Introduction

Actinorhizal plants usually form root nodules in association with the nitrogen fixing actinomycete Frankia. Because of their nitrogen fixing ability, actinorrhizal plants can thrive in infertile soils. Actinorhizal plants also form association with arbuscular mycorrhizal (AM) fungi, thus forming a tripartite symbiosis, which enhances the success of these plants under poor soil conditions (Wheeler et al. 2000). Mycorrhizas improve the nutrient uptake especially phosphorus (P) of the host plants (Jakobsen 1999). The extraradical mycelia of the mycorrhizal fungi may act as extensions of the root systems (Rousseau et al. 1994) and have a high nutrient mobilizing potential (Lapeyrie et al. 1991). The uptake of nutrients like P and micronutrients facilitated by AM fungi can help actinorhizal nodulation and function under stress conditions.

Phosphate solubilizing bacteria (PSB) are beneficial microorganisms in the plant rhizosphere, as they solubilize bound phosphorus (P) and increase their availability for the plant (Rodrìguez and Fraga 1999). In addition, some reports have shown that PSB have a strong stimulatory impact on the growth of mycorrhizal fungi (Artursson et al. 2006). Paenibacillus polymyxa [previously Bacillus polymyxa (Ash et al. 1993)], is a common soil bacterium. Activities of PSB include soil P solubilization, production of antibiotics, auxins, cytokinins, chitinase, and hydrolytic enzymes, as well as promotion of increased soil porosity (Rodrìguez and Fraga 1999). All these activities could account for or contribute to plant growth promotion at various times and in various environments during the life cycle of a plant. The organic acids produced by P. polymyxa accelerate solubilization of bound phosphates, thereby enhancing their availability to plants. Therefore, it is expected that increased P availability due to the PSB might stimulate the interaction of AM fungi and Frankia on host plants. It is well established that symbiotic N2-fixation is highly dependent on the uptake of P, calcium and micronutrients by the host (Sanginga et al. 1989; Walker et al. 1993). This leads to the hypothesis that simultaneous inoculation of PSB along with AM fungi and Frankia would enhance seedling growth through enhanced mycorrhization and nodulation. Thus, an increase in seedling quality should occur in response to greater N and P availability to the host.

The rate of nutrient absorption and nutrient use efficiency (NuUE): the efficiency with which a nutrient is utilized to produce dry matter are the two factors that control the rate of plant growth in low soil nutrient conditions (Koide et al. 2000). The NuUE is known to vary with nutrient availability and mycorrhizal colonization (Koide et al. 2000) and a very few studies has assessed this for tree species (Muthukumar et al. 2001; Muthukumar and Udaiyan 2006).

Casuarina equisetifolia Forst. is a fast growing actinorrhizal tree extensively used for fuel, land reclamation, wind breaks on farms, erosion control, afforestation, wasteland development in tropics and subtropics (Subba Rao and Rodriguez Barrueco 1995). Although C. equisetifolia can associate with both ecto- and endo-mycorrhizal fungi, the endomycorrhizal association with AM fungi is more common in C. equisetifolia than ectomycorrhiza (Duponnois et al. 2003; He and Critchley 2008). Casuarina equisetifolia also respond spontaneously to nutrient deficiency by developing cluster roots under non-mycorrhizal conditions (Zaïd et al. 2003). There is some evidence indicating the importance of bioinoculants on C. equisetifolia growth under nursery (Vasanthakrishna et al. 1994; Rajendran et al. 2003) and field (Rajendran and Devaraj 2004) conditions. The objective of this study was to determine whether simultaneous inoculation of AM fungus, PBS and Frankia could enhance growth, nodulation and seedling quality of C. equisetifolia under tropical nursery conditions. Further, we intended to determine the effect of bioinoculants on the efficiency of nutrient uptake and nutrient use efficiency of C. equisetifolia as no previous studies have determined these for an actinorhizal plant species.

Materials and methods

Nursery site and soil preparation

The study was conducted at the tree nursery of the Botany Department, Bharathiar University, Coimbatore (11°01′ N and 96°93′ E, altitude 410 m a.s.l), Tamilnadu, 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 and 21°C, respectively. Alfisol soil was used in this study. Topsoil (0–30 cm) of the soil was collected from a fallow field that had remained uncultivated for more than 7 years and represented areas where plantations are to be raised for protection purposes. The vegetation in this field was very sparse and dominated by grasses. Soil chemical analysis prior to experiments using standard procedures is presented in Table 1.

Table 1 Chemical and biological properties of the soil used in the study
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Assessment of indigenous microbial population

To assess the indigenous PSB populations in field soils, three 0.1-ml aliquots of soil diluted with sterile water (10−3–10−6) were spread on standard media for dilution plate counts for PSB (Pikovskaya 1948). The plates were incubated for 27°C for 3–5 days for PSB. The colonies that formed clear zones on the PSB medium were counted. The number of recorded PSB cells was expressed as log colony forming units (CFUs) per gram of soil. Infective propagules of indigenous AM fungi was assessed according to the Most Probable Number technique (Porter 1979).

Seed and inoculum

Casuarina equisetifolia seeds obtained from the Institute for Forest Genetics and Tree Breeding (IFGTB), Coimbatore, India, were scarified with 95% concentrated sulphuric acid for 2 min., rinsed thoroughly with running tap water for 30 min., soaked in distilled water overnight and sown in trays (30 × 22 × 6 cm) containing heat sterilized (121°C for 3 h) sand. The seedlings were maintained for 6 weeks. Two healthy and vigorously growing seedlings were transplanted into each black polythene bags (45 cm deep, 25 cm wide) containing 7.5 kg field soil. After establishment, it was thinned to one seedling per bag.

The AM fungus Glomus geosporum (Nicol. & Gerd.) Walker was selected as it has been reported to be associated with C. equisetifolia in different soil types of Tamilnadu, India (Sambandan et al. 1994). AM fungal inoculum consisted of soil containing spores and mycorrhizal roots from a pot culture of sorghum [Sorghum bicolor (L.) Moench.] colonized by G. geosporum (DBCC-72) and grown for 12 months. The AM fungal species originated from semi-arid grassland (Muthukumar and Udaiyan 2002). Five grams of AM fungal inoculum containing 258 propagules g−1 soil as assessed according to Porter (1979) was added to the planting hole at transplantation. Treatments not involving AM fungi received the same amount of sterile inoculum, which had been autoclaved at 121°C for 90 min., three times at regular intervals. Soil microbes in AM fungal inoculum were equalized across treatments by applying 25 ml ‘microbial cocktail’ to each bag. This ‘microbial cocktail’ was prepared by blending 100 g AM fungal inocula in 1 l deionized water and filtering it three times through a 25 μm sieve.

Frankia inoculum suspension was prepared from ca. 0.5 g of fresh nodule collected from C. equisetifolia trees growing in Bharathiar University Campus. The young nodule lobes were separated; surface sterilized (30% H2O2, 30 min.), rinsed in sterile distilled water and ground in a sterile mortar and pestle. The nodule suspension was made up to 500 ml with 2% sucrose solution (Reddell et al. 1988) prior to inoculation. Five ml of the nodule suspension (equivalent to 5 mg of nodule f. wt.) was pipetted around the roots of the seedlings. Treatments not involving Frankia received 5 ml of heat sterilized nodule suspension.

The PSB Paenibacillus polymyxa (isolate no. DBCC 005) was isolated and maintained as described earlier (Muthukumar et al. 2001). At transplantation 15 ml PSB inoculum grown in PSB medium without agar equivalent to 1.2 × 106 cells g−1 soil was pipetted into each bag specifying treatment. Seedlings not involving PSB received 15 ml of heat sterilized PSB inoculum.

Experimental design

There were seven treatments involving bioinoculants individually or in various combinations, and an uninoculated control. Each treatment consisted of ten replicates and measurements were made after destructive harvest at 60 and 120 days after transplantation (DAT). The experiment involved were 160 bags (8 × 10 × 2) set up in the nursery in a completely randomized design. The bags were rearranged every 15 days to ensure uniform growth conditions. Seedlings were watered to maintain field capacity and no nutrients were added. The maximum/minimum temperature range over the experiment period was 36°C/24°C, rainfall was 350 mm and relative humidity was between 58 and 70%.

Analyses

At harvest, the soil was washed from roots and a weighed portion of each root sample was preserved in formalin–acetic acid–alcohol (FAA) solution for the assessment of AM colonization. Shoot and roots were separated and oven dried at 70°C for 48 h for the determination of dry mass. The root samples were cleared in 2.5% KOH solution at 121°C for 30 min followed by acidification with 1.0% HCl and stained with 0.05% trypan blue in lactophenol (Koske and Gemma 1989). The extent of AM colonization was quantified according to the magnified intersection method (McGonigle et al. 1990). Infective propagules of AM fungi in the inoculum were determined by MPN method (Porter 1979).

Tissue nitrogen (N) in shoot and root samples was determined by the micro Kjeldahl digestion method using concentrated H2SO4 digest and selenium as catalyst. Total N was estimated using a Technicon Auto Analyser (Gedko International, UK). Tissue P concentration in seedling tissues was determined by the molybdenum blue method (Jackson 1971) using a Spectronic 20 electrophotocolorimeter after wet-ashing the plant samples in a nitric–sulphuric–perchloric acid mixture. Tissue potassium (K) was estimated by flame photometry (Davis 1962).

Nutrient–use efficiency (NuUE) was calculated according to Koide et al. (2000)

$$ {\text{NuUE}} = {\frac{{{\text{d}}W}}{{{\text{d}}t}}}\;{\text{mg mg}}^{ - 1} $$

(dW, increase in plant d. wt.; dt, time difference).

Relative growth rate (RGR) was calculated (Williams 1946) using the formula:

$$ {\text{RGR}} = {\frac{{{\text{Loge}}\,W2 - {\text{Loge}}\,W1}}{t2 - t1}}\;{\text{mg mg}}^{ - 1} {\text{day}}^{ - 1} $$

(W1, intial plant d. wt.; W2, final plant d. wt.; t2 − t1, time interval).

The efficiency of nutrient uptake (ENuU) defined as the amount of nutrients absorbed per unit root mass (Gray and Schlesinger 1983) was calculated as follows;

$$ {\text{ENuU}} = {\frac{{{\text{Plant\,nutrient\,content}}\,\left( {\upmu {\text{g}}} \right)}}{{{\text{Root biomass}}\,\left( {\text{mg}} \right)}}}\;\upmu {\text{g mg}}^{ - 1} $$

Microbial inoculation effect (MIE) was calculated according to Muthukumar and Udaiyan (2006):

$$ {\text{MIE}} = {\frac{{{\text{Dry\,weight\,of\,inoculated\,seedling}} - {\text{Dry\,weight\,of\,uninoculated\,seedling}}}}{\text{Dry\,weight\,of\,inoculated\,seedling}}} \times 100 $$

Seedling quality index (SQI) was calculated according to Dickson et al. (1960):

$$ {\text{SQI}} = {\frac{{{\text{Total\,dry\,weight }}\,\left( {{\text{g\,Plant}}^{ - 1} } \right)}}{{{\frac{{{\rm Height}\,\left({\rm cm} \right)}}{{{\text{Root\,collar\,diameter }}\left( {\text{cm}} \right)}}} + {\frac{{{\rm Shoot\,dry\,weight }\,\left( {{\text{g\,Plant }}^{ - 1} } \right)}}{{{\text{Root\,dry\,weight }}\left( {{\text{g\,Plant }}^{ - 1} } \right)}}}}}} $$

Statistical analysis

Analysis of Variance (ANOVA) was performed on all data to compare treatment effects and the influence of soil type and fertilizer application on treatments. Means were separated using Duncan’s Multiple Range Test (DMRT). Percentage data on mycorrhizal colonization were arcsine square root transformed prior to analysis. Pearson’s correlation analysis was used to assess the relationship between seedling growth and nutrient uptake, AM fungal colonization and components of nutrient efficiencies.

Results

Seedling growth and biomass

Seedlings inoculated with bioinoculants were taller and had increased stem girth and biomass both at 60 and 120 DAT (Table 2). Seedlings inoculated with bioinoculants were 11–48% taller compared to uninoculated seedlings at 60 DAT. Similarly, at 120 DAT bioinoculated seedlings were 14–48% taller than uninoculated seedlings. The stem girth of bioinoculated seedlings were respectively 4–56% and 6–55% higher compared to uninoculated seedlings at 60 and 120 DAT, respectively. Seedling biomass (shoot + root) of bioinocualted seedlings were 19–423 and 20–434% higher compared to uninoculated seedlings at 60 and 120 DAT, respectively. Bioinoculation significantly reduced the R/S ratio of the seedlings both at 60 and 120 DAT. However, seedlings inoculated with PSB or AM fungi along with Frankia recorded higher R/S ratios both at 60 and 120 DAT.

Table 2 Growth response of Casuarina equisetifolia to bioinoculant inoculation at 60 and 120 days after transplantation (DAT)
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Growth rate

Relative growth rate of non-inoculated C. equisetifolia seedlings was 0.025 mg d−1, and ranged between 0.034 and 0.059 mg d−1 for individual inoculations, 0.063 and 0.100 mg d−1 for dual inoculated seedlings and 0.132 mg d−1 for multi-microbial inoculated seedlings (Fig. 1). Thus, multi-microbial inoculated seedlings had growth rates 65–356% higher compared to other inoculated seedlings and 453% higher compared to uninoculated control.

Fig. 1
figure 1

Relative growth rate of Casuarina equisetifolia seedlings inoculated with arbuscular mycorrhizal fungi (AM), Frankia (F), phosphate solubilizing bacteria (PSB) individually or in combinations as assessed after 120 days of growth. Bars bearing same letter(s) do not significantly differ according to Duncan’s Multiple Range Test (p < 0.05)

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Seedling nutrients

Seedlings inoculated with bioinoculants had significantly higher concentration of nutrients in their tissues (Table 3). By the end of 120 DAT bioinocualted seedlings had 11–20% higher N, 50–192% higher P and 7–79% higher K in their shoots compared to uninoculated seedlings. Similarly, in roots the N, P and K concentration of bioinoculated seedlings were respectively 9–155%, 43–186% and 8–85% higher than uninoculated seedlings.

Table 3 Nutrient concentration of bioinoculated Casuarina equisetifolia at 60 and 120 days after transplantation (DAT)
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AM fungal colonization

Seedlings raised in natural soils had significantly low extent of their root colonized by AM fungi (Table 4). In contrast, bioinoculant inoculation significantly increased the extent of root length colonized by AM fungi and the inoculated seedlings had 2–6 fold higher colonization levels at 60 DAT and 1.5–4 fold higher colonization levels at 120 DAT compared to uninoculated seedlings.

Table 4 Nodulation and arbuscular mycorrhizal (AM) fungal colonization of bioinoculated Casuarina equisetifolia at 60 and 120 days after transplantation (DAT)
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Infective propagules of AM fungi

At 120 DAT, the infective propagules of AM fungi significantly varied among treatments (Fig. 2). Infective propagules were 55–130%, 144–193% and 210% higher in individual, dual and multimicrobial inoculations, respectively, compared to uninoculated control.

Fig. 2
figure 2

Infective propagules of arbuscular mycorrhizal (AM) fungi in Casuarina equisetifolia seedlings uninoculated (C) or inoculated with AM fungi (AM), Frankia (F), phosphate solubilizing bacteria (PSB) individually or in combinations as assessed after 120 days of growth. Bars bearing same letter(s) do not significantly differ according to Duncan’s Multiple Range Test (p < 0.05)

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Nodulation

Seedlings inoculated with bioinoculants had more numerous and heavier nodules compared to uninoculated seedlings (Table 4). The nodule numbers were 8–84% and 11–89% higher in seedlings inoculated with bioinoculants at 60 and 120 DAT, respectively compared to uninoculated seedlings. Similarly, nodule dry weight of inoculated seedlings was 191–385% higher at 60 DAT and 28–307% higher at 120 DAT compared to uninoculated seedlings. A significant positive correlation existed between nodulation and the extent of AM fungal colonization (Table 6).

Efficiency of nutrient uptake

Nutrient uptake per unit of root was higher for seedlings inoculated with bioinoculants (Table 5). The efficiency of nitrogen uptake (ENU) of multiple microbe-inoculated seedlings was 22–267% higher compared to other treatments including control. However, the efficiency of phosphorus uptake (EPU) of PSB and AM fungi inoculated seedlings was 52% higher than multiple microbe inoculated seedlings and 152–450% higher than other treatments. Likewise, the efficiency of potassium uptake (EKU) of PSB and AM fungi inoculated seedlings was 127–208% higher than other inoculations and 40% higher compared to multiple inoculation. Efficiency of nutrient uptake was correlated significantly and positively to nodulation, AM colonization, tissue nutrient content, and seedling biomass and growth rate (Table 6).

Table 5 Efficiencies of nutrient uptake by bioinoculated Casuarina equisetifolia at 60 and 120 days after transplantation (DAT)
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Table 6 Pearson’s correlation coefficients for efficiency of nitrogen (ENU), phosphorus (EPU), potassium (EPU) uptake, nodulation (NN), nodule dry weight (NDW), mycorrhizal colonization (AM), tissue nitrogen (N), phosphorus (P), potassium (K) and plant dry mass (PDW)
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Nutrient use efficiencies

Microbial inoculations significantly altered the nutrient use efficiencies of C. equisetifolia seedlings (Fig. 3). Nitrogen use efficiency (NUE) and phosphorus use efficiency (PUE) of bioinocualted seedlings were significantly lower compared to uninoculated seedlings. The NUE of bioinoculants inoculated seedlings was 8–196% lower compared to uninoculated seedlings. Similarly, PUE of bioinoculants inoculated seedlings was 68–273% lower compared to uninoculated seedlings. Uninoculated seedlings recorded 2–54% higher KUE compared to microbial inoculated seedlings. The NUE (r = −0.940; p < 0.01; n = 8) and KUE (r = −0.808; p < 0.05; n = 8) were significantly and negatively correlated to relative growth rate.

Fig. 3
figure 3

Nutrient use efficiency of Casuarina equisetifolia seedlings uninoculated (C) or inoculated with arbuscular mycorrhizal fungi (A), Frankia (F), phosphate solubilizing bacteria (P) individually or in combinations (AF, AP, FP, AFP) as assessed after 120 days of growth. Bars bearing same letter(s) do not significantly differ according to Duncan’s Multiple Range Test (p < 0.05)

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Microbial inoculation effect (MIE)

MIE of seedlings inoculated with all the microbes were 1.5–4.9 fold higher compared to seedlings inoculated with individual microbes and 1.1–1.4 fold higher compared to dual inoculated seedlings (Fig. 4).

Fig. 4
figure 4

Microbial inoculation effect in Casuarina equisetifolia inoculated with arbuscular mycorrhizal fungi (AM), Frankia (F), phosphate solubilizing bacteria (PSB) individually or in combinations as assessed after 120 days of growth. Bars bearing same letter(s) do not significantly differ according to Duncan’s Multiple Range Test (p < 0.05)

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Seedling quality index (SQI)

Calculated SQI of multi-microbial inoculated seedlings were 92–461% higher compared to other inoculations and 528% higher compared to uninoculated seedlings (Fig. 5). SQI was significantly and positively correlated to the ENU and relative growth rate (r = 0.884; p < 0.01; n = 8).

Fig. 5
figure 5

Seedling quality index of Casuarina equisetifolia inoculated with arbuscular mycorrhizal fungi (AM), Frankia (F), phosphate solubilizing bacteria (PSB) individually or in combinations as assessed after 120 days of growth. Bars bearing same letter(s) do not significantly differ according to Duncan’s Multiple Range Test (p < 0.05)

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Discussion

Bioinoculant application improved the growth of C. equisetifolia seedlings despite the general assumption that members of Casuarinaceae can thrive in soils of low fertility (Diem and Arahou 1996). This study further supports the positive response of C. equisetifolia seedlings in the nursery to bioinoculant application (Vasanthakrishna et al. 1994, 1995; Rajendran et al. 2003) and strengthens the microbial dependency of C. equisetifolia in soils of low fertility. In several studies (e.g., Ravikumar et al. 1997; Guissou et al. 1998; Gupta and Rahangdale 1999; Founoune et al. 2002; Gehring 2003; De Grandcourt et al. 2004; Lesueur and Duponnois 2005; Yamanaka et al. 2005) the plant growth promoting effects of microbes has been demonstrated in sterile substrates. However, the growth promoting effect in sterile soil may substantially differ from those in unsterile soil (Ortas 2003) possibly due to competition from resident microorganisms. Individual inoculation of G. geosporum or Frankia nodular suspensions improved growth of C. equisetifolia seedlings by 109 and 40%, respectively compared to uninoculated control. Similar results of lower intensities have been reported in other studies. Vasanthakrishna et al. (1994) and Rajendran et al. (2003) reported an increase in C. equisetifolia seedling dry weight of 3.45–39% in response to Glomus fasciculatum inoculation; and 3.45–25% in response to nodule suspension inoculation. However, the improved seedling growth in response to PSB inoculation contradicts the observation of Rajendran et al. (2003) where a reduction in seedling biomass (7%) has been reported due to PSB inoculation. Inoculation of Frankia along with G. geosporum resulted in better growth of C. equisetifolia than inoculation of Frankia alone. Several investigators have reported that dual inoculation of AM fungi and Frankia resulted in better growth of actinorhizal plants compared to individual inoculation with Frankia (Tian et al. 2002; Tiwari et al. 2003). We did not observe cluster roots in any of the root systems as cluster roots tend to occur under non-mycorrhizal condition (Zaïd et al. 2003).

In the present study inoculation of G. geosporum improved the nutrient content of nodulated C. equisetifolia. This contrasts the observation of Yamanaka et al. (2005), where Gigaspora margarita failed to improve the P content of nodulated Alnus sieboldiana. The AM effect on nutrient uptake may be due to the well-known ability of the external mycelium to extend the soil volume that the plants explore for P and other nutrient uptake although they do not solubilize nutrients (Yamanaka et al. 2003). This is confirmed by an increased nutrient uptake per given unit of root in G. geosporum inoculated seedlings. Improved growth of C. equisetifolia in response to G. geosporum inoculation may be a result of increased nutrient content in addition to plant hormones produced by AM fungi (Barea and Azcón-Aguilar 1982) or induced by AM fungi (Allen et al. 1982). Mycorrhizal inoculation is known to induce changes in root morphology (Berta et al. 1995), and our findings show that G. geosporum inoculation increased root dry weights, thus confirming previous findings, where inoculation of G. intraradices increased root dry weights in Prunus cerasifera (Berta et al. 1995), and Allium cepa (Toro et al. 1997) by increasing root diameter. Therefore, mycorrhizal roots are more efficient in taking up soil nutrients than non-mycorrhizal roots (Stribley et al. 1980). Most of the observed changes in growth of the host plant in response to mycorrhizal inoculation are consequences of this increased uptake of P and other nutrients as shown in the present and other studies (Raju et al. 1990; Kothari et al. 1991).

In this study, nodule suspension inoculation increased the nodule number and mass in C. equisetifolia. A similar response to crushed nodule suspension inoculation has been reported for Casuarina (Mansour 2003; Rajendran et al. 2003; Rajendran and Devaraj 2004), Alnus (Wheeler et al. 1991) and Coriaria nepalensis (Tiwari et al. 2003). When seedlings were inoculated with Frankia and P. polymyxa along with G. geosporum, additional nodule material was formed as evidenced by increased nodule mass. This increased nodular material provided sufficient fixed N2 to support higher growth rates than seedlings inoculated with Frankia and PSB either individually or in combinations. Further nodule biomass correlated positively with seedling biomass. A similar positive correlation between seedling biomass and nodule biomass has been reported in Alpova diplophloeus (Yamanaka et al. 2003), red alder (Alnus rubra) and snowbrush (Ceanothus velutinus) (Rojas et al. 2001, 2002). These correlations confirm the view that symbiotic nitrogen fixation is dependent on host photosynthesis (Arnone and Gordon 1990). It has been suggested that phosphate plays an important role in the regulation of nodulation in actinorhizal species (Wall and Huss Danell 1996; Wheeler et al. 2000) and it is conceivable that nodulation of C. equisetifolia by Frankia may be particularly susceptible to the improvement in P nutrition which would follow enhanced colonization by AM fungi.

Inoculation of P. polymyxa alone significantly increased the P content of C. equisetifolia. This is in accordance with the studies carried out in lowly weathered soils with a low P fixation capacity where inoculation of PSB alone increased the plant P uptake by 25–73% in different plant species (Kucey and Leggett 1989; Singh and Singh 1993). Further, P. polymyxa behaved as mycorrhizal helper bacteria because it promoted root colonization by mycorrhizal fungi, confirming previous findings involving other mycorrhizal and PSB combinations (Garbaye 1994). The mechanisms by which these bacteria stimulate mycorrhizal colonization are poorly understood. However, specialized bacterial activities such as production of vitamins, amino acids, and hormones may be involved in these interactions (Garbaye 1994).

Dual inoculation of Glomus geosporum and P. polymyxa did change the P concentration of nodulated C. equisetifolia. Plants inoculated with PSB are expected to improve P concentration in the plants by increasing the soluble P in the soil. Osorio and Habte (2001) showed that Leucaena leucocephala inoculated with Glomus aggregatum and Mortierella sp., took up more P than those with AM fungi or bacteria separately. These types of observations have also been reported in neem (Azadirachta indica) and bamboo (Dendrocalamus strictus) (Muthukumar et al. 2001; Muthukumar and Udaiyan 2006). Azcón et al. (1976) showed that the improved P uptake of mycorrhizal plants resulted from the increased availability of the sparingly soluble forms of P to the plant roots by AM fungi. Several studies have shown that PSB interact with AM fungi by releasing phosphate ions in the soil, which causes a synergistic interaction that allows for better exploitation of poorly soluble P sources (Piccini and Azcón 1987; Pramanik and Singh 2004; Yamanaka et al. 2005). It is likely that the seedlings through AM fungi could more effectively take up the phosphate solubilized by the bacteria (Jeffries and Barea 1994). This was evidenced in the present study by the existence of strong correlations between the EPU, AM colonization and plant P.

The dry weight of multiple microbe-inoculated seedlings was significantly higher than seedlings inoculated with bioinoculants individually or dually. This increased biomass results from the one to three fold increases in growth rates of the seedlings inoculated with all the microbes over other inoculations. The increased growth rates of multiple microbe-inoculated seedlings could be the result of increased nutrient inflow rates possibly through AM fungi as suggested by increased ENuU values along with increased nodular efficiency and nutrient availability. This is further supported by the existence of a strong positive correlation between these variables.

Uninoculated C. equisetifolia seedlings had the maximum NuUE, which indicates that these seedlings are under certain amount of resource limitation. Generally, plants respond to resource limitations by increasing the utilization efficiencies of the limiting resources (Chapin et al. 1987). Therefore, microbial inoculated C. equisetifolia seedlings with low NuUE are not inferior, since the quality index based on morphological qualities indicates them to be superior over uninoculated seedlings. The increased nutrient content of bioinoculated seedlings may be advantageous for these seedlings during out-planting as nutrient status of the seedlings influence resistance to various stresses (Ritchie 1984). Although AM fungi, PSB and Frankia naturally occur in soil, usually their numbers are not high enough to compete with other soil microbes commonly established in the rhizosphere. Thus, the amount of nutrients liberated or made available to seedlings by them is generally not sufficient for a substantial increase in situ seedling growth. Therefore, inoculation of seedlings by target microorganisms at a higher concentration than that normally found in the soil is necessary to take advantage of nutrient availability for better quality seedling production.