Rhizobium sp.

Rhizobium sp.

 

General Characteristics of Rhizobium sp.

• Gram’s classification – Gram negative
• Shape – Rod (Bacilli)
• Size – Measures 0.5 to 0.9 µm wide and 1.2 to 3.0 µm long
• Family - Rhizobiaceae
• Motility – Motile (due to Peritrichous flagella and Polar flagellum)
• Capsule – Absent
• Endospores – Absent
• Respiration – Aerobic respiration
• Optimum Temperature – In general, Rhizobium sp. have a poor growth at temperatures below 10 °C, but they are tolerant to 4° C and for most Rhizobium sp., the optimum temperature range for growth in culture is 28 °C to 31 °C, and many are unable to grow at 37 °C. 
• Optimum pH – 6.5 to 7.5
• Type of Nitrogen Fixation – Symbiotic Nitrogen Fixation
• Discovery – Martinus Beijerinck was the first to isolate and cultivate a microorganism from the nodules of legumes in 1888. He named it Bacillus radicicola, which is now placed in Bergey's Manual of Determinative Bacteriology under the genus Rhizobium. The first known species of Rhizobia was Rhizobium leguminosarum (identified in 1889).
• Rhizobium sp. is a bacterium (also called as the root nodule bacterium) which fixes atmospheric nitrogen in symbiotic association with leguminous plants (Symbiotic nitrogen fixation).
• Symbiotic associations between Rhizobium – legume plants are the primary biological contributors of fixed nitrogen in soil based ecosystem.
• Rhizobium sp. is relatively more effective and widely used biofertilizer.

Diazotrophs

• Rhizobium sp. are Symbiotic diazotrophs.
• Diazotrophy is the metabolic ability to fix atmospheric nitrogen into a biologically useful form (i.e., ammonia).
• Diazotrophs are Bacteria and Archaea that fix atmospheric Nitrogen gas into a more usable form such as Ammonia.
• A diazotroph is an organism that is able to grow without external sources of fixed nitrogen. Examples of organisms that do this are Rhizobium, Azospirillum and Frankia.
• All diazotrophs contain iron-molybdenum or -vanadium nitrogenase systems.

Common Rhizobium species

a)     Rhizobium leguminosarium

b)     Rhizobium meliloti

c)     Rhizobium loti

d)     Rhizobium fredii

e)     Rhizobium rhizogenes

f)      Azhorhizobium sp.

g)     Bradyrhizobium sp.

h)    Sinorhizobium sp.

Table – 1: Recent taxonomic classification of Rhizobia

Recognized genera	Recognized species
Bradyrhizobium	Bradyrhizobium japonicum
Rhizobium	Rhizobium leguminosarum
	Rhizobium meliloti
	Rhizobium loti
	Rhizobium galgae
	Rhizobium tropici
Azorhizobium	Azorhizobium caulinodans
Sinorhizobium	Sinorhizobium fredii
	Sinorhizobium xinjiangensis

 

 Crop Recommendation

Rhizobium sp. is recommended for Oilseed Groundnut and Pulses crops like Pea, Chick pea, Pigeon pea, Lentil, Bean, Soybean, Clover, Alfalfa and Lotus. Field application of Rhizobium sp. shows 10 – 35 % yield increase (50 – 200 kg N/ha).

Table – 2: Rhizobium species and its Host plants

Rhizobium species	Host plants
Rhizobium meliloti	Medicago, Melilotous and Trigonella spp.
Rhizobium leguminosarum bv viciae	Pisum, Vicia, Lathrus and Lens spp.
Rhizobium leguminosarum bv trifoli	Trifolium spp.
Rhizobium leguminosarum bv phaseoli	Phaseolus vulgaris
Rhizobium loti	Lotus spp.
Rhizobium cicero	Cicer arietinum and Tropical legumes Parasponia spp. (Non – legume)
Rhizobium tropici	Phaseolus vulgaris, Leucaena spp. and Macroptilium spp.
Rhizobium elti	Phaseolus vulgaris
Rhizobium galegae	Galega officinalis G. orientalis
Rhizobium fredii	Glycine max, Glycine soja and other legumes
Bradyrhizobium japonicum	Glycine max, Glycine soja and other legumes
Bradyrhizobium elkani	Glycine max, Glycine soja and other legumes
Bradyrhizobium sp. strain Parasponia	Parasponia
Azorhizobium caulinodans	Sesbania spp. (stem nodulating)

 

Isolation of Rhizobium sp.

            Rhizobium sp. are isolated from the Root nodules of Leguminous plants and Rhizosphere soil by Serial Dilution Method (Spread Plate Method or Pour Plate Method). In other method, Rhizobium sp. are also isolated from the Root nodules of the leguminous plants. The culture medium recommended for the isolation of Rhizobium sp. is Yeast Extract Mannitol Agar (YEMA) medium with Congo red as an indicator.

Figure - 1: Root nodules

Microscopic characteristics of Rhizobium sp.

• Gram staining – Rhizobium sp. was observed as Pink coloured medium sized rods. Uneven Gram staining is frequently encountered with rhizobia, depending on the age of the culture. Cells from a young culture and nodule bacteroids usually show even Gram staining while older and longer cells give a banded appearance with unstained areas. These unstained areas have been identified to be large granules of polymeric beta-hydroxybutyric acid (PHBA).
• Motility test (Hanging Drop Method) – Actively motile rods due to Peritrichous flagella and Polar flagellum.

Cultural characteristics of Rhizobium sp.

• In the laboratory, Rhizobium sp. are grown on a special Selective medium called Yeast Mannitol Agar (YMA) or Yeast Extract Mannitol Agar (YEMA) with Congo red as indicator.
• Rhizobia are grouped in two main genera (a) Fast growing Rhizobium sp. and (b) Slow growing Bradyrhizobium sp.
• When cultured on YEMA medium, the Rhizobium sp. produce visible growth in 2 to 3 days. They produce an acid growth reaction, which can be detected by adding a pH indicator, Congo red to the medium. The Bradyrhizobium sp. take 6 to 8 days to produce visible growth on YEMA and produce an alkaline reaction.
• In Yeast Extract Mannitol Agar (YEMA) medium with Congo Red, Rhizobium sp. produces Pink slimy colonies to distinguish itself from other bacterial contamination.

Figure – 2: Colony morphology of Rhizobium sp. in YEMA medium

Figure – 3: Colony morphology of Bradyrhizobium sp. in YEMA medium

Table – 3: Difference between Fast growing Rhizobium and Slow growing Bradyrhizobium

Character	Fast growing Rhizobium	Slow growing Bradyrhizobium
Generation time	< 6 hours	>6 hours
Carbohydrate nutrition	Uses pentoses, hexoses and mono, di and trisaccharides	Uses solely pentoses and hexoses.
Metabolic pathway	EMP low activity strain specific. ED main pathway TCA fully active PP pathway	EMP low activity ED main pathway TCA fully active Hexose cycle
Flagellation	Peritrichous	Sub polar
Symbiotic gene location	Plasmid and chromosome	Chromosome
Nitrogen fixation	nif H, nif D, nif K on same operon.	nif H, nif D & nifA on separate operon.
Intrinsic antibiotic resistance	Low	High

Biochemical characteristics of Rhizobium sp.

a)     Catalase test - Positive

b)     Oxidase test - Positive

c)     Urease test - Positive

d)     Indole test - Positive

e)     Methyl Red (MR) test - Positive

f)      Voges Proskauer (VP) test - Negative

g)     Citrate utilization test – Negative

h)    Starch hydrolysis - Positive

i)      Casein hydrolysis - Negative

j)      Gelatin hydrolysis – Negative

k)     Nitrate reduction – Positive

l)    Exopolysaccharide (EPS) production - Positive

Mass multiplication of Rhizobium sp.

Following are the steps of mass cultivation of Rhizobium sp.

a)     Prepare and sterilize the YEM Broth in an Erlenmeyer flask and inoculate with broth of mother culture prepared in advance.

b)     Incubate for 3 - 4 days at 30 – 32 °C

c)     Test the cultures for its purity and transfer to a large fermenter, wait for 4 - 9 days for bacterial growth (for good bacterial growth make the device for its aeration).

d)     Allow to grow the bacteria either in a large Bioreactor containing broth or in Erlenmeyer flasks as per demand.

e)     Check the quality of broth.

f)   Mix the YEM broth containing Rhizobium sp. with sterile carrier or liquid (peat, lignite, farmyard manure, charcoal powder and PVP). Good quality of carrier culture is that which contains sufficient amount of Rhizobial cells i.e. 1000 × 10⁶ to 4000 × 10⁶ Rhizobia/g carrier.

g)    Pack the culture in polyethylene bags (for Carrier based) or Bottles (for Liquid based) and keep at room temperature or 25 °C.

h)  Check the quality of the Rhizobium biofertilizer and supply to farmers.

Field application of Rhizobium sp.

• Seed inoculation with aqueous suspension of carrier culture during sowing has revealed the luxuriant nodulation and good yield of crops.
• Dissolve 10 per cent sugar or Jaggery in water by boiling it for some time. Leave the content to cool down. Gum arabic solution (10 %) may also be added to the solution. This serves as sticker for Rhizobium cells to seeds.
• Mix the culture of Rhizobium sp. to form the Inoculum slurry.
• For one hectare, 400 g charcoal based culture would be sufficient for mixing the seeds.
• Transfer the Inoculum slurry on seeds and mix properly. The number of Rhizobial cells/ seed should be between 10⁵ to 10⁶.
• Spread the seeds in shade for drying on cement floor or plastic sheets.
• While using Rhizobial cultures, certain precautions are taken into account. For example,

ü  Use of culture before expiry date

ü  Use of small amount of pesticides when required

ü  Immediate sowing of seeds after mixing (within 24 hours)

• Seeds must be stored at 4 °C when not used immediately to protect the Rhizobial cells.

BIS (Bureau of Indian Standards) specifications for Rhizobium inoculant

• Inoculation should contain a minimum of 10⁸ viable Rhizobium cells/g of the carrier on dry weight basis at the time of manufacture and 10⁷ cells on expiry date marked on the pocket. 
• No contaminants at 10⁶ dilution
• pH of inoculant should be 6.0 to 7.5
• Carrier material should pass through 10⁶ micron size sieve
• Packing low density polythene bags of 50 - 75 µ
• Each packet should be marked with the details – Name of the product, leguminous crop for which intended, strain number, date of manufacture, date of expiry, method of application and storage instructions.