134

DOI: 10.1002/jpln.200800153

J. Plant Nutr. Soil Sci. 2009, 172, 134139

Bacillus subtilis NRRL B-30408 inoculation enhances the symbiotic efficiency of Lens esculenta Moench at a Himalayan location
Rinu K.1 and Anita Pandey1*
1

Biotechnological Applications Group, G B Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora 263 643, Uttarakhand, India

Abstract
Field-based experiments were conducted to evaluate the promotion abilities of Bacillus subtilis NRRL B-30408 for growth of lentil (Lens esculenta Moench) at a mountain location of Indian Himalaya in two consecutive years. Observations were recorded for plant growth, yield, nodulation, root colonization by arbuscular mycorrhizal and endophytic fungi, and other related parameters. A positive influence of bacterial inoculation on plant biomass and yield-related parameters was recorded in both years. The significant increase in growth and nodule numbers as well as leghaemoglobin and protein concentrations of nodules indicated an enhancement in efficiency of the Rhizobiumlegume symbiosis due to bacterial inoculation. An increase in protein concentration was also recorded for shoots, leaves, and seeds. Due to bacterial inoculation, there was an increase in colonization by endophytic fungi and a simultaneous decrease in colonization by arbuscular mycorrhizal fungi in roots. Based on the results of this field study, inoculation with suitable plant growthpromoting rhizobacteria instead of dual inoculation is suggested as a better option for improving the yield and related attributes of a primary dietary legume such as lentil.
Key words: legumerhizobia symbiosis / PGPR / leghaemoglobin / arbuscular mycorrhizae / endophytic fungi

Accepted November 17, 2008

1 Introduction
Soil fertility is diminishing gradually due to soil erosion, loss of nutrients, accumulation of salts, water logging, unbalanced nutrient compensation, pollution, and contamination of soil. Fertility problems are greatly solved by addition of mineral fertilizers, but gaps between supply and demand and adverse effects on the environment have led agricultural scientists to look for alternative strategies (Rabie and Humiany, 2004). Legumerhizobia symbioses actively fix nitrogen and are important for N input into agroecosystems (Smith and Hume, 1987). Some rhizobacterial strains promote legume nodulation and nitrogen fixation by producing flavonoid-like compounds and/ or stimulating the host legume to produce more flavonoid signal molecules (Andrade et al., 1998; Schultze and Kondorosi, 1998; Parmar and Dadarwal, 1999). Plant growthpromoting rhizobacteria (PGPR) are classified into two groups. The first group includes bacterial strains that have the capability of synthesizing plant growthpromoting substances, fixing atmospheric nitrogen, solubilizing inorganic phosphate or iron, and of improving plant stress tolerance to drought, salinity, metal toxicity, and pesticide load. The second group includes the bacteria which are able to decrease or prevent the deleterious effects of phytopathogenic microorganisms (Bashan and Holguin, 1998; Lucy et al., 2004). Microorganisms are environment-specific. Ecological competence has been considered as one of the key factors in selection of suitable plant growthpromoting rhizobacterium. A promising strain of Bacillus subtilis NRRL B-30408, originally isolated from a temperate location in Indian Himalaya, has been evaluated for its plant growthpromoting and biocontrol abilities on several agricultural, forest, and micropropagated plant species and has been developed in form of a carrier-based bioinoculant (Pandey et al., 2000, 2004; Chaurasia et al., 2005; Trivedi et al., 2005, 2007). Lentil (Lens esculenta Moench) represents a primary dietary legume and is grown in the colder regions of Indian Himalaya. In the present study, inoculation of lentil with B. subtilis NRRL B-30408 was carried out to evaluate the influence on the legumerhizobial symbiosis for two consecutive years under field conditions. The influence of inoculation was analyzed on the basis of growth, nodulation, rhizosphere colonization, and yield-related parameters.

2 Material and methods

2.1 Bacterial inoculant and lentil seed
The bacterial inoculant (Bacillus subtilis, NRRL B-30408), originally isolated from the rhizosphere soil of established tea bushes cultivated at a temperate location of Indian Himalaya,

* Correspondence: Dr. A. Pandey; e-mail: anita@gbpihed.nic.in

2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.plant-soil.com

J. Plant Nutr. Soil Sci. 2009, 172, 134139 was used for inoculation. The bacterial strain has been reported as a strong antagonist and a moderate phosphate solubilizer (Chaurasia et al., 2005; Trivedi et al., 2007). The culture was maintained on tryptone yeast extract (TYE) agar slants at 4C in a refrigerator and in glycerol at 20C in a deep-freezer. The bacterial strain (NRRL B-30408) has been deposited in Agricultural Research Service (ARS) patent culture collection, United States Department of agriculture, Illinois, under the Budapest treaty. The lentil (Lens esculenta Moench, common name: Masoor Dal) seed was obtained from a local farmer, at a nearby village Kosi-Katarmal.

Bacillus subtilis inoculation affects symbiotic efficiency 135

2.5 Determination of leghaemoglobin and protein concentration

For determination of leghaemoglobin and protein concentration, three representative plants from control and inoculated plots at the peak flowering time were taken. Leghaemoglobin concentration was determined spectrophotometrically as described by Appleby and Bergerson (1980) with some modifications. One gram of fresh nodules in each case, collected from control as well as from inoculated plants at the peak flowering time, was crushed in liquid nitrogen and stored at 70C until use. Cryopreserved nodule powder (0.2 g) was mixed with 0.6 mL of phosphate buffer (pH 7.4), vortexed for 23 min, and centrifuged (Hitachi-Himac CR 22G) at 14,000 rpm for 20 min. The volume (reddish-brown precipitate) formed was estimated and made up to a definite volume with distilled water. The final solution was used for determination of leghaemoglobin concentration. For protein concentration, nodules (1 g), leaves (5 g), shoot (5 g), and seed (5 g) from control and inoculated plots were taken and macerated in liquid nitrogen and stored at 70C until use. Cryopreserved samples (0.1 g each) were resuspended in 750 lL of cold 50 mM Na-phosphate buffer (pH 7.5) containing 12 mM ethylene-diamine-tetra-acetic acid (EDTA), 1 mM phenylmethylsulfoxide fluoride (PMSF), 2 mM b-mercaptoethanol, and 10% polyvinylpolypyrolidone (PVPP). The resulting solution was centrifuged at 14,000 rpm for 20 min at 4C. The supernatant containing protein was determined by Lowrys method (Lowry et al., 1951). A standard curve was prepared by using bovine serum albumin (BSA) for determination of protein concentration. All the experiments were done in triplicate.

2.2 Bacterial inoculation of lentil

Bacillus subtilis NRRL B-30408 was grown on TY agar plates at 28C for 48 h. The bacterial cells were harvested in 100 mL of sterilized distilled water containing an approximate population 105 to 106 cells mL1. Fifty grams of lentil seed were mixed in 100 mL of culture suspension, 100 g of charcoal, and 10 g of sticker (gurraw sugar). The mixture was thoroughly stirred to facilitate even coating of lentil seed. Seed treated with charcoal slurry without bacteria served as control.

2.3 Experimental setup

The field experiments were set up in the third week of November 2005 (first-year trial) and 2006 (second-year trial) in the nursery plots of the institute situated at the Northern part of the state of Uttarakhand (293733 N to 79389 E; 1150 m above mean sea level). Five replicates were taken for each treatment (control and inoculated). Separate plots (plot size = 2.50 m 1.25 m) adjacent to each other and separated with a separate boundary were prepared. Soil type of these plots is sandy loam with pHH2 O 7.5 and 40% (w/w) moisture content. The soil nutrient analyses (C = 0.560%, N = 0.094%, P = 0.100%, and K = 0.022%) has been reported previously (Kumar et al., 2007)

2.6 Root colonization by arbuscular mycorrhizal fungi and other endophytes

These analyses were performed only in the second year. At the time of flowering, ten representative plants were selected randomly and harvested. The roots were washed thoroughly with running water. The root branches were taken for the study of fungal colonization. Twenty-five (randomly selected from five control and five inoculated plots) root samples were cut into small pieces of 1 cm and treated with 10% KOH at 90C for 1 h, then washed with tap water to remove KOH and treated with alkaline H2O2 for 15 min. After removing the H2O2 with tap water, the root samples were acidified with 1% HCl for 10 min. The HCl was drained, and root samples were stained with 0.1% trypan blue at 90C for 30 min and observed under a microscope (Nikon-Optiphot 2, Japan). The number of arbuscular mycorrhizal (AM) and other endophytic fungi counted in the root samples were recorded, and the percent colonization was calculated using the formula: number of positive root pieces 100 / total number of root pieces observed.

2.4 Plant growth, nodule number, yield, and harvest index

For taking measurements on growth parameters, ten plants were randomly harvested at their peak flowering time (4 months after sowing) from inoculated and control plots and washed several times with running water. First, root and shoot lengths were recorded. Then the roots were cut off, and roots and shoots were dried at 70C for 72 h separately, for each plant. For nodule number, another ten representative plants were harvested from control and inoculated plots. The nodules were separated from each plant, and dry weight was recorded after drying the nodules at 70C for 72 h. Towards the end of the growth period, another ten representative plants were randomly harvested from control and inoculated plots to determine yield parameters. The plants were dried at 70C for 72 h and weighed for calculating the biological yield. Seed weight (economic yield) was also recorded at the time of senescence. Harvest index was calculated according to the formula: harvest index = economic yield 100 / biological yield. 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.7 Statistics
The excel program of Microsoft Windows 2003 professional was used to calculate means and standard deviations. www.plant-soil.com

136

Rinu, Pandey

J. Plant Nutr. Soil Sci. 2009, 172, 134139 These species have already been recognized for their role in plant-growth promotion and biocontrol. Based on many screenings performed under greenhouse and field conditions, three strains of various bacterial species B. megaterium (MTCC 6521), B. subtilis (NRRL B-30408), and P. corrugata (NRRL B-30409) were found suitable for improving plant performance of agricultural and forest species of colder regions (Pandey et al., 2004). Bacillus subtilis NRRL B-30408 got preference over B. megaterium MTCC 6521 and P. corrugata NRRL B-30409 in rice (Trivedi et al., 2007). For maize grown in colder regions, P. corrugata NRRL B-30409 has been recommended (Kumar et al., 2007). In the present study, P. corrugata NRRL B-30409 inoculation was ruled out as it was not found compatible for some of the legume species (Pandey et al., 1999). For legumes, coinoculation with PGPR and rhizobia is preferred to improve plant performance (Sindhu et al., 2002; Garcia et al., 2004; Siddiqui et al., 2007). In the present study, only a PGPR inoculant, i.e., B. subtilis NRRL B-30408 was used instead of coinoculation. This attempt was made to evaluate the enhancement in symbiotic efficiency due to stimulation of native rhizobia associated with lentil. Stimulation of native rhizosphere microflora (such as pseudomonads and actinomycetes, in particular) has been considered as one of the important factors contributing to overall plant performance in inoculation experiments (Pandey et al., 1998; Kumar et al., 2007). Bacillus subtilis NRRL B-30408 used in this study is a native of temperate monsoonal Himalayan location and was isolated from rhizosphere soil following heavy snow fall (Pandey and Palni, 1996). The significant results obtained for nodulation-related parameters and protein concentration in seeds is of great importance. Food legumes are considered as a major source of dietary proteins. An increase in protein concentration with increasing altitude in Indian Himalayan region has been reported. Protein concentration in the plants at 500 m and

ANOVA was performed to determine significant differences between control and inoculated plants.

3 Results and discussion

Except for harvest index, there was a significantly positive influence of B. subtilis NRRL B-30408 on growth and yield parameters of lentil in both years (Tab. 1). The root length of lentil plants significantly increased 1.3 and 1.2 times in the first and second year of study, respectively, while shoot length increased 1.5 times and 1.1 times. Similarly, root biomass was increased significantly, 1.1 and 1.2 times in the first and second year of analysis. The shoot weight increased 1.4 and 1.6 times as a result of inoculation, which was found statistically significant. The dry weight of seeds was also found to be significantly enhanced in both the years of study. The bacterial inoculation enhanced the number, biomass, and leghaemoglobin concentration of nodules (Tab. 2). While an increase in nodule biomass was recorded in both the years, it was statistically significant in second year only. The study area occasionally experiences snow fall in winters. A heavy snowfall that occurred during the second year affected the growth parameters negatively. Nevertheless, the bacterial inoculation appeared to show positive influence on the growth parameters. A positive influence of bacterial inoculation on protein concentration in shoots, leaves, nodules, and seeds was also recorded, statistically significant in case of nodules in first year and in seed in both the years (Tab. 3). Protein concentration in nodules was increased 1.1 and 1.2 times in first and second year of study, which was statistically significant. In the case of seeds, the protein concentration significantly increased 1.5 times and 1.6 times. The present study is part of a long-term program that was initiated to develop bioinoculants for colder mountain regions. Earlier experiments showed a dominance of species of Bacillus and Pseudomonas in soil samples of higher altitudes.

Table 1: Influence of B. subtilis NRRL B-30408 inoculation on growth and yield-related parameters of lentil in field experiments of two consecutive years. Values are the means SD (n = 10). Year 200506 Treatment Control Inoculated ANOVA Shoot length (cm) Root length (cm) 25.6 3.73 38.94 4.68* F1, 18 = 49.66 p = 0.001 9.89 1.43 13.26 0.72* F1, 18 = 43.95 p = 0.001 Biomass production and yield (g dry weight) Shoot 10.37 1.10 14.62 2.90* F1, 18 = 18.71 p = 0.001 Root 4.66 0.20 5.31 0.72* F1, 18 = 7.48 p = 0.01 Seed 0.58 0.07 0.72 0.08* F1, 18 = 13.98 p = 0.001 Harvest index 5.76 5.83

Year 200607 Treatment Control Inoculated ANOVA Shoot length (cm) Root length (cm) 51.5 7.57 58.4 4.97* F1, 18 = 5.79 p = 0.03 * significant at p < 0.05% 17.25 3.12 21.6 4.83* F1, 18 = 5.71 p = 0.03

Biomass production and yield (g dry weight) Shoot 10.18 2.27 16.56 5.14* F1, 18 = 12.00 p = 0.002 Root 4.80 1.22 6.17 0.57* F1, 18 = 10.30 p = 0.004 Seed 0.85 0.15 1.04 0.48* F1, 18 = 12.38 p = 0.002 Harvest index 6.57 6.74

2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.plant-soil.com

J. Plant Nutr. Soil Sci. 2009, 172, 134139

Bacillus subtilis inoculation affects symbiotic efficiency 137

Table 2: Influence of B. subtilis NRRL B-30408 inoculation on nodule-based parameters of lentil in field experiments of two consecutive years. Values are the means SD. Treatment Year 200506 Control Inoculated ANOVA 44 17.01 86 12.82* F1, 18 = 39.60 p = 0.001 Year 200607 Control Inoculated ANOVA 20 19.60 41 20.23* F1, 18 = 5.93 p = 0.02
a n = 10, b Concentration of nodule tissue sap (n = 3) * significant at p < 0.05%

Nodule numbera

Dry weighta (g)

Leghaemoglobinb (mM)

Leghaemoglobin (% in nodule total protein)

0.04 0.03 0.05 0.04 F1, 18 = 0.07 p = 0.08

0.06 0.01 0.14 0.01* F1, 18 = 114.2 p = 0.001

29.63 59.15

0.004 0.06 0.03 0.01* F1, 18 = 20.13 p = 0.001

0.07 0.01 0.17 0.01* F1, 18 = 169.0 p = 0.001

36.20 70.83

Table 3: Influence of B. subtilis NRRL B-30408 inoculation on protein concentrations in various plant parts of lentil in field experiments of two consecutive years. Values are the concentration means of tissue sap SD of three individual plants. Treatment Nodule (mg L1) 2006 Control Inoculated ANOVA 40.5 3.5 2007 38.67 2.88 Shoot (mg L1) 2006 19.7 3.20 22.33 1.89 F1, 18 = 1.50 p = 0.28 2007 14.0 1.0 19.33 4.16 F1, 18 = 4.65 p = 0.09 Leaf (mg L1) 2006 31.67 2.02 31.83 4.65 F1, 18 = 0.003 p = 0.95 2007 28.33 2.52 31.33 (0.58) F1, 18 = 4.05 p = 0.11 Grains (mg L1) 2006 31.17 1.61 46.33 6.11* F1, 18 = 17.28 p = 0.01 2007 32.67 11.02 52.67 2.52* F1, 18 = 9.39 p = 0.03

47.33 1.25* 48.0 2.0* F1, 18 = 10.12 p = 0.03 F1, 18 =21.18 p = 0.01

* significant at p < 0.05%

3500 m altitude of Himachal hills was 4.5% and 9.8%, respectively. Similarly, in Uttar Pradesh hills the crude protein was estimated to be 6.5% and 9.8% in plants at 1550 m and 3600 m altitude, respectively (Mal, 1996). Protein increase due to bacterial inoculation has been reported by Romeiro et al. (2005). Another important nodule-related finding in the present study was the significant increase in the leghaemoglobin concentration in nodules. Leghaemoglobin has only been detected in the infected tissues of root and stem nodules of nitrogen-fixing plants (Arredondo-Peter et al., 1998). These proteins act as oxygen buffers for the rhizobia inside the root nodules. The rate of nitrogen fixation is closely correlated with the concentration of leghaemoglobin (Uheda and Syono, 1982). Since leghaemoglobin in nodules is essential for the respiring bacteroids (Appleby, 1984), an increase in leghaemoglobin concentration may be an indication of a positive effect of inoculation on metabolic activities such as nitrogenase enzyme activity associated with nitrogen fixation. Rhizobial colonization in roots of legumes is a chemotactic response and is probably mediated by chemical attractants, especially flavonoids. Flavonoids are positive regulators of the genes involved in nodulation (nod genes). Plant growth promoting rhizobacteria have been reported as inducers of 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

the flavonoid production in the host roots (Parmar and Dadarwal, 1999). Interesting observations were recorded for the colonization of AM fungi and other endophytes in lentil roots due to bacterial inoculation (Fig. 1). The endophytes in the root samples were filamentous structures with round spores growing along with the epidermal tissues. In some root samples, they were observed in vascular bundles as well. In control root samples, the percent colonization of AM was 52% and that of other endophytes was 56%, whereas in inoculated root samples, they were 2% and 78%, respectively. There was a significant increase in endophytic fungal colonization and a significant decrease in AM fungal colonization in B. subtilis NRRL B-30408inoculated plants. However, these preliminary observations need more attention. Endophytic microorganisms live within the host plants without causing any noticeable symptoms of disease. Since 1970, several reports have shown that these endophytes play an important role in protecting their host against predators and pathogens (Azevedo et al., 2000). Correa et al. (2006) have summarized the effects of mycorrhizal inoculation, including a decrease in plant productivity. Influence, positive or negative, of vesicular arbuscular mycorrhizae (VAM) inoculation on various legumes including lentil has been reported (Bala and Singh, www.plant-soil.com

138
100
% colonization

Rinu, Pandey

J. Plant Nutr. Soil Sci. 2009, 172, 134139

PGPB (plant growth-promoting bacteria) and PGPB. Soil Biol. Biochem. 30, 12251228. Chaurasia, B., Pandey, A., Palni, L. M. S., Trivedi, P., Kumar, B., Colvin, N. (2005): Diffusible and volatile compounds produced by an antagonistic Bacillus subtilis strain cause structural deformities in pathogenic fungi in vitro. Microbiol. Res. 160, 7581. Correa, A., Strasser, R. J., Loucao, M. A. M. (2006): Are mycorrhizal always beneficial? Plant Soil 279, 6573.

80 60 40 20 0
AM Fungi Endophytes

Figure 1: Percent colonization of AM and endophytic fungi in the roots of control and inoculated lentil. & Control, & B. subtilisinoculated (data are means of standard deviation).

El-Din, S. M. S. B., Moawad, H. (1988): Enhancement of nitrogen fixation in lentil, faba bean, and soybean by dual inoculation with Rhizobia and mycorrhizae. Plant Soil 108, 117123. Garcia, J. A. L., Probanza, A., Ramos, B., Barriuso, J., Manero, F. J. G. (2004): Effect of inoculation with plant growth promoting rhizobacteria (PGPRs) and Sinorhizobium fredii on biological nitrogen fixation, nodulation and growth of Glycine max cv. Osumi. Plant Soil 267, 143153. Kumar, B., Trivedi, P., Pandey, A. (2007): Pseudomonas corrugata: A suitable bacterial inoculant for maize grown under rainfed conditions of Himalayan region. Soil Biol. Biochem. 39, 30933100. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. (1951): Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265275. Lucy, M., Reed, E., Glick, B. R. (2004): Applications of free living plant growth-promoting rhizobacteria. Antonie Van Leeuwenhoek 86, 125. Mal, B. (1996): Forage production in temperate area in India Present Status and future prospects. 2nd meeting: Temperate Asia pasture and fodder working group, Dehradun, India, pp. 1419. Pandey, A., Palni, L. M. S. (1996): The rhizosphere effect of tea on soil microbes in a Himalayan monsoonal location. Biol. Fertil. Soils 21, 131137. Pandey, A., Sharma, E., Palni, L. M. S. (1998): Influence of bacterial inoculation on maize in upland farming systems of the Sikkim Himalaya. Soil Biol. Biochem. 30, 379384. Pandey, A., Durgapal, A., Joshi, M., Palni, L. M. S. (1999): Influence of Pseudomonas corrugata inoculation on root colonization and growth promotion of two important hill crops. Microbiol. Res. 154, 259266. Pandey, A., Palni, L. M. S., Bag, N. (2000): Biological hardening of tissue culture raised tea plants. Biotechnol. Lett. 22, 10871091. Pandey, A., Trivedi, P., Kumar, B., Chaurasia, B., Singh, S., Palni, L. M. S. (2004): Development of microbial inoculants for enhancing plant performance in the mountains, in Reddy, M. S., Khanna, S. (eds.): Biotechnological Approaches for Sustainable Development. Allied Publisher Pvt. Ltd., New Delhi, pp. 1320. Parmar, N., Dadarwal, K. R. (1999): Stimulation of nitrogen fixation and induction of flavonoid like compounds by rhizobacteria. J. Appl. Microbiol. 86, 3664. Rabie, G. H., Humiany, A. A. (2004): Role of VA mycorrhiza on the growth of cowpea plant and their associative effect with N2 fixing and P-solubilizing bacteria as biofertilizers in calcareous soil. J. Food Agric. Environ. 2, 186192. Romeiro, R. S., Filho, L., Junior, J. R. V., Silva, H. S. A., Pereira, M. C. B., Carvalho, M. G. (2005): Macromolecules released by a plant growth-promoting rhizobacterium as elicitors of systemic resistance in tomato to bacterial and fungal pathogens. J. Phytopathol. 2, 120123. Schultze, M., Kondorosi, A. (1998): Regulation of symbiosis root nodule development. Annu Rev. Gen. 32, 3357. Siddiqui, Z. A., Baghel, G., Akhtar, M. S. (2007): Biocontrol of Meloidogyne javanica by Rihzobium and plant growth promoting rhizobacteria on lentil. World J. Microbiol. Biotechnol. 23, 435441.

1985; El-Din and Moawad, 1988; Xavier and Germida, 2002). To the best of our knowledge, this is the first report on PGPR inoculation of lentil in a Himalayan location.

4 Conclusion
On the basis of the results in this study, it is concluded that B. subtilis NRRL B-30408 inoculation helped in stimulation of native rhizobia and subsequently of nitrogen fixation performed by the legume crop. While increase in nodule number and nitrogen concentration indicates the enhancement of symbiotic performance, an increase in root biomass may contribute to improved nutrient-uptake capability.

Acknowledgment
Director GBPIHED is thanked for extending the facilities. Uttarakhand State Council for Science and Technology, Government of Uttarakhand, and the Ministry of Environment and Forests, Govt. of India, New Delhi are acknowledged for financial support.

References
Andrade, G., De Leij, F. A., Lynch, J. M. (1998): Plant mediated interactions between Pseudomonas fluorescens, Rhizobium leguminosarum and arbuscular mycorrhizal on pea. Lett. App. Microbiol. 26, 311316. Appleby, C. A. (1984): Leghaemoglobin and Rhizobium respiration. Annu. Rev. Plant Physiol. 35, 443478. Appleby, C. A., Bergerson, F. J. (1980): Preparation and experimental use of leghaemoglobin, in Bergerson, F. J. (ed.): Methods for Evaluating Biological Nitrogen Fixation. John Wiley & Sons Ltd, New Jersey, pp. 315333. Arredondo-Peter, R., Hargrove, M. S., Moran, J. F., Sarath, G., Klucas, R. V. (1998): Plant haemoglobins. Plant Physiol. 118, 11211126. Azevedo, J. L., Macheroni Jr, W., Pereira, J. O., Araujo, W. L. (2000): Endophytic microorganisms: A review on insect control and recent advances on tropical plants. Eletronic J. Biotechnol. 3, 0136. Bala, S., Singh, O. S. (1985): Response of lentil to VA mycorrhizal inoculation and plant available P levels of unsterile soils. Pant Soil 87, 445447. Bashan, Y., Holguin, G. (1998): Proposal for the division of plant growth-promoting rhizobacteria into two classifications: biocontrol-

2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.plant-soil.com

J. Plant Nutr. Soil Sci. 2009, 172, 134139

Sindhu, S. S., Suneja, S., Goel, A. K., Parmar, N., Dadarwal, K. R. (2002): Plant growth promoting effects of Pseudomonas sp. on coinoculation with Mesorhizobium sp. Cicer strain under sterile and wilt sick soil conditions. Appl. Soil Ecol. 19, 5964. Smith, D. L., Hume, D. J. (1987): Comparison of assay methods for nitrogen fixation utilizing white bean and soybean. Can. J. Plant Sci. 67, 1119. Trivedi, P., Pandey, A., Palni, L. M. S. (2005): Carrier based preparations of plant growth promoting bacteria suitable for use in cooler regions. World J. Microbiol. Biotechnol. 26, 941945. Trivedi, P., Kumar, B., Pandey, A., Palni, L. M. S. (2007): Growth promotion of rice by phosphate solubilizing bioinoculants in a

Bacillus subtilis inoculation affects symbiotic efficiency 139

Himalayan location, in Velazqez, E., Rodriguez-Barrueco, C. (eds.): Plant and Soil. Developments in Plant and Soil Sciences, first international meeting on microbial phosphate solubilization. Springer, Heidelberg, pp. 291299. Uheda, E., Syono, K. (1982): Effects of leghaemoglobin components on nitrogen fixation and oxygen consumption. Plant Cell Physiol. 23, 8590. Xavier, L. J. C., Germida, J. J. (2002): Response of lentil under controlled conditions to co-inoculation with arbuscular mycorrhizal fungi and rhizobia varying in efficiency. Soil Biol. Biochem. 34, 181188.

2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.plant-soil.com