The how the relationship between nitrogen concentration on

The Effect that Nitrogen
Concentration in Fahraeus’ Medium has on the Rhizobium Nodulation of Alfalfa


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Nitrogen fixation of atmospheric nitrogen is essential for
all living organisms as it produces ammonia which is used to make amino acids,
proteins and nucleic acids which are the basic building blocks for life.
Leguminous plants have adapted a way of using the nitrogen-fixing bacteria,
rhizobia, which proliferates in root nodules when nitrogen concentrations are
low. The experiment carried out used Alfalfa (Medicago sativa) seeds sown on 5 different Fahraeus’ media containing different concentrations of nitrogen with
rhizobium solution surrounding them. There was 34-36 replicates and the data
recorded and analysed. This paper elaborates of the importance of root
nodulation in legumes and how the relationship between nitrogen concentration
on the growth of the Alfalfa plants.


Rhizobia bacteria are free-living within the surrounding
soil of the legumes and invade the plants roots (New Mexico State University,
2015). The plants supply the bacteria with necessary nutrients as they multiply
within the cortex of the plant and within a week nodules begin to form (New
Mexico State University, 2015). An age gradient is formed where a persistent
meristem develops in the distal end of the root nodule making differentiating
zones giving the nodules their irregular oval shape (Bianco et al. 2014). This involves the use of
phytohormones in particular the auxin indole-3-acetic acid (IAA) which is
produced by the rhizobium bacteria (Bianco et
al. 2014). The bacterially produced auxin can alter that auxin
balance within the plant and can interfere with plant auxin transport so
indirectly influencing auxin homeostasis, resulting in plant growth promotion
or inhibition (Spaepen and Vanderleyden, 2011; Defez et al. 2017).

Nitrogen cannot be directly used by organisms but
it is essential for growth therefor they need to have mechanisms for fixing
nitrogen into ammonia (Haag et al. 2013).
Only a few bacteria and archea are able to fix nitrogen and some plant have
been able to produce symbiotic interactions with these bacteria and us the
nitrogen for growth (Haag et al. 2013).
Within the nodules the rhizobium bacteria are used as endocellular
symbionts which convert atmospheric nitrogen into ammonia which is essential
for making amino acids, proteins etc. (Bianco et al. 2014). The nodules take a while to fully mature and the
rhizobia bacteria don’t carry out much nitrogen fixation in this time, when
they are fully mature they become pink inside indicating nitrogen fixation has
started as leghaemoglobin is controlling the oxygen flow to the bacteria (New
Mexico State University, 2015).

Legumes are one of the most abundant crops and as they have produced
this highly efficient symbiosis with the rhizobium bacteria it therefor reduces
costs and reduces soil/water pollution (ÍñIguez et al. 2016).

The aims of the paper are to describe the effects
of growth conditions, namely inorganic nitrogen concentrations, on nodulation
of Alfalfa roots. It is important to elaborate on this as is key for cutting
costs of agricultural crops and can help with poverty.


Firstly 40 alfalfa seeds are washed for 10 minutes in 80%
ethanol and stirring them occasionally; this sterilise their surfaces but makes
sure not to damage them. The seeds are then rinsed in sterile distilled water 3
times to remove excess ethanol.

Rhizobium bacteria in growth medium is centrifuged at 1000xg
for 10 minutes to pellet the bacteria and the supernatant is poured off and the
pellet re-suspended in the same volume of sterile water. This is then
re-centrifuged and the supernatant removed again and the pellet re-suspended in
5ml of sterile water.

30 surface-sterilised seeds are transferred aseptically into
a small volume of rhizobium suspension in sterile water and incubated for 20

5 separate
flasks are set up with Fahraeus’ medium and
different concentrations of nitrogen added to each; 0mM, 0.1mM, 1mM, 5mM, 10mM.
Then using aseptic techniques 6 incubated alfalfa seeds are sown on top of each
growth medium along with a small amount of bacterial suspension. The flasks are
the plugged with non-absorbent cotton wool and aluminium foil then kept in a
growth room in the light so the seeds can germinate; this should take about 2

1 mm


After 6 weeks the
alfalfa plants were then observed for successful inoculation, if they hadn’t
data was still collected but it was treated as a blind trial, non-inoculated
control set for comparison.

Before opening the
flask the number of plants that grew was recorded and then carefully removed
from the growth medium making sure not to damage the roots and any residual
agar removed from the roots by gently washing and blotting them. For each flask
the roots were cut off all the plants and weighed together getting a total
weight in grams (g); the same was then done for the shoots of each flask.

1 mm


Using a binocular microscope the roots of each flask were
examined for the presence of root nodules and then they were counted and

Figure B: Image of a root nodule at 0.1 mM NO-3


Once the nodules of each plant had been counted further examination
of the structure was carried out. This was achieved by cutting open the nodules
and looking at them under a binocular microscope as shown in figures A, B and C.
As seen in figure 1 the inside of the root nodule is pink and this is due to
the leghaemoglobin and shows that nitrogen fixation is taking place (New Mexico
State University, 2015).


Figure C: Image of a root nodule at 1.0 mM NO-3






Once the data had been collected and rearranged a
Shapiro-Wilk test is carried out to determine if the data is normally
distributed at p<0.05. This shows that the data is not normally distributed so the data is transformed and a Shapiro-Wilk test carried out again with the data still coming out at non-normally distributed. A QQ plot can also be used to support the fact that the data isn't normally distributed. A Bartlett's test of homogeneity of variances is used to test for a degree of heteroscedasticity which for this set of data comes out as heteroscedastic. Because the data is both highly non-normally distributed and heteroscedastic a Kruskal Wallis test was used to test is there is a significant difference between the data at p<0.05. Then a Mann Whitney U test used to see if the was a significant difference between the means (p<0.05). When the Kruskal Wallis test was carried out on number of rhizobium nodules pre-plant at different concentrations it showed that there was a significant difference between 0 and 10mM which is shown in figure 1. A Mann Whitney U test was then carried out which showed there was a significant difference of means from 0.1-5mM but no significant difference from 0-0.1mM.                     Preforming a Kruskal Wallis test on shoot weights at increasing nitrogen levels shows that there is a significant difference (figure 2). A Mann Whitney U test was carried out that confirmed that there is a significant difference between 0 and 5mM but there is no significant difference between 5-10mM. Kruskal Wallis test preformed on number of nodules per gram of root with increasing nitrate concentrations showed that there is no significant difference (figure 3). Mann Whitney U test was carried out that there was no significant difference between the means of 0-10mM   A Kruskal Wallis test carried out on number of nodules per gram of root with increasing nitrate concentrations showed that there is a significant difference (figure 4). A Mann Whitney U test shows that there is a significant difference between all concentrations from 0-10Mm.           By preforming a Kruskal-Wallis test of the ratio of shoot to root weight data it is evident that there is a significant difference (p<0.05) in increasing concentrations of nitrogen, this is shown clearly in figure 5. A Mann Whitney U test shows that there is a significant difference between all the nitrate concentrations 0-10Mm. Discussion Figure 2 shows that there is a relationship between shoot weights on increasing nitrogen concentration of growth medium. This is because the Alfalfa plants, at low nitrogen levels, are unable to use the atmospheric nitrogen for growth so begin to produce root nodules containing the rhizobium bacteria so they can use the nitrogen they fix for growth (Bianco et al. 2014). This is also supported by figures 1 and 2 which show that as nitrogen concentration increases rood nodulation decreases. Where as shown in figure 3 there is no correlation between root weight and nitrogen concentration. At lower concertation of nitrogen (0-0.1mM) the weight was quite low and this was because there wasn't enough nitrogen for growth. At higher concentrations (10mM) the weight was also lowered and this was because there was more nitrogen available to the plant so it didn't need more roots to go 'searching' for more nitrogen. Whereas at the middle concentrations (1-5mM) there is a peak in root weight and this is due to the fact that there is enough available nitrogen in the medium for growth but they need to go 'searching' for it. It's important to understand the correlation between root nodulation and nitrate concentration because it can benefit the agricultural community in the long run and can help to reduce costs on