuttings.

2 Materials and methods

2.1 Location of the study

The study was conducted at the Model Nursery of Institute of Forest Genetics and Tree Breeding (IFGTB), Coimbatore (11°01′ N and 96°93′ E; altitude 410 m a.s.l.), India. The climate is monsoonal, with an annual precipitation of 640 mm and a dry season between January and April. The maximum and minimum monthly temperatures are 31°C and 21°C, respectively.

2.2 Isolation and multiplication of Frankia

The Frankia used in this study was isolated from C. equisetifolia root nodules collected from coastal area, and the location and characteristics of collected nodules are shown in the table 1.

Table 1 Source of Frankia
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The nodules were collected in ice box and stored in frozen condition at −4°C. Afterwards, the nodules were surface-sterilized with 30% H2O2 and kept in a shaker for 30–40 min. Under aseptic conditions the nodules were rinsed in sterile water and 0.2 g of nodule was ground manually in sterile mortar and pestle. Then the nodule solutions were centrifuged at 1000 rpm for 20 min and the supernatant was filtered through Whatman No.1 filter paper. The suspension was then plated in P medium and incubated at 25°C for 3–4 weeks. One litre of P medium was prepared as follows (Shipton and Burgraff 1983): 10 g CaCl2,2H2O, 20 g MgSO4, 0.46 g propionic acid, 0.15 g H3BO3, 0.15 g ZnSO4.7H2O, 0.45 g MnSO4.H2O, 0.004 g CuSO4.5H2O, 0.028 g Na2MoO4.2H2O, 0.009 g CaCl2.6H2O, 0.04 g Biotin, 100 g K2HPO4, 67 g NaH2PO.2H2O, 0.1 g FeNa EDTA, and 8.g agar. The pH of the medium was adjusted to 6.8. After 21–30 days of incubation the Frankia growth was observed as fluffy white cloudy colonies in P media plates. These colonies were transferred in to P medium broth for mass multiplication. Under microscopic observations the Frankia showed pin-headed vesicles and septate hyphae (figure 1). The morphometric characters of the Frankia was also assessed and presented in table 2. The identification of Frankia was also confirmed at molecular level by16s RNA sequence and named as strain Ce Co1 (Karthikeyan et al. 2012).

Figure 1
figure 1

Microscopic structures of Frankia.

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Table 2 Morphometrics of Frankia
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2.3 Collection and propagation of C. equisetifolia stem cuttings

The stem cuttings of single elite Clone No. CE 100 were collected from the Casuarina germplasm bank at Model Nursery, IFGTB, Coimbatore, India, where 250 clones of Casuarina have been housed for research and breeding purpose. The stem cuttings were collected from the terminal ends of the twigs of the CE 100 during the month of November 2011 and preserved in ice box (4°C). Uniform sized stem cuttings with a length of 5 cm (±0.4.3) and a girth of 0.3 cm (±0.052) were taken for this study. The basal ends of the cuttings have been crossed cut to optimize the Indole Butyric Acid (IBA) uptake for better rooting. The stem girth was measured at the basal end where root initiation (root collar) will actually take place. The stem cuttings were treated with 0.1% carbendazim fungicide for 3 min and after 2000 ppm of IBA (40 mg of IBA + 20 g of talcum powder) at the basal end of the cuttings for 0.5 min by dip method. After the treatment with IBA the cuttings were placed in 100 cc root trainers containing the inert media vermiculite. The rooted cuttings were thereafter placed in polytunnels made of polythene sheets (180 cm × 90 cm) and maintained with temperature of 32–35°C and 60–65% relative humidity for 25 days. After 25 days the cuttings showed initiation of 2 to 3 lateral roots of 1 cm to 1.5 cm length. At this stage the rooted stem cuttings were transferred to shade house and watered regularly.

2.4 Inoculation of Frankia in C. equisetifolia rooted stem cuttings

An amount 0.1 mL of the cultured Frankia strain Ce Co1 in P medium broth was inoculated in conical flasks with 250 mL capacity. These liquid cultures were incubated at 32°C in the Orbitol Shaker (Gyromax 703 R, USA) at 20 rpm for 25 days. Later the cultures were homogenized in the centrifuge at the rate of 1000 rpm for 20 min. Thereafter the cultures of Frankia were inoculated in the root zone of rooted stem cuttings of C. equisetifolia at the rate of 5 mL−1 cutting that contained 2.2 μg protein mL−1. The inoculated rooted stem cuttings were maintained in the nursery with 15 replicates including uninoculated controls in the shade house and watered regularly. The initiation of root nodules and nodule numbers in each rooted stem cuttings were assessed. These planting stocks of C. equisetifolia were maintained for 3 months in the model nursery of IFGTB and harvested for analysis of growth and biomass. The dry weight of Frankia inoculated these planting stocks was determined after oven drying at 50°C to a constant weight.

2.5 Analyses of growth, biomass and tissue nitrogen content

A set of (5 replicates) of C. equisetifolia rooted stem cuttings were taken for harvest and analysis The growth of Frankia-inoculated rooted stem cuttings and uninoculated cuttings were analysed in terms of shoot length, root length, number of lateral roots, collar diameter, dry weights of shoot, root, number of nodules and nodule biomass. The dry weights were determined after oven drying at 50°C to a constant weight. The total N content was estimated in root and shoot sample using KELPLUS auto analyser to determine the N fixation by inoculation of Frankia in the rooted stem cuttings of C. equisetifolia The dried plant sample (0.25 gm) was digested with 3 gm of catalyst mixture (potassium sulphate and cupric sulphate in ratio of 5:1) and 10 mL of H2SO4 in Kjeldhal digestion system (KELFLOW) at 420°C for 1 h. Then the digested sample was diluted with 10 mL of distilled water before distillation. After distillation, the collected distillate was titrated against 0.1 N hydrochloric acid.

2.6 Field planting

The other 10 replicates were taken for field planting to test the effect of inoculated Frankia strain in field conditions. The rooted stem cuttings of C. equisetifolia were planted in a farm land located at coastal area of Karaikal, Pondicherry (India). The planting stocks were planted at an espacement of 1.5×1.5 met. The uninoculated control rooted stem cuttings were also planted in the same area in randomized block design. The growth of the rooted stem cuttings were monitored for 1 year under field conditions.

2.7 Analysis of soil nutrients

The major soil nutrients (N, P and K), soil pH and electrical conductivity of the soil were analysed before and after planting of C. equisetifolia inoculated with Frankia according Jackson (1973).

2.8 Statistical analysis

Each measured variable in the nursery and field experiments were statistically analysed using Duncan’s multiple range test (SPSS ver. 16).

3 Results

3.1 Morphological characteristics of Frankia isolate

Under optimal conditions (28–32°C), the growth of the isolate formed white fluffy colonies on the P media plates. They were examined under a light microscope and showed branched and septate hyphae and round vesicles. The morphometrics of Frankia are shown in table 2.

3.2 Growth and biomass of C. equisetifolia rooted stem cuttings

Nodulation of Frankia was observed 30 days after inoculation in the rooted stem cuttings of C. equisetifolia. The initial infection at 20 days showed clubbed roots in the rooted stem cuttings, and the nodule development occurred at 30 days. The rooted stem cuttings inoculated with Frankia strain showed significantly increased growth in shoot length, root length, collar diameter and biomass as compared with the uninoculated control seedlings. The rooted stem cuttings showed higher nodule biomass than the uninoculated control. Frankia-inoculated cuttings showed dense root nodules in the root region, whereas the uninoculated cuttings showed absence of nodules. The root nodules developed in the rooted stem cutting weighed up to 43 mg and the number of root nodules obtained was 12 per cutting. The R/S ratio was showed significantly lower in Frankia-inoculated rooted stem cuttings than the uninoculated control (table 3). A new finding was also derived from this study – successful nodulation establishment in the C.equisetifolia rooted stem cuttings in inert media without using soil (figure 2).

Table 3 Growth and bio mass of C. equisetifolia rooted stem cuttings to Frankia inoculation at 90 days under nursery conditions
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Figure 2
figure 2

Establishment root nodules in the rooted stem cuttings of C. equisetifolia inoculated with Frankia.

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3.3 Tissue N content

Significant differences in nitrogen concentration of Frankia-inoculated rooted stem cuttings of C. equisetifolia in comparison with uninoculated controls were observed. The total N concentration was found 3.30 mg/g dry weight in the rooted stem cuttings, whereas the uninoculated control rooted stem cuttings showed a mean value of 1.32 mg/g dry weight (figure 3).

Figure 3
figure 3

Tissue N content (mg g−1). Means followed by same letters are not significantly different at p<0.05 according to Duncan’s Multiple Range Test.

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3.4 Soil nutrients

The soil nutrients, particularly soil N, was highly increased after planting of C. equisetifolia rooted stem cuttings inoculated with Frankia. The electrical conductivity and soil pH was also changed after planting (table 4).

Table 4 Soil nutrient status of the planting site before and after planting of C. equisetifolia rooted stem cuttings inoculated with Frankia
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3.5 Field performance

Frankia inoculated rooted stem cuttings of C. equisetifolia significantly (p<0.05) increased their growth in terms of height and stem girth as compared with uninoculated controls. Three-fold increase of growth was observed due to inoculation of Frankia in the rooted stem cuttings of C. equisetifolia under field conditions (figure 4).

Figure 4
figure 4

Growth performance of C. equisetifolia rooted stem cuttings inoculated with Frankia under field conditions after one year of planting. Means followed by same letters are not significantly different at p<0.05 according to Duncan’s Multiple Range Test.

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4 Discussion

The results of this study have clearly shown that Frankia can improve plant growth through increased uptake of N. Frankia results in a positive effect on the rooted stem cuttings of C. equisetifolia growth through improvement of growth and biomass. Earlier studies also reported that the increase in growth and biomass of casuarinas due to inoculation of Frankia might be strongly correlated with improved accumulation of nitrogen due to Frankia (Reddell et al. 1988). This study further supports the positive response of C. equisetifolia rooted stem cuttings in the nursery to Frankia application and strengthens the Frankia dependency of C. equisetifolia in low fertility. Similar results were reported for Frankia (nodule suspension) inoculation employed in C. equisetifolia seedlings (Muthukumar and Udaiyan 2010). In several studies (Lesueur and Duponnois 2005; Yamanka et al. 2003) Frankia effects on plant growth promotion has been demonstrated in sterile soil substrates. However, the growth promoting effect of Frankia on C. equisetifolia rooted stem cuttings in inert media has not been reported. It has been repeatedly reported that spontaneous nodulation of the genera Casuarina is unlikely outside their natural habitat. This may be attributed to the fact that Frankia cannot transmit with the seed either within or on its surface (Torrey 1983). Inoculation experiments of this kind in nursery conditions is essential for C. equisetifolia rooted stem cuttings, which bring together the root system and nodulation as they propagated in inert media. In this study nodulation occurs in 30 days in the rooted stem cuttings of C. equisetifolia; however, Vergnaud et al. (1985) obtained axenic nodulation in Alnus glutinosa within 10 days. This also showed that there is a difference in nodulation behaviour between Alnus species and C. equisetifolia. Nodulation biomass and nodule number were increased the rooted stem cuttings of C. equisetifolia raised in inert medium. This reflects the symbiotic nitrogen fixation is dependent on host photosynthesis (Arnone and Gordon 1990), which affords energy in the form of ATP to Frankia in root nodules. The increased biomass in the rooted stem cuttings of both the clones could be the result of increased nutrient inflow rates through Frankia. The increased tissue N content of Frankia-inoculated rooted stem cuttings of C. equisetfolia raised in inert media as compared with the uninoculated control plants showed the influence of Frankia in N fixation. The field performance of Frankia-inoculated rooted stem cuttings showed increased growth, which may be due to increased uptake of N through Frankia. Earlier studies also showed that the growth of Alnus cordata inoculated with Frankia had increased growth and survival in field conditions due to the uptake of N (Lumini et al. 1994). Soil nutrients also improved due to the activity of Frankia, which supports the inoculation of C. equisetifolia with cultured Frankia for enhancement of growth, biomass, nutrient uptake and soil fertilization. It is essential to introduce potential Frankia in the rooted stem cuttings of C. equisetifolia as they propagated in inert media (vermiculite). The N fertilization through biological nitrogen fixation by Frankia improved significantly the growth and biomass in field conditions as compared with uninoculated C. equisetifolia. These results are also confirmed the earlier work on Alders inoculated with Frankia for reclamation of oil stand sites where Frankia-inoculated Alders produced significant biomass in the reclaimed oil stands in Canada (Lefroncois et al . 2010). From this study it was deduced that inoculation of Frankia in C. equisetifolia would be beneficial for early establishment in the field without additional chemical fertilizers.