The Effect Of Nitrogen On
Mycorrhizal Colonization Associated With
Populus grandidentata
Megan McLin
Tougaloo College
11/12/2013
Megan McLin
11/12/2013
The Effect Of Nitrogen On Mycorrhizal Colonization Associated With Populus
Grandidentata
Introduction
Mycorrhizal colonization occurs in 80% of all plant families. This symbiotic association
between roots and fungi is essentially what rules nature. Many terrestrial ecosystems depend on
Mycorrhizae that establish, grow, and promote the health of plants (Allison et al. 1157).
Mycorrhizal Fungi are crucial in forest ecosystems because they facilitate inorganic uptake of
Nitrogen (N) in trees. N is an essential plant nutrient and many ecosystems adapt to conditions
that are low on N availability. On the global scale, current N emissions in most regions bring
about an increase in rates of N deposition. This raises concern about what impact it might have
on plant diversity. Atmospheric nitrogen deposition has fallen from the air onto forest
ecosystems, which has partially fluctuations in nutrient availability in soils. The symbiotic fungi,
mycorrhizal, are vital to tree nutrient uptake, since tree roots are not able to uptake enough on
their own. For the 85-90% of land plants that form them, mycorrhizal represents the crucial link
between the root systems and soil and by creating resistances against parasites and other stress
factors (Smith and Read 1997). However, studies have shown that ecosystems have been found
to decline in abundances and diversity in response to nitrogen deposition, with possible
damaging effects on plant uptake of soil resources. In low nutrient areas, plants generally invest
more carbon in mycorrhizal fungi (Mosse & Phillips, 1971) But if nutrient availability rises
(fertilization), plants allocate carbohydrates elsewhere in the plant and a decline in mycorrhizal
abundance is expected with fertilization (Read, 1991) Under normal conditions, aspens are able
to colonize due to mutualistic relationships with mycorrhizal fungi (Cripps 1996, 2001). In
addition, aspen has been reported to transpire within phosphate mining dumps (Harris and
Jurgensen, 1977). Aspen can associate with over 60 different species of fungi, but only a portion
occurs on acidic soils (Cripps, 2001; Cripps and Miller, 1993). This paper aims at researching
what impact N has on Mycorrhizal colonization of Populus grandidentata. It also affirms an
increase in nitrogen availability translates to a decline in mycorrhizal biomass as the activity of
mycorrhizal reduces (Allison et al 1158).
Nitrogen Cycle
N cycling within the ecosystem is from biological N fixation, mineralization, and
atmospheric deposition. Normally without human interference, N’s atmospheric deposition is
relatively insignificant. With the industrial revolution, atmospheric deposition is now an
significant source that is now dominant. The major challenge in the N cycle is the increase of N
deposition due to human needs for food and energy. Anthropogenic N emissions into the
atmosphere are in the form of NH 3, and organic N. Major sources of NH3 are fertilizer emissions
and manure. Their addition into the ecosystem promotes plant growth. Major organic sources of
N include both anthropogenic and natural sources (Allison et al 1159). With the exception of
N2O, the entire N emitted to the atmosphere ends up on the earth’s surface after going through
the atmosphere. Subsequent deposition often represents the introduction of biologically active N,
into ecosystems. Both terrestrial and marine ecosystems have no way of obtaining
Anthropogenic N other than internal means. With the increase in N deposition over time, plant
communities shift to compositions of high N availability. This shift then results in loss of
diversity in plant species especially in areas with high N deposition. An increase in N deposition
results, in direct toxicity of nitrogen gases.
Figure 1 The Nitrogen Pool Cycle
Source: Nave, Lucas. Nitrogen Cycling in the Northern Hardwood Forest: Soil, Plant, and
Atmospheric Processes. Cleveland: Ohio State University Press, 2007. Print.
Figure 1 is a Conceptual diagram of N pools and fluxes influencing the distribution of
atmospheric N inputs within a forest ecosystem. Pools containing N are in boxes. Arrows
represent N transfer processes between pools. The atmospheric N retention in soil pools has a
direct effect on soil chemistry, organic matter turnover, and N-mineralization rates. Short-term
availability of N inputs to plants is low because of retention by SOM (soil organic matter), but
long-term increases in soil N stocks and cycling rates may lead to greater plant N uptake. N is
the mineral nutrient that most limits plant growth and microbial activity. Most of the N that
Populus grandidentata needs for growth goes through internal recycling in the N pool between
the soil and the plant, and by decomposition of organic matter. However, humans altered the N
cycle, and this increased the atmospheric N into forests. They are the natural habitat for Populus
grandidentata. Fossil fuel combustion, addition of Nitrogenous fertilizers and agriculture all
mobilize reactive N into the atmosphere. N addition is one factor of global change that has had
an influence on Mycorrhizal’s growth and abundance. This analysis also predicts that microbial
biomass would decline by 15% when there is an addition of N into the ecosystem. Moreover, a
decline in the numbers of microbes will be more evident in the study if the research is over a
long duration, and with an increase of N. In addition to this indication, the response of
Mycorrhizal biomass has a correlation with N fertilization. Nitrogen availability influences the
growth and abundance of Mycorrhizal. N is a crucial element that limits the growth of living
organisms on a wide range of ecosystems (Allison et al 1160). Humans increase the amount of N
that is available in the ecosystem. This increase has some far-reaching consequences that include
altering species’ composition, changes in biological diversity, the saturation of N in the
ecosystem and an increased N loss. In ecosystems where anthropogenic N additions are low,
changes like global warming increase the available N. This is by increasing the rate of nutrient
take up in the soil (Allison et al 1157). Mycorrhizal fungi are sensitive to the addition of N
because plants reduce carbon(C) allocation to Mycorrhizal symbionts when the available N is
high. N fertilization usually suppresses Mycorrhizal fungi and often reduces N cycling in
Mycorrhizal pool. Studies indicate that addition of N has a negative effect on microbial growth
(Allison et al 1161). This is an indication that other factors apart from N affect microbes. The
number of factors suggested for the reduction in microbes under N fertilization varies. This could
be because the osmotic potential in soil solutions becomes toxic because of the addition of ions
from the fertilizers. This addition of fertilizers inhibits microbial growth directly. N saturation
decreases soil pH, and this leads to leaching of calcium, magnesium, and aluminum. This results
in the microbes becoming calcium or magnesium-limited or the microbes suffering from
aluminum toxicity (Allison et al. 1161).
Figure 2: Effects of N
Deposition
Source: Allison, Gartner, Mack, McGuire, Treseder Kathleen. Nitrogen Alters Carbon
Dynamics During Early Succession In Boreal Forest. Journal of Soil Biology & Biochemistry,
42 (2010). 1157-1164. Print.
From figure 2, it is clear that excess N stops the production of ligninase by fungi. Lignin
blocks other compounds like cellulose and its presence in plant tissues stops the microbe from
accessing other compounds like Carbon. The addition of N reduces fine roots in plants and
Mycorrhizal fungi as these structures are not as effective as they should be. This way, they are
unable to maintain nutrient uptake. Nitrogenous compounds condense with carbohydrates and
the result is the production of melanoids. Mycorrhizae fungi thrive on Populus grandidentata
where Ectomycorrhizal symbiosis with the plant occurs. Populus grandidentata exist in many
habitats because of their symbiotic relationships with Mycorrihzae. Some species of Mycorrhizae
fungi can occur across a variety of forests. Previous studies indicate that Mycorrhizal fungi is
common in many forests across the Northern Rocky Mountains. One third of the Mycirrihzae
species occurred in three different soil types (Allison et al 1162).
Materials and Methods
Measuring Mycorrhizal abundance in the experiment was by collecting Populus
grandidentata roots. The level of colonization was by quantifying both AM (Arbuscular
mycorrhizae) and EM (Ectomycorrhizal) on each root with a microscope and staining
techniques.
Field Methods
Each root collection came from the C4 site nitrogen plots. However, there was no
availability of a control plot for P grandidentata. Therefore, a set of P grandidentata trees
outside of the nitrogen plots served the purpose of constant variability. Field sampling
procedures were as follows: selecting representative sites, sampling by horizon, and designating
and sampling a sub-horizon for root mass and morphology change. Examination of the 20 roots
was by the following experiment. Collection of the root samples from the trees using a soil
probe. This was for analysis and evaluation of Mycorrhization. There was also the manual
extraction of the roots from the soil. Zip lock bags stored each root, and each zip lock bag
labeled for the precise plot, to compare amounts of nitrogen. Sample collection involved
collecting three soil cores (subsamples) from different sides of the tree (North, South, East and
West). Fragments of delicate roots that are close to the soil surface and those that one can
unearth manually were the subsamples. 10% of the sample collection was from the organic soil
horizon. After one gathers the samples using zip lock bags for each root, the roots stayed in a
freezer for later analysis.
From the freezer, the separation of the roots and soil was by hand rinsing followed by
drying. It was imperative to isolate the soil for effective separation of the roots and plant excess
from the soil sample. Tap water, rather than distilled water, helped prevent dispersion problems.
Reporting of root biomass was in lbs/ac or kg/ha, over a given depth, and as a “root bulk density”
g/cc of oven dry root biomass in the soil from a certain horizon
Structures produced by AM fungi are indistinguishable in fresh roots since the cell
contents and natural pigments obscure internal structures. Conversely, the EM roots are
identifiable by direct examination through a dissecting microscope. A compound microscope
helped observe EM Mycorrhizae structures within roots.
Observation of fungal structures inhabiting the plant tissues was by the use of differential
stains that bond with fungal hyphae devoid of much background staining of plant material that
has been cleared. Stains like Chlorazol black E (CBE) or ink in vinegar help to stain Mycorrhizal
structures; they can differentiate the fungi from the root. For complete microscopic
examinations, staining of roots is by Chlorazol black E (CBE) diluted with lacto glycerol
solution. The prime stain concentration relied on the dye source and microscope procedure used.
There was also the heating of the roots for a given number of hours at 90° C, or 15 minutes
itemising an autoclave and opting for a liquids cycle at 121° C. Leaving them in staining
solutions state at standard room temperature for one or several days would improve staining. One
may reuse the staining solution several times after filtering through folded cheesecloth or a clear
nylon screen following each and every use (to get rid of root fragments), to make it translucent.
Lately, a more reliable staining method involving ink and vinegar has been developed. The
staining solution concentration is a 5% ink solution diluted in vinegar (5% acetic acid) or vice
versa. The process will entail staining with black or blue writing inks (Pelikan Blue).
Table 1: A Comparison of Staining Techniques
Visualization method
% of papers used
Trypan Blue
66.3
Acid Fuchsin
14.5
Chlorazol Black E
13.2
Aniline Blue
0.8
Other Methods
0.7
Source: Stapleton, Crout, Sawstrom C, Marshall W.A, Poulton P.R, Tye A.M. and LaybournParry J. “Microbial Carbon Dynamics In Nitrogen Amended Arctic Tundra Soil: Measurement
And Model Testing”. Journal of Soil Biology and Biochemistry. 37.1 (2005): 2088-2098. Print.
Table 1 compares different types of staining methods as recorded in 1399 manuscripts.
Other methods include molecular techniques, vital staining of enzyme activity and
electrophoresis. It is necessary to establish if the level of colonization recorded in a plant species
at any time is dependent on the visualization method used (Allison et al. 1157).
Materials:
Lab:
●
Dissecting microscope
●
Compound microscope
●
Microscope slides
●
Cover slips
●
Quarter liter of Differential staining dyes
●
1 bottle of Hydrogen peroxide
●
A liter of Glycerol
Figure 3: Timeline of Experimental Procedures
Task
Week
Collecting roots from each represented the plot following storage of roots.
1
Root separation from soil (sieving)
2
Staining of roots
3
Data and analysis
4
Data Analysis
Calculations for obtaining biomass
1. Moisture content.
Moist Soil (g) - Oven Dry Soil (g) = Soil moisture content prior to root washing
Moisture Content (g) / Oven Dry Soil (g)] x 100 = % Moisture Content
2. Soil bulk density of undisturbed soil.
Dry weight of soil / volume
3. Conversion of root weight to mass.
Oven Dry Roots (g) x Moisture Content Air Dry (g)] + Oven Dry Roots (g) = Air Dry
Roots (g)
Air Dry Roots (g) / Air Dry Soil (g) = Air Dry Mass
Oven Dry Roots (g) / Oven Dry Soil (g) = Oven Dry Mass (Allison et al. 1157).
4. Conversion of the mass of roots / g soil to volume.
Oven Dry Mass (g) x Soil Bulk Density (g/cc) = Root Content g/cc (Stapleton et al.
2090).
5. Converting the root mass of soil to a depth interval and multiply by the Soil Bulk
Density. Then Convert to the area and report as kg/ha or lbs/ac for a given depth (Allison
et al 1157).
Air Dry Roots (g) x Depth (cm) x Soil Bulk Density (g/cc) 100,000 (cm/ha) = Air Dry
Roots kg/ha depth interval (Franks and Going 2).
Discussion
Trees require N to maintain biochemical processes necessary for photosynthesis, nutrient
breakdown, protein synthesis and reproduction. The tree tissues with the highest N
concentrations use most of their energy in physiological processes. High Mycorrhizal inhibition
is in the roots because N investment in proteins and enzymes that are responsible for nutrient
assimilation takes place in the roots. Populus grandidentata obtain most of their N through root
uptake of N in the form of inorganic compounds (NH4 and NO3). The uptake of N compounds by
roots plays a minor role in the nutrition of Populus grandidentata. A sizeable fraction of N
entering roots goes through the Mycorrhizal fungi, which engage in a symbiotic relationship with
Populus grandidentata. NH4+ makes up the inorganic fraction of soil N and is the form of N that
flimsy roots take up (Allison et al. 1163). Populus grandidentata sacrifice 25% of their N every
year in the form of lost N through N- rich tissues leaves and delicate roots. Given the ability to
acquire N, the process of loosing N by a significant fraction is not possible every year. This
results in slow growth of Populus grandidentata in areas where N is not available in enough
quantities. Mycorrhizae in the soil use N to maintain their physiological processes. It is in their
growth that they keep the N cycle moving. Ninety percent of the N in habitats where Populus
grandidentata grow is in the soil. Most of N is not active in the N cycle. The N in these habitats
is within the plant and microbial detritus or Soil Organic Matter (SOM).
The release of N in inorganic from organic compounds is through Nitrogen
mineralization. This process occurs as the soil microbes break organic matter down. Mycorrhizae
have an active role in the N cycle. They are responsible for plant nutrition. As they colonize
plant and roots, they enter symbioses with their hosts. They receive C from the host plant while
they provide the host with N and phosphorus. Mycorrhizae add inorganic N from the soil into the
tree roots. Recent research indicates that some Mycorrhizal fungi have the capability to break
organic detritus and mineralize N in there free-living form (Allison et al 1157). Anthropogenic N
fertilization into the earth’s atmosphere is one of the ways that humans are influencing global
biogeological cycles. Farming activities like burning of biomass convert organic N into oxidized
and reduced N compounds. Farming activities, such as feedlot operations and field applications
of NH3 are pathways of NH3 volatilization to the atmosphere. Atmospheric N fertilization affects
ecosystems as there is an increase in vegetation N concentration. The vegetation senesces dies
and moves into the soil detritus. If the detritus is rich in N, it also affects the soil chemistry and N
cycling. Chronic N deposition leads to soil acidity, which in turns hastens the loss of nutrients in
the soil interfering with microbial production of enzymes that take part in decomposition. If N
deposition continues for enough time, it leads to N saturation. This is a state where the supply of
N exceeds the needs of the plants. This also leads to plant senescence and death. This results
from N getting into the ground water and the aquatic ecosystem, and the atmosphere. N can be a
limiting nutrient if it is available in quantities that are more than necessary. These large
quantities lead to toxic liability in the ecosystem.
Figure 3: Relative number and biomass of Microbial species at 0-15 cm depth of soil.
Microorganisms Number/gram Biomass
of soil
(g/m2)
Bacteria
108 - 109
40-500
Actinomycetes
107-108
40-500
Fungi
105-106
100-1500
Source: Rafiq, Islam and Hoorman, James. Understanding Soil Microbes and nutrient Cycle.
Cleveland: Ohio state university press, 2009. Print
Figure 3 shows the average distribution of microbes up to a depth of 15 centimeters. This
compared to the results gives a clear indication of the inhibition that N deposition has had on the
Mycorrhizal colonies. For the Mycorrhizal colony as a whole, biomass declined by an average of
15% under N fertilization. These results support the hypothesis of this research that N nitrogen
has an effect on Mycorrhizal colonization associated with Populus grandidentata. In this case,
there was an observation of the negative effects it has on the biomass. Numerous studies
highlight the negative effects of N fertilization on Populus grandidentata (Allison et al 1167). A
wide variation exists among the different systems used; this hinders the capability to arrive at a
conclusion on general patterns qualitatively. The negative relationship that exists between fungal
biomass and N load is because of progressive inhibition of growth of ligninase activity or fungi.
The high availability of N also induces “browning” of the plant material. This leads to
accumulation of material that is toxic to fungi. There is an adverse effect on Mycorrhizal fungi
by N fertilization. Populus grandidentata plants tend to allocate diminutive C to Mycorrhizal
fungi when N is readily available. However, other plant free fungi yield decreased fungal
biomass when there is an increase in of N. The inhibition of the Mycorrhizal may not explain the
decreased numbers of fungi fully. Changes in biomass and the composition of the groups of
fungal community can shift under N additions. A report by Allison et al (1160) suggests that the
past few decades witnessed a steady drop in fungal sporocarps produced in forests that have
exposure to large anthropogenic N depositions. The number of Ectomycorrhizal species and their
diversity usually decreases following N deposition. These changes are more visible below
ground than above ground (Allison et al 1157). There is an expectation that fungi would be Nlimited, given that their tissues have N concentration, combined with high growth rates and the
production of N extracellular enzymes. Each of these traits contributes to relatively high
demands of N, which then induce N-limitation if it is available in sufficient quantities. Other
considerations on the effect of N on Mycorrrhizal biomass do not reflect the growth rate. The
growth and death rate could have seen an increase while the standing biomass could have
dropped. Few studies have measured these rates in the soil under N fertilization. One example is
where Thymidine incorporation assessed bacterial growth. In this experiment, Stapleton et al.
‘’saw positive effects of N additions although standing biomass did not change’’ (p. 2090).
Demoling et al applied the same methodology and found a reduced number of in bacterial
growth. There was a positive effect of N additions although the standing biomass of the
Mycorrhizal did not change (377). If experimental measurements of microbial growth
experiments took place more often, it would be possible to determine the general pattern of
microbial productivity. A decrease in microbial biomass under N fertilization has consequences
on C fluxes. Studies have been indicating there are no significant effects on the addition of N
fertilizer type to Mycirrhizal response. Likewise, there were no significant differences among
calcium nitrate and ammonium sulfate fertilizers. Some studies proceed to indicate that some
species of Mycorrhiza are resistant to N addition. The studies report that there is a decline in the
numbers of Mycorrhizal and no change in overall microbial biomass. The emerging evidence
that Mycorrhizal biomass may increase in some ecosystems with N addition are not conclusive
(Allison et. al 1160). Roots have different ways of reacting to elevated N fertilization. Soil
composition as well as the plant community in the rhizosphere in close contact with roots affects
microbial community. In this research, Microbial biomass reduced in numbers under N
fertilization. The response is inconsistent among studies by several other researches (Demoling
et al.) As N load increased, the effects on microbial biomass developed to being very negative.
Studies further indicate that there are no significant effects on the addition of N fertilizer type to
Mycirrhizal response. There were no significant differences among calcium nitrate and
ammonium sulfate fertilizers. Some studies also show that few species of Mycorrhiza are
resistant to N addition. In addition, they report that there is a decline in the number of
Mycorrhizal and no change in overall microbial biomass. The emerging evidence that
Mycorrhizal biomass may increase in some ecosystems with N addition is not conclusive
(Allison et. al 1163). Roots have different ways of reacting to elevated N fertilization. It is
imperative to note that soil composition as well as the plant community in the rhizosphere that is
in close contact with roots affects microbial community. In this research, Microbial biomass
reduced in numbers under N fertilization. The response is inconsistent among studies by other
researches (Deng et al.378). As the N load increased, the effects on microbial biomass turned out
to be quite negative.
Conclusions
In summary, N depositions had a negative effect on the Mycorrhizal biomass and the
reduction of Mycorrhizal biomass became quite evident as the N deposition increased. The
results imply that anthropogenic N deposition leads to a decline in the release of greenhouse
gases from soils to the atmosphere. This is because of the inhibition of microbial activity. The
response turns out to be more evident in ecosystems with exposure to N over a long time. The
deposition of N results in the soil becoming acidic. This reduces the number of fungi in the soil.
The fungi play an essential role in the breakup of N compounds. However, the Mycorrhizal can
break down when N’s deposition exceeds the amount. Some of the N accumulates in the
ecosystem, and becomes toxic to the fungi within a certain duration.
Works Cited
Allison, Gartner, Mack, McGuire, Treseder Kathleen. Nitrogen Alters Carbon Dynamics
During Early Succession In Boreal Forest. Journal of Soil Biology & Biochemistry, 42
(2010). 1157-1164. Print.
Demoling, Nilsson, and Baath E. “Bacterial and Fungal Response to Nitrogen Fertilization In
Three Coniferous Forest Soil. Journal of Soil Biology And. Biocheistry 40.3 (2008):370379. Print.
Nave, Lucas. Nitrogen Cycling in the Northern Hardwood Forest: Soil, Plant, and Atmospheric
Processes. Cleveland: Ohio State University Press, 2007. Print.
Rafiq, Islam and Hoorman, James. Understanding Soil Microbes and Nutrient Cycle. Cleveland:
Ohio State University Press, 2009. Print
Stapleton, Crout, Sawstrom C, Marshall W.A, Poulton P.R, Tye A.M. and Laybourn-Parry J.
“Microbial Carbon Dynamics In Nitrogen Amended Arctic Tundra Soil: Measurement
And Model Testing”. Journal of Soil Biology and Biochemistry. 37.1 (2005): 2088-2098.
Print.
.