Enrichment Culture of Rhizobium meliloti from Trifolium repens

Discussion
    
Discussion of Results   
    The results obtained in the laboratory prove correct the hypothesis that Rhizobium can be isolated from the root nodule of the Trifolium repens (white clover plant). The above enrichment protocol allowed for the isolation of a pure culture of Rhizobium from the given environment. Observations of the isolated colonies were most telling as to the verification of our hypothesis. The colonies on the YMA plate were a milky white color, were opaque, raised, had irregular edges demonstrating motility, and were mucilaginous. The other tests including the Gram stain, simple stain, capsule stain, and wet mount further confirmed the isolation of Rhizobium.
    The Gram stain demonstrated that the bacterium were Gram negative and formed chains of cocci. Also, it was clear that a pure culture was obtained based on the consistency of bacterium color and shape throughout the slide.  The simple stain again demonstrated the consistency of the chain of cocci groupings and the presence of an endospore.  The capsule stain confirmed the presence of a capsule for the organism.
    Upon viewing the wet mount using dark field microscopy, it was clear that the organism was motile.  The result from the motility stab demonstrated that the organism was motile. Also, the organism only grew at the top of the media where oxygen was more readily available thus demonstrating that Rhizobium requires oxygen for respiration. This was consistent with Bergey's Manual of Determinative Microbiology.  
    All test results were consistent with what was expected for Rhizobium and therefore confirmed the hypothesis.
    Carbohydrate utilization tests using mannitol and dextrose were also run to confirm the isolation of the Rhizobium . Both tests were run twice and failed each time. This was due to the fact that Rhizobium was unable to obtain all the necessary growth factors from the tube media besides the carbohydrates. Also, the O/F tube test was run to determine the type of catabolism utilized by the bacterium. This resulted in a negative test because growth was not demonstrated in either tube. This was again due to the fact that the media did not support the growth of Rhizobium. From these results it was determined that no alteration to the given enrichment protocol is necessary. It must be added that when attempting to isolate the Rhizobium  the environmental isolate was inoculated on two types of media, YMA and TYA. This was done in order to determine which media would best support the growth of the Rhizobium for use during the entire enrichment culture project. Upon observation of the plates after inoculation, it was clear that the Rihizobium only grew on the YMA plates, not the TYA plates. Because of this, all future inoculations of the Rhizobium isolate were done on YMA plates.
   
Ecological Role
    Rhizobium plays an important role in the process of nitrogen fixation. Most nitrogen found in the atmosphere is in its diatomic (N2) form which can not be utilized by plants for biological processes. Rhizobium is one type of bacteria that possess enzymes allowing it to convert the atmospheric nitrogen to forms that can be utilized by plants, nitrate (NO3-) and ammonium (NH4+). Because of this, Rhizobium exist in a symbiotic relationship with certain legumes where both the bacterium and the plant benefit from one another. The plant provides carbohydrates and a living environment for the Rhizobium. The Rhizobium, in return provides the plant with useable forms of nitrogen. The Rhizobium is found within specialized structures called root nodules, found on the roots of the legume. These structures are formed through an interaction between the Rhizobium and young root hairs that protrude from the plant’s root system. The result is a structure attached to the root that completely encloses a colony of bacteria.    

Interesting Characteristics
    There are several interesting characteristics demonstrated by the isolated colonies of the Rhizobium. One characteristic is that each type of legume develops a symbiotic relationship with a specific species of Rhizobium. Because the Trifolium repens (white clover plant) was chosen as the enrichment source, one can be confident that the specific species of Rhizobium was in fact Rhizobium meliloti . Another interesting characteristic of the isolate was the mucilaginous nature of the colonies.
   
Significance
    The proper isolation of the Rhizobium is significant for agriculture. Nitrogen is one essential growth factor for all forms of life. Nitrogen makes up key components of amino acids, proteins and nucleic acids. Because of this, farmers often add chemicals to the soil that contain nitrogen to improve the quality and yield of their legume crops, such as alfalfa. These chemical are in a form that can be utilized by the crops. However, in time this nitrogen supplement will be depleted and will have to be reapplied. Rhizobium isolation and application can be used as an alternative to chemical treatment of crop soil. As already mentioned, the genus Rhizobium is essential to the fixation of nitrogen for crops. By treating soil with Rhizobium, the organism will form a symbiotic relationship with the alfalfa and undergo the normal nitrogen fixation process. This process will create a constant source of nitrogen for the legume and also lead to an overall increase in the quality of the soil and the crop. This method is advantageous for several reasons. First, it is an organic farming method and is healthier for the plant and for the environment than treatment of soil with chemicals. Also, the Rhizobium only need to be applied to the soil once for continuous nitrogen fixation whereas chemicals would need to be applied numerous times.
    A clear, precise method for the isolation of Rhizobium is important in terms of efficiency of soil treatment. Obtaining and applying a pure culture of Rhizobium to soil is most efficient because only the specific nitrogen fixing bacteria for that crop is applied and no other extraneous bacteria are present. A pure culture would also be safer for crop in that no disease causing bacteria could be transferred from the source soil to the crop soil.

Reference for Discussion: http://www.agwest.sk.ca/infosource/inf_nov99.pdf



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