Dear Alemayehu Getahun

I think, only applications of bacteria is not important, the conditions that the bacteria are living is more important in success of inoculation; soil type, environmental variables, soil management practices, soil chemical characteristics, appropriate microbial species and plant species also should be checked! 

I think, one time inoculation, especially seed inoculation is sufficient , but it is equally important to ensure the optimum conditions facilitating the maximum crop response , I agree with you Shokati. But while going for inoculation of growing medium, we could require inoculation , more than once..??

Most effective results with rhizobium inoculation depends on 1) the propagation and formulation onto peat material 2) avoiding storage extreme environments and having expiration dating 3) applying on of in the near vecinity of seed and seedling roots. The application of peat with a sticker can be limited by the limited material attached to the seed. Some greater population is possible with granular peat formulation applied near the seed row. If appropriate material with appropriate viable rhizobium are applied in soil and seed a single inoculation should be sufficient. The commercial inoculation usually hav3 6 to 12 strains of bacteria to ensure the bacteria can adapt to variable soil field environments.

We find for rhizobium seed inoculation is the best one. But if you find some environmental calamities during the inoculation and early growth period, then you may apply again as soil inoculation after two weeks . But most important thing is you have to be very careful about the cross inoculation groups of specific rhizobium with the leguminous plants you are using and the environmental condition for their survival as well as the optimum population for inoculation.

Dr Tarafdar and other learned colleagues , very often , it is difficult to ensure the distinction between different different cross inoculation groups  of rhizobium . What is your opinion about the  possibility developing a consortium of such cross inoculation groups of rhizobium to have more comprehensive crops response across crops  of leguminous nature..?? 

Soybean is often nodulated by distinct strains of R. japonicum. In Africa IITA has wanted to develop soybean varieties which will be well nodulated by cowpea strains which are more broad host plant range and adapt to acid soil environments. One of the most important aspects of bnf is having strains that both are good fixers of nitrogen but also have ability to maintain themselves in the soils of interest. 

Dear Alemayehu,

One of the considerations in seed inoculation of legumes by rhizpbium is to check the strain-host compatibility. Your strain should be compatible to your host legume. Secondly, you have to work on a live bacterial/rhizobium strain---should not be stored longer. Thirdly, while doing the inoculation just follow the specific requirements/guidelines set for that particular rhizobium-host combinations.  By then, you will see how effective nodulation has taken place.   

Dear Alemayehu,

Your question is not very specific in terms of which crop you would be applying the rhizobial strains. I have some experience working with lucerne crops in Argentina and overseas. In an experiment carried out in NZ, we analysed the effects of different types of carriers of Ensifer melilotii, and found that the most efficient formulation was using peat slurry as the carrier for bacteria. This carrier was superior to other commercial inoculants that usually come inside a coating that covers each seed.

For the inoculant to be effective, it needs not only to colonise the roots of the target crop but also to compete with other wild strains of bacteria that are usually present in the soil. Most of this wild strains are very competitive and can easily out-compete the commercial strains. However, these wild strains are very ineffective in terms of their ability to fix nitrogen.

I can't think of any way you could add more inoculant to the soil if the initial inoculation was not effective. In fact, it is likely that if the commercial inoculant failed to colonise roots of your target crop, some other bacteria is occupying that space. 

Hope this answer helps.