It is known that legumes enrich the soil by contributing nitrogen through symbiotic nitrogen fixation by Rhizobium through centuries. However, scientific demonstration of value of legumes in contributing nitrogen nutrition of plants was only done in 19th Century. This was established by the facts that nodules on legume roots are responsible for fixing atmospheric nitrogen through bacterium Rhizobium. Due to new technological development a substantial contribution in increasing production of legumes besides improving M. fertility of soil ,is made.
How to separate Rhizobium from nodules ?
Different types of legumes form various sizes and shapes of nodules on their roots. Differences have been even found within the same species of legumes. Hence, nodules are collected when plants are in flowering stage and one can make out an effective nodule which is large in size and red in colour. Such nodules are used for separation of Rhizobium in the laboratory. An isolation of Rhizobium is made by following usual techniques. Yeast monitol agur is the special medium used to grow Rhizobium. Colonies grown on a medium may not be only of Rhizobium they can be even of Agrobacterium. For definate conclusion one has to inoculate seeds of particular sequence and wait for formation of nodules on such plants. How to recognize Rhizobium? In order to confirm whether isolated colonies are of Rhizobium for this purpose one has to see that inoculated plants form effective nodules on roots. For further combination following tests are performed in the laboratory
Besides there are several tests of nodule formation on roots of legume, of which following are most important.
Estimation of nitrogen fixation: In order to estimate biological nitrogen fixation generally Jaldahls method is used. However, if the difference between two treatments is higher than some other methods are used viz. Label nitrogen N15. For this method, Marspectrometer and label nitrogen (N15) are must. One can estimate the nitrogen fixed by the organism by observing the N15 taken up by the plant.
At field level nitrogen estimation can be done by calculating the difference between nitrogen found in nodules of legume plant and the nitrogen found in non-nodulating legume plant variety. For legume a small amount of chemical nitrogen is applied and hence biological nitrogen fixation is found efficiently whereas for cereals higher doses of chemical nitrogen is applied. For cereals nitrogen from soil is only available on the other hand for legumes nitrogen from soil as well as from nodules is made available. Difference between two is the nitrogen fixed by the legumes.
During the nitrogen fixation nitrogenous enzyme converts nitrogen into ammonia, and then acetylene into ethylene. This method is most efficiently used to estimate the nitrogen fixed by plants. This method is simple, accurate and used on a large scale.
Cross innoculation groups: This team was reported by Nubbe (1891) and he described cross innoculation groups as a new concept that is separate Rhizobium species only nodulate specific related legumes. For example Braddy Rhizobium Japonicum nodulating soyabean cannot infect Groundnut plants and vice versa. The cross innoculation groups have waterbigul compartment and no exceptions are found. From the published information so far it is not known what are the reasons for existence of cross innoculation groups in the nature. Fred and his associates (1932) recognized eight cross innoculation group in legumes as mentioned below:-
Above cross innoculation groups are recognized everywhere and accordingly Rhizobium species is selected to innoculate a particular crop.
How Rhizobium enter the roots of legumes ?
Rhizobium enters the roots of the legumes either through root hair or directly at the point of emergence of lateral roots. Curling or controlled growth and branching of root hairs is the first visible plant response to Rhizobium. Although, legume nodules generally seem to harbour only one strain of Rhizobium a given root can certainly form nodules with more than one strain.
It is reported that Rhizobium strains capable of infecting a legume releases a specific polysachnarides that induces more pectolytic activity by the root that accounts for cross innoculation specificities. It is not known how Rhizobium initiates the infection thread. Some suggested mechanical rupture with Rhizobium entering a break in root hair wall. Rhizobium may also get trapped within the fold of growing deformed hair.
How a nodule is formed ?
The infection thread enters and penetrates the context of the root from cell to cell. Finally the thread bursts and liberates the rod shaped bacteria into a critical cells. This cell divides to form nodular tissue in which bacteria divide and multiply. Eventually, a demarcation develops, a centrally located bacteria containing, the tissue called the bacterial zone is marked out in the nodule from the surrounding bacteria-free tissue called the nodule cortex. The nodular tissue grown in a size, pushes itself through the root and then emerges as an appendage on the root system. Its size and shape depends on the species and legume.
There are two types of nodules effective and ineffective ones
Red Pigment Leghaemoglobin: A cross section of a mature nodule reveals a pink or red coloured central bacteriod zone surrounded by thin walled cells. The red colour is caused by the presence of a pigment called "leghaemoglobin". Similar to the one found in human blood, the prefix "leg" indicates its unique presence in root nodules of leguminous plants. The amount of red pigment in nodules is directly proportional to the amount of nitrogen fixed by nodules.
The red pigment in the nodules acts as a biological value in regulating the supply of oxygen into the bacteriod tissue. The supply of oxygen at an optimum rate to help the maximum activity of the enzyme "nitrogenose" which is key factor in the mechanism of nitrogen fixation.
Mechanism of N-fixation: The nodules is simply, a protective structure and bacteriods are the seats of N-fixation. Reduction of N2 to NH3 is mediated through enzyme `nitrogenose. Nitrogenose is made up of two components one with iron and molybdenum and the second without molybdenum. N-fixation is essentially anaerobic process. The oxygen supply to bacteriod is excluded due to presence of leg haemoglobin around it. This pigments limits oxygen supply and helps in providing low oxygen conditions near the bacteriods and thus protects the oxygen sensitive nitrogenous from damage. The enough oxygen is made available at the site for generation of ATP. The quantum of N-fixed is closely related to the amount of legheamoglobin and the extent of bacteriod tissues in nodules. The first stable intermediate in N-fixation is ammonia. This nitrogen is then converted into amino acids and proteins thereby plants are benefited.