**8. Rhizobium**

Rhizobium belongs to family Rhizobiaceae and the bacteria have the ability to reduce N2 and thereby "fix" atmospheric nitrogen using the enzyme nitrogenase. It colonizes the roots of species legumes to form tumor like growths called root nodules, which act as biofactories of ammonia production (**Figure 1**). The process of biological nitrogen fixation was discovered the Dutch microbiologist Martinus Beijerinck. Rhizobia (e.g., Rhizobium, Mesorhizobium, Sinorhizobium) fix atmospheric nitrogen or dinitrogen, N2 into inorganic nitrogen compounds such as ammonium, NH4, Which is then incorporated into amino acids, which can be utilized by the plant. Plants cannot fix nitrogen on their own, but need it in one form or another to make amino acids and protein. Because legumes form nodules with rhizobia, they have high levels of nitrogen available to them. Rhizobium is a soil habitat bacterium, which is able to colonize the legume roots and fixes the atmospheric

#### **Figure 1.**

*(a) Rhizobium root nodule, (b)* Frankia *root nodule, (c) AM fungi infection, (d) AM fungi spore, (e) phosphate solobilizing bacteria* Bacillus *sp., (f)* Paenibacillus polymyxa*, (g)* Azospirillum brasilense*.*

**279**

volume (20.397 cm3

*Sustainable Development of Horticulture and Forestry through Bio-Inoculants*

nitrogen. Rhizobium associated with nodulated legume trees have an outstanding potential for fixing atmospheric nitrogen (*Sesbania cannabina* and *Leucaena leuco-*

yr<sup>−</sup><sup>1</sup>

culture has been routinely recommended as an input in legume tree species cultivation. Rhizobium helps to boost up the tree growth by insoluble nutrients available for plant. Seedling treated with Rhizobium biofertilizer found to remarkable increase in

High nitrogen yield was estimated in the *Pongamia pinnata* seedling inoculated with Rhizobium + Phosphobacteria + VAM fungi [78]. Increased N content in the plant sample of various tree seedling, co-inoculated with different biofertilizers [102]. Similarly increase in biomass production due to VAM fungi inoculation with *Acacia* sp. [103] and in *Albizzia* sp. [104]. Rhizobium inoculation + PSB with 25% N significantly increase the average 53 nodule no./seedling was followed by only Rhizobium with 25% N (38 nodule/seedling) inoculation in *Acacia nilotica* shoot length increased from 58.50 to 78.75 cm, collar diameter from 5.05 to 6.15 mm and

Dual inoculation of *Azospirillum* and *Rhizobium* with legume plant has been found to increase plant-growth when compared with single inoculations. *Azospirillum* is considered a helper bacteria to *Rhizobium* by stimulating nodulation, nodule function, and possibly plant metabolism. Similarly, phytohormones produced by *Azospirillum* promoted epidermal-cell differentiation in root hairs that increased the number of potential sites for rhizobial infection and more nodule development [106]. Dual inoculation of AM fungi with *Rhizobium* improved nodulation, plant dry weight, N and P contents of *Leucaena leucocephala* in a P deficient

*Azotobacter* is a free living (non-symbiotic), aerobic, nitrogen fixing organism and these gram negative bacteria belongs to family Azotobacteriaceae. There are seven species of *Azotobacter* viz. *A. beijerinckii, A. chroococcum, A. vinelandii, A. paspali, A. agilis, A. insignis* and *A. macrocytogenes. A. chroococcum* appeared more in acidic soils and arable soils while *A. beijerinckii* in neutral and alkali soils. Apart from nitrogen, this organism is capable of producing antibacterial and antifungal compounds, hormones and siderophore [108]. Individual or combined inoculations stimulated the plant growth and significantly increased the concentrations of indole 3-acetic acid (IAA), P, Mg, N, and total soluble sugars in agri crop. Bioinoculants co-inoculation of nitrogen fixing organism *Azotobacter* and phosphate solubilizing microorganisms *Bacillus megaterium* showed a significant increase on the growth of

*Azotobacter* inoculated strawberry plants attained maximum height (24.92 cm)

ners per plant (18.70), heavier fruit (10.02gm), more fruit length (35.9 mm), and more fruit breadth (22.91 mm) as compared to all other treatment [109]. Similarly, combined application of manure + Azotobacter + wood ash + phosphorous solubilizing bacteria + oil cake improved significantly fruit diameter (3.11 cm), length (3.95 cm),

(9.01'B), acidity (0.857), TSS:acidity ratio (11:12) and yield (238.95 g/plant) [110].

), weight (11.11 g), total sugars (7.95%), total soluble solids

), number of run-

soil compared to single inoculation with either organism [107] .

teak and India red wood under nursery condition [96].

more number of leaves per plant (26.29), more leaf area (96.12 cm2

growth and nodulation of *D. sissoo* [100], *A. nilotica* [26] *Albizzia* sp. [101].

[99]. In recent year use of Rhizobium

*DOI: http://dx.doi.org/10.5772/intechopen.87148*

*cephala*) can fix up to 75–584 kg N ha<sup>−</sup><sup>1</sup>

**8.1 Rhizobium with helper microbes**

nodulation increased 0.071 to 0.342 g/seedling [105].

**8.2** *Azospirillum* **and** *Rhizobium* **interaction**

**8.3** *Azotobacter* **plant interaction**

*Sustainable Development of Horticulture and Forestry through Bio-Inoculants DOI: http://dx.doi.org/10.5772/intechopen.87148*

nitrogen. Rhizobium associated with nodulated legume trees have an outstanding potential for fixing atmospheric nitrogen (*Sesbania cannabina* and *Leucaena leucocephala*) can fix up to 75–584 kg N ha<sup>−</sup><sup>1</sup> yr<sup>−</sup><sup>1</sup> [99]. In recent year use of Rhizobium culture has been routinely recommended as an input in legume tree species cultivation. Rhizobium helps to boost up the tree growth by insoluble nutrients available for plant. Seedling treated with Rhizobium biofertilizer found to remarkable increase in growth and nodulation of *D. sissoo* [100], *A. nilotica* [26] *Albizzia* sp. [101].

#### **8.1 Rhizobium with helper microbes**

*Sustainable Crop Production*

Rhizobium belongs to family Rhizobiaceae and the bacteria have the ability to reduce N2 and thereby "fix" atmospheric nitrogen using the enzyme nitrogenase. It colonizes the roots of species legumes to form tumor like growths called root nodules, which act as biofactories of ammonia production (**Figure 1**). The process of biological nitrogen fixation was discovered the Dutch microbiologist Martinus Beijerinck. Rhizobia (e.g., Rhizobium, Mesorhizobium, Sinorhizobium) fix atmospheric nitrogen or dinitrogen, N2 into inorganic nitrogen compounds such as ammonium, NH4, Which is then incorporated into amino acids, which can be utilized by the plant. Plants cannot fix nitrogen on their own, but need it in one form or another to make amino acids and protein. Because legumes form nodules with rhizobia, they have high levels of nitrogen available to them. Rhizobium is a soil habitat bacterium, which is able to colonize the legume roots and fixes the atmospheric

*(a) Rhizobium root nodule, (b)* Frankia *root nodule, (c) AM fungi infection, (d) AM fungi spore, (e) phosphate solobilizing bacteria* Bacillus *sp., (f)* Paenibacillus polymyxa*, (g)* Azospirillum brasilense*.*

**8. Rhizobium**

**278**

**Figure 1.**

High nitrogen yield was estimated in the *Pongamia pinnata* seedling inoculated with Rhizobium + Phosphobacteria + VAM fungi [78]. Increased N content in the plant sample of various tree seedling, co-inoculated with different biofertilizers [102]. Similarly increase in biomass production due to VAM fungi inoculation with *Acacia* sp. [103] and in *Albizzia* sp. [104]. Rhizobium inoculation + PSB with 25% N significantly increase the average 53 nodule no./seedling was followed by only Rhizobium with 25% N (38 nodule/seedling) inoculation in *Acacia nilotica* shoot length increased from 58.50 to 78.75 cm, collar diameter from 5.05 to 6.15 mm and nodulation increased 0.071 to 0.342 g/seedling [105].

#### **8.2** *Azospirillum* **and** *Rhizobium* **interaction**

Dual inoculation of *Azospirillum* and *Rhizobium* with legume plant has been found to increase plant-growth when compared with single inoculations. *Azospirillum* is considered a helper bacteria to *Rhizobium* by stimulating nodulation, nodule function, and possibly plant metabolism. Similarly, phytohormones produced by *Azospirillum* promoted epidermal-cell differentiation in root hairs that increased the number of potential sites for rhizobial infection and more nodule development [106]. Dual inoculation of AM fungi with *Rhizobium* improved nodulation, plant dry weight, N and P contents of *Leucaena leucocephala* in a P deficient soil compared to single inoculation with either organism [107] .

#### **8.3** *Azotobacter* **plant interaction**

*Azotobacter* is a free living (non-symbiotic), aerobic, nitrogen fixing organism and these gram negative bacteria belongs to family Azotobacteriaceae. There are seven species of *Azotobacter* viz. *A. beijerinckii, A. chroococcum, A. vinelandii, A. paspali, A. agilis, A. insignis* and *A. macrocytogenes. A. chroococcum* appeared more in acidic soils and arable soils while *A. beijerinckii* in neutral and alkali soils. Apart from nitrogen, this organism is capable of producing antibacterial and antifungal compounds, hormones and siderophore [108]. Individual or combined inoculations stimulated the plant growth and significantly increased the concentrations of indole 3-acetic acid (IAA), P, Mg, N, and total soluble sugars in agri crop. Bioinoculants co-inoculation of nitrogen fixing organism *Azotobacter* and phosphate solubilizing microorganisms *Bacillus megaterium* showed a significant increase on the growth of teak and India red wood under nursery condition [96].

*Azotobacter* inoculated strawberry plants attained maximum height (24.92 cm) more number of leaves per plant (26.29), more leaf area (96.12 cm2 ), number of runners per plant (18.70), heavier fruit (10.02gm), more fruit length (35.9 mm), and more fruit breadth (22.91 mm) as compared to all other treatment [109]. Similarly, combined application of manure + Azotobacter + wood ash + phosphorous solubilizing bacteria + oil cake improved significantly fruit diameter (3.11 cm), length (3.95 cm), volume (20.397 cm3 ), weight (11.11 g), total sugars (7.95%), total soluble solids (9.01'B), acidity (0.857), TSS:acidity ratio (11:12) and yield (238.95 g/plant) [110].


#### **Table 1.**

**281**

(10 8

*Sustainable Development of Horticulture and Forestry through Bio-Inoculants*

Frankia is a genus of Actinomycetes, belongs to family Frankiaceae and an ability to fix the atmospheric nitrogen in symbiotic association with *Casuarina* species in tropical and temperate environmental condition. These microorganisms usually invade root hairs of Casuarina and developing within cortical cells in lobes of the resultant nodules. Frankia are able to convert the nitrogen gas in the atmosphere into amino acids, which are the building blocks of proteins. Frankia exchange nitrogen for carbohydrates from the plant. As the plant drop organic matter, or when the plants die, the nitrogen from their tissues is made available to other plants and organisms. This process of accumulating atmospheric nitrogen in plants and recycling it through organic matter is the major source of nitrogen in tropical ecosystems. Various agro

forestry practices such as alley cropping, improved fallow, and green manure/cover cropping exploit this natural fertility process by using nitrogen fixing plants. *Casuarina equisetifolia* seedling inoculated with Frankia strains showed improved growth, biomass and tissue N content over control seedlings [24, 111,

negatively correlated with a tissue N content [111]. Similarly, under nursery experi

ments, the growth and biomass of *C. equisetifolia* rooted stem cuttings inoculated with Frankia showed three times higher growth and biomass than uninoculated control and improved growth in height (8.8 m), stem girth (9.6 cm) and tissue nitrogen content (3.3 mg/g) than uninoculated controls in field condition [112]. Frankia inoculated Casuarina seedlings planted in farm forestry improve the tree growth and biomass in the field condition [2, 112] and improve the nutrient cycling of actinorrhizal plants through high amount of litter production and decomposi

tion [113]. Combined inoculation of Azospirillum, Phosphobacteria, AM fungi and *Frankia* produced excellent growth and biomass of *C. equisetifolia* seedlings due to co-inoculation with *Frankia* through improved nitrogen fixation [22, 114] (**Table**

**10.1 Inoculation methods of** *Azospirillum, Rhizobium* **and phosphobacteria**

Seed or nursery stage is best for application of bio-fertilizers. Suitable methods for forestry species is seed coating and inoculation in polythene bag. Two grams of

Inoculation requirement varies from the size of the seeds. Normally 200 g of lig

species. A slurry is formed by mixing the inoculant with cooled rice gruel (250 ml). The required quantity of seeds is added in the slurry and mixed thoroughly so that each seed is coated with the black colored inoculant. The treated seeds are then

Two hundred grams of lignite based carrier culture of *Rhizobium* or *Azospirillum*

cfu/g) is required for 4 m × 1 m mother bed. It has to be spread uniformly and

8

cfu/g) can be applied in rhizosphere of seedlings in the poly

cfu/g) is need for every 8–10 kg of seeds of the tree

112]. Nitrogenase activity of Frankia strains were significantly (*p*




**1**).



< 0.05) and

*DOI: http://dx.doi.org/10.5772/intechopen.87148*

**9. Frankia with actinorrhizal plants**

**10. Methods of inoculation**

− 8

carrier culture (10

thene bags in the nursery.

**10.2 Inoculation with seeds**

nite/peat soil based culture (10

shade dried for 30 min and sown.

**10.3 Inoculation in the nursery mother bed**

mixed thoroughly before sowing of seeds.

*Growth and biomass increases (percentage increased over control) of horticulture and forestry crops treated with bio-inoculants.*

*Sustainable Development of Horticulture and Forestry through Bio-Inoculants DOI: http://dx.doi.org/10.5772/intechopen.87148*
