I am studying the effects of pH on Biological Nitrogen Fixation and planning to use N15 natural abundance method for determining BNF. Can anyone suggest me if there is any other method more suitable for this particular study?
Hi,
I suggess ARA-test (Acetylen-Reductions-assay = ARA), the most common method i think.
I would suggest you to do acetylene reduction assay (ARA). Acetylene is converted to ethylene with nitrogenase activity. Ethylene can be detected by using gas chromatography
The problem with acetylene reduction method is that, it is not a good quantitative method to determine BNF. Also, I forgot to mention that I want to estimate the BNF in field condition.
Direct in field, impossible i think. You may take a sample and run to your lab doing ARA :-).
Seriously, i think you have to take samples and measure BNF in lab because in field i don't know any instrument able to do that.
ARA is a good quantitative method ! See some papers.
Hi Jordan,
Thanks for the response. I did some research on methods before asking this question. Many authors believe that the use of the acetylene reduction technique as a quantitative assay of Nitrogen fixation has been generally discouraged since the 1980s. You can check out articles by Unkovich et al.
When I say field condition it does not mean that I want to go to the field and measure BNF right there. What I mean is, I am not interested in nitrogen fixation from a single plant like we measure in acetylene reduction method. I would like to take plant samples from a fairly large area from my experimental plot and analyse them in the lab.
many details are are missing (plant species; duration, treatments,...). N15 is best, you can try also Total N (differences among treatments). For good are many samples are required.... and it us still comparative.
Hi Prashanta,
I agree, 15-N isotope techniques are the most reliable of the techniques available for measuring biological N2 fixation (BNF) by legumes under field conditions. There are two 15N based methods for the determination of BNF: (a) natural abundance method, (b) 15N enrichment methods.
Of these two methods, ‘natural abundance’ method is less costly, but needs very careful preparation of samples to avoid cross contamination. Also, soil ‘δ15N’ at your experimental site need to be significantly different from the ‘δ15N’ value of the atmospheric N (which considered as 0 ‰). A soil δ15N value around 2 ‰ is recommended by Unkovich et al. Both methods require a reference (non-nitrogen fixing plant) plant. The papers published by Unkovich et al. are good source for understanding the assumptions and principles related to the selection of a suitable reference plant for your study as well as general methodology of the two 15N methods.
Good luck.
-SJ
I agree with Susantha. ARA is a qualitative method, not quantitative. N15 is a good method, but dispendious and need a great accuracy in conduction, The comparison between plots with and without inoculation, with the measure of foliar or total N is a good method, to.
Together with the N content of inoculated and uninoculated plants (preferably the N content of shoots since root excavation can be very hard if the aim is to recover the entire root apparatus), you should also consider to simply quantify the shoot dry weight of inoculated and uninoculated plants grown in the differently treated plots. Depending on the experiment planned, this could be accompanied by a survey of the nodule number and appearance (e.g. if sterile soil is used to grow plants, the absence of root nodule in the uninoculated plants is indicative of a good experiment). This is a very simple, but very effective, method for the quantitative estimation of legume-rhizobia N2-fixation. There are really many papers in which you can find details on this method..... see for instance:
http://link.springer.com/article/10.1007/s11104-012-1153-3
http://link.springer.com/article/10.1007/s11104-005-0374-0
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