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dissertation.
Evaluating Trichoderma atroviride and water supply impacts on
Miscanthus x giganteus in New Zealand
A dissertation
submitted in partial fulfilment
of the requirements for the Degree of
Bachelor of Agricultural Science
at
Lincoln University
by
Victoria Shaw
Lincoln University
2015
Abstract of a dissertation submitted in partial fulfilment of the
requirements for the Degree of Bachelor of Agricultural Science.
Abstract
Evaluating Trichoderma atroviride and water supply impacts on Miscanthus x
giganteus in New Zealand
by
Victoria Shaw
Miscanthus x giganteus is one of the most promising biofuel feedstocks in the world. This secondgeneration biofuel source yields 40+ t DM/ha/yr without the need for high inputs of synthetic
nitrogen fertiliser. In New Zealand, M. x giganteus is primarily used for the replacement of tree
shelterbelts removed for centre pivot irrigation. The perennial, hybrid grass provides at least 16
ecosystem services to a landscape and therefore enhancing its production potential is desired.
Trichoderma atroviride is a soil fungus that often functions as a bio-control agent against soil-borne
plant pathogens and as a plant growth promoter. Past research indicates that there is potential to
use this fungus to enhance plant production. The objective of this glasshouse study was to evaluate
whether T. atroviride increases M. x giganteus production and some physiological parameters under
varied water conditions: Drought, intermediate to well-watered.
Results concluded that water significantly increased all plant variables, which was expected. Wellwatered M. x giganteus plants had a mean dry weight of 60.4 g plant-1, producing 238.7% and
756.0% more dry matter than intermediate and drought plants respectively. In contrast, the PR5 T.
atroviride strain mix had no effect on any variable compared with plants that were not inoculated.
However, there was an interaction between T. atroviride and water in inoculated plants under
drought. Inoculated drought plants had a 21.0% higher chlorophyll content and 3.6% higher
percentage of total dry matter than plants that were not inoculated.
Further research in the field is required to determine the effects of variable soil fertility treatments
on M. x giganteus plants with and without T. atroviride. Monitoring the abundance of T. atroviride in
roots of M. x giganteus under variable water treatments would also be valuable in determining the
competitiveness, effectiveness, growth and survival of the fungus in certain environments.
Keywords: Beneficial fungus, biofuel, C4 plant production, C4 plant physiology, ecosystem provision,
giant grass, second-generation feedstock.
i
Acknowledgements
I look back at my time at Lincoln University and realise how fortunate I am. I have met the most
amazing, supportive people. Friends that I will have for a lifetime and memories that I will never
forget. I have so many people to thank who have helped me keep my sanity through this journey of
my life.
First, I would like to thank the most caring, supportive person, who I’m lucky to call my fiancé.
Landon, I could not ask for anything more then you. You have been there for me in good times and in
bad, managing to stay by my side when I raged at my laptop because it would not do what I wanted
it to… I love you! Bring on our wedding in January!
Secondly, to my family. Your support is what gets me through life in general and I love you all. A
special thanks in particular to my mum, Natasha. I can see where I get my driven nature. If it were
not for your hard working attitude towards life, I probably would not be where I am today! I can’t
really say that you’ve kept me sane through this whole process because you’re pretty insane
yourself, but hey, two ‘negatives’ make a positive right?!
My supervisor Steve Wratten. You are one of a kind… in a good way of course! But seriously, I cannot
thank you enough for all the advice and help you’ve given me throughout this whole year. I’m going
to miss your sarcastic nature and correcting of my grammar even though it was a quick email or chat!
Lastly, to my friends that I’ve made here at Lincoln. You guys are awesome and should all be proud of
what you have achieved!
A few quick mentions:
Rainer Hofmann – My second supervisor. Thank you for helping me with all those little questions I
had at such short notice!
Dave Saville – Your help in understanding the crazy world of statistics.
All the kind people of Bio-Protection who have helped me with small things that are actually quite
important for my understanding of my dissertation.
I am going to miss Lincoln University, but bring on the next chapter in life!
ii
List of Tables
Table 2.1. Elemental nutrient analysis of Osmocote Exact and horticultural lime fertilisers (%).....12
Table 2.2. Treatments applied to M. x giganteus plantlets. .............................................................13
Table 3.1. Fresh weight composition (%) of M. x giganteus stem, leaves and roots relative to
aboveground fresh weight production under different treatments. Water treatments
are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I)) or wellwatered (watered until saturation (W)). Trichoderma treatments are: Not inoculated
with T. atroviride (-) or inoculated with T. atroviride (+). NS = not significant. *** = P <
0.001. LSD (5%) is the value for least significant difference of 5% among the means. 26
Table 3.2. Dry weight (g plant-1) of M. x giganteus plant components under different treatments.
Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3
days (I)) or well-watered (watered until saturation (W)). Trichoderma treatments are:
Not inoculated with T. atroviride (-) or inoculated with T. atroviride (+). NS = not
significant. *** = P < 0.001. LSD (5%) is the value for least significant difference of 5%
among the means. ........................................................................................................28
Table 3.3. Aboveground dry matter (%) of M. x giganteus stem, leaves and total (stem + leaves)
under different treatments. Water treatments are: Drought (50 ml per 3 days (D)),
intermediate (100 ml per 3 days (I)) or well-watered (watered until saturation (W)).
Trichoderma treatments are: Not inoculated with T. atroviride (-) or inoculated with
T. atroviride (+). NS = not significant. * = P < 0.05. ** = P < 0.01. LSD (5%) is the value
for least significant difference of 5% among the means. .............................................35
Table 3.4. Root disc cultures of M. x giganteus plants under different treatments. Water
treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I))
or well-watered (watered until saturation (W)). Trichoderma treatments are: Not
inoculated with T. atroviride (-) or inoculated with T. atroviride (+). NS = not
significant. * = P < 0.05. LSD (5%) is the value for least significant difference of 5%
among the means. ........................................................................................................37
vi
List of Figures
Figure 1.1. C3 carbon fixation and photorespiration pathway via RuBisCO catalyses of CO2 or O2.
From Bloom (2010). ........................................................................................................4
Figure 1.2. A simplified comparison diagram of the C3 and C4 photosynthetic pathways. From
Wang et al. (2012). .........................................................................................................5
Figure 2.1. Mean temperatures (°C) in an incubated glasshouse at different times of the day
throughout the experimental period. ..........................................................................11
Figure 3.1. Mean soil moisture content of M. x giganteus leaves under different treatments as
measured with a TDR on April 23, 2015. Water treatments are: Drought (50 ml per 3
days (D)), intermediate (100 ml per 3 days (I)) or well-watered (watered until
saturation (W)). Trichoderma treatments are: Not inoculated with T. atroviride (-) or
inoculated with T. atroviride (+). Least significant difference (error bar above: LSD 5%)
of the Trichoderma and water treatment interaction is 2.329. ...................................17
Figure 3.2. Mean plant growth (cm) of M. x giganteus plants under different treatments over
time. Treatments are: drought plants (50 ml/ 3 days) not inoculated with T. atroviride
, drought plants inoculated with T. atroviride
(100 ml/3 days) not inoculated with T. atroviride
inoculated with T. atroviride
, intermediate plants
, well-watered (watered until saturation) plants
not inoculated with T. atroviride
atroviride
, Intermediate plants
or well-watered plants inoculated with T.
. The least significant differences (LSD 5% (P=0.05)) of the
Trichoderma and water treatment interaction for each date are indicated with error
bars. ..............................................................................................................................19
Figure 3.3. Mean plant height (cm) of M. x giganteus leaves under different treatments at final
harvest. Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml
per 3 days (I)) or well-watered (watered until saturation (W)). Trichoderma
treatments are: Not inoculated with T. atroviride (-) or inoculated with T. atroviride
(+). Least significant difference (error bar above: LSD 5%) of the Trichoderma and
water treatment interaction is 16.81. ..........................................................................20
Figure 3.4. Mean tiller development per pot of M. x giganteus plants under different treatments
over time. Treatments are: Not inoculated with T. atroviride drought (50 ml/ 3 days)
, inoculated with T. atroviride drought
atroviride intermediate (100 ml/3 days)
intermediate
saturation)
, not inoculated with T.
, inoculated with T. atroviride
, not inoculated with T. atroviride well-watered (watered until
or inoculated with T. atroviride well-watered
. The least
vii
significant differences (LSD 5% (P=0.05)) of the Trichoderma and water treatment
interaction for each date are indicated with error bars. ..............................................21
Figure 3.5. Mean tiller number of M. x giganteus leaves under different treatments at final
harvest. Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml
per 3 days (I)) or well-watered (watered until saturation (W)). Trichoderma
treatments are: Not inoculated with T. atroviride (-) or inoculated with T. atroviride
(+). Least significant difference (error bar above: LSD 5%) of the Trichoderma and
water treatment interaction is 2.95. ............................................................................22
Figure 3.6. Mean stomatal conductance (mmol m-2s-1) of M. x giganteus leaves under different
treatments over the 10-week experimental measurement period. Water treatments
are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I)) or wellwatered (watered until saturation (W)). Trichoderma treatments are: Not inoculated
with T. atroviride (-) or inoculated with T. atroviride (+). Least significant difference
(error bar above: LSD 5%) of the Trichoderma and water treatment interaction is
38.83. ............................................................................................................................23
Figure 3.7. Chlorophyll content (SPAD units) of M. x giganteus leaves under different treatments.
Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3
days (I)) or well-watered (watered until saturation (W)). Trichoderma treatments are:
Not inoculated with T. atroviride (-) or inoculated with T. atroviride (+). Least
significant difference (error bar above: LSD 5%) of the Trichoderma and water
treatment interaction is 3.71........................................................................................24
Figure 3.8. Fresh weight composition (%) of M. x giganteus stem, leaves and dead material
relative to aboveground fresh weight production under different treatments. Water
treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I))
or well-watered (watered until saturation (W)). Trichoderma treatments are: Not
inoculated with T. atroviride (-) or inoculated with T. atroviride (+). ...........................25
Figure 3.9. Total dry matter (g plant-1) of M. x giganteus leaves, stem and roots under different
treatments. Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100
ml per 3 days (I)) or well-watered (watered until saturation (W)). Trichoderma
treatments are: Not inoculated with T. atroviride (-) or inoculated with T. atroviride
(+). Least significant difference (error bar above: LSD 5%) of the Trichoderma and
water treatment interaction is 2.513. ..........................................................................27
Figure 3.10. Mean leaf area (cm2) of M. x giganteus leaves under different treatments. Water
treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I))
or well-watered (watered until saturation (W)). Trichoderma treatments are: Not
inoculated with T. atroviride (-) or inoculated with T. atroviride (+). Least significant
viii
difference (error bar above: LSD 5%) of the Trichoderma and water treatment
interaction is 21.18. ......................................................................................................29
Figure 3.11. Mean photosynthetic rate (µmol CO₂ m-2 s-1) of M. x giganteus leaves under different
treatments. Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100
ml per 3 days (I)) or well-watered (watered until saturation (W)). Trichoderma
treatments are: Not inoculated with T. atroviride (-) or inoculated with T. atroviride
(+). Least significant difference (error bar above: LSD 5%) of the Trichoderma and
water treatment interaction is 8.35. ............................................................................30
Figure 3.12. Mean transpiration rate (mmol H2O m-2s-1) of M. x giganteus leaves under different
treatments. Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100
ml per 3 days (I)) or well-watered (watered until saturation (W)). Trichoderma
treatments are: Not inoculated with T. atroviride (-) or inoculated with T. atroviride
(+). Least significant difference (error bar above: LSD 5%) of the Trichoderma and
water treatment interaction is 0.6067. ........................................................................31
Figure 3.13. Water use efficiency (mmol CO2 mol-1 H2O m-2 s-1) of M. x giganteus leaves under
different treatments. Water treatments are: Drought (50 ml per 3 days (D)),
intermediate (100 ml per 3 days (I)) or well-watered (watered until saturation (W)).
Trichoderma treatments are: Not inoculated with T. atroviride (-) or inoculated with
T. atroviride (+). Least significant difference (error bar above: LSD 5%) of the
Trichoderma and water treatment interaction is 4.408. ..............................................32
Figure 3.14. Total (stem + leaf) aboveground dry matter (%) of M. x giganteus plants under
different treatments. Water treatments are: Drought (50 ml per 3 days (D)),
intermediate (100 ml per 3 days (I)) or well-watered (watered until saturation (W)).
Trichoderma treatments are: Not inoculated with T. atroviride (-) or inoculated with
T. atroviride (+). Least significant difference (error bar above: LSD 5%) of the
Trichoderma and water treatment interaction is 2.177. ..............................................34
Figure 3.15. Root: shoot dry weight ratio of M. x giganteus plants under different treatments.
Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3
days (I)) or well-watered (watered until saturation (W)). Trichoderma treatments are:
Not inoculated with T. atroviride (-) or inoculated with T. atroviride (+). Least
significant difference (error bar above: LSD 5%) of the Trichoderma and water
treatment interaction is 1.702......................................................................................36
Figure 4.1. An illustration of the variable fungal cultures isolated from the root sections of M. x
giganteus plants. Trichoderma spp. are recognised by the white downy like
appearance e.g. The bottom left disc contains five Trichoderma cultures from five
root sections. ................................................................................................................43
ix
Chapter 1
Introduction
1.1 Background
The repercussions of anthropogenic effects on the environment have become apparent;
deterioration of the environment which provides resources and services of ecosystems to humanity.
This comes at the expense of fuelling the life we have become accustomed to following the industrial
revolution. Humanity’s demand for resources already exhausted earth bio-capacity, subsequently
drawing into ecological deficit since the 1970s (Monfreda, Wackernagel, & Deumling, 2004). With a 7
billion and rising population, which is both a direct and indirect consequence of the revolution,
changes in the way we sustainably manage earth’s resources become priority.
Since the industrial revolution in 1750, the earth CO2 emissions have raised by 40%, with the earth’s
surface temperature set to increase by 1.7 °C to 4.8 °C by 2100 (IPCC, 2014). Approximately 2/3rds
of anthropogenic CO2 emissions result from fossil fuel burning and 1/3rd from land use changes
(IPCC, 2014). Therefore, an alternative source of energy is necessary. Any viable alternative to fossil
fuels has to provide a net energy gain, be environmentally and economically sustainable and produce
large quantities without reducing food supplies (Hill, Nelson, Tilman, Polasky, & Tiffany, 2006).
Biofuels are processed from plant and organic biomass and play an important role for a sustainable
future by reducing the dependence on oil and offsetting CO2 production (Naik, Goud, Rout, & Dalai,
2010). During plant growth, the CO2 absorbed as part of photosynthesis sanctions biofuels as ‘carbon
neutral’ sources of energy (Cannell, 2003). However, controversy has been raised surrounding the
production of first-generation biofuels, which are derived from food sources such as sugars, starch
and vegetable oil (Mohr & Raman, 2013; Naik et al., 2010). With first-generation biofuels, it is not
only a matter of ‘food vs fuel’ but also the negative impact on the environment and organisms within
the ecosystem during land use conversion and cultivation of these food crops. Fargione, Hill, Tilman,
Polasky, and Hawthorne (2008) suggested that first-generation biofuels could be much higher carbon
emitters than fossil fuels as the converted land causes a ‘carbon debt’. This debt is the time before
the biofuel’s greenhouse gas emissions are lower than those from fossil fuel burning. For a tropical
peatland rainforest that is converted into oil palm, this net release of carbon is approximately 6000
mega joules of CO2 ha-1 which would take 840 years to pay back (Fargione et al., 2008).
Second-generation biofuels are a more practical option as the derivative is lignocellulosic feedstocks
(plant biomass comprising of cellulose, hemicellulose and lignin) which are not sourced from food
1
Figure 1.1. C3 carbon fixation and photorespiration pathway via RuBisCO catalyses of CO2 or O2. From
Bloom (2010).
The C4 photosynthetic pathway occurs in the both the bundle sheath cells (BSC) and the mesophyll
cells which surround the BSC in C4 plants (Fig. 1.2.). Mesophyll cells contain phosphoenolpyruvate
(PEP) carboxylase, an enzyme that catalyses atmospheric CO2 and phosphoenolpyruvate to produce
the four carbon acid, oxaloacetate (Sage, 2004). Oxaloacetate is then converted into a more stable
four-carbon molecule called malate, which diffuses into the BSC. Malate is decarboxylated into
pyruvate and CO2 by NADP-malic enzymes after it enters the BSC, alleviating the abundance of CO2
within the cell (Doubnerova & Ryslava, 2011). Pyruvate continues the metabolic process when it is
regenerated into PEP by the enzyme, pyruvate, phosphate dikinase (PPDK) (Sage, 2004). In the BSC is
RuBP, which can now bind to CO2 to continue the Calvin Cycle as happens in the C3 photosynthesis
pathway. However, the photorespiration process does not occur because PEP is a selective enzyme
which does not fix O2 even in abundance, instead acting as a CO2 pump into the BSC (Sage, 2004;
Wang, Guo, Li, & Wang, 2012). Therefore, RuBP is not oxidised to the phosphoglycolate molecule
that is toxic to plants.
4
When C4 plants are exposed to cooler, temperate regions, the photosynthetic ability is often limited
by temperature. Photosynthesis has been recognised as one of the most temperature sensitive
processes in plants (Richards, Hagan, & McCalla, 1952; Yamori, Hikosaka, & Way, 2014).
Understanding the physiological responses of this process is important to recognise whether a plant
will grow and develop in the environment where it is established.
C4 plants have lower photosynthesis capacity in cooler temperatures because C4 cycle enzymes e.g.
PPDK (Pyruvate orthophosphate dikinase) that are important for pyruvate regeneration to PEP, are
temperature sensitivity (Naidu et al., 2003; Sage, 2004). The consequence of this is photoinhibition of
photosystem II (PSII) and therefore carbon assimilation (Beale, Bint, & Long, 1996; Farage, Blowers,
Long, & Baker, 2006; Yamori et al., 2014). As carbon assimilation diminishes, less CO2 from the
atmosphere is converted into carbohydrates for plant growth (Miguez, Zhu, Humphries, Bollero, &
Long, 2009).
M. x giganteus is unique in the fact that it can maintain high photosynthetic performance at
temperatures 6 °C lower than those tolerated by maize (Zea mays L.), which has been recognised as
one of the major cold-tolerant crops globally. When comparing maize and M. x giganteus
photosynthetic capacity of plants grown at 14 °C which were then exposed to 10 °C, M. x giganteus
plants continued to develop leaves and maintained a photosynthetic capacity approximately 6.5
times that of maize (10 µmol m-2 s-1 vs 1.5 µmol m-2 s-1 respectively) (Naidu et al., 2003). Not only was
this linked to the ability of M. x giganteus to maintain PPDK levels, but maize produced reactive
oxygen species (ROS) within the chloroplast which may act as a mechanism to protect PSII from
photoinactivation and damage (Farage et al., 2006; Naidu et al., 2003).
Under UK conditions (0 – 25 °C), the above ground production from intercepted photosynthetic
active radiation (PAR) was 0.060 kg m-2 for M. x giganteus; the equivalent to the conversion
coefficient 3.29 µg J-1 PAR. This shows that even in cool climates, M. x giganteus can exceed the
theoretical conversion coefficient maximum of C3 plants (2.8 µg J-1 PAR) (Monteith, 1978). The ability
to develop new leaves in combination with quick canopy development in temperate climates
recognises M. x giganteus capability of intercepting high radiation throughout a long growing season.
This provides M. x giganteus with another reason as to why it is not only the leading secondgeneration feedstock used for not just biofuel, but also a desirable plant for service provision in an
ecosystem.
1.3 New Zealand uses
The use of M. x giganteus is a relatively new prospect for New Zealand after it was introduced in
2010. Additional to the production of solid fuel for burning, the giant grass is used as a shelterbelt
7
replacement to plants that have been removed to allow for pivot irrigation. M. x giganteus can
rebound from the pivot moving over it and therefore can provide shelter to stock and pasture in a
range of agricultural systems (Littlejohn, Wratten, Curran, Hofmann, & Dennis, 2014). In Littlejohn et
al. (2014), M. x giganteus increased pasture production by approximately 18%. The pasture species
stomata remained open for longer because they were sheltered from the wind.
The added advantage of these shelter areas are an increase in biodiversity of beneficial insects,
which are important for pollination of crops, pest control and soil health; M. x giganteus provides 16
ecosystem services to an environment. Agriculture currently contributes to 46% of New Zealand’s
emissions profile and impacts largely on the environment even though it relies heavily on New
Zealand’s clean green image for export earnings (UNFCCC, 2012). M. x giganteus can play a
significant role in reducing the reliance on chemicals for agriculture due to the services it provides to
a landscape. Beneficial services may be enhanced further with the soil-borne beneficial fungus –
Trichoderma atroviride (Bissett).
1.4 Beneficial plant fungi - Trichoderma spp.
Trichoderma spp. are soil-borne, beneficial fungi that are used as a biological control agent against
plant pathogens that cause plant diseases (Harman, Bjorkman, Ondik, & Shoresh, 2008). They are
commercially available for this purpose and are specifically important in agricultural systems as an
alternative to chemical use (Harman, Howell, Viterbo, Chet, & Lorito, 2004; Saba, Vibhash, Manisha,
Prashant, Farhan, & Tauseef, 2012). Trichoderma spp. share a symbiotic relationship with the plant
by penetrating the epidermis and outer cortex and colonizing the root system, providing long term
benefits similar to mycorrhizae and rhizobia (Harman et al., 2004; Yedidia, Benhamou, & Chet, 1999).
The fungi induce systemic resistance mechanisms of the plant through chemical communication
leading to a barrier that permits further plant colonisation (Harman et al., 2004; Yedidia et al., 1999).
Additional modes of action include antibiotic production that inhibit growth through inactivation of
the enzyme system (Dennis & Webster, 1971) and parasitism through the release of lytic enzymes
that break down the hosts cell wall, allowing Trichoderma to consume its nutrients (Chet, 1990;
Howell, 2003).
The benefits on plants associated with Trichoderma are not limited specifically to biocontrol. Abiotic
stress tolerance, growth, and yield promotion have been directly related to the beneficial fungi
across a range of C3 and C4 plants (Harman et al., 2004; Saba et al., 2012; Salas-Marina, Silva-Flores,
Uresti-Rivera, Castro-Longoria, Herrera-Estrella, & Casas-Flores, 2011). Past research has suggested
that Trichoderma spp. can reduce the damage caused to cells by ROS, which are produced under
stressed environments (Mittler, 2006). The fungi cause an induced response mechanism whereby the
plant reprograms its transcriptome, proteome, and metabolomes (Martinez-Medina, Alguacil,
8
Pascual, & Van Wees, 2014). As a result, increased levels of anti-oxidative enzymes which code for
pathways convert ROS into non-toxic plant forms such as toxic superoxide (O2– ) to hydrogen
peroxide (H2O2) and O2 and then H2O2 to water (H2O) and O2 (Shoresh, Harman, & Mastouri, 2010).
Furthermore, Trichoderma spp. also have the ability to produce secondary metabolites which induce
plant growth through phytohormone production including indole-3-acetic acid (IAA), cytokinin and
auxin (Martinez-Medina et al., 2014; Vinale, Sivasithamparam, Ghisalberti, Marra, Barbetti, Li, Woo,
& Lorito, 2008). Consequently, the root system can spread to greater depths, increasing the nutrient
and water uptake availability for further growth (Harman et al., 2004; Martinez-Medina et al., 2014).
In New Zealand, Kandula, Jones, Stewart, McLean, and Hampton (2015) evaluated nine isolates (five
of T. atroviride and one each of Trichoderma hamatum (Bonord.), Trichoderma koningiopsis
(Samuels, Suárez & Evans), Trichoderma viride (Persoon) and Trichoderma virens (Rifai) on the
control of disease and plant physiology impacts of common New Zealand pasture species. This
included perennial ryegrass (Lolium perenne L.), red clover (Trifolium pratense L.) and white clover
(Trifolium repens L.). In the presence of disease, Trichoderma spp. increased emergence by 25-212%
compared to plants that had no Trichoderma spp. present. However, in some cases, emergence was
reduced in the presence of Trichoderma spp. because the fungi produced plant toxins. Therefore
rigorous stepwise antagonist screening programs are completed to determine the suitability of
Trichoderma isolates for commercial use (Kohl, Postma, Nicot, Ruocco, & Blum, 2011). Overall, T.
atroviride isolates (LU 584 and LU 633) had particular benefits, increasing seedling growth across the
three pasture species.
There has been little work done on the effect of Trichoderma spp. on second-generation feedstocks
(unpublished). However, Chirino-Valle, Kandula, Littlejohn, Hill, Walker, Hettiarachchi, and Wratten
(Unpublished) showed in glasshouse and field experiments that M. x giganteus plant height was
consistently taller in plants which had been inoculated with T. atroviride strain mixes than those that
were not inoculated. By inoculating M. x giganteus with appropriate strains of T. atroviride, an
increase in production would enhance the biofuel production and ecosystem services in New Zealand
and globally.
1.5 Dissertation aims
Past research suggests water requirements may be a limiting factor of M. x giganteus for biofuel
production (Ings et al., 2013; Lewandowski et al., 2000). However, the inoculation of M. x giganteus
with T. atroviride may provide an opportunity to reduce water requirements and increase plant
growth and production through physiological changes within the plant. Past literature has evaluated
T. atroviride strains can benefit M. x giganteus plant growth (Chirino-Valle et al., Unpublished;
9
Zonneveld, 2014), but there has been no research that evaluates the potential of T. atroviride under
different water supply levels on M. x giganteus physiology.
Therefore, the aim of this Honours dissertation is to:
-
Determine the water impacts on M. x giganteus under temperate glasshouse conditions.
-
Evaluate whether T. atroviride strain mix PR5 (isolates FCC 161, FCC 180, FCC 275 and FCC
327) will enhance M. x giganteus production and plant physiology under different water
supply levels. This mixture was selected as it is the most successful strain mixture at
increasing M. x giganteus plant production in past literature (Chirino-Valle et al.,
Unpublished; Zonneveld, 2014).
This Honours dissertation will contribute to a better understanding of the most suitable
environments of M. x giganteus when inoculated with the fungus, T. atroviride and define whether T.
atroviride benefits M. x giganteus under these conditions.
10
Chapter 2
Materials and Methods
2.1 Experimental site
The experimental site was located at the Aluminex Glasshouse on Farm Road at Lincoln University,
Canterbury (43°38'43.1" S 172°27'42.6" E). Glasshouse temperatures were automatically monitored
every two hours. Temperatures fluctuated from 13.9 °C to 26.8 °C, with a mean temperature of 17.5
°C during the experimental period (Fig. 2.1). The glasshouse had an inbuilt automatic fan and heating
system to manage temperatures to some extent.
Two high-pressure sodium lamps were placed approximately 2 m above the pots to duplicate
summer day length. This gave a 16 h day length to the M. x giganteus plants to ensure senescence
would not occur during the experiment.
25
Temperature (°C)
20
March
15
April
May
10
June
July
5
August
0
12:00 2:00
a.m. a.m.
4:00
a.m.
6:00
a.m.
8:00 10:00 12:00 2:00
a.m. a.m. p.m. p.m.
4:00
p.m.
6:00
p.m.
8:00 10:00
p.m. p.m.
Time
Figure 2.1. Mean temperatures (°C) in an incubated glasshouse at different times of the day
throughout the experimental period.
2.2 Experimental design
Pots of 5 L capacity were used for individual M. x giganteus plants and these were assigned to one T.
atroviride and one water treatment. T. atroviride treatments consisted of plantlet roots inoculated
either with the fungus or not (control). Water treatments applied were either ‘drought’ (50 ml every
third day), ‘intermediate’ (100 ml every third day) or ‘well-watered’ (watered to saturation). The
11
treatments were replicated ten times each and assigned in a randomised complete block design,
which gave a total of 60 M. x giganteus plants, each in individual pots.
2.3 Trichoderma treatment
On March 9 2015, Dr Ivan Chirino-Valle prepared a suspension of T. atroviride PR5 strain mixture at
Lincoln University. The strain mix contained New Zealand isolates FCC 161, FCC 180, FCC 275 and FCC
327. The specific isolates were selected based on best performance for plant growth in past
experiments (Chirino-Valle et al., Unpublished; Zonneveld, 2014).
That same day, 60 M. x giganteus plantlets were placed in two 30 cm x 30 cm trays at Lincoln
University’s Aluminex Glasshouse. Half of the plantlets were soaked in the prepared T. atroviride
suspension in the first tray (T+) and the additional half placed in the other with water as a control (T-)
to minimise any pre-planting variation. The plantlets were left to soak for 24 h to enable successful
inoculation of T. atroviride on the M x. giganteus roots.
2.4 Planting
On March 10 2015 following inoculation, plantlets were removed from the trays and planted into 5 L
pots (18 cm deep, 20 cm diameter). Pots contained 1.5 kg of dried soil media made up of 80%
composted bark and 20% pumice (grade 1 – 7 mm). Within the media mix were Osmocote Exact, 3 –
4 month release rate (3 g L-1) and horticultural lime (1 g L-1) which supplied nutrients to the plants nutrient analysis of fertilisers are shown in Table 2.1. A wetting agent, Hydraflo (1 g L-1), was also
applied to reduce the surface tension of the soil media, which increased the spread of the water
throughout the pot. All plantlets were watered to saturation (water dripping from the pot) every
third day for 28 days before water treatments were applied to allow equal establishment conditions.
Table 2.1. Elemental nutrient analysis of Osmocote Exact and horticultural lime fertilisers (%)
Nutrients
Nutrient (%) of fertilisers
Osmocote Exact
Horticultural lime
Nitrogen (N)
16.00
-
Phosphorous (P)
3.90
-
Potassium (K)
10.00
-
Magnesium (Mg)
1.20
-
-
36
Calcium (Ca)
12
Chapter 3
Results
3.1 Soil moisture
The moisture content of the soil in which M. x giganteus plants were planted was measured with a
TDR meter. The watering regime affected soil moisture content as expected. Plants under the wellwatered treatment had the highest soil moisture content (19.3%), which was 13.3% and 16.4% higher
than plants under intermediate and drought treatments (5.4% and 2.89% respectively) (Fig. 3.1.; P <
0.001).
Neither the Trichoderma treatment nor the interaction between the Trichoderma and water treatments
affected soil moisture content.
25
Soil moisture content (%)
20
15
10
+
5
0
D
I
W
Treatment
Figure 3.1. Mean soil moisture content of M. x giganteus leaves under different treatments as
measured with a TDR on April 23, 2015. Water treatments are: Drought (50 ml per 3 days (D)),
intermediate (100 ml per 3 days (I)) or well-watered (watered until saturation (W)).
Trichoderma treatments are: Not inoculated with T. atroviride (-) or inoculated with T.
atroviride (+). Least significant difference (error bar above: LSD 5%) of the Trichoderma and
water treatment interaction is 2.329.
17
3.2 Plant height
Water supply affected M. x giganteus plant growth after May 14. From April 30 until July 2, wellwatered, intermediate and drought plants grew by 47.2 cm, 17.3 cm and 1.8 cm respectively (Fig.
3.2.). Well-watered plants grew 29.9 cm and 45.4 cm more than intermediate and drought plants
respectively over the 10-week period (P < 0.001).
The Trichoderma treatment had no effect on plant growth. There was also no interaction effect
between the Trichoderma and water treatments.
Water affected M. x giganteus plant height by final harvest. Well-watered, intermediate and drought
plants were 128.8 cm, 74.6 cm and 38.8 cm tall respectively (Fig. 3.3.). Drought plants were 35.8 cm
and 90 cm shorter than intermediate and well-watered plants respectively (P < 0.001).
The Trichoderma treatment had no effect on plant height at final harvest. There was also no
interaction effect between the Trichoderma and water treatments.
18
100
90
Plant height (cm)
80
70
60
50
40
30
20
10
0
Date
Figure 3.2. Mean plant growth (cm) of M. x giganteus plants under different treatments over time.
Treatments are: drought plants (50 ml/ 3 days) not inoculated with T. atroviride
drought plants inoculated with T. atroviride
inoculated with T. atroviride
,
, Intermediate plants (100 ml/3 days) not
, intermediate plants inoculated with T. atroviride
, well-watered (watered until saturation) plants not inoculated with T. atroviride
well-watered plants inoculated with T. atroviride
or
. The least significant differences (LSD
5% (P=0.05)) of the Trichoderma and water treatment interaction for each date are indicated
with error bars.
19
160
140
Plant height (cm)
120
100
80
-
60
+
40
20
0
D
I
W
Water treatment
Figure 3.3. Mean plant height (cm) of M. x giganteus leaves under different treatments at final
harvest. Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3
days (I)) or well-watered (watered until saturation (W)). Trichoderma treatments are: Not
inoculated with T. atroviride (-) or inoculated with T. atroviride (+). Least significant difference
(error bar above: LSD 5%) of the Trichoderma and water treatment interaction is 16.81.
3.3 Tiller number
Water supply affected the mean tiller development of M. x giganteus plants. Over the 10 week
experiment period (from April 30 until July 2), well-watered, intermediate and drought plants
developed and average of 9.2, 2.3 and 0.8 tillers respectively (Fig. 3.4.). Well-watered plants
developed an average of 9.4 and 10.9 more tillers than intermediate and drought plants over 10
weeks (P < 0.001). However, tiller number under drought and intermediate treatments did not differ.
The Trichoderma treatment and interaction effect between the Trichoderma and water treatments
had no effect on tiller development.
Water supply affected the mean M. x giganteus plant tiller number at final harvest. Well-watered,
intermediate and drought plants had an average of 12.4, 6.0 and 4.0 tillers by the final harvest date
respectively (Fig. 3.5.). Well-watered plants had 6.8 and 10.8 more tillers than intermediate and wellwatered plants respectively (P < 0.001). Tiller number under drought and intermediate treatments
did not differ.
20
The Trichoderma treatment did not affect tiller number and neither did the interaction between the
Trichoderma and water treatments.
16
14
Tiller number
12
10
8
6
4
2
0
Date
Figure 3.4. Mean tiller development per pot of M. x giganteus plants under different treatments over
time. Treatments are: Not inoculated with T. atroviride drought (50 ml/ 3 days)
inoculated with T. atroviride drought
(100 ml/3 days)
, not inoculated with T. atroviride intermediate
, inoculated with T. atroviride intermediate
with T. atroviride well-watered (watered until saturation)
atroviride well-watered
,
, not inoculated
or inoculated with T.
. The least significant differences (LSD 5% (P=0.05)) of the
Trichoderma and water treatment interaction for each date are indicated with error bars.
21
14
12
Tiller number
10
8
-
6
+
4
2
0
D
I
W
Water treatment
Figure 3.5. Mean tiller number of M. x giganteus leaves under different treatments at final harvest.
Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I)) or
well-watered (watered until saturation (W)). Trichoderma treatments are: Not inoculated with
T. atroviride (-) or inoculated with T. atroviride (+). Least significant difference (error bar
above: LSD 5%) of the Trichoderma and water treatment interaction is 2.95.
3.4 Stomatal conductance
Water supply affected mean stomatal conductance of M. x giganteus leaves from April 30 – July 2
(Fig. 3.6.). Well-watered plants had the highest stomatal conductance of 255.80 mmol m-2s-1 , which
was 47.7 mmol m-2s-1 and 128.71 mmol m-2s-1 higher compared to intermediate and drought plants
(208.10 mmol m-2s-1 and 127.09 mmol m-2s-1 respectively; P < 0.001)
The Trichoderma treatment had no effect on stomatal conductance. Similarly, there was also no
interaction effect between the Trichoderma and water treatments on stomatal conductance.
22
Mean stomatal conductance (mmol m-2s-1)
300
250
200
-
150
+
100
50
0
D
I
W
Water treatment
Figure 3.6. Mean stomatal conductance (mmol m-2s-1) of M. x giganteus leaves under different
treatments over the 10-week experimental measurement period. Water treatments are:
Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I)) or well-watered (watered
until saturation (W)). Trichoderma treatments are: Not inoculated with T. atroviride (-) or
inoculated with T. atroviride (+). Least significant difference (error bar above: LSD 5%) of the
Trichoderma and water treatment interaction is 38.83.
3.5 Leaf chlorophyll content
Water supply affected the leaf chlorophyll content of M. x giganteus leaves. Plants under drought
conditions averaged a SPAD value of 35.09 units, which was 11.26 units and 10.34 units lower than
plants under intermediate and well-watered treatments from April 30 – July 2 (46.35 units and 45.43
units respectively) (Fig. 3.7.; P < 0.001). The chlorophyll content of plants under well-watered and
intermediate treatments did not differ.
The Trichoderma treatment had no effect on M. x giganteus leaf chlorophyll content. However, there
was an interaction effect between the Trichoderma and water treatments (Fig. 3.7.). Inoculated
plants under drought had 6.84 units more chlorophyll than control plants (38.51 SPAD units and
31.67 SPAD units respectively; P < 0.01). There was no interaction effect between the Trichoderma
and water treatments on the chlorophyll content of plants under intermediate and well-watered
treatments.
23
Mean chlorophyll content (SPAD units)
50
45
40
35
30
25
-
20
+
15
10
5
0
D
I
W
Water treatment
Figure 3.7. Chlorophyll content (SPAD units) of M. x giganteus leaves under different treatments.
Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I)) or
well-watered (watered until saturation (W)). Trichoderma treatments are: Not inoculated with
T. atroviride (-) or inoculated with T. atroviride (+). Least significant difference (error bar
above: LSD 5%) of the Trichoderma and water treatment interaction is 3.71.
3.6 Fresh weight
Water supply did not affect the composition of leaf fresh material as a percentage of aboveground
fresh weight i.e. total fresh weight of the stem, leaf and dead material. However, the composition of
stem and dead material was affected. Plants under well-watered treatments produced the highest
composition of stem relative to aboveground fresh weight (55.78%), which was 8.44% and 16.30%
more compared with plants under intermediate and drought treatments (47.34% and 39.48%
respectively) (Fig. 3.8. and Table 3.1.; P < 0.001). In contrast, plants under drought had the highest
composition of dead material relative to the fresh weight (23.88%), which was 9.80% and 16.84%
more than plants under the intermediate and well-watered treatments (14.80% and 7.04%
respectively; P < 0.001).
The Trichoderma treatment and interaction between the Trichoderma and water treatments had no
effect on the composition of either the stem, leaf or dead material relative to total aboveground
fresh weight.
24
Aboveground fresh weight composition (%)
100
90
80
70
60
50
Dead
40
Leaf
30
Stem
20
10
0
D-
D+
I-
I+
W-
W+
Treatment
Figure 3.8. Fresh weight composition (%) of M. x giganteus stem, leaves and dead material relative to
aboveground fresh weight production under different treatments. Water treatments are:
Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I)) or well-watered (watered
until saturation (W)). Trichoderma treatments are: Not inoculated with T. atroviride (-) or
inoculated with T. atroviride (+).
25
Table 3.1. Fresh weight composition (%) of M. x giganteus stem, leaves and roots relative to
aboveground fresh weight production under different treatments. Water treatments are:
Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I)) or well-watered (watered
until saturation (W)). Trichoderma treatments are: Not inoculated with T. atroviride (-) or
inoculated with T. atroviride (+). NS = not significant. *** = P < 0.001. LSD (5%) is the value for
least significant difference of 5% among the means.
Treatment
Aboveground dry weight composition (%)
Stem
Leaf
Dead
D-
39.13
34.79
26.08
D+
39.75
38.55
21.68
I-
46.50
37.54
15.60
I+
48.18
37.90
14.19
W-
55.52
37.56
6.86
W+
55.80
36.99
7.21
Trichoderma
NS
NS
NS
Water
***
NS
***
Trichoderma*water
NS
NS
NS
LSD (5%) Trichoderma
3.61
2.96
4.18
LSD (5%) Water
4.42
3.63
5.12
LSD (5%) Trichoderma*water
6.25
5.13
7.23
3.7 Total dry weight
Water supply affected the dry weight of all M. x giganteus plant components including the stem, leaf
and root. Well-watered, intermediate and drought plants had dry weight total of 60.35 g plant-1,
17.82 g plant-1 and 7.05 g plant-1 respectively (Fig. 3.9., Table 3.2.). Well-watered plants weighed
42.53 g and 53.3 g heavier than intermediate and drought plants respectively (P < 0.001). Drought
plants had 7.84 g and 1.31 g lighter stems, 10.07 g and 1.44 g lighter leaves and 35.4 g and 8.02 g
lighter roots than well-watered and intermediate plants respectively (P < 0.001).
The Trichoderma treatment and the interaction between the Trichoderma and water treatments had
no effect on the dry weight of any M. x giganteus plant components.
26
Aboveground dry matter (g plant-1)
70.00
60.00
50.00
40.00
Root
30.00
Leaf
Stem
20.00
10.00
0.00
D-
D+
I-
I+
W-
W+
Treatment
Figure 3.9. Total dry matter (g plant-1) of M. x giganteus leaves, stem and roots under different
treatments. Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3
days (I)) or well-watered (watered until saturation (W)). Trichoderma treatments are: Not
inoculated with T. atroviride (-) or inoculated with T. atroviride (+). Least significant difference
(error bar above: LSD 5%) of the Trichoderma and water treatment interaction is 2.513.
27
Table 3.2. Dry weight (g plant-1) of M. x giganteus plant components under different treatments.
Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I)) or
well-watered (watered until saturation (W)). Trichoderma treatments are: Not inoculated with
T. atroviride (-) or inoculated with T. atroviride (+). NS = not significant. *** = P < 0.001. LSD
(5%) is the value for least significant difference of 5% among the means.
Dry weight component (g plant-1)
Treatment
Stem
Leaf
Root
Total
D-
0.43
0.46
5.63
6.41
D+
0.37
0.35
6.86
7.68
I-
1.58
1.73
15.17
18.48
I+
1.83
1.95
13.37
17.15
W-
8.51
11.32
41.94
61.77
W+
7.96
9.62
41.35
58.93
Trichoderma
NS
NS
NS
NS
Water
***
***
***
***
Trichoderma*water
NS
NS
NS
NS
LSD (5%) Trichoderma
0.44
0.84
3.79
3.60
LSD (5%) Water
0.54
1.03
4.64
4.41
LSD (5%) Trichoderma*water
0.76
1.45
6.56
6.23
3.8 Leaf area
Water supply affected the mean leaf area of M. x giganteus leaves. Well-watered, intermediate and
drought plants had final mean leaf areas of 28.61 cm2, 62.03 cm2 and 98.65 cm2 respectively (Fig.
3.10.). Well-watered plants had a 36.62 cm2 and 70.04 cm2 larger leaf area than intermediate and
drought plants respectively (P < 0.001).
The Trichoderma treatment had no effect on leaf area and there was no interaction affect between
the Trichoderma and water treatments.
28
120
Laef area (cm2)
100
80
60
+
40
20
0
Drought
Intermediate
Well-watered
Water treatment
Figure 3.10. Mean leaf area (cm2) of M. x giganteus leaves under different treatments. Water
treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I)) or wellwatered (watered until saturation (W)). Trichoderma treatments are: Not inoculated with T.
atroviride (-) or inoculated with T. atroviride (+). Least significant difference (error bar above:
LSD 5%) of the Trichoderma and water treatment interaction is 21.18.
29
3.9 Photosynthetic rate
The photosynthetic rate of M. x giganteus plants was not affected by either the water or
Trichoderma treatments and there was no interaction affect between the Trichoderma and water
treatments at final harvest (Fig. 3.11.).
Photosynthetic rate (µmol CO₂ m-2 s-1)
25
20
15
10
+
5
0
Drought
Intermediate
Well-watered
Water treatment
Figure 3.11. Mean photosynthetic rate (µmol CO₂ m-2 s-1) of M. x giganteus leaves under different
treatments. Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3
days (I)) or well-watered (watered until saturation (W)). Trichoderma treatments are: Not
inoculated with T. atroviride (-) or inoculated with T. atroviride (+). Least significant difference
(error bar above: LSD 5%) of the Trichoderma and water treatment interaction is 8.35.
30
3.10 Transpiration rate
The transpiration rate of M. x giganteus plants was not affected by either the water or Trichoderma
treatments and there was no interaction affect between the Trichoderma and water treatments at
final harvest (Fig. 3.12.).
Transpiration rate (mmol H2O m-2s-1)
1.4
1.2
1
0.8
-
0.6
+
0.4
0.2
0
Drought
Intermediate
Well-watered
Water treatment
Figure 3.12. Mean transpiration rate (mmol H2O m-2s-1) of M. x giganteus leaves under different
treatments. Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3
days (I)) or well-watered (watered until saturation (W)). Trichoderma treatments are: Not
inoculated with T. atroviride (-) or inoculated with T. atroviride (+). Least significant difference
(error bar above: LSD 5%) of the Trichoderma and water treatment interaction is 0.6067.
31
3.11 Water use efficiency
The water use efficiency of M. x giganteus plants was not affected by either the water or
Trichoderma treatments and there was no interaction affect between the Trichoderma and water
treatments at final harvest (Fig. 3.13.).
Water use effeciency (mmol CO2 mol-1 H2O m-2 s1)
25
20
15
+
10
5
0
D
I
W
Water treatment
Figure 3.13. Water use efficiency (mmol CO2 mol-1 H2O m-2 s-1) of M. x giganteus leaves under
different treatments. Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100
ml per 3 days (I)) or well-watered (watered until saturation (W)). Trichoderma treatments are:
Not inoculated with T. atroviride (-) or inoculated with T. atroviride (+). Least significant
difference (error bar above: LSD 5%) of the Trichoderma and water treatment interaction is
4.408.
3.12 Dry matter
Water supply affected total (stem + leaf) dry matter percentage. Well-watered, intermediate and
drought plants had a total dry matter of 25.66%, 26.14% and 24.25% respectively (Fig. 3.14. and
Table 3.3.). M. x giganteus plants under intermediate water supply averaged 26.14% total dry matter,
which was 1.89% higher than plants under drought (24.25%; P < 0.05). However, plants under the
well-watered treatment did not differ in total dry matter percentage in comparison with plants under
intermediate or drought treatments.
32
The Trichoderma treatment affected total dry matter percentage. Plants inoculated with T. atroviride
averaged 24.61% total dry matter, which was 1.47% less than the control plants (26.08%; P < 0.05).
The interaction between the Trichoderma and water treatments also affected total dry matter
percentage. Inoculated plants under the drought treatment had a total dry matter of 22.43%, which
was 3.64% less than control plants under the same water treatment (26.07%; P < 0.05). However,
there was no interaction effect between the Trichoderma and water treatments on total dry matter
percentage under well-watered and intermediate water supply.
Water supply affected leaf dry matter percentage. At final harvest, well-watered, intermediate and
drought plants had a leaf dry matter percentage of 28.50%, 28.96% and 25.4% respectively (Table
3.3.). M. x giganteus plants under drought had 3.56% and 3.1% less leaf dry matter than plants under
intermediate and well-watered treatments respectively (P < 0.01). However, plants under wellwatered and intermediate treatments did not differ in the percentage of leaf dry matter.
The leaf dry matter was not affected by the Trichoderma treatments and there was no interaction
affect between the Trichoderma and water treatments.
Water supply did not affect M. x giganteus stem dry matter percentage. However, the Trichoderma
treatment did have an effect. Inoculated M. x giganteus plants averaged 22.56% stem dry matter,
which was 2.19% less than the control plants (22.56% and 24.75% respectively) (Table 3.3.; P < 0.05).
There was no interaction effect between the Trichoderma and water treatments on the percentage
of stem dry matter.
33
Total aboveground dry matter (%)
30
25
20
15
+
10
5
0
D
I
W
Water Treatment
Figure 3.14. Total (stem + leaf) aboveground dry matter (%) of M. x giganteus plants under different
treatments. Water treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3
days (I)) or well-watered (watered until saturation (W)). Trichoderma treatments are: Not
inoculated with T. atroviride (-) or inoculated with T. atroviride (+). Least significant difference
(error bar above: LSD 5%) of the Trichoderma and water treatment interaction is 2.177.
34
Table 3.3. Aboveground dry matter (%) of M. x giganteus stem, leaves and total (stem + leaves)
under different treatments. Water treatments are: Drought (50 ml per 3 days (D)),
intermediate (100 ml per 3 days (I)) or well-watered (watered until saturation (W)).
Trichoderma treatments are: Not inoculated with T. atroviride (-) or inoculated with T.
atroviride (+). NS = not significant. * = P < 0.05. ** = P < 0.01. LSD (5%) is the value for least
significant difference of 5% among the means.
Treatment
Dry matter percentage (%)
Leaf
Stem
Total
D-
26.22
25.28
26.07
D+
24.58
21.44
22.43
I-
28.26
23.87
25.74
I+
29.69
23.83
26.53
W-
28.50
25.09
26.43
W+
28.50
22.42
24.88
Trichoderma
NS
*
*
Water
**
NS
*
Trichoderma *water
NS
NS
*
LSD (5%) Trichoderma
1.81
1.78
1.26
LSD (5%) Water
2.22
2.18
1.54
LSD (5%) Trichoderma *water
3.14
3.08
2.18
3.13 Root: shoot dry weight ratio
Water supply affected the root: shoot dry weight ratio of M. x giganteus plants. For every 1 g of dry
shoot material (leaf and stem), plants under well-watered, intermediate and drought water supply
produced 2.41 g, 4.38 g and 8.02 g of dry root material respectively (Fig. 3.15.). Drought plants had
35
3.64 g and 5.61 g more root material per 1 g dry shoot material than intermediate and well-watered
plants (P < 0.001).
The Trichoderma treatment had no effect on root: shoot dry weight ratio. There was also no
interaction effect between the Trichoderma and water treatments on the root: shoot dry weight
ratio of M. x giganteus plants.
9
Root: shoot dry weight ratio
8
7
6
5
-
4
+
3
2
1
0
D
I
W
Water treatment
Figure 3.15. Root: shoot dry weight ratio of M. x giganteus plants under different treatments. Water
treatments are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I)) or wellwatered (watered until saturation (W)). Trichoderma treatments are: Not inoculated with T.
atroviride (-) or inoculated with T. atroviride (+). Least significant difference (error bar above:
LSD 5%) of the Trichoderma and water treatment interaction is 1.702.
3.14 Fungal cultures
Water supply did not affect the number T. atroviride fungal cultures from the 20 root sections taken
from M. x giganteus. However, as expected, the Trichoderma treatment did. Plants inoculated with T.
atroviride had 12 more T. atroviride cultures present than control plants overall (Table 3.4.; P < 0.05).
The interaction between the Trichoderma and water treatments also affected the number of T.
atroviride cultures. Inoculated plants under well-watered and intermediate water supply had more
cultures than control plants under the same water treatment (11 and 4 cultures compared with 1 and
36
2 cultures from 20 root sections respectively) (P < 0.05). However, the interaction between the
Trichoderma and water treatments did not differ under drought.
Table 3.4. Root disc cultures of M. x giganteus plants under different treatments. Water treatments
are: Drought (50 ml per 3 days (D)), intermediate (100 ml per 3 days (I)) or well-watered
(watered until saturation (W)). Trichoderma treatments are: Not inoculated with T. atroviride
(-) or inoculated with T. atroviride (+). NS = not significant. * = P < 0.05. LSD (5%) is the value
for least significant difference of 5% among the means.
Treatment
Disc number
Total/20
1
2
3
4
D-
0
2
0
0
2
D+
0
0
1
1
2
I-
0
1
0
1
2
I+
1
2
0
1
4
W-
0
0
0
1
1
W+
2
5
3
1
11
Trichoderma
*
Water
NS
Trichoderma *water
*
LSD (5%) Trichoderma
0.821
LSD (5%) Water
1.006
LSD (5%) Trichoderma *water
1.422
37
Chapter 4
Discussion
M. x giganteus is the one of the most suitable species for second-generation biofuel production
worldwide. In New Zealand, the grass is also a source for ecosystem service provision when it
replaces removed shelterbelts for irrigation. M. x giganteus has ability to produce upwards of 40 t
DM ha-1 yr-1 in temperate regions for 15 – 20 years and from relatively low inputs of nitrogen and
water. Increasing the production of this giant grass will enhance service provision and is therefore
desired.
Inoculating M. x giganteus with T. atroviride strain mixes have proved successful in increasing
production of the grass. However, no studies have shown how these fungi will perform under
variable water supply. This is important for understanding not only the fundamental range of M. x
giganteus, but also that of T. atroviride. Therefore, a glasshouse pot experiment was set-up to
understand this concept. M. x giganteus plants were grown under variable water and Trichoderma
treatments and plant physiology, growth and development measurements were taken.
4.1 Experimental set-up
The experimental methods were successfully implemented as shown from the soil moisture content
and fungal culture results. The soil moisture content significantly differed among water treatments.
However, the number of T. atroviride cultures taken from the roots of M. x giganteus plants did not.
Furthermore, the Trichoderma treatment had no effect on soil moisture, but plants inoculated with
T. atroviride had more cultures isolated on the discs than the control plants. These results dismiss the
possibility that if any results were not significant, the reason could be that the water supply between
treatments were not different, or that Trichoderma was not present.
4.2 Water
There was a clear linear correlation between leaf area and water stress (Fig. 3.10.). Drought and
intermediate plants had an 80% and 45% lower leaf area than well-watered plants. This is similar to
the findings of Ings et al. (2013), where leaf area in M. x giganteus plants under mild water stress
were 10% smaller than well-watered plants (192 cm2 and 212 cm2 respectively). Leaf cell expansion
and division can decline under water stress. Schuppler, He, John, and Munns (1998) reported that
mitotic activity of mesophyll cells in wheat plants under drought was reduced by 42% compared to
well-watered plants. Consequently, a smaller leaf area limits the ability of a plant to capture sunlight
radiation and convert it into energy for plant growth.
38
Leaf area was therefore a major contributor to the observed differences among water treatments on
M. x giganteus total dry matter production and overall plant height. Well-watered plants had 52.51 g
plant-1 and 42.53 g plant-1 greater dry weight production than drought and intermediate plants
respectively (Fig. 3.9.). Well-watered plants were also 72.65% and 231.96% taller than intermediate
and drought plants.
In this study, the first observed physiological response of M. x giganteus plants was that water stress
had no effect on the photosynthetic rate, transpiration rate or water use efficiency, proposing that
gas exchange factors do not play a major role in overall yield of the giant grass. Usually when a plant
is under environmental stress, phytohormones such as abscisic acid (ABA) regulate a plants ability to
tolerate these stressful situations. It interacts with the proteins (aquaporins) that serve as channels
in the transfer of water across the cell membrane of stomatal guard cells (Kaldenhoff, Ribas-Carbo,
Sans, Lovisolo, Heckwolf, & Uehlein, 2008). ABA alkalise the guard cell cytosol, which causes K+ and
anions to move out of the cell. As a result, the turgor of these guard cells is reduced (Kwak, Maser, &
Schroeder, 2008). The stomata closes when guard cells lose turgidity, thus reducing net assimilation
and transpiration of a plant. If drought occurs for long periods of time, the leaf eventually senesce.
There are three possible explanations as to why gas exchange variables did not differ in M. x
giganteus is: 1.) M. x giganteus uses the efficient C4 photosynthetic pathway that allows plants to
keep stomata closed in dry environments, thus reducing water loss and signalling of ABA. 2.) The LICOR system only read an instantaneous measurement of photosynthesis and transpiration, which
was not a true representation of the gas exchange parameters over the experimental period. 3.) The
area of the leaf that was measured with the LI-COR machine of drought plants were able to maintain
the same photosynthetic rate as those under intermediate and well-watered water supply.
After revising past literature, it is a combination of all factors. M. x giganteus is known to have a
higher water use efficiency per kg DM ha-1 compared to not only C3 plants, but also other C4
counterparts such as maize and switchgrass (Heaton et al., 2008; VanLoocke, Twine, Zeri, &
Bernacchi, 2012; Zhuang et al., 2013). In a glasshouse pot experiment by Clifton-Brown and
Lewandowski (2000), the water use efficiency of the M. x giganteus plants did not differ between
limited (< 12% soil moisture) and non-limited (> 12% soil moisture) water treatments. However, in
the same experiment, M. sinensis plants under non-limited water had a 30% lower water use
efficiency than plants under limited water. This suggests that M. x giganteus plants are able to
maintain photosynthetic ability under mild water stress and do not respond as quickly to water
deficit. However, the photosynthetic rate of M. x giganteus leaves in this study was under half of
those reported in Cosentino et al. (2007) (16 – 19 µmol CO₂ m-2 s-1 compared to 34 – 40 µmol CO₂ m-2
39
s-1). They also found that non-limited plants had a 35% lower water use efficiency than plants under
limited water.
Clifton-Brown, Lewandowski, Bangerth, and Jones (2002) reported in a pot trial that M. x giganteus
plants under limited water supply had 3.5 – 3.8 times higher ABA concentrations than plants under
non-limited water treatments. They quantified a 66% lower ABA concentration in M. sinensis
compared to M. x giganteus (20 ng g-1 DM and 60 ng g-1 DM respectively). As a result, M. x giganteus
plants had a significantly lower leaf area and greenness percentage (based on chlorophyll content)
than M. x sinensis, as ABA triggered leaf senescence. Furthermore, stomatal conductance was
approximately 50% lower in M. x giganteus plants under limited water compared to non-limited
ones. This is similar to findings from this study, where stomatal conductance in plants under drought
were 50% and 39% lower than well-watered and intermediate plants (Fig. 3.6.). This indicates that
less CO2 and water was exiting the leaf. Therefore, water does in fact influence gas exchange
variables of M. x giganteus plants. The above similarities and evidence from past literature also give
sufficient reason to believe that lower leaf area in drought plants compared with well-watered and
intermediate plants of this study was associated with ABA production (3.10). This indicates that the
machine used to measure gas exchange was not a true representation of M. x giganteus plants
overtime.
In this study, the transpiration and photosynthesis were measured with a LI-COR machine, which
measures the gas parameters of a leaf in a chamber. In a leaf chamber, the CO2 concentration around
the leaf varies with the photosynthetic rate. This would explaining why there was such low
photosynthetic rate readings and no differences in gas exchange parameters among water
treatments (Beale, Morison, & Long, 1999). Furthermore, gas exchange measurements where only
taken from the first fully unfolded leaf, which is one of the younger leaves on the plant. Chaves,
Maroco, and Pereira (2003) stated that younger leaves that survive drought are capable of
maintaining a similar photosynthetic rate and RuBisCO content per leaf area as the younger leaves of
non-stressed plants. In drought situations, the older leaves senesce and the nutrients from those
leaves relocate into the younger ones. This explains the high percentage of dead material to
aboveground fresh material in drought plants compared with well-watered and intermediate ones
(Fig. 3.8.).
Plants under well-watered supply had a higher percentage of stem than those of both intermediate
and drought. However, leaf percentage of aboveground fresh weight did not differ among
treatments (Fig. 3.8). This is an important concept in biofuel production of feedstocks because the
stem has a higher cellulose percentage and lower ash content than leaves, which is desired (Singh,
40
2013). Furthermore, a lower stem percentage could relate to the no-difference in average tiller
number of plants under drought and intermediate treatments.
Tiller number is an important concept as it correlates with the density of a crop (DeDatta, 1981).
Well-watered plants had 113.3% and 220% more tillers at final harvest than intermediate and
drought plants respectively (Fig. 3.5.). In a field situation, a density increase in M. x giganteus
shelterbelts would enhance the ecosystem provision from the plant. A denser crop reduces the
permeability of wind. This provides better shade and shelter to pastures, beneficial insects and
livestock from environmental stresses. As a consequence, pasture species can keep stomata open for
longer, thus increasing plant yield (Littlejohn et al., 2014). Livestock production also increases as
energy is allocated to growth instead of coping with environmental stresses (Gregory, 1995).
Plants under intermediate treatments did not differ in the number of tillers because they were under
mild water stress. As described above, plants under stress will close stomata, thus reducing
photosynthetic rate of leaves (Blum, 2011; Chen & Jiang, 2010). As a result, plants increase root to
shoot dry weight ratio in response to water stress. In drought plants, the root: shoot ratio was 3.3
times greater than plants under the well-watered treatment (Fig. 3.15.). Roots have a greater
osmotic adjustment compared with leaves (Blum, 2011). Solutes such as proline, move into the root
cells, which increase the movement of water into the cells from the soil so they can maintain turgor
for cell expansion (Chen et al., 2010). Furthermore, ABA plays a role in root extension. As described
previously, ABA inhibits leaf area expansion. However studies have shown that it promotes lateral
root extension by interacting with the plant growth hormone, auxin (Blum, 2011). Auxin activates the
proton pump, H+-ATPase, to release more protons to the root tip (Xu, Jia, Shi, Liang, Zhou, Li, &
Zhang, 2013). An accumulation of protons lower the pH of the cell which cause root elasticity for cell
expansion (Rayle & Cleland, 1992). This would benefit a plant in field conditions, as roots are able
exploit water and nutrients that are further down the soil profile.
Understanding water use efficiency between water treatments could be improved if water use
efficiency was calculated as the biomass produced from the water applied, instead of the ratio of
photosynthesis to transpiration. Biomass is a better representation than photosynthesis because it is
the actual production of a plant, instead of the ability of a plant to uptake CO2. However, the amount
of water applied to well-watered plants is unknown. Applying trays under the pots would allow the
biomass to water ratio to be calculated.
4.3 Trichoderma
Inoculating plants with T. atroviride had an effect on the number of fungal cultures as expected.
Inoculated plants had approximately three times more fungal cultures than control plants (Table
41
3.4.). When plants were inoculated for 24 hours, Trichoderma spp. were able to colonise the outer
cortex and epidermis of M. x giganteus roots (Harman et al., 2004; Yedidia et al., 1999). However,
although it was clear that the fungus was present, the relative abundance of isolated T. atroviride
cultures of inoculated roots was lower than expected. Each disc had five root sections per disc, with
four discs per treatment. Therefore, the abundance of cultures were 80% lower than the potential
number of fungal cultures that could have been present when added across all three water
treatments (12 comped to 60 cultures). Understanding why culture numbers were low is important
for future inoculation and management practices when dealing with T. atroviride in the field.
T. atroviride react in a similar way to mycorrhizae whereby the benefits on plants are more evident
under nutrient stress conditions (Harman et al., 2008; Shoresh et al., 2010). Trichoderma spp. secrete
chelating molecules such as siderophores that solubilise a range of unavailable nutrients so they
become available for plant uptake (Altomare, Norvell, Bjorkman, & Harman, 1999; Mastouri,
Bjorkman, & Harman, 2010; Shoresh et al., 2010). In a field trial, maize seed inoculated with
Trichoderma harzianum (Rifai) T22 grown under low soil conditions required 53% less nitrogen
fertiliser to reach the same yields than when seeds were not treated (Harman, 2001). In this study,
the soil media supplied sufficient nutrients to M. x giganteus plants, thus the production benefits
were not evidential as the nutrients were already available to the plants. Alternatively, ample
nutrients may have reduced the activity of root colonisation by T. atroviride. In nutrient-stressed
plants, soluble carbohydrates are released to the roots cells, consequently supporting more
mycorrhizae than those with ample nutrients (Johnson, 1993; Sylvia & Neal, 1990). Therefore,
although they may have been present in the root rhizosphere, they were not present in adequate
abundance within the epidermis and outer cortex. When the roots were surface sterilised during the
isolation culture process, many of the fungi were removed.
Fungal cultures that appeared in control plants may have contaminated the root sections following
sterilisation from the surrounding air. This was evident by the other pathogens that were present in
the fungal root cultures such as Fusarium spp. and Penicillium spp. (Fig. 4.1). To test this theory,
identifying the strains of Trichoderma cultures derived from the root sections would be valuable to
decipher between contamination and intentionally inoculated fungi. There are many pathogen
competitors in the root rhizosphere, so this contamination theory is a possibility even though
Trichoderma spp. are predatory fungi that are highly competitive for space and nutrients (Harman et
al., 2008). It would be valuable to identify the actual Trichoderma strains in the control. If T.
atroviride was present, then they have contaminated control plants either during the experiment,
e.g. from a water droplet, or from the air following root sterilisation.
42
Figure 4.1. An illustration of the variable fungal cultures isolated from the root sections of M. x
giganteus plants. Trichoderma spp. are recognised by the white downy like appearance e.g.
The bottom left disc contains five Trichoderma cultures from five root sections.
If the fungal culture Trichoderma strains differed between inoculated and control strains, then this
would explain why inoculated plants had a lower dry matter percentage than control plants (Fig. 3.14
and Table 3.3), but remained similar in other physiological, growth and development variables. A
higher dry matter percentage indicates that plants have less water within their cells. Water is
important for many metabolic process and chemicals functions within a cell. For example, cell
elongation, nutrient transportation, temperature regulation etc. Therefore, a lower dry matter
percentage indicates healthier plants. Although T. atroviride abundance was low, the fungi that were
present or those within the root rhizosphere did effect the water uptake of the plant enough to
detect a difference on the dry matter percentage. However, the dry matter percentage was only
different between Trichoderma treatments under drought, suggesting that water plays a role in the
effectiveness of T. atroviride. This theory will be discussed in ‘4.4 Trichoderma and water interaction’.
Trichoderma spp. produce secondary metabolites, which are chemicals that indirectly induce plant
growth by interacting with phytohormones such as indole-3-acetic acid (IAA), cytokinin and auxin
(Martinez-Medina et al., 2014; Vinale et al., 2008). Consequently, the root system can spread to
greater depths, increasing the nutrient and water uptake availability for further growth (Harman et
al., 2004; Martinez-Medina et al., 2014). As the M. x giganteus plants were in 5 L pots, the lateral
root growth would have been obstructed. If this experiment was repeated in a field trial, the roots
would have the ability to reach water further down into the soil profile, impacting on other variables
such as production.
43
A repeated field trial of the glasshouse experiment using the same PR5 T. atroviride strains under
variable water treatments is recommended. If there is a significant difference in plant physiological,
development or growth variables, then it is assumed that obstruction of roots influenced the
effectiveness of T. atroviride in this study. Furthermore, M. x giganteus plants should be grown in
variable fertility soils to show how T. atroviride will suffice under different soil conditions.
4.4 Trichoderma and water interaction
The inoculated plants had a higher number of isolated fungal cultures than control plants (Table 3.4).
As mentioned above, this number was lower than expected. However, water may play a significant
role in the survival and activity of T. atroviride.
Inoculated well-watered M. x giganteus plants had substantially higher numbers of T. atroviride
fungal cultures than inoculated plants under intermediate and drought treatments (Table 3.4).
Inoculated roots from the well-watered M. x giganteus plants had over 50% colonisation. The control
plants under the same water treatments had the lowest number of fungal cultures over all control
water treatments (5%), of which could have been due to contamination following sterilisation. This
suggests that as water stress intensified, the survival, competitive ability and growth of T. atroviride
became limited over time (by final harvest).
Trichoderma spp., like most soil-borne fungi, require free water for movement and growth. As the
soil moisture content decreased, T. atroviride growth also declined. The competitive ability was no
longer sufficient, which allowed for more xerotolerant pathogens such as other Trichoderma spp. or
Fusarium spp. to colonise the M. x giganteus roots. Kredics, Antal, and Manczinger (2000) measured
the effect of soil water potential on T. harzianum colony growth. They reported no growth at a water
potential below -11 MPa at 25 °C and -9 MPa at 10 °C. Dix and Webster (1995) concluded that nonxerotolerant pathogens will actively grow down to about -6 MPa, which is the equivalent of 14% soil
moisture, but some growth will occur down to about -14.5 MPa (8% soil moisture). However,
xerotolerant species such as Penicillium can continue to grow in water potentials as low as -30 MPa
(2-3% soil moisture). This suggests that the strains of T. atroviride used in this study were not
tolerant to dry conditions. Plants under both intermediate (5% soil moisture) and drought (2.5% soil
moisture) treatments had a lower abundance of T. atroviride cultures compared with well-watered
plants. Furthermore, the cultures that were isolated from the discs of intermediate and drought
plants were possibly other strains of xerotolerant Trichoderma spp.
Chlorophyll content is the amount of chlorophyll pigments (Chl a and Chl b) that are essential in the
conversion of captured light radiation to stored chemical energy (Gitelson, Gritz, & Merzlyak, 2003).
This is an important determinant of photosynthetic capacity, which contributes to the overall
44
productivity of a plant (Curran, Dungan, & Gholz, 1990). Plants that are under stress often have a
lower chlorophyll content because much of the leaf nitrogen is incorporated into the pigments
(Moran, Mitchell, Goodmanson, & Stockburger, 2000; Percival, Keary, & Noviss, 2008). When plants
are stressed, the nitrogen from leaves are translocated to newer ones as discussed previously.
Therefore, chlorophyll content is an indication of the health status of a plant (Moran et al., 2000).
In terms of the effect on physiological variables, chlorophyll content and dry matter percentage were
higher in inoculated drought plants than control ones (Fig. 3.7. and Fig. 3.14. respectively). Therefore,
T. atroviride has affected these two variables at some point in the experiment by pushing the content
of chlorophyll and dry matter towards those values of intermediate and well-watered plants.
Consequently, the inoculated drought plants did not differ in chlorophyll content or dry matter
percentage compared with all intermediate and well-watered plants. This suggests T. atroviride
maintained sufficient growth under drought conditions earlier on in the experiment to affect these
environmental variables. However, colonies were not present for long enough to have an effect on
growth and production variables.
In intermediate plants, inoculation with T. atroviride would have had no effect on physiological
variables because the plants were able to maintain similar chlorophyll content and dry matter
percentage as those of well-watered plants anyway. Therefore, the T. atroviride activity decreased,
as plants were neither water nor nutrient stressed enough to support T. atroviride colonies. In
drought plants, activity of fungi would have be observed because the solutes in the roots, due to
water stress, would have supported the activity of fungi. As the plants grew, the water requirements
for maintenance growth increased to a point where the water potential of soil was too high that it
could no longer support T. atroviride colonies.
To test whether the T. atroviride strains used in this study are non-xerotolerant fungi, cultures should
have been taken throughout the experiment. This would indicate whether the water supplied at the
beginning of the trial was able to support the growth and competiveness of the T. atroviride strains.
If there were higher abundancies of isolated Trichoderma cultures at the beginning of the pot trial, a
strain test would be valuable to ensure that those fungi are in fact the strains that were used for
intentional inoculation of M. x giganteus plants. This would also be valuable in determining what
strains had out competed the T. atroviride strain mix used in this study.
4.5 Conclusions
Main conclusions, contribution to the research, limitations and future prospects of M. x giganteus
will be discussed in this section. However, much of the detail is covered above in the sections of each
experimental chapter, so those aspects are not repeated in detail.
45
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