Faculty of Science and Bio-engineering Sciences

The role of species diversity, Nitrogen and soil microbes in plant physiology and growth

Author

Isaac Owuor Rayo la

Promoter

Prof.Dr.Harry Olde Venterink

Thesis submitted in partial fulfillement of the requirements for the Degree of Master of Science
in Biology: Specializaton Human Ecology

Academic Year 2013-2014

 Acknowledgements

My most sincere gratitude goes to Prof. Dr. Harry Olde Venterink who, as my promoter,
provided dedicated guidance throughout the process of working on my thesis. Particularly, I
appreciate his wealth of knowledge and the willingness to answer the many questions I had.
Also, I would like to recognize Tim Sierens for his guidance in the laboratory and Anne van Zon
for her counsel and encouragement. The VLIR-UOS programme provided funding for my
studies, while Martine Gheysen of IRMO provided much needed administrative support. Finally,
I appreciate the support of my entire family and friends during my stay and studies in Belgium.

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 Abstract

Human activities have led to changes in ecosystems and species composition of plant
communities. Particularly, agricultural intensification has resulted in the increased use of
synthetic fertilizers and accumulation of nutrients on various ecosystems. As a result, there has
been increased loss of plant species due to eutrophication of ecosystems. However, there is a
possibility of reversing the negative anthropological impacts by encouraging plant diversity
while maintaining productivity of natural and agricultural landscapes.

This thesis explored the impacts of plant species diversity (species richness), nitrogen levels and
microbes (mycorrhizae) on the growth and performance of plants. Two greenhouse experiments
were run for this purpose using six European grassland species. All the plant species used had
the ability to form association with mycorrhizae. The results from experiment 1 showed that
species diversity had an overall significant effect on PME activity (P = 0.009) and RMR (P =
0.022). Experiment 2 showed that elimination of mycorrhizae significantly increases PME
activity (P = 0.001) and biomass (P < 0.001). Also, N application significantly increased PME
activity (P < 0.001) and biomass (P < 0.001). The study concluded that though species diversity
increased plant root PME activity and biomass, the effect was significant at community level and
depended on some species within plant communities. Also, mycorrhizae has a more direct role in
regulating PME activity than N-fixing bacteria and is important in acquisition of P under natural
conditions.

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 Table of Contents

Acknowledgements .................................................................................................................................. i
Abstract................................................................................................................................................... ii
Table of Contents ................................................................................................................................... iii
List of Abbreviations ............................................................................................................................... v
Chapter one ............................................................................................................................................. 1
Introduction............................................................................................................................................. 1
1.1 Background ................................................................................................................................... 1
1.1.1 Intensification of Agriculture and its impacts on ecosystems.................................................... 1
1.1.2 Soil microbes and nutrient acquisition by plants ...................................................................... 3
1.2 Problem statement and aim of study ............................................................................................... 5
1.2.1 Objectives ............................................................................................................................... 5
1.2.2 Hypotheses ............................................................................................................................. 5
Chapter Two ........................................................................................................................................... 6
Materials and Methods ............................................................................................................................ 6
2.1 Location ........................................................................................................................................ 6
2.2 Experiment 1 ................................................................................................................................. 6
2.2.1Seedling preparation................................................................................................................. 6
2.2.2 Fungicide treatment................................................................................................................. 6
2.2.3 Soil collection and Preparation ................................................................................................ 7
2.2.4 Setup....................................................................................................................................... 7
2.2.5 Nutrients ................................................................................................................................. 7
2.3 Experiment 2 ................................................................................................................................. 8
2.3.1 Seedling preparation................................................................................................................ 8
2.3.2 Setup....................................................................................................................................... 8
2.3.3 Nutrients ................................................................................................................................. 8
2.4 Plant growth in the Greenhouse...................................................................................................... 9
2.4.1 Arrangement ........................................................................................................................... 9
2.4.2 Watering ................................................................................................................................. 9
2.4.3 Harvesting and sample preparation .......................................................................................... 9

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iii
 2.5 Measurements .............................................................................................................................. 10
2.5.1 Phosphomonoesterase (PME) activity.................................................................................... 10
2.5.2 Biomass and RMR ................................................................................................................ 10
2.6 Statistics ...................................................................................................................................... 11
Chapter Three........................................................................................................................................ 13
Results .................................................................................................................................................. 13
3.1 Effects of species diversity and microbes on PME activity and plant growth (Experiment 1) ........ 13
3.2 Effects of N supply and fungicide addition on root PME activity and plant growth (Experiment 2)
.......................................................................................................................................................... 20
Chapter four .......................................................................................................................................... 27
Discussion ............................................................................................................................................. 27
4.1 Species richness effects ................................................................................................................ 27
4.2 Role of microbes .......................................................................................................................... 28
4.3 Effect of N supply........................................................................................................................ 29
Chapter Five.......................................................................................................................................... 30
Conclusion and Recommendation .......................................................................................................... 30
References............................................................................................................................................. 31

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iv
 List of Abbreviations

AM Abuscular mycorrhiza

ANOVA Analysis of Variance

PME Phosphomonoesterase

pNPP para-Nitrophenylphosphate

RMR Root Mass Ratio

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 Chapter one

Introduction

1.1 Background

1.1.1 Intensification of Agriculture and its impacts on ecosystems

The Green Revolution has led to the intensification of agriculture (Borlaug 2007) with resultant
improvement in product of cereals which are the major sources of human and livestock food
globally (Tilman et al., 2001). However, improved agricultural production has also lead to great
environmental problems (Borlaug 2007). Modern agriculture has seen the popularization of
monocultures in food production, plantation forestry, management of pastures, production of
biofuels and fiber among other products. The result is that there are many large areas covered in
single species geared towards maximizing production (Nair 2007).

Increased use of synthetic fertilizers, a feature of agricultural intensification (Tilman 1998), has
been implicated in various environmental problems resulting from eutrophication (Mann 1999).
Global demand for cereals, which are the major source of human and livestock food, is the major
cause of increased synthetic fertilizer use (Borlaug 2007).This means that the use of synthetic
fertilizers is expected to increase over time alongside increased demand for cereals (Tilman et
al., 2001). Furthermore, projections indicate that fertilizer use is expected to increase from 176.8
million tons in 2011 to 194.1 million tons by the end of 2016 (FAO 2012). These estimates are
based on three main nutrients N, P and K since most fertilizers are composed of N with some P
and K (Campos 2011).

A major effect of increased eutrophication is the loss of species from ecosystems as has been
observed especially in fens and meadows in Western Europe (Olde Venterink 2000). A high
amount of N in the ecosystem has traditionally been blamed for loss of rare plant species in
temperate regions (Clark and Tilman 2008). Current research however, shows that P
accumulation may actually be the main cause of species losses (Olde Venterink et al., 2001;
Ceulemans et al., 2011).

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However, there is great deal of evidence that support the idea that agricultural production can be
enhanced in species mixtures. This is because in species mixtures, as opposed to monocultures
there is interspecific competition and niche differentiation. The result is that, the different species
use different nutrient pools within the soil profile leading to higher biomass production (Tilman
et al., 1997; Loreau and Hector 2001). For example, a study found that intercropping wheat
(Triticum aestivum) with a bean (Vicia faba), yielded higher dry weight than when the two
species were grown alone (Eskandari and Gharbani 2010). Hence, more studies on intercropping
using different species combinations are needed.

Apart from the problem of nutrient enrichment in ecosystems, there is growing pressure on
agricultural and natural ecosystems from grazing and monocultures for biofuel production
(Tilman et al., 2006). These monocultures have not only led to decrease in food production, but
have also seen the destruction of forests and other ecosystems (Ottinger 2007). The result is that
instead of combating climate change, for example, biofuel production has instead contributed to
climate change through release of carbon stored in these ecosystems into the atmosphere (Melillo
et al., 2009). This has happened despite evidence, that biofuel crops grown in species mixtures
actually yield as much as 238 % more bioenergy than those grown in monocultures (Tilman et
al., 2006).

A metanalysis of some 44 experiments show that species-mixtures have 1.7 times more biomass
than monocultures in 79 % of the studies (Cardinale et al., 2007). There is also evidence that
conservation of biodiversity could be an important way of removing excess nutrient from
ecosystems and reverse impacts of eutrophication (Tilman et al., 1996). For example, Cardinalle
(2011) shows a linear increase in nitrate uptake and biomass with species richness in experiments
with algae.

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1.1.2 Soil microbes and nutrient acquisition by plants

Plants use different strategies to acquire nutrients from soil, particularly when these nutrients
either exist in unavailable forms or are in low supply. Phosphorus exists in soil as available
inorganic or orthophosphate (Pi) and unavailable organic forms (Schachtman et al., 1998). Plants
readily take up Pi but organic P must be converted to Pi before roots can take it up (Bucher et al.
2001; Javot et al., 2007). To achieve the mineralization of organic P, plants produce root
phosphomonoesterase (PME) or phosphatase; phosphatase cleaves Pi from the organic P
compound (Juma and Tabatabai 1988). A low availability of Pi stimulates the activity would of
root PME in plants (Olde Venterink 2011).

Soil microbes such as fungi and N-fixing bacteria also play an important role in plant nutrition.
Ninety five percent of plants species for example, rely on mycorrhiza to obtain nutrients from
soil (Kour et al., 2011). The fungus obtains carbon compounds form the plant while the host
plant receives nutrients and water (Dell 2002; Javot et al., 2007; Kour et al., 2011). Arbuscular
mycorrhizae (AM), also known as endomycorrhiza (Ghorbani et al., 2012), are the most
common form (Finlay 2008; van der Heijden 2010; Smith and Smith 2011). Arbuscular
mycorrhizae form association with about 80% of plants in terrestrial ecosystems (Smith and
Smith 2011). At the same time, most fungi form associations with different species without
particular preference (Van der Heijden and Horton 2009).

Mycorrhizae appear particularly important for the uptake of P (Dodd 2000), though there is
evidence that N uptake is also facilitated by the presence of mycorrhizal association (Smith and
Smith 2011). Fuchs and Haselwandter (2004) observe that apart from P and N, AM help plants in
the uptake of other nutrients including Cu and Zn.

The significance of mycorrhiza in plant nutrition is further emphasized by observed differences

in plant P uptake due to high input of synthetic fertilizers, and use of certain fungicides (Dodd,
2000). Also, Kour et al., 2011, found that biomass production was significantly reduced,
compared to the control, in plants treated with thiophanate-methyl fungicide in a greenhouse
experiment. However, mycorrhizal plant interactions have not been studied extensively (Van der
Heijden et al., 1998).

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Legumes have the ability to fix atmospheric N in symbiosis with bacteria such as Rhizobium,
Bradyrhizobium, Mesorhizobim, Sinorhizobium, and Azorhizobium that live in root nodules. The
bacteria are collectively known as rhizobia (McLean et al., 2007). The efficiency of legumes to
fix N depends on a number of factors: Wall et al., 2000 observes that high levels of N inhibit
nodulation and N fixation in legumes. They further note that nodulation is inhibited by soil N: P
ratios above 7 and not at ratios equal to or less than 7. Olde Venterink (2011) found that PME
activity of Medicago lupulina (a legume) was not influenced by nodulation.

Generally legumes have higher PME activity than most forbs and grasses, though this is not true
for all legumes (Olde Venterink 2011). Therefore, legumes can survive better under P limitation;
a common phenomenon in most ecosystems (Elser et al., 2007). Higher investment of N in PME
activity by N-fixing bacteria has been observed, and attributed to high N in the soil (Wang et al.,
2007). However, this observation may likely hold true at the scale of plant communities (Olde
Venterink 2011).

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1.2 Problem statement and aim of study

There is need to adopt land management systems that create a balance between agricultural
productivity, biodiversity conservation and reducing impacts of climate change. However,
finding the balance still remains a major global challenge (Tilman et al., 2006). To address this
challenge, a better understanding is required in the underlying processes that drive relationships
between species diversity of plants, soil nutrient availabilities and the role of microbial
associations therein.

The central aim of this study was to investigate how species diversity, mycorrhizal associations
and nitrogen supply interact with each other in influencing plant growth and PME activity of
plants.

1.2.1 Objectives

i. To determine the effect of plant species diversity on PME activity and plant growth of six
European grassland species.
ii. To determine the effect of mycorrhizae on PME activity and plant growth
iii. To determine the effect of N supply on PME activity and plant growth

1.2.2 Hypotheses

i. Root PME activity of plants is higher when growing in species mixtures than in
monocultures
ii. Biomass production of plant communities is higher in species mixtures than in
monocultures
iii. Plants associated with mycorrhizae have lower root PME activity than plants without this
symbiotic association.
iv. Nitrogen application increases root PME activity

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 Chapter Two

Materials and Methods

2.1 Location

Two experiments were carried out at the Vrije Universiteit Brussel (VUB) greenhouse within the
Etterbeek campus with six European grassland species representing three functional groups;
legumes, non-legume forbs and grasses. Experiment 1 was carried out between November, 2013
to February, 2014 while experiment 2 ran between January-April, 2014. Additional light in the
greenhouse was provided by lamps.

2.2 Experiment 1

2.2.1Seedling preparation

Seeds of three different species were germinated in trays with soil for each of the three plant
functional groups, the extra species would act as backup in case of failure of germination. Lotus
corniculatus seeds were germinated one week earlier since the species needs more time to
germinate and reach the ideal size. Four weeks after germination, the seedlings were transplanted
into trays with sand to slow down the rate of growth since only one forb (Plantago lanceolata)
had germinated. This allowed the second forb (Taraxacum officinale), which was germinated
later to catch up.

2.2.2 Fungicide treatment

Thiophanate–methyl fungicide was formulated by mixing 124.7 mg of wettable Topsin powder,

70% active ingredient (from Bayer Crop Science SA-NV) with water in 100 ml bottles. This gave
an application rate of 25 mg active ingredient per 250g of soil (Boatman et al., 1978).

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2.2.3 Soil collection and Preparation

Soil was collected from under leguminous plants within the VUB Etterbeek campus. The soil
was passed through a 4mm sieve to remove large root material and other organic matter that may
pose a weed problem to the experiment. The sieved soil was mixed with quartz sand at a rate of 1
part of soil for 20 parts of sand (5% of the mixture was soil).The soil and sand mixture was
prepared once for the two experiments to ensure uniformity in the growth medium conditions

2.2.4 Setup

Experiment 1 used six plant species grown in 0.7 L pots. The species included two grasses,
Anthoxanthum odoratum and Holcus lanatus; two legumes, Lotus corniculatus and Trifolium
pratense and two non-legume forbs, Plantago lanceolata and Taraxacum officinale. A total of 78
pots all filled with soil and sand was used in this experiment. The pots were lined with cloth
while filling to prevent sand flushing out of the pots through holes in the bottom. Water was
poured on the pots to saturate the sand and the mixture in the pots. Immediately after applying
fungicide to 39 pots, the young seedlings were transplanted from the trays into the pots.

After 3 weeks for Taraxacum officinale and 10 weeks for the rest of the species, the plants were
transplanted into the pots. Each of the 78 pots had 4 plants with combinations of 1 species, 2
species or 4 species per pot, and the two fungicide treatments had similar plant combinations.
For each species there were three replicates for the monocultures (1 species per pot). The two
species-mixtures were not replicated, but all possible species combinations were included,
yielding 5 replicates of ‘2 species-mixtures ‘per species. For the pots with 4 species-mixtures,
four different species combinations were included, for every species. In total, there were 312
plants for the second experiment.

2.2.5 Nutrients

Chemical analysis of the sand showed that it contained P in a concentration of 7 mg P/kg soil
(Vanessa Muinden, pers.comm.); this corresponds to approximately 6.1 mg P per pot. Thus, the
plants received 0.5 mg P in solution (NaH2PO4) and 6.1 mg P from the sand per pot, N was
supplied as NaNO3 at the rate of 16 mg per pot. The pots also received K, Ca, Fe, Mg, Mn, Bo,
Mo, S, Cu and Zn in sufficient but non-toxic amounts. During the 8 weeks growth period,

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nutrient solutions were applied in weekly doses of 3.5 ml for the first 2 weeks, 5 ml for the next
3 weeks and 6 ml for the last 3 weeks.

2.3 Experiment 2

2.3.1 Seedling preparation

Legume, grass and non-legume forb seeds were germinated in cotton wool placed inside a petri
dish on the green house benches for two weeks. An extra species per functional group was
germinated as backup. The cotton wool was kept moist by applying deionized water at an
interval of two to three days. After the germination period, 50 seedlings each of Lotus
corniculatus (non-legume forb), Holcus lanatus (grass) and Plantagolanceolata were moved into
separate trays with sand so as to facilitate transplanting when setting up the experiment. Two
weeks before transplanting, the trays with plant seedlings were fertilized with a total of 50 ml
basic solution containing 32 mg N and 2.1 mg P. This was done to avoid severe nutrient
deficiency which was starting to manifest through yellowing of leaves. After 5 weeks, the
seedlings were transplanted from the trays and used to set up the experiment.

2.3.2 Setup

Ninety six 0.4 L pots, each with a water holding pan were obtained for the experiment. The pots
were divided into 4 groups of 24 pots, each with additions of soil and fungicide in a full factorial
design. Thereto, half of the pots were filled with quartz sand only, while the other half was filled
with sand and soil mixture. Additionally, half of the sand and sand + soil pots received the
fungicide, the other half did not. Each of the four treatments received 8 plants of each of the
three plant species and each pot had a single plant. Hence, the experiment had a total of 96
plants. Pots received fungicide immediately before planting (see experiment 1), and on the fifth
week of growth, a second fungicide application was made at the same rate.

2.3.3 Nutrients

Throughout the 10 week growth period, each of the four treatments received 8 levels of N; these
were 1, 2, 4, 8, 16, 32, 64 and 128 mg N per pot. Nitrogen was supplied in the form of NaNO 3
solution applied weekly at the rate of 2.5 ml for the first 3 weeks, 5 ml for the next 4 weeks and
7.5 ml for the last 4 weeks. The plants also received moderately limited P at the rate of 0.85 mg

8
per pot (Olde Venterink 2011) supplied in the form of NaH2PO4 plus 6.1 mg P per pot from the
sand (see experiment 1). Phosphorus was applied in a solution with stock solutions of K, Ca, Fe,
Mg, Mn, Bo, Mo, S, Cu and Zn.

2.4 Plant growth in the Greenhouse

2.4.1 Arrangement

Pots were randomly distributed on the greenhouse benches, to reduce bias in terms of light
conditions (Figure 1, a).

2.4.2 Watering

Plants in both experiments received water every 1-3 days. The frequency of watering was higher
for experiment 2 since it was running during the warmer months of the year when the rate of
transpiration and evaporation was higher compared to the cooler months. Watering was done
with de-ionized water from a hose into the water pans under the pots. Care was taken to avoid
contamination of the experiment from splashing.

2.4.3 Harvesting and sample preparation

At the end of 10 weeks for experiment 1 and 8 weeks for experiment 2, the plants were harvested
and processed for determining PME activity, biomass, root mass ratio (RMR) and nodulation.
All 96 plants from experiment 1 were harvested; however, 3 plants from experiment 2 were dead
1 week before harvesting leaving 309 plants. The plants were harvested at a rate of 40 – 60
plants per day. Before harvesting, the plants were assigned numbers, a second set of similar
numbers were randomly picked to determine plants to be harvested on a particular day.

The plants were removed from the pots by gently putting pressure on the sides of the pot using
hands to loosen the soil. The roots, still in soil were then immersed in a bucket of water and the
sand and soil washed off gently and the entangled roots separated for experiment two. The clean
roots were then scored for nodules (for the legumes), cut from the shoot and placed in a cup with
de-ionized water for every plant while the shoots were placed inside envelopes (Figure 1, b, c
and d).

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Water on the root samples was removed dried by pressing carefully between two sheets of tissue
paper. Subsamples of 100 mg were obtained from the each of the tissue-dried roots and placed
into test tubes; these were set aside for determining PME activity while the rest of the roots were
dried in the oven at 70oC for 48 hours. The same procedure was applied to Taraxacum officinale,
but the tap root and the small roots were weighed separately and the dry weights determined
separately. Tap roots were not used for the PME activity measurements.

2.5 Measurements

2.5.1 Phosphomonoesterase (PME) activity

To the 100 mg samples in test tubes, 5 ml tris (hydromethyl)-aminomethane/maleate buffer (pH

6) was added. These were placed in a shaker for one hour after which 0.5 ml sample was
obtained and added to 6 ml of 2 N NaOH. Using a spectrophotometer (HACH Lange DR 1300)
the concentration of para-nitrophenol was determined by measuring absorbance from the
resultant solution at 410 nm. The activity of PME was calculated as μmol para-nitrophenol
produced per g of fresh root per hour. This would correspond to the amount of pNPP cleaved
during this time period. (Johnson et al., 1999; Olde Venterink 2011).

2.5.2 Biomass and RMR

The root moisture content was determined by dividing the root fresh weight by the root dry
weight of the root samples that were oven dried. The total root fresh weight was then multiplied
by the moisture content to obtain the total root dry weight. Total plant biomass per plant was
obtained by adding total root dry weight and shoot dry weight. The RMR was calculated by
dividing total plant dry weight by the total root dry weight per plant.

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Figure 1: a) Plants on greenhouse benches b) counting root nodules c) root samples in de-ionized water
d) envelopes with plant material.

2.6 Statistics

The two data sets were separately analyzed using SPSS version 22 at 95% confidence interval;
however, each data set was checked for outliers using Box-and –Whisker plots. None of the data
sets had outliers.

In the ANOVA for experiment 1, fungicide addition, species and species number (species
richness) per pot were assigned as fixed factors. Biomass, PME activity, RMR and number of
nodules were assigned as dependent variables. The ANOVA was run for all the species at once
and for each species separately. To run the ANOVA per species, the data was split in terms of
species and the variable species removed as a fix ed factor. Apart from the main effects
(Fungicide, Species richness and Species), interactions were also included: Fungicide-Species
richness, fungicide-species, species richness-species and fungicide-species richness-species.

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The ANOVA for experiment 2 had soil, fungicide addition and species as fixed factors while N
supply was considered as a covariate. The ANOVA model was set to run main effects, two –
way, three-way and four-way interactions to capture all the 4 fixed factors. The main effects
were fungicide application, soil and N supply while interactions included fungicide-N supply,
fungicide-soil, soil-N supply and fungicide-soil-N supply. Species was included as a main effect
and in the interactions for combined ANOVA.

I used Levene’s test to determine the homogeneity of variance for both experiments. Log
transformed data was used in the ANOVAs only when Levene’s test was significant (P< 0.05);
otherwise the non-transformed data was used.

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 Chapter Th ree

Results

3.1 Effects of species diversity and microbes on PME activity and plant growth
(Experiment 1)

Overally, there was significant increase in PME activity with number of species per pot and with
fungicide application (Table 1). However, increase in PME activity with species number was
only significant for Anthoxanthum odoratum, Trifolium pratense and Taraxacum officinale
(Table 3). The first two species showing a positive effect of species richness on PME activity,
while the latter species showed a negative effect (Figure 2). Phosphatase activity was not
significantly affected by species richness (Table 3) for Holcus lanatus, Lotus corniculatus and
Plantago lanceolata. Fungicide application, significantly increased PME activity for Taraxacum
officinale and Plantago lanceolata, but tended towards significance for Lotus corniculatus and
Holcus lanatus. Fungicide effect on PME activity for Anthoxanthum odoratum and Trifolium
pratense was not significant. The interaction between species richness and fungicide application
was not significant for any of the six species or for the variables (Table 1).

Biomass (Figure 3) did not increase with number of species but significantly decreased with
fungicide application. Increase in biomass with species number was only significant for Holcus
lanatus and significantly decreased with fungicide application for Lotus corniculatus,
Taraxacum officinale and Trifolium pratense (Table 3). The overall interaction between species
richness and fungicide application was not significant for any of the species. However, fungicide
–species and species richness-species interactions were significant, but fungicide-species
richness-species interaction was not significant (Table 1). The biomass per pot did not increase
with species richness, but significantly reduced with fungicide application (Figure 4, Table 2).

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Figure 2: Effect of species number on phosphomonoesterase (PME) activity of a, b grasses c, d legumes and e, f
non-legume forbs with fungicide and without fungicide. The linear regressions were not significant except for
Anthoxanthum odoratum (P=0.005) treated with fungicide.

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Figure 3: Dry weight and species richness for six species. Linear regression for panel b (Holcus lanatus) was
significant for both fungicide treatments.

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Figure 4: Dry weight per pot with number of species. The weights of plants were taken per pot regardless of the
species. Linear regression was not significant for both fungicide treatments.

Figure 5: Nodulation of (a) Lotus corniculatus and (b) Trifolium pratense with species richness. Linear regression
for each of the species was not significant.

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Figure 6: Root Mass Ratio expresses as a percent with species richness. Linear regression for Taraxacum officinale
“Without fungicide” had a tendency towards significance (P=0.094), while the rest were not significant.

17
Table 1: Overall ANOVA results for PME activity, biomass, RMR for six species and Nodulation for the legumes.
The main effects and interactions are in the top row. The numbers are F values and the asterisks (*) represent level
of significance as indicated below the table. The F values are at one decimal place, but values less than one have

more decimal places.

There was a general increase in RMR (Table 1, Figure 6) with species richness (P=0.022) and
fungicide application (P<0.001). However, the fungicide- interaction was not significant for any
of the species except for Lotus corniculatus (P=0.003) Plantago lanceolata and Trifolium
pratense. The main effect of species richness, fungicide application and species on nodulation
was not significant (Table 1, Figure 5). None of the interactions had significant effect on
nodulation.

Table 2: Analysis of Variance results for dry weight per pot.

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Table 3: Species-specific ANOVA for PME activity, biomass, RMR and nodulation. The top row has the main
effects and interactions. The PME activity data for Lotus corniculatus and Plantago lanceolata were log10 -
transformed for this analysis. The numbers show F values at one decimal place though values less than 0.1 have
more decimal places. The asterisks (*) indicates the level of significance.

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3.2 Effects of N supply and fungicide addition on root PME activity and plant growth
(Experiment 2)

There was significant increase in PME activity with increase in N supply on average (Table 4)
and for Holcus lanatus, Lotus corniculatus and Plantago lanceolata (Table 5). However, PME
activity for Lotus corniculatus was less plastic compared to Holcus lanatus and Plantago
lanceolata (Figure 7). Fungicide-species and species-N supply interaction were generally,
significant for PME activity (Table 4). However, none of the interactions was significant for
individual species under PME activity (Table 5).

On average plant biomass increased with N supply and reduced with fungicide application
(Figure 8, Table 5). However, there was a decline in biomass past a certain level of N supply
which varied with species (Figure 8). Also, biomass for Lotus corniculatus did not increase with
N supply while soil did not have effect on plant biomass for any of the three species. Fungicide-
species and species-N supply interaction were generally significant for biomass (Table 4), but
none of the other interactions were significant for the individual species.

Root Mass Ratio significantly reduced with N supply, fungicide application and treatment with
soil. However, the RMR for Lotus corniculatus did not significantly change with fungicide or
soil application (Figure 9).

Soil induced nodulation while fungicide application had a negative effect on nodulation of Lotus
corniculatus. The number of nodules per plant reduced with increase in N supply (Figure 10).
Of the plants growing with soil, only 3 had nodules. Soil-fungicide and N supply-fungicide
interactions were significant while soil –fungicide –N supply interaction showed a tendency
towards significance. Also, there tended to be a negative interaction between nodulation and
PME activity for Lotus corniculatus (Figure 11).

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Figure 7: Phosphomonoesterase (PME) activity expressed per gram fresh root with N supply. Panels a, c & e are
sand only treatments while panels b, d &f are sand + soil treatments. The x –axis (N supply) is a logarithmic scale.
On panel b the value 181.12 is the actual value, but was changed to fit the scale. The panels show increase in PME
activity with N supply though panels c & d do not show a clear pattern. Linear regressions for panels a, b (Without
fungicide), d (With fungicide), e (Without fungicide) and f (Without fungicide) were significant.

21
Figure 8: Plant dry weight and N supply. Panels a, c & e are sand only treatments while panels b, d &f are sand +
soil treatments. The x –axis (N supply) is a logarithmic scale. The value 2.86 on panel e is an actual value for dry
weight, but was changed to fit the scale. Quadratic regression for panel a, b and f (Without fungicide) were
significant.

22
Figure 9: Plant root Mass ratio expressed as a percentage with N supply. Panels a, c & e are sand only treatments
while panels b, d & f are sand + soil treatments. The x–axis (N supply) is a logarithmic scale. Linear regressions for
panels a, b, d & f “Without fungicide” treatments were significant.

23
Figure10: Nodulation with N supply. Panels a is for sand only treatments while panels b is for sand + soil
treatments. The x–axis (N supply) is a logarithmic scale. Linear regression for panel b “Without fungicide” was
significant (P=0.009).

Figure 11: Phosphomonoesterace (PME) activity expressed per gram fresh weight with number of nodules. Linear
regression tended towards significance (P=0.068).

24
Table 4: Combined ANOVA results for PME activity, biomass, RMR and nodulation (Lotus corniculatus only) for
Holcus lanatus, Lotus corniculatus and Plantago lanceolata. The asterisks (*) indicate the level of significance as
indicated below the table. The numbers with asterisks are F values with only one decimal place, only values below
0.1 have more decimal places. The dashes (-) under “Nodulation” represent absence of values since there was a
single species (Lotus corniculatus) for this variable. Log10 - transformed data for PME activity and biomass data was
used the analysis.

25
Table 5: Individual species ANOVA results for PME activity, biomass, RMR and nodulation (only for Lotus
corniculatus). The top row indicates the main effects and interactions (Fung. = Fungicide). The numbers are F
values with the asterisk (*) indicating the level of significance. F values with are taken at one decimal places and
only values below 0.1 have more than one decimal place. The PME data for Plantago lanceolata and biomass data
for Lotus corniculatus and Plantago lanceolata were log 10 –transformed data for the analysis.

26
 Chapter fou r

Discussion

4.1 Species richness effects

There was no significant difference in biomass at community level between monocultures and
species mixtures, contrary to what I expected on the basis of other biodiversity manipulation
experiments (Cardinale et al., 2007; Hector et al., 1999; Tilman et al., 1996).The most likely
explanation for this observation is that plants in species mixtures did not benefit from niche
complementarity due to uniform growth conditions. This holds true for P supply; while P exists
in organic and inorganic forms in ecosystems, only organic P was supplied in this study. Also,
the pots had small space that limited roots to explore greater depths. Biomass production in
monocultures and species mixtures were not significantly different for most of the individual
species, but were highly significant for Holcus lanatus. Apparently, Holcus lanatus, being a fast
growing species, took advantage of interspecific competition and outcompeted the other species
in the species mixtures.

Despite that overall community biomass was not affected by species richness in the pots; I
observed a species richness effect on root PME activity. The overall PME activity increased with
number of species, although this was mainly due to the effects on two of the six species:
Anthoxanthum odoratum and Trifolium pratense. This species richness effect on PME activity is
consistent with results of Lannes (2012) who found the PME activity increased with number of
plants growing together. In contrast, Olde Venterink (2011) found no significant difference in
PME activity for plants growing in monocultures and those growing in mixtures. Therefore, this
experiment is the first to confirm the results by Lannes (2012) who also, observed a positive
correlation between number of species and root branching. Since there is higher PME activity in
apical meristems and cell surfaces, she concluded that this is the mechanism that leads to high
PME activity in species mixtures. Therefore, under natural conditions where organic P is more
abundant (Messier et al., 2009), high plant diversity is key in acquisition of P by stimulating high
root PME activity.

27
The species richness effect on PME activity did not depend on fungicide addition. This means
that in species rich plant communities, plants will benefit from P acquisition under limited (or
absence of) mycorrhoizal associations. However, this may hold true only if the soil is rich in
organic P. There was no clear pattern between diversity and RMR or nodulation in legumes.
Other studies have shown that this could be due to the fact that, though plants produce more
above above-ground biomass in mixtures than in monocultures, this may not happen within the
first two years of growth (Mommer et al.,2010).

4.2 Role of microbes

Plants treated with fungicide had higher PME activity than those not treated for Plantago
lanceolata and Lotus corniculatus, but not for Holcus lanatus in experiment 2. It is likely that the
fungicide eliminated mycorrhizae, which are important in P acquisition. Therefore, root PME
activity increased to acquire P. The lower response of Holcus lanatus (to fungicide addition)
compared to the forbs is consistent with the observation of Van der Heijden et al., (1998) who
found that grasses depended less on mycorrhizae than forbs. Application of soil did not have an
effect on PME activity, biomass and RMR, but it increased nodulation of Lotus lanatus in
experiment 2. This confirms that the soil used in this study had N-fixing bacteria which are
essential for nodulation (McLean et al., 2007). Biomass production in Holcus lanatus was,
however not significantly affected by lack of mycorrhizae confirming earlier expectation that
grasses are less dependent on mycorrhizae.

I expected that the RMR of plants would increase with fungicide application. This would be
because; elimination of mycorrhizae would trigger the plant to respond by investing more in the
roots (Chapin 1980) so as to have a larger surface area for acquisition of the limiting nutrient.
However, there was no effect of fungicide on RMR in experiment 1 for any of the species but the
fungicide effect was significant for experiment 2. The differences could be attributed to the fact
that experiment 2 received a higher treatment of fungicide than experiment 1. Boatman et al.,
(1978) observed that the percentage of mycorhizal infection of plant roots reduced with
increasing rate of fungicide application .They found that adding thiophanate-methyl fungicide at
20 mg active ingredient reduced infection by 90% and 95% at 25 mg active ingredient.

28
Adding soil had a clear effect on nodulation of Lotus corniculatus plants, but not on PME
activity or biomass. The results imply that Lotus corniculatus plants in the soil addition
treatments could fix atmospheric N, whereas plants in the sand-only treatments could not. The
presence of nodules in 3 out of 16 plants without soil may be due to contamination with bacteria
(from seeds or in the germination trays). This observation shows that PME activity of Lotus
corniculatus plants did not depend on their ability to fix atmospheric N.

4.3 Effect of N supply

Application of N increased significantly increased PME activity of the three species used in this
experiment. However, the legume Lotus corniculatus showed less plasticity on PME activity in
response to N supply. This finding is consistent with a previous study in which Trifolium
pratense did not show plasticity with N supply (Olde Venterink 2011). The increase in PME
activity is most likely to be the result of high amount of available N which is a component of
PME (Treseder and Vitousek, 2001). Also, increase in N leads to P limitation (Olde Venterink
2011) and stimulates P acquisition through increase in PME activity. However, Lotus
corniculatus responded in a less flexible pattern due to its ability to fix N while Holcus lanatus
and Plantago lanceolata are not able to fix N.

Being an essential nutrient for plants (Vance 2001), application of N significantly increased
plant biomass, though further increase in N past a threshold led to drop in biomass production
because of increased N-induced P limitation.

29
 Chapter Five

Conclusion and Recommendation

The results from this study shows that N influences availability of P, it can enhance P acquisition
through increased PME activity, but high amounts of N will lead to P limitation .Therefore, there
is need for a balance between these two nutrients for optimum plant growth and biomass
production. Furthermore, the absence of mycorrhizae, and not of bacteria induced higher root
PME activity when P or both P and N were limiting. This is clear since soil had no significant
effect on PME activity or biomass, but fungicide had significant effect on both PME and
biomass. Hence, the importance of mycorrhizae in improving plant nutrient acquisition was
emphasized. Also, species diversity has an effect on PME activity and biomass production, but
this may be significant over time than over a few weeks of the experiment

Understanding of how diversity affects root PME activity is key in explaining how P acquisition
may be enhanced in agricultural systems to improve yield. For example, intercropping cereals
with legumes would be an alternative to increased fertilizer use of P fertilizer. This would in turn
reduce eutrophication which would otherwise be a problem from high use of P fertilizers.

Future studies, should therefore use crop cereals such as maize, wheat or rice with legumes such
as beans; the results would be directly applicable in farming systems. The results from this study,
and similar studies would also be useful not only in informing cross-cutting policy making
process.

30
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